ALKALINE PHOSPHATASE (S.L) R2

Transcription

1 ALKALINE PHOSPHATASE (S.L) determination of Alkaline Phosphatase in serum or plasma. DGKC SCE recommended procedure Linear up to 700 U/L Working reagent can be prepared as per requirement Pack sizes to suit all types of laboratories. Alkaline phosphatase (ALP is widely distributed throughout the body, but clinically important one for diagnostic reasons are in bone, liver, placenta & intestine. Growing bone is associated with the release of ALP and so in childhood the level of ALP is around 3 times of that of adult. During pregnancy in 2 nd & 3 rd trimester the enzyme rises considerably due to placenta releasing ALP. It can be used to examine placental function. Elevated levels are seen in bone diseases, e.g. pagets disease, rickets, osteoblastic metastatic & in obstructive disease of biliary tract. Decreased levels are rarely seen. e.g. in Vitamin A resistant rickets. Kinetic determination of ALP according to the following reaction ALP Paranitrophenyl phosphate + H 2 O >p-nitrophenol+inorgnic phosphate ALP = Alkaline Phosphatase ALKALINE PHOSPHATASE (S.L) R1 2 x 24 ml / 2 x 40 ml Diethanolamine Buffer (ph 10.2) 125 mmol/l Magnesium Chloride mmol/l ALKALINE PHOSPHATASE (S.L) R2 2 x 6 ml / 2 x 10 ml P-Nitrophenyl phosphate 50 mmol /L when stored at C. The reagent is linear, up to 700 U/L. If the concentration is greater than linearity (700 U/L), dilute the sample with normal saline & repeat the assay. Multiply the result with dilution Women U/L Men U/L Children up to 15 yrs < 644 U/L Children up to 17 yrs < 483 U/L PREPARATION AND STABLITY OF WORKING REAGENT Mix 4 volume of Reagent 1 (R1) with 1 volume Reagent 2 (R2) The working reagent is stable for 30 days at C. Note : Discard the working reagent if the blank absorbance exceeds 1.00 at 405 nm. Avoid direct exposure of working reagent to light. Serum / plasma (free of haemolysis) Kinetic Wavelength 405 nm 37 0 C Factor 2750 DI water 700 U/L Delay time 60 sec No of readings 3 Interval 60 sec Sample volume 20 µl 2 x 30 ml, 2 x 50 ml , Working reagent 1000 µl Sample 20 µl Mix and incubate at 37 0 C for one minute. Measure the change in absorbance per minute ( OD/min) during 3 minutes. ALP Activity (U/L) = ( OD / min.) x Schlebusch, H.etal., Dtsch.med. Wschr. 99, 765 (1974) 2.Z. Klin. Chem. Klin Biochem. 8,658 (1980) 10, 182 (1972)

2 CHOLESTEROL (S.L) determination of Cholesterol in serum or plasma. CHOD-PAP methodology. Linear up to 600 mg/dl. Contains LCF (Lipaemic clearing factor) which minimizes rerun. It is the main lipid found in the blood, bile & brain tissues. It is also one of the most important steroids of the body & is a precursor of many steroid hormones. Two thirds of cholesterol present in the blood is esterified. The liver metabolizes the cholesterol & it is transported in the blood stream by lipoproteins. Increased levels are found in hypercholesterolemia, hyperlipidemia, hypothyroidism, uncontrolled diabetes, nephritic syndrome & cirrhosis. Decreased levels are found in malabsorption, malnutrition, hyperthyroidism, anaemia & liver diseases. Enzymatic colorimetric determination of total cholesterol according to the following reactions. Cholesterol esterase Cholesterol ester +H 2 O > Cholesterol + fatty acids Cholesterol Oxidase Cholesterol + O > 4-Cholesten-3- one + H 2 O 2 Peroxidase 2H 2 O 2 +Phenol+4-Aminoantipyrine > - Red quinone + 4H 2 O CHOLESTEROL (S.L) R1 5 x 25 ml / 5 x 100 ml / 4 x 250 ml Pipes buffer (ph 6.70) 50 mmol/l Phenol 24 mmol/l Sodium Cholate 0.5 mml/l 4-aminoantipyrine 0.5 mmol/l Cholesterol Esterase > 180 U/L Cholesterol Oxidase > 200 U/L Peroxidase > 1000 U/L CHOLESTEROL STANDARD 1x5 ml Cholesterol standard concentration 200 mg/dl Note: For 8 x 100 ml pack: Buffer will be provided separately. One reconstitution bottle will be provided. when stored at C. This reagent is linear up to 600 mg/dl. If the concentration is greater than linearity (600 mg/dl), dilute the sample with normal saline and repeat the assay. Multiply the result with dilution Serum / Plasma : mg/dl The reagent is ready to use. Avoid direct exposure of reagent to light. Serum / Plasma (free of haemolysis). 5 x 25 ml, 5 x 100 ml, 4 x 250 ml , , End point Wavelength I 505 ( ) nm Wavelength II 630 nm 37 0 C Standard Concentration 200 mg/dl Reagent 600 mg/dl Incubation time 5 min Sample volume 10 µl. Standard Sample Working Reagent 1000 µl 1000 µl 1000 µl Standard - 10 µl - Sample µl Mix, and incubate for 5 min. at 37 0 C. Measure the absorbance of sample and standard against reagent blank. Cholesterol Conc. (mg/dl) Absorbance of sample = x 200 Absorbance of standard Allain C.C.et al., Cain.Chem 20 (1974), 470

3 GLUCOSE (S.L) determination of Glucose in serum, plasma & CSF. GOD PAP methodology Linear up to 600 mg/dl Glucose is a major carbohydrate present in the blood & serves as a primary source of energy. It is usually obtained from ingested starch & sugar. The glucose concentration is normally maintained at constant level. Excessive glucose is stored as a inactive glycogen mainly in the liver & little in the muscles. Elevated blood glucose levels are found in diabetes mellitus, hyperthyroidism, hyperadrenalism & certain liver diseases. Decreased levels are found in Insulinoma, hypothyroidism, hypopituitarism. Enzymatic colorimetric determination of glucose according to the following reaction. Glucose Oxidase Glucose+ O 2 + H 2 O > Gluconic acid + H 2 O 2 Standard Sample Working Reagent 1000 µl 1000 µl 1000 µl Standard - 10 µl - Sample µl Mix and incubate for 10 minutes at 37 0 C. Read the absorbance of standard and sample against reagent blank. Absorbance of Sample Glucose Conc. (mg/dl) = x 100 Absorbance of standard 1.Trinder, P.Ann Clin Biochem. 6,24 (1969) 2.Dingeon, B.Ann.Bio.Clin 33,3 (1975) 3.Lott, J. Clin.Chem. 21, 1754 (1975) 5 x 100 ml, 1 X 1000 ml , Peroxidase 2H 2 O 2 +phenol + 4-Aminoantipyrine > Quinonimine + 4H 2 O GLUCOSE (S.L) R1 5 x 100 ml / 1 x 1000 ml Tris Buffer, (ph 7.40) 92 mmol/l Phenol 0.3 mmol/l Glucose Oxidase U/L 4- Aminophenazone 2.6 mmol/l GLUCOSE STANDARD 1 x 5 ml Glucose standard concentration 100 mg/dl when stored at C. This reagent is linear up to 600 mg/dl If the concentration is greater than linearity (600 mg/dl), dilute the sample with normal saline and repeat the assay. Multiply the result with dilution Serum / Plasma : mg/dl C S F : mg/dl The Reagent is ready to use. Avoid direct exposure of reagent to light. Serum / plasma (free of haemolysis) / CSF End Point Wavelength 505 ( nm) 37 0 C Standard Concentration 100 mg/dl 600 mg/dl Incubation Time 10 Minutes Reagent Sample volume 10 µl

