st 1

Block 16 Practical work

Department of Clinical Pathology Faculty of Medicine Universitas Gadjah Mada Yogyakarta

Glucose (GOD-PAP)
Principle:
Glucose concentration is determined after enzymatic oxidation by glucose oxidase. The colorimeteric indicator is quinoneimine, which is generated from 4-aminoantipyrine and phenol by hydrogen peroxide under the catalytic action of peroxidase (Trinders reaction).

Reaction
GOD Glucose + O2 POD 2 H2O2 + 4Aminoantipyrine + Phenol
Gluconic acid + H2O2

Quinoneimine + 4 H2O

Samples

Serum, heparinized plasma or EDTA plasma, spinal fluid Separate at the latest 1h after blood collection from cellular contents.

Reagents

Components and Concentration in the test

Phosphate buffer pH 7,5 250 mmol/L Phenol 5 mmol/L 4-Aminoantipyrine 0,5 mmol/L Glucose oxidase(GOD) 10 kU/L Peroxidase (POD) 1 kU/L

Standard:100 mg/dL (5,55 mmol/L)

10 UL water

1000 UL reagent

10 uL sample/std

1000 UL reagent

Blank
Mix well

Sample or standard
Mix well

incubate 20 minutes, at 20-25C or 10 min at 37C. Read absorbance against the blank within 60 minutes

 Calculation With standard or calibrator Glucose (mg/dL) = A Sample x Conc. Standard/Cal (mg/dL) A Std/Cal Conversion factor Glucose (mg/dL) x 0.05551 = Glucose (mmol/L) Normal range: Fasting 70 - 100 mg/dL (whole blood) 70 - 115 mg/dL (serum) 35 - 50 mg/dL (spinal fluid)

Glucose Challenge Test

Objective:
To know body response after glucose loading To screen DM among pregnant woman

Principle:
Patient body force to respond by glucose loading and the blood glucose are monitored. Abnormal result can be obtained after

Indicating patient:
Pregnancy 2nd trimester

Technical Requirement
Samples:
Serum or plasma (recommended) No finger prick blood samples

Reagents:
Glucose 50g Reagent kit for glucose assay (GOD-PAP method)

Assay Procedure
Patient is not fasting before test Patient is given 50 g glucose loading, dissolved in 200 ml water. The glucose should be drank in 5 minutes The patients venous blood sample is taken one hour after loading The glucose concentration is measured from the sample

Interpretation
Positive: venous glucose concentration >140 mg/dL. If the result is positive, the patient should be tested further by oral glucose tolerance test

Oral Glucose Tolerance Test

Objective:
To know body response after glucose loading and to screen DM among suspected person

Principle:
Patient body force to respond by glucose loading Blood glucose are monitored every hour for 3 hours. Blood glucose curve can show abnormal pattern

Indicating patient:
Screening test for DM is in a doubt

Technical Requirement
Samples:
Serum or plasma (recommended) No finger prick blood samples

Reagents:
Glucose 75g (PERKENI), or 100g (ADA) Reagent kit for glucose assay (GOD-PAP method)

Assay Procedure
Patient should fast for 8-12 hours before test In fasting condition, blood sample is taken Patient is given 75g or 100g glucose loading, dissolved in appropriate amount of water The patients venous blood sample is taken one hour, two hours and three hours after loading The glucose concentration is measured from the sample

Interpretation
Normal criteria (WHO, 2004):
Fasting blood glucose: <100 mg/dL 1 hour <190 mg/dL 2 hour <165 mg/dL 3 hour < 145 mg/dL

OGTT Interpretation in Gestational DM