MATERIAL AND METHODS -...

MATERIAL MATERIAL MATERIAL MATERIAL

AND METHODSAND METHODSAND METHODSAND METHODS

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In the present study, 50 subjects of Type 2 Diabetes mellitus (T2DM) and

50 subjects of Type 2 Diabetes mellitus with hypertension (T2DH) were

selected from diabetic research lab of B.J. Medical College and Civil

Hospital, Ahmadabad, Gujarat during the period of March 2012 to

December 2012. The 50 subjects in control group were selected from the

staff working in Biochemistry department and people coming for their

physical fitness at Civil Hospital, Ahmedabad.

Study Groups:

Group 1 – Control (n= 50)

Group 2 – Type- 2 Diabetes mellitus (n= 50)

Group 3 – Type -2 Diabetes mellitus with hypertension (n= 50)

The Diagnostic criteria for selection of subjects are as follow

• For Diabetes mellitus

– FBS ≥ 126 mg/dL

• For Hypertension

– Diastolic Blood pressure ≥ 90 mmHg

– Systolic Blood pressure ≥ 140 mmHg

The control subjects were apparently healthy. All subjects were >30

years but <79 years of age.

Exclusion criteria for selection subjects are as follow

1. Any acute or chronic infection 2. Heart disease 3. Pregnancy 4. Female taking oral contraceptive pills 5. Pneumonia 6. Any history of recent surgery 7. Hyperthyroidism 8. History of alcoholism

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9. Trauma: Surgical, Burns, Fractures 10. Malignancy: Lymphoma, Carcinoma, Sarcoma, Leukemia 11. Present or past smokers 12. Hepatic disease 13. Lipid lowering drugs 14. Antioxidant vitamin supplements 15. <30 and >79 years of age 16. Failed to give a written consent were excluded 17. Patients of type 2 DM, being managed with insulin 18. Any evidence of non-diabetic renal disease, severe renal

disease (serum creatinine >2.0 mg/dl).

EQUIPMENT:

• Fully Automated Biochemistry Analyzer: XL-640 (ERBA)

• ELISA Reader and Washer (TULIP)

• NycoCard Reader

SAMPLE TYPE:

• Serum (free of hemolysis)

• Plasma (free of hemolysis)

• Urine

SAMPLE COLLECTION:

A). BLOOD

All subjects were instructed, not to perform any strenuous exercise for at

least 24 hours prior to collection of samples.Venous blood was collected

after an overnight fasting of about 12-14 hours, from all subjects between

09:00AM to 10:00AM by venipuncture. Part of the blood sample was

placed into coagulating tubes and allowed to clot and serum was

separated by centrifugation at 3000 rpm for 10 minutes. The serum was

stored at -20°C until required. Portions (0.5 ml) of the blood collected

were placed in fluoride tubes for fasting plasma glucose estimation.

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B). URINE

The most sensitive standard of microalbuminuria measurement is the

collection of 24h urine. The disadvantage of 24h urines are collection

errors. Careful attention has to be paid on patient instructions because

collection of 24h urine is not an easy task. The collection of 24h urine

samples is cumbersome and time consuming. For this reason in present

study morning spot urine were collected into sterile containers from all

subjects for the analysis.

Laboratory Investigations

Samples analysis was done on fully auto analyzer XL-640 (ERBA),

ELISA reader and washer and NycoCard reader at Hi-Tech Biochemistry

Laboratory, Civil Hospital, B. J. Medical College, Ahmedabad.

Commercially available ready to use reagent kits were used for

estimation of various parameters. Following Laboratory Investigations

were done in study group and control group.

• GLUCOSE (Plasma) • TOTAL BILIRUBIN • GGT • ALT • AST • ALP • CREATININE • UREA • URIC ACID • TOTAL PROTEIN • ALBUMIN • MICROALBUMIN (Urine) • C- REACTIVE PROTEIN

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• HbA1c • INSULIN • TOTAL CHOLESTEROL • TRIGLYCERIDE • HDL-C • MAGNESIUM • COPPER • ZINC • SODIUM • POTASSIUM Care Taken

� Serum specimens should be free of fibrin, red blood cells and other

particulate matter.

� Human serum specimens to be tested should be protected from

light.

� For serum specimens ensured that complete clot formation has

taken place prior to centrifugation. (If the specimen is centrifuged

before a complete clot forms, the presence of fibrin may cause

erroneous results)

� If testing was delayed for more than 6 hours, serum specimens

were stored at -20°C and analyzed next day.

� Multiple freeze-thaw cycles must be avoided.

� Sample is mixed thoroughly after thawing, by low speed vortexing

or by gently inverting.

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CASE RECORD FORM

Reg. No. : Serial No.:

Date of Admission :

PERSONAL INFORMATION

Name :

Age/Sex :

Religion :

Address :

Contact no :

Occupation :

PATIENT HISTORY

Diagnosis :

Past history : HTN / TB / STROKE / MI

Family history :

Duration :

PERSONAL HISTORY

Habit : Smoking

Alcohol

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� ESTIMATION OF GLUCOSE METHOD: GOD/POD

PRINCIPLE

Glucose is oxidised to gluconic acid and hydrogen peroxide in the

presence of glucose oxidase. Hydrogen peroxide further reacts with

phenol and 4-aminoantipyrine by the catalytic action of peroxidase to

form a red coloured quinoneimine dye complex. Intensity of the colour

formed is directly proportional to the amount of glucose present in the

sample.

