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Product Inserts 2014
www.spectrum-diagnostics.com
The creative Approach to Bioscience
Egyptian Company For Biotechnology (S.A.E.) Obour city industrial area. block 20008 piece 19 A. Cairo. Egypt. Tel: +2 02 4665 1848 Fax: +2 02 4665 1847 E-mail: [email protected]
Contents A- Clinical Chemistry
7
i-Substrates 8 Albumin - BCG
10
Bilirubin (TOTAL AND DIRECT)
12
Pediatric Total BILIRUBIN
14
TOTAL Bilirubin
16
Bilirubin (DIRECT)
18
Bilirubin (TOTAL AND DIRECT)
20
Cholesterol – Liquizyme
22
CREATINE KINASE MB
86
g - Glutamyltransferase-(gGT)-Liquizyme (9+1)
88
g - Glutamyltransferase-(gGT)-Liquizyme (1+1)
90
Aspartate aminotransferase (AST/GOT)-Liquizyme (4+1) 92 Aspartate aminotransferase (AST/GOT)-Liquizyme (1 + 1) 94 Aspartate aminotransferase (AST/GOT)-Colorimetric
96
Aspartate aminotransferase
98
Alanine aminotransferase (ALT/GPT)-Liquizyme (4+1)
100
Alanine aminotransferase (ALT/GPT)-Liquizyme (1 + 1)
102
Alanine aminotransferase (ALT/GPT) - Colorimetric
104
Alanine aminotransferase (ALT/GPT) - Ultimate Single Reagent 106 Glucose-6-Phosphate dehydrogenase (G-6-PDH)
108
Rheumatoid Factor (RF)
170
D- turbidimetric immunoassay
173
Antistreptolysin O (ASO) Immuno-Turbidimetry
E- Infectious immunology, Bacteriology 2 34 Brucella A, Brucella M, Brucella Combo
236
174
Widal Test (Salmonella Ab)
238
Antistreptolysin O (ASO) Turbi Latex
176
RPR SYPHILIS TEST
240
ASO Standard Super High
178
Rose Bengal Brucella Antigen
242
ASO Control
179
TOXO LATEX KIT
244
b2 -MICROGLOBULIN (b2 -m)
180
b2 -MICROGLOBULIN Mono-Reagent Procedure
182
PT Reagent (SP-NORMOPLASTIN)
C REACTIVE PROTEIN (CRP) Immuno-Turbidimetry
184
NORMAL AND ABNORMAL PLASMA CONTROLS FOR COAGULATION ASSAYS 250
C REACTIVE PROTEIN (CRP) Turbi Latext
186
SP-Ultraplastin PT Reagent ISI 1.05
252
Ceruloplasmin Immuno-Turbidimetry
188
APTT Reagent (SP- UNICELIN)
254
CRP Standard Super High
189
CRP Control
190
CRP/hs(C- Reactive protein)
192
CYSTATIN-C Immunoturbidimetry
194
FERRITIN Turbi Latex
196
FIBRINOGEN Immuno - Turbidimetry
198
F- Hematology, Haemostasis Reagents 2 47 248
HDL CHOLESTEROL - Precipitant
24
HDL CHOLESTEROL
26
LDL CHOLESTEROL
28
Creatinine – Jaffè
30
Creatinine – Jaffè
32
Creatinine - Colorimetric
34
GLUCOSE - Liquizyme
36
Ammonia II (GLDH - UV - Test)
120
Fibrinogen Standard
200
GLYCOSYLATED HEMOGLOBIN (GHb)
38
Ammonia – Liquizyme Single Reagent
122
HAPTOGLOBIN (HAP) Immuno Turbidimetry
202
Direct Enzymatic HbA1c
40
Calcium O-CPC
124
Hemoglobin A1c Standard Set
203
CMV IgG/IgM Rapid Test - Cassette
Haemoglobin(Single Reagent)
42
Calcium Arsenazo III (Single Reagent)
126
Hemoglobin A1c (HbA1c) Immuno - Turbidimetry
204
H. pylori Ab Test Device (Serum / Plasma / Whole Blood) 272
Haemoglobin 44
Chloride Single Reagent
128
IMMUNOGLOBULIN E (IgE)
206
H. pylori Ag Test Device (Fecal Specimen)
274
Haemoglobin (Ready-To-Use)
46
Carbon Dioxide (CO2) (Colorimetric PEPC)
130
Immunology Control High
208
HAV IgM Rapid Test-Cassette (Serum / Plasma)
276
Lactate – Liquizyme
48
Copper 132
Immunology Control Low
209
HBsAg Hepatitis B Surface Antigen Test Device
278
Micrototal Protein (MT-P)
50
Total Iron Chromazurol B Single Reagent
134
Kappa light chain serum kit (KAP)
210
HBsAg/HCV Ab Rapid Test - Cassette
280
PYRUVATE 52
Total Iron and Total Iron Binding Capacity Reagent Set
136
Kappa light chain urine kit (KAP)
211
HCG One Step Pregnancy Test Device (Urine/Serum)
284
Total bile acids (TBA)
54
Total Iron Binding Capacity (TIBC)
138
Lambda light chain serum kit (LAM)
212
HCV Ab Hepatitis C Virus Ab Test Device (Serum/Plasma) 286
Total protein
56
Total Iron - Ferrozine
140
Lambda light chain urine kit (LAM)
213
HIV Ab 1/2/O
288
Triglycerides – Liquizyme
58
MAGNESIUM Xylidyl Blue Monoreagnt
142
LIPOPROTEIN (a) [Lp(a)] Mono-Reagent Procedure
214
Malaria Antigen Test (Whole Blood)
290
Urea/BUN - Liquizyme
60
Phosphorus, Inorganic
144
Microalbumin Standard
216
S.typhi-S.paratyphi “A” Direct Antigen Detection
292
Urea/BUN - LS
62
INORGNIC PHOSPHORUS
146
MICROALBUMIN (MAU) Immuno Turbidimetry
218
Syphilis Ab Combo Rapid Test
294
Urea/BUN – Liquizyme (UV)
64
Potassium 148
MICROALBUMIN (MAU) Turbi Latex
220
ToRCH 296
Urea/BUN – (UV)
66
Sodium Single Reagent
150
Microalbumin Control set
222
Toxo IgG/IgM Rapid Test- Cassette
298
Uric acid - Liquizyme
68
ZINC (Colorimetric Test with 5-Brom-PAPS)
152
Protein standard High
223
Troponin-I
300
Procalcitonin Assay
224
Troponin-I
302
RF Control
226
Typhoid IgG/IgM Rapid Test - Cassette
304
RF Standard Super High
227
URI-TRAK 306
RHEUMATOID FACTOR (RF) ImmunoTurbidimetry 3rd Generation 228
URI-TRAK-10 308
ii-Enzymes 71
Glucose-6-Phosphate 110 Lactate dehydrogenase (LDH)-Liquizyme (4+1)
112
Lactate dehydrogenase (LDH)-Liquizyme (1+1)
114
LIPASE-LS COLORIMETRIC (DGMRE)
116
iii-Electrolytes 119
B-Quality control
155
Acid Phosphatase (ACP)
72
SP-NORMOTROL 156
Alkaline phosphatase (ALP)
74
SP-PATHOTROL 158
Alkaline phosphatase (ALP)
76
SP-MULTICAL 160
ALKALINE PHOSPHATASE
78
HbA1c Calibrator
Alkaline Phosphatase - Colorimetric
80
Alpha Amylase
82
Antistreptolysin O Titre (ASOT)
166
Creatine Kinase (CK)
84
C Reactive Protein (CRP)
168
162
C-immunochemistry 165
RHEUMATOID FACTOR (RF) Turbi Latex
230
Specific Protein Control
232
Specific Protein standard
233
G-blood group
256
Spectrum Anti-A, Anti-B,Anti-AB Monoclonal (IgM)
258
Anti-D (Rho) Plus Monoclonal IgM & IgG
260
ANTI-HUMAN GLOBULIN SERUM (COOMBS)
262
BOVINE SERUM ALBUMIN (BSA)
264
Low Ionic Strength (LISS) Solution
266
H-Dry chemistry
269 270
A- Clinical Chemistry
6
7
i-Substrates
8
9
Albumin - BCG
SYMBOLS IN PRODUCT LABELLING
(Acetate Buffer) REF: 211 001 REF: 211 002 REF: 211 003 REF: 211 004
EC REP Authorised Representative
(2x100 ml) 200 test (4x100 ml) 400 test (2x500 ml) 1000 test (2x250 ml) 500 test
IVD LOT REF
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Specimen Collection and Preservation
Spectrum Diagnostics albumin reagent is intended for the in- v i t r o quantitative,diagnostic determination of albumin in human serum on both automated and manual systems.
The only acceptable anticoagulats are heparin and EDTA. Use preferably fresh serum, Serum should be separated immediately from the clot. The biological half-life of albumin in blood is 3 weeks. o o Stability: 1 day at 15 – 25 C; 4 weeks at 4 – 8 C; o 6 months at -20 C
Albumin is the major serum protein in normal individuals. It maintains the plasma colloidal osmotic pressure, binds and solubilizes many compounds such as calcium and bilirubin. Elevated serum albumin levels are usually the result of dehydration. Hyperalbuminemia is of little diagnostic significance. Hypoalbuminemia is very common in many diseases including malabsorption, liver diseases. kidney diseases, severe burns, infections, cancer and some genetic abnormalities. In severe hypoalbuminemia (less than 2.5 g/dL), the low plasma oncotic pressure allows water to move out of the blood capillaries into the tissues causing edema.
Method Modified bromocresol green colorimetric method.
Measurement of albumin is based on its binding to the indicator dye bromocresol green (BCG) in pH 4.1 to form a blue-green colored complex. The intensity of the blue-green color is directly proportional to the concentration of albumin in the sample. It is determined by monitoring the increase in absorbance at 623 nm, or 578 nm. Albumin + BCG
System Parameters
pH 4.1
Albumin-BCG Complex
Wavelength 623 nm ( or 578 nm ) Optical path 1 cm Assay type End-point Direction Increase Sample : Reagent Ratio 1 : 100 e.g.: Reagent volume 1 ml Sample volume 10 ml o Temperature 20 – 25 C o Incubation time 5 minutes at 20–25 C Zero adjustment Reagent Blank Sensitivity 1 g/dL Linearity 7 g/dL
Spectrum albumin reagents are supplied ready-to-use and stable up to the expiry date labeled on the bottles. The reagents are stable o at 2 – 8 C.
Deterioration Do not use the Spectrum albumin regents if precipitate forms. Failure to recover control values within the assigned range may be an indication of reagent deterioration. Specimen Collection and Preservation.
10
Grant GH, Silverman LM, Christenson RH. Amino acids and proteins. In:Tietz NW, ed. Fundamentals of Clinical Chemistry. 3rd ed. Philadelphia:WB Saunders;1987:291 345.
3.
Tietz NW, ed. Clinical Guide to laboratory tests. 2nd ed. Philadelphia: WB Saunders; 1990:26-29.
Linearity The reaction is linear up to an albumin concentration of 7.0 g/dL; specimens showing higher concentration should be diluted 1+1 with physiological saline and repeat the assay (result × 2).
ORDERING INFORMATION CATALOG NO.
QUANTITY
211 001 211 002 211 003 211 004
2 x 100 ml 4 x 100 ml 2 x 500 ml 2 x 250 ml
Expected Values Adults
Blank
Standard
specimen
Reagent (R)
1 ml
1 ml
1 ml
Standard
-------
10 ml
-------
Specimen
-------
-------
10 ml
18 – 60 y >60 y
3.5 – 5.5 g/dL 3.4 – 4.8 g/dL
(35 – 50 g/L) (34 – 48 g/L)
3.2-4.5 g/dL 3.8-5.4 g/dL
(32-45 g/L) (38-54 g/L)
Childern 14-18 y 4d-14 y
o
Calculation
Reagent Preparation, Storage and Stability
2.
Sensitivity
Lipemia No significant interference up to an intralipid level of 1000 mg/dL.
Standard albumin 4.0 g/dL.
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
Doumas BT,Watson WA, Biggs HG. Albumin standard and the measurement of serum albumin with bromocresol green Clin Chim Acta. 1971;31:87-96.
Icterus No significant interference up to a bilirubin level of 40 mg/dL.
Reagents
Precautions and Warnings
1.
Haemolysis A haemoglobin level of 800 mg/dL results in 13 % positive bias.
Mix , incubate for approximately 5 minutes at 20-25 C. Measure absorbance of specimen (Aspecimen) and standard (Astandard) against reagent blank within 60 minutes.
Reagent (R) Acetate Buffer 100 mmol/L Bromocresol green 0.27 mmol/L Detergent For further information, refer to the Albumin reagent material safety data sheet.
A comparison between Spectrum Diagnostics Albumin reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.97 was obtained.
Interfering Substances Serum, plasma
Procedure
Assay Principle
References
When run as recommended, the minimum detection limit of this assay is 1.0 g/dL.
Intended Use
Background
Methods Comparison
Albumin concentration (g/dL) =
Aspecimen Astandard
Quality Control
Newborns 0-4 day
2.8-4.4 g/dL
(28-44 g/L)
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure ; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
x4
Normal & abnormal commercial control serum concentrations should be analyzed with each run.
of
known
Analytical Range 1.0 – 7.0 g/dL.
Performance Characteristics Precision Within run (Repeatiblity)
Waste Disposal Level 1
Level 2
n
20
20
Mean (g/dL)
3.28
4.78
SD
0.8
0.12
CV%
2.66
2.68
Level 1
Level 2
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
Run to run (Reproducibility) n
20
20
Mean (g/dL)
3.4
4.9
SD
0.9
0.14
CV%
3.1
2.9
11
Bilirubin (TOTAL AND DIRECT) Jendrassik Grof
REF: 222 001 (255ml) 100 Test
EC REP Authorised Representative IVD
REF: 222 002 (750ml) 300 Test
LOT REF
R1 Sulphanilic Acid 1 x 45 ml R1 Sulphanilic Acid 2 x 65 ml R2 Nitrite
1 x 10 ml R2 Nitrite
2 x 15 ml
R3 Caffeine
1 x 100 ml
R3 Caffeine
3 x 100 ml
R4 Tartarate
1 x 100 ml
R4 Tartarate
3 x 100 ml
Intended Use Spectrum Diagnostics bilirubin reagent is intended for the in-vitro quantitative, diagnostic determination of bilirubin in human serum on both automated and manual systems.
Background The average level of the bilirubin produced in humans from different sources ranges between 250 to 300 mg/day, of which 85% is derived from the heme moiety of the haemoglobin released from senescent erythrocytes that are destroyed in the reticuloendothelial system. The remaining 15 % is produced from erythrocytes destroyed in the bone marrow and from catabolism of other heme containing proteins such as cytochromes and myoglobin. After it is produced in the peripheral tissues, bilirubin is transported to the liver in association with albumin. In the liver, bilirubin is conjugated with glucuronic acid for solubilization and subsequent transport through the bile duct and elimination via the digestive tract. Disease or conditions which, through hemolytic processes, produce bilirubin faster than the liver can metabolize it, cause the levels of unconjugated (indirect) bilirubin to increase in the circulation. Bile duct obstruction or damage to hepatocellular structure causes increases in the levels of both conjugated (direct) and unconjugated (indirect) bilirubin in the circulation.
Method
Colorimetric Diazo method. The total bilirubin concentration is determined in presence of caffeine by the reaction with diazotized sulphanilic acid to produce an intensely colored diazo dye (560-600 nm). The intensity of color of this dye formed is proportional to the concentration of total bilirubin. Direct bilirubin is determined in absence of caffeine by the direct reaction with diazotized sulphanilic acid to form red-colored azobilirubin, the color intenisity of which measured at 546 nm is proportional to the concentration of the direct bilirubin in the sample. HCL
Bilirubin + Diazotized sulfanilic acid
Diazotized sulfanilic acid pH 1.4
Azobilirubin
Reagents Reagent 1 (R1) Sulfanilic acid HCL
31.0 mmol/l 0.20 N
Reagent 2 (R2) Sodium nitrite
28.0 mmol/l
Reagent 3 (R3) Caffeine Sodium benzoate
0.28 mol/l 0.55 mol/l
Reagent 4 (R4) Tartarate Sodium hydroxide
0.99 mol/l 2.0 N
12
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use Temperature Limitation
Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by (C) - Corrosive
!
Reagent 1
Sample
blank Sample
200 ml
200 ml 1 drop
Reagent 2
-----
Saline 0.9% NaCl
2.0 ml
2.0 ml
Sample
200 ml
200 ml
Reagent 4 contains caustic material. Corrosive (C) R35 Causes severe burns. R41 Risk of serious damage to eyes. S26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. S28 After contact with skin, wash immediately with plenty of soap and water. For further information, refer to the •Bilirubin reagent material safety data sheet.
Precautions and Warnings Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
Reagent Preparation, Storage and Stability Spectrum bilirubin reagents are supplied ready-to-use and stable up to the expiry date labeled on the bottles when stored at room temperature.
Deterioration
Expected Values
Mix and incubate for exactly 5 minutes at 20 – 25 C. Measure absorbance of sample (Asample) against sample blank at 546 nm (530 - 560 nm).
Total Bilirubin Adults and infants>1 month < 0.2-1.0 mg/dL Newborns premature (3-5 d) 10-14 mg/dL
Calculation
Newborns: (3-5 d) (1.42
45 - 65 1.16 - 1.68 35 - 55 0.90 - 1.42
1 year at -20 C. Urine Thymol or toluene may be used for urine preservation. To determine creatinine concentration in urine, dilute 1 part sample with 49 parts isotonic saline prior to assay. Multiply result by 50 to compensate for dilution. o o Stability: 2 days at 15 - 25 C ; 6 days at 2 - 8 C o 6 months at -20 C away from light
System Parameters Wavelength 492 nm Optical path 1 cm Assay type Fixed Rate Direction increase Sample : Reagent Ratio 1 : 10 e.g.: Reagent volume 1 ml Sample volume 100 ml First read time 30 seconds delay time 120 seconds last read time 150 seconds o Temperature 30 C Zero adjustment Against Air Reagent Blank Limits Low 0.30 AU High 0.8 AU Sensitivity 0.31 mg/dL (0.027 mmol/L) Linearity 20 mg/dL (1.77 mmol/L)
Procedure Reagent (R) Standard or Specimen
1.0 ml 100 ml
Mix, and after 30 seconds. read the absorbance A1 of the standard or specimen. After exactly 2 minutes later, read absorbance A2 of standard or specimen.
Calculation
A2 – A1 = Aspecimen or Astandard. Concentration of creatinine in serum: (Aspecimen) Creatinine (mg/dL) = (Astandard) Concentration of creatinine in urine: (Aspecimen) Creatinine (mg/dL) = (Astandard)
Serum creatinine / min. per standard surface area = UCr x V PCr Where: UCr PCr V A 1.73/A
x
1.73 A
= Concentration of creatinine in urine( mg/dl) = Concentration of creatinine in plasma(mg/dl) = Volume of urine flow in mL/min. = Body surface area in square meter . = Factor normalizes clearance for average body surface.
Note: Body surface area can be determined from height weight via normograms in Tietz(6).
Quality Control
Normal & abnormal commercial control serum concentrations should be analyzed with each run.
of
known
Creatinine clearance (ml/minutes): mg creatinine / dl urine x ml urine / 24 hours
0.7-1.3 mg/dL 0.9-1.5 mg/dL
Urine(24 hrs) Females Males
0.9 – 1.6 g/24 hrs 1.1 – 2.8 g/24 hrs
62-115 mmol/L 80-133mmol/L
Creatinine clearance Females 75 – 115 ml / min. Males 85 – 125 ml / min. Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Waste Disposal
Within run (Repeatiblity)
Level 1
Level 2
20
20
Mean (mg/dL)
1.55
4.58
SD
0.069
0.1
CV%
4.45
2.2
Level 1
Level 2
n
20
20
Mean (mg/dL)
1.67
4.63
SD
0.081
0.19
CV%
4.58
2.7
Run to run (Reproducibility)
Methods Comparison
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References 1. 2. 3.
A comparison between Spectrum Diagnostics Creatinine Jaffè Single reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.991 was obtained.
4.
Sensitivity
5.
When run as recommended, the minimum detection of this assay is 0.31 mg/dL creatinine (0.027 mmol/L).
6.
Bowers LD, Wong ET: kinetic serum creatinine assays. II. A critical evaluation and review. Clin Chem 26:555, 1980. Doolan PD, Alpen EL, Theil GB: A clinical appraisal of the plasma concentration and endogenous clearence of creatinine. AM J Med 32:65, 1962. DI Giorgio J: Nonprotein nitrogenous constituents. In: clinical chemistry – principles and technics, 2 nd ed. RJ Henry, DC Cannon, JW Winkelman, editors, Harper and Row, Hagerstown (MD), 1974, pp 541-553. Spencer K, Price CP: A review of Non-enzyme mediated reaction and their application to centrifugal analyzers. IN centerfugal analyzers in clinical chemistry, CP Price, K Spencer, editors, Praeger publishers, New York, 1980, p231. Tobias GJ, Mclaughlin RF, Hopper J: Endogenous creatine clearence. N Engl j Med 266:317, 1962. Tietz NW: Textbook of clinical chemistry. WB saunders, philadelphia, 1986, pp 1271- 1281.
Linearity
The reaction is linear up to serum creatinine concentration of 20mg/ dL (1.77 mmol/L). Specimens showing higher concentration should be diluted 1+4 using physiological saline and repeat the assay (result×5). Serum, plasma
x 2 x 50
Serum, plasma Females Males
0.31 – 20 mg/dL (0.027-1.77 mmol/L).
Interfering Substances x2
Expected Values
Analytical Range
Performance Characteristics Precision
n
Pipette into test tubes 25 mmol/L 0.4 mol/L
Irritant (xi) R36/38: Irritating to eyes and skin. S26: In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. S37/39: Wear suitable gloves and eye/face protection.
Deterioration
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use Temperature Limitation
Correction for body surface area can be done using the following formula for creatinine clearance:
ORDERING INFORMATION CATALOG NO
QUANTITY
237 001 237 002 237 003
2 x 100 ml 4 x 100 ml 2 x 500 ml
Haemolysis Erythrocyte contamination doesn’t elevate results. Icterus Serum bilirubin levels higher than 5 mg/dL (85 mmol/L) decrease serum creatinine. Lipemia Lipemic specimens may cause high absorbance flagging. Diluted sample treatment may be recommended.
mg creatinine / dl serum x 1440
33
Creatinine - Colorimetric
EC REP Authorised Representative
REF: 235 001 REF: 235 002 REF: 235 003 REF: 235 004
IVD
2 x 100 ml 4 x 100 ml 2 x 800 ml 2 x 500 ml
LOT REF
Intended Use
Spectrum Diagnostics creatinine reagent is intended for the in-vitro quantitative, diagnostic determination of creatinine in human serum or urine on manual system.
Background
Creatine is synthesized in kidney, liver and pancreas. It is transported in blood to other organs such as muscle and brain where it is phosphorylated to phosphocreatine. Some free creatine in muscle is converted to creatinine daily, and the amount of creatinine produced is proportional to muscle mass. In the absence of renal disease, excretion rate of creatinine in an individual is relatively constant. Therefore, measurement of creatinine clearance is useful in detecting renal disease and estimating the extent of impairment of renal function. Both serum creatinine and urea levels are elevated in patients with renal malfunction, especially decreased glomerular filtration. In the erarly stage of kidney damage, increase in serum urea level usually precedes the increase in serum creatinine .However serum urea levels may be affected by dehydration, diet and protein metabolism. On the other hand serum creatinine levels tend to be constant and unaffected by such factors. Thus serum creatinine is a significantly more reliable renal function screening test than serum urea.
Method
Colorimetric method with deproteinization. Assay Principle
Reagents
Standard creatinine (ST) 2 mg/dL
177 mmol/L
Reagent 1 (R1)
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use Temperature Limitation
All reagents are stable until expiration date stated on label when stored at 15 - 25 oC. Working solution is stable for 5 hours at 15 – 25 oC away from light.
Deterioration
The creatinine reagents are not suitable for use if combined reagents have an absorbance greater than 0.6 at 492 nm measured in a 1-cm lightpath or if the reagents develop a hazy appearance.
Specimen Collection and Preservation Serum or plasma Both are suitable for analysis. The only acceptable anticoagulants are heparin and EDTA. Specimen should be promptly separated from cells after blood collection. The biological half-life of creatinine in blood is few minutes. Stability: 7 days 2 - 8 oC ; > 1 year at – 20 oC
Urine
Thymol or toluene may be used for urine preservation. To determine creatinine concentration in urine, dilute 1 part sample with 49 parts isotonic saline prior to assay. Multiply result by 50 to compensate for dilution. o o Stability: 2 days at 15 - 25 C ; 6 days at 2 - 8 C o 6 months at -20 C away from light
Procedure
Pipette into test tubes
Reagent 2 (R2)
Reagent 2 contains caustic material. Corrosive (C) R35 R41 S26 S28
cause severe burns. Risk of serious damage to eyes. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. After contact with skin, wash immediately with plenty of soap and water.
Serum creatinine / min. per standard surface area = UCr x V PCr
x
1.73
Blank
Standard
Sample
Urine
0.5 ml
-------
-------
-------
Standard
-------
0.5 ml
-------
-------
TCA
0.5 ml
0.5 ml
-------
0.5 ml
Supernatant
-------
-------
1.0 ml
-------
Urine (1+ 49)
-------
-------
-------
0.5 ml
Reagent mixture
1.0 ml
1.0 ml
1.0 ml
1.0 ml
o
Mix and let stand for 20 minutes. at 20–25 C. Measure the absorbance of specimen and standard against reagent blank at 546 nm.
Calculation
Concentration of creatinine in serum: (Aspecimen) Creatinine (mg/dL) = x 2 (Astandard)
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure;interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
A
Where: UCr = Concentration of creatinine in urine (mg/dL) PCr = Concentration of creatinine in plasma (mg/dL) V = Volume of urine flow in mL/min. A = Body surface area in square meter . 1.73/A = Factor normalizes clearance for average body surface. Note : Body surface area can be determined from height weight via normograms in Tietz(6).
Quality Control
Normal & abnormal commercial control serum concentrations should be analyzed with each run.
of
known
Methods Comparison
Distilled Water
1.6 mol/L
Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by (Xi) - Irritant (C) - Corrosive
!
Reagent Storage and Stability
Picric acid 38 mmol/L Reagent 1 contains a low concentration of picric acid, a chemical which, in its dry form, is flammable and potentially explosive. For this reason, it is recommended that drains be well flushed with water when discarding the reagent, spills be cleaned up at once, and dried material not be allowed to build up around the reagent bottle opening
Sodium hydroxide
Correction for body surface area can be done using the following formula for creatinine clearance:
SYMBOLS IN PRODUCT LABELLING
A comparison between Spectrum Diagnostics Creatinine colorimetric reagent and acommercial reagent of the same methodology was performed on 40 human sera. A correlation (r) of 0.996 was obtained.
Dynamic Range
0.4 - 15 mg/dL (0.035 - 1.32 mmol/L).
Waste Disposal
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References 1. 2.
Sensitivity
When run as recommended, the minimum detection limit of the assay is 0.4 mg/dL (0.035 mmol/L).
3.
Linearity
The reaction is linear up to a creatinine concentration of 15 mg/dL; specimens showing higher concentration should be diluted 1+4 using physiological saline and repeat the assay (result × 5).
Interfering Substances
4.
5.
Serum, plasma
6.
Haemolysis Erythrocyte contamination doesn’t elevate results. Icterus Serum bilirubin levels in the pathological range may interfere with the results. Lipemia Lipemic specimens may cause high absorbance flagging. Diluted sample treatment may be recommended.
Bowers LD, Wong ET: kinetic serum creatinine assays. II. A critical evaluation and review. Clin Chem 26:555, 1980. DI Giorgio J: Nonprotein nitrogenous constituents. In :clinical chemistry – principles and technics, 2 nd ed.RJ Henry, DC Cannon, JW Winkelman, editors, Harperand Row, Hagerstown (MD), 1974, pp 541-553. Doolan PD, Alpen EL, Theil GB: A clinical appraisal of the plasma concentration and endogenous clearence of creatinine. AM J Med 32:65, 1962. Spencer K, Price CP: A review of Non-enzyme mediated reaction and their application to centrifugal analyzers. In: Centerfugal analyzers in clinical chemistry , CP Price, K Spencer, editors, Praeger publishers, New York, 1980, p 231. Tobias GJ, Mclaughlin RF, Hopper J: Endogenous creatine clearence. N Engl J Med 266:317, 1962. Tietz NW: Textbook of clinical chemistry. WB saunders, philadelphia, 1986, pp 1271- 1281. ORDERING INFORMATION CATALOG NO
QUANTITY
235 001 235 002 235 003 235 004
2 x 100 ml 4 x 100 ml 8 x 100 ml 2 x 500 ml
Expected Values
Serum, plasma Females Males
0.7-1.3 mg/dL 0.9-1.5 mg/dL
Urine(24 hrs) Females Males
0.9 – 1.6 g/24 hrs 1.1 – 2.8 g/24 hrs
Creatinine clearance Females Males
75 – 115 mL / min 85 – 125 mL / min
62-115 mmol/L 80-133 mmol/L
Reagent Preparation
Prepare working solution as following: Combine one volume of R1 with one volume of R2, e.g. 1.0 ml R1 + 1.0 ml R2
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
34
Concentration of creatinine in urine: (Aspecimen) Creatinine (mg/dL) = (Astandard)
x 2 x 50
Creatinine clearance: mg creatinine / dL urine x mL urine / 24 hours mg creatinine / dL serum x 1440
35
GLUCOSE - Liquizyme GOD - PAP (Single Reagent) REF: 250 001 REF: 250 002 REF: 250 003 REF: 250 004 REF: 250 005 REF: 250 006 REF: 250 007
(2 x 100 ml) (4 x 100 ml) (8 x 100 ml) (2 x 500 ml) (2 x 250 ml) (4 x 250 ml) (1 x 250 ml)
EC REP Authorised Representative IVD
200 test 400 test 800 test 1000 test 500 test 1000 test 250 test
LOT REF
Spectrum Diagnostics liquizyme glucose reagent is intended for the in-vitro quantitative, diagnostic determination of glucose in human serum, plasma, urine and CSF on both manual and automated systems.
Background
Oxidation of glucose present in the peripheral blood represents the major source of cellular energy in the body. Dietary glucose is stored in the liver in the form of glycogen or converted to fatty acids and stored in the adipose tissues. The accurate estimation of glucose is important in the diagnosis and management of hyperglycemia & hypoglycemia. the most frequent cause of hyperglycemia is diabetes mellitus resulting from a deficiency in insulin secretion or action. Hypoglycemia may be the result of an insulinoma, insuline administration, inborn error of carbohydrate metabolism or fasting. The concentration of glucose in the blood is controlled within narrow limits by many hormones, the most important of which are produced by the pancreas. GLUCOSE measurement in urine is used as diabetes screening procedure and to aid in the evaluation of glucosuria to detect renal tubular defect and in the management of diabetes mellitus. GLUCOSE measurement in cerebrospinal fluid (CSF) is used for evaluation of meningitis, neoplastic involvement of meninges and other neurological disorders.
Method
GOD-PAP enzymtic colorimetric method.
Assay Principle
Glucose is determined after enzymatic oxidation in the presence of glucose oxidase. The formed hydrogen peroxide reacts under catalysis of peroxidase (PAP) with phenol and 4-aminoantipyrine to form a red violet quinoneimine dye as indicator. Glucose + 2H2O + O2
GOD
Gluconic acid + H2O2
2H2O2 +Phenol + 4-amino-antipyrine
PAP
4H2O + Quinoneimine
Reagents
Glucose standard (St) 100 mg/dL 5.55 mmol/L
Reagent (R)
Phosphate Buffer 100 mmol/L Phenol 4.0 mmol/L 4-amino-antipyrine 1.0 mmol/L Glucose oxidase > 20 KU /L Peroxidase > 2.0 KU/L Sodium Azide 8 mmol/L For further information, refer to the Glucose reagent material safety data sheet.
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately. Reagent (R) contains sodium azide which may react with copper or lead plumbing.
36
Glucose concentration (mg/dl) =
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Reagent Preparation, Storage and Stability
Intended Use
Calculation
SYMBOLS IN PRODUCT LABELLING
Spectrum Glucose liquizyme reagents are supplied ready-to-use and stable up to the expiry date labeled on the bottles when properly o stored refrigerated at 2 – 8 C. Once opened, the opened vial is stable for 3 months at the specified temperature.
Deterioration
The Spectrum glucose reagent is normally clear or pale pink. Do not use liquizyme Glucose reagent if it is turbid or if the absorbance is greater than 0.2 at 546 nm.
Quality Control
System Parameters
Wavelength Optical path Assay type Direction Increase Sample : Reagent Ratio e.g.: Reagent volume Sample volume Temperature Incubation time Zero adjustment Reagent Blank Limits Sensitivity Linearity
Urine Random 24 hours
× 100
Normal & abnormal commercial control serum concentrations should be analyzed with each run.
of
known
Within run (Repeatiblity)
Level 1
Level 2
n
20
20
Mean (mg/dL)
103
228
SD
1.12
1.19
CV%
1.09
0.83
Level 1
Level 2
n
20
20
Mean (mg/dL)
109
235
SD
1.23
1.27
CV%
1.17
0.98
Run to run (Reproducibility)
1 : 100 1 ml 10 ml o o 37 C or 20 – 25 C o 20 minutes at 20 – 25 C or o 10 minutes at 37 C Reagent Blank Low 0.00 AU High 0.15 AU 5 mg/dL (0.27 mmol/L) 500 mg/dL ( 27.7 mmol/L)
Procedure Blank
Standard
specimen
Reagent (R)
1.0 ml
1.0 ml
1.0 ml
Standard
-------
10 ml
-------
Specimen
-------
-------
10 ml
CSF Adults
40 - 75
mg/dL
(2.2-4.2 mmol/L)
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure;interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Dynamic Range
5 - 500 mg/dL (0.27 - 27.7 mmol/L).
Methods Comparison
A comparison between Spectrum Diagnostics Glucose reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.991 was obtained.
Sensitivity
When run as recommended, the minimum detection limit of the assay is 5 mg/dL (0.27 mmol/L).
Linearity
The reaction is linear up to glucose concentration of 500 mg/dl; specimens showing higher concentration should be diluted 1+2 using physiological saline and repeat the assay (result×3).
Interfering Substances
Waste Disposal
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References 1. 2. 3. 4. 5.
Caraway WT, Watts NB. Carbohydrates In : Tietz NW, ed.Fundamentals of Clinical Chemistry. 3ry ed. Philadephia WB saunde-rs 1987:422-447. Howanitz PJ, Howanitz JH. Carbohydrates. In: Henry JB,ed. Clinical diagnosis and mana-Gement by laboratory methods. 17th ed Philadelphia: WB saunders 1984:165-179 Trinder, P., Ann. Clin. Biochem. (1969), 6:24. Tietz NW, ed. Clinical guide to laboratory tests. 3rd ed.Philadelphia: WB saunders; 1995:268-273. Weissman M, klien B. Evaluation, of glucose determination In untreated serum samples. Clin Chem.1958;4:420-422.
Serum, plasma
Haemolysis No significant interference from haemoglobin up to 500 mg/dL.
546 nm (492 – 550 nm) 1 cm End-point
5.0 - 15 mg/dL (0.28 - 0.83 mmol/L) < 0.5 g/24 hrs ( 10.0 % It is recommended that each laboratory establish its own normal range representing its patient population.
Reagents
25 Tests
50 Tests
R1 Ion Exchange Resin (Predispensed Tubes)
25 x 3
50 x 3
ml
100 x 3 ml
R2 Lysing Reagent
1 x 12.5 ml
2 x 12.5 ml
4 X 12.5 ml
C Control (10% GHb)
1 x 1 ml
2 x 1 ml
4 x 1 ml
A Resin Separators
25 Nos
50 Nos
100 Nos
Reagents Preparation Storage and Stability
= Ratio of Control (RC) Ratio of Test (RT) = GHb in % =
ml
Blood samples with Hemoglobin greater than 18 g/dl should be diluted 1 + 1 with Normal saline before the assay. Samples from patients with Hemoglobinopathies, decreased red cell survival times, gross lipemia may show incorrect results. Do not use Ion Exchange Resin tubes in case of turbidity or visible discolouration. Diabetics with metabolic imbalance may have extremely high levels of the labile aldimine form. In such cases the incubation time during hemolysate preparation may be increased to 15 minutes to ensure elimination of this in stable fraction.
References 1.
Trivelli,L.A.,Ranney,H.M. and Lai,H.T., New Eng.J.Med. 284, 353 (1971).
2.
Nathan,D.M.,et al., New Eng.J.Med. 310, 341 - 346 (1984).
3.
Bunn,H.F., Diabetes 130, 613 (1981).
4.
Bates,H.M.,Lab Manag.,Vol 16 ( Jan.1978)
100 Tests
Contents are stable at 2-8°C till the expiry mentioned on the label. Do not freeze. The Resin separators can be removed on opening the kit and stored at R.T. The Ion Exchange Resin tubes & the Lysing Reagent are ready to use. Reconstitute the Control with 1 ml of distilled water. Allow to stand for 10 mins with occasional mixing. The reconstituted control is stable for at least 7 days when stored at 2-8°C tightly sealed, and at least 4 weeks when stored at -20°C. Do not thaw and refreeze. Specimens: Whole blood, preferably fresh collected in EDTA. GHb in whole blood is stable for week at 2-8°C
Procedure
Wavelength : 415 nm (Hg 405 nm) Temperature : R.T. Light path : 1 cm
A. Hemolysate Preparation 1. 2. 3.
Dispense 0.5 ml Lysing Reagent into tubes labeled as Control (C) & Test (T). Add 0.1ml of the reconstituted control & well-mixed blood sample into the appropriately labeled tubes. Mix until complete lysis is evident. Allow to stand for 5 minutes.
B. Glycosylated hemoglobin (GHb) Separation
Remove cap from the Ion-Exchange Resin tubes and label as Control & Test. Add 0.1 ml of the hemolysate from Step A into the appropriately labeled Ion Exchange Resin tubes. Insert a resin Separator into each tube so that the rubber sleeve is approximately 1 cm above the liquid level of the resin suspension. Mix the tubes on a rocker, rotator or a vortex mixer continuously for 5 minutes. Allow the resin to settle, then push the resin separator into the tubes until the resin is firmly packed. Pour or aspirate each supernatant directly into a cuvette and measure each absorbance against distilled water.
C. Total Hemoglobin (THb) fraction
Dispense 5.0 ml of distilled water into tubes labeled as Control & Test. Add to it 0.02 ml of hemolysate from Step A into the appropriately labeled tube. Mix well. Read each absorbance against distilled water
38
39
Direct Enzymatic HbA1c
EC REP Authorised Representative
REF: R1a: R1b: R2: LR:
255 001
IVD
60 Tests 1 x 7 ml 1 x 3 ml 1 x 4.4 ml 1 x 13 ml
LOT REF
GENERAL
The glycemic control in diabetes mellitus is performed mainly by the determination of Glucose, but also through quantitative determination of Hemoglobin A1c (HbA1c) in human blood : HbA1c is an indication for the actual glucose levels over the preceding 3 months. It was shown that HbA1c in diabetic subjects can be elevated 2-3 fold over normal and on the other hand approaches normal values when they are under metabolic control.
Hemolyzed blood is used as sample material. Through protease attack glycated valines are released and are subject to further enymatic reaction with fructosyl valine oxidase (FVO). The result is a measureable quantitative amount of hydrogen peroxide, which is determined through colorimetric reaction with a chromogen compound. The reaction product is proportional to the amount of HbA1c and is measured as absorbance A. The HbA1c value is derived from a calibration curve.
REAGENTS R1a:
MES-Buffer (pH 7.0) Protease Triton-X 100
5.0 mmol/l
R1b:
MES-Buffer (pH 6.3)
1.0 mmol/l
R2:
TRIS-Buffer (pH 8.0) Fructosyl Valine Oxidase Peroxidase Chromogen
5.0 mmol/l ≥ 9,5 kU/l ≥ 8,5 kU/l ≥ 0.7mmol/l
LR:
Lysing Reagent
0.5 %l
Storage and Stability
Store all reagents refrigerated at 2-8°C. Unopened reagents are stable up to the expiration date printed on the labels.
Preparation of Reagents
3-reagent-procedure: R1a, R1b and R2 are ready for use when the 3-reagentprocedure is applied. Stability after opening : At least 3 months when contamination is avoided 2-reagent-procedure: R1: Mix 7 parts R1a with 3 parts R1b Stability: At least 2 weeks when contamination is avoided
Important Note:
R1b and R2 are light sensitive ! Additional Reagents Control Set. Calibrator.
40
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use Temperature Limitation
!
Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by (Xn) - Harmful
Recommended Values are < 6% for non-diabetics (42 mmol/mol) 6 - 9% (42 – 75 mmol/mol) for diabetics under perfect glycemic control Up to 20% for diabetics out of glycemic control
Conversion from HbA1C % to mmol/mol HbA1c %
HbA1C mmol/mol
6.0
42
6.5
48
7.0
53
7.5
59
8.0
65
9.0
75
10
86
SAMPLES
Collect venous blood with EDTA. Storage and Stability Hemoglobin A1c in whole blood with EDTA is stable for one week at 2-8°C.5
2. 3.
Dispense 250µl of Lysing Reagent into cups or tubes and label as Controls, Patients, etc. Add 20ul of well mixed (!) whole blood samples respectively of Calibrators and Controls . (Note: Calibrators and Controls have to be treated exactly like the patient samples!) Let incubate at room temperature for minimum 10 min. Stability: Hemolysates may be stored up to 1 day at 2-8°C
PRECAUTIONS 1. 2.
1.The reagent is for in vitro diagnostic use only. 2.All human specimens should be regarded as potentially biohazardous. Therefore, universal precautions should be used in specimen handling (gloves, lab garments, avoid aerosol production, etc.)
ANALYTICAL PROCEDURE
Wavelength 700 nm Cuvette 1 cm light path Temperature 37 °C Specimen Blood Hemosylate Calibrator
Hemosylate Sample
R1 (μL)
200
200
Calibrator(μL)
30
-
-
30
Sample (μL)
Mix and incubate for 5 minutes exactly, then Read the absorbance (A1) of the samples and calibrator, and immediately add R2 R2(μL)
85
REFERENCES 1.
Trivelli, L.A., Ranney, H.M., and Lai, H.T., New Eng. J. Med. 284,353 (1971).
2. 3.
Gonen, B., and Rubenstein, A.H., Diabetologia 15, 1 (1978). Gabbay, K.H., Hasty, K., Breslow, J.L., Ellison, R.C., Bunn, H.F., and Gallop, P.M., J. Clin. Endocrinol. Metab. 44, 859 (1977). 4. Bates, H.M., Lab. Mang., Vol 16 (Jan. 1978). 5. Tietz, N.W., Textbook of Clinical Chemistry, Philadelphia, W.B. Saunders Company, p.794-795 (1999). 6. Corielo, A., et al, Diabetologia 22, p. 379 (1962). 7. Goldstein, D.E., et al, Clin. Chem. 32, pp. 364-370 (1986). 8. Fluckiger, R., et al, Med Intelligence 304 pp. 823-827 (1981). 9. Nathan, D.M., et al, Clin. Chem. 29, pp. 466-469 (1983). 10. American Diabetes Association: Clinical Practice Recommendations, Diabetes Care 24 (Suppl. 1):
ORDERING INFORMATION
Hemolysate Procedure: 1.
PRINCIPLE
EXPECTED VALUES
SYMBOLS IN PRODUCT LABELLING
85
Read the absorbance (A2) of the Samples and calibrator after 2 mins.
Note: Each laboratory should establish its own expected values. The given values can only be an average indication.
QUANTITY
255 000 255 001
30 Test 60 Test
LIMITATIONS 1. 2. 3.
Results may be inconsistent in patients e.g.with opiate addiction, lead-poisoning, alcoholism, ingestion of large doses of aspirin. Elevated levels( > 10%) of HbF may lead to underestimation of HA1c. Hemoglobin variants HbS, HbC and HbE do not interfere in this assay. There is also no interference by labile intermediates , and uremia does not interfere, too.
PERFORMANCE CHARACTERISTICS 1. Linearity: The Hemoglobin A1c assay range is 2.0% to 16.0%. Results in this range can be reported and used directly. 2.
Correlation: A study using 40 human specimens between this HA1c procedure and the reference method yielded a correlation coefficient of 0.9874 and a linear regression equation of y = 1.021 x + 0.014
3.
Precision: Within Run: The intra assay precision was established by assaying blood with two Hemoglobin A1c levels twenty times each. Level Medium High
Mean 5.7 10.3
% C.V. 1.0 0.7
INTERFERENCES 1.
CALCULATION (D A) Sample ────────── X Calibrator conc. = % HbA1c (D A) Calibrator
CATALOG NO
2.
Bilirubin to 15mg/dL, ascorbic acid to 10mg/dL, triglycerides to 3000mg/dL, Glucose to 4000mg/dL, carbamylated Hb to 5mmol/L and acetylated Hb to 5.0mmol/L do not interfere in the assay. It has been reported that results may be inconsistent in patients who have the conditions like opiate addiction, lead-poisoning, alcoholism, ingestion of large doses of aspirin.
QUALITY CONTROL
Use Spectrum HbA1C Control Set with assayed values.
41
Haemoglobin(Single Reagent) Drabkin’s Solution
SYMBOLS IN PRODUCT LABELLING EC REP Authorised Representative IVD
REF:610 001 (2 x 20 ml) 800 test REF:610 002 (5 x 20 ml) 2000 test
LOT REF
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use Temperature Limitation
!
Within run (Repeatiblity)
Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by (Xn) - Harmful
n
Level 1
Level 2
20
20
Mean (mg/dL)
10
14
CV%
2.3
1.3
CATALOG NO
QUANTITY
610 001 610 002
2 x 20 ml 5 x 20 ml
Run to run (Reproducibility)
Intended Use
Reagent Preparation Storage and Stability
Spectrum Diagnostics haemoglobin reagent is intended for the invitro quantitative, diagnostic determination of haemoglobin in human blood.
The reagent is stable until expiration date stated on label when stored o at 15 - 25 C. Prepare the working solution by diluting the contents of the contcentrated reagent (R) to 1000ml with distilled water (add one bottle of reagent (R) to 980 ml of dist.water), mix thoroughly. working reagent can be prepared according to the volume needed by mixing 1 ml of the concentrated reagent (R) plus 49 ml distilled water.
Background Haemoglobin (Hb) is the red pigmented protein located in the er-ythrocytes and consists of four subunits. Its main function is the transport of oxygen and carbon dioxide in blood. In normal humanadults, at least 96 % of the haemoglobin is HbA. HbA2 is usually about 2.5 – 3 % of total haemoglobin. Fetal hemoglobin (HbF) predominates during fetal life and diminishes rapidly during the first year of postnatal life. In normal adults less than 1 % is HbF.Blood haemoglobin concentration may be diminished as a consequence of hemorrhage or hemolysis or as a result of impaired blood formation in the bone marrow.
