Introduction:

G6PD is an enzyme called glucose-6-phosphate dehydrogenase. It is a type of housekeeping protein

that has many different activities in the body. G6PD is responsible for protecting your cells from
chemical and environmental damage, also known as oxidative damage.

A lack of G6PD causes your red blood cells to break down faster than they are made, which is called
hemolysis. When our body can’t make up for the quick loss, can get hemolytic anemia.

Hemolytic anemia can be dangerous because it causes a loss of oxygen to our organs and tissues.

Principle:
The G6PD released from the red cells catalyzes the Glucose-6-phosphate with reduction of NADP to
NADPH. The rate of reduction of NADP to NADPH is measured as an increase in absorbance at 340
nm produced in the reaction catalyzed by the enzyme which is proportional to the G6PD activity in
the sample.

Reagents:
• Methylene blue chloride 0.0004 M

(Add 150mg. methylene blue chloride + 1000ml of DW)

• Sodium nitrite solution 1.25%

Sample:

Fresh whole blood sample collected in EDTA or Heparin Tube

Procedure:

• Take three test tubes and label them Test, positive control and negative control
• Now add Sodium nitrite 0.1 ml to the test label tube, 0.1 ml to the positive control and nil to
the negative control label tube
• Add reagent methylene blue solution 0.1 ml to the test, nil to positive and negative control.
• Add sample blood 2 ml to the test, 2 ml to the negative control and 2 ml to the positive
control.
• Now place in a water bath at 37 degrees centigrade for 90 mins
• At the end of the 90mins, take out the tubes and mix by repeated inversions
• Take three test tubes and once again mark as Test, positive control and negative control.
• Add 10 ml of DW to each tube and 0.1ml of Ellicott’s from corresponding original tubes and
add to these new tubes
• Mix by repeated inversions and wait for 2 mins for colour to develop.
• Now match the colour of the test with the positive and negative control tube.

Interpretation:

Exact colour match with positive control Positive / Reactive – Complete G6PD deficiency
Exact colour match with negative control Negative / Non-reactive – no G6PD Deficiency
Intermediate colour development Intermediate