Compatibility Testing

Practical Blood Bank

Blood Transfusion Process

Pre-transfusion Transfusion Post-transfusion

What is compatibility testing? Also called pretransfusion testing

Purpose:

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 testing Proper specimen collection Reviewing patient transfusion history ABO, Rh, and antibody testing (screen/ID) Crossmatching Actual transfusion

Compatibility testing

Can be divided into 3 categories: Preanalytical procedures Serological testing Postanalytical procedures

Pre-analytical phases

Patient identification Specimen collection Review of patient history

Patient Identification

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

hospital number Name 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 additives If the venipuncture causes hemolysis, the

sample may be rejected True hemolysis in the patient is the result

of complement activation Samples 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/Rh Antibody detection/identification Crossmatch

ABO/Rh Typing

In the ABO typing, the forward and reverse MUST match

In the Rh typing, the control must be negative

Both 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) IS 37° (LISS) AHG

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

Proceed to the crossmatch…

CrossmatchingPurpose:

Prevent transfusion reactions Increase in vivo survival of red cells

Double checks for ABO errors Another method of detecting antibodies

Crossmatch

Two types of crossmatches Major – routinely performed in labs Minor – not required by AABB since 1976

Major vs Minor Crossmatch

Why is the minor crossmatch unnecessary? Donated units are tested for antibodies

Most 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 suspension

This fulfills the AABB standard for ABO incompatibility

This is an INCOMPLETE CROSSMATCH If antibodies ARE detected:

Antigen negative units found and X-matched

All phases are tested: IS, 37°, AHG This is a COMPLETE CROSSMATCH

Crossmatches…

Will Verify donor cell ABO

compatibility

Detect most antibodies against donor cells

Will Not Garantee normal survival of

RBCs

Prevent patient from developing an antibody

Detect all antibodies

Prevent delayed transfusion reactions

Detect ABO/Rh errors

Incompatible crossmatches

Antibody screen Crossmatch Cause Resolution

Positive NegativeAntibody directed against antigen on screening cell

ID antibody, select antigen negative blood

Negative Positive

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

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

Positive Positive

Antibodies directed against both screening and donor cells

Antibody ID, select antigen negative blood

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)

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 crossmatch According 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 bound 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. Washinganti-IgG-coated indicator red cells are added. centrifuge

SPAA

Post-analytical phase Involves labeling, inspecting, and issuing the

blood unit Labeling form includes patient’s full name, ID

number, Location, ABO/Rh(D) of patient and unit, donor #, compatibility results, and tech ID

Form is attached to the donor unit and only released for the recipient

The unit is visually inspected for abnormalities, such as bacterial contamination, clots, etc

Issuing blood

When it’s time to release a blood product to the nurse or physician, a few “checks” must be done Requisition form Comparing requisition form donor unit tag

blood product label Name of persons issuing and picking up blood Date and time of release Expiration date

What if the unit is unused? Blood can be returned to the blood bank if it is

not needed for transfusion Unit closure has to remain unopened Storage temperature must have remained in

the required range (1° to 10°C for RBCs) If not at correct temp, unit must be returned

within 30 minutes of issue

Special Circumstances

Emergency Release

In an emergency, there may not be enough time to test the recipient’s sample

In this case, blood is released only when signed by the physician (O negative)

The tag must indicate it is not crossmatched Segments from the released units should be

retained for X-matching Every detail is documented (names, dates..)

Emergency Release Once the specimen is received, ABO/Rh typing

and antibody screening should be performed Crossmatching the segments from the released

unit should be tested In addition, the lab may crossmatch additional

units as a precaution if more blood is needed If death should occur, testing should be

complete enough to show that the death was unrelated to an incompatibility

What can be given in an emergency? Group O Rh(D)-negative red cells or AB

plasma Emergency release Women below or of childbearing age

Group O Rh(D)-positive red cells Used as a substitution if O negative is not

available Male or elderly females

Massive transfusion Defined as a transfusion approaching or

exceeding the recipient’s own blood volume (about 5 liters or 10-12 units in an adult male) within 24 hour period

The original sample no longer represents the patient’s condition

Complete Crossmatch not necessary (if no antibodies were detected originally)

Give ABO identical units If antibodies were originally ID’s, continue to

give antigen negative units

Donor Selection: Appropriate donor units to give

Patient’s Type 1st Choice Other Choices

O O None

A A O

B B O

AB ABA, O, B only one of the

three should be used for a given patient

ABO specific blood should always be given first. When ABO-specific blood is not available or is in

less than adequate supply, alternative blood groups are chosen as summarized in the following table; (must be administered as red blood cells).

