Monday, September 17, 2007
Clinical Chemistry
G6PD is the most common enzyme deficiency in the world, affecting approximately 400 million people. It is an X-linked inherited disorder which affects Africans, Asians, Mediterraneans and people of Middle-Eastern descent.
G6PD catalyzes NADP to its reduced form, NADPH, through the pentose phosphate pathway, which prevents oxidative damage to cells.
Diagnosis of G6PD deficiency is made through 2 ways, a rapid fluorescence spot test (screening), or a quantitative spectrophotometric analysis.
I will elaborate more on the screening test in the lab I am working at. In Singapore, all newborns are screened for G6PD deficiency.
Glucose-6 Phosphate Dehydrogenase (G6PD) screening test
Procedure for G6PD screening
1. Pipette 100µL of lysing agent into the wells of a titration plate. The first 4 wells are for the 3 levels of controls (Deficient, Intermediate and Normal) and for a blank. Patient samples are placed in rows of 4s below the controls.
2. Blood in the EDTA tube is mixed by gently inverting the tube several times.
3. Pipette 5µL of whole blood/controls into the wells and shake the plate to mix the solution.
4. Incubate at room temperature for 10 minutes for complete lysis of the RBCs to release the enzyme.
5. Using a stick, take 2 drops of reaction mix and spot on to the absorbent filter paper.
6. Dry the spots completely for about 3 minutes with the aid of a hair-dryer.
7. View the spots under long-wave (365nm) UV light. Deficient samples will not show any fluorescence. Intermediate samples will show little or some fluorescence. Normal samples will fluoresce brightly.
8. The screening test for G6PD is based on the following principle:
Glucose-6-Phosphate + NADP+ --(G6PD)--> Gluconate-6-phosphate + NADPH + H+
The NADPH produced in the reaction fluoresces under long-wave UV light (365nm). No fluorescence will be observed if there is marked deficiency or lack of the enzyme G6PD. This screening test might be tricky at times, especially when there is little fluorescence. Some people may think it is deficient, while others think it is normal (i.e very subjective to which level of enzyme activity). Thus, if a technician is unsure of the result, he/she will consult another technician to confirm the result.
Ng Wei Shen Martin
0503312A
TG02
haha, why do u all still do screening since its so subjective, might as well do the quantitative method right away?
Jo-anne Loh
TG02
Not everyone is G6PD deficient. Only a small percentage of the population suffers from it. The screening method is also faster and cheaper to conduct than the quantitation.
Martin
TG02
are you gona repeat the test agn (like re-centrifuge the specimen and run into the machine) or report straight away?
Natalie
TG01
For samples from the hospital I am at, we will do the quantitative test immediately (the method is explained by Kent in his blog).
For samples from other labs, the staff will call them and ask if they want to continue with the quantitative test.
Martin
TG02
Subscribe to Post Comments [Atom]
<< Home
Subscribe to Posts [Atom]