Method colorimetric method using Drabkin’s solution.
Assay Principle Haemoglobin is oxidized by potassium ferricyanide which is converted into stable cyanomethaemoglobin by potassium cyanide. The absorbance of the cyanomethaemoglobin is monitored at 540 nm.
Reagents Reagent Potassium ferricyanide 31 mmol/l Potassium phosphate 52 mmol/l Potassium cyanide 77 mmol/l Surfactant 2% Harmful (Xn): R20/21/22: Harmful by inhalation, in contact with skin and if swallowed. S7: Keep container tightly closed. S28.1: After contact with skin, wash immediately with plenty of water. S45: In case of accident or if you feel unwell, seek medical advice immediately. The amount of cyanide present in one bottle of reagent is apreciably less than the minimum lethal dose for an adult. However, hydrogen cyanide is liberated by acidification. Never allow reagent to come in contact with acid. For further information, refer to the Haemoglobin reagent material safety data sheet.
Precautions and Warnings Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
o
Stability: 6 months in a brown glass bottle at 15-30 C
Specimen Collection and Preservation Anticoagulated venous or capillary blood . Blood may be anticoagulated with EDTA, or fluoride. Blood can be taken directly from a finger or heel puncture without use of anticoagulant. o
Stability : 7 days at 2 – 8 C o 4 days at 20 - 25 C
System Parameters Wavelength Optical path Assay type Direction Increase Sample : Reagent Ratio e.g .: Reagent volume Sample volume Temperature Incubation time Zero adjustment
Level 2
20
20
Mean (mg/dL)
11.1
14.1
CV%
2.9
2.1
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Methods Comparison A comparison between Spectrum Diagnostics Haemoglobin reagent and a commercial reagent of the same methodology was performed on 40 human blood samples. A correlation (r) of 0.983 was obtained.
Expected values 540 nm (Hg 546 nm) 1 cm End-point 1 : 250 2.5 ml 10 ml o 20 – 25 C 5 minutes Against reagent blank
Pipette into test tubes 2.5 ml 10 ml
Mix well and rinse the blood pipette several times with the reagents, o and incubate for 5 minuts at 20-25 C. Measure absorbance of specimen (Aspecimen) against reagent blank.
Calculation Haemoglobin concentration (g/dL)
= Aspecimen x 36.77
Haemoglobin concentration (mmol/L)= Aspecimen x 22.83
Quality Control Normal & abnormal commerical control blood concentrations should be analyzed with each run.
1- 6 days
15.2 – 23.5 g/dL
( 9.4 – 14.6 mmol/L)
14 – 50 days
10.3 – 16.6 g/dL
( 6.4 – 10.3 mmol/L)
2 - 10 months
10.0 – 12.9 g/dL
( 6.1 – 8.0 mmol/L)
1 – 15 years
11.0 – 14.3 g/dL
( 6.8 – 8.8 mmol/L)
Adults Women
12.0 – 16.0 g/dL
(7.5 – 9.9 mmol/L)
Men
14.0 – 18.0 g/dL
(8.7 – 11.2 mmol/L)
Waste Disposal
Procedure working solution Blood sample
Level 1 n
of
known
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References 1.
International committee for standardization in haematology. Brit.J. Haemat., 1967:13 (Suppl.) 71.
2.
Van Kampen, E. J. and Zijlstra, W.G., Clin. Chem. Acta., 1961:6:538 – 544.
3.
Tietz NW, Ed. Clinical guide to laboratory tests. 2ND ED. Philadelphia: WB Saunders; 1990:566. ORDERING INFORMATION
Performance Characteristics Precision
42
43
Haemoglobin Drabkin’s Solution
SYMBOLS IN PRODUCT LABELLING EC REP Authorised Representative IVD LOT REF
REF: 610 003 (2 x 50 ml) 1000 test REF: 610 004 (2 x 100 ml) 2000 test
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use Temperature Limitation
!
Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by (Xn) - Harmful
Intended Use
Precautions and Warnings
Spectrum Diagnostics haemoglobin reagent is intended for the in-vitro quantitative, diagnostic determination of haemoglobin in human blood .
Pay attention to all precautions and warnings listed in Spectrum Diagnostics catalogue available upon request .Haemoglobin reagent contains cyanide which is poisonous. Avoid contact with skin and never pipette by mouth .
Background Haemoglobin (Hb) is the red pigmented protein located in the erythrocytes and consists of four subunits . Its main function is the transport of oxygen and carbon dioxide in blood . In normal human adults , at least 96 % of the haemoglobin is HbA . HbA2 is usually about 2.5 – 3 % of total haemoglobin . Fetal haemoglobin (HbF) predominates during fetal life and diminishes rapidly during the first year of postnatal life . In normal adults less than 1 % is HbF.Blood haemoglobin concentration may be diminished as a consequence of haemorrhage or haemolysis or as a result of impaired blood formation in the bone marrow .
Method colorimetric method using Drabkin’s solution .
Assay Principle Haemoglobin is oxidized by potassium ferricyanide which is converted into stable cyanomethaemoglobin by potassium cyanide. The absorbance of the cyanomethaemoglobin is monitored at 540 nm .
Reagents Reagent (R1) Potassium ferricyanide Potassium phosphate
40 mmol/l 50 mmol/l
Reagent (R2) Potassium cyanide
77 mmol/l
Reagent Preparation Storage and Stability All reagents are stable until expiration date stated on label when o stored at 15 - 25 C . Prepare the working solution by diluting the reagents with bidistilled water as following : 1 ml(R1) + 1 ml (R2) + 48 ml H2O working solution is stable for 3 months and should be light protected .
Expected values 1- 6 days
15.2 – 23.5 g/dL
( 9.4 – 14.6 mmol/L)
14 – 50 days
10.3 – 16.6 g/dL
( 6.4 – 10.3 mmol/L)
2 - 10 months
10.0 – 12.9 g/dL
( 6.1 – 8.0 mmol/L)
1 – 15 years
11.0 – 14.3 g/dL
( 6.8 – 8.8 mmol/L)
Adults Women
12.0 – 16.0 g/dL
(7.5 – 9.9 mmol/L)
Men
14.0 – 18.0 g/dL
(8.7 – 11.2 mmol/L)
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure ; interpretation of the results is considered the responsibility of qualified medical personnel All indications of clinical significance are supported by literature references .
References 1.
International committee for standardization in haematology. Brit. J. Haemat., 1967:13 (Suppl.) 71 .
2.
Van Kampen, E. J. and Acta.,1961:6:538 – 544 .
3.
Tietz NW, Ed. Clinical guide to laboratory tests. 2ND ED. Philadelphia: WB Saunders; 1990:566 .
Zijlstra,
W.G.,
Clin.
Chem
Specimen Collection and Preservation Anticoagulated venous or capillary blood . Blood may be anticoagulated with EDTA , or fluoride . Blood can be taken directly from a finger or heel puncture without use of anticoagulant . Stability : 7 days 4 days
o
at 2 – 8 C o at 20 – 25 C
ORDERING INFORMATION CATALOG NO
QUANTITY
610 003 610 004
2 x 50 ml 2 x 100 ml
System Parameters Wavelength 540 nm (Hg 546 nm) Optical path 1 cm Assay type End-point Direction Increase Sample : Reagent Ratio 1 : 250 e.g . : Reagent volume 2.5 ml Sample volume 10 ml o Temperature 20 – 25 C Incubation time 5 minutes Zero adjustment Against reagent blank
Procedure Harmful (Xn): R20/21/22: Harmful by inhalation, in contact with skin and if swallowed. S7: Keep container tightly closed. S28.1: After contact with skin, wash immediately with plenty of water. S45: In case of accident or if you feel unwell, seek medical advice immediately. The amount of cyanide present in one bottle of reagent is apreciably less than the minimum lethal dose for an adult. However, hydrogen cyanide is liberated by acidification. Never allow reagent to come in contact with acid.
Pipette into test tubes working solution Blood sample
2.5 ml 10 µl
Mix well and rinse the blood pipette several times with the o reagents , and incubate for 5 minuts at 20-25 C . Measure absorbance of specimen (Aspecimen) against reagent blank .
Calculation For further information, refer to the Haemoglobin plus reagent material safety data sheet.
Haemoglobin concentration (g/dL) = Aspecimen x 36.77 Haemoglobin concentration (mmol/L)= Aspecimen x 22.83
Reagents also contain non-reactive stablizers and surfactants
44
45
Haemoglobin (Ready-To-Use) Drabkin’s Solution
EC REP Authorised Representative IVD
REF:611 001 (1 x 250 ml) 100 test REF:611 002 (2 x 250 ml) 200 test REF:611 003 (2 x 500 ml) 400 test
LOT REF
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use Temperature Limitation
!
Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by (Xn) - Harmful
Intended Use
Reagent Preparation Storage and Stability
Spectrum Diagnostics haemoglobin reagent is intended for the invitro quantitative, diagnostic determination of haemoglobin in human blood.
Reagent is supplied ready to Use.
Background Haemoglobin (Hb) is the red pigmented protein located in the er-ythrocytes and consists of four subunits. Its main function is the transport of oxygen and carbon dioxide in blood. In normal humanadults, at least 96 % of the haemoglobin is HbA. HbA2 is usually about 2.5 – 3 % of total haemoglobin. Fetal hemoglobin (HbF) predominates during fetal life and diminishes rapidly during the first year of postnatal life. In normal adults less than 1 % is HbF.Blood haemoglobin concentration may be diminished as a consequence of hemorrhage or hemolysis or as a result of impaired blood formation in the bone marrow.
Method colorimetric method using Drabkin’s solution.
Assay Principle Haemoglobin is oxidized by potassium ferricyanide which is converted into stable cyanomethaemoglobin by potassium cyanide. The absorbance of the cyanomethaemoglobin is monitored at 540 nm.
Reagents
Performance Characteristics
SYMBOLS IN PRODUCT LABELLING
References
Precision Within run (Repeatiblity)
o
Stability : 7 days at 2 – 8 C o 4 days at 20 - 25 C
2.
Van Kampen, E. J. and Zijlstra, W.G., Clin. Chem. Acta., 1961:6:538 – 544.
3.
Tietz NW, Ed. Clinical guide to laboratory tests. 2ND ED. Philadelphia: WB Saunders; 1990:566.
Level 2
n
20
20
Mean (mg/dL)
10
14
CV%
2.3
1.3
Level 1
Level 2
CATALOG NO
QUANTITY
n
20
20
Mean (mg/dL)
11.1
14.1
CV%
2.9
2.1
611 001 611 002 611 003
1 x 250 ml 2 x 250 ml 2 x 500 ml
Specimen Collection and Preservation Anticoagulated venous or capillary blood . Blood may be anticoagulated with EDTA, or fluoride. Blood can be taken directly from a finger or heel puncture without use of anticoagulant.
International committee for standardization in haematology. Brit. J. Haemat., 1967:13 (Suppl.) 71.
Level 1
Run to run (Reproducibility) Reagent is stable until expiration date stated on label when stored o at 15 - 25 C.Once opened, the opened vial is stable for 6 months at the specified temperature.
1.
ORDERING INFORMATION
Methods Comparison A comparison between Spectrum Diagnostics Haemoglobin reagent and a commercial reagent of the same methodology was performed on 40 human blood samples. A correlation (r) of 0.983 was obtained.
Expected values System Parameters Wavelength Optical path Assay type Direction Increase Sample : Reagent Ratio e.g .: Reagent volume Sample volume Temperature Incubation time Zero adjustment
Reagent Potassium ferricyanide 0.62 mmol/l Potassium phosphate 1.04 mmol/l Potassium cyanide 1.54 mmol/l Surfactant < 0.1 % Harmful (Xn): R20/21/22: Harmful by inhalation, in contact with skin and if swallowed. S7: Keep container tightly closed. S28.1: After contact with skin, wash immediately with plenty of water. S45: In case of accident or if you feel unwell, seek medical advice immediately. The amount of cyanide present in one bottle of reagent is apreciably less than the minimum lethal dose for an adult. However, hydrogen cyanide is liberated by acidification. Never allow reagent to come in contact with acid.
Procedure
For further information, refer to the Haemoglobin reagent material safety data sheet.
Quality Control
540 nm (Hg 546 nm) 1 cm End-point 1 : 250 2.5 ml 10 ml o 20 – 25 C 5 minutes Against reagent blank
15.2 – 23.5 g/dL
( 9.4 – 14.6 mmol/L)
14 – 50 days
10.3 – 16.6 g/dL
( 6.4 – 10.3 mmol/L)
2 - 10 months
10.0 – 12.9 g/dL
( 6.1 – 8.0 mmol/L)
1 – 15 years
11.0 – 14.3 g/dL
( 6.8 – 8.8 mmol/L)
Adults Women
12.0 – 16.0 g/dL
(7.5 – 9.9 mmol/L)
Men
14.0 – 18.0 g/dL
(8.7 – 11.2 mmol/L)
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Waste Disposal
Pipette into test tubes Reagent Blood sample
1- 6 days
2.5 ml 10 µl
Mix well and rinse the blood pipette several times with the reagents, o and incubate for 5 minuts at 20-25 C. Measure absorbance of specimen (Aspecimen) against reagent blank.
Calculation Haemoglobin concentration (g/dL)
= Aspecimen x 36.77
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
Haemoglobin concentration (mmol/L)= Aspecimen x 22.83
Normal & abnormal commerical control blood concentrations should be analyzed with each run.
of
known
Precautions and Warnings Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
46
47
Lactate – Liquizyme REF: 274 001 (5 x 20 ml)
o
SYMBOLS IN PRODUCT LABELLING EC REP Authorised Representative
100 test
IVD LOT REF
Intended Use
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Spectrum Diagnostics liquizyme Lactate reagent is intended for the in-vitro quantitative, diagnostic determination of lactate in human Plasma and CSF on both automated and manual systems.
Reagent (R2) contains sodium azide which may react with copper or lead plumbing.
Background
Reagent Preparation
Lactic acid, present in blood entirely as lactate is an intermediary product of carbohydrate metabolism and is derived mainly from muscle cells and erythrocytes. The blood lactate concentration is affected by its production in muscle cells and erythrocytes and its rate metabolism in the liver. During exercise, blood lactate can increase up to ten times of normal levels. Under normal conditions, the ratio between lactate and pyruvate is constant(10:1).The l i v e r can normally metabolize more lactate than is produced. In the case of decreased perfusion of the liver , however, removal of lactate by the liver may be significantly reduced.The amount of lactate in cerebrospinal fluid normally parallels blood levels. CSF lactate level is increased in bacterial meningitis, epilepsy, and intracranial hemorrhage. CSF lactate level may be an aid to distinguish between bacterial from viral meningitis.
Prepare working solution as following: REF:274 001:add 2 ml from R2 to one bottle of R1; mix gently. Or prepare the working solution according to the number of tests required by mixing 9 volumes of reagent 1 (R1) and 1 volume of reagent 2 (R2), e.g. 900 ml R1 +100 ml R2.
Reagent Storage and Stability
All reagents are stable until expiration date stated on label when o stored refrigerated at 2 – 8 C. Working solution is stable for 3 o o months at 2 – 8 C or 1 week at 15 – 25 C.
Enzymatic colorimetric method (LOX / PAP)with lactate oxidase and 4-aminoantipyrine.
Assay Principle
Specimen Collection and Preservation
Lactate is oxidized to pyruvate and hydrogen peroxide (H2O2) by lactate oxidase (LOX). In the presence of peroxidase (POD), hydrogen peroxide reacts with 2,4,6-tribromo-3-hydroxybenzoic acid (THB) and 4-aminoantipyrine (4-AAP) to form a red quinoneimine dye.
Lactate + O2
LOX
2H2O + 4-AAP + THB
POD
Pyruvate + H2O2 quinoneimine dye + 4H2O
The color intensity of the formed red quinoneimine dye is directly proportional to the lactate concentration. It is determined by measuring the increase in absorbance at 546 nm.
Reagents Standard lactate (ST) Reagent 1 (R1 Buffer) Tris buffer 2,4,6-tribromo-3-hydroxybenzoic acid 4-Amino antipyrine Reagent 2 (R2 Enzyme) Lactate oxidase Peroxidase Sodium Azide
10 mg/dL 100 mmol/L 2.0 mmol/L 0.8 mmol/L >20 U/L >15 U/L 0.02 %
For further information, refer to the Lactate reagent material safety data sheet.
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
48
Bailey EM, Domenico P,Cunha BA. Bacterial or viral meningitis? Measuring lactate in CSF can help you know quickly. Meningitis. 1990;88:217-223.
2.
Field M, Block JB, Levin R, Rall DP. Significance of blood lactate elevations amoung patients with acute leukemia and other neoplastic proliferative disorders. Am J Med. 1996;40:528-547.
3.
Klein TO. Nervensysteme. In:Greiling H, Gressner AM,eds. Lehrbuch der Klinischen Chemie und Pathobiochemie. Stuttgart:Schattauer; 1987:859-893.
A comparison between Spectrum Diagnostics Lactate reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.992 was obtained.
4.
Sacks DB. Carbohydrates. In: Burtis CA, Ashwood ER, eds. Tietz Fundamentals of Clinical Chemistry. 4 th ed. Philadelphia:WB Saunders;1996:351-374.
Sensitivity
5.
Sacks DB. Carbohydrates in: Burtis CA, Ashwood ER, eds. Tietz Textbook of Clinical Chemistry. 2 nd ed. Philadelphia:WB Sander; 1994;928-1001.
6.
Tietz NW, ed. Clinical Guide to laboratory tests. 3 rd ed. Philadelphia: WB Saunders;1995:351-374.
Calculation
Lactate conc. (mg/dL) =
(Aspecimen) (Astandard)
Quality Control
x 10
Normal & abnormal commercial control serum concentrations should be analyzed with each run.
of
known
When run as recommended, the minimum detection limit of the assay is 0.3 mg/dL (0.033 mmol/L).
Linearity
The reaction is linear up to lactate concentration of 90 mg/dL (9.99 mmol/L), specimens showing higher concentration should be diluted 1+1 using physiological saline, reassayed and the result multiplied by two (2).
Interfering substance
The working reagent is normaly clear or pale pink. Do not use liquizyme lactate reagent if it is turbid or if the absorbance is greater than 0.1 at 546 nm. Plasma and CSF. Do not use serum specimens. Avoid icteric and haemolytic specimens.The only acceptable anticoagulants are fluoride/heparin and iodoacetate/heparin. Collection of satisfactory specimen for lactate analysis requires special procedures to prevent changes of lactate both while and after the specimen is drawn. The patient should be fasting and at complete rest and exercise of the arm or hand should be avoided before or during collection of the specimens. The collected blood should be cooled on ice immediately and separated from the cells within 15 minutes. Once the plasma is separated from the cells, lactate values are stable. Use the CSF samples with addition of glycolysis inhibitor, e.g.sodium o fluoride. Lactate in CSF is stable for 3 hours at 20 – 25 C, for 24 o o hours at 4 – 8 C, and for 2 months frozen at -20 C, stable in plasma o o for 2 hours at 20 – 25 C and 2 days at 4 – 8 C. System Parameters Wavelength Optical path Assay type Direction Increase Sample : Reagent Ratio e.g.: Reagent volume Sample volume Temperature Zero adjustment Incubation time Reagent Blank Limits Sensitivity Linearity
Plasma
Procedure Blank
Standard
Sample
Working Reagent
1.0 ml
1.0 ml
1.0 ml
Standard
------
10 µl
------
Sample
------
------
10 µl
QUANTITY
274 001
5 x 20 ml
Icterus Bilirubin levels higher than 4.0 mg/dL (68 mmol/L) decrease apparent lactate concentration significantly. Lipemia No significant interference. Ascorbic acid Physiological ascorbic acid concentrations do not interfere with the test. Ascorbic Acid levels higher than 5 mg/dL (284 mmol/l) decrease the apparent lactate concentration significantly.
Expected Values
CSF
1 : 100 1 ml 10 ml o o 37 C or 15 – 25 C Reagent blank o 5 minutes at 37 C or o 10 minutes at 15 – 25 C Low 0.00 AU High 0.25 AU 0.3 mg/dL (0.033 mmol/L) 90 mg/dL (9.99 mmol/L)
ORDERING INFORMATION CATALOG NO
Haemolysis Haemoglobin levels higher than 2.5 g/L (0.16 mmol/L) increase the apparent lactate concentration significantly.
Plasma 546 nm 1 cm End-point
Reference 1.
Methods Comparison
Deterioration
Method
Mix and incubate for 5 minutes at 37 C or 10 minutes at 15 – 25 C. Measure absorbance of specimen (Aspecimen) and standard (Astandard) against reagent blank within 30 minutes.
o
Venous
4.5 – 19.8 mg/dL 0.5 – 2.2 mmol/L
Arterial
4.5 – 14.4 mg/dL 0.5 –1.6 mmol/L
Adult
10 – 22
mg/dL 1.1 – 2.4 mmol/L
Neonates
10 – 60
mg/dL 1.1 – 6.7 mmol/L
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Analytical Range
0.3 – 90 mg/dL ( 0.033 – 9.99 mmol/L).
Waste Disposal
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
49
Micrototal Protein (MT-P) Pyrogallol - Red REF:282 001 (2 x 50 ml) 100 test REF:282 002 (4 x100ml) 400 test REF:282 003 (2 x100ml) 200 test
Intended Use
Spectrum Diagnostics microprotein reagent is intended for the invitro quantitative, diagnostic determination of total protein in human cerebrospinal fluid (CSF) and urine on both automated and manual systems.
Background
Protein level in spinal fluid may be increased in a variety of diseases including tumors, meningitis, and polyneuritis. Most of the proteins found in CSF originate from plasma; only 20% originate from intrathecal synthesis. The two main proteins found in human urine are albumin and uromucoid. Increased urinary proteins may be associated with a number of diseases, among them are destructive lesion of the kidney, primary and secondary nephropathies and also during pregnancy.
Method
Colorimetric method (Pyrogallol–red molybdate complex).
Assay Principle
In acidic medium, protein in the specimen reacts with pyrogallol red in the presence of molybdate ions to form a purple color complex. This color complex absorbs maximally at 600 nm and the optical density is directly proportional to protein concentration of the test sample.
Reagents
Standard protein (ST) 150 mg/dL Reagent (R) Succinate buffer 100 mmol/L Sodium oxalate 4.0 mmol/L Sodium molybdate 60 mmol/L Pyrogallol red 80 mmol/L Harmful (Xn): R20/22: Harmful by inhalation and if swallowed. S24/25: Avoid contact with skin and eyes. For further information, refer to the Microprotein M-TP reagent material safety data sheet.
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
Reagent Preparation, Storage and Stability
EC REP Authorised Representative IVD LOT REF
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use Temperature Limitation
!
o
o
System Parameters
Wavelength 600 nm (578 is an optional) Optical path 1 cm Assay type End-point Direction Increase Sample : Reagent Ratio 1 : 50 e.g.: Reagent volume 1 ml Sample volume 20 ml o Temperature 15 – 25 C Zero adjustment Reagent blank o Incubation time 10 minutes ( 15– 25 C ) Sensitivity 6 mg/dL Linearity 500 mg/dL
Procedure Blank
Standard
Sample
Reagent
1.0 ml
1.0 ml
1.0 ml
Standard
------
20 µl
------
Sample
------
------
20 µl o
Level 2
20
20
Mean (mg/dL)
39
109
SD
0.79
1.36
CV%
2.7
1.9
A comparison between Spectrum Diagnostics Micrototal Protein reagent and a commercial reagent of the same methodology was performed on 20 human Urine samples. A correlation of 0.975 was obtained. When run as recommended, the minimum detection limit of the assay is 6 mg/dL.
Uric Acid
85 mg/dL
Oxalate
90 mg/dL
(10 mmol/L)
1.2 g /L
(39 mmol/L)
Calcium
130 mg/dL
( 32 mmol/L)
Creatinine
6 g/L
( 53 mmol/L)
Ascorbic acid
10 mg/dL
( 568 mmol/L)
Bilirubin
60 mg/dL
(1.0 mmol/L)
: < 10 mg/dL
CSF
: 15 – 45 mg/dL
Measure the urine volume and calculate the concentration of M-TP in 24hr urine as following: ml of urine Concentration of MTP /24hr urine = Concentration /dL x 100
Quality Control
Normal & abnormal control serum of known concentrations should be analyzed with each run.
n
CATALOG NO
QUANTITY
282 001 282 002 282 003
2 x 50 ml 4 x 100 ml 2 x 100 ml
( 5 mmol/ L)
Phosphate
Urine ( random)
Concentration / 24hr urine:
ORDERING INFORMATION
Avoid erythrocyte contamination. There is no significant interference from the following substances present in urine up to the following concentrations:
: 20 – 145 mg/day
x 150
Watanabe N., Kamei, S., Oh Kubo A., and Tok uda K., Clin, Chem.,1986., 32/8:1551.
Interfering Substances
Urine ( 24 hrs ) (Astandard)
2.
The reaction is linear up to microprotein concentration of 500 mg/ dL. Specimens showing higher concentration should be diluted 1+1 using physiological saline and repeat the assay (result × 2).
Calculation CSF or Urine protein conc. (mg/dL)=
Henry R.J., Cannon, D.C., Winkelman J.W., “Clinical Chemistry,Principles and Techniques.” Harper & Row, 2nd Ed.1974.
Linearity
Expected values
(Aspecimen)
1.
Methods Comparison
Mix, incubate for exactly 10 minutes at 15 - 25 C. Measure absorbance of specimen (Aspecimen) and standard(Astandard) against reagent blank within 10 minutes.
Deterioration
50
o
Level 1 n
Sensitivity
Within run (Repeatiblity)
Use Urine and CSF free from blood contamination. Urine:24 hour urine is the specimen of choice. Centrifuge urine specimen if turbidity is obvious. No special additives are required but keep the specimen cool during collection. To avoid enhanced albumin excretion, samples should not be collected after exertion or following acute ingestion of a fluid load. CSF: Avoid blood contamination since protein concentration in whole blood is 1000 times higher than normal CSF. Centrifuge CSF specimen if turbidity is obvious.
o
Stability (CSF): 1 day at 15 – 25 C; 4 weeks at 4 – 8 C; o 6 months at -20 C
Performance Characterstics Precision
Specimen Collection and preservation
Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by (Xn) - Harmful
Stability (urine): 1 day at 15 – 25 C; 8 weeks at 4 – 8 C; o 1 year at -20 C
Spectrum Microprotein reagents are supplied ready-to-use and stable up to the expiry date labeled on the bottles,when stored at o 2 – 8 C. Do not use the Spectrum microprotein reagents if turbid. Failure to recover control values within the assigned range may be an indication of reagent deterioration.
References
Run to run (Reproducibility)
SYMBOLS IN PRODUCT LABELLING
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretition of the results is considered the responsibality of qualified medical personnel. All indications of clinical significance are supported by literature references.
Analytical Range 6 – 500 mg/dL.
Waste Disposal
Level 1
Level 2
20
20
Mean (mg/dL)
37
105
SD
0.74
1.27
CV%
2.0
1.3
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
51
PYRUVATE
SYMBOLS IN PRODUCT LABELLING
(Quantitative Enzymatic UV–Test)
EC REP Authorised Representative IVD
REF: 335 001
LOT
100 test
REF
R1 : 2 x 50 ml R2 : 1 x 5 ml R3 : 1 x 5 ml St : 1 x 20 ml
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
CALCULATION with Factor : Pyruvate (mg/dL) = DA x 6,37 ( at 340 nm ) with Standard : Pyruvate (mg/dL) =
(DAsample) x 4.0 (DAstandard)
Expected values 0.3 – 0.7 mg/dL
Intended Use
Reagent Storage and Stability
Spectrum Diagnostics liquizyme Pyruvate reagent is intended for the in-vitro quantitative, diagnostic determination of pyruvate in human blood.
All reagents are stable until expiration date stated on label when o protected from light stored refrigerated at 2 - 8 C.
Note: It is recommended for each laboratory to establish and maintain its own reference values. The given data are only an indication.
Specimen Preparation
QUALITY CONTROL
Pipet 2,0 mL of freshly drawn blood into a centrifugation tube containing 4 mL of cold 0.6 m perchloric acid. Vortex for about 30 seconds. Keep the blood precipitate mixture for about 5 min in the cold to assure complete protein precipitation. Centrifuge 10 min at approximately 1500 x g. The protein free supernatant is ready for use. The Standard solution has to be diluted with perchloric acid, in the same ratio as the sample.
For quality control use adequate control materials, available from Spectrum Diagnostics.
Method Enzymatic UV - Test.
Assay Principle In the presence of an excess of NADH pyruvate is converted to lactate.The reduction of the absorbance = DA,at 340 nm, due to the oxidation of NADH to NAD+ , is a measure of the amount of pyruvate originally present : Pyruvate + NADH + H+
LDH
L-Lactate + NAD+
System Parameters
Reagents Standard (St.) Reagents:
4.0 mg/dl
Wavelength Optical path Assay type
340 nm ( 334 - 365 ) 1 cm End point o
R1 Buffer Reagent : (Tris buffer, pH 7.20)
R2 Coenzyme NADH R3 Start Reagent LDH
1.50 mol/L
o
Temperature 30 C or 37 C Zero adjustment Against Water Linearity 400 mg/dl (61.2 mmol/l)
Waste Disposal This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Procedure 10.0 mmol/L
1.50 kU/mL
Additional Reagent ( not provided with the kit ) Perchloric Acid 0.6m for deproteinization
Precautions and Warnings Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
Reagent Preparation
Sample
Standard
R1 Buffer Reagent
1 ml
1 ml
Supernatant Sample
2 ml
------
Diluted Standard
------
Mix and add R2 Coenzyme
50 ml
ORDERING INFORMATION
2 ml
CATALOG NO
QUANTITY
50 ml
335 001
100 Test
Mix and incubate for approximately 5 min ,pour into uvette,measure initial absorbance A1 R3 Start Reagent
50 ml
50 ml
mix, incubate for approximately 5 min and measure absorbance A2 DA = A2 - A1 For Sample and Standard
Spectrum Pyruvate reagents are supplied ready-to-use.
52
53
Total bile acids (TBA)
Enzymatic recycling method
EC REP Authorised Representative
REF: 305 001
LOT
100 test
IVD REF
Intended Use
Total bile acids (TBA) diagnostic reagent is used for quantitative in vitro determination of total bile acids in serum or plasma on photometric systems.
Background
Liver: Total bile acids are metabolized in the liver and hence serve as a marker for normal liver function. Total bile acids are increased in patients with acute hepatitis, chronic hepatitis, liver sclerosis and liver cancer, liver cancer, Cholestasis, Congenital and acquired vascular shunts. Pregnancy: Obestetric cholestasis is the most common liver disease seen during pregnancy and the test key diagnostics tool for investigating suspected cases. the main symptom is itching, but tiredness , nausea and jaundice may also be reported. in addition to the discomfort experience by the mother, high levels of bile acids also increased risk to the baby and may lead to premature birth or greater risk of passing meconium in the womb. Veterinary: In veterinary medicine the bile acids test is typically used to assess liver function in mammals such as cats, dogs, horses, pigs, cows and sheep. The test is also of use in the investigation of avian liver disease, where elevated liver enzymes do not always correlate with liver disease. In the most species a single, random sample is assessed but a dual sampling protocol is typically used with cats and dogs to investigate bile reabsorption. For the latter a fasting blood sample is taken and then a second, post prandial sample taken a few hours after being fed for comparison. Total bile acids are typically low in fasted state, whilst level will rise immediately after a meal. When the liver has normal mass and blood flow the bile acid levels will rapidly fall back to near fasting level as the liver removes them from the blood and returns them to the gall bladder.
Method
Enzymatic recycling method
Assay Principle
In the presence of Thio-NAD, the enzyme 3-á hydroxysteroid dehydrogenase (3-á HSD) converts bile acids to 3-keto steroids and Thio-NADH. The reaction is reversible and 3-á HSD can convert 3-keto steroids and Thio-NADH to blie acids and Thio-NAD. In the presence of excess NADH, the enzyme cycling occurs efficiently and the rate of formation of Thio-NADH is determined by measuring specific change of absorbance at 405nm.
Reagents Reagent 1 (R1) Buffer (pH 7.0) 65mmol/L Thio-NAD 1 g /L Reagent 2 (R2) Buffer (pH 7.0) 65 mmol/L Thio-NADH 6 g /L Sodium Azide 5000 U/L
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
54
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Reagent Preparation, Storage and Stability
Spectrum TBA reagents are supplied ready-to-use and stable up to the expiry date labeled on the bottles at 2 – 8 oC.
Deterioration
Failure to recover control values within the assigned range may be an indication of reagent deterioration.
Specimen Collection and Preservation
The only acceptable anticoagulat is EDTA. Use preferably fresh serum.
Level 2
1.
Abbassi-Ghanavati M, Greer LG, cunningham FG.
20
20
Mean (µmol/L)
85.16
228.18
2.
Wallach, J.,Eighth ed. lippincott Williams & Wilkins
SD
3.25
6.69
3.
CV%
3.82
2.93
H. Bergmeyer, K. Gawehn, and M. Grassl in Methods of Enzymatic Analysis (Bergmeyer H. U. ed) 2nd Volume I, 505507, Academic Press, Inc. New York, NY (1974).
4.
Drummond, G. I. & Masanobu, Y. In: The enzymes (boyer, P.D. (ed.), (3rd ed.), vol.4, pp. 337(1971).
Calibration
The assay requires the use of a total bile acids calibrator. Recalibration is recommended at anytime if one of the following events occurs: * The Lot number of reagents changes. * Preventative maintenance is performed or a critical component is replaced. * Control values have shifted or are out of range and a new vial of control does not rectify the problem.
ORDERING INFORMATION CATALOG NO
QUANTITY
305 001
100 test
Methods Comparison
Sensitivity
System Parameters
When run as recommended, the minimum detection limit of this assay 0.5 µmol/L.
Wavelength 405 nm Optical path 1 cm Assay type Fixed rate Direction Increase Temperature 37 oC Incubation time 5 minutes at 20–25oC Zero adjustment Reagent Blank Sensitivity 0.5 µmol/L Linearity 150 µmol/L
Linearity
The reaction is linear up to an albumin concentration of 180 µmol/L ; specimens showing higher concentration should be diluted 1+1 with physiological saline and repeat the assay (result × 2).
Interfering substances Serum, plasma
Procedure Blank
Calibrator
Specimen
300 ml
300 ml
300 ml
Calibrator
-----
5 ml
-----
Specimen
-----
-----
5 ml
Mix , incubate for 5 minutes at 37oC then add: Reagent2(R2)
Level 1 n
A comparison between Spectrum Diagnostics TBA reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.97 was obtained.
Stability: 1 week at 4 – 8 oC 2 months at -20 oC
Reagent1(R1)
References
Run to run (Reproducibility)
SYMBOLS IN PRODUCT LABELLING
100 ml
100 ml
100 ml
Mix, and after 1 minute read the absorbance A1 of the Calibrator or specimen. After 3 minutes later, read absorbance A2 of calibrator or specimen.
Calculation
Normal & abnormal commercial control serum concentrations should be analyzed with each run.
of
Performance Characteristics Precision Within run (Repeatiblity)
Level 1
Icterus No significant interference up to a bilirubin level of 30 mg/dL. Lipemia No significant interference up to an intralipid level of 2 %. Expected Values 0 -10.4 µmol/L It is recommended that each laboratory should establish its own reference interval. Analytical Range 0.5 – 180 µmol/L.
A2 – A1 = Aspecimen or Acalibrato or Ablank. (ASpecimen - Ablank) TBA(µmol/L) = x conc. of calibrator (ACalibrator - Ablank)
Quality Control
Haemolysis A haemoglobin level of 150 mg/dL results in 13 % positive bias.
Waste Disposal
known
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
Level 2
n
20
20
Mean (µmol/L)
45.54
171.62
SD
0.35
0.65
CV%
0.76
0.38
55
Total protein Biuret Reagent REF: 310 001 REF: 310 002 REF: 310 003 REF: 310 004 REF: 310 005
EC REP Authorised Representative IVD LOT
(2 x 100ml) 200 test (4 x 100ml) 400 test (8 x 100ml) 800 test (2 x 500ml) 1000 test (2 x 250ml) 500 test
REF
!
Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by (C) - Corrosive
Spectrum Diagnostics total protein reagent is intended for the invitro quantitative, diagnostic determination of total protein in human serum on both automated and manual systems.
Do not use The total protein regents if precipitate forms. Failure to recover control values within the assigned range may be an indication of reagent deterioration.
Plasma proteins are mainly synthesized in the liver and are involved in the maintenance of normal water distribution between tissues and blood, as well as acid-base balance. Due to some pathological conditions, both total protein level and the ratio of different fractions may change independently of one another. Hyperproteinemia may be detected during dehydration associated with diarrhea or vomiting. The total protein levels also increase in multiple myeloma. Hypoproteinemia may occur as a result of prolonged low protein diet and in some pathological conditions such as nephrotic syndrome, bleeding, sprue, and salt retention.
Method
Coloremetric method (Biuret reagent).
Assay Principle
In alkaline medium the copper reacts with the peptide bonds of proteins to form the characteristic pink to purple biuret complex. Sodium potassium tartarate prevents copper hydroxide precipitation, and potassium iodide prevents the autoreduction of copper. 2+
Alkaline pH
Cu – protein complex
The color intensity is directly proportional to the protein concentration. It is determined by measuring the increase in the absorbance at 546nm.
Reagents
Standard Total protein (ST) 6.0 g/dL Reagent (R) Sodium hydroxide Copper sulfate Sodium potassium tartarate Potassium iodide
Use serum or plasma ( EDTA or heparin ) for the test . Usually plasma results are higher due to fibrinogen . The serum or plasma should be separated from the cells within 4 hours . o o Stability : 1 day at 15 – 25 C ; 4 weeks at 4 – 8 C; o 1 year at -20 C
System Parameters
mmol/L mmol/L mmol/L mmol/L
56
0.21
CV%
2.53
2.4
Blank
Standard
Sample
Reagent (R)
1.0 ml
1.0 ml
1.0 ml
Standard
------
20 µl
------
Sample
------
------
20 µl
Serum protein conc. (g/dL) =
When run as recommended, the minimum detection limit of this assay is 1.0 g/dL.
Gornall AG, Bardawill CJ, David MM: Determination of serum protein by means of the biuret reagent. J Biol Chem 177:751, 1949 .
3.
Kaplan A, Szalbo J :Clinical chemistry :Interpretation and techniques, 2 nd ed. A Kaplan, J Szabo, editors, 1983, p 157.
4.
Schultze HE, Heremans JF:Molecular biology of human protein. Elsevier publishing company, Amsterdam, 1966.
5.
Tietz NW : Fundamentals of Clinical Chemistry: 2 nd ed. NW Tietz, editor, 1994, pp692 .
Linearity
The reaction is linear up to total protein concentration of 12 g/dL. Specimens showing higher concentration should be diluted 1+1 using physiological saline and repeat the assay (result × 2).
ORDERING INFORMATION CATALOG NO
QUANTITY
310 001 310 002 310 003 310 004 310 005
2 x 100 ml 4 x 100 ml 8 x 100 ml 2 x 500 ml 2 x 250 ml
Expected Values Adults
x6
6.6 – 8.7 g/dL
Children
(> 1 year) (< 1 year)
6.0 – 8.0 g/dL 4.8 – 7.6 g/dL
Newborns
(< 4 weeks)
4.6 – 6.8 g/dL
Prematures (Aspecimen) (Astandard)
2.
Drugs Sera from patients receiving dextran may cause artificially high levels due to turbidity during color development. This positive bias can be minimized by centrifuging the reaction mixture before reading the absorbance.
Procedure
Calculation
A comparison between Spectrum Diagnostics Total Protein reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.978 was obtained.
Cannon DC, Olitzky I, Inkpen JA :Proteins. In:Clinical chemistry, principles and technics, 2 nd ed. RJ Henery, DC Cannon, JW Winkelman, editors, Harper & Row, New York, pp 407 – 421,1974.
Lipemia No significant interference.
Normal & abnormal commercial control serum concentrations should be analyzed with each run.
Spectrum Total protein reagents are supplied ready-to-use and stable up to the expiry date labeled on the bottles. The reagents o are stable at 15 – 25 C. Only the standard is needed to be kept o refrigerated at (2 - 8 C).
7.32
0.19
1.
Icterus No significant interference up to a bilirubin level of 30 mg/dL.
For further information, refer to the Total Protein reagent material safety data sheet.
Reagent Preparation, Storage and Stability
5.7
SD
Hemolysis No interference up to hemoglobin level of 7.5 g/L.
Note: For turbid highly icteric sera, prepare a serum blank by adding 20 ml serum to 1 ml saline into a labeled test tube. Read absorbance of serum blank at 540 nm vs water and subtract serum blank absorbance from test absorbance before calculating results.
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
Mean (mg/dL)
Serum, plasma
(C)-Corrosive contains caustic materials. R34 Causes burns. S26-45 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice in case of accident or if you feel unwell, seek medical advice immediately.
Precautions and Warnings
20
Interfering Substances
Wavelength Hg 546 nm (530 – 570 nm) Optical path 1 cm Assay type End-point Direction Increase Sample : Reagent Ratio 1 : 50 e.g .: Reagent volume 1 ml Sample volume 20 ml o Temperature 15 – 25 C Zero adjustment Reagent blank o Incubation time 10 minutes at 15 – 25 C Sensitivity 1.0 g/dL Linearity 12 g/dL
Mix, Incubate for 10 minutes at room temp. Measure absorbance of specimen (Aspecimen) and standard (Astandard) against reagent blank within 30 minutes. 750 12.0 40.9 19.8
Level 2
20
Sensitivity
Specimen Collection and Preservation
Background
Level 1 n
Methods Comparison
Deterioration
Intended Use
Protein + Cu
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use Temperature Limitation
References
Run to run (Reproducibility)
SYMBOLS IN PRODUCT LABELLING
3.4 – 5.0 g/dL
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure;interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Analytical Range 1.0 – 12 g/dL.
Quality Control
of
Performance Characteristics Precision Within run (Repeatiblity)
Level 1
Level 2
n
20
20
Mean (mg/dL)
5.2
7.23
SD
0.12
0.15
CV%
2.47
2.2
known
Waste Disposal
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
57
Triglycerides – Liquizyme GPO-PAP (Single Reagent) REF: 314 001 REF: 314 002 REF: 314 003 REF: 314 004 REF: 314 005 REF: 314 006 REF: 314 007 REF: 314 008 REF: 314 009
( 2 x 25 ml) ( 4 x 25 ml) ( 3 x 40 ml) (10 x 15 ml) ( 4 x 50 ml) ( 4 x 100 ml) ( 8 x 100 ml) ( 5 x 100 ml) ( 4 x 6 0 ml)
50 100 120 150 200 400 800 500 240
test test test test test test test test test
Reagents
Triglycerides are the main lipids present in the human plasma; the others are the cholesterol, phospholipids and nonesterified fatty acids. They are formed in the intestinal mucosa by the esterification of glycerol and fatty acids. Triglycerides measurements are used in the diagnosis and treatment of patients with diabetes mellitus, liver obstruction, nephrosis and other diseases involving lipid metabolism. The measurement of serum triglycerides is important in the diagnosis of hyperlipoproteinemia and in the prediction, detection and monitoring of atherosclerosis.
Method
Assay Principle
The series of the reaction involved in the assay system is as follows:
Triglycerides are hemolyzed by lipoprotein lipase (LPL) to glycerol
3.
Glycerol-3-phosphate + ADP
GPO
Dihydroxyacetone phosphate + H2O2
In the presence of peroxidase (POD), hydrogen peroxide effects the oxidative coupling of 4-chlorophenol and 4-aminoantipyrine (4AAP) to form a red color quinoneimine dye which is measured at 546 nm. 2 H2O2+4-APP + 4-chlorophenol
58
GK
The oxidation of glycerol-3-phosphate is catalyzed by glycerol phosphate oxidase (GPO) to form dihydroxyacetone phosphate and hydrogen peroxiode (H2O2). Glycerol-3-phosphate + O2
4.
Glycerol + Fatty acids
Glycerol is then phosphorylated to glycerol-3-phosphate by ATP in a reaction catalyzed by glycerol kinase (GK). Glycerol + ATP
POD
Standard triglyceride (ST) 200 mg/dL Reagent (R) Pipes Buffer pH 7.0 4-chlorophenol Magnesium aspartate Lipase Peroxidase 4-Aminoantipyrine Glycerol-3-phosphate oxidase Glycerol kinase ATP Sodium Azide
2.29 mmol/L 50 mmol/L 6.0 mmol/L >0.5 mmol/L >10 K U/L >2.0 KU/L 1.0 mmol/L >3.5 K U/L >750 U/L 1.0 mmol/L 8.0 mmol/L
For further information, refer to the Triglycerides reagent material safety data sheet..
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately. Reagent (R) contains sodium azide which may react with copper or lead plumbing.
GPO-PAP-enzymatic colorimetric method.
2.
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
Blank
Standard
Sample
Reagent
1.0 ml
1.0 ml
1.0 ml
Standard
------
10 µl
------
Sample
------
------
10 µl
o
Background
LPL
IVD REF
Spectrum Diagnostics liquizyme triglycerides reagent is intended for the in-vitro quantitative, diagnostic determination of triglycerides in human serum on both automated and manual systems.
Triglycerides
EC REP Authorised Representative LOT
Intended Use
1.
Procedure
SYMBOLS IN PRODUCT LABELLING
Quinoneimine dye + 4H2O
Reagent Preparation, Storage and Stability
Spectrum triglyceride reagents are supplied ready-to-use and stable up to the expiry date labeled on the bottles when properly stored o refrigerated at ( 2 – 8 C). Once opened, the opened vial is stable for 3 months at the specified temperature.
Others physiological ascorbic acid concentration dosn’ t interfere with the test. Ascorbic acid levels higher than 114 mmol /l (2 mg /dL) decrease the apparent triglycerides concentration significantly.
Expected Values o
Mix and incubate for 5 minutes at 37 C or 10 minutes at 15 – 25 C. Measure absorbance of specimen (Aspecimen) and standard (Astandard) against reagent blank within 30 minutes.
Calculation Serum triglycerides conc. (mg/dL) =
Quality Control
(Aspecimen) (Astandard)
of
known
Performance Characteristics Precision Within run (Repeatiblity)
Level 1
Level 2
n
20
20
Mean (mg/dL)
155.1
245.8
SD
2.03
1.85
CV%
1.31
0.75
Level 1
Level 2
Run to run (Reproducibility) n
20
20
Mean (mg/dL)
156
246.5
SD
2.2
1.9
CV%
1.4
0.87
Methods Comparison
Deterioration
The reagent is normally clear or pale pink. Do not use liquizyme triglyceride reagent if it is turbid or if the absorbance is greater than 0.2 at 546 nm.
A comparison between Spectrum Diagnostics Triglyceride reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.967 was obtained.