Selection of Appropriate Donor Units. Rh-negative blood can be given to Rh-positive

patients, however, good inventory management should conserve this limited resource for use in Rh-neg recipients.

If Rh-neg units is near expiration, the unit should be given rather than wasted.

Selection of Appropriate Donor Units.

Rh-pos blood should not be given to Rh(D) -neg women of childbearing age.

Transfusion of Rh-neg male patients and female patients beyond menopause with Rh-pos blood is acceptable as long as no performed anti-D is demonstrable in the sera.

Major Crossmatch Tests

It is done both for IgM and IgG antibodies Requirement:

Recipient’s serum. Donor’s red cells taken from the tube attached to

the bag.

A-Saline technique

Saline 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

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 agglutination.

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.

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.

Method1.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/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.

Cross Match – Major Compatibility Test:

1. Label 3 tubes S1, S2 (Saline) and A1 (Albumin).

2. To each tube add 2 drops of fresh serum from recipient.

3. To each Tube add 2 drops of 5% saline suspension of donor's Cells.

4. To tube A1 add 2 drops of Bovine Albumin (22%).

5. Centrifuge both tubes S1 and A1 for 15 seconds at 3400 rpm.

6. Read Macroscopically for Haemolysis and/or agglutination and record results.

ABO incompatibility may be detected in this phase.

7. Incubate the Tube S1 at room temperature for 15 min (Optional).

8. Incubate the Tube S2 and A1 in the water bath for 30 min at 37o C.

9. When the incubation time finished centrifuge the tube/tubes for 15 second at 3400 rpm.

10. Read the tube/tubes macroscopically for Haemolysis and/or agglutination and record results.

11. Wash Tube A1 with saline 3 times.12. Add drops of Anti Human Globulin serum

and mix well.13. Centrifuge tube A for 15 second at 3400.14. Read for agglutination and record the

results. 

Interpretation:

If no agglutination of Haemolysis is present in corssmatch procedure, the blood is regarded compatible and reported as crossmatch Negative.

Cross Match – Minor Compatibility Test:

1. Label a test tube with donor number and recipient's initials.2. Add one drop of 2-5% suspension Recipient cells.3. Add 2 drops of Donor serum and 1 drop of 22% bovine

albumin to the tube.4. Centrifuge immediately 1 min at 1000 rpm.5. Read macroscopically for Haemolysis and agglutination.6. Incubate at 37o C for 30 minutes.7. Centrifuge 1 min at 1000 rpm.8. Read macroscopically for Haemolysis and agglutination.9. Wash the tube 3 times with saline.10. Add 2 drops of anti human globulin serum to the dry cell

button.11. Centrifuge 1 min at 1000 rpm.12. Read macroscopically for Haemolysis and agglutination.13. Add Check Cells to all negative tests; spin, read and record results.

Interpretation:

If no agglutination of Haemolysis is present in corssmatch procedure, the blood is regarded as serological compatible and reported as crossmatch Negative.

The Incompatible Crossmatch:

Although the majority crossmatches will indicates compatibility, problems still occur. Even if an incompatible is detected before crossmatch has been carried to the anti-globin stage, the procedure should be completed for investigational purpose. If blood is urgently needed, additional donor blood should be crossmatched before starting to investigate the problem.

Rather than continuing to crossmatch blindly, it is always advisable to try to determine the cause of the incompatibility. However, in emergency situations, it may be necessary to crossmatch many units of blood of appropriate ABO group and Rh Type, in the hope that a compatible unit will be found. In addition, the patient's blood relatives should be tested for compatibility since there is an increased chance of finding suitable donors among them.

The antibody should be identified, not only for the present transfusion, but also to protect the patient in any future transfusions when the antibody titer may have decreased or even disappeared.

The following Questions and Answers will help guide subsequent investigations:

1. Identification: Is the "Patient's Blood Specimen" really from the intended

recipient? Was a unit of blood with the correct ABO group and Rh Type

Selected? Does both the blood unit pilot tube have the same

identification?2. ABO grouping:

Recheck recipient and donor from original specimens using freshly prepared red cell suspensions. If anti-A1 or anti-H is identified, blood for transfusion should be selected on the basis of A subgroup.

3. Rh Type: Recheck recipient and donor, determination of the Rh phenotype may be

helpful in some cases.

4. Auto Control: Test Patients serum with his own cells to determine if the problems are due

to blood group isoantibody, autoantibody, or nonspecific reaction. This control should be run concurrently with crossmatch or antibody identification.