Glucose Oxidase Glucose + O2 + H2O Gluconate + H2O2 H2O2+ 4 Aminoantipyrine Red Quinoneimine dye +

H2O + Phenol

REAGENT

Glucose reagent

REAGENT PREPARATION

Reagents are ready to use. Protect from bright light.

SAMPLE TYPE

Plasma free from haemolysis.

SAMPLE STABILITY

Plasma is stable for 7 days at 2 to 80C.

Peroxidase

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PROCEDURE:

Wavelength 505 nm

Reaction Mode End Point Cuvette 0.6 cm light path Reaction Temperature 37°C or R.T. Measurement (Blanking) Reagent blank Sample Volume 3 µl Reagent Volume 300 µl Incubation 10 minutes at 37°C Linearity 500 mg/dL Standard Concentration 100 mg/dL

NORMAL REFERENCE VALUES Plasma (F): 70 - 110 mg/dl CALCULATION Glucose concentration (mg/dL) = -------------------------- X Std. conc.

� ESTIMATION OF TOTAL BILIRUBIN

METHOD: Modified Jendrassik & Grof’s Method PRINCIPLE

Bilirubin reacts with diazotised sulphanilic acid to form a coloured

azobilirubin compound. The unconjugated bilirubin couples with the

sulphanilic acid in the presence of a caffein- benzoate accelerator. The

Intensity of the colour formed is directly proportional to the amount of

total bilirubin present in sample.

Bilirubin + Diazotized sulphanilic acid Azobilirubin compound

REAGENTS

R1: Bilirubin Reagent

R2: Nitrite Reagent

Absorbance of Test Absorbance of Std.

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REAGENT PREPARATION


SAMPLE TYPE

Serum free from haemolysis.

SAMPLE STABILITY

Serum is stable for 1 day at 20 to 250C & 7 days at 4 to 80C.

(Darkness required when stored for > 8 hours).

PROCEDURE

Wavelength 546 nm Cuvette 0.6 cm light path Reaction temperature 37ºC Reaction mode End Point Blank Sample Sample Volume 20 µl Reagent 1Volume R1- 200 µl

Reagent 2Volume R2- 10 µl Incubation time 10 minutes Factor 13 Unit mg/dL Linearity High(mg/dL) 20

If result obtained was greater than linearity limit, sample was diluted 1:2

with normal saline and result was multiplied by 2.

INTERFERENCE

• Lipemic and Hemolysed sera interfere strongly with the measurement of

bilirubin.

• Serum for bilirubin estimation must be kept away from the bright light.

CALCULATION

Total bilirubin (mg/dL) = (Sample OD – Blank OD) × 13

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BIOLOGICAL REFERENCE INTERVAL

Bilirubin Total: Up to 1.0 mg/dL

� ESTIMATION OF GAMMA GLUTAMYL TRANSFERASE (GGT)

METHOD: Carboxy Substrate method

PRINCIPLE

GGT catalyzes the transfer of amino group between L-g-Glutamyl-3-

carboxy-4-nitroanilide and Glycylglycine to form L-g

Glutamylglycylglycine and 5-amino-2-nitrobenzoate. The rate of

formation of 5-amino-2- nitrobenzoate is measured as an increase in

absorbance which is proportional to the GGT activity in the sample.

L-g-Glutamyl-3-carboxy-4-nitroanilide + Glycylglycine L-g-Glutamylglycylglycine + 5-amino-2-nitrobenzoate

REAGENTS

R1: Buffer Reagent

R2: Substrate Tablets

STORAGE / STABILITY

Contents are stable at 2-8 ºC till the expiry mentioned on the label.

REAGENT PREPARATION

Working reagent: Dissolve 1 substrate tablet in 2.2 ml of buffer reagent.

This working reagent is stable for at least 15 days when stored at 2-8 ºC.

SAMPLE MATERIAL

Serum free from haemolysis. GGT is reported to be stable in serum for 3

days at 2-8 ºC.

GGT

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PROCEDURE



CALCULATION

GGT (U/L) = (∆ A/60 sec) × 1158

BIOLOGICAL REFERENCE INTERVAL:

SERUM (Men) : 10-50 U/L at 37°C

(Women) : 7-35 U/L at 37°C

� ESTIMATION OF ALT

METHOD: NADH, Kinetic UV, IFCC recommended

PRINCIPLE

Alanine aminotranferase (ALT) or Glutamate pyruvate transaminase

(GPT) catalyses the reversible transfer of an amino group from alanine to

α-ketoglutarate forming glutamate and pyruvate. The pyruvate produced

is reduced to lactate by lactate dehydrogenase (LDH) and NADH. The

Wavelength 405 nm Cuvette 0.6 cm light path Reaction temperature 37ºC Reaction mode Kinetic Reaction Direction Increasing Blank Distilled water Sample Volume 20 µl Reagent Volume 200 µl Delay time 30 sec Interval time 30 sec No of Readings 4 Factor 1158 Unit U/L Linearity High (U/L) 700

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rate of oxidation of NADH to NAD is measured as a decrease in

absorbance which is proportional to the SGPT (ALAT) activity in the

sample.