4 SGOT (S.L) determination of SGOT in serum or plasma. IFCC recommended procedure Linear up to 1000 U/L. Working reagent can be prepared as per requirements It is present in most of the tissues. Especially in cardiac muscle, liver cells, skeletal muscle & kidneys. Injury to these tissues results in the release of the enzyme in blood stream. Increased levels are found in myocardial infarction. The duration & extent of increase is related to the infract. GOT determination is of considerable value to differentiate myocardial infarction from other cardiac disorders. Increased levels are also found in various types of liver disease, skeletal muscle trauma & in renal diseases. Decreased levels may be found in pregnancy, Beri-Beri & Diabetic ketoacidosis. Kinetic determination of Aspartate Aminotrasferase (AST) based upon the following reaction. AST L- Asparate + α - ketoglutarate > Oxaloacetate + L-Glutamate. MDH Oxaloacetate + NADH + H > L- Malate + NAD + AST Aspartate aminotransferase. MDH : Malate dehydrogenase. SGOT (S.L) R1 2 x 24 ml / 3 x 40 ml / 4 x 100 ml Tris Buffer (ph 7.8) 88 mmol/l L-Aspartate 260 mmol/l LDH >1500 U/L MDH > 900 U/L SGOT (S.L) R2 2 x 6 ml / 3 x 10 ml / 4 x 25 ml α -ketoglutarate 12 mmol/l NADH 0.24 mmol/l when stored at C. This reagent is linear up to 350 U/L. If the concentration is greater than linearity (1000 U/L), follow the high linearity procedure. If the concentration is greater than linearity, dilute the sample with normal saline and repeat the assay. Multiply the result with dilution Serum up to : 46 U/L Mix 4 volumes of Reagent 1 (R1 ) with the volume of Reagent 2 (R2) The working reagent is stable for 30 days at C. NOTE: Discard the working reagent if the blank absorbance is less than 1.0 at 340 nm. Avoid direct exposure of working reagent to light. Serum / plasma (free of haemolysis) Normal procedure High procedure Kinetic Kinetic Decreasing Decreasing Wavelength 340 nm 340 nm 2 x 30 ml, 3 x 50 ml, 4 x 125 ml , , C 37 0 C Factor U/L 1000 U/L DI Water DI Water Delay 60 sec 60 sec No of reading 3 3 Interval 60 sec 20 sec Sample volume 100 µl 100 µl 1000 µl Working reagent 1000 µl Sample 100 µl Mix and incubate at 37 0 C for 1 minute. Measure the change in absorbance per minute ( OD/min) during 3 minutes. High Procedure Mix and incubate at for 1 minutes 37 0 C. Read the change in absorbance per 20 sec, ( OD/ 20 sec) during 1 minute. SGOT activity (U/L) = ( OD/min) x Clin. Clim, Acta. 70, (1976) 2.Thefeld, w.et al.dtsch. med wschr.99, 343 (1974)

5 SGPT (S.L) determination of SGPT in serum or plasma. IFCC recommended methodology Linear up to 1000 U/L. Working reagent can be prepared as per requirements It is present in most of the tissues, but mainly found in the liver. Increased levels are found in hepatitis, cirrhosis, obstructive jaundice & other hepatic disease. SGPT activity is markedly elevated even before clinical signs of jaundice become apparent in disease associated with hepatic necrosis. Slight elevations are also found in myocardial infarction. Kinetic determination of Alanine Aminotransferase (ALAT) according to the following reaction. ALT L-Alanine + α ketogutarate >Pyruvate +L-Glutamate LDH Pyruvate +NADH+ H > L-Lactate +NAD + ALT Alanine aminotranferase LDH - Lactate dehydrogenase SGPT (S.L) R1 2 x 24 ml / 3 x 40 ml / 4 x 100 ml Tris buffer (ph 7.5) 110 mmol/l L-Alanine 600 mmol/l LDH > 1500 U/L SGPT (S.L) R2 2 x 6 ml / 3 x 10 ml / 4 x 25 ml α-ketoglutarate 16 mmol/l NADH 0.24 mmol/l when stored at C. This reagent is linear up to 350 U/L. For getting linearity up to 1000 U/L, follow high linearity procedure. If the concentration is greater than linearity dilute the sample with normal saline and repeat the assay. Multiply the result with dilution Serum up to : 49 U/L Mix 4 volumes of Reagent 1 (R1) with 1 volume of Reagent 2 (R2) The working reagent is stable for 30 days at C. NOTE: Discard the working reagent if the blank absorbance is less than 1.00 at 340 nm. To avoid contamination use clean laboratory wares. Avoid direct exposure of working reagent to light. Serum / plasma (free of haemolysis) Normal procedure High procedure Kinetic Kinetic Decreasing Decreasing Wavelength 340 nm 340 nm 37 0 C 37 0 C Factor U/L 1000 U/L DI Water DI Water Delay 60 sec 60 sec No of reading 3 3 Interval 60 sec 20 sec Sample volume 100 µl 100 µl 1000 µl 2 x 30 ml, 3 x 50 ml, 4 x 125 ml , , Working reagent 1000 µl Sample 100 µl Mix and incubate at 37 0 C for 1 minute. Measure the change in absorbance per minute ( OD/min) during 3 minutes. High Procedure Mix and incubate for 1 minute at 37 0 C. Read the change in absorbance per 20 sec ( OD/20 sec) during 1 minutes. SGPT activity (U/L) = ( OD/min) x Clin. Clim, Acta. 105, (1780) 2.Thefeld, w.et al.dtsch. med wschr.99, 343 (1994)

