GLUCOSE

LIQUID STABLE REAGENT

Trinder’s Method, End Point

INTENDED USE
This reagent is intended for in vitro determination of Glucose in serum / plasma.

CLINICAL SIGNIFICANCE
Accurate measurement of glucose in body fluids is important in the diagnosis
and management of diabetes, hypoglycaemia, adrenal dysfunction and various
other conditions.

INCREASES
Due to diabetes mellitus, in patients receiving glucose containing fluids
intravenously, during severe stress and cerebrovascular accidents.

DECREASES
On insulin administration, as a result of insulinoma, in born errors of
carbohydrate metabolism or on fasting.

METHODOLOGY

Trinder’s Method.

PRINCIPLE

Glucose present in the sample is oxidized to yield gluconic acid and hydrogen
peroxide in the presence of Glucose oxidase. The enzyme peroxidase catalyses
the oxidative coupling of 4-aminoantipyrine with phenol to yield a coloured
quinoneimine complex, with absorbance proportional to the concentration of
glucose in sample.

SPECIMEN
Use fresh unhaemolysed serum. The st ability of glucose in specimen is reduced by
bacterial contamination and by glycolysis. Serum or plasma should be separated from the cells, as
soon as possible, to prevent glycolysis. The addition of sodium fluoride is recommended to inhibit
glycolysis. Other commonly used anticoagulants with concentration not causing interference are:
Fluoride 10 mg/ml blood
Oxalate 20 mg/ml blood
Edta 10 mg/ml blood
Citrate 30 mg/ml blood
Heparin 2 mg/ml blood

ASSAY PROCEDURE

Pipette into tubes marked Blank Calibrator Test

Glucose Reagent 1000 µl 1000 µl 1000 µl
Distilled Water 10 µl -- --
Standard` -- 10 µl --
Test -- -- 10 µl

Mix well after each addition and incubate at +37 °C for 5 minutes. Read
absorbance of Standard and test against reagent blank at 505 (505-520) nm or
506/670 nm on bichromatic analyses against reagent blank.

CALCULATION

Abs. of Test
Glucose x Concentration of
=
(mg/dl) Abs. of Calibrator (mg/dl)
calibrator

NORMAL VALUES (Reference for guidelines)

Fasting: 65-110 mg/dl
Post prandial: 90-130 mg/dl

CHOLESTEROL
LIQUID STABLE REAGENT
CHOD – PAP, End Point

INTENDED USE
This reagent is intended for in vitro determination of Cholesterol in serum /
plasma.

CLINICAL SIGNIFICANCE

Measurements of serum Cholesterol levels are useful in evaluation of risk of the

coronary arterial occlusion, atherosclerosis, myocardial infarction, liver function, biliary
function, intestinal absorption thyroid function and adrenal disease.

INCREASES

Increased levels are found in most characteristically in primary

hyperlipproteinaemias, in nephritic syndrome, myxodema, obstructive jaundice and in
diabetes mellitus.

DECREASES

Low values are frequently obtained in anaemias, in haemolytic jaundice, in

malabsorption syndrome, severe malnutrition acute infections and in terminal state,
 very low values occur in abetalipoproteinameia and to lesser extent in familial
hypobetalipoproteinaemias.

METHODOLOGY

This method is based on Trinder’s Methodology.

PRINCIPLE
CHE
1. Cholesterol ester + Cholesterol + Fatty acids
H20

CHOD
2. Cholesterol + O2 Cholest-4-en-3-one + H2O

POD
3. 2H2O2 + 4AAP + 4- Quinoneimine dye + 4H2O
HBA

SAMPLE

Unhaemolysed serum or Heparin or EDTA plasma from fasting patients. Plasma

cholesterol values have been reported to be 3% to 5% lower than serum cholesterol
values. Specimens should be analyzed on the same day of collection. When stored
between 4…8 °C, the stability is for 7 days and 3 months at – 20 °C.