Specimen Collection and Preservation
Sensitivity
Patients should be fasting for 10 to 14 hours before blood is drawn. Samples must be drawn in a soap and glycerol free collection device. Recommended anticoagulats are EDTA or heparin at levels of 1mg and 0.2 mg/dl whole blood, respectively.
Hg 546 nm (500 – 550 nm) 1 cm End-point 1 : 100 1 ml 10 µl o o 15 – 25 C or 37 C Reagent blank o 10 minutes at 15 – 25 C or o 5 minutes at 37 C Low 0.00 AU High 0.2 AU 5 mg/dL (0.057 mmol/L) 1000 mg/dL (11.45 mmol/L)
5 - 1000 mg/dL (0.057 - 11.45 mmol/L)
Waste Disposal
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References 1. 2. 3.
5.
Interfering Substances
Wavelength Optical path Assay type Direction Increase Sample : Reagent Ratio e.g.: Reagent volume Sample volume Temperature Zero adjustment Incubation time Reagent Blank Limits Sensitivity Linearity
Serum, plasma
Haemolysis No significant interference up to a haemoglobin level of 6.0 g/L (0.36 mmol/L). Icterus Bilirubin levels higher than 171 mmol/L (10 mg/dL) decrease the apparent triglycerides concentration significantly. Drugs Of the drugs tested in-vitro, methyldopa and levodopa cause artificially low triglyceride values at the tested drug Level.
(1.71 mmol/L) (2.28 mmol/L)
Dynamic Range
Linearity
System Parameters
above 150 mg/dL above 200 mg/dL
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure;interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
4.
Triglycerides in serum samples remain stable for 7 days at 4 C, for o o 3 months at -20 C, and for years at -70 C.
(0.4 – 1.54 mmol/L) (0.45 – 1.82 mmo/L)
The following limits are recommended for the recognition of the risk factor hypertriglyceridemia:
When run as recommended, the minimum detection limit of the assay is 5 mg/dL (0.057 mmol/L). The reaction is linear up to triglycerides concentration of 1000 mg/ dL; specimens showing higher concentration should be diluted 1+1 using physiological saline and repeat the assay (result × 2).
o
35 -135 mg/dL 40 -160 mg/dL
Suspicious Elevated
x 200
Normal & abnormal commercial control serum concentrations should be analyzed with each run.
Females Males
6.
Bucolo G, David H : Quantitative determination of serum triglycerides by the use of the enzymes. Clin Chem 19 : 475, 1973 Chowdhury RF, Rodman H, Bleicher SJ : Glycerol like contamination of commerical blood sampling tubes. J Clin Pathol 12: 116, 1971 MGowan MW, Artiss JD, Standbergh DR, Zak B. A peroxidasecoupled method for colorimetric determination of serum triglycerides. Clin Chem ;29:538-452 ;1983. Stein EA; Lipids , lipoproteins, and apolipoproteins. In : NW Tietz , ed. Fundamentals of clinical chemistry, 3 rd ed. Philadelphia : WB Saunders; 448 ; 1987. Tietz NW, Boden T, Stepleton JD : An improved method for the determination of lipase in serum. Am J Clin Pathol 31: 148, 1959 Young DS et al, Clin Chem. 21 ; 1975 ORDERING INFORMATION CATALOG NO 314 001 314 002 314 003 314 004 314 005 314 006 314 007 314 008 314 009
QUANTITY 2 x 25 ml 4 x 25 ml 3 x 40 ml 10 x 15 ml 4 x 50 ml 4 x 100 ml 8 x 100 ml 5 x 100 ml 4 x 60 ml
59
Urea/BUN - Liquizyme
o
SYMBOLS IN PRODUCT LABELLING
(Modified Urease-Berthlot Method) REF: 318 001
100 test
REF: 318 002
EC REP Authorised Representative
200 test
R1 Buffer 1 x 100 ml R1 Buffer 2 x 100 ml R2 Urease 1 x 6 ml R2 Urease 2 x 6 ml R3 Alkaline reagent 1 x 20 ml R3 Alkaline reagent 1 x 40 ml REF: 318 003
500 test
REF: 318 004
1000 test
R1 Buffer 5 x 100 ml R1 Buffer 4 x 250 ml 51 ml R2 Urease 2 x 15 ml R2 Urease R3 Alkaline reagent 2 x 50 ml R3 Alkaline reagent 1 x 210 ml
Intended Use
Spectrum Diagnostics colorimetric urea reagent is intended for the in-vitro quantitative, diagnostic determination of urea in human serum on both automated and manual systems.
Background
Urea is the major end product of protein nitrogen metabolism. It is synthesized by the urea cycle in the liver and excreted through the kidneys. The circulating levels of urea depend upon protein intake, protein catabolism and kidney function. Elevated urea levels can occur due to renal impairment or in some diseases such as diabetes, infection, congestive heart failure and during different liver diseases. Determination of blood urea nitrogen is the most widely used screening test for renal function together with serum creatinine.
Method
Urease-colorimetric method.
Assay Principle
The reaction involved in the assay system is as follows: Urea is hydrolyzed in the presence of water and urease to produce ammonia and carbon dioxide. Urea + H2O
Urease
2NH3 + CO2
The free ammonia in an alkaline pH and in the presence of indicator forms coloured complex proportional to the urea concentration in the specimen.
Reagents
Standard urea (ST) Aqueous primary standard 50 mg/dL Reagent 1 (R1 Buffer) Phosphate buffer pH 8.0 Sodium salicylate Sodium nitroprusside EDTA Reagent 2 (R2 Enzyme) Urease
8.33 mmol/l 100 mmol/l 80 mmol/l 6.0 mmol/l 30.0 mmol/l >6000 U/l
IVD LOT REF
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use Temperature Limitation
Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by (Xi) - Irritant
!
Do not use the reagent if it is turbid. Failure to recover control values within the assigned range may be an indication of reagent deterioration.
Specimen Collection and Preservation
Serum No special preparation of the patient is required. Use nonhaemolyzed serum or plasma only.The only acceptable anticoagulants are heprin, EDTA and fluoride. Do not use ammonium heparin plasma. o o Stability: 7 days at 15 –25 C ; 7 days at 2 – 8 C; o 1 year at -20 C Urine Urine samples are prediluted 1 : 50 with ammonium free water prior to assay. o o Stability: 2 days at 15 –25 C ; 7 days at 2 – 8 C; o 1 month at -20 C
System Parameters
Wavelength 578 nm (578-623 nm) Optical path 1 cm Assay type End-point Direction increase o o temperature 15-25 C or 37 C Zero adjustment Against Reagent blank Reagent Blank Limits Low 0.02 AU High 0.2 AU Sensitivity 0.6 md/dL (0.1 mmol/l) Linearity 200 mg/dL (33.3 mmol/l)
For further information, refer to the Urea/BUN reagent material safety data sheet.
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
Reagent Preparation, Storage and Stability
Spectrum coloremetric urea reagents are supplied ready-to-use and o stable up to the expiry date labeled on the bottles (2 – 8 C).
60
Normal & abnormal control serum of known concentrations should be analyzed with each run.
Performance Characteristics Precision Within run (Repeatiblity)
Specimen
1.0 ml
1.0 ml
1.0 ml
one drop (50 ml)
one drop (50 ml)
one drop (50 ml)
Standard
------
10 ml
------
Sample
------
------
10 ml
Mix and incubate for at least 3 minutes at 37 C or 5 minutes at o 20-25 C 200 ml
200 ml
200 ml
o
Mix and incubate for 5 minutes at 37 C or 10 minutes at o 20-25 C Measure absorbance of specimen (Aspecimen) and standard (Astandard) against reagent blank. Procedure 2 (Using working solution) Blank
Standard
Specimen
1.0 ml
1.0 ml
1.0 ml
Standard
------
10 ml
------
Sample
------
------
10 ml
Expected Values Urea(Serum) Adults ≤ 65 years Adults ≥ 65 years
: 15 – 50 mg/dL (2.5-8.33 mmol/L) : ≤ 70 mg/dL (≤11.66 mmol/L)
BUN(Serum) Adults ≤ 65 years Adults ≥ 65 years Children
: 7 – 23.5 mg/dL : 7 – 32.9 mg/dL : 5 – 18 mg/dL
Urine (24) hours Urea : 20 – 35 g/24hrs (330-580 mmol/24hrs) BUN : 9.3 – 16.4 g/24hrs Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Analytical Range
Level 2
20
20
0.6 – 200 mg/dL (0.1 - 33.3 mmol/L).
Mean (mg/dL)
60
144
SD
1.87
2.1
Waste Disposal
CV%
3.12
1.46
Level 1
Level 2
20
20
Mean (mg/dL)
62
146
SD
1.92
2.5
CV%
3.25
1.65
Run to run (Reproducibility)
Sensitivity Standard
suppressed by amines, thiols, steroids and ascorbic acid.
Level 1 n
A comparison between Spectrum Diagnostics Urea/BUN reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.97 was obtained.
Blank
Working solution
Urea Nitrogen: To convert the result from urea to urea nitrogen multiply the result by 0.467.
Methods Comparison
Procedure 1
R3(Alk.Reagent)
xn
Urine urea concentration is determined by multiplying the result by the dilution factor (50).
n
o
Reagent 3 (R3 Alkaline Reagent) Sodium hydroxide 400 mmol/l Sodium hypochlorite 20.0 mmol/l Irritant (xi) R36/38: Irritating to eyes and skin. S26: In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. S37/39: Wear suitable gloves and eye/face protection.
(Aspecimen) Serum urea concentration (mg/dl) = A ( standard)
Quality Control
Deterioration
R2(Enzyme)
Calculation
where n = 50.0 mg/dl (8.33 mmol/l)
NB: For megalabs having high numbers of patient specimens,working buffer reagent can be prepared .( Stability 1 week ) REF:318 001: add 5 ml from R2 to one bottle of R1; mix gently. REF:318 002: add 5 ml from R2 to one bottle of R1; mix gently. REF:318 003: add 5 ml from R2 to one bottle of R1; mix gently. REF:318 004:add 12.5 ml from R2 to one bottle of R1; mix gently.
R1(Buffer)
Mix and incubate for 5 minutes at 37 C or 10 minutes at o 20-25 C. Measure absorbance of specimen (Aspecimen) and standard (Astandard) against reagent blank.
When run as recommended, the minimum detection limit of the assay is 0.6 mg/dL.
Linearity
The reaction is linear up to a urea concentration of (200 mg/dl) 33.3 mmol/L. Specimens showing higher concentrations should be diluted 1+2 with physiological saline and repeat the assay (result×3).
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References 1. 2. 3. 4.
Batton, C. J & crouch, S.R : Anal. Chem., 1977,49:464-469. Shephard MD, Mezzachi RD : Clin Biochem Revs, 4:61-7, 1983. Tietz NW, ED. Clinical guide to Laboratory tests. 2ND ED. Philadelphia: WB Saunders; 1990:566. Tiffany to, jansen JM, Burtis CA,Overton JB, Scott CD. Enzymatic Kinetic Rate and end Point analyses of Substrate, By USE of A Gemsaec fast analyzer. Clin Chem. ORDERING INFORMATION CATALOG NO
QUANTITY
318 001 318 002 318 003 318 004
100 Test 200 Test 500 Test 1000 Test
Interfering Substances Serum, plasma
Haemolysis Erythrocyte contamination doesn’t elevate results. Icterus No significant interference. Lipemia Lipemic specimens interfere with the method of Berthlot. Anticoagulants Ammonium heparin should not be used.
o
Mix and incubate for at least 3 minutes at 37 C or 5 minutes at o 20 -25 C R3(Alk.Reagent)
200 ml
200 ml
200 ml
Others Ammonium ions should be avoided since it may cause erroneously elevated results. Color development in the Berthlot reaction is
61
Urea/BUN - LS (Modified UreaseBerthlot Method) REF: 321 001
90 Test
Quality Control
SYMBOLS IN PRODUCT LABELLING EC REP Authorised Representative IVD LOT
REF
REF: 321 002
180 Test
R1 Buffer R2 Urease R3 Alkaline reagent
1 x 90 ml 1 x 1.5 ml 1 x 20 ml
R1 Buffer R2 Urease R3 Alkaline reagent
2 x 90 ml 2 x 1.5 ml 1 x 40 ml
REF: 321 003
270 Test
REF: 321 004
400 Test
R1 Buffer R2 Urease R3 Alkaline reagent
3 x 90 ml 3 x 1.5 ml 1 x 65 ml
R1 Buffer R2 Urease R3 Alkaline reagent
4 x 100 ml 4 x 1.7 ml 2 x 40 ml
Intended Use
Spectrum Diagnostics colorimetric urea reagent is intended for the in-vitro quantitative, diagnostic determination of urea in human serum on both automated and manual systems.
Background
Urea is the major end product of protein nitrogen metabolism. It is synthesized by the urea cycle in the liver and excreted through the kidneys. The circulating levels of urea depend upon protein intake, protein catabolism and kidney function. Elevated urea levels can occur due to renal impairment or in some diseases such as diabetes, infection, congestive heart failure and during different liver diseases. Determination of blood urea nitrogen is the most widely used screening test for renal function together with serum creatinine.
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use Temperature Limitation
Normal & abnormal control serum of known concentrations should be analyzed with each run.
Use by/Expiration Date
! CAUTION. Consult instructions for use Manufactured by (Xi) - Irritant
Within run (Repeatiblity)
Reagent Preparation , Storage and Stability
To prepare the working solution add the content of one vial of urease (R2) to one bottle of buffer reagent (R1). o Stability : 1 Month at 2-8 C.
Deterioration
Do not use the reagent if it is turbid. Failure to recover control values within the assigned range may be an indication of reagent deterioration.
Specimen Collection and Preservation
Urease-colorimetric method.
Assay Principle
System Parameters
The reaction involved in the assay system is as follows: Urea is hydrolyzed in the presence of water and urease to produce ammonia and carbon dioxide. Urea + H2O
Urease
2NH3 + CO2
The free ammonia in an alkaline pH and in the presence of indicator forms coloured complex proportional to the urea concentration in the specimen.
Reagents Standard urea (ST) Aqueous primary standard
50 mg/dL 8.33 mmol/l
Reagent 1 (R1 Buffer)
Phosphate buffer pH 8.0 100 mmol/l Sodium salicylate 80 mmol/l Sodium nitroprusside 6.0 mmol/l EDTA 30.0 mmol/l
Reagent 2 (R2 Enzyme)
Urease >350000 U/l
Reagent 3 (R3 Alkaline Reagent)
Sodium hydroxide 400 mmol/l Sodium hypochlorite 20.0 mmol/l Irritant (xi) R36/38: Irritating to eyes and skin. S26: In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. S37/39: Wear suitable gloves and eye/face protection.
Wavelength 578 nm (578-623 nm) Optical path 1 cm Assay type End-point Direction increase o o temperature 15-25 C or 37 C Zero adjustment Against Reagent blank Reagent Blank Limits Low 0.02 AU High 0.2 AU Sensitivity 0.6 md/dL (0.1 mmol/l) Linearity 200 mg/dL (33.3 mmol/l) Specimen
1.0 ml
1.0 ml
1.0 ml
------
10 ml
------
Sample
------
------
10 ml
200 µl
o
o
Mix and incubate for 5 minutes at 37 C or 10 minutes at 20-25 C Measure absorbance of specimen (Aspecimen) and standard (Astandard) against reagent blank ..
Serum urea concentration (mg/dl) =
(Aspecimen) (Astandard)
xn
For further information, refer to the Urea/BUN reagent material safety data sheet.
where n = 50.0 mg/dl (8.33 mmol/l)
Precautions and Warnings
Urine urea concentration is determined by multiplying the result by the dilution factor (50).
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
62
Mean (mg/dL)
60
144
SD
1.87
2.1
CV%
3.12
1.46
Level 1
Level 2
n
20
20
Mean (mg/dL)
62
146
SD
1.92
2.5
CV%
3.25
1.65
Methods Comparison
A comparison between Spectrum Diagnostics Urea/BUN reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.97 was obtained.
Sensitivity
0.6 – 200 mg/dL (0.1 - 33.3 mmol/L).
Waste Disposal
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References 1. 2. 3. 4.
Batton, C. J & crouch, S.R : Anal. Chem., 1977,49:464-469. Shephard MD, Mezzachi RD : Clin Biochem Revs, 4:61-7, 1983. Tietz NW, ED. Clinical guide to Laboratory tests. 2ND ED. Philadelphia: WB Saunders; 1990:566. Tiffany to, jansen JM, Burtis CA,Overton JB, Scott CD. Enzymatic Kinetic Rate and end Point analyses of Substrate, By USE of A Gemsaec fast analyzer. Clin Chem.
When run as recommended, the minimum detection limit of the assay is 0.6 mg/dL. ORDERING INFORMATION
Linearity
The reaction is linear up to a urea concentration of (200 mg/dl) 33.3 mmol/L. Specimens showing higher concentrations should be diluted 1+2 with physiological saline and repeat the assay (result×3).
CATALOG NO
QUANTITY
321 001 321 002 321 003 321 004
1 x 90 ml 2 x 90 ml 3 x 90 ml 4 x 100 ml
Urea Nitrogen: To convert the result from urea to urea nitrogen multiply the result by 0.467.
Lipemia Lipemic specimens interfere with the method of Berthlot. Anticoagulants Ammonium heparin should not be used.
Mix and incubate for at least 3 minutes at 37 oC or 5 minutes o at 20-25 C .
Calculation
20
Icterus No significant interference.
Standard
200 µl
20
Analytical Range
Haemolysis Erythrocyte contamination doesn’t elevate results.
Blank
200 µl
n
Serum, plasma
Standard
R3(Alk)
Level 2
Interfering Substances
Procedure
Working solution
Level 1
Run to run (Reproducibility)
Serum No special preparation of the patient is required. Use nonhaemolyzed serum or plasma only.The only acceptable anticoagulants are heprin, EDTA and fluoride. Do not use ammonium heparin plasma. o o Stability: 7 days at 15 –25 C ; 7 days at 2 – 8 C; o 1 year at -20 C Urine Urine samples are prediluted 1 : 50 with ammonium free water prior to assay. o o Stability: 2 days at 15 –25 C ; 7 days at 2 – 8 C; o 1 month at -20 C
Method
Performance Characteristics Precision
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Others Ammonium ions should be avoided since it may cause erroneously elevated results. Color development in the Berthlot reaction is suppressed by amines, thiols, steroids and ascorbic acid.
Expected Values Urea(Serum) Adults ≤ 65 years Adults ≥ 65 years
: 15 – 50 mg/dL (2.5-8.33 mmol/L) : ≤ 70 mg/dL (≤11.66 mmol/L)
BUN(Serum) Adults ≤ 65 years Adults ≥ 65 years Children
: 7 – 23.5 mg/dL : 7 – 32.9 mg/dL : 5 – 18 mg/dL
Urine (24) hours Urea : 20 – 35 g/24hrs (330-580 mmol/24hrs) BUN : 9.3 – 16.4 g/24hrs
63
Urea/BUN – Liquizyme (UV) REF: 319 001 REF: 319 002 REF: 319 003 REF: 319 004
(3 x 50 ml) (3 x 90 ml) (4 x 100 ml) (4 x 50 ml)
150 270 400 200
EC REP Authorised Representative
test test test test
IVD LOT REF
Intended Use
Spectrum Diagnostics liquizyme urea reagent is intended for the invitro quantitative, diagnostic determination of urea in human serum or urine on both automated and manual applications.
Background
Urea is the major product of protein nitrogen metabolism. It is synthesized by the urea cycle in the liver and excreted through the kidneys. The circulating levels of urea depend upon protein intake, protein catabolism and kidney function. Elevated urea levels can occur due to renal impairment or in some diseases such as diabetes, infection, congestive heart failure and during different liver diseases. Determination of blood urea nitrogen is the most widely used screening test for renal function together with serum creatinine.
urease-UV fixed rate (enzymatic method).
Assay Principle
The series of reactions involved in the assay are as follows : Urea is hydrolyzed in the presence of water and urease to produce ammonia and carbon dioxide. Urea + H2O 2.
Urease
2NH3 + CO2
In the presence of glutamate dehydrogenase (GLDH) and reduced nicotinamide adenine dinucleotide (NADH),the ammonia combines with a-ketoglutarate (a-KG) to produce L-glutamate. 2NH4 + 2a-KG + 2 NADH
GLDH
2 L-Glutamate + 2 NAD+ + H2O
The rate decrease in the NADH concentration is directly proportional to the urea concentration in the specimen. It is determined by measuring the absorbance at 340 nm.
Reagents
Standard urea (ST) BUN Urea
50 107
mg/dL mg/dL
Reagent 1 (R1 Buffer) Tris Buffer ( pH 8.5) a-Ketoglutarate GLDH Urease Sodium azide
50 10 8.0 5.0 8.0
mmol/L mmol/L K U/L K U/L mmol/L
Reagent 2 (R2 Starter) NADH Sodium azide
>0.20 mmol/L 8 mmol/L
For further information, refer to the Urea/Bun reagent material safety data sheet.
Reagent Preparation Prepare working solution as following:
REF: 319 001 : add 5 ml from R2 to one bottle of R1; mix gently REF: 319 002 : add one bottle from R2 to one bottle of R1 ; mix gently REF: 319 003 : add one bottle from R2 to one bottle of R1 ;mix gently REF: 319 004 : add 5 ml from R2 to one bottle of R1; mix gently
64
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Or prepare the working solution according to the number of tests required by mixing 9 volumes of reagent 1 (R1) and 1volume of reagent 2 (R2) eg. 900 ml R1 +100 ml R2.
Expected Values
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately. Both reagents (R1) and (R2) contain sodium azide which may react with copper or lead plumbing.
Reagent Storage and Stability
Urea (Serum) Adults 65 years
D A specimen = A1 specimen – A2 specimen D A standard = A1 standard – A2 standard (D Aspecimen) Serum urea concentration (mg/dl) = xn (D Astandard) where
n = 107 mg/dL
Urine urea concentration is determined by multiplying the result by the dilution factor (50).
Quality Control
All reagents are stable until expiration date stated on label when o stored refrigerated at 2 – 8 C. o Working solution is stable for 1 month at 2 – 8 C or 8 days at o 15 – 25 C.
Method
1.
Calculation
SYMBOLS IN PRODUCT LABELLING
Normal & abnormal commercial control serum concentrations should be analyzed with each run.
of
Specimen Collection and Preservation
No special preparation of the patient is required. Use nonhemolyzed serum or plasma only.The only acceptable anticoagulants are heprin, EDTA and fluoride. Do not use ammonium heparin plasma. o o Stability: 7 days at 15 –25 C ; 7 days at 2 – 8 C; o 1 year at -20 C Urine samples are prediluted 1 : 50 with ammonium free water prior to assay. o o Stability: 2 days at 15 – 25 C ; 7 days at 2 – 8 C; o 1 month at -20 C
System Parameters
Wavelength Optical path Assay type Direction Decrease Sample : Reagent Ratio e.g.: Reagent volume Sample volume First read time Delay time Last read time Temperature Zero adjustment Reagent Blank Limits Sensitivity Linearity
Performance Characteristics Precision Within run (Repeatiblity)
Level 1
Level 2
n
20
20
Mean (mg/dL)
45
150
SD
0.7
2.7
CV%
1.5
1.95
Level 1
Level 2
Run to run (Reproducibility) n
20
20
Mean (mg/dL)
47
153
SD
0.82
2.81
CV%
1.63
2.15
Methods Comparison
A comparison between Spectrum Diagnostics Urea (UV) reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.992 was obtained.
340 nm 1 cm Fixed Rate
When run as recommended, the minimum detection limit of the assay is 0.9 mg/dL.
1 : 100 1 ml 10 ml 30 seconds 60 seconds 90 seconds o 37 C Against Air Low 1.00 AU High 2.0 AU 0.9 mg/dL (0.15 mmol/L) 300 mg/dL (49.8 mmol/L)
The reaction is linear up to a urea concentration of 200 mg/dL. Specimens showing higher concentration should be diluted 1+2 with physiological saline and repeat the assay (result × 3).
Linearity
Analytical Range
0.9 – 200 mg/dL (0.15 - 49.8 mmol/L).
Waste Disposal
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
Referances 1. 2. 3. 4.
Batton, C. J & Crouch, S.R: Anal. Chem. , 1977, 49:464-469 . Shephard MD, Mezzachi RD: Clin Biochem Revs, 4:61-7, 1983. Tiffany TO, jansen JM, Burtis CA,Overtion JB, SCOTT CD. Enzymatic kinetic rate and end point analyses of substrate, by use of a gemsaec fast analyzer. Clin Chem. 1972;18:829-840. Tietz NW, Ed.Clinical guide to laboratory tests. 2ND. Philadelphia: WB Saunders;1990:566. ORDERING INFORMATION CATALOG NO
QUANTITY
319 001 319 002 319 003 319 004
3 x 50 ml 3 x 90 ml 34 x 100 ml 4 x 50 ml
Haemolysis Erythrocyte contamination doesn’t elevate results.Haemolytic specimens may cause high absorbance flagging. Icterus No significant interference.
1 ml
1 ml
Lipemia Lipemic specimens may cause high absorbance flagging Diluted sample treatment may be recommended.
Standard
10 µl
------
Anticoagulants Ammonium heparin should not be used.
Specimen
------
10 µl
Mix, and after 30 seconds read the absorbance A1 of the standard or specimen. Exactly 1 minute later, read the absorbance A2 of standard or specimen.
: 20-35 g/24hrs (330-580 mmol/24hrs) : 9.3-16.4 g/24hrs
Serum, plasma
Specimen
solution
Urine (24) hours Urea BUN
Interfering Substances
Standard Working
BUN (Serum) Adults 65 years : 7-32.9 mg/dL Children : 5-18 mg/dL
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure;interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Sensitivity
Procedure
mg/dL (2.5-8.33 mmol/L) mg/dL (0.20 mmol/L Sodium azide 8 mmol/L The reagent also contains additives required to maintain NADH in its reduced forrm. For further information, refer to the Urea/Bun reagent material safety data sheet.
Reagent Preparation
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
Calculation
D A specimen = A1 specimen – A2 specimen D A standard = A1 standard – A2 standard DAspecimen Serum urea concentration (mg/dl) = DAstandard where
n = 107.0 mg/dL
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
Quality Control
Reagent (R) contains sodium azide which may react with copper or lead plumbing.
Performance Characteristics Precision
Spectrum Urea UV Single reagent is supplied ready-to-use and stable up to the expiry date labeled on the bottles, Once opened, the opened vial is stable for 3 months at the specified temperature.
Deterioration
Do not use Spectrum Ultimate Urea reagent if it is turbid or if the absorbance of the working reagent is less than 0.9 at 340 nm. Failure to recover control values within the assigned range may be an indication of reagent deterioration.
Specimen Collection and Preservation
No special preparation of the patient is required. Use nonhemolyzed serum or plasma only.The only acceptable anticoagulants are heprin, EDTA and fluoride. Do not use ammonium heparin plasma. o o Stability: 7 days at 15 –25 C ; 7 days at 2 – 8 C; o 1 year at -20 C Urine samples are prediluted 1 : 50 with ammonium free water prior to assay. o o Stability: 2 days at 15 – 25 C ; 7 days at 2 – 8 C; o 1 month at -20 C 340 nm 1 cm Fixed Rate
of
known
Level 1
Level 2
Standard
Specimen
1 ml 10 ml ------
1 ml -----10 ml
Mix, and after 30 seconds read the absorbance A1 of the standard or specimen. Exactly 1 minute later, read the absorbance A2 of standard or specimen.
: 15-50 : < 70
BUN (Serum) Adults 65 years Children
: 7-23.5 mg/dL : 7-32.9 mg/dL : 5-18 mg/dL
Urine (24) hours Urea BUN
: 20-35 g/24hrs (330-580 mmol/24hrs) : 9.3-16.4 g/24hrs
mg/dL (2.5-8.33 mmol/L) mg/dL (3000 U/L > 500 U/L
For further information, refer to the Uric acid reagent material safety data sheet.
Reagent Preparation
Spectrum Uric acid liquizyme Single reagent is supplied ready-touse and stable up to the expiry date labeled on the bottles when o properly stored refrigerated at 2 – 8 C. Once opened, the opened vial is stable for 3 months at the specified temperature.
68
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
Quality Control
Normal & abnormal commercial control serum concentrations should be analyzed with each run.
Deterioration
Uric Acid Single reagent is normally clear or pale pink. Do not use liquizyme uric acid reagent if it is turbid or if the absorbance is greater than 0.15 AU at 546 nm.
System Parameters Wavelength Optical path Assay type Direction Increase Sample : Reagent Ratio e.g. : Reagent volume Sample volume Temperature Reaction Time Zero adjustment Reagent Blank Limits Sensitivity Linearity
Hg 546 nm (500 – 550 nm) 1 cm Endpoint 1 : 50 1 ml 20 ml o o 37 C or 15 – 25 C o 5 minutes at 37 C o 10 minutes 15 – 25 C Reagent blank Low 0.00 AU High 0.15 AU 1.0 mg/dL (0.06 mmol/L) 20 mg/dl (1.19 mmol/L)
Blank
Standard
Specimen
Reagent (R)
1.0 ml
1.0 ml
1.0 ml
Standard
------
20 µl
------
Specimen
------
------
20 µl
o
Mix and incubate for 5 minutes at 37 C or 10 minutes at o 15 –25 C .Measure absorbance of specimen (Aspecimen) and standard (Astandard) against reagent blank within 30 minutes.
Calculation Serum uric acid concentration (mg/dL) = Concentration of uric acid in urine =
(Aspecimen) x6 (Astandard)
known
Expectd Values Child
2.0 -5.5 mg/dL
(0.119 – 0.327 mmol/L)
Adult male
3.5 -7.2 mg/dL
(0.208 – 0.428 mmol/L)
Adult female
2.6 -6.0 mg/dL
(0.155-0.357
Urine
250 -750 mg/day
(14.8-44.6
mmol/L) mmol/day)
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Within run (Repeatiblity) Level 1
Level 2
n
20
20
Mean (mg/dL)
4.46
11.42
SD
0.15
0.21
CV%
3.38
1.88
Level 1
Level 2
Run to run (Reproducibility)
Specimen Collection and Preservation
The only acceptable anticoagulants are heparin and EDTA. Uric acid in serum & plasma samples remains stable for 3 days at room o temperature; 3 to 5 days if kept at 4oC and for 6 months at -20 C. Urine samples should be diluted 1:10 before assay with physiological saline. It is recommended that 15 ml of sodium hydroxide 2 mol/l, be added to the urine samples to keep urine alkaline and prevent ureate percipitation. upon receipt, urine sample pH should be checked and kept over 8.0.
of
Performance Characteristics Precision
Reagent Storage and Stability
All reagents are stable until expiration date stated on label when o stored refrigerated at 2 - 8 C.
Procedure
Reagents
Standard uric acid (ST) 6 mg/dL
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Precautions and Warnings
Intended Use
Method
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
Serum blank: Extremely lipemic samples may give falsely elevated results and a serum blank must be run. Add 20 ml serum to 1 ml water. Zero the spectrophotometer with water.Read and record absorbance and subtract reading from test absorbance.
n
20
20
Mean (mg/dL)
4.51
11.59
SD
0.23
1.32
CV%
3.46
1.97
Methods Comparison
A comparison between Spectrum Diagnostics Uric Acid reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.979 was obtained.
Analytical Range
1.0 – 20 mg / dL ( 0.06 – 1.19 mmol/l).
Waste Disposal
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References 1. 2. 3. 4.
Sensitivity
When run as recommended, the minimum detection limit of this assay is 1 mg/dL (0.06 mmol/L).
Linearity
The reaction is linear up to a uric acid concentration of 20 mg/dl. Specimens showing higher concentration should be diluted 1+1 using physiological saline, reassayed and the result multiplied by two.
Interfering Substance
Haemoglobin No interference up to a haemoglobin level of 200 mg/dl. Icterus No significant interference from free bilirubin up to a level of 8mg/dl and from conjugated bilirubin up to a level of 12mg/dl.
5.
Barham D.and Trinder P., Analyst 97,142-145 (1972). Fossati P.,Prencipe L.,and Berti G., Clin. Chem . 26/2,227-273 (1980). Richterich R, colombo JP. Klinische Chemie. 4th ed.basel:karger s;1978 :319-324 . Tiffany to, jansen JM, Burtis CA,Overton JB, scott cd.Enzymatic kinetic rate and end point analyses of substrate, by use of a GEMSAEC fast analyzer. Clin Chem. 1972; 18 : 829-840. Tietz NW, ED. Clinical guide to laboratory tests. 2nd ED. philadelphia: WB Sauners; 1990: 566.
ORDERING INFORMATION CATALOG NO 323 001 323 002 323 003 323 004 323 005 323 006
QUANTITY 4 4 4 4 2 4
x 30 x 50 x 100 x 60 x 500 x 250
ml ml ml ml ml ml
Lipemia No significant interference with mild to moderate lipemia. Drugs Of the drugs tested in vitro,methyldopa and noramidopyrine cause artificially Low uric acid values at the tested drug Level. Others Physiological ascorbic acid concentration does not interfere with the test. Ascorbic Acid levels higher than 170 mmol/l (3.0 mg/dl) decreases the apparent uric acid concentration significantly.
(Aspecimen) x 6 x 10 (Astandard)
69
ii-Enzymes
70
71
Acid Phosphatase (ACP)
SYMBOLS IN PRODUCT LABELLING
(Colorimetric Test with α-Naphthylphosphate)
EC REP Authorised Representative IVD LOT REF
REF 229 001
50 Tests
R1 Substrate R2 For Total ACP R3 For Non-Prostatic ACP R4 Stabilizer
5 x10 ml 1 x 50 ml 1 x 50 ml 1 x 2 ml
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
LINEARITY:
The reaction is linear up to ACP concentration 74 U/L, Specimen Shows higher concentration should be diluted 1+3 with physiological saline and repeat assay (Result x 3).
Expected Values Total Acid Phosphatase Men £ 4,7 U/l Women £ 3,7 U/l Prostatic Acid Phosphatase £ 1,6 U/l
LITERATURE 1.Hillmann G. Z.Klin.Chem.Klin.Biochem.9, p.273.
METHOD / REACTION PRINCIPLE:
Stability of Working reagents
α-Naphthylphosphate is hydrolysed by ACP to phosphate and α-naphthol, which is converted with FRTR-salt into an azo dye. The increase of absorbance at 405 nm is proportional to the total ACP activity in the sample. The prostatic acid phosphatase (PACP) can be blocked by tartrate and can be determined indirectly (through the non-prostatic ACP) by calculation of the activity difference.
α-Naphthylphosphate + H2O α-Naphthol + FRTR-salt
ACP
phosphate + 1-naphthol
Reagent A Sample
1.0 ml (Pre-heated At 37°C) 100 µl
Mix, Wait 5 minutes then measure absorbance (at 405 nm) each one minute for 3 minutes. Determine ΔA /min Calculation: ΔA /min x 743 = TACP Activity in sample
The sealed reagents are stable up to the indicated expiry date o if stored at 2 - 8 C.
II-Manual assay for Non-Prostatic ACP Reagent B Sample
PREPARATION AND STABILITY OF WORKING REAGENTS
1.0 ml (Pre-heated At 37°C) 100 µl
Mix, Wait 5 minutes then measure absorbance (at 405 nm) each one minute for 3 minutes. Determine ΔA /min
R2 and R3 are ready for use Reagent A (determination of Total ACP):
72
Serum, no plasma! Avoid hemolysis! Use immediately or stabilize : Add 1 drop of 0.1% acetic acid to 1 ml of serum: o ACP is stable for 3 days at 2-8 C .
I-Manual assay for Total ACP
R1 : 1-Naphthyl phosphate 10 mmol/l Fast Red TR-salt 1.5 mmol/l (4-chloro-2-methylphenyl diazonium salt) R2 : Citrate buffer pH 5.2 100 mmol/l R3 : Citrate buffer pH 5.2 100 mmol/l Tartrate 135 mmol/l R4 : Stabilizer ( Acetic acid) 0.8 mol/l
Calculation: ΔA /min x 743 = NPACP Activity
Dissolve the contents of R1 (substrate) in 10 ml of buffer solution R2. Mark label with „A“.
CALIBRATORS AND CONTROLS
Reagent B (determination of Non-Prostatic ACP): Dissolve the contents of R1 (substrate) in 10 ml of tartrate solution R3.Mark label withB
SAMPLES :
PROCEDURE:
Azo dye
REAGENTS: (Concentrations in the test)
o
3 days at 2 - 8 C o 1 day at 18 - 25 C
For the calibration of automated analyzers Spectrum Multicalibrator is recommended, for quality control use Spectrum Normotrol and pathotrol.
73
Alkaline phosphatase (ALP) Liquizyme (9 + 1) IFCC E.C.3.1.3.1. REF: 214 001 REF: 214 002 REF: 214 003 REF: 214 004 REF: 214 005
( 4 x 20 ml) (10 x 10 ml) ( 9 x 20 ml) ( 4 x 60 ml) ( 5 x 20 ml)
80 test 100 test 180 test 240 test 100 test
Spectrum Diagnostics liquizyme Alkaline Phosphatase reagent is intended for the in-vitro quantitative, diagnostic determination of ALP in human serum on both automated and manual systems.
Background
Alkaline phosphatase (ALP) catalyzes the hydrolysis of a wide variety of physiologic and non-physiologic phosphoric acid esters in alkaline medium (pH optimum 10). The liver and biliary tract are the source of alkaline phosphatase in normal sera. Normal alkaline phosphatase levels are age dependent being higher in children and adolescents in comparison to adults. ALP is one of the tests of choice for evaluating cholestasis and obstructive juandice. Elevated levels are found in many diseases including hepatitis, cirrhosis, malignancy, and in bone diseases.
Method
Kinetic method according to the International Federation of Clinical Chemistry (IFCC) (3). Alkaline phosphatase (ALP) hydrolyzes p-Nitrophenylphosphate (p-NPP) to p-Nitrophenol and phosphate. p-Nitrophenol+ Phosphate
The increase of absorbance per minute at 405 nm is proportional to the enzyme activity.
Reagents Reagent 1 (R1 Buffer) 2-Amino-2-Methyl-1-Propanol (pH 10.3)
2.0
MgCl2
2.0 mmol/L
mol/L
Reagent 2 (R2 Substrate) 16 mmol/L
For further information, refer to the Alkaline phosphatase reagent material safety data sheet.
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
Reagent Preparation
Prepare working solution as following: REF: 214 001: add 2 ml from R2 to one bottle of R1; mix gently. REF: 214 002: add 1 ml from R2 to one bottle of R1; mix gently. REF: 214 003: add 2 ml from R2 to one bottle of R1; mix gently. REF: 214 004: add 6 ml from R2 to one bottle of R1; mix gently. REF: 214 005: add 2 ml from R2 to one bottle of R1; mix gently. Or prepare the working solution according to the number of tests required by mixing 9 volumes of reagent 1 (R1) and 1volume of reagent 2 (R2),e.g. 900 ml R1 + 100 ml R2.
74
IVD LOT REF
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
All reagents are stable until expiration date stated on label when o stored refrigerated at 2 - 8 C. o Working solution is stable for 4 weeks at 2 – 8 C or 5 days at o 15 - 25 C. Do not use liquizyme ALP reagent if it is turbid or if the absorbance of the working reagent is more than 1.0 at 405 nm. Failure to recover control values within the assigned range may be an indication of reagent deterioration.
Specimen Collection and Preservation Serum and Plasma
Nonhaemolyzed fresh serum is the preferred specimen. Heparin is the only acceptable anticoagulant. Complexing anticoagulants such as citrate, oxalate, and EDTA must be avoided. Alkaline phosphatase activity may slowly increase in serum samples stored at room temperature. Previously frozen or lypholized sera may show a marked decrease in values immediately upon thawing or reconstitution.The activity then increases to the initial values, and the rate of this increase is time and temperature dependent. o
Waste Disposal
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
Within run (Repeatiblity) Level 1
Level 2
n
20
20
Mean (U/L)
177.7
359.7
SD
1.71
1.5
CV%
0.96
0.43
References
Level 1
Level 2
n
20
20
Mean (U/L)
178.5
365.5
SD
1.82
1.86
CV%
1.15
0.55
Methods Comparison
A comparison between Spectrum Diagnostics ALP reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.988 was obtained.
2 months at -20 C ; 4 weeks at 4 – 8 C; o 7 days at 20 – 25 C
When run as recommended, the minimum detection limit of this assay is 5.0 U/L. The reaction is linear up to alkaline phosphatase concentration of 750 U/L; specimens showing higher concentration should be diluted 1+5 with physiological saline and repeat the assay (result × 6).
System Parameters
Wavelength 405 nm (400 – 420 nm) Optical path 1 cm Assay type Kinetic Direction Increase Sample : Reagent Ratio 1 : 100 e.g.: Reagent volume 1 ml Sample volume 10 ml o o Temperature 37 C or 30 C Equilibration time 1 minute Read time 1 to 3 minutes Zero adjustment Against air Reagent Blank Limits Low 0.2 AU High 1.0 AU Sensitivity 5 U/L Linearity 750 U/L
Moss DW, Henderson AR, Kachmar JF. Enzymes in:Tietz NW, ed. Fundamentals of clinical chemistry. 3 rd ed. Philadelphia: WB Saunders; 1987:346-421.
3.
Tietz NW, Rinker AD, Shaw LM. IFCC methods for the measurement of catalytic concentration of enzymes. Part 5. IFCC method for alkaline phosphatase . J Clin Chem Clin Biochem. 1983;21:731-748.
4.
Zawta B, Klein G, Bablok W. Temperaturumrechnung in der Klinischen Enzymologie? Klin lab. 1994:40:23-32. Sensitivity
CATALOG NO
QUANTITY
214 001 214 002 214 003 214 004 214 005
4 x 20 ml 10 x 10 ml 9 x 20 ml 4 x 60 ml 5 x 20 ml
Haemolysis A 200 mg/dL haemoglobin results in a 10 % negative bias. Icterus No significant interference up to bilirubin level of 40 mg/dL. Lipemia No significant interference from lipemia up to 1000 mg/dL. Expected Values o
o
30 C
37 C
Males
(20 - 50) years
30 - 90 U/L
53-128 U/L
Males
(≥ 60)
years
30 - 90 U/L
56-119 U/L
Pipette in a test tube:
Females
(20 - 50) years
20 - 80 U/L
42-98 U/L
Working solution
1.0 ml
Females
(≥ 60)
40 - 111 U/L
53-141 U/L
Specimen
10 ml
Mix, read initial absorbance after 1 minute. and start timer simultaneously. Read again after 1, 2 and 3 minutes. Determine the mean absorbance change per minute (DA/min).
Calculation
To calculate the alkaline phosphatase (ALP) activity, Use the following formula U/L = 5454 ×DA 405 nm/min Normal & abnormal commercial control serum concentrations should be analyzed with each run.
2.
Interfering Substances Serum, plasma
Procedure
Quality Control
Moss DW. Alkaline phosphatase isoenzymes. Clin Chem. 1982;28:2007-2016 .
ORDERING INFORMATION
Sensitivity
Linearity
o
1. Run to run (Reproducibility)
Deterioration
Stability:
Assay Principle
p-Nitrophenylphosphate
EC REP Authorised Representative
Performance Characteristics Precision
Reagent Storage and Stability
Intended Use
p-Nitrophenylphosphate + H2O ALP
SYMBOLS IN PRODUCT LABELLING
of
known
years
Childern (1 - 12) years ≤ 350 U/L Temperature conversion factor is 1.22 (25 o 1.52 (25 37 C ).
≤ 460
U/L
o
30 C ) and
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Analytical Range 5 – 750 U/L.
75
Alkaline phosphatase (ALP) Liquizyme (1 + 1) IFCC E.C.3.1.3.1.
SYMBOLS IN PRODUCT LABELLING EC REP Authorised Representative IVD LOT REF
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
REF: 215 001 (2 x 25 ml) 50 test REF: 215 002 (4 x 25 ml) 100 test
Intended Use
Spectrum Diagnostics liquizyme Alkaline Phosphatase reagent is intended for the in-vitro quantitative, diagnostic determination of ALP in human serum on both automated and manual systems.
Background
Alkaline phosphatase (ALP) catalyzes the hydrolysis of a wide variety of physiologic and non-physiologic phosphoric acid esters in alkaline medium (pH optimum 10). The liver and biliary tract are the source of alkaline phosphatase in normal sera. Normal alkaline phosphatase levels are age dependent being higher in children and adolescents in comparison to adults. ALP is one of the tests of choice for evaluating cholestasis and obstructive juandice. Elevated levels are found in many diseases including hepatitis, cirrhosis, malignancy, and in bone diseases.
Method
Kinetic method according to the International Federation of Clinical Chemistry (IFCC) (3).
Assay Principle
Alkaline phosphatase (ALP) hydrolyzes p-Nitrophenylphosphate (p-NPP) to p-Nitrophenol and phosphate. p-Nitrophenylphosphate + H2O
ALP
p-Nitrophenol + Phosphate
The increase of absorbance per minute at 405 nm is proportional to the enzyme activity.
Reagents
Nonhaemolyzed fresh serum is the preferred specimen. Heparin is the only acceptable anticoagulant. Complexing anticoagulants such as citrate, oxalate, and EDTA must be avoided. Alkaline phosphatase activity may slowly increase in serum samples stored at room temperature. Previously frozen or lyophilized sera may show a marked decrease in values immediately upon thawing or recon-stitution.The activity then increases to the initial values, and the rate of this increase is time and temperature dependent. Stability:
o
2-Amino-2-Methyl-1-Propanol (pH 10.3)
2.0
mol/L
MgCl2
2.0 mmol/L
Reagent 2 (R2 Substrate) 16 mmol/L
For further information, refer to the Alkaline phosphatase reagent material safety data sheet.
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
Reagent Preparation Storage and Stability
All reagents are stable until expiration date stated on label when o stored refrigerated at 2 - 8 C . Working solution can be prepared by adding equal volumes o o from R1 and R2; Stability: 1 month at 2 – 8 C or 5 days at 15-25 C.
Within run (Repeatiblity) Level 1
Level 2
n
20
20
Mean (U/L)
177.7
359.7
SD
1.71
1.5
CV%
0.96
0.43
Level 1
Level 2
Wavelength 405 nm (400 – 420 nm) Optical path 1 cm Assay type Kinetic Direction Increase Sample : Reagent Ratio 1 : 100 e.g.: Reagent volume 1 ml Sample volume 10 ml o o Temperature 37 C or 30 C Equilibration time 1 Minute Read time 1 to 3 minutes Zero adjustment Against air Reagent Blank Limits Low 0.2 AU High 1.0 AU Sensitivity 5 U/L Linearity 750 U/L
n
20
20
178.5
365.5
SD
1.82
1.86
CV%
1.15
0.55
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References 1.
Moss DW. Alkaline phosphatase isoenzymes. Clin Chem.1982;28:2007-2016.
2.