6 TRIGLYCERIDES (S.L) determination of triglycerides in serum or plasma. GPO-PAP methodology Linear up to 1000 mg/dl. Contains LCF (Lipaemic clearing factor) which minimizes rerun. Triglycerides are simple lipids, formed in the liver by glycerol & fatty acids. They are transported by VLDL, LDL & constitute about 95% of fat, stored as source of energy in the tissue & plasma. Increased levels are found in hyperlipidemias, diabetes, nephrotic syndrome & hypothyroidism. Increased levels are risk factor for arteriosclerotic coronary disease, peripheral vascular disease, acute pancreatitis & hyperlipoproteinaemia. Decreased levels are found in malnutrition & hyperthyroidism. Enzymatic determination of triglyceride is based on following reactions: LPL TGL+H 2 O >Glycerol + Fatty acid GK Glycerol + ATP > Glycerol-3-phosphate + ADP Mg ++ GPO Glycerol-3-phospahte+O > Dihydroxyacetone phosphate +H 2 O 2 POD 2H 2 O 2 +4-Aminoantipyrine+p-Chlorophenol > Red quinonemine GPO = Glycereol-3-phosphate Oxidase. LPL = Lipoprotein Lipase GK = Glycerol Kinase TRIGLYCERIDES(S.L) R1 5 x 25 ml / 4 x 50 ml / 5 x 100 ml Pipes buffer (ph 7.00) 50 mmol/l p-chlorophenol 5.3 mmol/l Potassium ferrocynate 10 mmol/l Magnesium Salt 17 mmol/l 4-Aminoantipyrine 0.9 mmol/l ATP 3.15 mmol/l Lipoprotein Lipase > 1800 U/L Glycerol Kinase > 450 U/L Glycerol 3- phosphate oxidase > 3500 U/L Peroxidase > 450 U/L TRIGLYCERIDES STANDARD 1 x 5 ml Triglycerides standard concentration 200 mg/dl when stored at C. This reagent is linear up to 1000 mg/dl. If the concentration is greater than linearity (1000 mg/dl), dilute the sample with normal saline and repeat the assay. Multiply the result with dilution Male : mg/dl Female : mg/dl The reagent ready to use. Avoid direct exposure of working reagent to light. Serum / plasma (free of haemolysis) 5 x 25 ml, 4 x 50 ml, 5 x 100 ml , , End Point Wavelength I 505 nm ( nm) Wavelength II 630 nm 37 0 C Standard Concentration 200 mg/dl 1000 mg/dl Reagent Incubation time 5 min Sample volume 10 µl Standard Sample Working Reagent 1000 µl 1000 µl 1000 µl Standard - 10 µl - Sample µl Mix and incubate for 5 minutes at 37 0 C. Measure the change in absorbance of standard and sample against reagent blank. Absorbance of Sample Triglycerides Con. (mg/dl) = x 200 Absorbance of Standard 1.Schettler G. Nussel, E,Arav. Med 10, 25 (1975) 2.Jacobs, N.J. VanDemark, P.J.Arch, Biochem, Biophy. 88, (1960)

7 UREA - B (S.L) determination of urea in serum, plasma & urine. Modified Berthelot methodology Linear up to 300 mg/dl Highly convenient stable liquid reagent. Non staining dye, does not affect the cuvettes. Proteins cannot be stored in human body, so excess should be broken down. Amino acids which from the components of proteins, break down to give ammonia. This is toxic & so through a series of chemical reactions (urea cycle) non toxic urea is produced & this is released into the blood which is filtered in the kidney & excreted in the urine. Elevated levels are seen during increased protein in breakdown, dehydration, vomiting, diarrhea. Also seen in any kind of renal disorder like Glomerular nephritis, chronic nephritis & Nephritic syndrome. Decreased levels are found in liver failure & pregnancy. Enzymatic determination of Urea according to the following reaction. Urease Urea + H 2 O > 2NH 3 + CO 2 Nitroprusside NH 3 + salicylate > 2-2-Dicarboxy Indophenol Hypochlorite UREA-B (S.L) R1 2 x 50 ml/ 2 x 100 ml Buffer (ph 7) 120 mmol/l Sodium Salicylate 60 mmol/l Sodium nitroprusside 5 mmol/l UREA-B (S.L) R2 2 x 50 ml/ 2 x 100 ml Phosphate buffer, (ph <13) 120 mmol/l Sodium hypochlorite 10 mmol/l UREA-B (S.L) R3 2 x 0.5 ml, 2 x 1.0 ml Enzyme concentrate Urease > 500 K U/L UREA-B (S.L) STANDARD 1 x 5 ml Urea-B Standard concentration 40 mg/dl when stored at C. This reagent is linear up to 300 mg/dl. If the concentration is greater than linearity (300 mg/dl), dilute the sample with normal saline and repeat the assay. Multiply the result with dilution Serum(urea) : mg/dl Urine(urea) : gm/24 hrs Reagent 2 & standard are ready to use. The working reagent is prepared by mixing one bottle of Reagent 3 (R3) with one bottle of Reagent 1 (R1) Working reagent is stable for 120 days at C. *If any turbidity appears in the enzyme concentrate (R3), it does not effect the performance of the test result. The turbidity will disappear after mixing with Reagent 1. Avoid direct exposure of working reagent to light. Serum / plasma (free of haemolysis) / Urine (1/100 Diluted) 4 x 50 ml, 4 x 100 ml , End point Wavelength 578 nm ( nm) 37 0 C Reagent Standard Concentration 40 mg/dl 300 mg/dl Incubation time 5+5 min Sample volume 10 µl µl Standard Sample Working Reagent 1000 µl 1000 µl 1000 µl Standard - 10 µl - Sample µl Mix and incubate for 5 minutes at 37 0 C Reagent R2 (R2) 1000 µl 1000 µl 1000 µl Mix and incubate 5 minutes at 37 0 C. Read the absorbance of the sample and the standard against the reagent blank. Urea Con. (mg/dl) Absorbance of Sample = x 40 Absorbance of Standard 1. Berthelot, M, Report chem. Applique 1, 2 & 4 (1859) 2. Tobacco, A. et al. Clin chem. 25 (2) 336 (1979)

8 URIC ACID (S.L) determination of uric acid in serum, plasma & urine. Uricase PAP methodology Linear up to 25 mg/dl Fast incubation, just 5 minutes at 37 0 C. Uric acid is the end product of purine metabolism. Uric acid is excreted by the kidneys. Increased levels are found in Gout, arthritis, impaired renal functions & starvation. Decreased levels are found in yellow atrophy of the liver. Enzymatic determination of uric acid according to the following reactions. Uricase Uric acid + 2H 2 O+O > Allantoine +CO 2 +H 2 O 2 Peroxidase 2H 2 O Aminoantipyrine +EHSPT > Red quinone EHSPT = N-Ethyl N-(2-Hydroxy-3-Sulfoproyl) n-toluidine URIC ACID R1 2 x 30 ml / 2 x 50 ml / 2 x 100 ml Phosphate Buffer (ph 7.0) 100 mmol/l EHSPT 1.10 mmol/l Ferrocyanure 50 mmol/l Amino-4-antipyrine 0.37 mmol/l Uricase > 140 U/L Peroxidase > 3000 U/L URIC ACID STANDARD 1 x 5 ml Uric acid standard concentration 8 mg/dl when stored at C. This reagent is linear up to 25 mg/dl. If the concentration is greater than linearity (25 mg/dl), dilute the sample with normal saline and repeat the assay. Multiply the result with dilution Serum / Plasma Men : mg/dl Women : mg/dl The reagent is ready to use Note : Discard the reagent if the blank absorbance exceeds 0.3 Avoid direct exposure of reagent to light. Serum / EDTA or Heparin plasma (free of haemolysis) End point Wavelength I 546 nm ( ) Wavelength II 630 nm 37 0 C Standard Concentration 8 mg/dl Reagent 25 mg/dl Sample volume 25 µl 2 x 30 ml, 2 x 50 ml, 2 x 100 ml , , Standard Sample Working Reagent 1000 µl 1000 µl 1000 µl Standard - 25 µl - Sample µl Mix and incubate 5 min. at 37 0 C. Measure absorbance of sample and standard against the reagent blank. Urea Con. (mg/dl) Absorbance of Sample = x 8 Absorbance of Standard 1. Barham, D., Trinder, Analyst 97, 142(1972)

