WEEK XVII ATTACHMENT
Hello all once again!
Time really does fly doesn’t it? And we’re suddenly finding ourselves staring at the final few numbered weeks of our attachments. Hope the projects are getting along fine there. Anyway, this would be my final blog entry regarding my attachment, but nonetheless, I hope you guys would enjoy it all the same.
For this entry, I’d be touching on G-6PD screening on neonatal blood. Basically, G-6PD is the abbreviation for the enzyme Glucoes-6-phosphate dehydrogenase. It is indirectly involved in the prevention of oxidant damage to the RBC, in that it is necessary for the maintaining of adequate quantities of Glutathione, an important buffer to oxidants within a RBC (see diagram below).
In the event that the production of NADPH is impaired, insuffiecient quantities of Glutathione would be regenerated, allowing cellular oxidants to accumulate, thereby resulting in erythrocyte injury and hemolysis.
This screening procedure makes use of a buffer(provided in the kit) which contains Glucose-6-P and NADP+, both of which would react with G6PD(if present in the erythrocyte) to give Gluconate-6-P, NADPH and H+. In turn, NADPH produced in the reaction would fluoresce under long-wave UV-light. However, in the event that there is a marked deficiency or complete absence of the enzyme, no fluoresce would occur. In short, the below equation describes the reaction taking place in the screening test.
The procedure for this screening is as follow:
1. Fill up well(s) with 50mL of buffer(provided in kit), one well per patient.
2. Using an applicator stick, add <1>
3. Allow the mixtures to incubate at r.t.p for 10mins. Meanwhile, label on the filter paper the necessary details such as that of date and time of experiment, and identity of each sample at the area where the sample would be placed on the filter paper.
4. Using a micropipette, place a drop(10-20mL) of each of the incubated sample into each respective the circles on the filter paper.
5. Incubate the filter paper containing the samples in the oven(42°C) for a further 15mins, or until the filter paper has dried.
6.View the filter paper under a long-wave UV-lamp in a dark room. Samples obtained from normal/ slightly reduced G-6PDH activity will show strong fluorescence. Failure to fluoresce, as mentioned above, suggests a total or marked deficiency of the enzyme G-6PD.
7. Mark out on the paper “present” for samples that fluoresce. Samples that do not fluoresce would be sent to a biochemist to obtain a titre count for any G-6PD present.
Alrights. That’d be all for this entry of mine. Thanks all for reading once again. All the best to your projects.
Alexander Soo TG02
0608122H
posted by THE CODEC 5 @ 10:30 AM
7 Comments:
hi alex.
i was just wondering..do you incubate the filter paper in the oven at 42 degrees celcius for 15 mins simply to allow the filter paper to dry up or is it to enhance the reaction?
thanks C:
nur azeimah
0607060A
tg 02
Hi there
U mentioned that this test is screen on neonatal blood. What about adults?
Sharon
Tg01
HI
Since this is a screening test on neonatal blood, what is the confirmatory test used in your lab?
Xin Ni
TG02
Hey alex,
I also want to know why is a 10 min incubation needed before placing the it under the UV light. Btw what is the wavelength??
Thanks.
Yvonne Teo
0605109H
Hi!
In our lab, we do G6PD qualitative and quantitative, does your lab provide services for both too?
And does controls come in Normal, deficient etc?
Tq~
Hi Alex,
I did this screening test in my lab. Just wondering, when the sample does not fluoresce, the first step is to send to biochemist for titre count is it? As for my lab, we check whether there is blood clt as it will affect the results.
And what type of controls do you use?
Hardina
Hi guys,
Thanks for reading. Here's to your questions.
To nur azeimah:
QUES:
do you incubate the filter paper in the oven at 42 degrees celcius for 15 mins simply to allow the filter paper to dry up or is it to enhance the reaction?
ANS:
The incubation's just to dry the filter paper. An alternative method is using a hair drier(not kidding).
To Sharon:
QUES:
U mentioned that this test is screen on neonatal blood. What about adults?
ANS:
Well, the enzyme would still continue to be present in the adults since their childhood as it is encoded in their DNA/ hereditary. So a second test would not be necessary(hope that's what your question was asking).
To Xinni:
QUES:
Since this is a screening test on neonatal blood, what is the confirmatory test used in your lab?
ANS:
Well i guess you'd missed it as it wasn't exactly that obvious in the entry. But we'd do a titre count on the quantity of the enzyme found as a confirmatory test.
To Yvvone:
QUES:
I also want to know why is a 10 min incubation needed before placing the it under the UV light. Btw what is the wavelength??
ANS:
The incubation is just a drying process as somehow any G-6PDH present doesn't fluoresce under the UV-light if the filter paper is still wet during observation. And to your second question, the wavelength of UV-light or black light if you will is 400 nm – 315 nm.
To tq:
QUES:
In our lab, we do G6PD qualitative and quantitative, does your lab provide services for both too?
And does controls come in Normal, deficient etc?
ANS:
Nope,we don't. We primarily check to see if G6PD is present, meaning quality. It'd probably be only in special cases where there is G6PD present but not so much where the deterining of the quantity might become necessary in aiding the doctor with his perspective about the situation.
Also, as you'd suggested, the controls would come in "presnt" änd "deficient". I doubt there's any other possible ways for it to be tested.
To Hardina:
QUES:
I did this screening test in my lab. Just wondering, when the sample does not fluoresce, the first step is to send to biochemist for titre count is it? As for my lab, we check whether there is blood clt as it will affect the results.
ANS:
Well, i guess that depends if you mean the "official" next step, or the next step "technically". There are many reasons why a sample may bot floresce(one of these is incompetent skills on the part of the lab tech). So technically, our next step would be to run a repeat test for the same sample, this time much much more careful to make any mistakes. But if it still turns up to be G6PD-deficient, then we'd carry out the official next step, and that is to carry out a titre count.
Alrights. Thanks all for your questions. Hope the answers quech your doubts. Outta here.
Alexander Soo TG02
0608122H
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