SUBJECT TITLE: Chemistry
Name of Test: G6PD Screening Test
Principle
Glucose-6-P reacts with NADP+ to produce gluconate-6-P and NADPH and H+ in the presence of G6PD. The NADPH produced in the reaction fluoresces under long-wave UV-light. If there is a marked deficiency of this enzyme, or if G-6-PD is lacking entirely, no fluorescence will be observed.
Materials
1. Timer (not necessary)
2. G6PD Reagent
3. Negative Control (Commercial Blood)
4. Pipette
5. Pipette tips
6. Filter paper (absorbent paper)
7. Gloves
8. Sample in EDTA tube
9. Hitachi Cups
10. Tube rack (to hold the sample tube & Hitachi cups)
11. Marker (for labelling purposes)
Method (using 1 patient’s sample)
1. One Hitachi cup was labelled with “Negative Control”.
2. Another Hitachi cup was labelled with the patient’s laboratory request number e.g. “08-48”.
3. “Negative Control” and “08-48” were written below respective circles on the filter paper.
4. 100ml of G6PD Reagent was pipetted into patient’s tube labelled e.g. “08-48” and “Negative Control”.
5. 5ml of patient’s EDTA Blood and Negative Control were pipetted into the labelled cups “08-48” and “Negative Control” respectively and mixed.
6. The pipette tips were remained in the cups.
7. Using the same pipette tips, 5ul of mixture from each cup was pipetted onto each circle on filter paper respectively as shown below as the 1st drop.
8. It was left at room temperature for 5 minutes.
9. After 5 minutes, the 2nd drop was pipetted and left for another 5 minutes.
10. After 5 minutes, the 3rd drop was pipetted and incubated for 10 minutes at 35°C.
11. After incubation, the filter paper was observed under long wave UV light using equipment shown below.
12. Result was recorded.
Note: EDTA tube would be passed to Haematology Section for ABO typing if the patient is an infant. Otherwise, the tube would be filed back.
Results and Interpretation
If fluorescence is seen under UV light, the result is recorded as “present” on the worksheet. For the Negative Control, there should not be any fluorescence seen. However, if fluorescence is seen using the patient’s blood, it indicates that the patient has the enzyme G6PD and therefore, it shows that the person is not suffering from G6PD Deficiency.
Before reading the results under UV light, ensure that the filter paper is dry. The sample obtained from a normal or slightly reduced G6PD activity will show a strong fluorescence. If there is absolutely no fluorescence after 10 minutes of incubation in all the 3 drops, it suggests that there is a total or significant deficiency of G6PD. If there is fluorescence in any of the 3 drops but the other 2 drops do not fluorescence, incubate for another 5 minutes and observe again.
During the test, ensure that the blood is well mixed with G6PD reagent. Otherwise, it can result in false positive result. In other words, the patient does not have G6PD Deficiency, but the result shows that he/she has.
By: Rebecca (0703363B)
Hi Rebecca. What is the significance of a person having G6PD deficiency? Thanks.
ReplyDeleteIndah
0705361D
hi rebecca,why does the sample on the filter paper need to be dry before reading the results?will it effect the results?thank you :)
ReplyDeletenyzah
To Indah:
ReplyDeleteG6PD is an enzyme that helps the red blood cells to function normally. Thus, a person with G6PD Deficiency, either their RBCs do not make enough G6PD or the G6PD that is produced cannot function properly. Without enough G6PD to protect them RBCs can be damaged. This deficiency can cause haemolytic anaemia.
To Nyzah:
Basically, if the filter paper is wet,there would not be any fluorescence seen even if G6PD is present.
Hi rebecca,
ReplyDeleteu mentioned in ur post that basically 3 drops of the mixture are needed. i wana ask, if all 3 drops are done at 3 different spots or the 2nd drop is pippetted exactly onto the 1st drop to produce a thicker solution and subsequently the 3rd drop is pippetted exactly onto the same section to produce even thicker solution...?
if it is 3 different spots, i wana ask why stop only at 3...? why not 4 or 5 drops...?? hehehe
thanks
nadiah
tg02
Hi. Erm my first is same as nyzah, why the filter paper need to be dry before reading the result and what is hitachi paper?
ReplyDeleteJennifer.
Why must the same pipette tip be used?
ReplyDeleteIf clotted blood is used for this test, will the result be still the same? Or the blood used here must be unclotted?
Alvin
To Nadiah:
ReplyDeletehaha! 3 separate drops but in a circle on the filter paper. Basically, the filter paper is a rectangular piece of filter paper. And on this filter paper, there are 3 circles already drawn/printed on it. ONE circle is only needed for ONE sample ONLY. And for one sample, 3 drops are taken. So, that means after doing the experiment, you'll see 3 small circles (drops) in the one circle. You can actually go to http://alsubs.blogspot.com/2007/07/biochemistry-immunology.html for the picture.
And for your 2nd qn..hmm...I'm not sure of the answer. In fact, I don't know the answer. haha. i'll try and ask my colleagues.
To Jennifer:
Basically, if the filter paper is wet,there would not be any fluorescence seen even if G6PD is present.
Hitachi paper? I did not type "hitachi paper". I typed "hitachi cup". Hitachi cup is just a very small little cup/container to put serum to run for tests.
To Alvin:
Actually, there's no need to use the same pipette tip. It's just for convenience sake. But of cos, different pipette tips are used for neg control and sample respectively haha.
The blood here should be mixed thoroughly, which means unclotted blood should be used. If clotted blood was used or if the blood is not mixed thoroughly, flourescence would not be seen and it would get a false positive result.