In my major project, i am tasked to evaluate a serum protein electrophoresis system.
Within the evaluation process,i intend to demonstrate the influence of haemolysed samples on the results of the tests. This portion of experiment is conducted for interference study of evaluation process.
As stated, my system separates serum proteins according to their charges along an agarose gel. It separates the proteins into 2 major fractions known as albumin and globulin. The fractions can be further catergorized into 5 minor distinct fractions, known as albumin, alpha-1, alpha-2, beta and gamma.
As you known, when a blood sample is lysed, haemoglobin is released into the serum. Haemoglobin is a protein and therefore may appear as an artefact on the gel presentation, increasing the concentration of alpha-2 and beta protein. Haemoglobin should not appear as a band, as only serum is used to be tested on the analyser.
Therefore, i prepared haemolysed samples to identify the extent of influence of haemolysis on the results. In this posting, i will explain the steps taken to prepare haemolysate. Haemolysate is the preparation of the resulting product of erythrocyte lysis.
HOW DO WE GET HAEMOLYSATE?
1. Obtain a normal blood sample collected in EDTA tube and spin down for 10mins, 3500rpm.
2. Remove plasma and add 10ml of saline to red cell pellet.
3. Resuspend red cells in saline by inverting.
4. Spin for 5mins,3500rpm and decant saline. Step 2-4 constitutes washing of red cells.
5. Conduct washing of red cells for 3 times.
6. Remove saline and add equal volume of deionised water. Mix well by inverting.
7. Freeze cells overnight in freezer.
8. Thaw cells til rtp.
9. Centrifuge mixture for 30mins.
10. Save supernatant. Supernatant = Stock Haemolysate
Stock Haemolysate will be used to spike normal serum to produce different levels of haemolysis status. We achieved 3 levels of serum status (mildly lysed, lysed and severely lysed). These samples will be used to identify the extent of haemolysis on the results of electrophoresis.
Here are some pictures to improve yr imagination of the above information.
These are the fractions along with some specific proteins found within the fractions. (above)
Concentrate on lanes 8-11. 8 is normal serum, 9 is mildly lysed sample, 10 is lysed sample, 11 is severely lysed sample. Look at the alpha-2 and beta region. You will notice increasing concentration.Tng Wess Lee
0702570C
wess.
ReplyDeleteWhat is the significance of using agarose gel?
Is there any other types of gel that can be used in serum protein electrophoresis?
siti shahimah :)
0702717j
wess.
ReplyDeletewhat is inteference study?
thanks!
stella
hello shah.
ReplyDeletefirtly, there are several types of gels that can be used for serum protein electrophoresis, such as SDS-page. however, we are evaluating the serum protein electrophoresis system. the company commercially produces the gel for us, and therefore we use agarose gel.
the significance of using agarose gel is that its not toxic and cheaper.
next is lala.
when doing evaluation, it comprises of several parameters to be examined/evaluated. one of which is interference study. interference study is aimed at identifying any possible interferents which may alter the integrity of the results. Some examples of common interferents in serum samples are haemoglobin, bilirubin and lipoproteins/lipids.
hello wess,
ReplyDeletey presence of hemoglobin will increase the concentration of alpha 2 and beta protein? also also from your experiment, is there any conclusion of presence hemoglobin will affect the test results?
thanks in advance~!
yaNLing xD
hemoglobin is a protein. most prolly it has characteristics such as charge which make it similar to alpha-2/beta region.
ReplyDeletei don't understand yr last qns.
but i'll try to answer yr qns.
my project is evaluating a SPE machine.
so we are able to use urine/serum samples to test for proteins.
as you can see from the pics, presence of haemoglobin would increase alpha-2/beta region.
therefore, we would recommend the institution using this machine to not accept running haemolysed samples as it would produce false results.
ooo.. i see.. thanks alot~
ReplyDeletehave fun with ur remaining 6 wks of sip!
yaNLing xD