9 AMYLASE determination of amylase in serum, plasma & urine. CNPG 3 methodology. Linear up to 2000 U/L Amylase occurs in the salivary glands, fallopian tubes & in pancreas. α -amylase is secreted by the pancreas from where it enters the duodenum, through the pancreatic duct. Any obstruction to these ducts causes α-amylase enzyme to enter the blood stream. Elevated levels seen in acute pancreatitis, peptic ulcers, biliary disease, parotitis & other intestinal obstructions. Decreased levels are seen in chronic pancreatic disorders having pancreatic cell destruction. Amylase 5CNPG > 3 CNP +2CNPG 2 +3 Maltotriose + 2 Glucose. 4 x 5 ml, 4 x 10 ml , Interval 60 sec Sample volume 25 µl Working reagent 1000 µl Sample 25 µl Mix, and incubate for 1 min, at 37 0 C. Measure the change in absorbance per minute ( OD/min) during 3 minutes. α-amylase activity (U/L) = ( OD/ min.) x Junge, w.,et al., clin. Biochem. 22, 109(1989) 2.Hohenwallnern, w., J.Clin. chem. Clin. Biochem. 27,97(1989) CNP = 2-Chloro-4-nitrophenol CNP-G2= 2-chloro -4-nitrophenyl-α-maltoside ALPHA AMYLASE (S.L)R1 4 x 5 ml / 4 x 10 ml MES Buffer (ph6.0) 50mmol/L CNPG mmol/l Calcium chloride 60 mmol/l Sodium chloride 70 mmol/l Activator 900 mmol/l when stored at C. The reagent is linear, up to 2000 U/L. If the concentration is greater than linearity (2000 U/L), dilute the sample with normal saline & repeat the assay. Multiply the result with dilution Serum / plasma : < 86 U/L Urine : < 470 U/L PREPARATION AND STABLITY OF WORKING REAGENT The reagent is ready to use. Avoid direct exposure of reagent to light. This reagent is very sensitive to external contamination, ie Saliva, Sweat etc which contains α-amylase. Handle with gloves & keep vial tightly sealed after use. Discard reagent if it turns cloudy. Fresh serum / plasma (free of haemolysis) / Urine (1/3 diluted) Kinetic Wavelength 405 nm 37 0 C Factor U/L DI water Delay time 60 sec No.of readings 3

10 BILIRUBIN TOTAL & DIRECT determination of Bilurubin in serum, plasma & urine. Modified DMSO methodology Linear up to 20 mg/dl Fast incubation 5 minutes at room temperature. Sample volume only 50 µl. Bilirubin is formed by the break down of RBC s in the spleen, liver & bone marrow. Small amount of bilirubin circulates in the plasma loosely bound to albumin, which is not water soluble. This is referred to as indirect or unconjugated bilirubin. In the liver bilirubin is conjugated with glucuronic acid, which forms a soluble compound. This is referred to a direct bilirubin. Elevated levels are found in Hepatitis, Cirrhosis, Haemolytic jaundice, obstruction of biliary tract & drug induced reactions. Sulfanilic acid reacts with sodium nitrite to form in the presence of diazotized sulfanilic acid to form azobilirubin. In the absence of dimethyl sulfoxide, only the direct bilirubin reacts to give azobilirubin. TOTAL BILIRUBIN REAGENT 2 x 50 ml Sulfanilic acid 28.9 mmol/l Hydrochloric acid 165 mmol/l Dimethyle sulfoxide 7 mmol/l DIRECT BILIRUBIN REAGENT 2 x 50 ml Sulfanilic acid 28.9 mmol/l Hydrochloric acid 165 mmol/l TOTAL BILIRUBIN ACTIVATOR 1 x 4mL DIRECT BILIRUBIN ACTIVATOR 1 x 4mL when stored at RT. The activator should be stored at C. This reagent is linear up to 20 mg/dl. If the concentration is greater than linearity (20 mg/dl), dilute the sample with normal saline and repeat the assay. Multiplythe result with dilution Total Bilirubin : upto 1.2 mg/dl Direct Bilirubin : upto 0.4 mg/dl Reagent and standard are ready to use. Avoid direct exposure of reagent to light. Serum/plasma (free of haemolysis) End point Wavelength 546 nm or 532 nm Room Factor (Total ) 20.5 / 23.5 (Direct) 16.0/ 18.0 Sample blank 20 mg/dl Reaction time 5 min Sample volume 50 µl Activator 20 µl 4 x 50 ml Total Bilirubin Direct Bilirubin Sample Test Sample Test Total Bilirubin Re 1000 µl 1000 µl - - Total Bilirubin Rea µl 1000 µl Activator Total/Direct - 20 µl - 20 µl Serum 50 µl 50 µl 50 µl 50 µl Mix well and incubate for exactly 5 minutes. Measure the absorbance of the sample against respective sample blank at 546 or 532 nm. Multiplication Factor Filter Total Bilirubin Direct Bilirubin 546 nm nm For colorimeter Customers Read the optical density (OD) Bilirubin standard at 546 directly. OD of sample test (T) OD of sample (T) T.Bili = x 10 OD of Artificial Std OD of sample test (D) OD of sample (D) D.Bilil = x 7.7 OD of Artificial Std For semi auto analyser Customers T.Bili Conc. in mg/dl = (OD of sample (T) OD of Sample blank (T) ) x Multiplication factor D. Bili.Conc. in mg/dl = ( OD of sample (D)- OD of sample blank (D) ) x Multiplication of 1.Walter M., Gerard H.: MICROCHEM JM 15, 231(1980) 2.Annino J. S.: C.C. Principles and procedure, A.A. A.C.C.: Cain Chem 8 : 405,196