L-Alanine + α-Ketoglutarate ALT Glutamate + Pyruvate Pyruvate + NADH + H

+ LDH Lactate + NAD+

REAGENTS

R1: Enzyme Reagent

R2: Starter Reagent

REAGENT PREPARATION


SAMPLE TYPE


SAMPLE STABILITY

Serum is stable for 3 day at 2 to 80C.

PROCEDURE

Wavelength 340 nm Cuvette 0.6 cm light path Reaction temperature 37ºC Reaction mode Kinetic Reaction Direction Decreasing Blank Distilled water Sample Volume 20 µl Reagent 1Volume R1- 160 µl Reagent 2Volume R2- 40 µl Delay time 60 sec Interval time 60 sec No of Readings 3 Factor 1746 Unit U/L Linearity High(U/L) 350



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CALCULATION

ALT (U/L) = ∆OD/min × 1746


Men: 0-45 U/L

Women: 0-34 U/L

� ESTIMATION OF AST

METHOD: NADH, Kinetic UV, IFCC recommended

PRINCIPLE

Aspartate aminotransferase (AST) formerly called glutamate oxaloacetate

(GOT) catalyses the reversible transfer of an amino group from aspartate

to α-ketoglutarate forming glutamate and oxalacetate. The oxalacetate

produced is reduced to malate by malate dehydrogenase (MDH) and

NADH. The rate of oxidation of NADH to NAD is measured as a

decrease in absorbance which is proportional to the SGPT (ALAT)

activity in the sample.

L-Aspartate + α-Ketoglutarate AST Glutamate + Oxaloacetate Oxaloacetate + NADH + H

+ MDH Malate + NAD+

REAGENTS

R1: Enzyme Reagent

R2: Starter Reagent

REAGENT PREPARATION


SAMPLE TYPE


SAMPLE STABILITY


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PROCEDURE




CALCULATION

AST (U/L) = ∆OD/min × 1745

BIOLOGICAL REFERENCE INTERVAL Men: 0-35 U/L Women: 0-31 U/L

� ESTIMATION OF ALP

METHOD : p- Nitrophenylphosphate, Kinetic, DGKC recommended

PRINCIPLE

Alkaline phosphatase (ALP) catalyses the hydrolysis of p-nitrophenyl

phosphate at pH 10.4, liberating p-nitrophenol and phosphate. The rate of

formation of p-Nitrophenol is measured as an increase in absorbance

which is proportional to the alkaline phosphatase activity present in the

sample.

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p-Nitrophenylphosphate + H2O ALP p-Nitrophenol + Phosphate REAGENTS

R1: Buffer Reagent

R2: Substrate Reagent

REAGENT PREPARATION


SAMPLE TYPE


SAMPLE STABILITY


PROCEDURE




CALCULATION

ALP (U/L) = ∆OD/min × 2713

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Children (1-14 yrs): 250-700 U/L

Adults: 100-250 U/L

� ESTIMATION OF CREATININE METHOD: Mod. Jaffe’s kinetic method

PRINCIPLE

Picric acid in an alkaline medium reacts with creatinine to form an orange

coloured complex with the alkaline picrate. Intensity of the colour formed

is directly proportional to the amount of creatinine present in the sample.

Creatinine + Alkaline Picrate Orange Coloured Complex

REAGENTS

Picric acid reagent

Buffer reagent

REAGENT PREPARATION


SAMPLE TYPE


SAMPLE STABILITY

Serum is stable for 7 days at 2 to 80C

PROCEDURE

Wavelength 520 nm Reaction Mode Fixed time kinetic Cuvette 0.6 cm light path Reaction Temperature 37°C or R.T. Measurement (Blanking) Distilled water Sample Volume 20 µl Reagent 1Volume 100 µl Reagent 1Volume 100 µl Incubation 3 minutes at 37°C Linearity 20 mg/dL Standard Concentration 2 mg/dL

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CALCULATION Creatinine concentration (mg/dL) = ----------------------- X Std conc. BIOLOGICAL REFERENCE INTERVALS

Male : 0.6-1.2 mg/dL

Female : 0.5-1.1 mg/dL

� ESTIMATION OF UREA

METHOD: Urease / GLDH, Enzymatic, U.V. Kinetic method.

PRINCIPLE

Urease hydrolyzes urea to ammonia and CO2 . The ammonia formed

further combines with a Ketoglutarate and NADH to 2form Glutamate

and NAD. The rate of oxidation of NADH to NAD is measured as a

decrease in absorbance in a fixed time, which is proportional to the urea

concentration in the sample.