Moss DW, Henderson AR, Kachmar JF. Enzymes in: Tietz NW, ed. Fundamentals of Clinical Chemistry. 3 rd ed. Philadelphia: WB Saunders; 1987:346-421.
3.
Tietz NW, Rinker AD, Shaw LM. IFCC methods for the measurement of catalytic concentration of enzymes. Part5. IFCC method for alkaline phosphatase . J Clin Chem Clin Biochem.1983;21:731-748.
4.
Zawta B, Klein G, Bablok W. Temperaturumrechnung in der Klinischen Enzymologie? Klin lab. 1994:40:23-32.
Sensitivity
When run as recommended, the minimum detection limit of this assay is 5.0 U/L.
Linearity
The reaction is linear up to alkaline Phosphatase concentration of 750 U/L; specimens showing higher concentration should be diluted 1+5 with physiological saline and repeat the assay (result×6).
Interfering Substances Serum, plasma
ORDERING INFORMATION
Haemolysis A 200 mg/dL haemoglobin results in a 10 % negative bias.
Pipette in a test tube:
Icterus No significant interference up to bilirubin level of 40 mg/dL.
Working solution
1.0 ml ( or add 0.5 ml R1 + 0.5 ml R2 )
Lipemia No significant interference from lipemia up to 1000 mg/dL.
Specimen
10 ml
CATALOG NO
QUANTITY
215 001 215 002
2 x 25 ml 4 x 25 ml
Expected Values
Mix, read initial absorbance after 1 minute. and start timer simultaneously. Read again after 1, 2 and 3 minutes. Determine the mean absorbance change per minute (DA/min).
Calculation
To calculate the alkaline phosphatase (ALP) activity. Use the following formulae U/l = 5454 × DA 405 nm /min Normal & abnormal commercial control serum concentrations should be analyzed with each run.
Waste Disposal
A comparison between Spectrum Diagnostics ALP reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.990 was obtained.
System Parameters
Quality Control
5 – 750 U/L.
Run to run (Reproducibility)
Mean (U/L)
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Analytical Range
Methods Comparison
o
2 months at – 20 C ; 4 weeks at 4 – 8 C; o 7 days at 20 – 25 C
Procedure
Reagent 1 (R1 Buffer)
p-Nitrophenylphosphate
Specimen Collection and Preservation Serum and Plasma
Performance Characteristics Precision
of
known
o
o
30 C
37 C
Males
(20 - 50) years
30 - 90 U/L
53 - 128 U/L
Males
(> 60)
30 - 90 U/L
56 - 119 U/L
Females
(20 - 50) years
20 - 80
U/L
42 - 98 U/L
Females
(> 60)
40 - 111 U/L
53 - 141 U/L
Childern
(1 - 12) years
< 350
60)
years
56-119 U/L
Females
(20 - 50) years
42-98 U/L
Females
(> 60)
53-141 U/L
Childern
(1 - 12) years
0.600 at 405 nm. Quality control data sheet of the reagent are available upon request. Refer to the batch number on the label.
Up to 100 U/L
Random Urine
Up to 273 U/L
Up to 365 U/L
Up to 450 U/L
24hrs Urine
Up to 205 U /24h
Up to 295 U /24h
Up to 410 U/24h
Interferences
The following substances doesn’t interfere up to the concentration of: Bilirubin conjugated 20 mg/dL Bilirubin free 20 mg/dL Hemoglobin 500 mg/dL NaF 500 mg/dL Ascorbic acid 500 mg/dL Glucose 5,0 g/dL Maltose 5,0 g/dL
mL
Mean value [U/L]
Standarddeviation [U/L]
CV [%]
------
mL
Sample 1
77,7
1,009
1,298
25
mL
Sample 2
196
1,834
0,935
Sample 3
72,1
1,700
0,934
SAMPLE
1000
1000
25 ------
Procedure2 (Fixed rate colorimetric method) Wavelength l = 405 nm Light path 1 cm Temperature 37°C Reagent Blank Against distilled water Reaction Fixed rate (increase) Allow reagents to reach working temperature before use. Sample Reagent
1000 µl
Sample
25
µl
Mix well and incubate exactly for 1 minute at 37°C then Read the Absorbance A1. After exactly 4 minutes later read the Absorbance A2. ∆E = A2 – A1 Alpha amylase (U/L) = ∆E x 765
37°C
Up to 73 U/L
Within series n = 20
BLANK
Alpha amylase (U/L) - ∆E/min x 3060
Calculation:
30°C
Up to 55 U/L
Within-run reproducibility
Calculation:
The reagent is stable liquid ready to use
25°C Serum / Plasma
- Precision
Mix thoroughly and incubate for 1 minute at 37°C. Record initial absorbance and at 1 minute intervals thereafter for 3 minutes against reagent blank. Calculate the difference between consecutive absorbance’s, and the average absorbance difference per minute (DE/min).
Reagent Preparation
Precaution and warning
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
IVD LOT
REF: 219 001 REF: 219 002
Expected Values (37°C)
SYMBOLS IN PRODUCT LABELLING
Between-run reproducibility Day to day n = 20
Mean value [U/L]
Standarddeviation [U/L]
CV [%]
Sample 1
79,5
0,934
1,176
Sample 2
195
0,674
0,345
Sample 3
72,5
0,934
1,288
LINEARITY: SENSITIVITY: MEASURED RANGE:
1500 2 2 - 1500
U/L U/L U/L
References 1.
Henry, R.J., Chiamori, N., Clin. Chem., 6;434, (1961)
2.
Winn-Deen et Al., Clin. Chem. 24-10 (1989)
3.
Lorentz, K., Clin. Chem. Clin. Biochem. 17,499 (1979)
ORDERING INFORMATION CATALOG NO
QUANTITY
219 001 219 002
2 x 25 ml 4 x 25 ml
83
Creatine Kinase (CK) Liquid Reagent REF: 238 001 REF: 238 002 REF: 238 004
SYMBOLS IN PRODUCT LABELLING EC REP Authorised Representative LOT
( 6 x 5 ml) 30 Test ( 6 x 20 ml) 120 Test ( 6 x10 ml ) 60 Test
REF
Intended Use
Spectrum Diagnostics Creatine Kinase (CK) reagent is intended for the in-vitro quantitative, diagnostic determination of Creatine kinase in human serum on both automated and manual systems.
Background
Creatine kinase (CK) is an enzyme which is contained in heart, brain and skeletal muscles. Thus, an increase of circulating level of CK may be associated to myocardial infarction, acute cerebrovascular disease, trauma or diseases of skeletal muscles. After a myocardial infarct, CK level begins raising between 4th and 6th hour after first acute symptoms, reaching the peak between 18th and 30th hour and coming back to normal values during the 3rd day. CK is present in three different isoenzymatic forms, which could be separated by electrophoresis or column chromatography; each form is originated in different body tissues, paying off their diagnostic determinations. The formula of present reagent is based on DGKC and IFCC recommendations.
Method
According to the recommendations of the International Federation of Clinical Chemistry (IFCC).
Assay Principle
Creatine kinase (CK) catalyzes the phosphorylation of ADP, in the presence of creatine phosphate, to form ATP and creatine. The catalytic concentration is determined from the rate of NADPH formation, measured at 340 nm, by means of the hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH) coupled Reactions1,2. CK
Creatine phosphate + ADP ATP + Glucose
HK
Glucose-6- phosphate+NADP
+
Creatine + ATP
6-Phosphogluconate+NADPH +H
REF: 238 001 add 1 ml from R2 to one bottle of R1; mix gently REF: 238 002 add 4 ml from R2 to one bottle of R1; mix gently REF: 238 004 add 2 ml from R2 to one bottle of R1; mix gently
Specimen Collection and Preservation
Serum free of haemolysis or heparin plasma. Stability 2 days at 20-25 oC, 7 days at 2-8oC, 4 weeks at -20oC protected from light.
System Parameters
Wavelength 340 nm (334-365 nm) Optical path 1 cm Assay type Kinetic Direction Increase Sample: Reagent Ratio 1:25 e.g.: Reagent volume 1 ml Sample volume 40 ml o Temperature 37 C Equilibration Time 60 seconds Read time 1 to 3 minutes Zero adjustment against air Reagent blank limits Sensitivity 1 U/L Linearity 2000 U/L
Reagent 1 (pH 6.7) (Buffer / Coenzyme) Imidazol D-Glucose N-Acetyl-L-Cysteine Magnesium acetate NADP EDTA
125 25 25 12.5 2.5 2
mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L
Reagent 2 (Enzymes) ADP AMP P1,P5-di (adenosine-5’-) penta-phosphate Glucose-6-phosphate Dehydrogenase (G6PDH) Creatine phosphate Hexokinase (HK)
15.2 25 103 9 250 3
mmol/L mmol/L mmol/L KU/L mmol/L KU/L
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
Level 2
20
20
Mean (U/L)
86
616
CV%
2.8
1.0
Level 1
Level 2
20
20
Mean (U/L)
77
624
CV%
2.5
0.8
Run to run (Reproducibility) n
Methods Comparison
A comparison between Spectrum Diagnostics CK reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.983 was obtained.
Sensitivity
When run as recommended, the minimum detection limit of the assay is 1 U/L.
Linearity
The reaction is linear up to CK concentration of 2000 U/l; specimens showing higher concentration should be diluted 1+2 using physiological saline and repeat the assay (result×3).
Interferences:
No interferences were observed with haemoglobin until 5 g/L, bilirubin 20 mg/dL and triglycerides 7 mmol/L. Other drugs and substances may interfere3,4.
Procedure
References 1.
Pipette into a thermostatized cuvette:
+
Level 1 n
Or prepare the working solution according to the number of test required by mixing 4 volumes of R1 with 1 volume of R2. Stability: 4 weeks at 2-8oC away from light sources.
1.
Reagents
Within run (Repeatiblity)
Reagent preparation, Storage, and Stability
ADP + Glucose-6-phosphate
G6PDH
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
IVD
Performance Characterstics Precision
Working solution
1 ml
Serum
40 mL
2. 3.
2.
Mix and incubate 60 seconds.
3.
Read initial absorbance (A) of the sample, start the stopwatch and read absorbance at 1 minute intervals thereafter for 3 minutes.
4.
Calculate the difference between absorbances and the average absorbance differences per minute (DA/min).
Calculation
DA/min x 4127 = U/L CK Units: One international unit (IU) is the amount of enzyme that transforms 1 mmol of substrate per minute, in standard conditions. The concentration is expressed in units per liter of sample (U/L).
4.
IFCC methods for the measurement of catalytic concentration of enzymes. Part 7: IFCC method for creatine kinase. JIFCC 1989; 1: 130-139. Tietz Textbook of Clinical Chemistry, 3rd edition. Burtis CA, Ashwood ER. WB Saunders Co., 1999. Young DS. Effects of drugs on Clinical Lab. Tests, 4th ed AACC Press, 1995. Young DS. Effects of disease on Clinical Lab. Tests, 4th ed AACC 2001. ORDERING INFORMATION CATALOG NO
QUANTITY
238 001 238 002 238 004
5 x 6 ml 6 x 20 ml 6 x 10 ml
Expected values Men
24 - 204 U/L
Women
24 - 173 U/L
Quality Control
Normal & abnormal commercial control serum concentrations should be analyzed with each run.
of
known
Storage and Stability
The reagents are stable up to the expiration date specified when stored at 2 – 8 oC.
84
85
CREATINE KINASE MB (CK-MB) REF: 239 001 REF: 239 002 REF: 239 003 REF: 239 004
(6 (6 (5 (6
SYMBOLS IN PRODUCT LABELLING EC REP Authorised Representative IVD LOT
x 5 ml) 30 Test x 10 ml) 60 Test x 25 ml) 125 Test x 20 ml) 120 Test
REF
Intended Use
Spectrum Diagnostics Creatine Kinase MB (CK-MB) reagent is intended for the in-vitro quantitative, diagnostic determination of Creatine kinase MB in human serum on both automated and manual systems. Creatine kinase (CK) is an enzyme which is contained in heart, brain and skeletal muscles. Thus, an increase of circulating level of CK may be associated to myocardial infarction, acute cerebrovascular disease, trauma or diseases of skeletal muscles. After a myocardial infarct, CK level begins raising between 4th and 6th hour after first acute symptoms, reaching the peak between 18th and 30th hour and coming back to normal values during the 3rd day. CK is present in three different isoenzymatic forms, which could be separated by electrophoresis or column chromatography; each form is originated in different body tissues, paying off their diagnostic determinations. CK exists in serum in dimeric forms as CK-MM, CK-MB, and CK-BB and as macro-enzymes. Measurement of CK-MB is a quite specific test for detection of cardiac muscle damage and is therefore used for diagnosis and monitoring of myocardial infarction.
Method
After immunoinhibition with antibodies to the CK-M subunit, the CK-B activity is determined with a method according to the recommendations of the International Federation of Clinical Chemistry (IFCC).
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
A specific antibody inhibits the M subunits of CK-MM and CK-MB, and thus allows determination of the B subunit of CK-MB (assuming the absence of CK-BB or CK-1). CK-B catalytic concentration, which corresponds to half of CK-MB concentration, is determined from the rate of NADPH formation, measured at 340 nm, by means of the hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH) coupled reactions1,3. CK
Creatine phosphate + ADP HK
Glucose-6- phosphate+NADP
+
Creatine + ATP
ADP + Glucose-6-phosphate
G6PDH
6-Phosphogluconate+NADPH + H
+
Reagents
All the components of the kit are stable until the expiration date on the label when stored tightly closed at 2-8oC, protected from light and contaminations prevented during their use. Signs of reagent deterioration: Presence of particles and turbidity.
Reagent preparation, Storage, and Stability
REF: 239 001 add 1 ml from R2 to one bottle of R1; mix gently REF: 239 002 add 2 ml from R2 to one bottle of R1; mix gently REF: 239 003 add 5 ml from R2 to one bottle of R1; mix gently REF: 239 004 add 4 ml from R2 to one bottle of R1; mix gently Or prepare the working solution according to the number of test required by mixing 4 volumes of R1 with 1 volume of R2. Stability: 2 weeks at 2-8oC away from light sources.
Specimen Collection and Preservation
Serum free of hemolysis is the preferred specimen. Plasma containing heparin, EDTA, citrate or fluoride may produce unpredictable reaction rates. Stable for 2 hours at 20-25 oC, 5 days at 4-8 oC.Total CK concentration in the sample must be lower than 1000 U/L. Dilute the serum 1/2 if necessary, with NaCl (150 mmol/L).
Imidazol D-Glucose N-Acetyl-L-Cysteine Magnesium acetate NADP EDTA
Wavelength 340 nm (334-365 nm) Optical path 1 cm Assay type Kinetic Direction Increase Sample: Reagent Ratio 1:25 e.g.: Reagent volume 1 ml Sample volume 40 ml o Temperature 37 C Equilibration Time 60 seconds Read time 1 to 5 minutes Zero adjustment against air Reagent blank limits Sensitivity 2 U/L Linearity 2000 U/L
125 25 25 12.5 2.5 2
mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L
1.
ADP AMP P1,P5-di (adenosine-5’-) penta-phosphate Glucose-6-phosphate Dehydrogenase (G6PDH) Creatine phosphate Hexokinase (HK) Anti-human-CK-M.
15.2 25 103 9 250 3
mmol/L mmol/L mmol/L KU/L mmol/L KU/L
Pipette into a thermostatized cuvette: Working solution
1 ml
Serum
40 mL
2.
Mix and incubate 60 seconds.
3.
Read initial absorbance (A) of the sample, start the stopwatch and read absorbance at 1 minute intervals thereafter for 5 minutes.
4.
Calculate the difference between absorbances and the average absorbance differences per minute (DA/min).
Reagent 2 (Enzymes)
1.
Units: One international unit (IU) is the amount of enzyme that transforms 1 mmol of substrate per minute, in standard conditions. The concentration is expressed in units per liter of sample (U/L).
IFCC methods for the measurement of catalytic concentration of enzymes. Part 7: IFCC method for creatine kinase. JIFCC 1989; 1: 130-139.
2.
Tietz Textbook of Clinical Chemistry, 3rd edition. Burtis CA, Ashwood ER. WB Saunders Co., 1999.
Expected values
3.
Friedman and Young. Effects of disease on clinical laboratory tests, 3th ed. AACC Press, 1997.
4.
Urdal P and Landaas S. Clin Chem 1979; 25: 461-465.
5.
Young DS. Effects of drugs on clinical laboratory tests, 3th ed. AACC Press, 1997.
DA/min x 8254 = U/L CKMB
The discrimination value for myocardial infarction is around 25 U/L. However, an index higher than 6% of total CK concentration discriminates better. These values are for orientation purpose; each laboratory should establish its own reference range. Normal & abnormal commercial control serum concentrations should be analyzed with each run.
of
known ORDERING INFORMATION CATALOG NO
Performance Characterstics Precision Within run (Repeatiblity) Level 1
Level 2
20
20
Mean (U/L)
45
129
CV%
3.5
3.2
Level 1
Level 2
20
20
Mean (U/L)
40
130
CV%
2.8
2.3
n
239 001 239 002 239 003 239 004
QUANTITY 6 6 5 6
x 5 ml x 10 ml x 25 ml x 20 ml
Run to run (Reproducibility) n
Methods Comparison
A comparison between Spectrum Diagnostics CK-MB reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.959 was obtained.
Sensitivity
When run as recommended, the minimum detection limit of the assay is 2.0 U/L.
Linearity
The reaction is linear up to CK-MB concentration of 2000 U/l; specimens showing higher concentration should be diluted 1+2 using physiological saline and repeat the assay (result×3).
Interferences:
Procedure
Reagent 1 (pH 6.7) (Buffer / Coenzyme)
References
Quality Control
System Parameters
Assay Principle
86
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Storage & Stability
Background
ATP + Glucose
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
Calculation
Haemoglobin (< 2.5 g/L) ,lipemia (Lipids < 900 mg/dL) and Bilirubin (< 25 mg/dL) do not interfere. Presences in the sample of above normal concentrations of CK-BB or adenilate kinase, and of macro or mitochondrial CK interfere. Other drugs and substances may interfere3,4.
87
g - Glutamyltransferase(gGT)-Liquizyme (9+1) E.C.2.3.2.2. REF: 246 001 REF: 246 002 REF: 246 003 REF: 246 004 REF: 246 005
( 4 x 20 ml) (10 x 10 ml) ( 9 x 20 ml) ( 4 x 60 ml) ( 5 x 20 ml)
SYMBOLS IN PRODUCT LABELLING EC REP Authorised Representative IVD LOT REF
80 test 100 test 180 test 240 test 100 test
Precautions and Warnings
Intended Use
Spectrum Diagnostics liquizyme g-glutamyltransferase reagent is intended for the in-vitro quantitative, diagnostic determination of g-glutamyltransferase in human serum on both automated and manual systems.
Background
g- Glutamyltransferase (gGT) is usually most significantly elevated by obstructive disease and has good specificity for the liver. It is not elevated in bone diseases or pregnancy (as ALP) or in skeletal muscle diseases (as AST). gGT can also help to differentiate between mechanical and viral from drug induced cholestasis. The highest concentration of gGT is found in the luminal membrane of the proximal tubules of the kidney. Other sources are the pancreas, prostate, and liver. High gGT activity is found in prostate tissue, which may account for the increased gGT activity seen in some sera from men compared with sera from women.
Method
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately. Both reagents (R1) and (R2) contain sodium azide which may react with copper or lead plumbing.
Reagent Storage and Stability
All reagents are stable until expiration date stated on label when o stored refrigerated at 2 - 8 C. Working solution is stable for 3 months o o at 2 – 8 C or 2 weeks at 15 to 25 C when stored in a dark bottle.
Deterioration
Do not use liquizyme gGT reagent if it is turbid or if the absorbance of the working reagent is greater than 1.0 at 405 nm. Failure to recover control values within the assigned range may be an indication of reagent deterioration.
Specimen Collection and Preservation
Use serum and plasma, free from haemolysis. Heparin is the only acceptable anticoagulant. The biological half-life of gGT in serum is 3 – 4 days.
Kinetic colorimetric according to Szasz method. (5)
Assay Principle Determination of g-Glutamyltransferase (g-GT) according to the following reaction: L-g-Glutamyl-3-carboxy-4-nitroanilide + Glycylglycine g-GT L-g-Glutamyl- glycylglycine + 5-amino-2-nitrobenzoate The rate of liberation of yellow coloured indicator 5-amino-2nitrobenzoate is directly proportional to g-GT activity in the sample and is quantitated by measuring the increase in absorbance at 405nm.
Reagents Reagent 1 (R1 Buffer) Tris buffer pH 8.2 Glycylglycine Sodium Azide
80 mmol/L 130 mmol/L 8.0 mmol/L
Reagent 2 (R2 Starter) L-g-Glutamyl-3-carboxy-4-nitroanilide Sodium Azide
4.0 mmol/L 8.0 mmol/L
For further information, refer to the g-Glutamyltrasferase reagent material safety data sheet.
Reagent Preparation
Prepare working solution as following: REF: 246 001: add 2 ml from R2 to one bottle of R; mix gently. REF: 246 002: add 1 ml from R2 to one bottle of R; mix gently. REF: 246 003: add 2 ml from R2 to one bottle of R; mix gently. REF: 246 004: add 6 ml from R2 to one bottle of R; mix gently. REF: 246 005: add 2 ml from R2 to one bottle of R; mix gently. Or prepare the working solution according to the number of tests required by mixing 9 volumes of reagent 1 (R1) and 1volume of reagent 2 (R2) ,e.g. 900 ml R1 +100 ml R2.
88
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
Stability:
o
o
7 days at 4 – 8 C ; 7 days at 20 – 25 C ; o 1 year at -20 C
System Parameters
Wavelength 405 nm (400 – 420 nm) Optical path 1 cm Assay type Kinetic Direction Increase Sample : Reagent Ratio 1 : 10 e.g.: Reagent volume 1 ml Sample volume 100 ml o o Temperature 37 C or 30 C Equilibration time 30 seconds. Read time 1 to 3 minutes Zero adjustment Against air Reagent Blank Limits Low 0.2 AU High 1.0 AU Sensitivity 2.0 U/L Linearity 600 U/L
Analytical Range
Within run (Repeatiblity)
Waste Disposal
Pipette in a test tube:
Macro
Semi-Micro
Working Solution
1.0 ml
500 ml
Specimen
100 ml
50 ml
Mix, read initial absorbance after 30 seconds and start timer simultaneously. Read again after 1, 2 and 3 minutes. Determine the mean absorbance change per minute (DA/min).
Calculation
To calculate the g-glutamyl transferase (g-GT) activity, use the following formula: U/L = 1158 × DA 405 nm/min
2 – 600 U/L.
Level 1
Level 2
n
20
20
Mean (U/L)
44.75
120.2
SD
2.07
2.2
CV%
4.63
1.84
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References
Run to run (Reproducibility) Level 1
Level 2
n
20
20
Mean (U/L)
45.1
121.3
SD
2.19
2.29
CV%
4.72
1.92
Methods Comparison
A comparison between Spectrum Diagnostics g-GT and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.969 was obtained.
1.
Heersink W, Hafkenscheid JCM, Siepel H, van der venjongekryg J, Dijt CCM. Temperature – converting factors for enzymes: comparison of methods. Enzyme. 1980;25:333341.
2.
Moss DW, Henderson AR, Kachmar IF. Enzymes In:Tietz NW, ed. Fundamentals of clinical chemistry. 3 rd ed.
3.
Persjn JP, van der slike W. A new method for the determination of g-glutamyl transferase in serum. J Clin Chem Clin Biochem. 1976;14421-427.
4.
Saw M, Stromme JH, london JL, Theodorsen L. IFCC method for g-glutamyl transferase[(g-glutamyl ) – peptide:ammino acid g-glutamyl transferase, EC 2.3.2.2]. Clin Chem Acta. 1983; 135:315F-338F.
5.
Szasz, G., Persijn JP. Clin. Chem. Clin. Biochem. 1974;12:228.
Sensitivity
When run as recommended, the minimum detection limit of this assay is 2.0 U/L.
Linearity
The reaction is linear up to g-Glutamyltransferase concentration of 600 U/L; specimens showing higher concentration should be diluted 1+5 with physiological saline and repeat the assay (result×6).
Interfering Substances Serum, plasma Haemolysis No significant interference up to a haemoglobin level of 5 g/L.
ORDERING INFORMATION CATALOG NO
QUANTITY
246 001 246 002 246 003 246 004 246 005
4 x 20 ml 10 x 10 ml 9 x 20 ml 4 x 60 ml 5 x 20 ml
Icterus No significant interference. Lipemia Lipemic specimens may cause high absorbance flagging. Diluted sample treatment may be recommended. Anticoagulants Citrate, EDTA and fluoride inhibit the enzyme activity.
Expected Values o
Procedure
Quality Control
Performance Characterstics Precision
37 C Females Males
7 -32 U/L 11-50 U/L
(0.12 -0.53 mkat/L) (0.18 -0. 82 mkat/L)
o
5-24 U/L 8-37 U/L
(0.08-0. 4 mkat/L) (0.1 -0. 6 mkat/L)
o
4-18 U/L 6-28 U/L
(0.07-0. 3 mkat/L) (0. 1-0. 5 mkat/L)
30 C Females Males 25 C Females Males
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Normal & abnormal control serum of known concentrations should be analyzed with each run.
89
g - Glutamyltransferase(gGT)-Liquizyme (1+1) E.C.2.3.2.2.
EC REP Authorised Representative IVD LOT REF
REF: 247 001 (2 x 25 ml) 50 test REF: 247 002 (4 x 25 ml) 100 test
Intended Use
Spectrum Diagnostics liquizyme g- glutamyltransferase reagent is intended for the in-vitro quantitative, diagnostic determination of g-glutamyltransferase in human serum on both automated and manual systems.
Background
g-Glutamyltransferase (gGT) is usually most significantly elevated by obstructive disease and has good specificity for the liver. It is not elevated in bone diseases or pregnancy (as in ALP) or in skeletal muscle diseases (as AST). gGT can also help to differentiate between mechanical and viral from drug induced cholestasis. The highest concentration of gGT is found in the luminal membrane of the proximal tubules of the kidney. Other sources are the pancreas, prostate, and liver. High gGT activity is found in prostate tissue, which may account for the increased gGT activity seen in some sera from men compared with sera from women.
Method
Kinetic colorimetric according to Szasz method. (5)
Assay Principle
Determination of g-Glutamyltransferase (gGT) according to the following reaction: L-g-Glutamyl-3-carboxy-4-nitroanilide + Glycylglycine g-GT L-g-Glutamyl- glycylglycine + 5-amino-2-nitrobenzoate The rate of liberation of yellow coloured indicator 5-amino-2nitrobenzoate is directly proportional to g-GT activity in the sample and is quantitated by measuring the increase in absorbance at 405nm.
Reagents Reagent 1 (R1 Buffer) Tris buffer pH 8.2 Glycylglycine Sodium Azide
120 mmol/L 300 mmol/L 12 mmol/L
Reagent 2 (R2 Starter) L-g-Glutamyl-3-carboxy-4-nitroanilide Sodium Azide
1.0 mmol/L 8 mmol/L
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Do not use liquizyme gGT reagent if it is turbid or if the absorbance of the working reagent is greater than 1.0 at 405 nm. Failure to recover control values within the assigned range may be an indication of reagent deterioration.
Specimen Collection and Preservation
Use serum and plasma, free from hemolysis. Heparin is the only acceptable anticoagulant. The biological half-life of gGT in serum is 3 – 4 days. o o Stability: 7 days at 4 – 8 C ; 7 days at 20 – 25 C ; o 1 year at -20 C
System Parameters
Wavelength Optical path Assay type Direction Increase Sample : Reagent Ratio e.g.: Reagent volume Sample volume Temperature Equilibration time Read time Zero adjustment Reagent Blank Limits Sensitivity Linearity
405 nm (400 – 420 nm) 1 cm Kinetic
90
Mean (U/L)
45.1
121.3
SD
2.19
2.29
CV%
4.72
1.92
A comparison between Spectrum Diagnostics g-GT reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.969 was obtained.
1.
Heersink W, Hafkenscheid JCM, Siepel H, van der venjongekryg J, Dijt CCM. Temperature – converting factors for enzymes: comparison of methods. Enzyme. 1980;25: 333341.
2.
Moss DW, Henderson AR, Kachmar IF. Enzymes In :Tietz NW, ed. Fundamentals of clinical chemistry. 3 rd ed.
3.
Persjn JP, van der slike W. A new method for the determination of g-glutamyl transferase in serum. J Clin Chem Clin Biochem. 1976;14421-427.
4.
Saw M, Stromme JH, london JL, Theodorsen L. IFCC method for g-glutamyl transferase[(g-glutamyl ) – peptide:ammino acid g- glutamyl transferase, EC 2.3.2.2]. Clin Chem Acta. 1983; 135:315F-338F.
5.
Szasz, G., Persijn JP. Clin. Chem. Clin. Biochem. 1974;12:228.
Sensitivity
When run as recommended, the minimum detection limit of this assay is 2.0 U/L. Linearity The reaction is linear up to g-Glutamyltransferase concentration of 600 U/L; specimens showing higher concentration should be diluted 1+5 with physiological saline and repeat the assay (result×6).
ORDERING INFORMATION CATALOG NO
QUANTITY
247 001 247 002
2 x 25 ml 4 x 25 ml
Hemolysis No significant interference up to a hemoglobin level of 5 g/L.
1 : 10 1 ml 100 ml o o 37 C or 30 C 30 seconds. 1 to 3 minutes Against air Low 0.2 AU High 1.0 AU 2.0 U/L 600 U/L
Icterus No significant interference. Lipemia Lipemic specimens may cause high absorbance flagging Diluted sample treatment may be recommended. Anticoagulants Citrate, EDTA and fluoride inhibit the enzyme activity.
Procedure
Expected Values
Pipette in a test tube: working solution
1.0
ml (or add 0.5 ml R1 + 0.5 ml R2)
Specimen
100
ml
Mix, read initial absorbance after 30 sec. and start timer simultaneously. Read again after 1, 2 and 3 minutes. Determine the mean absorbance change per minute (DA/min).
Calculation
To calculate the g-glutamyl transferase (gGT) activity, use the following formula: U/L = 1158 × DA 405 nm /min
Quality Control
Performance Characterstics Precision
All reagents are stable until expiration date stated on label when o stored refrigerated at 2 - 8 C. Working solution can be prepared by adding equall volumes from o o R1 and R2 ; Stability : 3 months at 2 - 8 C or 2 weeks at 15 -25 C when stored in a dark bottle.
20
Serum, plasma
Precautions and Warnings
Reagent Preparation, Storage and Stability
Level 2
20
Interfering Substances
Normal & abnormal control serum of known concentrations should be analyzed with each run.
Both reagents (R1) and (R2) contain sodium azide which may react with copper or lead plumbing.
Level 1 n
Methods Comparison
Deterioration
For further information, refer to the g-Glutamyltrasferase reagent material safety data sheet. Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
References
Run to run (Reproducibility)
SYMBOLS IN PRODUCT LABELLING
7 -32 U/L 11-50 U/L
(0.12 -0.53 mkat/L) (0.18 -0. 82 mkat/L)
o
5-24 U/L 8-37 U/L
(0.08-0. 4 mkat/L) (0.1 -0. 6 mkat/L)
o
4-18 U/L 6-28 U/L
(0.07-0. 3 mkat/L) (0. 1-0. 5 mkat/L)
30 C Females Males 25 C Females Males
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Analytical Range 2 – 600 U/L.
Waste Disposal
Within run (Repeatiblity) Level 1
o
37 C Females Males
Level 2
n
20
20
Mean (U/L)
44.75
120.2
SD
2.07
2.2
CV%
4.63
1.84
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
91
Aspartate aminotransferase (AST/GOT)-Liquizyme (4+1) E.C.2.6.1.1. REF: 291 001 REF: 291 002 REF: 291 003 REF: 291 004 REF: 291 005 REF: 291 006 REF: 291 007 REF: 291 008
( 4 x 20 ml) 80 test (10 x 10 ml) 100 test ( 9 x 20 ml) 180 test ( 4 x 60 ml) 240 test ( 5 x 20 ml) 100 test ( 4 x 50 ml) 200 test ( 5 x100 ml) 500 test ( 6 x100 ml) 600 test
Spectrum Diagnostics liquizyme AST reagent is intended for the invitro quantitative, diagnostic determination of AST in human serum on both automated and manual systems.
Background
The enzyme aspartate aminotransferase (AST) is widely distributed in erythrocytes and tissues, principally heart, liver, muscle, and kidney. Elevated serum levels are found in diseases involving these tissues such as myocardial infarction,viral hepatitis and muscular dystrophy. Following myocardial infarction, serum AST is elevated and reaches a peak two days after onset. Two isoenzymes of AST have been detected, cytoplasmic and mitochondrial. Only the cytoplasmic isoenzyme occurs in normal serum, while the mitochondrial, together with the cytoplasmic isoenzyme, has been detected in the sera of patients with coronary and hepatobiliary diseases.
Method
Kinetic method according to the International Federation of ClinicalChemistry (IFCC) (3).
Assay Principle
The series of the reaction involved in the assay system is as follows: The amino group is enzymatically transferred by AST present in the sample from L-aspartate to the carbon atom of 2-oxoglutarate yielding oxaloacetate and L-glutamate. L-Aspartate + 2-Oxoglutarate
2.
Oxaloacetate + L-Glutamate
Oxaloacetate in presence of NADH and malate dehyrogenase (MDH), is reduced to L-malate. In this reaction NADH is oxidized to NAD. The reaction is monitored by measuring the rate of decrease in absorbance at 340 nm due to oxidation of NADH to NAD. Oxaloacetate + NADH + H+
3.
AST
MDH
L-Lactate + NAD+
Addition of lactate dehydrogenase (LDH) to the reagent is necessary to achieve rapid and complete reduction of endogenous pyruvate so that it does not interfere with the assay. Sample pyruvate + NADH + H+
LDH
L-Lactate + NAD+
Reagents
Reagent 1 (R1 Buffer / Enzymes) Tris buffer (pH 7.7 ) L- Aspartate MDH LDH Sodium Hydroxide Sodium Azide
92
EC REP Authorised Representative IVD LOT REF
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use Temperature Limitation
!
Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by (Xi) - Irritant
Irritant (Xi): R36/38: Irritating to eyes and skin. S26: In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. S37/39: Wear suitable gloves and eye/face protection.
Intended Use
1.
SYMBOLS IN PRODUCT LABELLING
80 mmol/L 240 mmol/L ≥ 450 U/L ≥ 1200 U/L 220 mmol/L 8 mmol/L
Reagent 2 (R2 Coenzyme) NADH ≥ 0.18 mmol/L 2 – Oxoglutarate 18 mmol/L Sodium Azide 8 mmol/L For further information, refer to the Aspartate aminotransferase reagent material safety data sheet.
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately. Both reagents (R1) and (R2) contains sodium azide which may react with copper or lead plumbing.
Reagent Preparation
Prepare working solution as following: REF:291 001 : add 4 ml from R2 to one bottle of R1; mix gently. REF:291 002 : add 2 ml from R2 to one bottle of R1; mix gently. REF:291 003 : add 4 ml from R2 to one bottle of R1; mix gently. REF:291 004 : add 12 ml from R2 to one bottle of R1; mix gently. REF:291 005 : add 4 ml from R2 to one bottle of R1; mix gently. REF:291 006 : add 10 ml from R2 to one bottle of R1; mix gently. REF:291 007 : add one bottle of R2 to one bottle of R1; mix gently. REF:291 008 : add one bottle of R2 to one bottle of R1; mix gently. Or prepare the working solution according to the number of tests required by mixing 4 volumes of reagent 1 (R1) and 1volume of reagent 2 (R2), e.g. 400 ml R1 +100 ml R2.
Reagent Storage and Stability
All reagents are stable until expiration date stated on label when o stored refrigerated at 2 - 8 C. Working solution is stable for 4 weeks o o at 2 – 8 C or 2 days at 15 - 25 C.
Deterioration
Do not use liquizyme AST reagent if it is turbid or if the absorbance of the working reagent is less than 1.0 at 340 nm. Failure to recover control values within the assigned range may be an indication of reagent deterioration.
Specimen Collection and Preservation
Use nonhemolyzed serum. Heparin and EDTA are the only acceptable anticoagulants. The biological half-life of AST in serum is 17 hours. o o Stability: 1 day at 15 – 25 C; 7 days at 4 - 8 C; o 12 weeks at -20 C
System Parameters
Wavelength 340 nm (334 – 365 nm) Optical path 1 cm Assay type Kinetic Direction decrease Sample : Reagent Ratio 1 : 10 e.g.: Reagent volume 1 ml Sample volume 100 ml o o Temperature 37 C or 30 C Equilibration time 60 seconds. Read time 1 to 3 minutes Zero adjustment Against air Reagent Blank Limits Low 1.00 AU High 2.5 AU Sensitivity 5 U/L Linearity 400 U/L
Procedure Macro
Semi-Micro
Working solution
1.0 ml
500 ml
Specimen
100 ml
50 ml
Drugs Calcium dobesilate and doxycycline HCL cause artificially low AST values at the tested drug level.
Expected values
Mix, read initial absorbance after 60 seconds. and start timer simultaneously. Read again after 1, 2 and 3 minutes. Determine the mean absorbance change per minute (DA/min).
Quality Control
Normal & abnormal control serum of known concentrations should be analyzed with each run.
up to 31 U/l up to 37 U/l
(up to 0.52 mkat/L) (up to 0.62 mkat/L)
o
up to 21 U/l up to 25 U/l
(up to 0.35 mkat/L) (up to 0.42 mkat/L)
30 C Females Males
Temperature conversion factor is 1.37 (25 o 2.04 (25 37 C ).
Calculation
To calculate the AST/GOT activity use the following formulae: U/l = 1780 x DA 334 nm /min U/l = 1746 x DA 340 nm /min U/l = 3235 x DA 365 nm /min
o
37 C Females Males
o
30 C ) and
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Analytical Range
Performance Characterstics Precision
5 – 400 U/L.
Waste Disposal
Within run (Repeatiblity) Level 1
Level 2
n
20
20
Mean (U/L)
32.6
133
SD
1.3
1.3
CV%
4.08
0.97
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References
Run to run (Reproducibility) Level 1
Level 2
n
20
20
Mean (U/L)
33.1
135.5
SD
1.5
1.42
CV%
4.25
1.13
Methods Comparison
A comparison between Spectrum Diagnostics AST (4+1) reagent and a commercial reagent of the same methodology was performed on 20 human serum.A correlation of 0.991 was obtained.
1.
Breuer J, Report on the symposium “drug effects in clinicalchemistry methods”. Eur J Clin Chem Clin Biochem. 1996;34:385-386.
2.
ECCLS. Determination of the catalytic activity concentration in serum on L- aspartate aminotransferase (EC 2.6.1.1,AST) Clin Chem. 1989;20:204-211.
3.
IFCC expert panel on enzymes part 3. J Clin Chem Clin Biochem 1986;24:481-95.
4.
Henry RJ, et al. Am j Clin Path 1960 :34:381
5.
Sherwin JE. Liver function. In:kaplan LA, PESCE AJ, eds.Clinical chemistry, theory, analysis, and correlation. Stlouis:mosby;1984:420- 438.
6.
Young DS. Effects of drugs on clinical laboratory tests.Third edition. 1990 :3:6-12.
7.
Zilva JF, pannall PR : Plasma enzymes in diagnosis inclinical chemistry in diagnosis and treatment lioyd- luke london 1979:chap 17: 338.
Sensitivity
When run as recommended, the minimum detection limit of this assay is 5.0 U/L.
Linearity
The reaction is linear up to AST concentration of 400 U/L; specimens showing higher concentration should be diluted 1+5 with physiological saline and repeat the assay (result×6).
Interfering Substances Serum, plasma
Hemolysis Erythrocyte contamination elevates results, since AST activities in erythrocytes are 15 times higher than those in normal sera. Icterus No significant interference. Lipemia Lipemic specimens may cause high absorbance flagging. Diluted sample is recommended.
ORDERING INFORMATION CATALOG NO
QUANTITY
291 001 291 002 291 003 291 004 291 005 291 006 291 007 291 008
4 x 20 ml 10 x 10 ml 9 x 20 ml 4 x 60 ml 5 x 20 ml 4 x 50 ml 5 x 100 ml 6 x 100 ml
Anticoagulants Citrate and fluoride inhibit the enzyme activity.
93
Aspartate aminotransferase (AST/GOT)-Liquizyme (1 + 1) E.C.2.6.1.1. REF: 259 001 (2 x 25 ml) 50 test REF: 259 002 (4 x 25 ml) 100 test REF: 259 003 (2 x 100 ml) 200 test
Intended Use
Spectrum Diagnostics liquizyme AST reagent is intended for the invitro quantitative, diagnostic determination of AST in human serum on both automated and manual systems.
Background
The enzyme aspartate aminotransferase (AST) is widely distributed in erythrocytes and tissues, principally heart, liver, muscle, and kidney. Elevated serum levels are found in diseases involving these tissues such as myocardial infarction, viral hepatitis and muscular dystrophy. Following myocardial infarction, serum AST is elevated and reaches a peak two days after onset. Two isoenzymes of AST have been detected, cytoplasmic and mitochondrial. Only the cytoplasmic isoenzyme occurs in normal serum, while the mitochondrial, together with the cytoplasmic isoenzyme, has been detected in the sera of patients with coronary and hepatobiliary diseases.
Method
Kinetic method according to the International Federation of Clinical Chemistry (IFCC) (3). The series of the reaction involved in the assay system is as follows: The amino group is enzymatically transferred by AST present in the sample from L-aspartate to the carbon atom of 2-oxoglutarate yielding oxaloacetate and L-glutamate. L-Aspartate + 2-Oxoglutarate 2.
3.
AST
Oxaloacetate + L-Glutamate
Oxaloacetate in presence of NADH and malate dehydrogenase (MDH), is reduced to L-malate. In this reaction NADH is oxidized to NAD. The reaction is monitored by measuring the rate of decrease in absorbance at 340 nm due to oxidation of NADH to NAD. Oxaloacetate + NADH + H+
MDH
L-Lactate + NAD+
Addition of lactate dehydrogenase (LDH) to the reagent is necessary to achieve rapid and complete reduction of endogenous pyruvate so that it does not interfere with the assay. Sample pyruvate + NADH + H+
Reagents
LDH
L-Lactate + NAD+
Reagent 1 (R1 Buffer/Enzymes) Tris buffer (pH 7.7) 80 mmol/L L- Aspartate 450 mmol/L MDH ≥ 900 U/L LDH ≥ 2000 U/L Sodium Hydroxide 300 mmol/L Sodium Azide 8 mmol/L Irritant (Xi): R36/38: Irritating to eyes and skin. S26: In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. S37/39: Wear suitable gloves and eye/face protection.
94
EC REP Authorised Representative IVD LOT REF
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use Temperature Limitation
!
Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by (Xi) - Irritant
Reagent 2 (R2 Coenzyme) NADH 2-Oxoglutarate Sodium Azide
≥ 0.06 mmol/L 4 mmol/L 8 mmol/L
For further information, refer to the Aspartate aminotransferase reagent material safety data sheet.
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately. Reagent (R1) contains sodium azide which may react with copper or lead plumbing.
Reagent Preparation, Storage and Stability
All reagents are stable until expiration date stated on label when o stored refrigerated at 2 - 8 C. Working solution can be prepared by adding equal volumes from R1 o and R2; Stability: 2 days at 2 – 8 C.
Deterioration
Assay Principle 1.
Calculation
SYMBOLS IN PRODUCT LABELLING
Do not use liquizyme AST reagent if it is turbid or if the absorbance of the working reagent is less than 1.0 at 340 nm. Failure to recover control values within the assigned range may be an indication of reagent deterioration.
Specimen Collection and Preservation
Use nonhemolyzed serum. Heparin and EDTA are the only acceptable anticoagulants. The biological half-life of AST in serum is 17 hours. o o Stability: 1 day at 15 – 25 C; 7 days at 4 - 8 C; o 12 weeks at -20 C
System Parameters
Expected values
To calculate the AST/GOT activity use the following formulae U/l = 1780 x DA 334 nm /min U/l = 1746 x DA 340 nm /min U/l = 3235 x DA 365 nm /min
Quality Control
Normal & abnormal commercial control serum concentrations should be analyzed with each run.
of
known
Within run (Repeatiblity)
Level 2
n
20
20
Mean (U/L)
32
135
SD
1.2
1.27
CV%
4.1
0.99
Level 1
Level 2
20
20
Mean (U/L)
34
136
SD
1.6
1.43
CV%
4.5
1.16
Methods Comparison
A comparison between Spectrum Diagnostics AST (1+1) reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.98 was obtained.
Sensitivity
When run as recommended, the minimum detection limit of this assay is 5.0 U/L.
Linearity
The reaction is linear up to AST concentration of 400 U/L;specimens showing higher concentration should be diluted 1+5 with physiological saline and repeat the assay (result×6).
Interfering Substances
Wavelength 340 nm (334 – 365 nm) Optical path 1 cm Assay type Kinetic Direction decrease Sample: Reagent Ratio 1 : 10 e.g.: Reagent volume 1 ml Sample volume 100 ml o o Temperature 37 C or 30 C Equilibration time 30 seconds. Read time 1 to 3 minutes Zero adjustment Against air Reagent Blank Limits Low 1.00 AU High 2.5 AU Sensitivity 5 U/L Linearity 400 U/L
Serum, plasma
Procedure
Drugs Calcium dobesilate and doxycycline HCL cause artificially low AST values at the tested drug level.
Pipette in a test tube: Working solution
1.0 ml ( or 0.5 ml R1 + 0.5 ml R2)
Specimen
100 ml
(up to 0.52 mKat/L) (up to 0.62 mKat/L)
o
up to 21 U/l up to 25 U/l
(up to 0.35 mKat/L) (up to 0.42 mKat/L) o
Temperature conversion factor is 1.37 ( 25 o and 2.04 ( 25 37 C ).
30 C )
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Analytical Range 5 – 400 U/L.
Waste Disposal
Run to run (Reproducibility) n
up to 31 U/l up to 37 U/l
30 C Females Males
Performance Characteristics Precision Level 1
o
37 C Females Males
Haemolysis Erythrocyte contamination elevate results, since AST activities in erythrocytes are 15 times higher than those in normal sera. Icterus No significant interference. Lipemia Lipemic specimens may cause high absorbance flagging. Diluted sample is recommended. Anticoagulants Citrate and fluoride inhibit the enzyme activity.
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References 1.
Breuer J, report on the symposium “drug effects in clinical chemistry methods”. Eur J Clin Chem clin Biochem. 1996;34:385-386.
2.
ECCLS. Determination of the catalytic activity concentration in serum on L- aspartate aminotransferase (EC 2.6.1.1,AST) Clin Chem. 1989;20:204-211.
3.
IFCC expert panel on enzymes part 3. J Clin Chem Clin Biochem 1986;24:481-95.
4.
Henry RJ et al. Am J Clin Bath 1960 :34:381
5.