ASSAY PROCEDURE

Pipette into tubes marked Blank Calibrator Test

Cholesterol Reagent 1000 µl 1000 µl 1000 µl
Distilled Water 10 µl -- --
Calibrator -- 10 µl --
Test -- -- 10 µl
Mix well and incubate for 5 min. at 37 °C or 10 min. at 20…25 °C. Read the absorbance of
the Test & calibrator against reagent blank.

CALCULATION
Abs. of Test
Cholesterol x Concentration of
=
(mg) Abs. of calibrator (mg/dl)
calibrator

UNIT CONVERSION
mg/dl x 0.026 = mmol/l
REFERENCE VALUES
Serum / Plasma mg/dl
Children 4 weeks 50 - 170
2-12 60 – 190
months
1 year 110 – 230
Adults <200

TRIGLYCERIDES
LIQUID STABLE REAGENT
GPO – PAP Method, End Point

INTENDED USE
This reagent is intended for in vitro determination of Triglycerides in serum /
plasma.

CLINICAL SIGNIFICANCE
Triglycerides are a family of lipids absorbed from the diet and produced of
endogenously from carbohydrates. Measurement of triglycerides is important in the
diagnosis and management of hypoerlipidemias. These diseases can be genetic or
secondary to other disorders including nephrosis, diabetes mellitus and endocrine
disturbances. Elevation of triglycerides has been identified as a risk factor for
atherosclerotic disease.

METHODOLOGY
Colorimetric, enxymatic method with glycerophosphate oxidase. This reagent is
based on the method of Wako and the modifications by Mc Gowan et al and Fossati et
al.

PRINCIPLE
LPL
Triglycerides +H2O Glycerol + Free Fatty Acids

GK
Glycerol + ATP Glycerol-3-phosphate + ADP

GPO
Glycerol-3-phosphate DAP + H2O2
+ O2

Peroxidase
H2O2 + 4 AAP + 3,5- Quinoneimine dye + 2H2O
DHBS
LPL : Lipoprotein Lipase
GK : Glycerol Kinase
GPO : Glycerol Phosphate Oxidase
DAP : Dihydroxy Acetone Phosphate
ATP : Adenosine Triphosphate
4-AAP : 4-Aminoantipyrine
DHBS : 3,5-Dichloro – 2 Hydroxy Benzene SULFONATE
The intensity of chromogen (Quinoneimine) formed is proportional to the triglycerides
concentration in the sample when measured at 505 nm (500 – 540 nm).

SAMPLE

Unhaemolysed serum or heparinised plasma collected after 12 - 16hrs fast are

suitable for triglycerides estimation. Triglycerides in the serum is stable for 3 days at
4°C. Storage at room temperature may cause the release of glycerol from
phospholipids with a resulting apparent increase in triglycerides and hence it is not
recommended.

ASSAY PROCEDURE

Pipette into tubes marked Blank Calibrator Test

Triglyceride Reagent 1000 µl 1000 µl 1000 µl
Distilled Water 10 µl -- --
Standard -- 10 µl --
Test -- -- 10 µl
Mix well and incubate for 10 mins at 37 °C. Read the absorbance of standard and each
sample at 505 / 670 nm on bichromatic analyzers against reagent blank.

CALCULATION

Abs. of Test
Triglycerides= x Concentration of
(mg/dl) Abs. of calibrator (mg/dl)
calibrator
UNIT CONVERSION
mg/dl x 0.011 = mmol/l

REFERENCE VALUES
Male: 40 – 160 mg/dl
Female: 35 – 135 mg/dl
It is recommended for each laboratory to establish its own reference range for the
population it serves.

HDL CHOLESTEROL
LIQUID STABLE REAGENT
Phosphotungstic Acid Method, End Point
CLINICAL SIGNIFICANCE
High density lipoproteins (HDL) contain particles of different density including
lipid and highest concentration of proteins amongst the different lipoproteins. It
includes free and esterified cholesterol, triglycerides, phospholipids and apoproteins
A, C and E. HDL Cholesterol values are about 1/5 th of the total cholesterol values and
can be expressed as percentage of total cholesterol.

