Blood Transfusion Cross match test (Saline and Indirect Coomb Techniques)

Crossmatch MLTGEEKS

Though used interchangeably, cross matching is only a part of compatibility testing. A full compatibility testing comprises of the following

Cross matching

Major and Minor Crossmatch
Major and Minor Crossmatch

The purpose of blood match or blood crossmatch is to exclude the presence of any antibody  that is autoantibody or alloantibody in the recipient’s serum that will react with donor cells when transfused. There are two types of Crossmatch

  • Major crossmatch
  • Minor Crossmatch

Major crossmatch involves testing of recipient’s serum with donor’s red cells.

Minor crossmatch involves the testing of donor’s serum with recipient’s red cells. If the donor’s serum has been screened for irregular antibodies with a panel of cells and no antibody has been detected,minor crossmatch can be excluded.

Compatibility tests are therefore done to ensure safe blood transfusion

How to wash Red blood cells

In order to prepare 5% red cells,you just need to mix 5 part whole blood with 95 part normal saline or phosphate buffered saline.

  1. Start by Centrifuging the whole blood at 3000 rpm for 5 minutes  
  2. Then  remove the plasma and buffy coat layer using a pipette
  3. Resuspend the red cells in normal saline (0.9% NaCl) with approximately 2 times the volume of the red cells, and gently invert the tube to mix.
  4. Now centrifuge for 5 minutes at 2000 rpm and discard the supernatant.
  5. Repeat Steps 3 and 4 twice (Now centrifuge for 5 minutes at 2000 rpm and discard the supernatant) for a total of 3 washes or until the supernatant is clear
  6. Now you can completely remove  the supernatant!That’s Washed Red cells
  7. To store the washed red cells,you can freeze the red cells at -20˚C, preferably at -80˚C. Ship frozen.
Also read  Types of Blood transfusion

Saline Technique for Cross match

saline crossmatch
saline crossmatch

This technique detects IgM antibodies in the recipient’s serum that react at room temperature or lower against the donor red cells.

Materials/Reagents Required

  • Test tube
  • Incubator
  • Centrifuge
  • Microscope
  • Recipients serum
  • Donor Red Cells

Saline Technique for Cross match procedure

  1. Prepare 5% Washed RBC suspension of the donor (Major Crossmatch) OR Recipient Red cells (Minor Crossmatch) in saline in properly labelled tubes.In this case we are doing a Major crossmatch
  2. Put 2 drops of recipient serum in a labeled test tube.
  3. Now add 1 drop of 5% donor red cells suspended in saline to the tube
  4. Mix the tube and incubate for 5-10 minutes at room temperature.
  5. Centrifuge at 1000 rpm for 1 minute (Alternatively, the tube may be incubated for a period of 30-60 minute at room temperature and the result read. In this case;centrifugation  becomes optional)
  6. Make a wet mount of the mixture
  7. Observe for hemolysis or agglutination using a microscope at 10 – 40X objectives and confirm all negative results microscopically

Interpretation of result

CompatibleIncompatible
No Agglutination or Hemolysis indicates compatibility of donor and recipient blood.Therefore,Can be transfuseHemolysis or Agglutination indicates incompatibility of donor and recipient blood.Therefore,Blood can not be Transfuse

Limitation of Saline technique

  • This technique does not clinically detect significant IgG antibodies.Therefore,as this technique does not detect Immunoglobulin G antibodies, it is inadequate as a complete compatibility test.
  • Also Incubation improves the sensitivity of this technique.

Antiglobulin compatibility Technique (Indirect Coombs Test)

Indirect coombs technique
Indirect coombs technique

Indirect anti human globulin test (AHG) is one of the most widely used technique to detect compatibility between donor red cells and recipient serum. This technique  detects IgG antibodies in the recipient serum which may react with the donor red cells.

Also read  How to perform Direct and Indirect coombs test

Indirect coombs technique Procedure

  1. Prepare 5% Washed RBC suspension of the donor (Major Crossmatch) OR Recipient Red cells (Minor Crossmatch) in saline in properly labelled tubes.In this case we are doing a Major crossmatch
  2. Pipette 2 drops of patient’s serum in a labeled test tube.
  3. Now,add 1 drop of  the 5% donor red cells suspended in saline to the tube
  4. Incubate the mixture at 37°C for 30-60 minutes.
  5. Then centrifuge at 1000 rpm for 1 minute
  6. Examine macroscopically and microscopically for hemolysis or agglutination
  7. If there is no agglutination or hemolysis, wash the cells three times with normal saline then decant the supernatant after each wash.
  8. If there’s hemolysis or agglutination,don’t proceed further.The agglutination at this stage can be die to IgG.If partial haemolysis is observed,record and proceed further.
  9. Now add 2 drops of  the polyspecific  anti-human globulin (AHG) serum
  10. Mix and centrifuge the tube at 1000 rpm for 1 minute.
  11. Examine the tubes macroscopically and microscopically for agglutination and record your result
  12. If there is no agglutination,leave the tube at room temperature for 5 minutes the re-centrifuge and examine again for agglutination
  13. If the test is still negative,Add  1 drop IgG coated red cells to negative AHG test then centrifuge and check for agglutination which must be present at this stage.
  14. If there is no agglutination, the test is considered invalid.
  15. It is necessary to run a positive control using known antibody in parallel with the test

Result and Interpretation

  • A positive reaction or agglutination in step 12 indicates that the negative reaction in step 11 is invalid and the test should be recorded as positive indirect coombs test
  • A positive reaction in step 13 indicates a negative indirect coombs test
  • A negative reaction in step 11,12 and 13 indicates that the test is invalid and must be repeated.
Also read  Red Blood Cell Indices (MCV,MCH and MCHC) Clinical Significance

The Saline and Antiglobulin crossmatch can be done in separate tubes.


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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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