Urease

Urea + H2O + 2 H+ 2NH4++ CO2

GLDH 2NH4

++ 2α Ketoglutarate + 2NADH 2L-glutamate + 2NAD+ + 2H2O REAGENTS

Enzyme reagent

Starter reagent

REAGENT PREPARATION


SAMPLE TYPE


SAMPLE STABILITY



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PROCEDURE

Wavelength 340 nm Reaction Mode Fixed time kinetic Cuvette 0.6 cm light path Reaction Temperature 37°C or R.T. Measurement (Blanking) Distilled water Sample Volume 3 µl Reagent 1Volume 300 µl Incubation 3 minutes at 37°C Linearity 250 mg/dL Standard Concentration 40 mg/dL

BIOLOGICAL REFERENCE INTERVALS Serum / Plasma : 14 - 40 mg/dl CALCULATION Urea concentration (mg/dL) = -------------------------- X Std. conc.

� ESTIMATION OF URIC ACID

METHOD: Uricase/ PAP method.

PRINCIPLE

Uricase converts uric acid to allantoin and hydrogen peroxide. The

hydrogen peroxide formed further reacts with aphenolic compound and 4

aminoantipyrine by the catalytic action of peroxidase to form a red

coloured quinoneimine dyecomplex. Intensity of the colour formed is

directly proportional to the amount of uric acid present in the sample.

Uricase Uric Acid + H2O Allantoin + H2O2 H2O2+ 4 Aminoantipyrine Red Quinoneimine dye +

H2O + Phenolic Compound

REAGENTS

Buffer reagent


Peroxidase

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Enzyme reagent

REAGENT PREPARATION


SAMPLE TYPE


SAMPLE STABILITY


PROCEDURE

Wavelength 520 nm Reaction Mode End point Cuvette 0.6 cm light path Reaction Temperature 37°C or R.T. Measurement (Blanking) Distilled water Sample Volume 4 µl Reagent 1Volume 160 µl Reagent 2Volume 40 µl Incubation 5 minutes at 37°C Linearity 20 mg/dL Standard Concentration 8 mg/dL CALCULATION Uric acid concentration (mg/dL) = X X Std. conc. BIOLOGICAL REFERENCE INTERVALS Serum / Plasma : Male: 3.4-7.0 mg/dl Female: 2.5-6 mg/dl

� ESTIMATION OF TOTAL PROTEIN

METHOD: Biuret, Colorimetric

PRINCIPLE

Proteins give an intensive violet-blue complex with copper salts in an

alkaline medium. Iodide is included as an antioxidant. The intensity of

Absorbance of Test Absorbance of Test

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the color formed is proportional to the total protein concentration in the

sample.

REAGENTS

Biuret reagent

REAGENT PREPARATION


SAMPLE TYPE


SAMPLE STABILITY


PROCEDURE

Wavelength 540 nm Cuvette 0.6 cm light path Reaction temperature 37ºC Reaction mode End point Blank Reagent Sample Volume 4 µl Reagent Volume 200 µl Incubation time 10 minutes Unit g/dL Linearity High(g/dL) 15



CALCULATION

Total Protein (g/dL) = Sample OD × Factor


Adults: 6.6 – 8.3 g/dL

� ESTIMATION OF ALBUMIN

METHOD: Bromocresol green, Colorimetric

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PRINCIPLE

Albumin in the presence of bromocresol green at a slightly acid pH

produces a colour change of the indicator from yellow-green to green-

blue. The intensity of the color formed is proportional to the albumin

concentration in the sample.

REAGENT

Bromocresol green, pH 4.2

REAGENT PREPARATION


SAMPLE TYPE


SAMPLE STABILITY


PROCEDURE

Wavelength 630 nm Cuvette 0.6 cm light path Reaction temperature 37ºC Reaction mode End point Blank Reagent Sample Volume 2 µl Reagent Volume 220 µl Incubation time 10 minutes Unit g/dL Linearity High(g/dL) 7



CALCULATION

Albumin (g/dL) = Sample OD × Factor


3.5 to 5.0 g/ dL

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� ESTIMATION OF MICROALBUMINURIA

METHOD: TURBIDIMETRIC IMMUNOASSAY PRINCIPLE

In this method determination of microalbumin is based on the principle of

agglutination reaction. The test specimen is mixed with the activation

buffer (R1) and anti-human antibody solution (R2) and allowed to react.

Presence of albumin in the test specimen forms an insoluble complex

producing a turbidity, which is measured at wavelength 340 nm. The

resulting turbidity corresponds to the concentration of albumin in the test

specimen.

REAGENT

1. Activation Buffer (R1): Ready to use.

2. Anti-human albumin Reagent (R2): Ready to use solution of anti-

human albumin antibody.

3. Calibrator: Ready to use albumin solution and is equivalent to the

stated amount of albumin on mg/L basis.

REAGENT STORAGE AND STABILITY

1. Store the reagents at 2-8°C. DO NOT FREEZE. 2. The mixed stability of working reagent (R1+R2) is 7 days when

stored at 2-8°C.

PROCEDURE

Wavelength 340 nm Cuvette 0.6 cm light path Reaction mode 2-point Blank Distilled water Sample Volume 25 µl Reagent 1 Volume 250 µl Reagent 2 Volume 50 µl Incubation time 7 minutes Unit mg/L Detection limit (mg/L) 20

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PREPARATION OF MA CALIBRATION CURVE

Dilute the calibrator serially as mentioned below for preparation of

calibration curve.

