One of the routine work I had to do was sample processing. These are the few steps I had to follow:
1. Collect samples from the Haem reception counter.
2. Label request form and sample lab no, ensure patients particulars match with sample, initial.
3. Record patient's data (as well as all results) in Hb Electro worksheet.
4. Set up cellulose acetate electrophoresis.
5. Set up HbH inclusion bodies test.
6. Record patient's FBC from LIS
7. If no result in LIS, run FBC using the analyzer and file results.
Other than sample processing, I had to perform this test called Cellulose Acetate Electrophoresis. This test helps to detect common Hb variants present in the blood, if present. Cellulose Acetate is used as it is easily available, provides sharp resolution of Hb bands in a short time, and permits clearing.
Materials needed:
1. Supre-Heme buffer
2. Helena Hemolysate reagent
3. Ponceau S stain
4. 5% glacial acetic acid
Preparation:
1. Prepare Hemolysate
- pipette 25ul of washed packed cells into a 10x75mm test tube
- add 150ul of hemolysate agent
- mix and stand for 5 min
2. Prepare Titan cellulose acetate plate
- label
- slowly lower to rack containing supreme heme buffer (leave it for 5 min)
3. Prepare electrophoresis chamber
- pour 100ml of Supre-Heme buffer into each of the oter section of the zip zone chamber
- wet 2 disposable wicks in the buffer and drape over each support bridge and verify contact with buffer
Procedures:
1. Add 200ul of patients' sample into 10x75mm test tubes
2. Wash the cells by adding 0.9% saline till 3/4 full
3. Centrifuge at 3000rpm for 10 min
4. Repeat steps 3 & 4 to obtain packed cells
5. Dispense 25ul of hemolysate reagent in another set of 10x75mm test tubes
6. Add 25ul of packed cells into each of the test tubes containing hemolysate reagent (Note: Pipette up and down to ensure complete haemolysis)
7. Place 10ul of packed cells containing hemolysate reagent into each sample well of prime applicator
8. Prime applicator by depressing the tips gently into samples wells and blot on filter paper
9. Remove wet titan cellulose acetate plate from buffer and blot once between 2 blotters
10. Prime applicator again and transfer to the wet titan cellose acetate plate
11. Transfer the plate to the electrophoresis chamber with the acetate faced down
12. Electrophoresis the plate for 25 min at 350 volts
13. Staining of Hb bands occurs after 25 mins by placing the plates into Ponceau S stain for 5 min
14. Destain in 3 successive washes of 5% acetic acid (2 min for each wash)
Evalutation of Hb bands:
Hb inspected visually for abnormal Hb bands. Helena Hemo controls act as markers for band identification.
Clinical Significance:
Many Hb variants exhibit similar electrophoretic mobility. Hbs A2, E, C & O cannot be differentiated in this medium. However, HbA2, fraction never exceed 10% of the total whereas HbE fraction of heterozygote amounts to 30-40%. When HbE traits is combined with alpha-thalassemia, the percentage of HbE is lower than the usual 30-40% range. Hb S, D, G, H and I cannot be distinguished in the medium. Therefore, additional confirmatiory tests eg. Citrate Agar Electrophoresis at pH 6.3, solubility test and unstable Hb test are necessary to establish the Hb variant.
Overall, this is one of the common tests I've been doing so far. I also tried many other tests such as Hbh inclusion bodies test etc. But I guess I'll leave that for my other posts! Over the 3 weeks, I've been learning and polishing up my lab skills in Hb Electrophoresis. This has already given me confidence in my lab skills and I'm sure there'll be more chances for me to learn more as I rotate to other labs!
Till the next post! :)
Hey Fel!
ReplyDeleteWhat is the rationale behind detecting the common Hb variants? What kind of information does it tell us about the thalassemia?
Yvonee, Group 8.
Hey yvonee!
ReplyDeleteThe rationale behind detecting the common Hb variants to determine if there are any mutations in our Hb.
From what we've learnt in Haem, we know that:
HbA - >95%
HbA2 - 2.2-3.5%
HbF - <1%
Any abormal/mutated findings of the different Hb levels will be indicated in the gel plate after staining. The thickness of the bands on the gel plate will determine the amount of Hb present. The Hb levels will be compared with Hb E, S, F and A controls to see if they are within or out of range.
Hb variants are not acquired, but congenital. The purpose of cellulose acetate alkaline electrophoresis is mainly to detect alpha-thalessemia, whereby there is a decrease in the production of the alpha globin chains, and will affect the results of Hb A, A2 and F.
Hope that answers to your question!
Fel
Hey Felicia,
ReplyDeleteWhat colour does the Ponceau S stain produce on the gel ? What is the purpose of a de-staining step ? (step no 14 in procedures)
Thanks,
Ng Tze Yang Justin
0703747F
Hey Fel!
ReplyDeletesounds so interesting to do electrophoresis!
:)
anyway, what's the purpose of Supre-Heme buffer?
Thanks!
Stella,
0701059H
To Justin,
ReplyDeletePonceau S produces a dark red stain on the gel. The purpose of the destaining step is to get rid of excessive dye in the gel so that the bands might not appear thicker should the dye be excessive. ;)
To Stella,
The Supre-Heme buffer helps in the separation of the Hb bands on the gel during electrophoresis. :)
Hope the answers are useful!
Fel
Hi Felicia, I have a question in regard to your answer to yvonee. Does it mean that the thicker the bands, the higher the amount of hemoglobin? Besides that, are there any particular precautions that have to be taken in order to avoid inaccurate results? Thanks!
ReplyDeleteZi shuang :)
0703383J
Hello Zi Shuang!
ReplyDeleteYes. If the bands are thicker, it indicates that large amount of that certain Hb is present. ;)
There are indeed a few precautions one has to take while doing this test as the results are very sensitive to the way the test is being carried out. Here are some of the examples of the inaccuracy of the test;
1. The bands are not visible enough. Reason being that the RBCs are not lysed properly together with the hemolysate reagent. Therefore, it is important for the cells to have sufficient lysing time (10-15 min).
2. Bands on the gel are too thick. This is usually because of the rapid elevation of the gel from the buffer/stain. Due to the rapid elevation, the stain might smear on the Hb bands too rapidly. Thus, a slow elevation is needed.
3. The bands on the gel are too thin. This is also another reason for the cells not being lysed properly. Due to that, the amount of Hb released from the cells are few and it is not enough to form a visible band on the gel. Another reason would be because the gel was not left in the stain for long. Thus, the staining process might not be effective.
Hope my answers are clear enough for you to understand! :)
Fel