Friday, September 26, 2008
Campus discussion week's blog
For this week, we are supposed to post an entry on any test that we have done. Therefore, I will share with all of you a test to detect for G6PD deficiency.
Introduction
G6PD (Glucose-6 Phosphate dehydrogenase) is a key enzyme in needed for the formation of NADPH in the hexose monophosphate pathway. HADPH is important for maintaining the integrity of the RBC(erythrocyte) membrane and functioning properly.
G6P-DH
Glucose-6-P + NADP+ <======> Gluconate-6-P + NADPH + H+
The lack of G6PD is the cause of hemolytic disease in newborn. It is an inherited condition where the body does not have enough G6PD. It can be also be transmitted as X-linked recessive. It can cause hemolytic anemia, usually after exposure to certain drugs (e.g. some oxidant drug) or food etc.
Principle of test
The name of this test is called the Fluorescent spot test. It is basically mixing the working substrate with the blood sample. After that, it is pipetted onto a filter paper. If NADPH is produced in the reaction, when the filter paper is placed under long wave UV light, the spot of blood will fluorescence.
Methods
1) G6PD working stock is removed from the refrigerator
It contains
- 1 tube of positive control
- 1 bottle of working substrate
2) One micro tube of intermediate control and one deficient control is taken out from a
-20degree Celsius freezer and then thawed.
3) For every specimen, checked that the name and the IC number on the form and the specimen bottle tallies. Also, check that the test ordered is correct as indicated on the sticky label.
4) The tubes were to be prepared for loading of specimen according to the number of specimen and label accordingly. Also, label 4 other tubes; blank, intermediate, deficient and positive
5) 100ul of substrate was pipetted into each tube at room temperature.
6) 5ul of the patient’s specimen is added into the specific tubes. Also, 5ul of intermediate, deficient and positive is allocated into the specific tubes. The mixture is pipetted up and down several times to mix it.
7) Allow 10minutues for the reaction to take place. Time is taken using a timer.
8) The filter paper that is provided in the kit is to be labelled with the number
9) After 10minutes is up, 10ul of the mixture from each tube of the patient’s samples are added to the filter paper. Also, 10ul of the blank, intermediate, deficient and positive are also added to the filter paper.
10) The filter paper is then placed into the incubator to allow it to dry completely (approximately 15mins)
11) After 15mins, take out the filter paper and view it under UV light.
12) Patient’s sample with normal G6PD should show strong fluorescence while patients. Samples that fail to fluoresce may indicate marked deficiency or total lack of G6PD enzyme.
13) It is reported as positive for samples that fluoresces and test is repeated for weak or no fluorescence.
For the specimen that test is needed to be repeated, check the specimen tube to see if there are any clots present, if there are no clots, then the test repeated over again to confirm. If there are clots, then inform the ward for a repeat of test with a new specimen.
That's about all =)
Zhenling
TG02
