Compatibility Testing

practical NO 4

Dr: Dalia Kamal Eldien

Compatibility Testing

Also called pretransfusion testing, each compatibility test is a unique experiment in which an unknown (patient) serum and (donor) red cells are tested for the detection of unexpected antibodies which are directed against antigens found on the cells. Negative results indicate compatibility. This is one of the most important tests performed by a transfusion service

Purpose:To select blood components that will not cause harm to the

recipient and will have acceptable survival when transfused

Compatibility Testing

There are several components of compatibility testingProper specimen collectionReviewing patient transfusion historyABO, Rh, and antibody testing CrossmatchingActual transfusion

Sample Identification

The sample should also have the full patient name, hospital number, and physician

Date and time of collectionAll of this should be on the

request form and the sample

Specimen Collection

Collected in tube with EDTA or no additivesIf the venipuncture causes hemolysis, the sample may be

rejectedSamples are labeled at the bedside (pre-labeling is not

recommended)A record of individuals who collect (or test) the specimens

should be documented in order to “backtrack” in case of an error

Specimen Tubes

Pink Top - EDTA Red Top – no additives

Specimen Collection

If the sample is drawn from an IV line, the IV infusion should be stopped 5-10 minutes prior to blood drawing and the first 10 mL discarded

Testing should be performed on samples less than 72 hours or else complement dependent antibodies may be missed (complement can become unstable)

Getting the history

Look at recipient’s records for any prior unexpected antibodies

Previous transfusion reactions

Serological Testing

3 tests:ABO/RhAntibody detection/identificationCrossmatch

Antibody screening test

The antibody screen will detect the presence of any unexpected antibodies in patient serum

If antibodies are detected, identification should be performed using panel cells (with an autocontrol)IS37° (LISS)AHG

Antibody screening is used to test: 1 -Donor plasma to make sure no unexpected antibodies will

be transfused. 2-Patient serum before transfusion to make sure patient

has no unexpected antibodies to react with donor cells.3 -Maternal serum to make sure pregnant mother has no

antibodies to react with fetal cells.

Testing of the patient sample

Abs Regarded as always being potentially clinically significantABO- Rh -Kell -Duffy -Kidd - S s &U

Abs that may sometime be clinically SignificantLea-p - Lua &Lub Cartwright.

Abs that rarely, if ever, are clinically significantLeb Chido/Rodgers (Cha/Rha) York, Sd Xg& Bg

Procedure for Antibody ScreeningAntibody Screening will involve various phases to allow for antibody-antigen agglutination.Phase 1: Immediate Spin:.

3 tubes– 1 -Recipient serum plus saline suspension Screening Cell I

2-Screening Cell II 3 -the recipient's own cells for the auto control.

Centrifuge these three tubes and read for agglutinationDetects IgM antibodies

Phase 2: 37oC Incubation:37oC phase is required since IgG are warm-acting antibodies .Can add enhancement media if desired (LISS or albumin) .

LISS is Low Ionic Strength Solution composed of NaCl, glycine and phosphate buffer along sodium preservative. This solution speeds up antigen-antibody reaction but unfortunately enhances "nuisance" antibodies, so add after immediate spin step.

Albumin is added to lower zeta potential so cells can agglutinate without Coombs step and may detect Rh antibodies .

Whether adding an enhancement media or not we must do 37oC incubation, but we do not need to read at this step. We can proceed directly to Coombs (AHG or AGT) phase.

Phase 3: Coombs phaseThe Coombs phase is required since a number of these clinically significant

antibodies may only show up at this phase  .After 37oC incubation, wash the cells 3-4 times

Remove the saline and add AHG.Mix and centrifugeRead for agglutination.Add Coombs Control Cells to all negative results to confirm negative reactions.

This phase detects IgG antibodies, most of which are considered clinically significant and capable of causing Hemolytic Disease of the Newborn or Hemolytic Transfusion Reactions.

Correct ABO grouping results are much more critical to transfusion safety than Ab screening.

Most Abs, other than anti-A and anti-B do not cause severe hemolytic transfusion reactions. Thus the vast majority of patients would not suffer grave consequences if transfused with blood from ABO group compatible donor without the benefit of Ab screening tests.

Crossmatching

Purpose:Prevent transfusion reactionsIncrease in vivo survival of red cellsDouble checks for ABO errorsAnother method of detecting antibodies

Crossmatch

Two types of crossmatchesMajor – routinely performed in labsMinor –

Crossmatch

Donor RBCs (washed)

Patient serum

No agglutination ~ compatible

Agglutination ~ incompatible

The procedure

Donor cells are taken from segments that are attached to the unit itself

Segments are a sampling of the blood and eliminate having to open the actual unit

Major Crossmatch Tests

It is done both for IgM and IgG antibodiesRequirement:1. Recipient’s serum.2. Donor’s red cells taken from the tube attached to the

bag.

Method

1. Label 1 tube for each donor sample to be tested.2. Put 2 drop of patient’s serum in labeled tube.3. Add 1 drop of 2-5% saline suspended red cells of donor4. Mix and incubate for 5-10 min. (spin method) or incubate for

30-60 min (sedimentation method) at RT.5. Centrifuge at 1000 rpm for 1 min. in spin method (after 5-10

min. incubation);centrifugation is optional in sedimentation method.

6. Read the result, observe for hemolysis and agglulination.7. Negative result should be confirmed under microscope. Interpretation Agglutination or hemolysis indicates a positive result

(incompatible)

Crossmatch Test for IgG Antibody

B. Anti -Human Globulin Test (IAT) Indirect anti human globulin test (IAT) is the most important

and widely used serological procedure in modern blood banking to test the IgG compatibility

between recipient’s serum and donor’s cells. The majority of incomplete antibodies are IgG and are detected by AHG test.

Method

1. Put 2 drops of patient’s serum in a labeled tube.2. Add 1 drop of 2-5 % saline suspended red cells of donor.3. Incubate for 30-60 min at 37° C4. Centrifuge at 1000 rpm for 1 min, check for

hemolysis/agglutination5. If there is no hemolysis/agglutination, wash the cells three

times with normal saline.

6. Perform IAT test• Add 2 drops of polyspecific AHG serum to washed cells• Centrifuge at 1000 rpm for 1 minute• See for agglutination

7. Add IgG coated red cells to negative AHG test.8. Centrifuge and check for agglutination - if there is no

agglutination test is invalid.

Interpretation

Hemeolysis or agglutination at any stage indicates incompatibility.

Good luck