Sherwin JE. Liver function. In:kaplan LA, PESCE AJ, eds. Clinical chemistry, theory,analysis, and correlation. St louis:mosby;1984:420- 438.
6.
Young DS. Effects of drugs on clinical laboratory tests. Third edition. 1990 :3:6-12.
7.
Zilva JF, pannall PR : Plasma enzymes in diagnosis in clinical chemistry in diagnosis and treatment lioydluke london 1979:chap 17 : 338.
ORDERING INFORMATION CATALOG NO
QUANTITY
259 001 259 002 259 003
2 x 25 ml 4 x 25 ml 2 x 100 ml
Mix, read initial absorbance after 30 seconds. and start timer simultaneously. Read again after 1, 2 and 3 minutes. Determine the mean absorbance change per minute (DA/min).
95
Aspartate aminotransferase (AST/GOT)-Colorimetric
EC REP Authorised Representative IVD LOT
REF: 260 001 (2 x 50 ml) 100 test REF: 260 002 (2 x100 ml) 200 test
REF
Intended Use
Spectrum Diagnostics colorimetric AST reagent is intended for the in-vitro quantitative, diagnostic determination of AST in human serum.
Background
The enzyme aspartate aminotransferase (AST) is widely distributed in erythrocytes and tissues, principally heart, liver, muscles, and kidneys. Elevated serum levels are found in diseases involving these tissues such as myocardial infarction, viral hepatitis and muscular dystrophy. Following myocardial infarction, serum AST is elevated and reaches a peak two days after onset.Two isoenzymes of AST have been detected, cytoplasmic and mitochondrial. Only the cytoplasmic isoenzyme occurs in normal serum, while the mitochondrial, together with the cytoplasmic isoenzyme, has been detected in the sera of patients with coronary and hepatobiliary diseases.
Method
AST – (Colorimetric method).
Assay Principle
The reaction involved in the assay system is as follows: The amino group is enzymatically transferred by AST present in the sample from L-aspartate to the carbon atom of 2-oxoglutarate yielding oxaloacetate and L-glutamate. L-Aspartate + 2-Oxoglutarate
AST
Oxaloacetate + L-Glutamate
AST activity is measured by monitoring the concentration of oxaloacetate hydrazone formed with 2,4-dinitrophenylhydrazine.
Reagents
Reagent 1 (R1 Buffer) Phosphate buffer 100 mmol/L L- aspartate 100 mmol/L 2–Oxoglutarate 5 mmol/L Sodium Hydroxide 140 mmol/L Sodium Azide 12 mmol/L Harmful (Xn): R20/22: Harmful by inhalation and if swallowed. S24/25: Avoid contact with skin and eyes.
Reagent 2 (R2)
2,4-dinitrophenyl-hydrazine 2 mmo/L HCl 8.4 % (C)-Corrosive contains caustic materials. R35 Causes severe burns. R41 Risk of serious damage to eyes. S26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. S28 After contact with skin, wash immediately with plenty of soap and water. For further information, refer to the Aspartate aminotransferase reagent material safety data sheet.
Additional Reagent
Sodium hydroxide 0.4 mol/L.
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
96
Calculation
SYMBOLS IN PRODUCT LABELLING For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use Temperature Limitation
!
Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by (Xi) - Irritant (C) - Corrosive
Reagent (R1) contains sodium azide which may react with copper or lead plumbing.
Obtain the AST activity from the following table Absorbance
U/L
Absorbance
U/L
0.020 0.030 0.040 0.050 0.060 0.070 0.080 0.090
7 10 13 16 19 23 27 31
0.100 0.110 0.120 0.130 0.140 0.150 0.160 0.170
36 41 47 52 59 67 76 89
Reagent preparation, Storage and Stability
Quality Control
Deterioration
Sensitivity
The reagents are supplied ready-to-use and stable up to the expiry o date labeled on the bottles when stored at 2 – 8 C. Do not use The AST regents if precipitate forms. Failure to recover control values within the assigned range may be an indication of reagent deterioration.
Specimen Collection and Preservation
Use only non haemolyzed serum. The only acceptable anticoagulants are heparin and EDTA. The biological half-life of AST in serum is 17 hours. o o Stability: 1 day at 15 – 25 C ; 7 days at 4 - 8 C ; o 12 weeks at -20 C
System Parameters
Wavelength 546 nm (530-550 nm) Optical path 1 cm Assay type Endpoint Direction Increase Sample : Reagent Ratio 1 : 60 o o Temperature 37 C and 20 – 25 C Zero adjustment Reagent or Sample blank Sensitivity 7 U/L Linearity 89 U/L
Procedure
1. Measurement against Reagent Blank Pipette into test tubes R1(buffer) Sample Distilled water
Reagent blank
Sample
0.5 ml -----100 ml
0.5 ml 100 ml -----o
Mix and incubate for exactly 30 minutes at 37 C R2
0.5 ml
0.5 ml o
Mix and incubate for exactly 20 minutes at 20 – 25 C Sodium hydroxide
5.0 ml
Sample blank
Sample
0.5 ml -----
0.5 ml 100 ml o
Mix and incubate for exactly 30 minutes at 37 C 0.5 ml 100 ml
of
Henry RJ et al. Am J Clin Path 1960 :34:381.
2.
Reitman S and Frankel S.Am .J.Clin.Path, 1975 ;28;65.
3.
Sherwin JE. Liver function. In:kaplan LA, PESCE AJ, eds. Clinical chemistry, theory,analysis, and correlation. St louis:Mosby;1984:420- 438.
4.
Young DS. Effects of drugs on clinical laboratory tests. Third edition. 1990 :3:6-12.
ORDERING INFORMATION
known
CATALOG NO
QUANTITY
260 001 260 002
2 x 50 ml 2 x 100 ml
If run as recommended, the minimum detection level is 7 U/L.
Linearity
The assay is linear up to 89 U/L. If the absorbance exceeds 0.170 at 546 nm ( 89 U/L ), samples should be diluted 1 + 9 using sodium chloride and repeat the assay (result × 10).
Interfering Substances Serum, plasma
Haemolysis Erythrocyte contamination elevates results, since AST activities in erythrocytes are 15 times higher than those in normal sera. Icterus No significant interference. Lipemia Lipemic specimens may cause high absorbance flagging. Diluted sample is recommended. Note High concentration of aldehydes, ketones, or oxo-acids in some sera may cause false high transaminases levels. Measurement aganist a serum blank instead of a reagent blank avoids the risk of finding such artifacts.
Expected values Up to 12 U/L.
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the reults is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Analytical Range 7 – 89 U/L.
Waste Disposal
2. Measurement against Sample Blank
R2 Sample
Normal & abnormal commercial control serum concentrations should be analyzed with each run.
1.
5.0 ml
Mix, measure absorbance of specimen against reagent blank at 546 nm after 5 minutes.a
R1(buffer) Sample
References
0.5 ml ----o
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
Mix and incubate for exactly 20 minutes. at 20 – 25 C Sodium hydroxide
5.0 ml
5.0 ml
Mix, measure absorbance of specimen against sample blank at 546 nm after 5 minutes.
97
Aspartate aminotransferase
(AST/GOT) - Ultimate Single Reagent E.C.2.6.1.1. t REF: 261 001 REF: 261 002 REF: 261 003 REF: 261 004 REF: 261 005
( 2 x 20 ml) ( 6 x 20 ml) ( 2 x 100 ml) ( 4 x 50 ml) ( 2 x 50 ml)
40 120 200 200 100
test test test test test
Spectrum Diagnostics Ultimate AST reagent is intended for the invitro quantitative, diagnostic determination of AST in human serum on both automated and manual systems.
Background
The enzyme aspartate aminotransferase (AST) is widely distributed in erythrocytes and tissues, principally heart, liver, muscle, and kidney. Elevated serum levels are found in diseases involving these tissues such as myocardial infarction,viral hepatitis and muscular dystrophy. Following myocardial infarction, serum AST is elevated and reaches a peak two days after onset. Two isoenzymes of AST have been detected, cytoplasmic and mitochondrial. Only the cytoplasmic isoenzyme occurs in normal serum, while the mitochondrial, together with the cytoplasmic isoenzyme, has been detected in the sera of patients with coronary and hepatobiliary diseases.
Kinetic method according to the International Federation of ClinicalChemistry (IFCC)(3).
Assay Principle
The series of the reaction involved in the assay system is as follows: The amino group is enzymatically transferred by AST present in the sample from L-aspartate to the carbon atom of 2-oxoglutarate yielding oxaloacetate and L-glutamate. L-Aspartate + 2-Oxoglutarate
MDH
L-Lactate + NAD+
Addition of lactate dehydrogenase (LDH) to the reagent is necessary to achieve rapid and complete reduction of endogenous pyruvate so that it does not interfere with the assay. Sample pyruvate + NADH + H+
Reagent (R)
Tris buffer (pH 7.7 ) L- Aspartate MDH LDH Sodium Hydroxide Sodium Azide NADH 2 - Oxoglutarate Sodium Azide
98
AST
Oxaloacetate + L-Glutamate
Oxaloacetate in presence of NADH and malate dehyrogenase (MDH), is reduced to L-malate. In this reaction NADH is oxidized to NAD. The reaction is monitored by measuring the rate of decrease in absorbance at 340 nm due to oxidation of NADH to NAD. Oxaloacetate + NADH + H+
3.
IVD LOT
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use Temperature Limitation
!
Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by (Xi) - Irritant
LDH
L-Lactate + NAD+
80 mmol/L 240 mmol/L ≥ 450 U/L ≥ 1200 U/L 220 mmol/L 8 mmol/L > 0.18 mmol/L 18 mmol/L 8 mmol/L
Calculation
To calculate the AST/GOT activity use the following formulae: U/l = 1780 x DA 334 nm /min U/l = 1746 x DA 340 nm /min U/l = 3235 x DA 365 nm /min
Irritant (Xi): R36/38: Irritating to eyes and skin. S26: In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. S37/39: Wear suitable gloves and eye/face protection. The reagent also contains additives required to maintain NADH in its reduced forrm. For further information, refer to the •Aspartate aminotransferase reagent material safety data sheet.
The Reagent (R) contain sodium azide which may react with copper or lead plumbing. Spectrum Ultimate AST reagent is supplied ready-to-use and stable up to the expiry date labeled on the bottles Once opened, the opened vial is stable for 3 months at the specified temperature.
Deterioration
Do not use Spectrum Ultimate AST reagent if it is turbid or if the absorbance of the working reagent is less than 0.9 at 340 nm. Failure to recover control values within the assigned range may be an indication of reagent deterioration.
Specimen Collection and Preservation
Use nonhemolyzed serum. Heparin and EDTA are the only acceptable anticoagulants. The biological half-life of AST in serum is 17 hours. o o Stability: 1 day at 15 – 25 C; 7 days at 4 - 8 C; o 12 weeks at -20 C
System Parameters
Wavelength 340 nm (334 – 365 nm) Optical path 1 cm Assay type Kinetic Direction decrease Sample : Reagent Ratio 1 : 10 e.g.: Reagent volume 1 ml Sample volume 100 ml o o Temperature 37 C or 30 C Equilibration time 60 seconds. Read time 180 seconds Zero adjustment Against air Reagent Blank Limits Low 0.9 AU High 2.5 AU Sensitivity 5 U/L Linearity 400 U/L
Procedure Macro
Semi-Micro
Reagent (R)
1.0 ml
0.5 ml
Specimen
100 ml
50 ml
o
37 C Females Males o
Quality Control
Performance Characterstics Precision Within run (Repeatiblity)
Level 1
Level 2
n
20
20
Mean (U/L)
32.6
133
SD
1.3
1.3
CV%
4.08
0.97
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
Expected values
30 C Females Males
Normal & abnormal control serum of known concentrations should be analyzed with each run.
Reagent Preparation, Storage and Stability
Method
2.
EC REP Authorised Representative
REF
Intended Use
1.
SYMBOLS IN PRODUCT LABELLING
Run to run (Reproducibility) Level 1
Level 2
n
20
20
Mean (U/L)
33.1
135.5
SD
1.5
1.42
CV%
4.25
1.13
Methods Comparison
A comparison between Spectrum Diagnostics AST reagent and a commercial reagent of the same methodology was performed on 20 human serum.A correlation of 0.991 was obtained.
Linearity
The reaction is linear up to AST concentration of 400 U/L; specimens showing higher concentration should be diluted 1+5 with physiological saline and repeat the assay (result×6).
Interfering Substances Serum, plasma Hemolysis Erythrocyte contamination elevates results, since AST activities in erythrocytes are 15 times higher than those in normal sera. Icterus No significant interference. Lipemia Lipemic specimens may cause high absorbance flagging. Diluted sample is recommended. Anticoagulants Citrate and fluoride inhibit the enzyme activity.
(up to 0.52 mKat/L) (up to 0.62 mKat/L)
up to 21 U/l up to 25 U/l
(up to 0.35 mKat/L) (up to 0.42 mKat/L) o
Temperature conversion factor is 1.37 (25 o (25 37 C ).
30 C ) and 2.04
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Analytical Range 5 – 400 U/L.
Waste Disposal
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References 1.
Breuer J, Report on the symposium “drug effects in clinicalchemistry methods”. Eur J Clin Chem Clin Biochem. 1996;34:385-386.
2.
ECCLS. Determination of the catalytic activity concentration in serum on L- aspartate aminotransferase (EC 2.6.1.1,AST) Clin Chem. 1989;20:204-211.
3.
IFCC expert panel on enzymes part 3. J Clin Chem Clin Biochem 1986;24:481-95.
4.
Henry RJ, et al. Am j Clin Path 1960 :34:381
5.
Sherwin JE. Liver function. In:kaplan LA, PESCE AJ, eds.Clinical chemistry, theory, analysis, and correlation. Stlouis:mosby;1984:420- 438.
6.
Young DS. Effects of drugs on clinical laboratory tests.Third edition.1990 :3:6-12.
7.
Zilva JF, pannall PR : Plasma enzymes in diagnosis inclinical chemistry in diagnosis and treatment lioyd- luke london 1979:chap 17: 338.
Sensitivity
When run as recommended, the minimum detection limit of this assay is 5.0 U/L.
up to 31 U/l up to 37 U/l
ORDERING INFORMATION CATALOG NO 261 001 261 002 261 003 261 004 261 005
QUANTITY 2 6 2 4 2
x 20 ml x 20 ml x 100 ml x 50 ml x 50 ml
Drugs Calcium dobesilate and doxycycline HCL cause artificially low AST values at the tested drug level.
Mix, read initial absorbance after 60 seconds. and start timer simultaneously. Read again after 60, 120 and 180 seconds. Determine the mean absorbance change per minute (DA/min).
99
Alanine aminotransferase (ALT/GPT)-Liquizyme (4+1) E.C.2.6.1.2. REF: 292 001 REF: 292 002 REF: 292 003 REF: 292 004 REF: 292 005 REF: 292 006 REF: 291 007 REF: 291 008
( 4 x 20 ml) 80 test (10 x 10 ml) 100 test ( 9 x 20 ml) 180 test ( 4 x 60 ml) 240 test ( 5 x 20 ml) 100 test ( 4 x 50 ml) 200 test ( 5 x100 ml) 500 test ( 6 x100 ml) 600 test
Background
The enzyme alanine aminotransferase ALT is widely distributed with high concentrations in the liver and to a lesser extent in kidneys, heart, skeletal muscles, pancreas and lungs. Elevated serum ALT is found in hepatitis, cirrhosis, obstructive jaundice, carcinoma of the liver, and chronic alcohol abuse. ALT is only slightly elevated in patients who have an uncomplicated myocardial infarction. Although both serum aspartate aminotransferase AST and ALT become elevated whenever disease processes affect liver cell integrity, ALT is the more liver specific enzyme. Moreover, elevations of ALT activity persist longer than elevations of AST activity.
Method
Kinetic method according to the International Federation of Clinical Chemistry (IFCC) (3).
Assay Principle
The series of the reaction involved in the assay system is as follows: The amino group is enzymatically transferred by ALT present in the sample from alanine to the carbon atom of 2-oxoglutarate yielding pyruvate and L-glutamate.
2.
Pyruvate + L-Glutamate
Pyruvate is reduced to lactate by LDH present in the reagent with the simultaneous oxidation of NADH to nicotinamide adenine dinucleotide (NAD). The reaction is monitored by measuring the rate of decrease in absorbance at 340 nm due to the oxidation of NADH. Pyruvate + NADH + H+
3.
ALT
LDH
L-Lactate + NAD+
Endogenous sample pyruvate is rapidly and completely reduced by LDH during the initial incubation period so that it does not interfere with the assay. Sample pyruvate + NADH + H+
LDH
L-Lactate + NAD+
Reagents Reagent 1 (R1 Buffer / Enzyme) Tris buffer (pH 7.4) L- Alanine LDH Sodium Azide
100
IVD LOT REF
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
≥ 0.18 18 8
mmol/L mmol/L mmol/L
For further information, refer to the Alanine aminotransferase reagent material safety data sheet.
Spectrum Diagnostics liquizyme ALT reagent is intended for the invitro quantitative, diagnostic determination of ALT in human serum on both automated and manual systems.
L-Alanine + 2-Oxoglutarate
EC REP Authorised Representative
Reagent 2 (R2 Coenzyme) NADH 2 – Oxoglutarate Sodium Azide
Intended Use
1.
Procedure
SYMBOLS IN PRODUCT LABELLING
100 800 ≥ 2000 8
mmol/L mmol/L U/L mmol/L
Reagent preparation
Prepare working solution as following: REF:292 001 REF:292 002 REF:292 003 REF:292 004 REF:292 005 REF:292 006 REF:292 007 REF:292 008
: : : : : : : :
add 4 ml from R2 to one bottle of R1; mix gently. add 2 ml from R2 to one bottle of R1; mix gently. add 4 ml from R2 to one bottle of R1; mix gently. add12 ml from R2 to one bottle of R1; mix gently. add 4 ml from R2 to one bottle of R1; mix gently. add10 ml from R2 to one bottle of R1; mix gently. add one bottle of R2 to one bottle of R1;mix gently. add one bottle of R2 to one bottle of R1; mix gently.
Or prepare the working solution according to the number of tests required by mixing 4 volumes of reagent 1 (R1) and 1volume of reagent 2 (R2), e.g. 400 ml R1 + 100 ml R2.
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately. Both reagents (R1) and (R2) contain sodium azide which may react with copper or lead plumbing.
Reagent Storage and Stability
All reagents are stable until expiration date stated on label when o stored refrigerated at 2 - 8 C. Working solution is stable for 4 weeks o o at 2 – 8 C or 2 days at 15 - 25 C.
Deterioration
Do not use liquizyme ALT reagent if it is turbid or if the absorbance of the working reagent is less than1.0 at 340 nm. Failure to recover control values within the assigned range may be an indication of reagent deterioration.
Specimen Collection and Preservation
Use nonhemolyzed serum or plasma. Heparin and EDTA are the only acceptable anticoagulants; avoid other anticoagulants. The biological half-life of ALT in serum is 47 hours. o Stability: 3 days at 15 - 25 C or 7 days o o at either 4- 8 C or at -20 C
System Parameters
Wavelength 340 nm (334 – 365 nm) Optical path 1 cm Assay type Kinetic Direction decrease Sample : Reagent Ratio 1 : 10 e.g .: Reagent volume 1 ml Sample volume 100 ml o o Temperature 37 C or 30 C Equilibration time 60 seconds. Read time 1 to 3 minutes Zero adjustment Against air Reagent Blank Limits Low 1.00 AU High 2.5 AU Sensitivity 5 U/L Linearity 400 U/L
Macro
Semi-Micro
Working solution
1.0 ml
500 ml
Specimen
100 ml
50 ml
Anticoagulants Citrate and fluoride inhibit the enzyme activity. Drugs Calcium dobesilate and doxycycline HCL cause artificially low ALT values at the tested drug level.
Mix, read initial absorbance after 60 seconds. and start timer simultaneously. Read again after 1, 2 and 3 minutes. Determine the mean absorbance change per minute (DA/min).
Calculation
Expected values o
up to 31 U/l up to 41 U/l
(up to 0.52 mKat/L) (up to 0.68 mKat/L)
o
up to 22 U/l up to 29 U/l
(up to 0.37 mKat/L) (up to 0.48 mKat/L)
37 C Females males
To calculate the ALT/GPT activity use the following formula
30 C Females males
U/l = 1780 x DA 334 nm /min U/l = 1746 x DA 340 nm /min U/l = 3235 x DA 365 nm /min
Temperature conversion factor is 1.32 (25 o 1.85 (25 37 C )
Quality Control
Normal & abnormal control serum of known concentrations should be analyzed with each run.
Performance Characterstics Precision
o
30 C) and
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Analytical Range
Within run (Repeatiblity)
5 – 400 U/L. Level 1
Level 2
n
20
20
Mean (U/L)
24.6
105.9
SD
0.93
0.94
CV%
3.78
0.89
Run to run (Reproducibility) Level 1
Level 2
n
20
20
Mean (U/L)
25.2
106
SD
1.1
1.05
CV%
3.9
0.95
Waste Disposal
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References 1.
Breuer J, report on the symposium “drug effects in clinical chemistry methods”. Eur J Clin Chem Clin Biochem. 1996;34:385-386.
2.
ECCLS. Determination of the catalytic activity concentration in serum on L- alanine aminotransferase (EC 2.6.1.2,ALAT) Clin chem. 1989;20:204-211.
A comparison between Spectrum Diagnostics ALT (4+1) reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.983 was obtained.
3.
IFCC expert panel on enzymes part 3. J Clin Chem Clin Biochem 1986;24:481-95 .
4.
Henry RJ, et al. Am J clin Path 1960 :34:381
Sensitivity
5.
Sherwin JE. Liver function. In:kaplan LA, PESCE AJ, eds Clinical chemistry, theory, analysis, and correlation. St louis:mosby;1984:420-438.
6.
Young DS. Effects of drugs on clinical laboratory tests. Third edition. 1990 :3:6-12.
7.
Zilva JF, pannall PR : plasma enzymes in diagnosis in clinical chemistry in diagnosis and treatment lioydluke london 1979:chap 17 : 338.
Methods Comparison
When run as recommended, the minimum detection limit of this assay is 5.0 U/L.
Linearity
The reaction is linear up to ALT concentration of 400 U/L; specimens showing higher concentration should be diluted 1+5 with physiological saline and repeat the assay (result×6).
Interfering Substances Serum, plasma
Hemolysis Erythrocyte contamination elevates results, since ALT activities in erythrocytes are 3 to 5 times higher than those in normal sera. Icterus No significant interference. Lipemia Lipemic specimens may cause high absorbance flagging. Diluted sample is recommended.
ORDERING INFORMATION CATALOG NO
QUANTITY
292 001 292 002 292 003 292 004 292 005 292 006 292 007 292 008
4 x 20 ml 10 x 10 ml 9 x 20 ml 4 x 60 ml 5 x 20 ml 4 x 50 ml 5 x 100 ml 6 x 100 ml
101
Alanine aminotransferase (ALT/GPT)-Liquizyme (1 + 1) E.C.2.6.1.1. REF: 263 001 REF: 263 002 REF: 263 003
(2 x 25 ml) 50 test (4 x 25 ml) 100 test (2 x 100 ml) 200 test
Intended Use
Spectrum Diagnostics liquizyme ALT reagent is intended for the invitro quantitative, diagnostic determination of ALT in human serum on both automated and manual systems.
Background
The enzyme alanine aminotransferase ALT is widely distributed with high concentrations in the liver and to a lesser extent in kidn- e y s , heart, skeletal muscles, pancreas and lungs. Elevated serum ALT is found in hepatitis, cirrhosis, obstructive jaundice, carcinoma of the liver, and chronic alcohol abuse. ALT is only slightly elevated in patients who have an uncomplicated myocardial infarction. Although both serum aspartate aminotransferase AST and ALT become elevated whenever disease processes affect liver cell integrity, ALT is the more liver specific enzyme. Moreover, elevations of ALT activity persist longer than elevations of AST activity.
Method
Kinetic method according to the International Federation of ClinicalChemistry (IFCC) (3).
Assay Principle
The series of the reaction involved in the assay system is as follows: 1.
The amino group is enzymatically transferred by ALT present in the sample from alanine to the carbon atom of 2-oxoglutarate yielding pyruvate and L-glutamate. L-Alanine + 2-Oxoglutarate
ALT
Pyruvate + L-Glutamate
EC REP Authorised Representative IVD LOT REF
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
≥ 0.06 4 8
mmol/L mmol/L mmol/L
For further information, refer to the Alanine aminotransferase reagent material safety data sheet.
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately. Both reagents (R1) and (R2) contain sodium azide which may react with copper or lead plumbing. All reagents are stable until expiration date stated on label when o stored refrigerated at 2 - 8 C. Working solution can be prepared by adding equal volumes o from R1 and R2 , Stability: 2 days 2 – 8 C.
Deterioration
Do not use liquizyme ALT reagent if it is turbid or if the absorbanceof the working reagent is less than 1.0 at 340 nm. Failure to recover control values within the assigned range may be an indication of reagent deterioration.
Specimen Collection and Handling
Use nonhaemolyzed serum or plasma. Heparin and EDTA are the only acceptable anticoagulants; avoid other anticoagulants. The biological half-life of ALT in serum is 47 hours. o
Stability: 3 days at 15 - 25 C or 7 days at o either 4- 8oC or at - 20 C 2.
Pyruvate is reduced to lactate by LDH present in the reagent with the simultaneous oxidation of NADH to nicotinamide adenine dinucleotide (NAD). The reaction is monitored by measuring the rate of decrease in absorbance at 340 nm due to the oxidation of NADH. Pyruvate + NADH + H+
3.
LDH
L-Lactate + NAD+
Endogenous sample pyruvate is rapidly and completely reduced by LDH during the initial incubation period so that it does not interfere with the assay. Sample pyruvate + NADH + H+
LDH
L-Lactate + NAD+
102
Wavelength 340 nm (334 – 365 nm) Optical path 1 cm Assay type Kinetic Direction decrease Sample: Reagent Ratio 1 : 10 e.g .: Reagent volume 1 ml Sample volume 100 ml o o Temperature 37 C or 30 C Equilibration time 30 seconds. Read time 1 to 3 minutes Zero adjustment Against air Reagent Blank Limits Low 1.00 AU High 2.5 AU Sensitivity 5 U/L Linearity 400 U/L
Working solution Specimen 100 mmol/L 1.4 mol/L ≥ 3500 U/L 0.06 mmol/L
up to 31 U/l up to 41 U/l
(up to 0.52 mKat/L) (up to 0.68 mKat/L)
o
Females males
up to 22 U/l up to 29 U/l
(up to 0.37 mKat/L) (up to 0.48 mKat/L)
known
Within run (Repeatiblity) Level 1
Level 2
n
20
20
Mean (U/L)
103
190
SD
6.1
13
CV%
6
7.4
Level 1
Level 2
20
20
Mean (U/L)
103
190
SD
14.8
16
CV%
14.3
8
Run to run (Reproducibility)
o
Temperature conversion factor is 1.32 (25 o (25 37 C )
30 C ) and 1.85
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Analytical Range 5 – 400 U/L.
Waste Disposal
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References 1.
Breuer J, report on the symposium “drug effects in clinical chemistry methods”. Eur J Clin Chem Clin. 1996;34:385-386.
2.
ECCLS. Determination of the catalytic activity concentration in serum on L- alanine aminotransferase (EC 2.6.1.2,ALAT) Clin chem. 1989;20:204-211.
3.
IFCC expert panel on enzymes part 3. J Clin Chem Clin Biochem 1986;24:481-95.
4.
Henry RJ, et al. Am J clin Path 1960 :34:381.
5.
The reaction is linear up to ALT concentration of 400 U/L; specimens showing higher concentration should be diluted 1+5 with physiological saline and repeat the assay (result×6).
Sherwin JE. Liver function. In:kaplan LA, PESCE AJ, eds. Clinical chemistry, theory,analysis, and correlation. St louis:mosby;1984:420- 438.
6.
Young DS. Effects of drugs on clinical laboratory tests. Third edition. 1990 :3:6-12.
Interfering Substances
7.
Zilva JF, pannall PR : plasma enzymes in diagnosis in clinical chemistry in diagnosis and treatment lioyd-luke london 1979:chap 17 : 338.
Methods Comparison
A comparison between Spectrum Diagnostics ALT (1+1) reagent and a commercial reagent of the same methodology was performed on 20 human sera.A correlation of 0.997 was obtained.
Sensitivity
When run as recommended, the minimum detection limit of this assay is 5.0 U/L.
Linearity
Serum, plasma
Haemolysis Erythrocyte contamination elevates results, since ALT activities in erythrocytes are 3 to 5 times higher than those in normal sera. Icterus No significant interference. Lipemia Lipemic specimens may cause high absorbance flagging. Diluted sample is recommended.
ORDERING INFORMATION CATALOG NO
QUANTITY
263 001 263 002 263 003
2 x 25 ml 4 x 25 ml 2 x 100 ml
Anticoagulants Citrate and fluoride inhibit the enzyme activity.
Procedure Pipette in a test tube:
Reagents Reagent 1 (R1 Buffer / Enzyme) Tris buffer ( pH 7.4) L- Alanine LDH Sodium Azide
System Parameters
of
Performance Characterstics Precision
n
Females males
30 C
Quality Control
Reagent Preparation, Storage and Stability
o
37 C
U/l = 1780 x DA 334 nm /min U/l = 1746 x DA 340 nm /min U/l = 3235 x DA 365 nm /min Normal & abnormal commercial control serum concentrations should be analyzed with each run.
Reagent 2 (R2 Coenzyme) NADH 2-Oxoglutarate Sodium Azide
Expected values
Calculation To calculate the ALT/GPT activity use the following formula
SYMBOLS IN PRODUCT LABELLING
1 ml 100 ml
(Or 0.5 ml R1 + 0.5 ml R2)
Mix, read initial absorbance after 30 seconds. and start timer simultaneously. Read again after 1, 2 and 3 minutes. Determine the mean absorbance change per minute (DA/min).
Drugs Calcium dobesilate and doxycycline HCL cause artificially low ALT values at the tested drug level.
103
Alanine aminotransferase (ALT/GPT) - Colorimetric
SYMBOLS IN PRODUCT LABELLING EC REP Authorised Representative IVD LOT
REF: 264 001 ( 2 x 50 ml ) REF: 264 002 ( 2 x 100 ml )
100 test 200 test
REF
Intended Use
Spectrum Diagnostics ALT reagent is intended for the in-vitro quantitative, diagnostic determination of ALT in human serum.
Background
The enzyme alanine aminotransferase (ALT) is widely distributedwith high concentrations in liver and to a lesser extent in kidneys, heart, skeletal muscles, pancreas and lungs. Elevated serum ALT is found in hepatitis, cirrhosis, obstructive jaundice, carcinoma of the liver, and chronic alcohol abuse. ALT is only slightly elevated in patients who have an uncomplicated myocardial infarction. Although both serum aspartate aminotransferase (AST) and ALT become elevated whenever disease processes affect liver cell integrity, ALT is the more liver specific enzyme. Moreover,elevations of ALT activity persist longer than elevations of AST activity.
Method
Assay Principle
The reaction involved in the assay system is as follows: The amino group is enzymatically transferred by ALT present in the sample from alanine to the carbon atom of 2-oxoglutarate yielding pyruvate and L-glutamate. ALT
Pyruvate + L-Glutamate
ALT activity is measured by monitoring the concentration of pyruvate hydrazone formed with 2,4-dinitrophenylhydrazine.
Reagents Reagent 1 (R1 Buffer) Phosphate buffer DL- Alanine 2 – Oxoglutarate Sodium Azide
100 mmol/L 200 mmol/L 6 mmol/L 12 mmol/L
Reagent 2 (R2) 2,4-dinitrophenylhydrazine 2.0 mmo/L (C)-Corrosive contains caustic materials. R35 Causes severe burns. R41 Risk of serious damage to eyes. S26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. S28 After contact with skin, wash immediately with plenty of soap and water. For further information, refer to the Alanine aminotransferase reagent material safety data sheet.
Precautions and Warnings
Reagent Preparation, Storage and Stability
The reagents are supplied ready-to-use and stable up to the expiry o date labeled on the bottles when stored at 2 – 8 C.
Deterioration
Do not use the ALT regents if precipitate forms. Failure to recover control values within the assigned range may be an indication of reagent deterioration. Specimen Collection and Preservation Use only non haemolyzed serum. The biological half-life of ALT in serum is 47 hours. The only acceptable anticoagulants are heparin and EDTA o Stability: 3 days at 15 - 25 C or 7 days o o at either 4 - 8 C or at -20 C Wavelength 546 nm (530-550 nm) Optical path 1 cm Assay type End-point Direction Increase Sample: Reagent Ratio 1 : 60 o o Temperature 37 C and 20 – 25 C Zero adjustment Reagent or Sample blank Sensitivity 4 U/L Linearity 94 U/L
1. Measurement against Reagent Blank
0.5 ml
0.5 ml o
Mix and incubate for exactly 20 minutes at 20 – 25 C Sodium hydroxide
5.0 ml
5.0 ml
Mix, measure absorbance of specimen against reagent blank at 546 nm after 5 minutes. 2. Measurement against Sample Blank R1(buffer) Sample
Sample blank
Sample
0.5 ml -----
0.5 ml 100 ml
U/L
Absorbance
U/L
0.025 0.050 0.075 0.100 0.125 0.150 0.175 0.200 0.225 0.250
4 8 12 17 21 25 29 34 39 43
0.275 0.300 0.325 0.350 0.375 0.400 0.425 0.450 0.475 0.500
48 52 57 62 67 72 77 83 88 94
Quality Control
Normal & abnormal commercial control serum concentrations should be analyzed with each run.
of
1.
Henry RJ et al. Am J Clin Path 1960 :34:381.
2.
Reitman S and Frankel S . Am . J.Clin.Path., 1975 ;28;65.
3.
Sherwin JE. Liver function. In:kaplan LA, Pesce AJ, eds. Clinical chemistry, theory, analysis, and correlation. St louis:Mosby;1984:420-438.
4.
Young DS. Effects of drugs on clinical laboratory tests. Third edition. 1990 :3:6-12.
ORDERING INFORMATION CATALOG NO
QUANTITY
264 001 264 002
2 x 50 ml 2 x 100 ml
known
Sensitivity
When run as recommended, the sensitivity of this assay is 4 U/L.
Linearity
The assay is linear up to 94 U/L. If the absorbance exceeds 0.5 at 546 nm (ALT 94 U/L) samples should be diluted 1 + 9 using sodium chloride and repeat the assay (result × 10).
Interfering Substances Serum, plasma
Haemolysis Erythrocyte contamination may elevate results, since ALT activities in erythrocytes are three to five times than those in normal sera.
Note High concentration of aldehydes, ketones, or oxo-acids in some sera may cause false high transaminase levels. Measurement aganist a serum blank instead of a reagent blank avoids the risk of finding such artifacts.
Expected Values
Serum : up to 12 U/L. Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Analytical Range 4 – 94 U/L.
o
Mix and incubate for exactly 30 minutes at 37 C
Reagent (R1) contains sodium azide which may react with copper or lead plumbing.
Sodium hydroxide
104
Sample 0.5 ml 100 ml -----o
R2 Sample
Sodium hydroxide 0.4 mol/L.
Reagent blank 0.5 ml -----100 ml
Mix and incubate for exactly 30 minutes at 37 C R2
Absorbance
References
Lipemia Lipemic specimens may cause high absorbance flagging. Diluted sample is recommended.
Pipette into test tubes R1(buffer) Sample Distilled water
The ALT activity in the serum can be determined from the following table:
Icterus No significant interference.
Procedure
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
Additional Reagent
!
System Parameters
ALT – (colorimetric method).
L-Alanine + 2-Oxoglutarate
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use Temperature Limitation
Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by (C) - Corrosive
Calculation
0.5 ml 100 ml
0.5 ml ----o
Mix and incubate for exactly 20 minutes at 20 – 25 C 5.0 ml
5.0 ml
Mix, measure absorbance of specimen against sample blank at 546 nm after 5 minutes.
Waste Disposal
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
105
Alanine aminotransferase
(ALT/GPT) - Ultimate Single Reagent E.C.2.6.1.2. REF: 265 001 REF: 265 002 REF: 265 003 REF: 265 004 REF: 265 005
(2 (6 (2 (4 (2
x 20 ml) x 20 ml) x 100 ml) x 50 ml) x 50 ml)
40 test 120 test 200 test 200 test 100 test
Background
The enzyme alanine aminotransferase ALT is widely distributed with high concentrations in the liver and to a lesser extent in kidneys, heart, skeletal muscles, pancreas and lungs. Elevated serum ALT is found in hepatitis, cirrhosis, obstructive jaundice, carcinoma of the liver, and chronic alcohol abuse. ALT is only slightly elevated in patients who have an uncomplicated myocardial infarction. Although both serum aspartate aminotransferase AST and ALT become elevated whenever disease processes affect liver cell integrity, ALT is the more liver specific enzyme. Moreover, elevations of ALT activity persist longer than elevations of AST activity.
Method
Kinetic method according to the International Federation of Clinical Chemistry (IFCC) (3). The series of the reaction involved in the assay system is as follows: The amino group is enzymatically transferred by ALT present in the sample from alanine to the carbon atom of 2-oxoglutarate yielding pyruvate and L-glutamate.
3.
ALT
LDH
L-Lactate + NAD+
Endogenous sample pyruvate is rapidly and completely reduced by LDH during the initial incubation period so that it does not interfere with the assay. Sample pyruvate + NADH + H+
Reagent (R)
Tris buffer (pH 7.4) L- Alanine LDH Sodium Azide NADH 2 – Oxoglutarate Sodium Azide
LDH
L-Lactate + NAD+
100 800 ≥ 2000 8 ≥ 0.18 18 8
mmol/L mmol/L U/L mmol/L mmol/L mmol/L mmol/L
The reagent also contains additives required to maintain NADH in its reduced forrm.
106
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
Normal & abnormal control serum of known concentrations should be analyzed with each run.
Performance Characterstics Precision
Spectrum Ultimate ALT reagent is supplied ready-to-use and stable up to the expiry date labeled on the bottles. Once opened, the opened vial is stable for 3 months at the specified temperature.
Level 1
Level 2
n
20
20
Mean (U/L)
24.6
105.9
SD
0.93
0.94
CV%
3.78
0.89
Run to run (Reproducibility)
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
Level 1
Level 2
n
20
20
Mean (U/L)
25.2
106
SD
1.1
1.05
CV%
3.9
0.95
The reagent (R) contain sodium azide which may react with copper or lead plumbing.
Deterioration
Do not use Spectrum Ultimate ALT reagent if it is turbid or if the absorbance of the working reagent is less than 0.9 at 340 nm. Failure to recover control values within the assigned range may be an indication of reagent deterioration. Use nonhemolyzed serum or plasma. Heparin and EDTA are the only acceptable anticoagulants; avoid other anticoagulants. The biological half-life of ALT in serum is 47 hours. o Stability: 3 days at 15 - 25 C or 7 days o o at either 4- 8 C or at -20 C Wavelength 340 nm (334 – 365 nm) Optical path 1 cm Assay type Kinetic Direction decrease Sample : Reagent Ratio 1 : 10 e.g .: Reagent volume 1 ml Sample volume 100 ml o o Temperature 37 C or 30 C Equilibration time 60 seconds Read time 180 seconds Zero adjustment Against air Reagent Blank Limits Low 0.9 AU High 2.5 AU Sensitivity 5 U/L Linearity 400 U/L
Procedure: Macro
Semi-Micro
Reagent (R)
1.0 ml
500 ml
Specimen
100 ml
50 ml
Mix, read initial absorbance after 60 seconds. and start timer simultaneously. Read again after 60, 120 and 180 seconds. Determine the mean absorbance change per minute (DA/min).
Calculation
To calculate the ALT/GPT activity use the following formula U/l = 1780 x DA 334 nm /min U/l = 1746 x DA 340 nm /min U/l = 3235 x DA 365 nm /min
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Analytical Range
Within run (Repeatiblity)
Reagent Preparation, Storage and Stability
System Parameters
Pyruvate + L-Glutamate
Pyruvate is reduced to lactate by LDH present in the reagent with the simultaneous oxidation of NADH to nicotinamide adenine dinucleotide (NAD). The reaction is monitored by measuring the rate of decrease in absorbance at 340 nm due to the oxidation of NADH. Pyruvate + NADH + H+
LOT
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Specimen Collection and Preservation
Assay Principle
2.
IVD
Quality Control
For further information, refer to the Alanine aminotransferase reagent material safety data sheet.
Spectrum Diagnostics Ultimate ALT reagent is intended for the invitro quantitative, diagnostic determination of ALT in human serum on both automated and manual systems.
L-Alanine + 2-Oxoglutarate
EC REP Authorised Representative
REF
Intended Use
1.
SYMBOLS IN PRODUCT LABELLING
5 – 400 U/L.
Waste Disposal
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References 1.
Breuer J, report on the symposium “drug effects in clinical chemistry methods”. Eur J Clin Chem Clin Biochem. 1996;34:385-386.
2.
A comparison between Spectrum Diagnostics ALT reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.983 was obtained.
ECCLS. Determination of the catalytic activity concentration in serum on L- alanine aminotransferase (EC 2.6.1.2,ALAT) Clin chem. 1989;20:204-211.
3.
Sensitivity
IFCC expert panel on enzymes part 3. J Clin Chem Clin Biochem 1986;24:481-95 .
4.
Henry RJ, et al. Am J clin Path 1960 :34:381
5.
Sherwin JE. Liver function. In:kaplan LA, PESCE AJ, eds. Clinical chemistry, theory, analysis, and correlation. St louis:mosby;1984:420-438.
6.
Young DS. Effects of drugs on clinical laboratory tests. Third edition. 1990 :3:6-12.
7.
Zilva JF, pannall PR : plasma enzymes in diagnosis in clinical chemistry in diagnosis and treatment lioydluke london 1979:chap 17 : 338.
Methods Comparison
When run as recommended, the minimum detection limit of this assay is 5.0 U/L.
Linearity
The reaction is linear up to ALT concentration of 400 U/L; specimens showing higher concentration should be diluted 1+5 with physiological saline and repeat the assay (result×6).
Interfering Substances Serum, plasma
Hemolysis Erythrocyte contamination elevates results, since ALT activities in erythrocytes are 3 to 5 times higher than those in normal sera. Icterus No significant interference. Lipemia Lipemic specimens may cause high absorbance flagging. Diluted sample is recommended.
ORDERING INFORMATION CATALOG NO 265 001 265 002 265 003 265 004 265 005
QUANTITY 2 6 2 4 2
x 20 x 20 x 100 x 50 x 50
ml ml ml ml ml
Anticoagulants Citrate and fluoride inhibit the enzyme activity. Drugs Calcium dobesilate and doxycycline HCL cause artificially low ALT values at the tested drug level.
Expected values o
Females males
up to 31 U/l up to 41 U/l
(up to 0.52 mKat/L) (up to 0.68 mKat/L)
o
Females males
up to 22 U/l up to 29 U/l
(up to 0.37 mKat/L) (up to 0.48 mKat/L)
37 C 30 C
Temperature conversion factor is 1.32 (25 o 1.85 (25 37 C )
o
30 C) and
107
Glucose-6-Phosphate dehydrogenase (G-6-PDH) REF: 253 001
10 tests
Reagent 1: Assay Reagent Reagent 2 : Substrate Reagent
10 ml 20 ml
SYMBOLS IN PRODUCT LABELLING EC REP Authorised Representative IVD LOT REF
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
Glucose-6-Phosphate-Dehydrogenase (G6PDH) deficiency is one of the most common human enzyme deficiencies in the world. During G6PD deficiency, the red cells are unable to regenerate reduced Nicotineamide adenine dinucleotide phosphate (NADPH), a reaction that is normally catalyzed by the G6PD enzyme. Since the X chromosome carries the gene for G6PD enzyme, this deficiency mostly affects the males. The two major conditions associated with G6PD deficiency are hemolytic anaemias and neonatal jaundice, which may result in neurological complications and death. Screening and detection of G6PD deficiency helps in reducing such episodes, through appropriate selection of treatment, patient counseling and abstinence from disease precipitating drugs such as anti malarials and other agents.
The integrity of erythrocytes collected in ACD is preserved even after prolonged storage so that obtaining accurate red cell counts usually poses no problem. However, red cell counts on specimens collected in heparin become unreliable after about 2 days. Thus, for heparinized samples, results are best reported in terms of hemoglobin concentration. Both copper, which completely inhibits the enzyme at a concentration of 100ìmol/L, and sulfate ions (0.005 mol/L) will decrease observed values of G-6-PDH activity, certain drugs and other substances are known to influence circulating levels of G-6-PDH. Reticulocytes have higher G-6-PDH levels than mature red cells. Therefore it is not recommended that assays be performed after a severe hemolytic crisis, since G-6-PDH levels appear falsely elevated. Under those conditions, detection of deficiency may require family studies. Testing may be more helpful after the level of mature red cells has returned to normal. Under normal circumstances, activity contributed by leucocytes, platelets and serum is relatively small. However, in cases of extreme anemia, grossly elevated white counts or very low levels of red cell G-6-PDH activity, the contribution to the total made under these conditions may be significant. See “Use of Buffy-Coat-Free Samples” section.
Method
Reagent preparation
Assay Principle
System Parameters
Intended Use
Spectrum-Diagnostics G-6-PDH reagent is intended for the in-vitro quantitative UV diagnostic estimation of G-6-PDH in human blood.
Background
G-6-PDH Reagents are supplied ready to use.
UV-Kinetic Method. G6PDH in the RBC’s is released by a lysing agent present in the reagent. The G6PDH released catalyzes the oxidation of Glucose 6 phosphate with the reduction of NADP to NADPH. The rate of reduction of NADP to NADPH is measured as an increase in absorbance which is proportional to the G6PDH activity in the sample. G-6-P +NADP
G-6-PDH
Gluconate -6-P+ NADPH+H
Reagents
Reagent 1 (R1) : Assay Reagent Reagent 2 (R2) : Substrate Reagent G6PDH Control (Vial)
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
Reagent Storage and Stability
o
3.
Read and record absorbance (A) of Test at 340nm vs water or Pottasium Dichromate solution. This is INITIAL A. (if using the water bath or incubator, return the cuvet to it)
4.
Exactly 5 minutes later, again read and record absorbance. This is FINAL A.
5.
To determine G-6-PDH activity, refer to “calculations” section.
CALCULATION A per min =
FINAL A - INITIAL 5
G-6-PDH activity is expressed as U/1012 erythrocytes (RBC) or U/g hemoglobin (Hb). G-6-PDH (U/1012 RBC) = A per min x
48,390 N
N = Red cell count divided by 106 TCF = Temperature correction factor (1 at 30 °C) G-6-PDH (U/g Hb) = A per min x
48390 x TCF Hb(g/dL)
EXAMPLE :
Assay of a specimen which had a red cell count of 4.6 x /mm3 and a hemoglobin concentration 15.2 g/dL resulted in a A per min at 30°C of 0.026. 48,390 = 295 4.6 4839 = 8.9 15.2
Procedure
USE OF BUFFY-COAT-FREE SAMPLE
The temperature of the reaction mixture should be maintained at 30°C or some other constant temperature (see “Temperature Correction” section). Prepare reaction mixture :
b)
Add 0.01ml blood to 1.0 ml of G-6-PDH Assay reagent and mix thoroughly to completely suspend erythrocytes. Let stand at room temperature (18-26°C) for 5-10 minutes.
