Name of test: G6PD screening test
Principle:
- G6PD test carried out in our lab is only a QUALITATIVE and SCREENING test
- G6PD is an enzyme which catalyzes the reduction of NADP+ to NADPH
- NADPH keeps glutathione in its reduced form
- Reduced glutathione or GSH protects red blood cells from oxidant damage and hence prevents the lysis of RBCs
- The reagent used in this test contains NADP
- Test principle is as follows:
G6PD (this is supposed to be on top of the arrow in the equation..but i juz can't move it)
Glucose-6-P + NADP+ ----->Gluconate-6-P + NADPH+H+
- The NADP in the reagent will be converted to NADPH if G6PD is present
- NADPH produced in the reaction will fluoresce under long-wave UV-light
- If there is G6PD deficiency or total absence of it, no fluorescence will be observed
Materials:
1. Filter paper
2. Hitachi cup
3. G6PD reagent
4. UV light
5. Incubator
6. Patient's sample in EDTA tube
Method:
1. Pipette 100uL of reagent solution into the hitachi cup which has been labelled with the patient's lab no
2.Add about 2.5uL of patient's sample into the same cup
3. Mix well and pipette 5uL onto the filter paper(1st spot)
4. After 5 mins, pipette another 5uL onto the filter paper(2nd spot) and wait for another 5 mins to pipette again 5uL of patient's sample(3rd spot)
5. Then, put the filter paper into the incubator
6. After 5 mins, observe the filter paper under long-wave UV light
ok the filter paper will look something like this, u would have three spots of blood which are made in 5 mins interval by pipetting 5ul of the patient's sample
Results:
1. Fluorescence indicates presence of G6PD
- usually the 1st spot of blood would not fluoresce well, the 2nd and 3rd ones will
2. Absence of fluorescence indicates the absence of G6PD
Note:
- Deficient cases will be followed up with G6PD quantitative test which is a send-out test
- Negative control is done before our first sample of the day
Nur Farhana Binte Ramlan
0604834B
15 comments:
Hi can i ask whats the content inside the G6PD reagent? and what is the function of that reagent? e.g why must it be added?haha thanks a lot:)
Rachael
Tg01
hey Farhana!
I heard that if a person suffers from G6PD it will not really affects your life.. only that you need to watch your diet.. is that true?
THANKS!
cheers,
huimin=)
tg01
Hello.
May I know what is a Hitachi cup? What is the purpose of using it?
And why must there be a 5 minute interval before pipetting the mixture into each spot?
Thank you =).
-Li Ping-
TG o2
Hi Rachael,
The contents of the reagents are as follows:
1.Glucose-6-phosphate
2.NADP
3.Oxidized glutathione
4.Saponin
5.Tris
the most important component here would be NADP as it will be changed to NADPH IF G6PD is present. without the reagent, there will be no reaction at all. (remember the equation?)
hope ive cleared ur doubts, thanks =)
Nur Farhana
0604834B
Hi Farhana
May I know how NADPH will fluoresce under UV light?
Thanks!
LeeJin
TG02
Hey Hui Min!
Yupzz u will have to avoid certain drugs like antimalarial agents, sulphonamides, aspirins, Nonsteroidal Anti-Inflammatory Drugs, Nitrofurantoin, Quinidine and Quinine. These drugs can give oxidant stress to the red blood cells and hence making them lysed as there is an absence or deficient reduced glutathione to protect the red blood cells. Other things they have to avoid would be fava beans and moth balls.
thanks for the qn =)
Nur Farhana
0604834B
Hi Farhana,
Y is Positive control not done?
Thanks
shihui
0607135A
HIHI
you mentioned that G6PD test is only a qualitative and screening test and the Deficient cases will be followed up with G6PD quantitative test, so what kind of quantitative test will be done ? how is it carry out and what is the priniciple of the quantitative test ?
thank you=)
tingjie
tgo2 0608495h
Hi Li Ping,
i will add the image of the hitachi cup in my entry. it just serves as a cup for us to mix the reagent and the sample. u may use other things i suppose if u would like to heheh.
there must be intervals to allow the blood and the reagent to react.
thanks for the qns =)
Nur Farhana
0604834B
Hi LeeJin,
i am not very sure. but from what i know, fluorescence is produced when a substance, NADPH here, absorbs a photon of the electromagnetic radiation from the long wave UV light, causing an electron to move from a low energy state to high energy state.
Nur Farhana
0604834B
Hi shihui,
i think this really depends on the individual lab. other labs may carry out pos control or even both.
however, the neg control is very useful in the sense that if u are not sure whether ur sample has fluorescence or not, u can compare it with the neg control. u can see difference well.
thanks for the qn =)
Nur Farhana
0604834B
Hello tinjie,
im not very sure about the quantitative test coz it is not done in our lab. instead we send the sample out to another lab to do this test. but what i know is that the test is a spectrophotometric analysis.
thanks =)
Nur Farhana
0604834B
hello fana
During incubation, does incubation temperature affects the results in any way? is the procedure light sensitive, as in incubation in the dark?
thanks
rusydiana
tg02
hey rusy..
the temp of incubator is 37 degrees celsius which is similar to our body temp so i guess it enhances the reaction. nopez it is not light sensitive =)
thanks for the qn!
Nur Farhana
0604834B
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