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
0702862D
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Siewming,
ReplyDelete(1) What is the final end colour if enzyme G6PD is present?
(2) Other than rerunning the test, is there a more accurate method to determine whether the newborn is G6PD deficient?
Li Yinliang Alex 0704894E
TG02 Group 8
26 October 2009
Hi alex,
ReplyDelete(1) I'm sorry that I've made a mistake there. I meant a fluorescence will be produced in the presence of the enzyme.
(2) Currently, this quantitative assay is still under evaulation. However, right now a qualitative screening test (fluorescence spot test) is being done in the lab to screen infants for G6PD deficiency. For any doubtful cases, they will be sent to another hospital for confirmation.
As both qualitative and quantitative test use the same cord blood samples, we can compare the results to see if they tally.
Siew Ming
Hello Siewming
ReplyDeleteI will like to ask, how long is the incubation time for this test? And is there a specfic timing for you to do the absorbance reading after the addition of the fluoresence compound?
Vanessa Chua
0702099C
TG02
Hi there, vanessa!
ReplyDeleteBasically, the incubation time of the samples is 15mins long at 37 degrees celsius/
And as for the timing.. This test is fully automated. But right after the addition of the colour reagent, a initial absorbance reading will be taken. After incubation, a second absorbance reading is then taken at 550nm.
Siew Ming (:
Hi Siew Ming!
ReplyDeletedo you guys only do cord blood samples?
how about adult samples?
thanks!
stella
0701059H
Hey stellaaaaa! :D
ReplyDeleteIn our lab, we only process cord blood samples. I think this is part of the screening program that is provided for all neonates. We don't process adult samples because they should already know if they have the deficiency.
Siew Ming