DECREASES
There exists an inverse relationship between HDL cholesterol and coronary
heart diseases. Low concentration i.e. below 30 mg/dl is one of the risk factors for
cardiac ailments.

METHODOLOGY
Burstein et.al.

PRINCIPLE
CHylomicrons, LDL and VLDL (low and very low density lipoproteins) are
precipitated from serum by phosphotungstate in the presence of divalent cations such
as magnesium. The HDL cholesterol remains unaffected in the supernatant and is
estimated using ERBA Cholesterol reagent.

Phosphotungstate
Serum/plasma HDL + (LDL + VLDL + Chylomicrons)
2+
Mg (Supernatent) (Precipitate)

SAMPLE
Unhaemolysed serum or EDTA Plasma. Citrate and Heparin should not be used
as anticoagulants. Samples are stable for seven days at 2…8°C or one month at
-20°C, when freezed once.

ASSAY PROCEDURE

Pipette into tubes marked Blank Calibrator Test

Cholesterol Working Reagent 1000 µl 1000 µl 1000 µl
Distilled Water 50 µl -- --
Calibrator -- 50 µl --
Supernatent -- -- 50 µl
Mix well, incubate for 10 min. at 37 °C or 12 min. at 30 °C. Read the absorbance of the
CALIBRATOR AND EACH TEST AT %)% nm or 505/670 nm for biochromatic analysers
against reagent blank.

CALCULATION

Abs. of
Test
HDL x Concentration x dilution factor
Cholesterol = of
(mg/dl) Abs. of calibrator
Cal. (mg/dl)

Abs. of
Test
= x 25 x 3
Abs. of
Cal.

Abs. of
Test
= x 75
Abs. of
Cal.

UNIT CONVERSION
mg/dl x 0.026 = mmol/l

NORMAL VALUES (Reference for guidelines)

In male: (30 – 65) mg/dl

In female: (35 – 80) mg/dl
It is recommended that each laboratory to establish its own normal range depending on
population it serves.

URIC ACID
LIQUID STABLE REAGENT
Uricase – PAP, End Point

INTENDED USE
This reagent is intended for in vitro determination of Uric Acid in serum /
plasma.

CLINICAL SIGNIFICANCE

Uric Acid is a metabolite of purines, nucleic acids and nucleoproteins.

Consequently, abnormal levels may be indicative of a disorder in the metabolism of
these substances.
INCREASES

Increased levels of serum uric acid are observed in renal dysfunction, gout,
leukemia, polycythemia, atherosclerosis, diabetes, hypothyroidism, or in some genetic
diseases.

DECREASES

Uric acid concentration decreases in patients with Wilson’s diseases.

METHODOLOGY
The reagent is based on Trinder’s reaction, enzymatic and Colorimetric method.

PRINCIPLE

Uricase
Uric Acid + O2 + H2O Allantoin + CO2 + H2O2

Peroxidase
DHBS + 4AAP + Quinoneimine dye + 4H2O
2H2O2

The intensity of colour formed is proportional to the uric acid concentration.

SAMPLE

Unhaemolysed serum or heparinised plasma. Do not use EDTA and fluoride as

anticoagulants. Uric Acid in serum is stable for three days at 2…8°C. It is
recommended to perform assay with freshly collected samples.

ASSAY PROCEDURE
Pipette into tubes marked Blank Calibrator Test
Uric Acid Reagent 1000 µl 1000 µl 1000 µl
Distilled Water 25 µl -- --
Standard -- 25 µl --
Test -- -- 25 µl
Mix and incubate for 5 minutes at 37 °C. Read the absorbance of standard and each sample
at 505 / 670 nm on bichromatic analyzers against reagent blank.