Test tube No. 1 2 3 4 5 Calibrator dilution No. D1 D2 D3 D4 D5 Volume of Saline in µl - 100 100 100 100 Volume of Calibrator (S) in µl

100 100 100 100 100

Conc. of albumin in mg/L 400 200 100 50 25 CALCULATION

Instrument calculates ∆A for each diluted calibrator for preparing the

calibration curve and plots a graph of ∆A versus concentration of MA. By

interpolating ∆A for each sample on calibration curve, instrument

measured MA concentration.


Adults: ≤ 20mg/L

� ESTIMATION OF C-REACTIVE PROTEIN

METHOD: TURBIDIMETRIC IMMUNOASSAY

PRINCIPLE

In this method determination of C-reactive protein in human serum is

based on the principle of agglutination reaction. The test specimen is

mixed with activation buffer (R1), reagent (R2) is then added and allowed

to react. Presence of CRP in the test specimen results in the formation of

an insoluble complex producing a turbidity, which is measured at 340 nm

wavelength. The increase in turbidity corresponds to the concentration of

CRP in the test specimen.

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REAGENT 1. Activation Buffer (R1): Ready to use.

2. Reagent (R2): Ready to use solution of anti-CRP antibody.

3. Calibrator: A lyophilized preparation of serum equivalent to the stated

amount of CRP on a mg/dl basis, when hydrated appropriately.


1. Store the reagents at 2-8°C. DO NOT FREEZE.

2. The shelf life of the reagent, activation buffer and the calibrator is as

per the expiry date mentioned on the respectivevial label.

3. The reconstituted calibrator is stable for 7 days at 2-8°C and 48 hours

at 25°C-30°C (RT.).

PROCEDURE

Wavelength 340 nm Cuvette 0.6 cm light path Reaction mode 2-point Blank Distilled water Sample Volume 25 µl Reagent 1 Volume 250 µl Reagent 2 Volume 250 µl Incubation time 10 minutes Unit mg/dL Detection limit (mg/dl) 0.3

PREPARATION OF CRP CALIBRATION CURVE The calibrator must be reconstituted exactly with 1.0 ml of distilled

water, wait for 10 minutes , gently swirl the vial till the solution attains

homogeneity. Once reconstituted it is ready to use for preparing the CRP

calibration curve. The Concentration of CRP (S) in the reconstituted

calibrator is as mentioned at the end of the package insert. Dilute the

calibrator serially as mentioned below for preparation of calibration

curve.

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Test tube No. 1 2 3 4 5 Calibrator dilution No. D1 D2 D3 D4 D5 Volume of Saline in µl - 100 375 880 940 Volume of Calibrator (S) in µl 100 100 125 120 60 Conc. Of CRP in mg/dl 10 5 2.5 1.2 0.6

CALCULATION

Instrument calculates ∆A for each diluted calibrator for preparing the

calibration curve and plots a graph of ∆A versus concentration of CRP.

Interpolate ∆A for each sample on calibration curve and obtain CRP

concentration.


Adults: < 0.6 mg/dL

� ESTIMATION OF HbA1c

METHOD: NycoCard method

PRINCIPLE

NycoCard HbA1c is a boronate affinity assay. When blood is added to

the reagent, the erythrocytes immediately lyse. All hemoglobin

precipitates. The boronic acid conjugate binds to the cis-diol

configuration of glycated hemoglobin. An aliquot of the reaction mixture

is added to the test device, and all the precipitated hemoglobin,

conjugate-bound and unbound, remains on top of the filter. Any excess of

coloured conjugate is removed with the washing solution. The precipitate

is evaluated by measuring the blue and red colour intensity with the

NycoCard reader, the ratio between them being proportional to the

percentage of HbA1c in the sample.

135

REAGENTS

Test device (TD): Plastic device containing a membrane filter.

Reagent 1: Glycinamide buffer containing dye-bound boronic acid and

detergents.

Washing solution (R2): Morpholine buffered NaCl solution and

detergents.


1. Store the R1 reagent at 2-8°C. DO NOT FREEZE.

2. Test device can be stored at room temperature (15-20°C). Store the test

devices in the original bag and avoid humidity below 20% and above

70%.

3. Washing solution (R2) can be stored at room temperature (15-20C).

PROCEDURE

Add 5µl whole blood to the test tube with R1 reagent. Mix well. Leave

the tube for 2-3 minutes. Remix to obtain a homogenous suspension.

Apply 25 µl of the mixture to a TD. Allow the mixture to soak

completely into the membrane. Wait for minimum 10 seconds. Apply 25

µl washing solution to the TD. Allow the reagent to soak completely into

the membrane. Wait for minimum 10 seconds. Read the test result within

5 minutes using Card reader.


