hello hello hello!
stella here for the final blog posting!
and i apologize for the late entry.
G6PD (Glucose-6-Phosphate Dehydrogenase) test is performed mainly on patients who develop symptoms of hemolytic anemia and neonatal jaundice. Hemolytic anemia takes place when the bone marrow is not able to compensate for the destruction by elevating the RBC production. G6PD is necessary for the maintenance of an intact red cell membrane because G6PD deficiency may lead to hemolysis of red cells. During the abnormal state, the RBCs will not be able to regenerate NADPH, a reaction that will be catalysed by G6PD in normal situations.
Both G6PD Quantitative and Qualitative (screening) assays are performed to enable differential diagnosis in G6PD deficiency to be made.
The principle of both G6PD assays is based on this reaction:
Glucose-6-Phosphate + NADP (no fluorescence) ---(G6PD)---> Gluconate-6-Phosphate + NADPH (fluorescence)
The NADPH produced will fluoresce under long-wave UV light during the reaction.
There is maerked deficiency or lack of enzyme G6PD when no fluorescnece is observed.
If Glucose-6-Phosphate is oxidised to Gluconate-6-Phosphate, the coenzyme NADP is reduced to NADPH with a corresponding elevation in fluorescence.
The procedures for both assays are different, with the screening assay much simpler as compared to the quantitative assay.
Steps for screening assay:
1) The EDTA consisting of patient's blood sample is inverted gently for several times for thorough mixing
2) 5ul of whole blood is added, 100ul of working reagent and 100ul of reagent blank into the wells of the titration plates
3) Working reagent is added and start timing
4) After 10 minutes, a small amount of reaction is taken and i spotted onto filter paper
5) The spots are dried for 5 mins using a hair-dryer
6) The spots are then viewed under long-wave UV light
Steps for quantitative assay:
1) The EDTA blood is washed 1 time with 0.9% saline
2) It is then sent for centrifugation to pack the cells at 3000rpm for 10 mins
3) The saline and the buffer coat are removed completely
4) 200ul of washed packed cells are pipetted into 200ul normal saline to obtain 1:1 rbc suspension
5) 50ul of this 1:1 suspension is pipetted into 250ul of !% saponin for lysis
6) It is then mixed well and left to stand for 10 mins at 2-8 degree celsius
7) The hemolysate is ready for testing
Reporting of results for Qualitative (screening) assay:
When fluorescence is observed, enzyme activity is indicated. This explains the presence of G6PD.
Reporting of results for Quantitative assay:
Reference ranges for adults/children = 7.2-17.4 ug/Hb
newborns/cord blood = 13.4-25.4 ug/Hb
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6 comments:
Hey Stella,
With regards to the quantitative assay, do you actually count G6PD? Sounds retarded but I'm quite lost when I read the post :$ (Or did I missed any point already mentioned?)
Yvonee
0703189A
Stella,
(1) For screening, how does the spot looks like under UV?
(2) Is it a quantitative or qualitative test (2nd part)since you mentioned to detect flouorscence to determine the presence of G6PD.
Li Yinliang Alex 0704894E
TG02 Group 8
26 October 2009
heelloo stellaa~
you mention that this G6PD test employs both qualitative and quantitative test. when do you need to do either one of them first? for example, do you have to do qualitative test first then quantitative, or vice versa?
siti shahimah
0702717J
:)
Hi Stellaaaa! :D
I was wondering if letting the mixture to stand for 10 mins at 2-8 degree celsius is to aid the lysis of the RBCs? This is because my lab let the mixture stand for 10 mins at room temperature. (:
Siew Ming
0702862D
Hey Yvonee!
yea we count the G6PD using the analyzer :)
Hi Alex!
1) there will be bright spots when G6PD is present. but when its not, then dat means G6PD is not present. anyway i'm trying to find images for you to see. but i still cant find. hahah
2) its qualitative :)
Hi Siti!
yep, usually they'll perform qualitative first and then quantitative :)
Hi Siew Ming!
yea its to aid in the lysis of the RBCs. hee.
My daughter had the quantitative test, which was reported as high out of range at 13.6 - Ref. range 4.6 - 13.5 - what would this indicate - sorry if this is a class.
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