11 BILIRUBIN TOTAL TAB determination of Bilurubin in serum or plasma. Modified TAB method Linear up to 25 mg/dl Fast incubation 5 minutes at Room temperature. Sample volume only 50 µl. Without sample blank procedure also included. Bilirubin is formed by the break down of RBC s in the spleen, liver & bone marrow. Small amount of bilirubin circulates in the plasma loosely bound to albumin, which is not water soluble. This is referred to as indirect or unconjugated bilirubin. In the liver bilirubin is conjugated with glucuronic acid, which forms a soluble compound. This is referred as direct bilirubin. Elevated levels are found in Hepatitis, Cirrhosis, Haemolytic jaundice, obstruction of biliary tract & drug induced reactions. Sulfanilic acid reacts with sodium nitrite to form diazotized sulfanilic acid. Total Bilirubin reacts with diazotized sulfanilic acid in the presence of TAB form azobilirubin. TOTAL BILIRUBIN REAGENT 4 x 50 ml Sulfanilic acid 28.9 mmol/l TAB 9 mmol/l Preservatives and Stabilizers TOTAL BILIRUBIN ACTIVATOR 2 x 4 ml BILIRUBIN CALIBRATOR Not provided along with the Kit, recommended Agappe multicalibrator product code : when stored at RT. The standard & activator should be stored at C. This reagent is linear up to 25 mg/dl. If the concentration is greater than linearity (25 mg/dl), dilute the sample with normal saline and repeat the assay. Multiply the result with dilution Total Bilirubin - up to 1.2 mg/dl Reagents are ready to use. Avoid direct exposure of reagent to light. Serum/Plasma (free of haemolysis) End point Wavelength 546 nm Room Factor (Total ) 25 Sample blank 25 mg/dl Reaction time 5 min Sample volume 50 µl Activator 20 µl 4 x 50 ml Total Bilirubin Sample Test Total bilirubin Reagent 1000 µl 1000 µl Activator Total - 20 µl Serum/Calibrator 50 µl 50 µl Mix well and incubate for 5 minutes at room temperature. Measure the absorbance of calibrator and test against respective blank at 546 nm. With factor : Total Bilirubin = OD test OD sample blank x 25 With calibrator : OD test OD sample Bilirubin Concentration = x n OD calibrator OD Calibrator n = Calibrator concentration Alternative Method without sample blank End point Wavelength I 546 nm Wavelength II 630 nm Room Factor (Total ) 29 Reagent blank 25 mg/dl Reaction time 5 min Sample volume 50 µl Activator 20 µl Total Bilirubin Test Total bilirubin Reagent 1000 µl 1000 µl Activator Total 20 µl 20 µl Serum/Calibrator - 50 µl Mix well and incubate for exactly 5 minutes. Measure the absorbance of calibrator and test against reagent blank at 546/ 630 nm. With Factor : Total Bilirubin = OD Test OD Reagent blank x With calibrator : OD Test OD Reagent Bilirubin = x n OD Calibrator OD Reagent n = Calibrator concentration 1.Walter M., Gerard H.: MICROCHEM JM 15, 231.(1980) 2.Annino J. S.: C.C. Principles and procedure, A.A. A.C.C.: Cain Chem 8 : 405,196

12 CALCIUM determination of calcium in serum, plasma & urine. Modified OCPC methodology Linear up to 15 mg/dl Calcium is an important ion present in the body. Mainly it is found in bones. In serum calcium exists equally in a free ionized form & also in a bound form with albumin. Calcium helps in enzyme activation, muscle contraction, coagulation of blood, regulation of some hormonal secretions & cell membrane permeability. Increased levels are found in hyperthyroidism, malignant tumors, acute osteoporosis & adrenal insufficiency. Decreased levels found in hypoparathyrodism, osteomalacia, rickets, renal failure & tetanus. Colorimetric measurement with ortho-cresolphtalein complex. The 8-hydroxy-quinoline prevents Mg +2 from interference up to 4 mmol/l. CALCIUM DYE REAGENT 2 x 25 ml Diethylamine 360 mmol/l CALCIUM BASE REAGENT 2 x 25 ml O-Cresolphalein complex 0.15 mmol/l 8-Hydroxyquinoline 17.2 mmol/l CALCIUM STANDARD 1 x 4 ml Calcium standard concentration 10 mg/dl STORAGE & STABILITY when stored at C. This reagent is linear up to 15 mg/dl. If the concentration is greater than linearity (15 mg/dl), dilute the sample with normal saline and repeat the assay. Multiply the result with dilution Serum : mg/dl Urine : mg/24 hrs Mix reagent 1 (R1) and Reagent 2 (R2) in the ratio 1:1 stability of working is 14 days at C. Avoid direct exposure of working reagent to light. (Use acid washed (50 % HNO 3 ) glass wares & tips) Serum / plasma (free of haemolysis) / Urine (1/3 diluted) End point Wavelength 578 nm ( nm) Room Standard Concentration 10 mg/dl 15 mg/dl Reagent Incubation time 5 min Sample volume 10 µl Standard Sample Working Reagent 1000 µl 1000 µl 1000 µl Standard - 10 µl - Sample µl Mix and incubate for 5 min. at room temperature. Read the absorbance of standard and sample against reagent blank. 4 x 25 ml Absorbance of sample Calcium Conc. (mg/dl) = x 10 Absorbance of Standard INTEREFERENCE Bilirubin concentrations higher than 20 mg/dl and phosphate higher than 40 mg/dl, will interfere with the assay. 1.Schwarzenbach G., (1955), Analyst., 80, Kessler G., Wolfman M., (1964).Clin.Chem., 10, Connerty, H. V., Briggs, A.R., (1965). Am.J.Clin.pathol., 45, Gitelmann H.J. (1967) Anal Biochem 18, Biggs H.G. Moorehead, W. R. (1974), Clin.Chem., 20,

13 CREATININE 4 x 50 ml determination of creatinine in serum, plasma & urine. Modified Jaffe s method Linear up to 24 mg/dl No Bilirubin interference up to 10 mg/dl It is formed in muscles from phospho creatinine. It is important form of energy being a store of high-energy phosphate. Creatinine determinations have one advantage over Urea determination that it is not affected by a high protein diet. Serum creatinine is more specific & sensitive indicator of renal function. Simultaneous estimations of serum urea & creatinine provides better information. Serum urea nitrogen, creatinine ratio is > 15 in pre renal failure, & < 10 in renal failure. Decreased levels are found in muscle dystrophy. Creatinine reacts with picric acid to produce a colored compound, creatinine alkaline picrate. The change in absorbance is proportional to the creatinine concentration. CREATININE DYE REAGENT 2 x 50 ml Picric Acid 8.73 mmol/l Surfactant CREATININE BASE REAGENT 2 x 50 ml Sodium hydroxide 300 mmol/l Sodium Phosphate 25 mmol/l CREATININE STANDARD 1 x 5 ml Creatinine standard concentration 2 mg/dl STORAGE & STABILITY when stored at room temperature & standard at C. This reagent is linear up to (24 mg/dl). If the concentration is greater than linearity (24 mg/dl), dilute the sample with normal saline and repeat the assay. Multiply the result with dilution Serum :Men : mg/dl Female : mg/dl Urine : gm/24 hr Mix 1 volume of Reagent 1 (R1) with 1 volume of Reagent 2 (R2). This working reagent is stable for 30 days at room temperature. Avoid direct exposure of working reagent to light. Serum / plasma (free of haemolysis) / Urine (diluted 1/100 with distilled water) Fixed time Wavelength 492 nm/ 505 nm 37 0 C Standard Concentration 2 mg/dl 24 mg/dl D I water Delay time 60 sec Interval 60 sec Sample volume 100 µl Standard Sample Working Reagent 1000 µl 1000 µl Standard 100 µl - Sample µl Mix and read the optical density (T 1 ) 60 seconds after the sample or standard addition. Exactly 60 seconds after the first reading take second reading (T 2 ) (T 2 - T 1 ) of sample Creatinine conc. (mg/dl) = x 2 (T 2 - T 1 ) of Standard 1. Allen. L.C.: Clin chem.vol.28 No.3, 1982, Haeckel, R. et al. Chlin. Chem. 27/ (1981). 3. Tanganelli, E. Prencipe, L; Bassi, D. Cambiaghi, S. and Murador,E.: Clin.Chem 28/7, (1982)