Non-diabetic reference range is 6.4% HbA1c

� ESTIMATION OF INSULIN

METHOD: ELISA

136

PRINCIPLE

Insulin ELISA kit is based on the sandwich principle. The microtiter

wells are coated with a monoclonal antibody directed towards a unique

antigenic site on the insulin molecule. An aliquot of patient samle

containing endogenous insulin is incubated in the coated well with

enzyme conjugate, which is an anti-insulin antibody conjugated with

biotin. After incubation the unbound conjugate is washed off. During

second incubation step streptavidin peroxidase enzyme complex binds to

the biotin-anti-insulin antibody. The amount of bound HRP complex is

proportional to the concentration of insulin in the sample. Having added

the substrate solution, the intensity of colour developed is proportional to

the concentration of insulin in the patient sample.

REAGENTS

1. Microtiterwells: Wells coated with anti-insulin antibody (monoclonal).

2. Zero standard: Ready to use 0µIU/mL

3. Standard (1-5): Ready to use, Concentrations:

6.25,12.5,25,50,100µIU/mL

4. Enzyme conjugate: Ready to use, Mouse monoclonal anti-insulin

conjugated to biotin

5. Enzyme complex: Ready to use, Streptavidin-HRP complex

6. Substrate solution: Ready to use, Tetramethylbenzidine (TMB)

7. Stop solution: Ready to use, 0.5M H2SO4

8. Wash solution: 40X concentration

Dilute 30 ml of concentrated wash solution with 1170 ml deionized water

to a final volume of 1200mL.


1. Store the reagent at 2-8°C. DO NOT FREEZE.

137

2. Opened kits retain activity for 8 weeks.

PROCEDURE

Dispense 25 µl of each standard, control and samples with new

disposable tips into appropriate wells. Dispense 25 µl enzyme conjugate

into each well. Thoroughly mix for 10 seconds. Incubate for 30 minutes

at room temperature. Briskly shake out the contents of the wells. Rinse

the wells 3 times with diluted wash solution (400µl per well). Strike the

wells sharply on absorbent paper to remove residual droplets. Add 50 µl

of enzyme complex to each well. Incubate for 30 minutes at room

temperature. Briskly shake out the contents of the wells. Rinse the wells 3

times with diluted wash solution (400µl per well). Strike the wells

sharply on absorbent paper to remove residual droplets. Add 50 µl of

substrate solution to each well. Incubate for 15 minutes at room

temperature. Stop the enzymatic reaction by adding 50 µl of stop solution

to each well. Determine the absorbance (OD) of each well at 450nm with

a microtiter plate reade within 10 minutes.

CALCULATION

Instrument calculates average absorbance values for each set of standards,

controls and patient samples. A standard curve is constructed

automatically by the instrument. The concentration of the samples can be

read directly from this standard curve.


Adults : 2 -25µIU/mL

� ESTIMATION OF CHOLESTEROL

METHOD: CHOD / PAP

138

PRINCIPLE

Cholesterol esters are hydrolyzed by cholesterol esterase to produce

cholesterol. This cholesterol is then oxidized by Cholesterol oxidase to

produce hydrogen peroxide which in turn reacts with 4 aminoantipyrine

and phenolic compound in presence of peroxidase to yield a red coloured

complex. Intensity of the colour formed is directly proportional to the

amount of cholesterol present in the sample.

REAGENTS

R1: Enzyme Reagent 1


REAGENT PREPARATION


SAMPLE TYPE


SAMPLE STABILITY

Serum is stable for 7 days at 2 to 80C.

PROCEDURE

Wavelength 520 nm Reaction Mode End Point Cuvette 0.6 cm light path Reaction Temperature 37°C or R.T. Measurement (Blanking) Against Reagent Blank Sample Volume 2 µl Reagent Volume 200 µl Incubation 5 minutes at 37°C Linearity 750 mg/dL Standard Concentration 200 mg/dL

CALCULATION

Cholesterol concentration (mg/dL) = ----------------------- X Std con.


139


150 to 200 mg/dL

� ESTIMATION OF TRIGLYCERIDE

METHOD: GPO/PAP

PRINCIPLE

Lipoprotein Lipase hydrolyses serum triglycerides to glycerol & free fatty

acids. Glycerol, in turn is converted to glycerol 3-phosphate in presence

of ATP and glycerokinase. Glycerol-3-phosphate is then oxidized by

glycerolphosphate oxidase to yield hydrogen peroxide which is further

broken down by peroxidase to give a purple coloured complex. The

intensity of colour is measured photometrically at 545 nm. The intensity

of colour is directly proportional to the triglyceride concentration in the

sample.