CALCULATION

Abs. of Test
Uric Acid= x Concentration of
(mg/dl) Abs. of calibrator (mg/dl)
 calibrator

UNIT CONVERSION
mg/dl x 0.060 = mmol/l

REFERENCE VALUES
Serum / Plasma mg/dl
Women 2.5 – 6.8
Men 3.6 – 7.7
It is recommended for each laboratory to establish its own reference range for the population
it serves.

UREA (BUN)
LIQUID STABLE REAGENT
UREASE – GLDH – FIXED TIME

INTENDED USE

This reagent is intended for in vitro determination of Urea in serum / plasma.

CLINICAL SIGNIFICANCE

Urea is the major end product of protein metabolism in humans. It consumes

the largest fraction of the non-protein nitrogen component of the blood. Urea is
produced in the liver and excreted through the kidneys in the urine. Consequently the
circulating levels of urea depend upon protein intake, protein catabolism and kidney
function.

INCREASES

Elevated serum urea concentrations are observed in impaired kidney function,

liver diseases, congestive cardiac failure, diabetes, infections and diseases which
impair kidney function.

METHODOLOGY
Kinetic, enzymatic method. Talke and Schubert, Tiffany et al.

PRINCIPLE
Urease
UREA + 2 NH4+ + CO32-
2H2O
 GLDH
NH4+ + 2-Oxoglutarate + L-Glutamate + NAD+ + H2O
NADH

The rate of absorbance changing at 340 nm is directly proportional to UREA Concentration in

the serum.

SAMPLE
Unhaemolysed serum, EDTA or heparinised plasma from fasting patients. Do
not use heparin ammonium salt and fluoride as anticoagulants. Urea in serum is stable
for 7 days at 2…8°C. It is recommended to perform assay with freshly collected
samples.

ASSAY PROCEDURE

Pipette into tubes marked Calibrator Test

Working Reagent 1000 µl 1000 µl
Calibrator 10 µl --
Test -- 10 µl
Mix well and aspirate calibrator followed by samples. READ initial absorbance (A 1) 20
seconds after mixing and final absorbance (A 2) 80 seconds after mixing.

CALCULATION

Calculate the result as follows:

A = A2 – A 1

A of Test
Urea x Concentration of
(mg/dl)=
A of calibrator (mg/dl)

Standard

REFERENCE VALUES
Serum / Plasma 13 – 45 mg/dl

CREATININE (CRE)
Jaffe’s Method, Initial Rate
CLINICAL SIGNIFICANCE

Creatinine is a waste product formed in muscle from the high energy storage
compound, creatinine phosphate. The amount of creatinine produced is fairly constant
(unlike Urea) and is primarily a function of muscle mass. IT is re-excreted in the urine
without any appreciable reabsorption by the tubules. Creatinine is a useful indicator of
renal function.

INCREASES

Elevated creatinine level in serum is usually associated with various renal

diseases. In the earlier stage of renal disease, creatinine clearance test is a sensitive
index of impaired renal function.

METHODOLOGY
Modified Jaffe’s reaction.

PRINCIPLE

Creatinine reacts with alkaline picrate to produce an orange-yellow colour

(Jaffe’s reaction). Specificity of the assay has been improved by the introduction of an
initial rate method. However, Cephalosporin antibiotics are still major interferants.
The absorbance of the orange-yellow colour formed is directly proportional to
creatinine concentration and is measured photometrically at 500-520 nm.

SAMPLE

Serum is preferred, but heparinised plasma may also be used. AVOID USING
severly haemolysed sample as haemolysis increases non-creatinine chromogens. For
urine samples, dilute the fresh urine sample 1/50 with distilled water.
Specimens are stable for 24 hours at room temperature (15-25°C) and 1 week
at 2-8°C.

ASSAY PROCEDURE

Pipette Calibrator Test

Working Reagent 1000 µl 1000 µl
Calibrator 100 µl --
Test -- 100 µl
Mix well and read initial absorbance (A 1) 30 seconds after mixing and final absorbance (A 2)
90 seconds after mixing.