Sample collection and preparation
c)
Add 2.0ml G-6-PDH Substrate Reagent and mix gently by inverting several times.
d)
Transfer contents to cuvette labeled Test & proceed with Step2.
x TCF
Where :
G-6-PDH(U/1012 RBC) = 0.026 x
1.
108
Place cuvette in constant temperature compartment or water bath and incubate for approximately 5 minutes to obtain thermal equilibrium.
Wavelength 340 nm Optical path 1 cm Assay type UV-Kinetic Direction Increase Sample: Reagent Ratio 1:100 o Temperature 30 C Measurement Against distilled water Delay/Lag/Time 300 sec Interval Time 60 sec NO. OF READINGS 05 Blank Absobance Limit < 0.8 Factor 4839 Low Normal at 30°C 4.6 m/g Hb High Normal at 30°C 13.5 m/g Hb Linearity at 30°C 19.5 m/g Hb
Reagents and standard are ready-to-use. When stored at 2 – 8 C; they are stable up to the expiry date stated on the label. Recontituted G6PDH Assay solution is stable for 8hrs at room temp. (15 - 25 °C) or 5 days refrigerated (2 - 8 °C). Whole blood collected in EDTA, Heparin or ACD is satisfactory. Red cell G-6-PDH is stable in whole blood for one week refrigerated (28°C), but is unstable in red cell hemolysates. Freezing of blood is not recommended. Since activity is reported in terms of number of red cells or grams of hemoglobin. The red cell count or hemoglobin concentration should be determined prior to performing the G-6PDH assay.
2.
G-6-PDH(U/g Hb) = 0.026 x
Note: If A per min is greater than 0.060, repeat determination (3) using 5ìL blood and multiply results by 2
Linearity
The assay is linear up to 19.5 m/g Hb
Expected Values
G6PDH Activity (U/g Hb.): 4.6 -13.5 at 30°C 6.4 -18.7 at 37°C (U/10¹² RBC’s): 146 - 376 at 30°C 202 - 522 at 37°C
Note:
It is recommended for each laboratory to establish and maintain its own reference values. The given data are only an indication. Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Waste Disposal
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References
S.K. Sood et al., The Indian journal of path and micro,, 24 (1981), 89. Lubin, B.H. and Oski, F.A., J. Pediatr. 70 (1967), 788. ORDERING INFORMATION CATALOG NO
QUANTITY
253 001
10 Test
CALIBRATION
The procedure is standardized on the basis of the milimolar absorptivity of NADPH, which is 6.22 at 340nm. The oxidative conversion of G-6-P by G-6-PDH leads to reduction of NADP to NADPH on a molar equivalent basis. Measurement of the rate of increase in absorbance ( A) at340nm serves to quantitate enzymatic activity. The maximum G-6-PDH activity which may be measured by this procedure is approximately 650 U/1012 RBC or 19.5 U/g Hb. Under normal circumstances G-6-PDH activity contributed by leucocytes, platelets and serum is relatively small. However, as reported by Echler and others, more accurate measurement of G-6-PDH activity, specially in the presence of anaemia and / or leucocytosis, can be achieved by using buffy coat-free blood samples for assay. Thus in case of a boderline value obtained with whole blood, it may be warranted to repeat the assay on a buffy coat-free sample.
TEMPERATURE CORRECTION
When temperature of 30°C, no temperature correction factor (TCF) is required in the calculations. If assay is performed at a room temperature other than 30°C, a TCF must be used. When the temperature is 37° C, the TCF is 0.66.
109
Glucose-6-Phosphate
dehydrogenase plus (G-6-PDH plus) REF: 372 001 (10 x1.1 ml) Reagent 1: G6PDH (Co-enzyme-substrate) Reagent 2: G6PDH (Buffer) Reagent 3 : G6PDH (Lysis reagent) REF: 372 002 (20 x1.1 ml) Reagent 1: G6PDH (Co-enzyme-substrate) Reagent 2: G6PDH (Buffer) Reagent 3 : G6PDH (Lysis reagent)
10 tests 10 vials 12 ml 5.5 ml 20 tests 20 vials 24 ml 11 ml
Intended Use
Spectrum-Diagnostics G-6-PDH reagent is intended for the in-vitro quantitative estimation of G-6-PDH in erythrocytes.
Background
Glucose-6-Phosphate-Dehydrogenase (G6PDH) deficiency is one of the most common human enzyme deficiencies in the world. During G6PD deficiency, the red cells are unable to regenerate reduced Nicotineamide adenine dinucleotide phosphate (NADPH), a reaction that is normally catalyzed by the G6PD enzyme. Since the X chromosome carries the gene for G6PD enzyme, this deficiency mostly affects the males. The two major conditions associated with G6PD deficiency are hemolytic anaemias and neonatal jaundice, which may result in neurological complications and death. Screening and detection of G6PD deficiency helps in reducing such episodes, through appropriate selection of treatment, patient counseling and abstinence from disease precipitating drugs such as anti malarials and other agents.
Method
UV-Kinetic Method.
Assay Principle
G6PDH in the RBC’s is released by a lysing agent present in the reagent. The G6PDH released catalyzes the oxidation of Glucose 6 phosphate with the reduction of NADP to NADPH. The rate of reduction of NADP to NADPH is measured as an increase in absorbance which is proportional to the G6PDH activity in the sample. G-6-P +NADP
G-6-PDH
Gluconate -6-P+ NADPH+H
Reagents
Reagent 1 (R1): G6PDH ( Co-enzyme-substrate) Reagent 2 (R2): G6PDH (Buffer) Reagent 3 (R3) : G6PDH (Lysis reagent)
Precautions and Warnings
The Lysing Reagent (G6PDH) (R3) should be used cold (0 - 8 °C) to avoide the decrease in enzyme activity.
Reagent Storage and Stability
G6PDH reagents are stable until the expiry date stated on the label. Reconstituted reagent is stable for 1 week at (2 - 8 °C).
Sample collection and preparation
Fresh whole blood is the specimen required.Collection of blood by using any one of the anticoagulants such as citrate,oxalate or heparin is recommended. Determine the Hemoglobin content of the whole blood and the RBC’s count prior to lysis of the cells.
110
SYMBOLS IN PRODUCT LABELLING EC REP Authorised Representative IVD LOT REF
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Preparation of Red cell hemolysate
Wash 0.1 ml of whole blood with 2 ml aliquots of physiological saline (0.9 %) 3 times and suspend the washed, packed and centrifuged erythrocytes in precooled 0.5 ml of G-6-PDH Lysing reagent (R3), Mix well and keep in the refrigerator (2 - 4 °C) for at least 15 minutes and maximum for 2 hours. Centrifuge the lysate at 3000 r.p.m, for 5 minutes prior to use.
2.G6PDH Activity (U/g Hb) = DA /Min X = DA /Min X Where
EXAMPLE :
Assay of a specimen which had a red cell count of 4.6 x106 /mm3 and a hemoglobin concentration 15.2 g/dL resulted in a DA /Min at 30°C of 0.028. 36013 = 219 4.6 3601 G-6-PDH(U/g Hb) = 0.028 x = 6.83 15.2 G-6-PDH(U/10¹² RBC) = 0.028 x
System Parameters
CALIBRATION
Wavelength 340 nm Optical path 1 cm Assay type Kinetic/ Increasing OD Direction Increase Temperature 30 °C Measurement Against distilled water Delay/Lag/Time 90 sec Interval Time 60 sec NO. of reading 4 sample volume 25 ml (0.025 ml) working reagent volume 1.0 ml Low Normal at 30°C 4.6 m/g Hb High Normal at 30°C 13.5 m/g Hb Linearity at 30°C 19.5 m/g Hb
Procedure Pipette into test tubes Working solution
1.0
ml
Hemolysate
0.025 ml
Add 1ml of working solution to 0.025 ml of hemolysate , mix and aspirate. After the intial delay of 90 seconds,record the absorbance of test at the interval of 1 minute for the next 3 minute at 340 nm. Determine the mean change in absorbance per minute and calculate test results.
CALCULATION
= DA /Min X Where 224 1012 6.22 Nx106 1000
The procedure is standardized on the basis of the milimolar absorptivity of NADPH, which is 6.22 at 340nm. The oxidative conversion of G-6-P by G-6-PDH leads to reduction of NADP to NADPH on a molar equivalent basis. Measurement of the rate of increase in absorbance ( A) at 340nm serves to quantitate enzymatic activity. The maximum G-6-PDH activity which may be measured by this procedure is approximately 650 U/10¹² RBC or 19.5 U/g Hb.
USE OF BUFFY-COAT-FREE SAMPLE
224 x 1012 6.22 x N x 106 x 1000 36013 N
= Total assay volume to sample volume = Factor for expressing activity in 1012 cells = Millimolar absorptivity of NADPH at 340 nm = Number of erythrocytes/cmm = Conversion of cell count from count per cmm to count per ml.
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Waste Disposal
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References 1. 2.
Gross R.T. Hurwitz R.E.Marks P.A. Red cell Glucose-6phosphate dehydrogenase deficiency, J.Clin.Invest (1978) 37.176. Kachmar J.F Moss.D.W,Enzymes,In Fundamentals of clinical chemistry Ed, by N.W. Teitz,Saunders philadelphia 1976 pp 666-672.
ORDERING INFORMATION CATALOG NO
QUANTITY
372 001 372 002
10 x 1.1ml 20 x 1.1ml
Under normal circumstances G-6-PDH activity contributed by leucocytes, platelets and serum is relatively small. However, as reported by Echler and others, more accurate measurement of G-6-PDH activity, specially in the presence of anaemia and / or leucocytosis, can be achieved by using buffy coat-free blood samples for assay. Thus in case of a borderline value obtained with whole blood, it may be warranted to repeat the assay on a buffy coat-free sample.
TEMPERATURE CORRECTION
When temperature of 30°C, no temperature correction factor (TCF) is required in the calculations. If assay is performed at a room temperature other than 30°C, a TCF must be used. When the temperature is 37°C, the TCF is 0.66.
Linearity
The assay is linear up to 19.5 m/g Hb
Expected Values
1. G6PDH Activity (U/10¹² RBC’s): = DA /Min X
3601 Hb (g/dl)
100 = Factor to convert to 100 ml 224 = Total assay volume to sample volume 6.22 = Millimolar absorptivity of NADPH at 340 nm Hb (g/dl) = Hemoglobin concentration
preparation of Reagent
Add 1.1 ml G-6-PDH buffer (R2) to the vial labelled G-6-PDH (R1). Mix well and use after 5 minutes.
224 x 100 6.22 x Hb (g/dl)
G6PDH Activity (U/g Hb.): 4.6 6.4 (U/10¹² RBC’s): 146 202
Note:
-
13.5 at 30°C 18.7 at 37°C 376 at 30°C 522 at 37°C
It is recommended for each laboratory to establish and maintain its own reference values. The given data are only an indication. If the G6PDH activity is very low , measure the absorbance change for 5 minute after addition of buffered substrate and divide by 5 to obtain DA /minute and calculate the test results.
111
Lactate dehydrogenase (LDH)-Liquizyme (4+1) E.C.1.1.1.27. REF: 283 001 REF: 283 002 REF: 283 003 REF: 283 004 REF: 283 005
( 4 x 20 ml) 80 test (10 x 10 ml) 100 test ( 9 x 20 ml) 180 test ( 4 x 60 ml) 240 test ( 5 x 20 ml) 100 test
Intended Use
Spectrum Diagnostics liquizyme LDH reagent is intended for the invitro quantitative, diagnostic determination of LDH in human serum on both automated and manual systems.
Background
The lactate dehydrogenase (LDH) enzyme is widely distributed in heart, liver, muscle, and kidney. LDH catalyzes the conversion of lactate to pyruvate. The enzyme is a tetrameric protein and gives rise to five isoenzymes. Heart, kidney, brain and erythrocytes have the highest proportion of LD-1 and LD-2. Liver and skeletal muscle have highest percentage of LD-5. LDH is significantly increased during myocardial infarction. A maximum value is reached 48 hours after the onset of manifestation and persists up to 10 days .Elevated serum levels of LDH have also been observed in patients with megaloblastic anemia, disseminated carcinoma, leukemia, and trauma. Mild increases in LDH activity has been reported in cases of haemolytic anemia, muscular dystrophy, pulmonary infarction, hepatitis, nephrotic syndrome, and cirrhosis.
SYMBOLS IN PRODUCT LABELLING EC REP Authorised Representative IVD LOT REF
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Or prepare the working solution according to the number of tests required by mixing 4 volumes of reagent 1 (R1) and 1 volume of reagent 2 (R2),e.g. 400 ml R1 +100 ml R2.
Reagent Storage and Stability
All reagents are stable until expiration date stated on label when o stored refrigerated at 2 - 8 C. o Working solution is stable for 2 months at 2 – 8 C or 1 week o at 15 -25 C.
Deterioration
L- Lactate + NAD+
The initial rate of the NADH oxidation is directly proportional to the catalytic LDH activity. It is determined by measuring the decrease in absorbance at 340 nm.
Reagents Reagent 1 (R1 Buffer) Phosphate buffer (pH 7.5) Pyruvate Sodium Azide Reagent 2 (R2 Coenzyme) NADH Sodium azide
50 mmol/L 3.0 mmol/L 8.0 mmol/L > 0.18 mmol/L 8.0 mmol/L
For further information, refer to the Lactate dehydrogenase reagent material safety data sheet.
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately. Both reagents (R1) and (R2) contain sodium azide which may react with copper or lead plumbing.
Reagent Preparation
REF:283 001: add 4 ml from R2 to one bottle of R1; mix gently. REF:283 002: add 2 ml from R2 to one bottle of R1; mix gently. REF:283 003: add 4 ml from R2 to one bottle of R1; mix gently. REF:283004:add 12 ml from R2 to one bottle of R1; mix gently. REF:283 005: add 4 ml from R2 to one bottle of R1; mix gently.
112
Wavelength 340 nm (334 – 365 nm) Optical path 1 cm Assay type Kinetic Direction decrease Sample : Reagent Ratio 1 : 50 e.g.: Reagent volume 1 ml Sample volume 20 ml o Temperature 37 C Equilibration time 30 seconds. Read time 1 to 3 minutes Zero adjustment Against air Reagent Blank Limits Low 1.00 AU High 2.5 AU Sensitivity 10 U/L Linearity 1200 U/L
Procedure Pipette into cuvette
o
(37 C):
Macro
Semi-Micro
Working solution
1 ml
500 ml
Specimen
20 ml
10 ml
Mix, read initial absorbance after 30 secnods. and start timer simultaneously. Read again after 1, 2 and 3 minutes. Determine the mean absorbance change per minute (DA/min).
Calculation
To calculate the LDH activity use the following formula U/L = 8095 x DA 340 nm/min. U/L = 15000 x DA 365 nm/min.
Quality Control
Normal & abnormal control serum of known concentrations should be analyzed with each run.
10 - 1200 U/L.
Level 1
Level 2
n
20
20
Mean (U/L)
433
923
SD
6.8
6.64
CV%
1.57
0.71
Level 1
Level 2
n
20
20
Mean (U/L)
439
935
SD
7.1
6.71
CV%
1.62
0.79
Run to run (Reproducibility)
Specimen Collection and Preservation
System Parameters
LDH
Waste Disposal
Sensitivity
Assay Principle
Pyruvate + NADH + H+
Within run (Repeatiblity)
Methods Comparison
Kinetic ultraviolet method. LDH catalyzes the reaction between pyruvate and NADH to produce NAD+ and L-Lactate:
Analytical Range:
Do not use liquizyme LDH reagent if it is turbid or if the absorbance of the working reagent is less than 1.0 at 340 nm. Failure to recover control values within the assigned range may be an indication of reagent deterioration. Use nonhaemolyzed serum. Heparin is the only acceptable anticoagulant. Sodium citrate and EDTA have an inhibitor effect and must not be used.The biological half-life of LDH in serum is 10 - 54 hours. o o Stability: 6 weeks at 4 – 8 C ; 4 days at 20 – 25 C Freezing of the samples is not recommended.
Method
Performance Characterstics Precision
A comparison between Spectrum Diagnostics LDH reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.967 was obtained. When run as recommended, the minimum detection limit of this assay is 10 U/L.
Linearity
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References 1.
Dito WR. Lactate dehydrogenase: A brief review. In: Griffiths JC, ed. Clinical Enzymology. New York :masson publishing USA; 1979:18
2.
Kachmar JF, Moss DW: Enzymes. In Fundamentals of clinical chemistry. NW Tietz, editor, saunders, philadelphia, 1976 pp 652-6603.
3.
Van der heiden C, B Ais, Gerh Ardt W,Rosallsis. Approved recommendation on IFCC methods for the measurement of catalytic concentration of enzymes. Part 8. IFCC method for LDH.Eur J Clinical Chem Clin Biochem. 1994;32:639-655
4.
young DS.Effects of drugs on clinical laboratory tests. AACC press, Washington D.C., 1990
5.
Zimmerman HJ, henery JB: Clinical enzymology. In: Clinical diagnosis and management by laboratory methods, 16 th., JB Henery, editor, saunders, philadelphia,1979, pp 365-368.
The reaction is linear up to LDH concentration of 1200 U/L; specimens showing higher concentration should be diluted 1+5 with physiological saline and repeat the assay (result×6). ORDERING INFORMATION
Interfering substances Serum, plasma
Haemolysis Erythrocyte contamination elevates results significantly since LDH activities in erythrocytes are 150 times higher than those in normal sera.
CATALOG NO
QUANTITY
283 001 283 002 283 003 283 004 283 005
4 x 20 ml 10 x 10 ml 9 x 20 ml 4 x 60 ml 5 x 20 ml
Icterus No significant interference.
Lipemia
Lipemic specimens may cause high absorbance flagging. Diluted sample may be recommended. Anticoagulants EDTA and citrate may inhibit the reaction.
Expected values (at 37 oC) Adults at
240-480 U/L
(4.0- 8.0 mkat/L)
Children (7-12 Years) Female : Male : Premature :
< 580 U/L < 764 U/L < 1103 U/L
(< 9.65 mkat/L) (< 12.7 mkat/L) (< 18.4 mkat/L)
Calculate for temperature conversion factor of o o 0.5 (37 25 C) and 0.67 (37 30 C).
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure ; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
113
Lactate dehydrogenase (LDH)-Liquizyme (1+1) E.C.1.1.1.27.
EC REP Authorised Representative IVD LOT REF
REF: 279 001 (2 x 25 ml) 50 test REF: 279 002 (4 x 25 ml) 100 test
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Level 1
Level 2
n
20
20
Mean (U/L)
439
935
SD
7.1
6.71
CV%
1.62
0.79
Deterioration
Intended Use
Spectrum Diagnostics liquizyme LDH reagent is intended for the invitro quantitative, diagnostic determination of LDH in human serum on both automated and manual systems.
Do not use liquizyme LDH reagent if it is turbid or if the absorbance of the working reagent is less than 1.0 at 340 nm. Failure to recover control values within the assigned range may be an indication of reagent deterioration.
Methods Comparison
Background
Specimen Collection and Preservation
The lactate dehydrogenase (LDH) enzyme is widely distributed in heart, liver, muscle, and kidney. LDH catalyzes the conversion of lactate to pyruvate. The enzyme is a tetrameric protein and gives rise to five isoenzymes. Heart, kidney, brain and erythrocytes have the highest proportion of LD-1 and LD-2. Liver and skeletal muscle have highest percentage of LD-5. LDH is significantly increased during myocardial infarction. A maximum value is reached 48 hours after the onset of manifestation and persists up to 10 days .Elevated serum levels of LDH have also been observed in patients with megaloblastic anemia, disseminated carcinoma, leukemia, and trauma. Mild increases in LDH activity has been reported in cases of haemolytic anemia, muscular dystrophy, pulmonary infarction, hepatitis, nepherotic syndrome, and cirrhosis.
Method
Kinetic ultraviolet method.
Assay Principle
LDH catalyzes the reaction between pyruvate and NADH to produce NAD and L-Lactate: Pyruvate + NADH + H+
LDH
L- Lactate + NAD+
The initial rate of the NADH oxidation is directly proportional to the catalytic LDH activity. It is determined by measuring the decrease in absorbance at 340 nm.
Reagents Reagent 1 (R1 Buffer) Phosphate buffer (pH 7.5) Pyruvate Sodium Azide Reagent 2 (R2 Enzyme) NADH Sodium azide
50 3.0 8.0
mmol/L mmol/L mmol/L
> 0.06 8.0
mmol/L mmol/L
Use nonhemolyzed serum. Heparin is the only acceptable anticoagulant. Sodium citrate and EDTA have an inhibitor effect and must not be used.The biological half-life of LDH in serum is 10 - 54 hours. o o Stability: 6 weeks at 4 – 8 C ; 4 days at 20 – 25 C Freezing of the samples is not recommended.
System Parameters
Wavelength 340 nm (334 – 365 nm) Optical path 1 cm Assay type Kinetic Direction decrease Sample : Reagent Ratio 1 : 50 e.g.: Reagent volume 1 ml Sample volume 20 ml o Temperature 37 C Equilibration time 30 seconds. Read time 1 to 3 minutes Zero adjustment Against air Reagent Blank Limits Low 1.00 AU High 2.5 AU Sensitivity 10 U/L Linearity 1200 U/L
All reagents are stable until expiration date stated on label when o stored refrigerated at 2 - 8 C. Working solution can be prepared by adding equal volumes from R1 and R2 . o
o
Stability: 2 months at 2 – 8 C or 1 week at 15 – 25 C.
Dito WR. Lactate dehydrogenase :A brief review. In : Griffiths JC, ed. Clinical Enzymology. New York :masson publishing USA; 1979:18.
Sensitivity
2.
Kachmar JF , Moss DW: Enzymes. In Fundamentals of clinical chemistry. NW Tietz, editor, saunders, philadelphia, 1976 pp 652- 6603.
Linearity
3.
Van der heiden C, B AIS, Gerh Ardt W,Rosallsis. Approved recommendation on IFCC methods for the measurement of catalytic concentration of enzymes. Part 8. IFCC method for LDH.Eur J Clinical Chem Clin Biochem . 1994;32:639-655.
4.
young DS. Effects of drugs on clinical laboratory tests. AACC press, Washington D.C., 1990.
5.
Zimmerman HJ, Henery JB : Clinical enzymology. In Clinical diagnosis and management by laboratory methods, 16 th ed., JB henery, editor, saunders, philadeLphia,1979, pp 365-368.
When run as recommended, the minimum detection limit of this assay is 10 U/L. The reaction is linear up to LDH concentration of 1200 U/L; specimens showing higher concentration should be diluted 1+5 with physiological saline and repeat the assay (result×6).
Haemolysis Erythrocyte contamination elevates results significantly since LDH activities in erythrocytes are 150 times higher than those in normal sera. Icterus No significant interference.
ORDERING INFORMATION
Lipemia Lipemic specimens may cause high absorbance flagging.Diluted sample may be recommended.
o
Working solution
1 ml ( or add 0.5 ml R1 + 0.5 ml R2 )
CATALOG NO
QUANTITY
279 001 279 002
2 x 25 ml 4 x 25 ml
Specimen
20 ml
Expected value (at 37 oC) Adults
240-480 U/L
(4.0- 8.0 mkat/L)
Children (7-12 Years)
Mix, read initial absorbance after 30 secnods. and start timer simultaneously. Read again after 1, 2 and 3 minutes. Determine the mean absorbance change per minute (DA/min).
Calculation
Quality Control
Reagent Preparation, Storage and Stability
Reference 1.
Serum, plasma
Pipette into cuvette ( 37 C )
Precautions and Warnings
Both reagents (R1) and (R2) contain sodium azide which may react with copper or lead plumbing.
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
Anticoagulants EDTA and citrate may inhibit the reaction.
To calculate the LDH activity use the following formula U/L = 8095 x DA 340 nm/min.
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
A comparison between Spectrum Diagnostics LDH reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.967 was obtained.
Interfering substances
Procedure
For further information, refer to the Lactate dehydrogenase reagent material safety data sheet.
114
Waste Disposal
Run to run (Reproducibility)
SYMBOLS IN PRODUCT LABELLING
Normal & abnormal control serum of known concentrations should be analyzed with each run.
Performance Characterstics Precision Within run (Repeatiblity) Level 1
Level 2
n
20
20
Mean (U/L)
433
923
SD
6.8
6.64
CV%
1.57
0.71
Female : Male : Premature :
< 580 U/L < 764 U/L < 1103 U/L
(< 9.65 mkat/L) (< 12.7 mkat/L) (< 18.4 mkat/L)
Calculate for temperature conversion factor of o o 0.5 (37 25 C) and 0.67 (37 30 C).
Spectrum diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Analytical Range: 10 - 1200 U/L.
115
LIPASE-LS COLORIMETRIC (DGMRE)
EC REP Authorised Representative IVD LOT
REF:281 001
40 test
REF
Intended use
Spectrum diagnostics Lipase-LS reagent is intended for in-vitro quantitative determination of Lipase in human serum, heparinized or EDTA plasma.
Background
Pancreatic lipase in serum is closely associated with pancreatic diseases. The activity of this enzyme has been measured as an important marker for diagnosing pancreatic diseases and the associated monitoring of therapeutic effects.Pancreatic lipase test kits currently available include a turbidimetric method using triglyceride as substrate and a colorimetric method using synthetic substrates.
METHOD
Lipase catalyzes the following reaction : 1,2-o-Dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin)-ester Lipase /Colipase 1,2-o-Dilauryl-rac-glycerol + Glutaric acid-(6-methylresorufin)-ester Glutaric acid -(6-methylresorufin)-ester
Cleavage
Glutaric acid + Methylresorufin
A synthetic substrate (DGMRE) is split by Lipase to yield the colored final product Methylresorufin. The increasing absorbance of the red Methylresorufin is measured photometrically . The reaction is highly specific on the human enzyme.
REAGENTS Reagents Composition (concentrations in the test): Reagent 1: Goods Buffer pH 8,0 40 mmol/l Taurodesoxycholate 3,4 mmol/l Desoxycholate 2,6 mmol/l Calciumchloride 12 mmol/l Colipase 1 mg/l Reagent 2: Tartrate Buffer pH 4,0 1,5 mmol/l Taurodesoxycholate 3,4 mmol/l DGMRE 0,13 mmol/l Coemulgator
Precautions
- For in vitro diagnostic use only.
Stability
When stored at 2-8° C and protected from light, the reagents are stable up to the expiry date printed on the labels.
Preparation and stability of Working reagents
The reagents are ready to use. Stability : 3 months at 2-8°C, if contamination is avoided
116
3 U/l - 300 U/l.
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
SAMPLES
Serum free of hemolysis, Heparin plasma. Stability : 24 hrs at 15 - 25 °C 5 days at 2 - 8 °C 1 year at -20 °C
This reagent can be used manually (see method below) and on most analyzers. Applications are available on request. Wavelength Cuvette Temperature Measure
Within run n = 40
Mean [U/l]
SD [U/l]
CV [%]
Sample 1 Sample 2 Sample 3
13,4 58,9 103
0,24 0,60 1,50
1,81 1,01 1,45
Mean [U/l]
SD [U/l]
CV [%]
13,4 58,9 103
0,24 0,49 0,65
1,81 0,82 0,63
Sample 1 Sample 2 Sample 3
Correlation
A comparative study has been performed between the Spectrum method and another commercial reagent on 67 human serum samples.
580 nm, Hg 578 nm 1 cm 37 °C Against air Reagent Blank
Sample / Calibrator
------
10 µl
Sample /Calibrator
Precision
Beween run n = 40
PROCEDURE
Colorimetric Test, Kinetic
PRINCIPLE
Analytical range
SYMBOLS IN PRODUCT LABELLING
dist. water
10 µl
------
Reagent 1
500 µl
500 µl
The parameters of linear regression are as follows: y = 0,96 x – 1,15 U/l r = 0,999
INTERFERING SUBSTANCES
- Ascorbic Acid: - Bilirubin: - Hemoglobin: - Triglycerides:
no interference up to 30 mg/dL no interference up to 60 mg/dL no interference up to 500 mg/dL no interference up to 1000 mg/dL
Mix carefully (do not shake), incubate 1 to 5 min, then add R2 to start the reaction :
Refrences
Reagent 2
1.
Lorentz K. Lipase. In: Thomas L, editor. Clinical laboratory diagnostics.1st ed. Frankfurt: TH-Books Verlags-gesellschaft; 1998. p. 95-7.
2.
Moss DW, Henderson AR. Digestive enzymes of pancreatic origin. In: Burtis CA, Ashwood ER, editors. Tietz Textbook of Clinical Chemistry. 3rd ed. Philadelphia: W.B Saunders Company; 1999. p. 689-708.
3.
Tietz N, Shuey DF. Lipase in serum – the elusive enzyme: an overview. Clin Chem 1993;39:746-56.
4.
Lott J, Patel ST, Sawhney AK, Kazmierczak SC, Love JE. Assays of serum lipase: analytical and clinical considerations. Clin Chem 1986;32:1290-1302.
5.
Note: It is recommended for each laboratory to establish and maintain its own reference values. The given data are only an indication.
Leybold A, Junge W. Importance of colipase for the measurement of serum lipase activity. Adv Clin Enzymol 1986;4:60-7.
6.
Borgström B. The action of bile salts and other detergents on pancreatic lipase and the interaction with colipase. Biochimica et Biophysika Acta 1977;488:381-91.
CALIBRATOR & CONTROLS
7.
Gargouri Y, Julien R, Bois A, Verger R, Sarda L. Studies on the detergent inhibition of pancreatic lipase activity. J of Lipid Research 1983;24:1336-42.
125 µl
125 µl
Mix carefully (do not shake), incubate for 2 min at 37 °C, read absorbance and start stopwatch. After 1 min and after 2 min read absorbance again.
CALCULATION DA/min = [ A/minSample / Calibrator ] – [ A/minReagent Blank ] Lipase (U/l) =
DA/minSample DA/minCalibrator
Expected Values
6 kU/L > 2 kU/L 5.3 mmol/L 0.18 mmol/L
Precautions and Warnings
Prepare R1 Mixture as following: REF: 221 001:add 2 ml from R1b to one bottle of R1a;mix gently Or prepare the working solution according to the number of tests required by mixing 9 volumes of reagent 1a (R1a) and 1volume of reagent 1b (R1b); eg. 900 ml R1a +100 ml R1b.
Reagent Storage and Stability
All reagents are stable until expiration date stated on label when o stored refrigerated at 2 - 8 C. o R1 Mixture is stable for 1 month at 2– 8 C.
Calculation
Sodium cefoxitin causes artificially high ammonia values at the tested drug level.
A1 – A2 = DAspecimen or DAstandard .
18
mmol/L
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
Ammonia (mg/dl) =
Do not use Ammonia II reagent if it is turbid or if the absorbance of the working reagent is less than 1.0 at 340 nm. Failure to recover control values within the assigned range may be an indication of reagent deterioration. EDTA is the only acceptable anticoagulant because it reduces red cell ammonia production. Other anticoagulants produce sponteniously high results. Collect blood from stasis-free vein of fasting patient. Smoking should be avoided prior to sample. Tubes should be filled completely and kept tightly stoppered at all times. Place immediately o on ice and centrifuge, preferrable at 4 C. Perform analysis within 30 minutes of venipuncture. Note: avoid contamination of samples by ammonia from smoking or traffic in laboratory or patient’s room, glassware, or water. One known source of spontaneous ammonia formation is an increased ∝-glutamyl-transferase activity leading to decomposition of glutamine. o o Stability: 15 minutes. at 15 – 25 C; 2 hours at 4 – 8 C; o 3 weeks at -20 C
System Parameters
Wavelength 340 nm Optical path 1 cm Assay type Fixed Rate Direction Decrease First read time 2 seconds Delay time 300 seconds last read time 5 Minutes o Temperature 37 C Zero adjustment Reagent blank Reagent Blank Limits Low 1.00 AU High 2.0 AU Sensitivity 9 mg/dL (5.3 mmol/L) Linearity 1700 mg/dL (1000 mmoL/L)
Procedure Specimen
1ml
1ml
Reagent blank 1ml
Standard
100 ml
-----
-----
Specimen
-----
100 ml
-----
Dist. H2O
-----
-----
100 ml
Drugs
EDTA plasma Adults Females 19- 87 mg/dL Males 27-102 mg/dL Children < 81.5 mg/dL Neonates (1- 6 days)
x 521
Quality Control
Normal & abnormal commercial control of known concentrations should be analyzed with each run.
Performance Characteristics Precision
(11-51 mmol/L) (16-60 mmol/L) ( < 48 mmol/L) < 228 mg/dL (< 134 mmol/L)
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Analytical Range Level 1
Level 2
n
20
20
Mean (mg/dL)
1.8
3.5
SD
0.04
0.06
CV%
2.3
1.3
Level 1
Level 2
Specimen Collection and Preservation
R1 mixture
DAspecimen DAstandard
Fluoride, citrate, and heparin must not be used.
Expected Values
Within run (Repeatiblity)
Deterioration
Standard
For further information, refer to the Ammonia reagent material safety data sheet.
120
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Anticoagulants
Concentration of ammonia in Plasma:
Intended Use
NH4+ + ∝-KG + NADH
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
Mix, and immediately read the absorbance A1 of the standard or specimen against reagent blank. Exactly 5 minutes. later, read absorbance A2 of standard or specimen.
run to run (Reproducibility)
9 – 1700 mg/dL.
Waste Disposal
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
n
20
20
References
Mean (mg/dL)
1.8
3.5
1.
SD
0.07
0.14
Burtis CA, Ashwood ER, eds. Tietz fundamentals of Clinical Chemistry. 4 th ed. Philadelphia: WB saunders: 1996:755.
CV%
3.4
4.1
2.
Dewan JG: the L[+]-glutamic dehydrogenase of animal tissue Biochem J 32”1378,1938.
3.
A comparison between Spectrum Diagnostics Ammonia reagent and a commercial reagent of the same methodology was performed on 20 human plasma. A correlation of 0.976 was obtained.
Diamond EG: Inhibitory efect of heparin upon adenylic deaminase. J lab Clin Med 46:807,1955.
4.
Howanitz JH, Howanitz PJ, Skrodzki CA, Iwanski JA. Influences of specimen processing and storage condition on results for plasma ammonia. Clin Chem. 1984;30:906-908.
Sensitivity
5.
Olson JA, anfinsen CB: kinetic and equilibrium studies on crystalline L-glutamate acid dehydrogenase. J Biol Chem 202:841, 1953
Linearity
6.
Vananken HC, Scphorst ME. A kinetic determination of ammonia in plasma. Clin Chem Acta.1974;56:151-157.
7.
Young DS:et al. Clin Chem. 1975; 21.
Methods Comparison
When run as recommended, the minimum detection limit of this assay is 9.0 mg/dL.
The reaction is linear up to ammonia concentration of 1700 mg/dL.
Interfering Substances plasma
Haemolysis Avoid haemolyzed specimen since RBCs contain three times the ammonia content of plasma. Icterus Bilirubin levels higher than 30 mg/dL increase the ammonia concentration significantly.
ORDERING INFORMATION CATALOG NO
QUANTITY
333 001
100 Tests
∝-globulin Elevated ∝-globulin levels (more than 3 g/dL) may increase the apparent ammonia concentration values.
o
Incubate for 3 minutes at 37 C, Then add R2
100 ml
100 ml
100 ml
Lipemia
Lipemic samples should be centrifuged and the analysis performed on the supernatent.
121
Ammonia – Liquizyme Single Reagent REF: 220 001 REF: 220 002
EC REP Authorised Representative IVD LOT
(2 x 20 ml) (5 x 20 ml)
REF
Intended Use
Spectrum Diagnostics liquizyme ammonia single reagent is intended for the in-vitro quantitative, diagnostic determination of ammonia in human plasma on both automated and manual systems.
Background
Ammonia enters the body in nitrogen-containing foods via the gastrointestinal tract and is excreted largely as urea in urine and as bacterial protein in feces. Ammonia, the end product of nitrogen metabolism is absorbed into the portal venous blood and, after passing through the liver enters the systemic circulation. Normally about half the ammonia is extracted from the body by the skeletal muscle and about 16 % by the liver and brain. Clinically, the extraction of ammonia by individual organs has different implications. The hepatic conversion of ammonia to urea represents the primary mechanism of eliminating ammonia from the body. Conversely, the excessive uptake of ammonia by the brain results in ammonia intoxication, increased intracranial pressure and hepatic encephalopathy. Hyperammonemia in infants may be due to inherited deficiencies of the urea cycle enzymes or acquired through acute (as in Reye’s syndrome) or chronic (as in cirrhosis) liver disease.
Method
Kinetic enzymatic method with glutamate dehydrogenase.
Assay Principle
∝ – ketoglutarate reacts with ammonium ions in presence of glutamate dehydrogenase and the coenzyme NADPH to produce L-glutamate and NADP+ NH4 + ∝-KG + NADPH +
GLDH
Calculation
SYMBOLS IN PRODUCT LABELLING
L-glutamate + NADP+ + H2O
The concentration of The NADP+ formed is directly proportional to the ammonia concentration. It is determined by measuring the decrease in absorbance at 340 nm.
Reagents
Standard ammonia (ST) 521 mg/dL
307 mmol/L
Reagent (R) Bicine buffer (pH 8.5) ∝ – ketoglutarate Sodium Azide GLDH (microbial) NADPH Sodium Azide
100 mmol/L 7.5 mmol/L 0.05% 500 Ku/L 0.2 mmol/L 8 mmol/L
For further information, refer to the Ammonia reagent material safety data sheet.
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately. The reagent (R) contain sodium azide which may react with copper or lead plumbing.
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
Aspecimen Final = Aspecimen – Aspecimen blank.
Aspecimen Final Ammonia (mg/dl) = x 521 Astandard
Deterioration
Quality Control
Do not use liquizyme Ammonia reagent if it is turbid or if the absorbance of the working reagent is less than 1.0 at 340 nm. Failure to recover control values within the assigned range may be an indication of reagent deterioration.
Specimen Collection and Preservation
EDTA is the only acceptable anticoagulant because it reduces red cell ammonia production. Other anticoagulants produce sponteniously high results. Collect blood from stasis-free vein of fasting patient. Smoking should be avoided prior to sample. Tubes should be filled completely and kept tightly stoppered at all times. Place immediately o on ice and centrifuge, preferrable at 4 C. Perform analysis within 30 minutes of venipuncture. Note: avoid contamination of samples by ammonia from smoking or traffic in laboratory or patient’s room, glassware, or water. One known source of spontaneous ammonia formation is an increased ∝-glutamyl-transferase activity leading to decomposition of glutamine. o o Stability: 15 minutes. at 15 – 25 C; 2 hours at 4 – 8 C; o 3 weeks at -20 C
System Parameters
Wavelength 340 nm Optical path 1 cm Assay type Fixed Rate Direction Decrease Sample : Reagent Ratio 1 : 10 e.g.: Reagent volume 1 ml Sample volume 100 ml First read time 30 seconds Delay time 150 seconds last read time 180 seconds o Temperature 37 C Zero adjustment Against Air Reagent Blank Limits Low 1.00 AU High 2.0 AU Sensitivity 9 mg/dL (5.3 mmol/L) Linearity 1700 mg/dL (1000 mmoL/L)
Procedure Reagent blank
Standard
Specimen
Reagent (R)
1.0 ml
1.0 ml
1.0 ml
Standard
---------
100 ml
---------
Specimen
---------
---------
100 ml
Mix, and after 30 seconds. read the absorbance A1 of the standard or specimen or specimen blank. Exactly 2.5 minutes. later, read absorbance A2 of standard or specimen or specimen blank. o
*Note: It is recommended to Incubate reagent at 37 C for 3 minutes. then add 100 ml of the serum or standard to each 1 ml and complete the procedure as above.
Anticoagulants Fluoride, citrate, and heparin must not be used. Drugs Sodium cefoxitin causes artificially high ammonia values at the tested drug level.
Concentration of ammonia in serum:
All reagents are stable until expiration date stated on label when o stored refrigerated at 2 - 8 C.
Reagent Storage and Stability
Lipemia Lipemic samples should be centrifuged and the analysis performed on the supernatent.
A1 – A2 = Aspecimen or Astandard or Aspecimen blank.
Expected Values
Normal & abnormal commercial control serum concentrations should be analyzed with each run.
of
known
Performance Characteristics Precision Within run (Repeatiblity) Level 1
Level 2
n
20
20
Mean (mg/dL)
1.8
3.5
SD
0.04
0.06
CV%
2.3
1.3
run to run (Reproducibility) n
Level 1
Level 2
20
20
Mean (mg/dL)
1.8
3.5
SD
0.07
0.14
CV%
3.4
4.1
Methods Comparison
A comparison between Spectrum Diagnostics Ammonia single reagent and a commercial reagent of the same methodology was performed on 20 human sera. A correlation of 0.978 was obtained.
Sensitivity
When run as recommended, the minimum detection limit of this assay is 9.0 mg/dL.
EDTA plasma Adults Females 19- 87 mg/dL Males 27-102 mg/dL Children < 81.5 mg/dL Neonates (1- 6 days)
Spectrum Diagnostics does not interpret the results of a clinical laboratory procedure; interpretation of the results is considered the responsibility of qualified medical personnel. All indications of clinical significance are supported by literature references.
Analytical Range 9 – 1700 mg/dL.
Waste Disposal
This product is made to be used in professional laboratories. Please consult local regulations for a correct waste disposal. S56: dispose of this material and its container at hazardous or special waste collection point. S57: use appropriate container to avoid environmental contamination. S61: avoid release in environment. refer to special instructions/ safety data sheets.
References 1.
Burtis CA, Ashwood ER, eds. Tietz fundamentals of Clinical Chemistry. 4 th ed. Philadelphia: WB saunders: 1996:755.
2.
Dewan JG: the L[+]-glutamic dehydrogenase of animal tissue. Biochem J 32”1378,1938.
3.
Diamond EG: Inhibitory efect of heparin upon adenylic deaminase. J lab Clin Med 46:807,1955.
4.
Howanitz JH, Howanitz PJ, Skrodzki CA, Iwanski JA. Influences of specimen processing and storage condition on results for plasma ammonia. Clin Chem. 1984;30:906-908.
5.
Olson JA, anfinsen CB: kinetic and equilibrium studies on crystalline L-glutamate acid dehydrogenase. J Biol Chem 202:841, 1953
6.
Vananken HC, Scphorst ME. A kinetic determination of ammonia in plasma. Clin Chem Acta.1974;56:151-157.
7.
Young DS:et al. Clin Chem. 1975; 21.
Linearity
The reaction is linear up to ammonia concentration of 1700 mg/dL.
Interfering Substances plasma Haemolysis Avoid haemolyzed specimen since RBCs contain three times the ammonia content of plasma. Icterus Bilirubin levels higher than 30 mg/dL increase the ammonia concentration significantly.
∝-globulin
Elevated µ-globulin levels (more than 3 g/dL) may increase the apparent ammonia concentration values.
(11-51 mmol/L) (16-60 mmol/L) ( < 48 mmol/L) < 228 mg/dL (< 134 mmol/L)
ORDERING INFORMATION CATALOG NO
QUANTITY
220 001 220 002
2 x 20 ml 5 x 20 ml
Reagent preparation
Spectrum Ammonia single reagent is supplied ready-to-use and stable up to the expiry date labeled on the bottles Once opened, the opened vial is stable for 3 months at the specified temperature.
122
123
Calcium O-CPC
o
SYMBOLS IN PRODUCT LABELLING EC REP Authorised Representative IVD LOT
REF: 226 001 REF: 226 002 REF: 226 003 REF: 226 004
(2 x 30 ml) 60 test (2 x100 ml) 200 test (4 x100 ml) 400 test (2 x 50 ml) 100 test
REF
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use Temperature Limitation
!
Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by (Xi) - Irritant
Reagent Preparation, Storage and Stability
Spectrum Diagnostics calcium reagent is intended for the in-vitro quantitative, diagnostic determination of calcium in human serum on both automated and manual systems.
Spectrum Calcium reagents are supplied ready-to-use and stable up to the expiry date labeled on the bottles when stored sealed at o 15 – 25 C.
Background
Deterioration
Method
O-cresolphthaline complexone colorimetric method.
Assay Principle
Calcium ions react with O-cresolphthalein complexone (O-CPC) under alkaline conditions to form a violet colored complex. magnesium and iron. Ca2+ + O-CPC
Alkaline pH
calcium-O-CPC complex
The color intensity of the complex formed is directly proportional to the calcium concentration. It is determined by measuring the increase in absorbance at 578 nm.
Reagents Standard Calcium (ST) 10 mg/dL Reagent 1 (R1 Buffer) 2-Amino-2-methyl-1-propanol (pH 10.5)
0.3 mol / L
Reagent 2 (R2 Chromogen) O-cresolphthalein complexone 8-hydroxyquinoline
0.16 mmol/L 7.0 mmol/L
Irritant (Xi) R20/21/22 Harmful by inhalation, in contact with skin and if swallowed. R38 Irritating to skin. R41 Risk of serious damage to eyes. S24/25 Avoid contact with skin and eyes. S26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. For further information, refer to the Calcium reagent material safety data sheet.
Precautions and Warnings
Do not ingest or inhalate. In case of contact with eyes or skin; rinse immediately with plenty of soap and water. In case of severe injuries; seek medical advice immediately.
124
Do not use the Spectrum Calcium reagents if turbid. Failure to recover control values within the assigned range may be an indication of reagent deterioration.
Specimen Collection and Preservation Serum and plasma
Use nonhemolyzed serum.Heparin is the only acceptable anticoagulant. No other anticoagulant can be used. Fresh serum collected in the fasting state is the perferred specimen. Serum or plasma should be separated from cells as soon as possible, because prolonged contact with the clot may cause lower calcium values. Sera from patients receiving EDTA (treatment of hypercalcemia) are unsuitable for analysis, since EDTA will chelate the calcium and render it unavailable for reaction with O-cresolphthalein complexone. The biological half-life of calcium in blood is few hours. Urine Specimens should be collected in acid washed bottles. 24 hour Specimens should be collected in containers containing 5 ml of 6 mol/L HCl. If the specimen is collected without acid, the pH should be adjusted < 3 with 6 mol/L HCl. Dilute urine specimen 2 times with bidistilled water (1volume urine + 1volume distilled water) before assay. o
(Aspecimen) (Astandard)
x 10
x10 x10*x 2**x V***
* The factor “10” converts mg/dl to mg/litre ** The factor “2” represents the dilution factor *** “V” represents the 24-hour urine volume in litres
o
Stability (urine): 2 days at 15 – 25 oC; 4 days at 4 – 8 C; o 3 weeks at -20 C
System Parameters
Wavelength Optical path Assay type Direction Increase Sample : Reagent Ratio e.g.: Reagent volume Sample volume Temperature Zero adjustment Reagent Blank Limits Sensitivity Linearity
Normal & abnormal control serum of known concentrations should be analyzed with each run.
