This is one of the simpler and less expensive test to screen for G6PD deficiency.Deficiency or a reduction in G6PD activity in red blood cells can cause an acute intravascular haemolysis following exposure to oxidant agents such as fava beans(favism),neonatal jaundice and less commonly chronic haemolytic anaemia.The severity of clinical symptoms is mainly dependent on the variant of defective G6PD gene inherited.
Causes of G6PD Deficiency
G6PD deficiency is a genetic condition that it is passed along from either one or both parents to their child. This defective gene causes G6PD deficiency on the X chromosome, which is one of the two sex chromosomes.
Men have only one X chromosome, while women have two X chromosomes. In the males, one altered copy of the gene is enough to cause G6PD deficiency while In females, a mutation would have to occur in both copies of the gene. Since it is unlikely for females to have two altered copies of this gene.
Male are most affected by G6PD deficiency than females.
Symptoms of G6PD deficiency
- shortness of breath
- rapid heart rate
- urine that is dark or yellow-orange
- jaundice, or yellowing of the skin and whites of the eyes
Principle of Methaemoglobin reduction test
- Haemoglobin is oxidized to methaemoglobin (Hi) when treated with sodium nitrite.
- Methylene blue, a redox dye activate the pentose phosphate pathway resulting in the enzymatic conversion of methaemoglobin back to haemoglobin in those red cells with normal G6PD activity.
- In G6PD deficient red blood cells,there is no enzymatic conversion of methaemoglobin to haemoglobin
Reagent required for Methaemoglobin reduction test
- Methylene blue
- Sodium nitrite-glucose reagent (0.5 g sodium nitrite and 2.0 g Glucose dissolved in 40 ml distilled water or deionized water)
Specimen used for Methaemoglobin reduction test
- Venous blood sample collected in an EDTA tube is suitable and should not be collected during haemolytic crisis
- Blood sample must be tested within 8 hours
Methaemoglobin reduction test Procedure
- Use 3 small glass tube labelling them test,normal and deficient
- Pipette into each of those tube as follow
|Tube||Test||Normal Control||Deficient Control|
|Sodium nitrite-glucose reagent||0.1ml||——–||0.1ml|
|Methylene Blue reagent||0.1ml||———–||———-|
- Now stopper these tubes and gently mix then incubate all three tubes at a temperature of 35-37°C for 90 minutes
- Take another 3 large tubes of about 15 ml capacity and label them as Test,normal and deficient as described above
- Pipette 10 ml of distilled water into each of those large tubes
- Then transfer 0.1 ml of the well mixed incubated sample from the test,normal and deficient tubes to each large tubes containing 10 ml distilled water respectively.
- Mix the contents of each large tubes
- Then examine the colour of the solution in each tube
Observation and result
|Color of test solution is similar to the red color of the normal tube||Normal G6PD activity|
|Colour of test solution is similar to the brown colour of the deficient tube||Reduced G6PD activity(G6PD deficiency in homozygote)|
- Monica Cheesbrough Part 2,p334