Pretranfusion Compatibility Testing

Mr. Mohammed A. Jaber

Blood Transfusion Process

Pre-transfusion Transfusion Post-transfusion

What is compatibility testing?

Also called pretransfusion testingPurpose:

To select blood components that will not cause harm to the recipient and will have acceptable survival when transfused

If properly performed, compatibility tests will confirm ABO compatibility between the component and the recipient and will detect the most clinically significant unexpected antibodies

Compatibility testing?

There are several components of compatibility testingProper specimen collectionReviewing patient transfusion historyABO, Rh, and antibody testing (screen/ID)CrossmatchingActual transfusion

Compatibility testing

Can be divided into 3 categories:Preanalytical proceduresSerological testingPostanalytical procedures

Pre-analytical phases

Patient identificationSpecimen collectionReview of patient history

Patient Identification

Must confirm recipient’s ID from bracelet ON the patientFull patient name and

hospital numberName of physician

Sample Identification

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

Date and time of collection, phlebotomist’s initials

All of this should be on the request form and the sample

Specimen Tubes

Pink Top - EDTA Red Top – no additives

Specimen Collection

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

be rejectedTrue hemolysis in the patient is the result of

complement activationSamples 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 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

ABO/Rh Typing

In the ABO typing, the forward and reverse MUST match

In the Rh typing, the control must be negativeBoth of these will indicate what type of blood should

be given

Antibody screen and/or ID

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

If an antibody is present, units negative for the antigen must be given

Proceed to the crossmatch…

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 – not required by AABB since 1976

Major vs Minor Crossmatch

Why is the minor crossmatch unnecessary?Donated units are tested

for antibodiesMost blood is transfused

as packed cells, having little antibodies

The plasma volume is small, and Abs will be diluted in recipient circulation

Crossmatches

The crossmatch “shall use methods that demonstrate ABO incompatibility and clinically significant antibodies to

red cell antigens and shall include an antiglobulin phase”

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

Units of whole blood with segments attached

Procedure

ABO/Rh typing is FIRST performed

Antibody Screen is performed next….

Crossmatch Procedure

if antibodies are NOT detected:Only immediate spin (IS) is performed using patient serum

and donor blood suspensionThis fulfills the AABB standard for ABO incompatibilityThis is an INCOMPLETE CROSSMATCH

If antibodies ARE detected:Antigen negative units found and X-matchedAll phases are tested: IS, 37°, AHGThis is a COMPLETE CROSSMATCH

Crossmatches …

WillVerify donor cell ABO compatibility

Detect most antibodies against donor cells

Will NotGuarantee normal survival of RBCs

Prevent patient from developing an antibody

Detect all antibodies

Prevent delayed transfusion reactions

Detect ABO/Rh errors

Incompatible crossmatches

Antibody Antibody screenscreen

CrossmatchCrossmatchCauseCause ResolutionResolution

PosPosNegNegAntibody directed Antibody directed against antigen on against antigen on screening cellscreening cell

ID antibody, select ID antibody, select antigen negative antigen negative bloodblood

NegNegPosPosAntibody directed Antibody directed against antigen on against antigen on donor cell which may donor cell which may not be on screening not be on screening cell cell OROR donor unit donor unit may have IgG may have IgG previously attachedpreviously attached

ID antibody, select ID antibody, select antigen negative antigen negative blood blood OROR perform perform DAT on donor unitDAT on donor unit

PosPosPosPosAntibodies directed Antibodies directed against both against both screening and donor screening and donor cellscells

Antibody ID, select Antibody ID, select antigen negative antigen negative bloodblood

Additional Information on Types of Compatibility Tests

Manual (IS and IAT)Gel TechnologyElectronic (Computerized) Cross matchRed cell Affinity Column Technology (ReACT) Solid Phase Adherence Assays (SPAA)

Manual (IS and IAT)

IS detect RT reactive antibodies (Auto, Alloantibody, Naturally occuring)IAT detect IgG antibodies (Auto & alloantibody)

Antibody

Naturally occuring

(Cold agglutinin)

Acquired

AutoantibodyAlloantibody

Gel Technology

Patient serum, and 1% of suspended RBCs in LIM are dispensed into the microtube and incubated at 37oC for 15 minutes.The card containing the microtubes is then centrifuged at a controlled speed for 10 minutes.At the start of centrifugation the cells are separated from the serum; then they meet the AHG contained in the microtube. Finally the cells are trapped by the gel (if agglutinated) or pellet to the bottom of the tube.

New Technologies…

The electronic crossmatchAccording to the AABB, the following must be

fulfilled:Critical elements of the information system have been

validated on-site. No clinically significant antibodies are detected in the current

blood sample and there is no record of clinically significant antibodies in the past

Computer crossmatch (cont’d)

The patient's ABO group and Rh type has been done twice and entered in the computer

The donor ABO/Rh have been confirmed and entered in the computer. The donor unit identification number, component name, and ABO/Rh type must also be entered in the computer

The computer system will alert the technologist to ABO & Rh discrepancies between information on the donor label and results of donor confirmatory testing

Red Cell Affinity Column Technology

(ReACT)

Based on affinity adherence of coated red cells in an immunologically active matrix. Antibody- sensitized red cells bind or adsorbed to ligands attached to an agarose matrix.The main ligand is Protein G (prepared from Group C or G Streptococcus or by recombinant technology), which has high affinity for all four IgG subclasses.Another ReACT ligand is Protein A (from Group A Staphlococcus), which binds to IgG 1, 2, and 4.

Red Cell Affinity Column Technology (ReACT)

Positive reaction: the coated red blood cells with IgG are boud to immunoreactive gel particles, occurs mostly at the top of the gel column.

Negative reaction: the red blood cells are not coated with antibody and pass through to the bottom of the gel column.

Solid Phase Adherence Assays (SPAA)

Uses red cell membrane bound to the surfaces of polystyrene microtitration strip wells, capturing IgG antibodies (if present) in patient sera.

Patient serum is added to wells coated with screen cells

Incubated at 37oC for 15 min.

Washing

anti-IgG-coated indicator red cells are added.

centrifuge

SPAA

Result:

Positive

Negative

dispersed cells

indicator cells forming distinct ring

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.

A.Saline techniqueSaline technique is designed to detect compatibility of

IgM antibody(ies) in patient’s serum against antigens on donor’s red cells.

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 donor

4. 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) Note: In emergency spin technique is acceptable. Saline technique is inadequate as a complete

compatibility test because it is inadequate to detect clinically significant IgG antibodies.

Crossmatch Test for IgG Antibody(ies)

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° C

4. Centrifuge at 1000 rpm for 1 min, check for hemolysis/agglutination

5. 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.

Note: Cross-match can be done by two tubes technique for IgM and IgG separately as described above or by one tubes in which donor’ cell and the patient’s serum after step 5 in saline technique is incubated at 37°C for 20-30 minutes and then do IAT.

In major-cross for IgG antibodies albumin or enzyme or LISS can be used with IAT to increase sensitivity. For techniques see chapter on Antiglobulin Test.