Performance Characterstics Precision
Level 1
Level 2
n
20
20
Mean (mg/dL)
9.58
13.97
SD
0.12
0.207
CV%
1.33
1.48
Level 1
Level 2
Run to run (Reproducibility) n
20
20
Mean (mg/dL)
9.62
14.15
SD
0.23
0.221
8.8-10.2 mg/dl 8.4- 9.7 mg/dl
(2.20-2.55 mmol/L) (2.09-2.42 mmol/L)
Children 4 -18years >4 weeks
9.2-11.0 mg/dl 7.2-11.2 mg/dl
(2.30-2.75 mmol/L) (1.80-2.8 mmol/L)
Urine (24 h) Females Males Childern
99% of the time.
BIBLIOGRAPHY
1. Janssen RS, Satten,GA, Stramer SL, Rawal BD, O’Brien TR, Weiblen BJ, Hecht FM, Jack N, Cleghorn FR, Kahn JO, Chesney MA and Busch MP. New testing strategy to detect early HIV-1 infection for use in incidence estimates and for clinical and prevention purposes. JAMA (1998) 280(1):42-48. 2. Chang SY, Bowman BH, Weiss JB, Garcia RE and White TJ. The Origin of HIV-1 isolate HTLVIIIB. Nature (1993) 3:363:4669. 3. Greenberg AE, Wiktor SZ, DeCock KM, Smith P, Jaffe HW and Dondero TJ Jr. HIV-2 and natural protection against HIV-1 infection. Science (1996) 272:1959-1960. 4. Travers K, Mboup S, Marlink R, Gueye-Nidaye A, Siby T, Thior I, Traore I, Dieng-Sarr A, Sankale JL and Mullins C. Natural protection against HIV-1 infection provided by HIV-2. Science (1995) 268:1612-1615. 5. Arya SK, Beaver B, Jagodzinski L, Ensoli B, Kanki PJ,Albert J, Fenyo EM, Biberfeld G, Zagury JF and Laure F. New human and simian HIV-related retroviruses possess functional transactivator (tat) gene. Nature (1987) 328:548-550. 6. Caetano JA. Immunologic aspects of HIV infection. Acta Med Port (1991) 4 Suppl 1:52S-58S. ORDERING INFORMATION CATALOG NO.
QUANTITY
1168 001 1168 002
25 test 50 test
289
Malaria Antigen Test (Whole Blood) For Plasmodium falciparum & vivax
MATERIALS:
Note
Materials Provided: Test Device Assay Buffer Sample Dropper
DIRECTIONS FOR USE
Optimal assay performance requires strict adherence to the assay procedure described in this instruction sheet and any deviations from the procedure may lead to aberrant results.
1. REF: 1150 001 REF: 1150 002
25 Card Test 50 Card Test
INTENDED USE For the rapid qualitative determination of Malaria P.falciprum & P. vivax specific lactate dehydrogenase (pLDH) in human blood for the diagnosis of Malaria infection.
Background Malaria is a serious parasitic disease characterized by fever, chills and anemia and is caused by a parasite that is transmitted from one human to another by the bite of infected Anopheles mosquitoes. There are four kinds of malaria that can infect humans: Plasmodium falciparum, P. vivax, P. Ovale, and P. Malaria. In humans, the parasites (called sporozoites) migrate to the liver where they mature and release another form, the merozoites. The disease now occurs in more than 90 countries worldwide, and it is estimated that there are over 500 million clinical cases and 2.7 Million malaria-caused deaths per year. At present, malaria is diagnosed by looking for the parasites in a drop of blood. Blood will be put into a Microscope slide and stained so that the parasites will be visible under a Microscope.
Assay Principle Spectrum Malaria Antigen Test contains a membrane strip, which is pre-coated with two monoclonal antibodies as two separate lines across a test strip. One monoclonal antibody (test line 1) is specific to the P. vivax (pLDH) and another monoclonal antibody (test line 2) is specific to (pLDH) of P. Ovale. Conjugate pad is dispensed with monoclonal antibodies conjugated to the colloidal gold, which are specific to P. vivax & P. Ovale (pLDH).
Materials Not Provided:
Add 5μl of whole blood into sample well (“S” small well) (Do not use excess blood). Add two drops or 60-80 μLs of assay buffer into developer well (“A”). Read the test result at 20 mins.
5μl Pipette
2.
SPECIMEN COLLECTION & PREPARATION
3.
(Collection by venipuncture) 1. Collect whole blood into a collection tube containing EDTA, citrate or heparin) by venipuncture. 2. If specimens are not immediately tested, they should be refrigerated at 2~8°C. For storage periods greater than three days, freezing is recommended. They should be brought to room temperature prior to use. Using the specimen after longterm storage more than three days can cause non-specific reaction. 3. When stored at 2~8°C, the whole blood sample should be used within three days.
INTERPRETATION OF RESULTS 1.
P. falciparum Positive reaction The presence of two color bands (C and 1) indicates a positive result for P.falciparum.
2.
P.vivax Positive reaction The presence of two color bands (C and 2) indicates a positive result for P. vivax. The pLDH present in the sample reacts with the pan anti-pLDH conjugate and moves through the test strip where the pLDH is captured by Ovale specific anti-pLDH.
3. (Collection using a lancet) 1. Clean the area to be lanced with an alcohol swab. 2. Squeeze the end of the fingertip and pierce with a sterile lancet provided. 3. Wipe away the first drop of blood with sterile gauze or cotton. 4. Take a sample pipette provided, and while gently squeezing the tube, immerse the open end in the blood drop and then gently release the pressure to draw blood into the sample pipette up to the black line. 1) Gently squeeze the tube
2) Immerse open end in blood
4.
Negative reaction The presence of only one band within the result window indicates a negative result. Invalid The test is invalid if the C line does not appear. If this occurs, the test should be repeated using a new cassette.
References 1.
Leonard K. Basco, Frederique Marquet, Michael M. Makler, and Jacques Le Bras : Plasmodium falciparum and Plasmodium vivax: Lactate Dehydrogenase Activity and its Application for in vitro Drug Susceptibility Assay. Experimental Parasitology 80, 260-271 (1995)
2.
David L. Vander, Jagt, Lucy A. Hunsaker and John E. Heidrich: Partial Purification and Characterisation of Lactate Dehydrogenase from Plasmodium falciparum. Molecular and Biochemical Parasitology, 4 (1981) 255-264.
3.
David J. Bzik, Barbara A, Fox and Kenneth Gonyer : Expression of Plasmodium falciparum Lactate Dehydrogenase in Escherichia coli Molecular and Biochemical Parasitology, 59 (1993) 155-166
4.
Cameron R. Dunn, Mark J. Banfield, John J. Barker, Christopher W. Highm, Kathleen M. Moreton, Dilek TurgutBalik, R. Leo Brady and J. John Holbrook. The Structure of Lactate Dehydrogenase from Plasmodium falciparum reveals a new target for anti-malarial design. Nature Structural Biology 3(11) 1996, 912-915
5.
Howard, RJ, et al. The secretion of a Malaria Histidine-rich Protein (Pf HRP-2) from Plasmodium falciparum-infected Erythrocytes. J. Cell Biol., (1986) 103, 1269-1277
6.
Rock, EP, et al. Comparative Analysis of Plasmodium falciparum Histidine-Rich Protein - 2, HRP-I, HRP-II, and HRPIII in Malaria Parasitology Diverse Origin. Parasitol., (1987) 95:209-27.
7.
Panna, ME, et al. Identification of Plasmodium falciparum Histidine-Rich Protein - 2 in the of Humans with Malaria. J. Clin. Microbiological 29:1629-1634.
8.
Rodriguez-del Valle, M. et al, Detecting Antigens and Antibodies in the Urine with Plasmodium falciparum Malaria. Slit Microbiol., (1991) 29:1236-1242
3) Gently release to draw blood
LIMITATIONS The Spectrum Malaria Antigen Test is designed for the differential diagnosis between Plasmodium falciparum and Plasmodium Vivax.
1. 2.
Precautions and Warnings For professional in vitro diagnostic use only. Do not use after expiration date. l Do not eat, drink or smoke in the area where the specimens or kits are handled . l Handle all specimens as if they contain infectious agents. Observe established precautions against microbiological hazards throughout testing and follow the standard procedures for proper disposal of specimens. Wear protective clothing such as laboratory coats, disposable gloves and eye protection when specimens are being tested. Humidity and temperature can adversely affect results.
STORAGE & STABILITY
3.
Directions for use: Add whole blood (5 ml) Into sample well “S”
Add 2 drops or 60-80 ml of assay buffer to well “A”
Read Result in 20 mins.
4. 5.
The test procedure, precautions and interpretation of results for this test must be followed when testing. Anti-coagulants. heparin, EDTA, and citrate do no affect the test result This test kit detects Plasmodium lactate dehydrogenase in patient whole blood and is useful as a screening procedure for malaria diagnosis. Do not mix reagent of different lots. The test is limited to the detection of antigen of Malaria Plasmodium vivax & ovale. Although the test is very accurate in detecting pLDH, a low incidence of false results can occur. Other clinically available tests. are required if questionable results are obtained. As with all diagnostic tests, a definitive clinical diagnosis should not be based on the results of a single test, but should only be made by the physician after all clinical and laboratory findings have been evaluated
The kit can be stored at room temperature or refrigerated (4-30°C). The test device is stable through the expiration date printed on the sealed pouch. The test device must remain in the sealed pouch until use. DO NOT FREEZE. Do not use beyond the expiration date.
290
291
S.typhi-S.paratyphi “A” Direct Antigen Detection REF: 1154 001 REF: 1154 002
25 Card Test 50 Card Test
Reagents The test cassette contains anti-salmonella conjugated to gold particles and anti-salmonella coated on the membrane. Material Provided: Test Cassette, Droppers, 25 ml Phosphate Buffer Saline and Instruction for Use
2.
Storage and Stability
Introduction Typhoid fever is a life threatening illness caused by the bacterium Salmonella typhi, and was observed by Eberth (1880) in the mesenteric nodes and spleen of fatal cases of typhoid fever. It is common in developing countries where it affects about 12.5 million persons annually. The infection is acquired typically by ingestion. On reaching the gut, the bacilli attach themselves to the epithelial cells of the intestinal villi and penetrate to the lamina and submucosa. They are then phagocytosed there by polymorphs and macrophages. The ability to resist intracellular killing and to multiply within these cells is a measure of their virulence. They enter the mesenteric lymph nodes, where they multiply and, via the thoracic duct, enter the blood stream. A transient bacteremia follows, during which the bacilli are seeded in the liver, gall bladder, spleen, bone marrow, lymph nodes and kidneys, where further multiplication takes place. Towards the end of the incubation period, there occurs a massive bacteremia from these sites, heralding the onset of the clinical symptoms. The diagnosis of typhoid consists of isolation of the bacilli and the demonstration of antibodies. The isolation of the bacilli is very time consuming and antibody detection is not very specific. Other tests include the Widal reaction. has developed a test that takes only 1020 minutes and requires only a small quantity of stool or one drop of serum* to perform. It is the easiest and most specific method for detecting S.typhi-S.paratyphi infection.
Principle Spectrum-Diagnostics S.typhi-S.paratyphi rapid test is a qualitative one step immunochromatographic assay. The test employs a conation of monoclonal antibody/colloidal gold dye conjugate and a polyclonal antibody immobilized on the solid phase. This will selectively identify S.typhi-S.paratyphi antigen associated with typhoid infection with a high degree of sensitivity and specificity. As the specimen flows through the absorbent pad in the sample well and through the antibody/colloidal gold complex any S.typhi-S. paratyphi antigen present in the sample binds to the conjugate forming an antigen/antibody complex. The sample and dye complex continue to migrate along the membrane to the immobilized monoclonal antibody. In the presence of S.typhi-S.paratyphi, the monoclonal antibody captures the complex. This forms a visible pink/purple band in the (B) or test area of the card. If no antigen is present, there is no line formation in the (B) area. The remaining complex continues to migrate to another immobilized antibody on the membrane in the (C) or Control area of the card, and is captured which then forms a band indicating proper performance of the test.
292
Spectrum-Diagnostics S.typhi-S.paratyphi test kit is stable at any room temperature between 8 – 30 °C when in the unopened pouches. DO NOT FREEZE the kit or expose to temperature extremes. Stability of the kit is 24 months from the date of manufacture – dating is indicated on the kit label.
Precautions • •
• •
•
The test is for In Vitro Diagnostic Use only. Appropriate infection control and handling procedures should be followed – do not smoke, eat or drink in the area where the test is to be performed. Use suitable clothing and gloves when handling samples and when performing the test. Do not pipette any samples or reagents by mouth. All materials should be considered as potentially infectious. To disinfect, either autoclave materials at 121.5°C for 1 hour or treat with Sodium hypochlorite (1 percent solution). Do not use test beyond the expiration date indicated.
Sample Collection Spectrum-Diagnostics S.typhi-S.paratyphi test can be run on stool or serum samples. The test works best on fresh samples. If testing cannot be done immediately, they should be stored at 2-8’C after collection for up to 3 days. If testing cannot be done within 3 days, serum can be stored frozen at -20 °C or colder. Shipment of samples should comply with local regulations for transport of etiologic agents.
Procedure 1.
Remove as many test cards from the pouches as needed. Lay on a clean flat surface. For stool samples: Add about 0.5 gm stool to approximately 1 ml of Phosphate Buffered Saline PBS. Mix well and allow to sit for 5 minutes or so to allow the large particles to settle. Then add 200 ml from the upper layer of the extract to the well (A). For serum samples: Using the pipette, add 100 ml of serum/ plasma to the sample well (A) The amount of S.typhi-S.paratyphi antigens present in serum is typically less than that in stool. This may decrease the sensitivity of the test when using serum depending how soon after the onset of the infection the test is performed. Early infection typically exhibits greater levels of the antigen in the serum than in later infection.
To confirm serum results: The use of a stool sample is recommended if serum is used first and a negative result is obtained and typhoid is still suspected A second test run on a stool sample should be performed.
Sensitivity: Spectrum-Diagnostics S.typhi-S.paratyphi assay was run using serum and stool samples versus culture positive samples and found to give positive results in all cases.
Results are then read in as little as10 minutes for strong positives or up to 20 minutes for weaker positives and to make sure negatives are confirmed.
Reading the Test Results Negative Only one pink/ purple band appears in the C (Control) area of the test card.
POSITIVE
S. typhi
S. paratyphi
S.typhi & Paratyphi
Indeterminate If only one band appears in the 1 or 2 – Test area, or no band appears at all in the C well – Control area. It is then recommended that a fresh device be used and the test repeated carefully following the directions in this insert.
Quality Control A known positive and negative control should be run to ensure proper performance. All controls should be handled in the same manner as patient samples.
Limitations of the Test The instructions for use and reading of the test instructions must be followed carefully for the test to perform properly. The Spectrum-Diagnostics S.typhi-S.paratyphi test is designed to detect S.typhi-S.paratyphi antigen in stool or serum samples. Testing of any other body fluids has not been validated and may not yield appropriate results. For samples that test positive (reactive) by the Spectrum-Diagnostics S.typhi-S.paratyphi test, more specific confirmatory testing should be done. A clinical evaluation of the patient’s situation and history should also be made before a final diagnosis is established. The use of a rapid test alone is not sufficient to diagnose S.typhi-S.paratyphi infection even if antigen is present. Also, a negative result does not preclude the possibility of infection with S.typhi-S.paratyphi.
Performance Characteristics Specificity: The antibodies used in the Spectrum-Diagnostics S.typhi-S. paratyphi assay were developed specifically against Salmonella typhi and Salmonella paratyphi LPS antigen.
293
Syphilis Ab Combo Rapid Test (Serum / Plasma / Whole Blood) REF: 1204 001
30 test
SYMBOLS IN PRODUCT LABELLING EC REP Authorised Representative IVD LOT REF
INTENDED USE
The Spectrum Syphilis Ab Combo Rapid Test is a lateral flow chromatographic immunoassay for the qualitative detection of antibodies including IgG, IgM, and IgA to Treponema pallidum (Tp) in human serum, plasma or whole blood. It is intended to be used as a screening test and as an aid in the diagnosis of infection with Tp. Any reactive specimen with the Spectrum Syphilis Ab Combo Rapid Test must be confirmed with alternative testing method(s) and clinical findings.
Serological detection of anti-Tp antibody has been long recognized in the diagnosis of syphilis since the natural course of the infection was characterized by periods without clinical manifestations. Both IgM and IgG antibodies were detected in sera from patients with primary and secondary syphilis. The IgM antibody may be detectable towards the second week of infection, while IgG antibody appears later, at about 4 weeks. These antibodies could last for several years or even decades in the serum of a patient with untreated latent syphilis. Antigens such as Rapid Plasma Cardiolipin antigen (RPR) and Tp bacterial extracts have been used in the syphilis serological tests for decades. However, RPR antigen is a non-treponema antigen, derived from bovine heart. Antibody to RPR antigen does not develop until 1-4 weeks after the appearance of the chancre, thus this antigen lacks of sensitivity to primary syphilis. The Tp extracts are prepared from inoculated rabbit testis and contain a certain amount of contaminated materials such as flagella, which can lead to cross reactions with borreliae and leptospires in the serological test. In addition, the composition of extracts may vary from lot to lot. Recently, several highly immunogenic Tp specific antigens have been identified and used as an alternative to the traditional antigens with the advantages of high specificity and reproducibility. The Spectrum Syphilis Ab Combo Rapid Test permits the measurement of antibodies (IgM, IgG and IgA) to recombinant antigens of Tp in blood rapidly and reliably without instrumentation.
TEST PRINCIPLE
The Spectrum Syphilis Ab Combo Rapid Test is a lateral flow chromatographic immunoassay. The test cassette consists of: 1) a burgundy colored conjugate pad containing recombinant Tp antigens conjugated with colloidal gold (Tp conjugates) and rabbit IgG-gold conjugates, 2) a nitrocellulose membrane strip containing a test band (T band) and a control band (C band). The T band is pre-coated with non-conjugated recombinant Tp antigens, and the C band is pre-coated with goat anti-rabbit IgG antibody.
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
an internal control (C band) which should exhibit a burgundy colored band of the immunocomplex of goat anti-rabbit IgG/rabbit IgG-gold conjugate regardless of color development on the T band. Otherwise, the test result is invalid and the specimen must be retested with another device.
REAGENTS AND MATERIALS PROVIDED 1.
SUMMARY AND EXPLANATION OF THE TEST
Tp, a spirochete bacterium, is the causative agent of the venereal disease syphilis. Although syphilis rates are declining in the United States after an epidemic outbreak between 1986 and 1990, the incidence of syphilis in Europe has increased since 1992, especially in the countries of the Russian Federation, where peaks of 263 cases per 100,000 have been reported. In 1995, WHO reported 12 million new cases of syphilis. Currently, the positive rate of syphilis serological tests in HIV-infected individuals has been rising recently.
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
2. 3.
Individually sealed foil pouches containing: a. One cassette device. b. One plastic dropper. c. One desiccant. Sample diluent (1 vial, 5 mL) One package insert (instruction for the use). Positive Control Negative Control
MATERIALS REQUIRED BUT NOT PROVIDED 1. 2.
Clock or Timer Lancing device for whole blood test
WARNINGS AND PRECAUTIONS
For in Vitro Diagnostic Use 1. This package insert must be read completely before performing the test. Failure to follow the insert gives inaccurate test results. 2. Do not open the sealed pouch, unless ready to conduct the assay. 3. Do not use expired devices. 4. Bring all reagents to room temperature (15°C-30°C) before use. 5. Do not use the components in any other type of test kit as a substitute for the components in this kit. 6. Do not use hemolized blood specimen for testing. 7. Wear protective clothing and disposable gloves while handling the kit reagents and clinical specimens. Wash hands thoroughly after performing the test. 8. Users of this test should follow the US CDC Universal Precautions for prevention of transmission of HIV, HBV and other blood-borne pathogens. 9. Do not smoke, drink, or eat in areas where specimens or kit reagents are being handled. 10. Dispose of all specimens and materials used to perform the test as biohazardous waste. 11. Handle the Negative and Positive Control in the same manner as patient specimens. 12. The testing results should be read within 15 minutes after a specimen is applied to the sample well or sample pad of the device. Reading result after 15 minutes may give erroneous results. 13. Do not perform the test in a room with strong air flow, ie. an electric fan or strong air-conditioning.
REAGENT PREPARATION INSTRUCTIONS
AND
Avoid multiple freeze-thaw cycles. Prior to testing, bring frozen specimens to room temperature slowly and mix gently. Specimens containing visible particulate matter should be clarified by centrifugation before testing. Do not use samples demonstrating gross lipemia, gross hemolysis or turbidity in order to avoid interference on result interpretation. Blood Drops of whole blood can be obtained by either finger tip puncture or veinpuncture. Do not use any hemolized blood for testing. Whole blood specimens should be stored in refrigeration (2°C-8°C) if not tested immediately. The specimens must be tested within 24 hours of collection.
MATERIALS MAY BE REQUIRED AND NOT PROVIDED 1. 2.
no anticoagulants in Vacutainer®) by veinpuncture. 2. Allow the blood to clot. 3. Separate the serum by centrifugation. 4. Carefully withdraw the serum into a new pre-labeled tube. Test specimens as soon as possible after collecting. Store specimens at 2°C-8°C if not tested immediately. Store specimens at 2°C-8°C up to 5 days. The specimens should be frozen at -20°C for longer storage.
ASSAY PROCEDURE
Step 1: Bring the specimen and test components to room temperature if refrigerated or frozen. Mix the specimen well prior to assay once thawed. Step 2: When ready to test, open the pouch at the notch and remove device. Place the test device on a clean, flat surface. Step 3: Be sure to label the device with specimen’s ID number. Step 4: Fill the plastic dropper with the specimen. Holding the dropper vertically, dispense 1 drop (about 30-45 µL) of serum/plasma or 1 drop of whole blood (about 40-50 µL) into the sample well making sure that there are no air bubbles. Immediately add1 drop (about 35-50 µL) of Sample Diluent to the sample well.
Absence of the T band suggests a negative result. The test contains
294
Samples with positive results should be confirmed with alternative testing method(s) and clinical findings before a positive determination is made. 3.
INVALID: If no C band is developed, the assay is invalid regardless of color development on the T band as indicated below. Repeat the assay with a new device.
PERFORMANCE CHARACTERISTICS
Clinical Performance A total of 1055 samples from susceptible subjects were tested by the Spectrum Syphilis Ab Combo Rapid Test and by a TPHA test. Comparison for all subjects is shown in the following table. Spectrum Syphilis Ab Combo Rapid Test TPHA
Positive
Negative
Total
Positive
318
0
318
Negative
2
735
737
Total
320
735
1055
Relative Sensitivity: 100%, Relative Specificity: 99.7%, Overall Agreement: 99.8% Precision Within run and between run precisions have been determined by testing 15 replicates with three of the samples: a negative, a weak positive, and a strong positive sample. The negative, weaker positive, and strong positive samples were correctly identified in all of the tests each time. 1.
Step 5: Set up timer. Step 6: Results can be read in 15 minutes. Positive results can be visible in as short as 1minute. Don’t read result after 15 minutes. To avoid confusion, discard the test device after interpreting the result.
QUALITY CONTROL 1.
2.
STORAGE
All reagents are ready to use as supplied. Store unused test device unopened at 2°C -30°C. If stored at 2°C -8°C, ensure that the test device is brought to room temperature before opening. The test device is stable through the expiration date printed on the sealed pouch. Do not freeze the kit or expose the kit over 30°C. Consider any materials of human origin as infectious and handle them using standard biosafety procedures. Plasma 1. Collect blood specimen into a lavender, blue or green top collection tube (containing EDTA, citrate or heparin, respectively in Vacutainer® ) by veinpuncture. 2. Separate the plasma by centrifugation. 3. Carefully withdraw the serum into a new pre-labeled tube. Serum 1. Collect blood specimen into a red top collection tube (containing
POSITIVE RESULT: If both C and T bands are developed, the test indicates for the presence of anti-Tp antibody in the specimen. The result is positive.
LIMITATIONS OF TEST
SPECIMEN COLLECTION AND HANDLING When an adequate volume of test specimen is dispensed into the sample well of the cassette, the specimen migrates by capillary action across the cassette. Anti-Tp antibody, if present in the specimen will bind to the Tp conjugates. The immunocomplex is then captured on the membrane by the pre-coated Tp antigen, forming a burgundy colored T band, indicating a Tp antibody positive test result.
2.
Internal Control: This test contains a built-in control feature, the C band. The C line develops after adding specimen and sample diluent. Otherwise, review the whole procedure and repeat test with a new device. External Control: Good Laboratory Practice recommends using external controls, positive and negative, to assure the proper performance of the assay, particularly under the following circumstances: a. New operator uses the kit, prior to performing testing of specimens. b. A new lot of test kit is used. c. A new shipment of kits is used. d. The temperature used during storage of the kit falls outside of 2-30°C. e. The temperature of the test area falls outside of 15-30°C. f. To verify a higher than expected frequency of positive or negative results. g. To investigate the cause of repeated invalid results.
INTERPRETATION OF ASSAY RESULT 1.
NEGATIVE RESULT: If only the C band is developed, the test indicates that no detectable anti-Tp antibody is present in the specimen. The result is negative.
2.
3.
4.
5. 6.
The Assay Procedure and the Assay Result Interpretation must be followed closely when testing the presence of antiTp antibody in serum, plasma or whole blood from individual subjects. Failure to follow the procedure may give inaccurate results. The Spectrum Syphilis Ab Combo Rapid Test is limited to the qualitative detection of anti-Tp antibody in human serum or plasma. The intensity of the test band does not linear correlation with the antibody titer in the specimen. A negative result for an individual subject indicates absence of detectable anti-Tp antibody. However, a negative test result does not preclude the possibility of exposure to or infection with Tp. A negative result can occur if the quantity of the anti-Tp antibody present in the specimen is below the detection limits of the assay, or the antibodies that are detected are not present during the stage of disease in which a sample is collected. Some specimens containing unusually high titer of heterophile antibodies or rheumatoid factor may affect expected results. The results obtained with this test should only be interpreted in conjunction with other diagnostic procedures and clinical findings.
REFERENCES 1. 2. 3.
Tichonova, L., K. Borisenko, H.Ward, A.meheus, et al. Epidemics of syphilis in the Russian Federation: Trends, origins, and priorities for control. Lancet 1997; 350:210-213. Bailey MJ, Thomas CM, Cockayne A, Strugnell RA, Penn CW. Cloning and expression of Treponema pallidum antigens in Escherichia coli. J Gen Microbiol 1989; 135 ( Pt 9):2365-78. Rufli T. Syphilis and HIV infection. Dermatologica 1989; 179:113-117.
295
ToRCH
SYMBOLS IN PRODUCT LABELLING
Toxo/Rubella/CMV/HSV 1/2 IgM Antibodies Combo Rapid Test Device (Serum/Plasma) REF: 1204 001
EC REP Authorised Representative IVD LOT
25 test
A rapid test for the qualitative detection of IgM antibodies to Toxoplasma gondii (Toxo), Rubella virus (Rubella), Cytomegalovirus (CMV), and Herpes simplex virus 1/2 (HSV 1/2) in serum or plasma. For professional in vitro diagnostic use only.
INTENDED USE
The ToRCH IgM Antibodies Combo Rapid Test Device (Serum/ Plasma) is a rapid chromatographic immunoassay for the qualitative detection of IgM antibodies to Toxoplasma gondii (Toxo), Rubella virus (Rubella), Cytomegalovirus (CMV), and Herpes simplex virus 1/2 (HSV 1/2) in serum or plasma to aid in the diagnosis of ToRCH.
REF
SPECIMEN COLLECTION AND PREPARATION • • •
SUMMARY
ToRCH is an acronym for a group of infectious diseases that, while infecting the pregnant women, may cause birth defects in their newborns.1 ToRCH stands for 4 different infections that can adversely affect the pregnant women and the fetus, newborn children including birth defects and often leading to abortion. The four infections are Toxomplasma gondii (A spirochete), Rubella (Virus), CMV - Cytomegalovirus (Virus), HSV 1/2 - Herpes Simplex Virus 1 and/or 2 (Virus). The infections usually cause few, if any, symptoms in the pregnant woman, but pose greater risks of serious birth defects for neonates. Infections caused by ToRCH Toxoplasma, Rubella Virus, Cytomegalo Virus (CMV) and Herpes Simplex Virus (HSV) - is the major cause of BOH (Bad Obstetric History).2 Risks are severe, if the mother gets the infection in the first trimester as the baby’s organs start to form in this stage. General symptoms include premature birth, growth retardation, neurological abnormalities, damage of the eye, liver, heart and ear as well as bone lesions. Microcephaly, hydrocephaly, seizures and psychomotor retardation accompany these malformations. The ToRCH IgM Antibodies Combo Rapid Test Device (Serum/ Plasma) is a rapid chromatographic immunoassay for the qualitative detection of IgM antibodies to Toxo, Rubella, CMV, and HSV 1/2 in serum or plasma specimens.
PRINCIPLE
The ToRCH IgM Antibodies Combo Rapid Test Device (Serum/ Plasma) is a qualitative, lateral flow immunoassay for the detection of IgM antibodies to Toxo, Rubella, CMV, and HSV 1/2 in serum or plasma specimens. In this test, antigens of Toxo, Rub, CMV and HSV 1/2 are coated in the test line regions of each section in the test. During testing, the serum or plasma specimen reacts with Goat anti-human IgM coated particles in the test strip. The mixture then migrates upward on the membrane by capillary action and reacts with the Toxo, Rub, CMV and HSV 1/2 specific antigens on the membrane in the test line regions of the respective sections. The presence of a colored line in the test line region of a particular section indicates a positive result for the corresponding infection, viz. Toxo, Rub, CMV, HSV 1/2, while its absence indicates a negative result for that infection. To serve as a procedural control, a colored line will always appear in the respective control line regions of all the four strips indicating that proper volume of specimen has been added and membrane wicking has occurred.
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
•
•
The ToRCH IgM Antibodies Combo Rapid Test Device (Serum/ Plasma) can be performed using either serum or plasma specimen. Separate the serum or plasma from blood as soon as possible to avoid hemolysis. Only clear, non-hemolyzed specimens can be used. Testing should be performed immediately after the specimens have been collected. Do not leave the specimens at room temperature for prolonged periods. Specimens may be stored at 2-8°C for up to 3 days. For long term storage, specimens should be kept below -20°C. Bring specimens to room temperature prior to testing. Frozen specimens must be completely thawed and mixed well prior to testing. Specimens should not be frozen and thawed repeatedly. If specimens are to be shipped, they should be packed in compliance with local regulations for the transportation of etiologic agents.
Materials Provided • Test devices • Disposable droppers • Buffer • Package insert Materials Required But Not Provided • Specimen collection container • Centrifuge (for plasma only) • Timer
DIRECTIONS FOR USE
Allow the test device, specimen, and/or controls to equilibrate to room temperature (15-30°C) prior to testing. 1. Bring the pouch to room temperature before opening it. Remove the test device from the sealed pouch and useit as soon as possible. Best results will be obtained if the assay is performed within one hour. 2. Place the test device on a clean and level surface. Hold the dropper vertically and transfer 1 full drop of serum or plasma (approximately 10 µL) and 2 drops of buffer (approximately 80 µL) to each specimen well of the test device respectively, and then start the timer. Avoid trapping air bubbles in the specimen well. See illustration below. 3. Wait for the colored line(s) to appear. The results should be read at 15 minutes. Do not interpret the results after 20 minutes.
• • •
• • •
For professional in vitro diagnostic use only. Do not use after expiration date. Do not eat, drink or smoke in the area where the specimens or kits are handled. Do not use test if the package is damaged. Handle all specimens as if they contain infectious agents. Observe established precautions against microbiological hazards throughout testing and follow the standard procedures for proper disposal of specimens. Wear protective clothing such as laboratory coats, disposable gloves and eye protection when specimens are being tested. Humidity and temperature can adversely affect results. The used test should be discarded according to local regulations.
STORAGE AND STABILITY
Store as packaged in the sealed pouch either at room temperature or refrigerated (2-30°C). The test device is stable through the expiration date printed on the sealed pouch. The test device must remain in the sealed pouch until use. DO NOT FREEZE. Do not use beyond the expiration date.
296
A procedural control is included in the test individually for all the four sections. Four colored lines appearing in control line regions (C) of all four sections is the internal procedural control. It confirms sufficient specimen volume and correct procedural technique. Control standards are not supplied with this kit; however, it is recommended that positive and negative controls be tested as good laboratory practice to confirm the test procedure and to verify proper test performance.
LIMITATION 1.
2.
3. 4.
The ToRCH IgM Antibodies Combo Rapid Test Device (Serum/ Plasma) is for in vitro diagnostic use only.This test should be used for the detection of IgM antibodies to Toxo, Rubella, CMV and HSV 1/2 in serum or plasma specimens. Neither the quantitative value nor the rate of increase in the concentration of IgM antibodies to Toxo, Rubella, CMV and HSV 1/2 can be determined by this qualitative test. The ToRCH IgM Antibodies Combo Rapid Test Device (Serum/ Plasma) will only indicate the presence of IgM antibodies to Toxo, Rubella, CMV and HSV 1/2 in the specimen and should not be used as the sole criteria for the diagnosis of any of the ToRCH infections for which the positive result is obtained. As with all diagnostic tests, all results must be considered with other clinical information available to the physician. If the test result is negative and clinical symptoms persist, additional follow-up testing using other clinical methods is suggested. A negative result for any one out of the four infections of ToRCH at any time does not preclude the possibility of that particular infection.
EXPECTED VALUES
The ToRCH IgM Antibodies Combo Rapid Test Device (Serum/ Plasma) has been compared with leading commercial EIA Toxo, Rubella, CMV, and HSV 1/2 tests, demonstrating an overall accuracy of 98.4% for Toxo, 99.1% for Rubella, 98.8% for CMV, and 99.0% for HSV 1/2.
PERFORMANCE CHARACTERISTICS
Sensitivity and Specificity The ToRCH IgM Antibodies Combo Rapid Test Device (Serum/ Plasma) was compared with leading commercial EIA Toxo, Rubella, CMV, and HSV 1/2 tests, the results show that the ToRCH IgM Antibodies Combo Rapid Test Device (Serum/Plasma) has a high sensitivity and specificity for each of its sections. Toxo Rapid Test Device vs. EIA Method Toxo Rapid Test
The test contains Goat anti-human IgM coated particles and Toxo antigens, Rub antigens, CMV antigens, and HSV 1/2 antigens coated on the membrane. •
QUALITY CONTROL
MATERIALS
REAGENTS
PRECAUTIONS
NEGATIVE: One colored line appears in the control line region (C) of every section. Non-appearance of a visible line in the test line region (T) of any section is indicative of a negative test result for that specific section, viz. Toxo, Rub, CMV, and HSV 1/2. INVALID: Control line fails to appear in any section. Insufficient specimen volume or incorrect procedural techniques are the most likely reasons for control line failure. Review the procedure and repeat the test with a new test device. If the problem persists, discontinue using the test kit immediately and contact your local distributor.
Results
Positive
Negative
Total Results
Positive
54
12
66
Negative
3
846
849
57
858
915
Total Results
INTERPRETATION OF RESULTS
(Please refer to the illustration above)
POSITIVE: Toxo Positive: *Two colored lines appear in the ‘Toxo’ Section. One line should be in the control line region (C) and another line should be in the test line region (T). Rubella Positive: *Two colored lines appear in the ‘Rub’ Section. One line should be in the control line region (C) and another line should be in the test line region (T). CMV Positive: *Two colored lines appear in the ‘CMV’ Section. One line should be in the control line region (C) and another line should be in the test line region (T). HSV 1/2 Positive: *Two colored lines appear in the ‘HSV 1/2’ Section. One line should be in the control line region (C) and another line should be in the test line region (T). *NOTE: The intensity of the color in test line regions (T) will vary depending on the concentrations of IgM antibodies present in the specimen. Therefore, any shade of color in test line regions (T) should be considered positive.
EIA
Relative Sensitivity: 94.7% (85.4%-98.9%)* Relative Specificity: 98.6% (97.6%-99.3%)* Relative Accuracy: 98.4% (97.3%-99.1%)* * 95% Confidence Interval EIA Positive 19 1 20
Negative 2 298 300
Relative Sensitivity: 95.0% (75.1%-99.9%)* Relative Specificity: 99.3% (97.6%-99.9%)* Relative Accuracy: 99.1% (97.3%-99.8%)* * 95% Confidence Interval
Method Results CMV Rapid Positive Test Negative Total Results
EIA Positive 18 1 19
Negative 5 461 466
Total Results 23 462 485
Relative Sensitivity: 94.7% (74.0%-99.9%)* Relative Specificity: 98.9% (97.5%-99.7%)* Relative Accuracy: 98.8% (97.3%-99.5%)* * 95% Confidence Interval HSV 1/2 Rapid Test Device vs. EIA Method Results HSV 1/2 Positive Rapid Test Negative Total Results
EIA Positive 12 2 14
Negative 1 300 301
Total Results 13 302 315
Relative Sensitivity: 85.7% (57.2%-98.2%)* Relative Specificity: 99.7% (98.2%-100.0%)* Relative Accuracy: 99.0% (97.2%-99.8%)* * 95% Confidence Interval Precision Intra-Assay Within-run precision has been determined by using 10 replicates of three specimens containing negative, low positive and high positive of Toxo, Rubella, CMV or HSV 1/2. The negative and positive values were correctly identified >99% of the time Inter-Assay Between-run precision has been determined by using the same three specimens of negative, low positive and high positive of Toxo, Rubella, CMV and HSV 1/2 in 3 independent assays. Three different lots of the ToRCH IgM Antibodies Combo Rapid Test Device (Serum/Plasma) have been tested using negative, low positive and high positive specimens. The specimens were correctly identified >99% of the time. Cross-Reactivity Sera containing known amounts of antibodies to Toxo, Rubella, CMV and HSV 1/2 have been tested with HIV, HCV, SYP, HBV, and RF. No cross-reactivity was observed, indicating that the ToRCH IgM Antibodies Combo Rapid Test Device (Serum/Plasma) has a high degree of specificity for IgM antibodies to Toxo, Rubella, CMV, and HSV 1/2. Interfering Substances The ToRCH IgM Antibodies Combo Rapid Test Device (Serum/ Plasma) has been tested and no interference was observed in specimens containing 110 mg/mL human albumin, 1 mg/mL bilirubin, 10 mg/mL hemoglobin, 0.2mg/mL cholesterol and 15 mg/ mL triglycerides. The following compounds have also been tested using the ToRCH IgM Antibodies Combo Rapid Test Device (Serum/Plasma) and no interference was observed Acetaminophen Caffeine Gentisic acid
Acetylsalicylic Ascorbic Acid Bilirubin Acid Ethanol EDTA Glucose Phenothiazine Phenylpropanolamine Salicylic Acid
BIBLIOGRAPHY 1.
Rubella Rapid Test Device vs. EIA Method Results Rubella Positive Rapid Test Negative Total Results
CMV Rapid Test Device vs. EIA
Total Results 21 299 320
2.
S M Kadri, Torch Test: Test & Inference, INDIAN JOURNAL OF THE PRACTISING DOCTOR, January 2005, Vol. I, No. 4 : P16-18 Rajendra B Surpam, Usha P Kamlakar, RK Khadse, MS Qazi, & Suresh V Jalgaonkar, Serological study for TORCH infections in women with bad obstetric history, The Journal of Obstetrics and Gynecology of India, January/February 2006, Vol. 56, No. 1 : P 41-43 ORDERING INFORMATION CATALOG NO.
QUANTITY
1204 001
25 test
297
Toxo IgG/IgM Rapid TestCassette
SYMBOLS IN PRODUCT LABELLING EC REP Authorised Representative
(Serum / Plasma)
IVD
REF: 1196 001
REF
30 test
INTENDED USE
The Spectrum Toxo IgG/IgM Rapid Test is a lateral flow chromatographic immunoassay for the simultaneous detection and differentiation of IgG and IgM anti-Toxoplasma gondii (T. gondii) in human serum or plasma. This kit is intended to be used as a screening test and as an aid in the diagnosis of infection with T. gondii. Any reactive specimen with the Spectrum Toxo IgG/IgM Rapid Test must be confirmed with alternative testing method(s) and clinical findings.
SUMMARY AND EXPLANATION OF THE TEST
T. gondii is an obligate intracellular protozoan parasite with a worldwide distribution. Serological data indicates that approximately 30% of the population of most industrialized nations is chronically infected with the organism. A variety of serological tests for antibodies to T. gondii have been used as an aid in diagnosis of acute infection and to assess previous exposure to the organism. These tests are: the Sabin-Feldman dye test, direct agglutination, indirect hemagglutination, latex agglutination, indirect immunofluorescence and ELISA. Recently, lateral flow chromatographic immunoassay such as the Spectrum Toxo IgG/IgM Rapid Test has been introduced to the clinic for the instant detection of T. gondii infection.
TEST PRINCIPLE
The Spectrum Toxo IgG/IgM Rapid Test is a lateral flow chromatographic immunoassay. The test cassette consists of: 1) a burgundy colored conjugate pad containing recombinant T. gondii antigens conjugated with colloidal gold (T. gondii conjugates) and rabbit IgG-gold conjugates, 2) a nitrocellulose membrane strip containing two test bands (M and G bands) and a control band (C band). The M band is pre-coated with monoclonal anti-human IgM for detection of IgM anti-T. gondii antibody, G band is pre-coated with reagents for detection of IgG anti-T.gondii antibody, and the C band is pre-coated with goat anti-rabbit IgG.
When an adequate volume of test specimen is dispensed into the sample well of the test cassette, the specimen migrates by capillary action across the cassette. IgM anti-T. gondii if present in the specimen will bind to the T. gondii conjugates. The immunocomplex is then captured on the membrane by the pre-coated anti-human IgM antibody forming a burgundy colored M line, indicating a T. gondii IgM positive or reactive test result. IgG anti-T. gondii if present in the specimen will bind to the T. gondii conjugates. The immunocomplex is then captured by the precoated reagents on the membrane forming a burgundy colored G line, indicating a T. gondii IgG positive or reactive test result. Absence of any T lines (M and G) suggests a negative or nonreactive result. The test contains an internal control (C band) which should exhibit a burgundy colored line of the immunocomplex of goat anti-rabbit IgG/rabbit IgG-gold conjugate regardless of color development on any of the T lines. Otherwise, the test result is invalid and the specimen must be retested with another device.
REAGENTS AND MATERIALS PROVIDED 1. 2. 3. 4.
Individually sealed foil pouches containing: a. One cassette device b. One desiccant Plastic droppers Sample diluent (1 bottle, 5 mL) One package insert (instruction for use)
MATERIALS MAY BE REQUIRED AND NOT PROVIDED 1. 2.
298
Positive Control Negative Control
LOT
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
assay, once thawed. Step 2: When ready to test, open the pouch at the notch and remove device. Place the test device on a clean, flat surface. Step 3: Be sure to label the device with the specimen’s ID number.Step 4: Fill the plastic dropper with the specimen. Holding the dropper vertically, dispense 1 drop (about 30-45 µL) of specimen into the sample well. Then add 1 drop (about 30-45 µL) of Sample Diluent immediately.
MATERIALS REQUIRED BUT NOT PROVIDED 1.
WARNINGS AND PRECAUTIONS
REAGENT PREPARATION AND STORAGE INSTRUCTIONS
All reagents are ready to use as supplied. Store unused test devices unopened at 2°C-30°C. Do not freeze the kit. Do not expose the kit over 30°C. The positive and negative controls should be kept at 2°C-8°C or the temperature indicated. If stored at 2°C-8°C, ensure that the test device is brought to 15°C-30°C before opening. The test device is stable through the expiration date printed on the sealed pouch.
SPECIMEN COLLECTION AND HANDLING
Consider any materials of human origin as infectious and handle them using standard bio-safety procedures. Plasma 1. Collect blood specimen into a lavender, blue or green top collection tube (containing EDTA, citrate or heparin, respectively in Vacutainer®) by veinpuncture. 2. Separate the plasma by centrifugation. 3. Carefully withdraw the plasma into new pre-labeled tube. Serum 1. Collect blood specimen into a red top collection tube (containing no anticoagulants in Vacutainer®) by veinpuncture. 2. Allow the blood to clot. 3. Separate the serum by centrifugation. 4. Carefully withdraw the serum into a new pre-labeled tube. Test specimens as soon as possible after collecting. Store specimens at 2°C-8°C if not tested immediately. Store specimens at 2°C-8°C for up to 5 days. The specimens should be frozen at -20°C for longer storage. Avoid multiple freeze-thaw cycles. Prior to testing, bring frozen specimens to room temperature slowly and mix gently. Specimens containing visible particulate matter should be clarified by centrifugation before testing. Do not use samples demonstrating gross lipemia, gross hemolysis or turbidity in order to avoid interference on result interpretation.
ASSAY PROCEDURE
Step 1: Bring the specimen and test components to room temperature if refrigerated or frozen. Mix the specimen well, prior to
INVALID: If no C line is developed, the assay is invalid regardless of any burgundy color in the test bands as indicated below. Repeat the assay with a new device.
PERFORMANCE CHARACTERISTICS
Clock or Timer
For in Vitro Diagnostic Use 1. This package insert must be read completely before performing the test. Failure to follow the insert gives inaccurate test results. 2. Do not open the sealed pouch unless ready to conduct the assay. 3. Do not use expired devices. 4. Bring all reagents to room temperature (15°C-30°C) before use. 5. Do not use the components in any other type of test kit as a substitute for the components in this kit. 6. Do not use hemolized blood for testing. 7. Wear protective clothing and disposable gloves while handling the kit reagents and clinical specimens. Wash hands thoroughly after performing the test. 8. Users of this test should follow the US CDC Universal Precautions for prevention of transmission of HIV, HBV and other blood-borne pathogens. 9. Do not smoke, drink or eat in areas where specimens or kit reagents are being handled. 10. Dispose of all specimens and materials used to perform the test as bio-hazardous waste. 11. Handle the negative and positive control in the same manner as patient specimens. 12. The test results should be read within 15 minutes after a specimen is applied to the sample well or sample pad of the device. Reading the results after 15 minutes may give erroneous results. 13. Do not perform the test in a room with strong air flow, i.e. an electric fan or strong air-conditioning.
3.
Step 5: Set up timer. Step 6: Results can be read in 15 minutes. Positive results can be visible in as short as 1 minute. Don’t read result after 15 minutes. To avoid confusion, discard the test device after interpreting the result.
QUALITY CONTROL 1.
2.
