G6PD Methaemoglobin Reduction Test Principle and Interpretation

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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
  • fever
  • fatigue
  • dizziness
  • paleness
  • 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
Also read  How to stain peripheral blood and interpret blood film (Giemsa stain)

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
TubeTestNormal ControlDeficient Control
Sodium nitrite-glucose reagent0.1ml         ——–0.1ml
Methylene Blue reagent0.1ml         ———–  ———-
Patient’s blood2ml2ml2ml
  • 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 tubeNormal G6PD activity
Colour of test solution is similar to the brown colour of the deficient tubeReduced G6PD activity(G6PD deficiency in homozygote)
Normal blood → clear red color. Deficient blood → brown color. 


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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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