Haematology laboratory

Second week of SIP

Hello everyone… I was posted to haematology department for the whole of 20 weeks. This week I was scheduled to thalassaemia lab and will be there for the next two weeks. Well, I had learnt quite a lot at the lab dealing with the blood samples that were sent in for Hb electrophoresis. Firstly, I will collect patient’s blood samples at the reception sent to them by telelift, transporter or pneumatic tubes. After which, I will do labeling on the samples and check that the patient’s data tally with the request form. Next, key the specimen no. into LIS.

Test performed: Cellulose acetate alkaline electrophoresis

Type of specimen: a minimum of 3.0ml of EDTA blood
EDTA preferred but heparin is acceptable.
Reject clotted blood and lyse specimen except for clotted cord blood (very difficult to get the blood)

Purpose: it is used as the initial procedure to screen for hemoglobin variants. The technique is sensitive to blood samples throughout the human life span (from newborns, cord blood, to old age)

Principle: Hb has a net negative charge at pH8.6 and move in an electrical filed towards the anode (positive) due to the variation in the amino acid content off different Hb, the net charge of each Hb types varies and this will determine their rate of mobility. Various supporting media such potato starch, paper and polyacrylamide gel have been used in electrophoresis. Cellulose acetate is used because it is easily available and provides a sharp resolution of the Hb bands in a short time; allow cleaning, densitometric quantitiation and permanent storage of the transparent film.

Procedures:

1) Pipette 200microlitires of blood to tubes and wash 2x
2) Pipette 25microlitres of washed packed red cells into the tube.
3) Add 150microlitres of hemolystate reagent and allow standing for 20 minutes.
4) Label the plate with specimen no. using water proof marker and place in carrying rack. ( the other side of the plate is the cellulose acetate)
5) Slowly, lower the rack into heme buffer and soak for 5 minutes. ( it must be done slowly to prevent any air bubbles)
6) Pipette 10microlitres of the patient’s blood and hemolystate into each sample well. Control is pipette into the first well. Control used is ESFA. E and S are abnormal variants. F and A represent fetal and adult respectively.
7) Prime the applicator by depressing gently into samples well 1 to 2 times and blot on a piece of filter paper.
8) Remove the wetted cellulose acetate and blot dry
9) Place the plate in the aligning base with cellulose acetate side up
10) Transfer the applicator to the aligning base
11) Apply samples to plate (on the cellulose acetate) by the contacting the tips (“teeth”) of the applicator to the plate and hold for 5 seconds.
12) Place the plate in electrophoresis chamber with cellulose acetate side down
13) Electrophoresis the plate for 25 minutes at 350volts


Staining of bands

1) The plate is stained in ponceau S stain for 5 minutes
2) Destain in 3 successive washes of 5% acetic acid (2 minutes for each wash)

Evaluation of bands

For qualitative evaluation, the haemoglobin is inspected visually for the presence of abnormal haeomglobin bands. EFSA control provides a marker for band identification

For quantative evaluation, the relative percentage of each haemoglobin band is determined by scanning the plate in the densitometer using 525nm filter.

Record results

taken from http://www.dokkyomed.ac.jp/dep-k/cli-path/a-super/h25.html

Test performed: sickling test

Purpose: used to demo the sickling phenomenon in test samples and a confirmatory test following alkaline cellulose acetate electrophoresis


Procedure:

1) 25microlitres of reagent is added to 5microlitres of EDTA blood used the pipette tip to mix to a coin shaped
2) cover with cover glass and left dry
3) seal with grease and incubate at 37degrees for about 2 hours
4) examine microscopically
5) record results


Quality control: include a negative and positive control to check that reagents are okay.

That’s all… have fun for SIP yea :)

kai lin

0503211E

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