Internal Control: This test contains a built-in control feature, the C line. The C line develops after adding the specimen and the sample diluent. If the C line does not develop, review the whole procedure and repeat the test with a new device. External Control: Good Laboratory Practice recommends using external controls, positive and negative, to assure the proper performance of the assay, particularly under the following circumstances: a. New operator uses the kit, prior to performing the testing of specimens. b. A new lot of test kits is used. c. A new shipment of kits is used. d. The temperature used during storage of the kits fall outside of 2-30°C. e. The temperature of the test area falls outside of 15-30°C. f. To verify a higher than expected frequency of positive or negative results. g. To investigate the cause of repeated invalid results.
1. Clinical Performance For IgM Test A total of 302 samples from susceptible subjects were tested by the Spectrum Toxo IgG/IgM Rapid Test and by a commercial IgM EIA kit. Comparison of the results for all subjects is shown in the following table. Spectrum Toxo IgG/IgM Rapid Test IgM EIA
Positive
Negative
Total
Positive
2
0
2
Negative
2
298
300
Total
4
298
302
Relative Sensitivity: 100% , Relative Specificity: 99.3%, Overall Agreement: 99.3% 2. Clinical Performance For IgG Test A total of 324 samples from susceptible subjects were tested by the Spectrum Toxo IgG/IgM Rapid Test and by a commercial IgG EIA kit. Comparison of the results for all subjects is shown in the following table. Spectrum Toxo IgG/IgM Rapid Test IgG EIA
Positive
Negative
Total
Positive
22
2
24
Negative
3
297
300
Total
25
299
324
INTERPRETATION OF ASSAY RESULT 1.
2.
NEGATIVE RESULT: If only the C line is present, the absence of any burgundy color in both the test lines (M and G) indicates that no anti-T. gondii antibodies are detected in the specimen. The result is negative or non-reactive
POSITIVE RESULT: a. In addition to the presence of the C line, if only the M line is developed, the test indicates the presence of IgM anti-T. gondii in the specimen. The result is positive or reactive.
Relative Sensitivity: 91.6% , Relative Specificity: 99.0%, Overall Agreement: 98.5%
LIMITATIONS OF TEST 1.
2.
3.
4. b.
In addition to the presence of the C line, if only the G line is developed, the test indicates the presence of IgG anti-T. gondii in the specimen. The result is positive or reactive.
5. 6.
7. c.
In addition to the presence of the C line, if both the M and the G lines are developed, the test indicates the presence of both IgG and IgM anti-T. gondii in the specimen. The result is also positive or reactive.
REFERENCES 1.
2. Samples with positive results should be confirmed with alternative testing method(s) and clinical findings before a positive determination is made.
The Assay Procedure and the Interpretation of Assay Result sections must be followed closely when testing for the presence of antibodies to T. gondii in serum or plasma from individual subjects. Failure to follow the procedure may give inaccurate results. The Spectrum Toxo IgG/IgM Rapid Test is limited to the qualitative detection of antibodies to T. gondii in human serum or plasma. The intensity of the test line does not have a linear correlation with the antibody titer in the specimen. A negative result for an individual subject indicates absence of detectable anti-T. gondii antibodies. However, a negative test result does not preclude the possibility of exposure to or infection with T. gondii. A negative result can occur if the quantity of the anti-T. gondii antibodies present in the specimen is below the detection limits of the assay or the antibodies that are detected are not present during the stage of disease in which a sample is collected. Some specimens containing unusually high titers of heterophile antibodies or rheumatoid factor may affect expected results. If the symptoms persist when the result from Spectrum Toxo IgG/IgM Rapid Test is negative or non-reactive, it is recommended to re-sample the patient a few days later or test with an alternative test device. The results obtained with this test should only be interpreted in conjunction with other diagnostic procedures and clinical findings.
3.
Pyndiah N, Krech U, Price P and Wilhelm J: Simplified chromatographic separation of immunoglobulin M from G and its application to Toxoplasma indirect immunofluorescence. J. Clin. Micro.1979, 9:170-174 Fraser KB, Shirodaira PV, and Stanford CF: Fluorescent staining and human IgM Br. Med. J. 1971, 3:707 Montoya JG, Rosso F. Diagnosis and management of toxoplasmosis. Clin Perinatol. 2005, 32(3):705-26.
299
Troponin-I
Troponin I Test Device (Serum/Plasma/Whole Blood) REF: 1172 001 REF: 1172 002
PERFORMANCE CHARACTERISTICS
SYMBOLS IN PRODUCT LABELLING EC REP Authorised Representative
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
IVD LOT
25 test 50 test
REF
Sensitivity:
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
The Spectrum Troponin I Test Device can detect cTnI in serum or plasma with concentration of 1.0 ng /mL or greater. Interference Substances: Levels of the following substances do not appear to interfere with the Cardiac Troponin I Assay.
A rapid test for the qualitative detection of Cardiac Troponin I in Human serum, plasma or whole blood. For professional in vitro test use only.
INTENDED USE
For the rapid qualitative determination of Cardiac troponin I (cTnI) in human whole blood, serum and plasma as an aid in the diagnosis of myocardial infarction
SUMMARY
Troponin I (TnI) is part of the troponin complex which, together with tropomyosin, forms the main component that regulates the Ca+2sensitive ATP-ase activity of actomyosin in striated muscle (skeletal and cardiac). The troponin complex consists of three subunits, troponin T(TnT), troponin I(TnI), and troponin C (TnC). Each subunit has a distinct function with TnC as the site of Ca+2 binding, TnT the tropomyosin binding, and TnI as the inhibitory subunit. Different isoforms of TnI exist in the skeletal and cardiac muscles (sTnI and cTnI, respectively) with distinct immunologic epitopes that allow the production of cardiac-specific TnI antibodies. The cardiac marker, troponin I has been established as useful tools in the diagnosis of acute myocardial infarction (AMI). Troponin I is found in blood at elevated concentrations approximately 4- 6 hours after the onset of chest pain and peak at 12-24 hours. Troponin I levels remain elevated for up to 14 days. The use of this marker is an aid in the diagnosis of AMI after myocardial infarction.
PRINCIPLE
The Troponin I Rapid Test Device employs a solid-phase chromatographic immunoassay technology to qualitatively detect the elevation of troponin I in human blood samples. When a sample of blood is dispensed into the sample well, red blood cells are removed by the built in separation system. Troponin I in the specimen makes a complex with the specific dye conjugate and biotinylated anti-troponin I antibody. This complex migrates through the test area containing immobilized streptavidin. The antibody dyetroponin I-biotinylated antibody complex bind to the immobilized streptavidin in the Test area. Unbound dye complexes migrate out of the Test area and are later captured in the Control area. Visible pinkish-purple bands will appear in the Test and Control areas if the concentrations of troponin I is above established cutoff values. If the troponin I concentration in the specimen is 0.6 ng/ml or greater, a band is present in the troponin I area. If a band is present only in the Control area, the test result is read as negative, indicating that the Troponin I concentrations are all below the cutoff values. If no band is present in the Control area, the test is invalid and another test must be run, regardless of the presence or absence of band(s) in the Test Area.
PRECAUTIONS • • • • • •
• •
300
For in vitro diagnostic use only. Do not use beyond the expiration date. Use separate syringe or clean pipette tips for different specimens. Do not pipette by mouth. Do not smoke, eat or drink in areas in which specimens or kits are handled. Wear disposable gloves while handling specimens and running the tests, and thoroughly wash hands afterwards. All patient samples should be handled as if they are capable of transmitting diseases. Observe established good laboratory procedures for proper disposal of specimens, used pipette tips or syringes, and used test devices. The test device should remain in its sealed pouch until ready for use. Humidity and temperature can adversely affect results.
STORAGE AND STABILITY
Store as packaged in the sealed pouch at 4-30 °C. The test device is stable through the expiration date printed on the sealed pouch. The test device must remain in the sealed pouch until use. Do not freeze.
SPECIMEN COLLECTION AND PREPARATION
Whole blood, plasma or serum may be used as samples for this procedure. Collect blood in a tube containing heparin as the anticoagulant. Guidelines recommended by the National Committee for Clinical Laboratory Standards (NCCLS) should be followed when collecting, transporting and processing patient samples. Since cTnI is relatively unstable, it is recommended that fresh samples be used as soon as possible to collect critical patient information. Heat inactivation of samples may lead to hemolysis or protein denaturation and therefore should be avoided. Whole blood samples should be tested within 2 hours of collection. If specimens are to be shipped, they should be packed in compliance with federal regulations covering the transportation of etiologic agents.
MATERIALS
Materials Provided • Test device(s) • Instructions for use Materials Required But Not Provided • Vacutainer tube containing heparin as an anticoagulant • Timer • Positive and Negative Controls • Calibrated Pipette
DIRECTIONS FOR USE
1. Open the foil pouch, remove the Cardiac test device, and lay the device on a level surface. Label the device with the patient’s name or control number. 2. Using a micropipette, add 80 ml of whole blood or 60 ml of serum or plasma specimen into the Sample well. 3. Read the test result in 15 minutes. NOTES: •
•
If the test has been stored in the refrigerator, allow it to return to room temperature before testing. Do not open the foil pouch until you are ready to perform the test. When the specimen is dispensed, do not position the pipette tip too high from the device’s Sample area, in order to prevent the samples from splashing.
QUALITY CONTROL
Using individual Spectrum Troponin I Test Device cassettes as described in the Assay Procedure above, run 1 Positive Control and 1 Negative Control (provided upon request) under the following circumstances to monitor test performance: 1. A new operator uses the kit, prior to performing testing of specimens. 2. A new test kit is used. 3. A new shipment of kits is used. 4. The temperature used during storage of the kit falls outside of 2°C-30°C. 5. The temperature of the test area falls outside of 15°C-30°C.
Human Albumin Bilirubin (unconjugated) Free Hemoglobin Triglycerides
Serum samples (n=121) collected from individuals after being admitted to a hospital emergency department with chest pain. The samples were tested with the Troponin I test and with FDA approved cardiac troponin I test kit. The correlation between the tests is shown below: FDA approved Troponin I test Positive
INTERPRETATION OF RESULTS
Negative result: A color band will appear only in the control window, which indicates a negative result. Positive result: Two bands will appear in the control and test window, which indicates a positive result. Notes for Result Interpretation: • The color intensity of the Test and Control bands may increase beyond 15 minutes. As the membrane in the reading window dries up, the color intensity of the bands and background change and thus may interfere with reading the test results. • For best results, the test result should be read at 15 minutes. The result, particularly a result which is negative before 15 minutes, should not be read beyond 15 minutes. • The test bands will appear before the control band in most strong positive cases. The test bands may be darker than the control band. • The test bands may appear after the control band in weak positive cases, and the test band may be weaker than the control band.
LIMITATION
The results of the Cardiac TnI Assay are to be used in conjunction with other clinical information such as clinical signs and symptoms and other test results to diagnose myocardial infarction. A negative result obtained from a patient’s sample 16 hours after the onset of chest pain may help in ruling out AMI. A positive assay result from a patient suspected of AMI may be used as an indicative of myocardial damage and requires further confirmation. Serial sampling of patients suspected of AMI is also recommended due to the delay between the onset of symptoms and the release of protein markers into the bloodstream.Samples containing an unusually high titer of certain antibodies, such as human anti mouse or human anti goat antibodies, may affect the performance of the test.
16 g/dL 60 mg/dL 4 g/dL 1,300 mg/dL
Method Comparison
Spectrum Invalid: A distinctive colored band in the control window should always appear. If no pink band is present in the Control window within 15 minutes, the test is invalid, and the sample should be run again with a new test device.
Negative
Total
Positive
31
1
32
Negative
1
88
89
Total
32
89
121
Comparative Sensitivity: 96.9 % (31/32) Comparative Specificity: 98.9% (88/89) Overall Agreement: 98.35 % (119/121)
BIBLIOGRAPHY 1.
Hamm, C.W., et al. Emergency room triage of patients with acute chest pain by means of rapid testing for cardiac troponin T or troponin I. New Eng.J.Med.337:1648 (1997). 2. Mehegan, J.P. And Tobacman, L.S. Cooperative interaction between troponin molecules bound to the cardiac thin filament. J.Biol.Chem. 266:966 (1991). 3. Antman, E.M., et al. Cardiac-specific troponin I levels to predict the risk of mortality inpatients with acute coronary syndromes. New Eng.J.Med. 335:1342 (1996). 4. Ebashi,S., Ca2+ and the contractile proteins. J.Mol.Cell. Cardiol. 16:129 (1984). 5. Bodor, S.G. et al., Development of monoclonal antibody for an assay of cardiac troponin I and preliminary results in suspected cases of myocardial infarction. Clin.Chem. 38:2203 (1992). 6. Ellis, A.K. Serum protein measurements and the diagnosis of acute myocardial infarction. Circulation 83:1107 (1991).
ORDERING INFORMATION CATALOG NO.
QUANTITY
1172 001 1172 002
25 test 50 test
EXPECTED VALUES
The Cardiac Troponin I Assay is designed to yield a positive result for cTnI concentrations at or more than 0.6 ng/mL
301
Troponin-I
Troponin I Test Device (Serum/Plasma/Whole Blood)
EC REP Authorised Representative
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
IVD LOT
REF: 1173 001
LIMITATION
SYMBOLS IN PRODUCT LABELLING
REF
30 test
The results of the Cardiac TnI Assay are to be used in conjunction with other clinical information such as clinical signs and symptoms and other test results to diagnose myocardial infarction. A negative result obtained from a patient’s sample 16 hours after the onset of chest pain may help in ruling out AMI. A positive assay result from a patient suspected of AMI may be used as an indicative of myocardial damage and requires further confirmation. Serial sampling of patients suspected of AMI is also recommended due to the delay between the onset of symptoms and the release of protein markers into the bloodstream.Samples containing an unusually high titer of certain antibodies, such as human anti mouse or human anti goat antibodies, may affect the performance of the test.
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
A rapid test for the qualitative detection of Cardiac Troponin I in Human serum, plasma or whole blood. For professional in vitro test use only.
INTENDED USE
For the rapid qualitative determination of Cardiac troponin I (cTnI) in human whole blood, serum and plasma as an aid in the diagnosis of myocardial infarction
SUMMARY
Troponin I (TnI) is part of the troponin complex which, together with tropomyosin, forms the main component that regulates the Ca+2sensitive ATP-ase activity of actomyosin in striated muscle (skeletal and cardiac). The troponin complex consists of three subunits, troponin T(TnT), troponin I(TnI), and troponin C (TnC). Each subunit has a distinct function with TnC as the site of Ca+2 binding, TnT the tropomyosin binding, and TnI as the inhibitory subunit. Different isoforms of TnI exist in the skeletal and cardiac muscles (sTnI and cTnI, respectively) with distinct immunologic epitopes that allow the production of cardiac-specific TnI antibodies. The cardiac marker, troponin I has been established as useful tools in the diagnosis of acute myocardial infarction (AMI). Troponin I is found in blood at elevated concentrations approximately 4- 6 hours after the onset of chest pain and peak at 12-24 hours. Troponin I levels remain elevated for up to 14 days. The use of this marker is an aid in the diagnosis of AMI after myocardial infarction.
PRINCIPLE
The Troponin I Rapid Test Device employs a solid-phase chromatographic immunoassay technology to qualitatively detect the elevation of troponin I in human blood samples. When a sample of blood is dispensed into the sample well, red blood cells are removed by the built in separation system. Troponin I in the specimen makes a complex with the specific dye conjugate and biotinylated anti-troponin I antibody. This complex migrates through the test area containing immobilized streptavidin. The antibody dyetroponin I-biotinylated antibody complex bind to the immobilized streptavidin in the Test area. Unbound dye complexes migrate out of the Test area and are later captured in the Control area. Visible pinkish-purple bands will appear in the Test and Control areas if the concentrations of troponin I is above established cutoff values. If the troponin I concentration in the specimen is 0.6 ng/ml or greater, a band is present in the troponin I area. If a band is present only in the Control area, the test result is read as negative, indicating that the Troponin I concentrations are all below the cutoff values. If no band is present in the Control area, the test is invalid and another test must be run, regardless of the presence or absence of band(s) in the Test Area.
PRECAUTIONS • • • • • •
• •
302
For in vitro diagnostic use only. Do not use beyond the expiration date. Use separate syringe or clean pipette tips for different specimens. Do not pipette by mouth. Do not smoke, eat or drink in areas in which specimens or kits are handled. Wear disposable gloves while handling specimens and running the tests, and thoroughly wash hands afterwards. All patient samples should be handled as if they are capable of transmitting diseases. Observe established good laboratory procedures for proper disposal of specimens, used pipette tips or syringes, and used test devices. The test device should remain in its sealed pouch until ready for use. Humidity and temperature can adversely affect results.
STORAGE AND STABILITY
Store as packaged in the sealed pouch at 4-30 °C. The test device is stable through the expiration date printed on the sealed pouch. The test device must remain in the sealed pouch until use. Do not freeze.
EXPECTED VALUES
The Cardiac Troponin I Assay is designed to yield a positive result for cTnI concentrations at or more than 0.6 ng/mL
PERFORMANCE CHARACTERISTICS
SPECIMEN COLLECTION AND PREPARATION
Whole blood, plasma or serum may be used as samples for this procedure. Collect blood in a tube containing heparin as the anticoagulant. Guidelines recommended by the National Committee for Clinical Laboratory Standards (NCCLS) should be followed when collecting, transporting and processing patient samples. Since cTnI is relatively unstable, it is recommended that fresh samples be used as soon as possible to collect critical patient information. Heat inactivation of samples may lead to hemolysis or protein denaturation and therefore should be avoided. Whole blood samples should be tested within 2 hours of collection. If specimens are to be shipped, they should be packed in compliance with federal regulations covering the transportation of etiologic agents.
MATERIALS
Materials Provided • Test device(s) • Sample diluent (1 x 5 mL) • One plastic dropper. • Instructions for use Materials Required But Not Provided • Vacutainer tube containing heparin as an anticoagulant • Timer • Positive and Negative Controls • Calibrated Pipette
ASSAY PROCEDURE 1.
Bring the specimen and test components to room temperature if refrigerated or frozen. Mix the specimen well prior to assay once thawed. 2. When ready to test, open the pouch at the notch and remove device. Place the test device on a clean, flat surface. 3. Be sure to label the device with specimen’s ID number. 4. For whole blood test Apply 1 drop of whole blood (about 40-50 µL) into the sample well. Then add 1 drop (about 35-50 µL) of Sample Diluent immediately. For serum or plasma test Fill the pipette dropper with the specimen. Holding the dropper vertically, dispense 1 drop (about 30-45 µL) of specimen into the sample well making sure that there are no air bubbles. Then add 1 drop (about 35-50 µL) of Sample Diluent immediately 5. Set up timer. 6. Results can be read in 15 minutes. Positive results can be visible in as short as 1 minute. NOTES: •
•
If the test has been stored in the refrigerator, allow it to return to room temperature before testing. Do not open the foil pouch until you are ready to perform the test. When the specimen is dispensed, do not position the pipette tip too high from the device’s Sample area, in order to prevent the samples from splashing.
Sensitivity: The Spectrum Troponin I Test Device can detect cTnI in serum or plasma with concentration of 1.0 ng /mL or greater. Interference Substances: Levels of the following substances do not appear to interfere with the Cardiac Troponin I Assay.
QUALITY CONTROL
Using individual Spectrum Troponin I Test Device cassettes as described in the Assay Procedure above, run 1 Positive Control and 1 Negative Control (provided upon request) under the following circumstances to monitor test performance: 1. A new operator uses the kit, prior to performing testing of specimens. 2. A new test kit is used. 3. A new shipment of kits is used. 4. The temperature used during storage of the kit falls outside of 2°C-30°C. 5. The temperature of the test area falls outside of 15°C-30°C.
INTERPRETATION OF RESULTS
Invalid: A distinctive colored band in the control window should always appear. If no pink band is present in the Control window within 15 minutes, the test is invalid, and the sample should be run again with a new test device. Negative result: A color band will appear only in the control window, which indicates a negative result. Positive result: Two bands will appear in the control and test window, which indicates a positive result. Notes for Result Interpretation: • The color intensity of the Test and Control bands may increase beyond 15 minutes. As the membrane in the reading window dries up, the color intensity of the bands and background change and thus may interfere with reading the test results. • For best results, the test result should be read at 15 minutes. The result, particularly a result which is negative before 15 minutes, should not be read beyond 15 minutes. • The test bands will appear before the control band in most strong positive cases. The test bands may be darker than the control band. • The test bands may appear after the control band in weak positive cases, and the test band may be weaker than the control band.
Human Albumin Bilirubin (unconjugated) Free Hemoglobin Triglycerides
16 g/dL 60 mg/dL 4 g/dL 1,300 mg/dL
Method Comparison
Serum samples (n=121) collected from individuals after being admitted to a hospital emergency department with chest pain. The samples were tested with the Troponin I test and with FDA approved cardiac troponin I test kit. The correlation between the tests is shown below: FDA approved Troponin I test Spectrum
Positive
Negative
Total
31
1
32
Negative
1
88
89
Total
32
89
121
Positive
Comparative Sensitivity: 96.9 % (31/32) Comparative Specificity: 98.9% (88/89) Overall Agreement: 98.35 % (119/121)
BIBLIOGRAPHY 1.
Hamm, C.W., et al. Emergency room triage of patients with acute chest pain by means of rapid testing for cardiac troponin T or troponin I. New Eng.J.Med.337:1648 (1997). 2. Mehegan, J.P. And Tobacman, L.S. Cooperative interaction between troponin molecules bound to the cardiac thin filament. J.Biol.Chem. 266:966 (1991). 3. Antman, E.M., et al. Cardiac-specific troponin I levels to predict the risk of mortality inpatients with acute coronary syndromes. New Eng.J.Med. 335:1342 (1996). 4. Ebashi,S., Ca2+ and the contractile proteins. J.Mol.Cell. Cardiol. 16:129 (1984). 5. Bodor, S.G. et al., Development of monoclonal antibody for an assay of cardiac troponin I and preliminary results in suspected cases of myocardial infarction. Clin.Chem. 38:2203 (1992). 6. Ellis, A.K. Serum protein measurements and the diagnosis of acute myocardial infarction. Circulation 83:1107 (1991). ORDERING INFORMATION CATALOG NO.
QUANTITY
1173 001
30 test
303
Typhoid IgG/IgM Rapid TestCassette
EC REP Authorised Representative
(Serum / Plasma)
IVD
REF: 1200 001
REF
30 test
INTENDED USE
The Spectrum Typhoid IgG/IgM Rapid Test is a lateral flow immunoassay for the qualitative detection and differentiation of IgG and IgM anti-Salmonella typhi (S. typhi) and paratyphi in human serum or plasma. It is intended to be used as a screening test and as an aid in the diagnosis of infection with S. typhi and paratyphi. Any reactive specimen with the Spectrum Typhoid IgG/IgM Rapid Test must be confirmed with alternative testing method(s).
SUMMARY AND EXPLANATION OF THE TEST
Typhoid fever and paratyphi fever are bacterial infections caused by Salmonella typhi and paratyphi A, B, and C respectively, which are transmitted through the ingestion of tainted food and water. Worldwide an estimated 17 million cases and 600,000 associated deaths occur annually1. Patients who are infected with HIV are at significantly increased risk of clinical infection. 1-5% of patients become chronic carriers harboring S. typhi in the gallbladder. The clinical diagnosis of infections depends on isolation of S. typhi and paratyphi from blood, bone marrow or a specific anatomic lesion. In facilities that can not afford to perform this complicated and time-consuming procedure, Filix-Widal test is used to facilitate diagnosis. However, many limitations lead to difficulties in the interpretation of the Widal test3,4. In contrast, the Spectrum Typhoid IgG/IgM Rapid Test is a simple, fast laboratory test that simultaneously detects and differentiates IgG and IgM antibodies to S. typhi and paratyphi antigen5 thus aiding in the determination of current or previous exposure to S. typhi and paratyphi. IgM positive or IgM /IgG both positive suggest current infection, while IgG positive suggests late stage of infection, previous infection, or latent infection.
TEST PRINCIPLE
The Spectrum Typhoid IgG/IgM Rapid Test is a lateral flow chromatographic immunoassay. The test cassette consists of: 1) a burgundy colored conjugate pad containing recombinant H antigen and O antigen conjugated with colloidal gold (HO conjugates) and rabbit IgG-gold conjugates, 2) a nitrocellulose membrane strip containing two test bands (G and M bands) and a control band (C band). The M band is pre-coated with monoclonal anti-human IgM for the detection of IgM anti-S. typhi and paratyphi, G band is pre-coated with reagents for the detection of IgG anti-S. typhi and paratyphi , and the C band is pre-coated with goat anti rabbit IgG.
When an adequate volume of test specimen is dispensed into the sample well of the cassette, the test specimen migrates by capillary action across the test cassette. IgM antibodies if present in the patient specimen will bind to the HO conjugates. The immunocomplex is then captured on the membrane by the precoated anti-human IgM antibody, forming a burgundy colored M band, indicating a S. typhi or paratyphi IgM positive test result. IgG antibodies if present in the patient specimen will bind to the HO conjugates. The immunocomplex is then captured by the precoated reagents on the membrane, forming a burgundy colored G band, indicating a S. typhi or paratyphi IgG positive test result. Absence of any test bands (M and G) suggests a negative result. The test contains an internal control (C band) which should exhibit a burgundy colored band of the immunocomplex goat anti-rabbit IgG/rabbit IgG-gold conjugate regardless of the color development on any of the test bands. Otherwise, the test result is invalid and the specimen must be retested with another device.
REAGENTS AND MATERIALS PROVIDED 1. 2. 3. 4.
Individually sealed foil pouches containing: a. One cassette device. b. One desiccant. Plastic droppers. Sample Diluent (1 bottle, 5 mL) One package insert (instruction for use).
MATERIALS MAY BE REQUIRED AND NOT PROVIDED 1. 2.
304
Positive Control Negative Control
PERFORMANCE CHARACTERISTICS
SYMBOLS IN PRODUCT LABELLING
LOT
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
1. Clinical Performance For IgM Test A total of 334 samples from susceptible subjects were tested by the Spectrum Typhoid IgG/IgM Rapid Test and by a commercial S. typhi IgM EIA. Comparison for all subjects is shown in the following table.
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
Spectrum Typhoid IgG/IgM Rapid Test
MATERIALS REQUIRED BUT NOT PROVIDED
Step 5: Set up timer. Step 6: Results can be read in 15 minutes.
WARNINGS AND PRECAUTIONS
Don’t read result after 15 minutes. To avoid confusion, discard the test device after interpreting the result.
1.
Clock or Timer
For in Vitro Diagnostic Use 1. This package insert must be read completely before performing the test. Failure to follow the insert gives inaccurate test results. 2. Do not open the sealed pouch, unless ready to conduct the assay. 3. Do not use expired devices. 4. Bring all reagents to room temperature (15°C-30°C) before use. 5. Do not use the components in any other type of test kit as a substitute for the components in this kit. 6. Do not use hemolized blood specimen for testing. 7. Wear protective clothing and disposable gloves while handling the kit reagents and clinical specimens. Wash hands thoroughly after performing the test. 8. Users of this test should follow the US CDC Universal Precautions for prevention of transmission of HIV, HBV and other blood-borne pathogens. 9. Do not smoke, drink, or eat in areas where specimens or kit reagents are being handled. 10. Dispose of all specimens and materials used to perform the test as biohazardous waste. 11. Handle the Negative and Positive Control in the same manner as patient specimens. 12. The testing results should be read within 15 minutes after a specimen is applied to the sample well or sample pad of the device. Reading the test after 15 minutes may give erroneous results. 13. Do not perform the test in a room with strong air flow, ie. an electric fan or strong air-conditioning.
REAGENT PREPARATION INSTRUCTIONS
AND
Positive
Negative
Total
Positive
31
3
34
Negative
2
298
300
Total
33
302
334
Relative Sensitivity: 91%, Relative Specificity: 99.3%, Overall Agreement: 98.5%
QUALITY CONTROL 1.
2.
Internal Control: This test contains a built-in control feature, the C band. The C line develops after adding specimen and sample diluent. Otherwise, review the whole procedure and repeat test with a new device. External Control: Good Laboratory Practice recommends using external controls, positive and negative, to assure the proper performance of the assay, particularly under the following circumstances: a. New operator uses the kit, prior to performing testing of specimens. b. A new lot of test kit is used. c. A new shipment of kits is used. d. The temperature used during storage of the kit falls outside of 2-30°C. e. The temperature of the test area falls outside of 15 -30°C. f. To verify a higher than expected frequency of positive or negative results. g. To investigate the cause of repeated invalid results.
INTERPRETATION OF ASSAY RESULT 1.
NEGATIVE OR NON-REACTIVE RESULT: If only the C band is present, the absence of any burgundy color in the both test bands (M and G) indicates that no anti-S. typhi or paratyphi antibody is detected in the specimen. The result is negative or non-reactive.
2. Clinical Performance For IgG Test A total of 314 samples from susceptible subjects were tested by the Spectrum Typhoid IgG/IgM Rapid Test and by a commercial S. typhi IgG EIA kit. Comparison for all subjects is shown in the following table. Spectrum Typhoid IgG/IgM Rapid Test IgG EIA
Positive
Negative
Total
Positive
13
1
14
Negative
2
298
300
Total
15
299
314
Relative Sensitivity: 92.9% , Relative Specificity: 99.3%, Overall Agreement: 99.0% 3. Performance comparison with blood culture Nine (9) S. paratyphi A and eleven (11) S. typhi specimens confirmed with the blood culture were tested with the Spectrum Typhoid IgG/IgM Rapid Test. The Spectrum Typhoid IgG/IgM Rapid Test correctly indentified 9 S. paratyphi A and 10 S. typhi specimens. The agreement was 95%.
LIMITATIONS OF TEST 1.
STORAGE
All reagents are ready to use as supplied. Store unused test devices unopened at 2°C-30°C. If stored at 2°C-8°C, ensure that the test device is brought to room temperature before opening. The test device is stable through the expiration date printed on the sealed pouch. Do not freeze the kit or expose the kit over 30°C.
IgM EIA
2.
SPECIMEN COLLECTION AND HANDLING
Consider any materials of human origin as infectious and handle them using standard biosafety procedures. Plasma 1. Collect blood specimen into a lavender, blue or green top collection tube (containing EDTA, citrate or heparin, respectively in Vacutainer® ) by veinpuncture. 2. Separate the plasma by centrifugation. 3. Carefully withdraw the plasma into a new pre-labeled tube. Serum 1. Collect blood specimen into a red top collection tube (containing no anticoagulants in Vacutainer®) by veinpuncture. 2. Allow the blood to clot. 3. Separate the serum by centrifugation. 4. Carefully withdraw the serum into a new pre-labeled tube.
POSITIVE OR REACTIVE RESULT: a. In addition to the presence of C band, if only M band is developed, the test indicates for the presence of anti- S. typhi or paratyphi IgM in the specimen. The result is IgM positive or reactive.
2.
3. b.
In addition to the presence of C band, if only G band is developed, the test indicates for the presence of anti- S. typhi or paratyphi IgG in the specimen. The result is IgG positive or reactive.
4.
5. c.
Test specimens as soon as possible after collecting. Store specimens at 2°C-8°C if not tested immediately.
In addition to the presence of C band, both M and G bands are developed, the test indicates for the presence of anti-S. typhi or paratyphi IgG and IgM in the specimen. The result is both IgG and IgM positive or reactive.
6. 7.
Store specimens at 2°C-8°C for up to 5 days. The specimens should be frozen at -20°C for longer storage. Avoid multiple freeze-thaw cycles. Prior to testing, bring frozen specimens to room temperature slowly and mix gently. Specimens containing visible particulate matter should be clarified by centrifugation before testing. Do not use samples demonstrating gross lipemia, gross hemolysis or turbidity in order to avoid interference on result interpretation.
ASSAY PROCEDURE
Step 1: Bring the specimen and test components to room temperature if refrigerated or frozen. Mix the specimen well prior to assay once thawed. Step 2: When ready to test, open the pouch at the notch and remove device. Place the test device on a clean, flat surface. Step 3: Be sure to label the device with specimen’s ID number. Step 4: Fill the plastic dropper with the specimen. Holding the dropper vertically, dispense 1 drop (about 30-45 mL) of specimen into the sample well making sure that there are no air bubbles. Then add 1 drop (about 35-50 µL) of Sample Diluent immediately.
The Assay Procedure and the Test Result Interpretation must be followed closely when testing the presence of antibodies to S. typhi or paratyphi in serum or plasma from individual subjects. Failure to follow the procedure may give inaccurate results. The Spectrum Typhoid IgG/IgM Rapid Test is limited to the qualitative detection of antibodies to S. typhi or paratyphi in human serum or plasma. The intensity of the test band does not have linear correlation with the antibody titer in the specimen. A negative result for an individual subject indicates absence of detectable anti-S. typhi or paratyphi antibodies. However, a negative test result does not preclude the possibility of exposure to S. typhi or paratyphi . A negative result can occur if the quantity of anti-S. typhi or paratyphi antibodies present in the specimen is below the detection limit of the assay, or the antibodies that are detected are not present during the stage of disease in which a sample is collected. If the symptom persists, while the result from Spectrum Typhoid IgG/IgM Rapid Test is negative or non-reactive result, it is recommended to re-sample the patient few days late or test with an alternative test method, such as bacterial culture method. Some specimens containing unusually high titer of heterophile antibodies or rheumatoid factor may affect expected results. The results obtained with this test should only be interpreted in conjunction with other diagnostic procedures and clinical findings.
REFERENCES Samples with positive or reactive results should be confirmed with alternative testing method(s) and clinical findings before a positive determination is made. 3.
INVALID: If no C band is developed, the assay is invalid regardless of any burgundy color in the test bands as indicated below. Repeat the assay with a new device.
1.
2. 3. 4. 5.
Gotuzzo E, Frisancho O, Sanchez J, Liendo G, Carillo C, Black RE, Morris JG. Association between the acquired immunodeficiency syndrome and infection with Salmonella typhi or Salmonella paratyphi in an endemic typhoid area. Archives of Internal Medicine 1991; 151: 381-2. Ivanoff BN, Levine MM, Lambert PH. Vaccination against typhoid fever: present status. Bulletin of the World Health Organization 1994; 72: 957-71. Ismail A, Hai OK, Kader ZA. Demonstration of an antigenic protein specific for Salmonella typhi. Biochem Biophys Res Commun. 1991;181(1):301-5. Pang T. False positive Widal test in nontyphoid Salmonella infection. Southeast Asian Journal of Tropical Medicine and Public Health 1989; 20: 163-4.
305
URI-TRAK Test strips for rapid determination of Glucose, Ketones, Protein and pH-value in Urine
SYMBOLS IN PRODUCT LABELLING EC REP Authorised Representative IVD LOT REF
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
REF: 1100 010 (Uri-trak 1) for Glucose REF: 1100 020 (Uri-trak 2) for Glucose & Ketones REF: 1100 030 (Uri-trak 3) for Glucose, Protein & pH
pH: The pH value of fresh urine of healthy people varies between pH 5 and pH 6. The colour scale gives a clear distinction of pH value between pH 5 and pH 9.
Reagent Storage and Stability
o
Store at room temperature (15 to 30 C) out of direct sunlight. Do not use after expiration date.
Precautions The Kit contain a non-poisonous and harmless desiccant. In case this desiccant is swallowed accidently, then drink plenty of water.
Intended Use
Instructions for use
Disposal
Spectrum Uri-trak is a screening test strips for detection of glucose, ketones, protein and pH-value in urine. Certain configuration of strips may be read instrumentally, using the appropriate Urine Chemistry Analyzers.
Dip the test strip for approximately 1 second into the fresh urine. Draw it across the rim of the container to remove excess urine. After 30 to 60 seconds compare the test strip with the colour scale. The best time for ncomparison is after 30 seconds. Colour changes that take place after more than 2 minutes are of no significance. When tested the urine should not be older than 2 hours.
Please dispose all used dipsticks in accordance with your local laws and regulations.
Principle Glucose: The detection is based on the glucoseoxidase-peroxidasechromogen reaction. Apart from glucose, no other compound in urine is known to give a positive reaction. Ketones: The test is based on the principle of Legal’s test. Acetoacetic acid and acetone form with sodium nitroprusside in alkaline medium a violet coloured complex. Protein: The test is based on the „protein error“ principle of indicators. The test zone is buffered to a constant pH value and changes colour from yellow to greenish blue in the presence of albumin. Other proteins are indicated with less sensitivity. pH: The test paper contains indicators which clearly change colour between pH 5 and pH 9 (from orange to green to turquoise).
Reactive Ingredients Glucose: 2% w/w glucose oxidase; 1% w/w peroxidase; 10% w/w TMB; 70% w/w Buffer; 17% w/w nonreactive ingredients. Ketones: 5% w/w sodium nitroprusside; 95% w/w Buffer. Protein: 0.2% w/w tetrabromophenol blue; 97.4% w/w Buffer; 2.4% w/w nonreactive ingredients. pH: 0.2% w/w methyl red; 2.8% w/w bromothymol blue; 97% w/w nonreactive ingredients.
Specimen Collection Use a fresh urine samples that is less than 2 hours old and place it in a clean, dry container. Do not centrifuge. The presence of usual urine preservatives will not affect the test results.
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Evaluation – Sources of Error Glucose: Pathological glucose concentrations are indicated by a colour change from green to bluish green. Yellow or greenish test fields should be considered negative or normal. The colour fields correspond to the following ranges of glucose concentrations: neg. (yellow), neg. or normal (greenish), 50, 150, 500 and 1000 mg/ dl or neg. (yellow), neg. or normal (greenish), 2.8, 8.3, 27.8 and 55.5 mmol/l The influence of ascorbic acid (vitamin C) has been largely eliminated. An inhibitory effect is produced by gentisic acid. Falsely positive reactions can be produced by a residue of peroxide containing cleansing agents.
ORDERING INFORMATION CATALOG NO
QUANTITY
1100 010 1100 020 1100 030
100 Test Strip for each
Ketones: The test is more sensitive to acetoacetic acid than to acetone. Values of 10 mg/dl acetoacetic acid or 50 mg/dl acetone are indicated. The colour fields correspond to the following acetoacetic acid values: 0 (negative), 25(+), 100(++) and 300(+++) mg/dl or 0 (negative), 2.5(+), 10(++) and 30(+++) mmol/l Phenylketones in higher concentrations interfere with the test, and will produce variable colours. b-Hydroxybutyric acid is not detected. Phthalein compounds interfere by producing a red colouration. Protein: The minimum sensitivity of the test strip is 10 mg protein/ dl urine. The colour fields correspond to the following ranges of albumin concentrations: negative, 30, 100 and 500 mg/dl or negative, 0.3, 1.0 and 5.0 g/l Falsely positive results are possible in alkaline urine samples (pH L 9), after infusions with polyvinylpyrrolidone (blood substitute), after intake of medicaments containing quinine and also by disinfectant residues in the urine sampling vessel. The protein colouration may be masked by the presence of medical dyes (e.g. methylene blue) or beetroot pigments.
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URI-TRAK-10
REAGENT STRIPS FOR URINALYSIS
SYMBOLS IN PRODUCT LABELLING EC REP Authorised Representative IVD
REF: 1100 100 (Uri-trak -10) for Ten Parameters
Summary and Explanation
URI-TRAK-10 Reagent Strips are dip-and-read test strips for In Vitro Diagnostic Use only for testing ten parameters in urine. Test result may provide information regarding the status of carbohydrate metabolism, kidney and liver function, acid-base balance, and urinary tract infection. It is measured by comparison of test paper attached to a plastic strip with the color chart blocks printed on the vial label. The strips may be read visually. They can also be read instrumentally, using urine chemistry analyzers.
Principle
Blood: The test is based on the Pseudo-peroxidase activity of the haem moiety of hemoglobin and myoglobin. The chromogen is oxidized by a hydroperoxide in the presence of haem and changes color from yellow (or greenish yellow) to blue. Ingredients: diisopropylbenzene dihydro peroxide 26.0%W/W tetramethylbenzidine 1.5%W/W Bilirubin: Azo-coupling reaction of bilirubin with a diazonium salt in an acid medium to form an azodye. Color changes from light tan to beige or light pink. Ingredients: 2,4-dichloroaniline diazonium salt 0.6%W/W Urobilinogen: The test is based on the Ehrlich’s reaction. Color changes from light orange-pink to dark pink. Ingredients: fast blue B salt 0.2%W/W Ketone: Legal’s test-nitroprusside reaction. Acetoacetic acid in an alkaline medium reacts with nitroferricanide to produce a color change from beige to purple. Ingredients: sodium nitroprusside 5.7%W/W Glucose: Glucose oxidase catalyzes the oxidation of glucose to form hydrogen peroxide. The hydrogen peroxide thus formed then oxidizes a chromogen on the reaction pad by the action of peroxidase. Ingredients: glucose oxidase (microbial.123U) 1.7%W/W peroxidase(horseradish.203U) 0.2%W/W tetramethylbenzidine 0.1%W/W Protein: Protein “error of indicators.” When pH is held constant by a buffer, indicator dyes release H+ ions because of the protein present and change color from yellow (or greenish yellow) to bluegreen. Ingredients: tetrabromophenol blue 0.1%W/W Nitrite: The test is based on the diazotization reaction of nitrite with an aromatic amine to produce a diazonium salt. It is followed by an azo-coupling reaction of this diazonium salt with an aromatic compound on the reaction pad. The azo dye produced causes a color change form white to pink. Ingredients: p-arsanilicacid-N-(1-Naphthol)-ethylenediamine 1.3%W/W tetrahydroquinoline 0.9%W/W Leukocyte: This test pad contains an indoxyl ester and diazonium salt. It is followed by an azo-coupling reaction of the aromatic amine formed by leukocytes esterase with a diazonium salt on the reaction pad. The azo dye produced causes a color change from beige to violet. Ingredients: indoxyl ester 4.3%W/W phenyl diazonium salt 0.4%W/W pH: Double indicator system. Indicator’s methyl red and bromothymol blue are used to give distinct color changes from orange to green to blue. (pH 5.0 to 9.0) Ingredients: methyl red 3.3%W/W bromthymol blue 55.0%W/W
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LOT REF
For in-vitro diagnostic use Batch Code/Lot number Catalogue Number Consult instructions for use
1
3
Temperature Limitation Use by/Expiration Date CAUTION. Consult instructions for use Manufactured by
Blood: Normally, no hemoglobin is detectable in urine (0.010mg/dl; 3 RBC/µl). When hemoglobin appears in urine it indicates kidney disease or a urinary tract disorder. Blood may often be found in the urine of menstruating females.
Quality control
Storage and Handling
Limitations of procedure
Store in a cool, dry place at temperatures between 2OC ~ 30OC. Do not store the strips in a refrigerator or freezer. Store away from moisture and light. When stored in the original container, the product is stable up to the expiry date printed on the label and (or) vial box. Replace the bottle cap immediately and tightly after removing test strips, and keep the vial tightly closed between tests. Do not remove desiccant from bottle. Do not touch test areas of urine reagent strips. Do not open container until ready to use. Discoloration or darkening of the test pads may indicate deterioration. If this is evident, or if test results are questionable or inconsistent with expected finding, confirm that the product is within its expiration date and is reacting properly using known negative and positive control materials. Do not use after the expiry date. Note once the canister has been opened, the remaining strips remain stable for up to 6 months.
Specimen Collection and Preparation
Collect urine in a clean, dry container that allows complete immersion of all the fields on the test strip. Do not add preservatives. Test the specimen as soon as possible, with the sample well mixed but not centrifuged. The use of fresh morning urine is recommended for optimal nitrite tests, as well as for the valid determination of bilirubin and urobilinogen, since these compounds are unstable when exposed to light. If immediate testing is not possible, the sample should be stored in the refrigerator, but not frozen, and then brought to room temperature before used in the test. Unpreserved urine at room temperature may undergo pH changes due to microbial proliferation, which may interfere with protein determination. If cleanly voided specimens are not collected from females, positive results for leukocytes may be found due to contamination from outside the urinary tract. Skin cleansers containing chlorhexidine may affect protein test results if specimen contamination occurs. The procedure must be followed exactly to achieve reliable results. Do not compare strips with color chart before the strip is dipped in urine. 1. Dip the strip into the urine up to the test area for no more than two second. 2. Draw the edge of the strip along the brim of the vessel to remove excess urine; at this time, don’t make the test areas touched to the brim of the vessel.Turn the strip on its side and tap once on a piece of absorbent material to remove any remaining urine; Excessive urine on the strip may cause the interaction of chemicals between adjacent reagent pads, so that an incorrect result may occur. 3. Compare the colours of the reagent pads exactly after 60 seconds (Leukocytes after 90~120 seconds) with the color chart on the vial label under good light. While comparing, keep the strip horizontally to prevent possible mixing of chemicals when excessive urine is present.
Specific Gravity (SG): High-buffered alkaline urine may cause diminished result, whereas high-buffered acidic urine may cause slightly elevated result.
Expected values
Specific Gravity (SG): Ionic solutes present in the urine cause protons to be released from a polyelectrolyte. As the protons are released the pH decreases and produces a color change of bromothymol blue from blue-green to yellow-green. Ingredients: bromthymol blue 4.8%W/W poly(methyl vinyl ether-alt- maleic anhydride) 90.2%W/W
Instructions for use
2
For best results, performance of reagent strips should be confirmed by testing known negative and positive specimen or controls whenever a new bottle is first opened. Each laboratory should establish its own goals for adequate standards of performance. Each lab worker should ensure that it complies with government and local requirements. As with all laboratory tests, definitive diagnostic or therapeutic decisions should not be based on any single result of method. Substances that cause abnormal urine color may affect the readability of test pads in urinalysis reagent strips. Blood:. Elevated specific gravity or protein in urine may reduce the reactivity of the blood test portion. Microbial peroxidase associated with urinary tract infection may cause false positive results. Ascorbic acid concentrations (>30 mg/dl) may cause false negatives at the low level of blood. Bilirubin: Metabolites of drugs, such as pyridum and selenium, which give a color at low pH, may cause false positives. Indican (indoxyl sulfate) can produce a yellow-orange to red color response, which may interfere with the interpretation of negative or positive bilirubin readings. Ascorbic acid (> 30mg/dl) may cause false negative result. Urobilinogen: The absence of urobilinogen in the specimen cannot be determined. The test area will react with interfering substances known to react with Ehrlich’s reagent, such as p-aminosalicylic acid. Drugs containing azo gantrisin may give a masking golden color. The test is not reliable method for the detection of porphobilinogen. Ketone: Positive results (trace or less) may occur with highly pigmented urine specimens or those containing large amounts of levodopa metabolites. Some high SG and low pH urine may give false positive result. Phenosulfonphthalein may cause false positive result. Glucose: High SG (>1.020) with high pH urine and ascorbic acid (more than 40mg/dl) may cause false negative result at the low level of glucose. Protein: False positive results may be found in strongly basic urine (pH 9). The interpretation of results is also difficult in turbid urine specimens. Nitrite: Ascorbic acid (>30mg/dL) may cause false negative result with low level of nitrite containing (
