DETECTION OF GLUCOSE-6PHOSPHATE DEHYDROGENASE (G-6-PD) DEFICIENCY

DETECTION OF GLUCOSE-6PHOSPHATE DEHYDROGENASE (G-6-PD) DEFICIENCY

G6PD Screening Test 

Reagents for G6PD Screening Test 

Procedure for G6PD Screening Test 

Reading results

Introduction:- G6PD deficiency is one of the most commonly inherited disorders. The inheritance is sex-linked, affecting males, while females act as carriers. Most individuals are symptom free, but acute hemolytic anemia may occur by exposure to certain hemolytic agents listed . It is necessary to detect the deficiency so that the hemolytic agents can be avoided.

G-6-PD Screening Test

When whole blood or haemolysate is incubated with an excess of glucose-6-phosphate (the substrate) and nicotinamide adenosine diphosphate (NADP), the enzyme G-6-PD converts NADP to the reduced form of NADP (NADPH). The NADPH reduces the dye nitro blue tetrazolium

(NBT) in presence of phenazine methosulphate (PMS), to a brown colored compound. The reduction of NBT in a defined period of time indicates normal G-6-PD activity. 

Specimen 

Capillary blood or venous blood anticoagulated with EDTA.

A control specimen from a known normal individual should also be collected. 

Reagents

1. Tris (2-amino-hydroxy-methyl propane 1-3 diol) buffer, pH 8.5 (0.07M) Dissolve 8.95g of tris in about 90 ml distilled water. Adjust the pH to 8.5 with 0.1 N hydrochloric acid and make the volume upto 100ml with distilled water. 

2. Buffer-substrate solution 

Glucose-6-phosphate 40 mg 

NADP 25 mg 

Nitro blue tetrazolium 25 mg

Distilled water 20 ml 

Tris buffer 5 ml 

This reagent is stable for one month at 4°C.

3. Phenazine methosulphate (PMS) Prepare freshly before use by dissolving 1mg per ml distilled water. 

Technique

(1) Into a test-tube, pipette 0.9 ml of the buffer substrate solution.

(2) Add 0.01 ml whole blood. 

(3) Add 0.05 ml PMS solution

(4) Mix well and allow standing at room temperature for 30 minutes, away from direct sunlight.

(5) Read the color change macroscopically.

(6) Prepare a control tube in the same way as above using the control specimen.

(7) Prepare reagent blank using distilled water instead of whole blood. 

Reading results 

Dark brown color with or without a blue Normal G-6-PD level 

Precipitate 

No change in color- G-6-PD deficient malesor homozygous females 

Tan color or brown color-Heterozygous females