14 HAEMOGLOBIN determination of Haemoglobin in blood. Based on cyanmethaemoglobin method Linear up to 20 g/dl A decrease in haemoglobin below normal range is an indication of anaemia. An increase in haemoglobin concentration occurs in haemoconcentration due to loss of body fluid in severe diarrhea and vomiting. High values are also observed in congenital heart disease (due to reduced oxygen supply) in emphysema and also in poly cythemia. Haemoglobin concentration drops during pregnancy due to haemodilution The Haemoglobin (oxyhaemoglobin,methemoglobin, Carboxyhaemoglobin) is converted to cyanmethaemoglobin according to the following reactions. K3 Fe(CN)6 Haemoglobin > Methemoglobin KCN Methemoglobin > cyanmethemoglobin The intensity of the color is proportional to haemoglobin concentration and is compared to known cyan methaemoglobin standard at 540 nm (green filter) HAEMOGLOBIN REAGENT 1000 ml Potassium Phosphate 2.0 mmol/l Potassium ferricyanide 0.60 mmol/l Potassium cyanide 0.90 mmol/l Sodium chloride 1.4 mmol/l HAEMOGLOBIN STANDARD 1 x 4 ml Cyanmethaemoglobin standard con. 60 mg/dl when stored at room temperature & standard at C. This reagent is linear up to 20 gm/dl. New born : gm/dl Adult (male) : gm/dl Adult (Female) : gm/dl The reagent is ready to use. Avoid direct exposure of reagent to light. Do not pipette the reagent with mouth. Fresh whole blood. End point Wavelength 546nm ( nm) Room Reagent 20 mg/dl Standard concentration 15 gm/dl (60x0.251) (Factor = 35)* Incubation time 5min Sample volume 20 µl Reagent volume 5000 µl *NOTE : Analyzer users directly enter given Factor without running standard ml Sample Hb Reagent 5000 µl 5000 µl Sample - 20 µl Mix well and incubate at room temperature for 5 minutes. Measure the absorbance of sample against reagent blank and measure the absorbance of standard directly against blank. Haemoglobin Conc. (gm/dl) Absorbance of sample = x 60 x Absorbance of standard OR Dilution factor Where, = Convertion factor 15 = 60 x Absorbance of sample = x 15 Absorbance of standard 1.Drabkin. D.L., et al.j.bio.chem, 98 (1932), Zijlstra N.C.Clin.Chem.Acta, 5,(1960) 719

15 HDL CHOLESTEROL determination of HDL in serum or plasma. Precipitation method, Phosphotungstate magnesium acetate reagent Linear up to 125 mg/dl Lipoproteins are the proteins, which mainly transport lipids in the blood stream. They are (HDL) High density lipoproteins, (LDL) Low density lipoproteins, (VLDL) Very low density lipoproteins & chylomicrons. LDL carries cholesterol to the peripheral tissues where it can be deposited & increase the risk of atherosclerotic heart & peripheral vascular disease. Hence high levels of LDL are atherogenic. HDL transports cholesterol from peripheral tissues to the liver & then for excretion, hence HDL has a protective effect. Hence the determination of serum HDL cholesterol is a useful food in identify patients at risk of developing coronary heart disease. The chylomicrons, Very low density lipoproteins (VLDL) and Low density lipoproteins (LDL) of serum are precipitated by phosphotungstic acid and magnesium ions. After centrifugation, High density lipoproteins (HDL) are in the supernatant. HDL content of supernatant is measured by an enzymatic Method. HDL CHOLESTEROL R1 4 x 25 ml Phosphotungstate 14 mmol/l Magnesium Chloride 1 mmol/l Preservative HDL CHOLESTEROL STANDARD 1 x 5 ml HDL Cholesterol concentration 50 mg/dl STORAGE & STABILITY when stored at C. The reagent is linear up to 125 mg/dl. If the concentration is greater than linearity (125 mg/dl), dilute the sample with normal saline and repeat the assay. Multiply the result with dilution The following values may be used as guide line. HDL Cholesterol Men : mg/dl Women : mg/dl LDL Cholestrol Suspicious : 150 mg/dl Elevated : 190 mg/dl Reagent is ready to use. Avoid direct exposure of reagent to light. Serum / Plasma (free of haemolysis). End point Wavelength I 505 ( nm) Wavelength II 630nm 37 0 C Standard Concentration 50 mg/dl Cholesterol Reagent 125 mg/dl Incubation time 5 min Sample volume 50 µl 1. PRECIPITATION 4 x 25 ml Sample 300 µl HDL reagent 300 µl Mix well, allow to stand for 10 min. at room temperature, mix again and centrifuge for 10 min, at 4000 rpm. After centrifugation separate the clear supernatant from the precipitate within 1 hour and determine the HDL Cholesterol concentration using the cholesterol reagent. 2.HDL CHOLESTEROL DETERMINATION : Standard Sample Cholesterol Reagent 1000 µl 1000 µl 1000 µl Standard(HDL) - 50 µl - HDL supernatant µl Mix and incubate for 5 min. at 37 0 C. Measure the absorbance of the standard & sample against the reagent blank. HDL Cholesterol Conc. In mg/dl = Absorbance of sample x N x 2 Absorbance of standard where, 2= dilution factor of the sample. N= Standard concentration (50 mg/dl) LDL-Chol conc in mg/dl = Total Cholesterol (HDL Chol. + Triglycerides / 5) Assmann, G.: Intermist 20 (1979), 559 Gordon. T.et al.: Med 62 (1977), 707 friedewald, W.T et al.: clin.chem.18 (1972), 499.