CALCULATION
Calculate the results as follows:
 A = A2 – A 1

A of Test
Creatinine x Concentration of
=
(mg/dl) A of calibrator (mg/dl)

calibrator

NORMAL VALUES (Reference for guidelines)

Males 0.7 – 1.4 mg/dl
Females 0.6 – 1.2 mg/dl
It is recommended that each laboratory establishes its own normal range depending on
population it serves.

SGOT / ASAT
LIQUID STABLE REAGENT
IFCC Method, Kinetic without Pyridoxal Phosphate

INTENDED USE

This reagent is intended for in vitro determination of ASAT / SGOT in serum.

CLINICAL SIGNIFICANCE

ASAT/SGOT occurs in all human tissues and is present in large amounts in

liver, renal, cardiac, and skeletal muscle tissue.

INCREASES

Increased levels are associated with liver diseases or damage, myocardial

infarction, muscular dystrophy and cholecystitis.

DECREASES

Decreased levels are observed in patients undergoing renal dialysis and those
with B6 deficiency. Monitoring the change in levels over a period of time is beneficial to
the physician evaluating myocardial infarction or following chronic or resolving
hepatitis.

METHODOLOGY
International Federation of Clinical Chemistry (IFCC), without Pyridoxal Phosphate.
PRINCIPLE

ASAT/SGOT
L-Aspartate + 2- Oxaloaxetate + L-Glutamate
Oxoglutarate

MDH
Oxaloacetate + Malate + NAD+
NADH

LDH
Sample pyruvate + L-Lactate + NAD
NADH
ASAT: Aspartate aminotransferase
LDH: Lactate dehydrogenase
MDH: Malate dehydrogenase

The rate of absorbance change at 340 nm is directly proportional to SGOT (ASAT) activity in
the specimen.

SAMPLE
Unhaemolysed serum or HEPARINISED PLASMA> Anticoagulants such as
heparin or EDTA are suitable. AST is stable for 3 days at 2…8°C. It is recommended to
perform assay with freshly collected samples.

ASSAY PROCEDURE

Pipette Test
Working Reagent 1000 µl
Sample 100 µl
Mix well and aspirate.

CALCULATION
ASAT / SGOT activity (IU/I) = A/min. x Factor (1746)
Λ 37°C
340 nm 1746

REFERENCE VALUES
Serum / Plasma at 37°C
Women Up to 31 IU/I
Men Up to 37 IU/I
It is recommended for each laboratory to establish its own reference range for the population
it serves.
 SGPT / ALAT
LIQUID STABLE REAGENT
IFCC Method, Kinetic without P’5’ - P

INTENDED USE

This reagent is intended for in vitro determination of SGPT (ALT) in serum.

CLINICAL SIGNIFICANCE

ALT is present in high concentration in the liver and to a lesser extent in kidney,
heart, skeletal muscle, pancreas, spleen and lungs.

INCREASES

Increased levels are generally a result of primary liver diseases such as

cirrhosis, carcinoma, viral or toxic hepatitis and obstructive jaundice.

DECREASES

Decreased levels may be observed in renal dialysis patients and those with
vitamin B6 deficiency.

METHODOLOGY
International federation of Clinical Chemistry (IFCC), without Pyridoxal Phosphate.

PRINCIPLE
ALAT/SG
PT
L-Alanine + 2- Pyruvate + L-Glutamate
Oxoglutarate

LDH
Pyruvate + NADH L-Lactate + NAD+
ALT: Alanine aminotransferase
LDH: Lactate dehydrogenase
The rate of absorbance change at 340 nm is directly proportional to ALAT / SGPT activity in
the specimen.

SAMPLE

Unhaemolysed serum or heparinised plasma. Anticoagulants such as Heparin

or EDTA are suitable. ALT is stable for 3 days at 2 to 8°C. It is recommended to
perform assay with freshly collected samples.
ASSAY PROCEDURE

Pipette Test
Working Reagent 1000 µl
Test 100 µl
Mix well and aspirate.

