Immunochemistry- G6PD quantification

Hey guys, sorry for the late posting! This will be the last time I'm sharing! (:
This time I am going to talk about the quantitative determination of G6PD on an analyzer, which is currently undergoing evaulation.

Principle
The principle of the assay is to utilize G6PD (that is present in the RBCs) that catalyzes the oxidation of glucose-6-phosphate (G6P) to 6-Phosphogluconolactone (6PG). This takes place in the presence of NADP, which will be reduced to NADPH. The NADPH produced will then react with a colour reagent to produce a fluorescence that will be measured colourimetrically at 550nm.

What is G6PD
G6PD is an important cytoplasmic enzyme that is found in cells and plays a crucial role in reducing the oxidative effect of the free radicals on the RBCs. It is involve in the first step of the hexose-monophosphate pathway (HMP) to produced NADPH, which is essential in maintaining the integrity of the RBCs membrane. Hence, with a deficiency of this enzyme, the RBCs are prone to lysis due to the oxidative stress. And this may lead to hemolytic anaemia.

Why do this test?
An awareness of G6PD deficiency had been raised many years back in Singapore. Hence, all newborns will be screened for this deficiency. And of course, the samples used for this test will be all cord blood samples.

How does the assay work?
First, all the samples will be transferred manually into eppendorf tubes and loaded onto the analyzer. Next, an elution buffer will then be added to the blood to lyse the RBCs. This is to bring out the enzyme, if it is present. The reagent solution is then added for the production of NADPH in the presence of G6PD. After which, the colour reagent will be added for colour production if NADPH is produced. Incubation will take place before the absorbance reading is taken.

The quantitative results will then be calculated after the readings are entered onto Excel. For deficient samples, they will be re-run again to confirm whether if the enzyme is really absent.

That should be all! (: Feel free to ask me any questions, I will try my best to answer them.

Posted by: Tan Siew Ming
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