REAGENTS

R1: Enzyme Reagent

REAGENT PREPARATION


SAMPLE TYPE


SAMPLE STABILITY


140

PROCEDURE

Wavelength 505 nm Reaction Mode End Point Cuvette 1 cm light path Reaction Temperature 37°C Measurement (Blanking) Reagent Blank Sample Volume 3 µl Reagent Volume 300 µl Incubation 5 min. Linearity 1000 mg/dL Standard Concentration 200 mg/dL

CALCULATION

Triglyceride concentration (mg/dL) = -------------------------- X Std con. BIOLOGICAL REFERENCE INTERVAL

Men : 60 – 165 mg/dL (0.68 – 1.18 mmol/L) Women : 40 – 140 mg/dL (0.40 – 1.58 mmol/L)

� ESTIMATION OF HDL CHOLESTEROL

METHOD: Direct Enzymatic Method

PRINCIPLE

Direct determination of serum HDL-C (high-density lipoprotein

cholesterol) levels without the need for pretreatment or centrifugation of

the sample. The method depends on the properties of a detergent which

solubilizes only the HDL-C so that the HDL-C is released to react with

the cholesterol esterase, cholesterol oxidase and chromogens to give

colour. The non HDL-C lipoproteins LDL-C, VLDL and chylomicrons

are inhibited from reacting with the enzymes due to absorption of the

detergents in their surfaces. The intensity of the color formed is

proportional to the HDL-C concentration in the sample.


141

REAGENTS



REAGENT PREPARATION


SAMPLE TYPE


SAMPLE STABILITY


PROCEDURE

Mode of reaction End point Slope of reaction Increasing Wavelength 630 nm Temperature 37ºC Linearity Up to 150 mg/dL Blank Reagent Incubation time 5 min Sample Volume 3µl Reagent 1volume 225µL Reagent 2volume 75µl Cuvette 0.6 cm light path Linearity 150mg/dl Standard Concentration 46 mg/dL

CALCULATION

For Calibrator ∆ AC = A2C – A1C

For Test ∆ AT = A2T – A1T

HDL-C in mg/dL = X Concentration of Standard


Male Female Low risk > 50 mg/dL > 60 mg/dL Normal Risk 35 - 50 mg/dL 45-60 mg/dL High Risk < 35 mg/dL < 45 mg/dL

∆ AC ∆ AT

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� ESTIMATION OF VLDL-CHOLESTEROL VLDL is calculated by following Friedwald formula VLDL = TG/5 BIOLOGICAL REFERENCE INTERVAL Upto 35 mg/dL

� ESTIMATION OF LDL-CHOLESTEROL LDL is calculated by following Friedwald equation: LDL-Cholesterol = Total Cholesterol – (VLDL+ HDL-Cholesterol) BIOLOGICAL REFERENCE INTERVAL Upto 100 mg/dL TC/HDL and LDL/HDL Ratio were thereby calculated.

� ESTIMATION OF MAGNESIUM

METHOD: Calmagite Method

PRINCIPLE

Magnesium combines with Calmagite in an alkaline medium to form a

red coloured complex. Interference of calcium and proteins is eliminated

by the addition of specific chelating agents and detergents. Intensity of

the colour formed is directly proportional to the amount of magnesium

present in the sample.

Alkaline Magnesium + Calmagite Red coloured complex

Medium

REAGENTS

R1: Buffer Reagent

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R2: Colour Reagent

STORAGE / STABILITY

Contents are stable at 2-8 ºC till the expiry mentioned on the label.

REAGENT PREPARATION


REAGENT PREPARATION

It was prepared by mixing equal volumes of R1 (Buffer Reagent) and R2

(Colour reagent). The Working reagent is stable at 2-8 ºC for at least one

month.

SAMPLE MATERIAL


PROCEDURE

CALCULATION Magnesium concentration (mg/dL) = ----------------------------- X Std. con.

BIOLOGICAL REFERENCE INTERVAL Serum (Children): 1.83 - 2.44 mg/dL

(Adults): 1.59 – 3.05 mg/dL

Wavelength 546 nm Cuvette 0.6 cm light path Reaction temperature 37ºC Reaction mode Endpoint Reaction Direction Increasing Blank Reagent Sample Volume 2µl Reagent Volume 200 µl Incubation Time 300 sec Unit mEq/L Linearity High (mEq/L) 10

Absorbance of Test

Absorbance of Std.

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� ESTIMATION OF COPPER

METHOD: Colorimetric

PRINCIPLE

Copper, released from ceruloplasmin in an acidic medium, reacts with Di-

Br-PAESA to form a coloured complex. Intensity of the complex formed

is directly proportional to the amount of Copper present in the sample.

Copper + Di-Br-PAESA Coloured Complex REAGENTS

R1: Buffer Reagent

R2: Colour Reagent

REAGENT PREPARATION


SAMPLE TYPE

Serum, free from haemolysis.

SAMPLE STABILITY


PROCEDURE

Wavelength 580 nm Reaction Mode End Point Cuvette 0.6 cm light path Reaction Temperature 37°C Measurement (Blanking) Reagent Blank Sample Volume 10 µl Reagent Volume 200 µl Incubation 10 min. Linearity 500 µg/dL Standard Concentration 200 µg/dL

CALCULATION

Copper concentration (µg/dL) = ----------------------------- X Std. con.

Acidic Medium

Absorbance of Test

Absorbance of Std.

