Compatibility Testing

When patient requires transfusion or blood on standby, compatibility testing will have to be done.
There are 2 kinds of compatibility testing. Namely, Abbreviated crossmatch (AXM) or extended crossmatch (EXM)

Extended crossmatch will have to be performed when:
1) patient has history of significant red cell Ab
2)Ab screening is positive
3)Patient has a recent episode of adverse transfusion reaction
4)Patient is <4 months old

AXM is performed when patient all 4 EXM criteria does not apply to patient.

For EXM, there are 3 phases. Namely the saline phase, LISS phase and AHG phase.

Saline Phase
1) Add 3d patient serum
2) add 1d 3-5% washed donor cells
3)incubate for 20mins at room temperature
4)Spin down at 1000rpm for 15secs and read
Check for haemolysis and then re-suspend and read under microscope for agglutination.

LISS Phase
1) Add 3d patient serum
2) add 1d 3-5% washed donor cells
3) Add 2d LISS. Mix well
4)incubate for 10mins at 37 degree celcius
5)Spin down at 1000rpm for 15secs and read
Check for haemolysis and then re-suspend and read under microscope for agglutination.
Then we'll proceed to AHG phase

AHG Phase
1) Wash the cells in the LISS tube in 0.9% saline 3 times.
2)Add 2d AHG to the red cell button in test tube
3)Spin down at 1000rpm for 15secs and read
4)re-suspend and read under microscope for agglutination.
If the tube gives negative results, add 1d control cells and then spin again. Should it yield negative results again, test will have to be repeated.


For AXM, these are the steps. It's much easier and takes a shorter time
AXM
1)Label 1 tube for the donor sample to be tested.
2) Wash this donor sample in 0.9% saline.
3)Add 2 drops of patient serum to a clean, labelled tube
4)Add 1 drop of 3-5% washed donor cells to the labelled tube
5)Spin down at 1000rpm for 15secs and read
6)Check for haemolysis and then re-suspend and read under microscope for agglutination.

Purpose of compatibility testing is to select a unit of compatible blood for each potential recipient which will have acceptable survival and will not cause clinically significant destruction of the recipient's own cells. As long as it's possible, units of the same blood group and Rh(D) group will have to be selected for patient.

Since EXM consist of 3 phases, and includes incubation at 37deg, it can better detect IgG antibodies .

yanhong 0703979e

Microplate Technique

Hello everyone. I am sorry for the late post.

In this final post, I am going to share about one technique that I used in my project. I am designing antibody screening panels. This would require a lot of phenotyping of donor cells to create a good quality panel. One of the antigens that must be expressed in an antibody screening panel is the S antigen. This antigen is rare in the local population, with a frequency of 6%. Hence, the objective of this microplate technique is to screen for cells that are negative for the antithetical little s antigen. This means I would select cells that are either homozygous or heterozygous for S antigen to do full phenotyping.

Each week I would receive a batch of samples of about 60 and above. I need to screen all for little s negative antigen. Since the manufacturer insert of my antisera reagent does not recommend to be used for indirect coombs test, I cannot use the Techno TwinStation machine for this phenotyping. Instead, I used the microplate technique at room temperature, which allows screening of huge amount of samples at one go.

Procedures
1. Label all donor samples and position them according to the wells numbering in a rack.
2. Prepare 0.8% red cells suspension of each donor samples. The lower concentration would make it easier to read for agglutination.
3. Add 50ųl of red cells suspension into each well using a disposable pipette.
4. Add 50ųl of diluted antisera (1:1000) using the multi-dispenser pipette. The dilution factor of the antisera is derived from titration prior to the test.
5. Leave the microplate to stand without disturbance at room temperature for 30 to 40 minutes.
6. Record results.

Result Interpretation
If a well were positive for little s antigen, the red cells would form a monolayer suspension with no button formation observed. If a well were negative for little s antigen, the red cells would form a clear and compact button at the center of the well. To ensure valid results, positive and negative controls are used.

Picture retrieved from
http://www.hpa-midas.org.uk/images/page_images/agglutination_assay.gif

Advantages of microplate technique
1. It serves as a good preliminary screening method for phenotyping rare antigens.
2. It can handle large volume of donor samples at one time.
3. Produce rapid and reliable results.
4. Reduce wastage of disposable equipments in comparison to the tube technique.
5. Reduce cost, as the method is reliable for diluted antisera reagent.

Thank you.

Indah.