CALCULATION
ALAT / SGPT activity (IU/I) = A/min. x Factor (1746)
λ 37°C
340 nm 1746

REFERENCE VALUES
Serum / Plasma at 37°C
Women Up to 32 IU/I
Men Up to 42 IU/I

(BioMed Diagnostics)
ALBUMIN
LIQUID STABLE REAGENT
Colorimetric, End Point

INTENDED USE
For the quantitative determination of Albumin in serum.

CLINICAL SIGNIFICANCE
Albumin determination is useful in the diagnosis of hepatic and renal
pathologies.

PRINCIPLE
Albumin is bound by the BCG dye to produce an increase in the blue green
color in a pH 3.8 acid medium.
The color increase is proportional to the concentration of albumin present in the sample.

SAMPLE

Non hemolysed fresh serum, plasma with heparin.

ASSAY PROCEDURE
Blank Standard Sample
Reagent (R2) 2000 µl 2000 µl 2000 µl
Distilled Water 8 µl -- --
Standard -- 8 µl --
Sample -- -- 8 µl
Mix well and incubate for 5 min. at room temperature (+15-25 °C) Read the absorbance of
standard and sample tubes.
Volumes can be proportionally modified.
This methodology describes the manual procedure to use the kit.
For automated procedure, ask for specific application.

CALCULATION

(A) Sample
Albumin = x 4.0
(g/dl) (A) Standard

UNIT CONVERSION
g/dl x 144.9 = mol/µl

REFERENCE VALUES

Serum / Plasma 3.5 – 5.3 g/dl

The above mentioned values are to be considered as a reference. It is strongly
recommended that each laboratory establish its own normal range according to its
geographic area, according to IFCC protocol.

(Crescent Diagnostics)
TRIGLYCERIDE
Enzymatic, liquid, colourimetric, GPO-PAP method

PRINCIPLE
Lipases catalyze the hydrolysis of triglycerides to yield glycerol and free fatty
acids. The glycerol concentration is determined enzymetically with the Trinder reaction
using glycerol kinase (GK), glycerol-3-phosphate oxidase (GPO) and peroxidase
(POD). The end product is a quinoneimine dye the concentration of which at 546 nm is
directly proportional to the concentration of triglycerides in the sample.

Lipases
Triglyceride + H2O Glycerol + Free fatty acids

GK
Glycerol + ATP Glycerol-3-phosphate + ADP

GPO
Glycerol-3-phosphate + O2 H2O2 + Dihydroxyacetone phosphate

POD
H2O2 + 4-Aminoantipyrine + Quinoneimine dye
chlorophenol
SPECIMEN

Serum, plasma.
Fresh, clear, non haemolysed, from patient’s fasting at least 12 hours; Triglycerides in serum
are stable for 3 days at 2-8°C. With prolonged storage at room temperature, glyceride-
containing compounds hydrolyze to yield free glycerol with an apparent increase in
triglyceride levels.

ASSAY PROCEDURE

Wavelength: 546nm
Optical path: 1cm
Temperature: 25 or 37°C
Measurement: against reagent blank

Micro Method Macro Method

Pipette into Cuvettes Blank Standard Sample Blank Standard Sample
Sample ml - - 0.01 - - 0.025
Standard ml - 0.01 - - 0.025 -
Distilled Water ml 0.01 - - 0.025 - -
Working Reagent ml 1.0 1.0 1.0 2.5 2.5 2.5
Mix and incubate for 10 minutes at 25°C. or 5 min. at 37°C.
Measure the absorbance of the sample (As) and the standard (Astd) against the reagent
blank ( A) with an hour.

CALCULATION
As
Triglyceride x Concentration of
=
(mg/dl) Astd the standard(mg/dl)

UNIT CONVERSION
mg/dl 88.5 = mmol/l

REFERENCE VALUES
36 – 165 mg/dl
Serum / Plasma
0.4 – 1.86 mmol/l
Elevated >200mg/dl