16 TOTAL PROTEIN 4 x 50 ml determination of Total Protein in serum or plasma. Direct Biuret Method up to 15 gm/dl. Long shelf life, 18 months at room temperature Proteins form the major portion of dissolved substances in the plasma. They form the basic structural components of the body. They constitute the enzymes present in our body & also act as secondary source of energy. The other functions include distribution of water, buffering, transport of various components, defense & coagulation of blood in our body. Increased levels are found in dehydration & myeloma. Decreased levels are found in liver disorders, Nephrotic syndrome, malnutrition & protein due to haemorrhage. Colorimetric determination of total protein based on the principle of the Biuret reaction (copper salt in an alkaline medium). Protein in plasma or serum sample forms a blue colored complex when treated with cupric ions in alkaline solution. The intensity of the blue color is proportional to the protein concentration. TOTAL PROTEIN REAGENT 4 x 50 ml Potassium iodide 6 mmol/l Potassium sodium tartarate 21 mmol/l Copper Sulphate 6 mmol/l Sodium hydroxide 58 mmol/l TOTAL PROTEIN STANDARD 1 x 5 ml Total protein standard Concentration 6gm/dL STORAGE & STABILITY when stored at room temperature and standard at C. The procedure is linear up to 15 gm/dl. If the concentration is greater than linearity (15 gm/dl), dilute the sample with normal saline and repeat the assay. Multiply the result with dilution Serum : gm/dl Reagent is ready to use. Avoid direct exposure of working reagent to light. Serum / plasma (free of haemolysis) End point Wavelength 546 nm 37 0 C Standard Concentration 6 gm/dl 15 mmol/l Reagent Incubation time 10 min Sample volume 20 µl Standard Sample Reagent 1000 µl 1000 µl 1000 µl Standard - 20 µl - Sample µl Mix and incubate for 10 minutes at 37 0 C. Measure the absorbance of standard and sample against reagent blank. Absorbacne of sample Total Protein Conc. (gm/dl) = x 6 Absorbance of standard Gomall, A.J.Biol. Chem, 177 C (1949) 751

17 ASO 50 T, 100 T , INTENDED USE: This reagent is intended for in vitro qualitative & semi quantitative determination of Anti Streptolysin O (ASO) in serum. High sensitivity 200 IU/mL Optimized antigen concentration; no prozone effect. Rapid procedures; test time only 2 minutes. Excellent clarity; clear agglutination. Streptolysin O is a toxic exoenzyme produced by β-haemolytic Streptococcus group A, C and G. ASO is useful for the diagnosis of rheumatoid fever, acute glomerulonephritis and streptococcal infections. Rheumatic fever is an inflammatory disease affecting connective tissue from several parts of human body as skin, heart, joints etc. and acute glomerulonephritis is a renal infection that affects manly glomerulus. The ASO is a rapid agglutination procedure for the direct detection and semi-quantitation (on slide) of antistreptolysin-o (ASO). The antigen, latex particles suspension coated with Streptolysin-O, agglutinates in the presence of specific antibodies present in the sera of patients with streptococcal â- haemolytic infection (group A and C). ASO LATEX 1 x 2.5 ml/ 1 x 5 ml Suspension of polystyrene particles coated with Streptolysin-O. ASO POSITIVE CONTROL 1 x 0.5 ml Human pooled serum ASO NEGATIVE CONTROL 1 x 0.5 ml Human pooled serum GLYCINE SALINE BUFFER CONCENTRATE 1 x 5 ml Dilute 1:20 (v/v) with distilled water ACCESSORIES: for 50T for 100 T 1.Reaction slide Serum droppers Applicator sticks Rubber teat (blue) 1 1 Reagent components of origin have been tested and found to be negative for the presence of antibody to HIV (1/2) as well as for HBsAg and HCV antibody. However, handle cautiously as potentially infectious. The reagent and controls contain less than 0.1% sodium azide. When stored at C and protected from light, the reagent and the controls are stable until the expiry date stated on the label. Preparation of glycine saline buffer: Prepare glycine saline buffer by adding 95mL of distilled water to 5 ml of concentrate glycine saline buffer (provided). It is stable up to expiry date, when stored at room temperature. SENSITIVITY The ASO latex sensitivity has been adjusted to detect a minimum of 200 IU/mL ASO in the undiluted samples. Fresh serum (free of haemolysis) QUALITATIVE TEST Allow all reagents as well as the sample to reach room temperature. Mix well before use. 1.Place 1 drop of serum sample on to the slide with help of disposable serum dropper. 2.Add one drop of ASO-latex reagent to the above drop and mix with disposable applicator stick. 3.Rock the slide gently to and fro for 2 minutes and then examine under a good light source for agglutination, do not examine beyond 2 minutes. For positive & negative controls follow the same procedures as mentioned above by taking control serum from respective vials. RESULT AND INTERPRETATION Positive result: The presence of agglutination indicated concentration of ASO in the sample equal or greater than 200 IU/mL (above normal). Negative result: The lack of agglutination indicates ASO level lower than 200 IU/mL in the sample, (within the normal range). SEMI QUANTITATIVE TEST In the cases in which it is desired to find out the titre of a positive sample, it is feasible by the serial dilution methodology. 1.Place 50 µl diluted glycine saline Buffer onto each of five circles of the slide 2.Using a 50 µl (0.05 ml) micro pipette add 50 µl (0.05m/L) of the serum sample to the drop of glycine saline buffer in 1 st circle. 3.Using the same micro pipette, mix the sample with saline by aspirating back & forth several times. Aspirate 50 µl (0.05 ml) from 1 st circle and transfer to 2 nd circle. Repeat the same operation up to 5 th circle. Aspirate 0.05 ml from 5 th circle and discard. Following dilutions obtained. Dilution 1/2, 1/4 1/8, 1/16, 1/32 Then add 1 drop of ASO latex to the above circles. Mix and rock the slide gently to add fro for 2 minutes; observe the agglutination under good source of light. ASO Conc. (IU/mL) = sensitivity x Titre (Highest dilution serum showing agglutination) Where, ASO sensitivity = 200 IU/mL 1.CURTIS G. D.W, KRAAK W.A.G., MITCHELL R.G. Comparison of latex and hemolysis test determination of antistreptolysin O (ASO) antibodies. J. Clin. Pathol. 41, 1331 (1988) 2.TADZYNSKY L.A., RYAN M.E. Diagnosis of rheumatoid fever. A guide to criteria and manifestations. Post grad Med. 79, 295 (1986) 3.D ANGELO W.A. Rheumatic Diseases. Diagnostic tests and procedures. in: The Laboratory in Clinical Medicine (Halsted J.A. Ed.) Saunders Company, Philadelphia, chapter 29 (1976)