5. Stage of incompatibility: Procedure will be determined to a large extent by the

stage at which the incompatibility is most pronounced, as suggested by the following table.

If some donors are incompatible in an early stage of the crossmatch but other donors are not incompatible until a later stage, this might indicate two or more antibodies.

Stage of apparent incompatibility

Possible Cause

Saline or serum at RT - ABO Error.- Cold

autoagglutinin or irregular agglutinin

Saline, Serum or High protein at 37o C

- Irregular Antibody- Autoagglutinin- Rouleaux- Other serum

direct Antiglobulin test

Antiglobulin or Enzyme - Irregular Antibody- Autoantibody- Positive Direct

Antiglobulin test

6. Percentage of incompatible donors: The approximate percentage of incompatible donors may help in

elucidate the problem, for example, with an antibody reaction n the Antiglobulin phase: six or seven bloods positive out of 10 bloods tested suggests anti-Fya; one blood positive out of 10 tested suggests anti-K.

7. Grading donor reactions: Are the reactions of incompatible bloods all of the same strength? If

not There may be two or more antibodies of varying strength. The antibody may be exhibiting a dosage phenomenon.

8. What was the patient diagnosis?

9. Is the direct Antiglobulin test of either recipient or donor positive?a) If recipient has a positive direct Antiglobulin tests:

Serum may or may not contain autoantibody. If an autoantibody is present, the serum may react with all donor samples tested. The technique by which the incompatibility is detected depends upon the type of antibody (cold or warm).

All minor crossmatches will be incompatible. If the recipient has been recently transfused, the positive Antiglobulin test may

indicate incompatibility of infused donor red cells, specially if the appearance is that of a mixed filed reaction

b) If donor has a positive direct Antiglobulin test. Major Crossmatch will be incompatible. Minor crossmatch may or may not be incompatible. Other donor units will crossmatch satisfactorily.

10. Abnormal proteins, autoagglutinin and cold agglutinins. Factors relating to disease or medication may cause agglutination or pseudo agglutination.

If Rouleaux occurs: Check patient's diagnosis and serum protein level. Autologous red cells and serum at 22o C and 37o C

should give the same reactions as in the compatibility test.

Compatibility testing with strong Rouleaux, the saline anti-globulin crossmatch may be the only reliable test since the Antiglobulin reactions is not affected by properties of serum that cause Rouleaux. High protein techniques are affected.

Cold agglutinins are the most common cause of difficulty in compatibility testing. Although they react best at 4o C, they may cause agglutination in the room temperature phase of the crossmatch and on immediate centrifugation of the high protein test. They also may cause a positive Antiglobulin test, especially in autoimmune disease. Strong cold autoagglutinin, especially those with wide thermal

amplitude, must be absorbed from the patient's serum since they may mask the presence of specific blood group antibodies. If the autoantibody is active at 22o C or lower, it can usually be removed from the serum by placing a fresh recipient blood specimen in ice and allowing it to clot in the refrigerator.

After the cold active antibody is adsorbed onto autologous red cell, the absorbed serum is used for antibody detection and compatibility testing. A suspension of the red cells (For Control) should be prepared from blood that has not been refrigerated.

11) Does the serum contain irregular antibodies? Test with reagent blood cells (DiaCell), if this has not been

done as part of the compatibility test, identify any antibodies present.

If the crossmatch is incompatible only with one donor, and antibody detection tests are negative, the recipient's serum should be tested for antibodies directed against low-incidence antigens.

12) Technical Causes of apparent incompatibility (False Positive):

Dirty Glassware Bacterial Contamination. Chemical or other contamination or reagents, including

saline. Fibrin clots. Over-centrifugation.  

What to Do With Crossmatch Clues

Crossmatch for Newborns and infants:

In case of Erythroblastosis: The corssmatch should include a crossmatch with the mother's serum.

If mother's serum is not available crossmatch with baby's serum. In ABO incompatibility: choose blood compatible with mother's or

group O cells suspended in-group specific plasma. Perform major and minor crossmatch.

In Rh incompatibility: Mother and infant are of the same blood group, transfer with compatible group specific Rh Negative

In Rh incompatibility: Mother and infant are of different blood group, choose O Rh Negative cells suspended in-group specific fresh plasma.

In Erythroblastosis due to (c): use blood which is c/c also Rho(D). In case transfusion is to be repeated, use the same group and method

as for the first transfusion In Case of No Erythroblastosis:

When Infant's RBC is compatible with mother's serum; do crossmatch with mother's serum.

When infant's RBC's are incompatible with mother's serum use infant's serum for crossmatch.

Selection of Blood for Exchange transfusion