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BIOLOGICAL REFERENCE INTERVAL Male : 80 – 140 µg/dL Female : 80 – 155 µg/dL

� ESTIMATION OF ZINC

METHOD: Colorimetric

PRINCIPLE

Zinc in an alkaline medium reacts with Nitro-PAPS to form a purple

coloured complex. Intensity of the complex formed is directly

proportional to the amount of Zinc present in the sample.72, 73

Zinc +Nitro-PAP Purple Coloured Complex REAGENTS

R1: Buffer Reagent

R2: Colour Reagent

REAGENT PREPARATION


SAMPLE TYPE


SAMPLE STABILITY


PROCEDURE

Wavelength 570 nm Reaction Mode End Point Cuvette 0.6 cm light path Reaction Temperature 37°C or R.T. Measurement (Blanking) Against Reagent Blank Sample Volume 10 µl Reagent Volume 200 µl Incubation 5 min. Linearity Up to 700 µg/dL Standard Concentration 200 µl

Alkaline Medium

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CALCULATION Zinc concentration (µg/dL) = ------------------------- X Std con. BIOLOGICAL REFERENCE INTERVAL 60 – 120 µg /dL

� ESTIMATION OF SODIUM AND POTASSIUM

METHOD: Ion Selective Electrode Method

PRINCIPLE:

The electrolyte measurement system measures sodium and potassium

ions in biological fluids using ion selective electrode technology the flow

through electrodes use selective membrane tubing specially formulated to

be sensitive to the respective ion. The potential of each electrode is

measured relative to a fixed stable voltage established by the double

junction Silver / Silver – Chloride reference electrode. And ion selective

electrode develops a voltage that varies with the concentration of the ion

to which it responds. The relationship between the voltages develop and

the concentration of the sensed ion is logarithmic, as expressed by the

Nernst equation.

RT log (αC) E=E0 + --------------

nf Where E = the potential of the electrode in sample solution. E0 = the potential developed under standard condition. RT/nf =A temperature dependent “constant”, termed the slope. Log = base ten logarithm function. α = Activity coefficient of the measured ion in the solution. C = concentration of the measured ion in the solution. REAGENTS

Following reagents are used in the XL-640 ISE module. • Calibrant A • Calibrant B • Cleaning solution

Absorbance of Test

Absorbance of Std.

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PROCEDURE

• When the electrode calibration slopes are in the acceptable range the electrolyte measurement system is ready for the sample analysis.

• For serum sample 70 µl of sample is required for the electrolyte measurement.


• SODIUM: Adult : 136 to 145 mEq/L

• POTASSIUM: Adult: 3.5 to 5.1 mEq/L

� HOMA-IR (Homeostasis Model of Assessment - Insulin Resistance) HOMA-IR originally described by Mathews DR et al. (1985) HOMA-IR was calculated using the following formula:

HOMA-IR = Fasting Glucose (mg/dl) × Fasting Insulin (µU/ml) /405

� eGFR (Estimated Glomerular Filteration Rate) eGFR was calculated using the following formula: Cockcroft and Gault (CG) method:

eGFR (male) = (140-age) x wt. / sCr x 72 eGFR (female) = eGFR (male) x 0.85 age = age (years) wt. = weight (Kg) sCr = serum creatinine concentration (mg%) This result was then adjusted for body surface area (BSA). The Mosteller

formula is used because it is much simpler and easily calculated with a

hand-held calculator.

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The Mosteller formula: BSA (m²) = ([Height (cm) x Weight (kg)]/ 3600)½

Formula used for eGFR calculation in present study was:

eGFR = eGFR from CG Method x 1.73/BSA


> 90 ml/min/1.73m2

� BLOOD PRESSURE

After 30 minutes of resting, blood pressure was measured by standard

mercury sphygmomanometers twice at a gap of 1–2 minutes. The average

of the two readings was used to classify high blood pressure or

hypertension.

� ANTHROPOMETRIC MEASUREMENTS

Weight, height, Waist circumference (WC), Hip circumference (HC)

were measured as per WHO guidelines (WHO-2008)

� WEIGHT

Weight of subjects wearing light clothing without shoes was

measured using a calibrated weighing machine. Weight was recorded to

the nearest 0.1 Kg.

� HEIGHT

The subjects were asked to stand erect without shoes against a wall

and their topmost point of vertex was identified with the help of a plastic

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ruler. Thus height was measured at this point. Height was recorded to the

nearest cm.

� BODY MASS INDEX (BMI)

BMI was calculated using following formula (Garrow JS and

Webster J-1985).

BMI (Kg. /m2) = 2meters)in(Height

Kg.inWeight

Categorization of subjects on the basis of BMI is given in Table G

(WHO-1997).

� WAIST CIRCUMFERENCE

The waist circumference was measured at the midpoint between the lower

margin of the last palpable rib and the top of the iliac crest, using a

stretch‐resistant tape. The measurement is made at the end of a normal

expiration.

� HIP CIRCUMFERENCE

Hip circumference was measured at the widest portion of the buttocks,

with the tape parallel to the floor. The waist to hip ratio (W/H) was

thereby calculated.

� STATISTICAL ANALYSIS Numerical variables are reported in terms of mean and standard

deviation. Data were evaluated by analysis of variance (ANOVA) with

bonferroni’s post hoc test, adjusted for multiple comparisons. P-values of

<0.05 were considered statistically significant. Correlations were

determined by Pearson’s test. Analysis was carried out using Graphpad

Instat version 3 statistical software.

MATERIAL AND METHODS -...

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