18 CRP 50 T, 100 T , determination of C-reactive protein (CRP) in serum High sensitivity 0.6 mg/dl Optimized antigen concentration; No prozone effect. Rapid procedure, only 2 minutes test. Excellent clarity, clear agglutination. CRP is a protein present in normal serum, which increases significantly after most forms of tissue injuries, bacterial & viral infections, inflammation and malignant neoplasia. CRP is a rapid agglutination procedure for the direct detection and semiquantitation (on slide) of C-reactive protein (CRP). The reagent, a latex particles suspension coated with specific antihuman C-reactive protein antibodies, agglutinates in presence of CRP in patient serum. REAGENTS CRP LATEX 1 x 2.5 ml/ 1 x 5 ml Suspension of polystyrene particles coated with anti human CRP goat antibodies. CRP POSITIVE CONTROL 1 x 0.5 ml Human pooled serum CRP NEGATIVE CONTROL 1 x 0.5 ml Human pooled serum GLYCINE SALINE BUFFER CONCENTRATE 1 x 5 ml Dilute 1:10 (v/v) with distilled water ACCESSORIES: For 50 T For 100 T 1.Reaction slide Plastic droppers Applicator sticks Rubber teat (blue) 1 1 Reagent components of human origin have been tested and found to be negative for the presence of antibody to HIV (1/2) as well as for HBsAg and HCV antibody. However, handle cautiously as potentially infectious. The reagent and controls contain less than 0.1% sodium azide. When stored at C and protected from light, the reagent and the controls are stable until the expiry date stated on the label. Preparation of glycine saline buffer: Prepare glycine saline buffer by adding 45mL of distilled water to 5mL of concentrate glycine saline buffer (provided). It is stable up to expiry date, when stored at room temperature. SENSITIVITY The CRP latex sensitivity has been adjusted to detect a minimum of 20.6 mg/dl in the undiluted samples. Fresh serum (free of haemolysis) QUALITATIVE TEST Allow all reagents as well as the sample to reach room temperature. Mix well before use. 1.Place 1 drop of serum sample on to the slide with the help of disposable serum dropper. 2.Add one drop of CRP-latex reagent to the above drop and mix well with disposable applicator stick. 3.Rock the slide gently to and fro for 2 minutes examine under good light source for agglutination, do not examine beyond 2 minutes. 4.For positive & negative controls follow the same procedures as mentioned above by taking control serum from respective vials. RESULT AND INTERPRETATION Positive result: The presence of agglutination indicated concentration of CRP in the sample equal or greater than 0.6 mg/dl (above normal) Negative result: The lack of agglutination indicates CRP level lower than 0.6 mg/dl in the sample, (within the normal range) SEMI QUANTITATIVE TEST In the case in which it is desired to find out the titre of a positive sample, it is feasible by the serial dilution methodology. 1.Place 50 µl diluted glycine saline Buffer onto each of five circles of the slide 2.Using a 50 µl (0.05 ml) micro pipette add 50 µl (0.05m/L) of the serum sample to the drop of glycine saline buffer in 1 st circle. 3.Using the same micro pipette, mix the sample with saline by aspirating back & forth several times. Aspirate 50 µl (0.05 ml) from 1 st circle and transfer to 2 nd circle. Repeat the same operation up to 5 th circle. Aspirate 0.05 ml from 5 th circle and discard. Following dilutions obtained. Dilution 1/2, 1/4, 1/8, 1/16, 1/32 4.Then add 1 drop of CRP latex reagent to the above circles. Mix and rock the slide gently to and fro for 2 minutes; observe the agglutination under good source of light. Concentration of CRP in serum can be calculated as follows: CRP Conc. (mg/dl) = sensitivity x titre (highest dilution serum showing agglutination) Where, CRP sensitivity = 0.6 mg/dl 1.Wadsworth, C.Wadsworth, E., Efficacy of latex agglutination methods for determination of C - reactive protein in Pediatric sera. Clin. Chem. Acta, 138, (1984), Ballou S.P, Kushner, I.C. Reactive Protein and acute phase response. ADV Int. Med, 37, (1992), 313.

19 RF 50 T, 100 T , INTENDED USE: This reagent is intended for in vitro qualitative & semi quantitative determination of Rheumatoid factor (RF) in serum. High sensitivity 8 IU/mL Optimized antigen concentration; No prozone effect. Rapid procedure; only 2 minutes test. Excellent clarity; crystal clear agglutination. Rheumatoid factors are a group of antibodies directed to determinants in the Fc portion of the immunoglobulin G molecule (IgG). Although rheumatoid factors are found in a number of Rheumatoid disorders, such as systemic lupus erythematosus (SLE) and Sjogrens syndrome as well as in non rheumatic condition, its central role in clinic lies in its utility as an aid in the diagnosis of rheumatoid arthritis (RA). RF is a rapid agglutination procedure for the detection and semi-quantitation (on slide) of Rheumatoid Factors (RF). The antigen, a latex particles suspension coated with human ã-globulin, agglutinates in presence of rheumatoid factors in the patient serum REAGENTS RF LATEX 1 x 2.5 ml/ 1 x 5 ml Suspension of polystyrene particles coated with anti human γ -globulin. RF POSITIVE CONTROL 1 x 0.5 ml Human pooled serum RF NEGATIVE CONTROL 1 x 0.5 ml Human pooled serum GLYCINE SALINE BUFFER CONCENTRATE 1 x 5 ml Dilute 1:10(v/v) with distilled water ACCESSORIES: For 50 T For 100 T 1.Reaction slide Plastic droppers Applicator sticks Rubber teat (blue) 1 1 Reagent components of human origin have been tested and found to be negative for the presence of antibody to HIV (1/2) as well as for HBsAg and HCV antibody. However, handle cautiously as potentially infectious. The reagent and controls contain less than 0.1% sodium azide. When stored at C and protected from light, the reagent and the controls are stable until the expiry date stated on the label. Preparation of Glycine be saline buffer: Prepare glycine saline buffer by adding 45 ml of distilled water to 5 ml of concentrate glycine saline buffer (provided). It is stable up to expiry date, when stored at room temperature. SENSITIVITY The RF sensitivity has been adjusted to detect a minimum of 8.0 IU/mL RF in the undiluted samples. Fresh serum (free of haemolysis) QUALITATIVE TEST Allow all reagents as well as the sample to reach room temperature. Mix well before use. 1.Place 1 drop of serum sample on to the slide with disposable serum dropper. 2.Add one drop of RF-latex reagent to the above drop and mix well with disposable applicator stick. 3.Rock the slide gently to and fro for 2 minutes and then examine under good light source for agglutination, do not examine beyond 2 minutes. For positive & negative controls follow the same procedures as mentioned above by taking control serum from respective vials. RESULT AND INTERPRETATION Positive result: The presence of agglutination indicated concentration of RF in the sample equal or greater than 8 IU/mL (above normal). Negative result: The lack of agglutination indicates RF level lower than 8 IU/mL in the sample, (within the normal range). SEMI QUANTITATIVE TEST In the case in which it is desired to find out the titre of a positive sample, it is feasible by the serial dilution methodology. 1.Place 50 µl diluted glycine saline Buffer onto each of five circles of the slide 2.Using a 50 µl (0.05 ml) micro pipette add 50 µl (0.05m/L) of the serum sample to the drop of glycine saline buffer in 1 st circle. 3.Using the same micro pipette, mix the sample with saline by aspirating back & forth several times. Aspirate 50 µl (0.05 ml) from 1 st circle and transfer to 2 nd circle. Repeat the same operation up to 5 th circle. Aspirate 0.05 ml from 5 th circle and discard. Following dilutions obtained. Dilution ½, ¼, 1/8, 1/16, 1/32 Then add 1 drop of RF latex reagent to the above circles, mix and rock the slide gently to and fro for 2 minutes; observe the agglutination under good source of light. The Rheumatoid Factor (RF) level in serum can be calculated as follows: RF conc. (mg/dl) = sensitivity x titre (highest dilution of serum showing agglutination) Where, RF Sensitivity = 8.0 IU/mL 1.Frederick Wolf et al-arthritis and Rheumatism 1991: 34: Robert W Dorner et al. Clinica Chemica Acta 1987; 167: 1-21