Compatibility Testing - PowerPoint Presentation

Slide1

Compatibility Testing

Practical

Blood BankSlide2

Blood Transfusion Process Pre-transfusion Transfusion

Post-transfusionSlide3

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 antibodiesSlide4

Compatibility testing?There are several components of compatibility testingProper specimen collection

Reviewing patient transfusion historyABO, Rh, and antibody testing (screen/ID)

Crossmatching

Actual transfusionSlide5

Compatibility testingCan be divided into 3 categories:Preanalytical procedures

Serological testingPostanalytical proceduresSlide6

Pre-analytical phasesPatient identificationSpecimen collectionReview of patient historySlide7

Patient IdentificationMust confirm recipient’s ID from bracelet ON the patientFull patient name and hospital numberName of physicianSlide8

Sample IdentificationThe sample should also have the full patient name, hospital number, and physicianDate and time of collection, phlebotomist’s initials

All of this should be on the request form and the sampleSlide9

Specimen Tubes

Pink Top - EDTA

Red Top – no additivesSlide10

Specimen CollectionCollected in tube with EDTA or no additivesIf the venipuncture causes hemolysis, the sample may be rejected

True 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 errorSlide11

Specimen CollectionIf 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)Slide12

Getting the historyLook at recipient’s records for any prior unexpected antibodiesPrevious transfusion reactionsSlide13

Serological Testing3 tests:ABO/RhAntibody detection/identification

Crossmatch Slide14

ABO/Rh TypingIn the ABO typing, the forward and reverse MUST matchIn the Rh typing, the control must be negative

Both of these will indicate what type of blood should be givenSlide15

Antibody screen and/or IDThe 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…Slide16

CrossmatchingPurpose:Prevent transfusion reactions

Increase in vivo

survival of red cells

Double checks for ABO errors

Another method of detecting antibodiesSlide17

CrossmatchTwo types of crossmatchesMajor – routinely performed in labs

Minor – not required by AABB since 1976Slide18

Major vs Minor CrossmatchWhy is the minor crossmatch unnecessary?Donated units are tested for antibodies

Most blood is transfused as packed cells, having little antibodiesThe plasma volume is small, and Abs will be diluted in recipient circulationSlide19

Crossmatches

The crossmatch “shall use methods that demonstrate ABO incompatibility and clinically significant antibodies to red cell antigens and shall include an antiglobulin phase”Slide20

Crossmatch

Donor RBCs (washed)

Patient serum

No agglutination ~ compatible

Agglutination ~ incompatibleSlide21

The procedureDonor 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 unitSlide22

Units of whole blood with segments attachedSlide23

ProcedureABO/Rh typing is FIRST performed

Antibody Screen is performed next….Slide24

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 CROSSMATCHSlide25
Slide26

Crossmatches… WillVerify 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 errorsSlide27

Incompatible crossmatches

Antibody screen

Crossmatch

Cause

Resolution

Positive

Negative

Antibody 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 bloodSlide28

Additional Information on Types of Compatibility TestsManual (IS and IAT)

Gel TechnologyElectronic (Computerized) Cross match

Red cell Affinity Column Technology (ReACT)

Solid Phase Adherence Assays (SPAA)Slide29

Manual (IS and IAT)IS detect RT reactive antibodies (Auto, Alloantibody, Naturally occuring)

IAT detect IgG antibodies (Auto & alloantibody)Slide30

Gel TechnologyPatient serum, and 1% of suspended RBCs in LIM are dispensed into the microtube and incubated at 37

oC 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.Slide31

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 pastSlide32

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 Slide33

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.Slide34

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. Slide35

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 37

o

C for 15 min.

Washing

anti-IgG-coated indicator red cells are added.

centrifugeSlide36

SPAASlide37

Post-analytical phaseInvolves labeling, inspecting, and issuing the blood unitLabeling 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, etcSlide38

Issuing bloodWhen it’s time to release a blood product to the nurse or physician, a few “checks” must be done

Requisition formComparing requisition form



donor unit tag

 blood product label

Name of persons issuing and picking up blood

Date and time of release

Expiration dateSlide39

What if the unit is unused?Blood can be returned to the blood bank if it is not needed for transfusionUnit 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 issueSlide40

Special CircumstancesSlide41

Emergency ReleaseIn an emergency, there may not be enough time to test the recipient’s sampleIn 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..)Slide42

Emergency ReleaseOnce the specimen is received, ABO/Rh typing and antibody screening should be performed

Crossmatching the segments from the released unit should be testedIn 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 incompatibilitySlide43

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

Women below or of childbearing ageGroup O Rh(D)-positive red cells

Used as a substitution if O negative is not available

Male or elderly femalesSlide44

Massive transfusionDefined 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 conditionComplete Crossmatch not necessary (if no antibodies were detected originally)

Give ABO identical units

If antibodies were originally ID’s, continue to give antigen negative unitsSlide45

Donor Selection: Appropriate donor units to give

Patient’s Type

1

st

Choice

Other Choices

O

O

None

A

A

O

B

B

O

AB

AB

A, 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

). Slide46

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. Slide47

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

. Slide48

Major Crossmatch TestsIt is done both for IgM and IgG antibodiesRequirement:

Recipient’s serum.Donor’s red cells taken from the tube attached to the bag.Slide49

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:

Label 1 tube for each donor sample to be tested.

Put 2 drop of patient’s serum in labeled tube.

Add 1 drop of 2-5% saline suspended red cells of donor

Mix and incubate for 5-10 min. (spin method) or incubate for 30-60 min (sedimentation method) at RT.

Centrifuge at 1000 rpm for 1 min. in spin method (after 5-10 min. incubation);centrifugation is optional in sedimentation method.Slide50

Read the result, observe for hemolysis and agglutination.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.Slide51

B- Anti -Human Globulin Test (IAT)Indirect anti human globulin test (IAT) is the most important and widely used serological procedurein 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.Slide52

MethodPut 2 drops of patient’s serum in a labeled tube.

Add 1 drop of 2-5 % saline suspended red cells of donor.

Incubate for 30-60 min at 37° C

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

If there is no hemolysis /agglutination, wash the cells three times with normal saline.Slide53

Perform IAT testAdd 2 drops of polyspecific AHG serum to washed cells

Centrifuge at 1000 rpm for 1 minuteSee for agglutination

Add IgG coated red cells to negative AHG test.

Centrifuge and check for agglutination - if there is no agglutination test is invalid.Slide54

InterpretationHemeolysis 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.Slide55

Cross Match – Major Compatibility Test:

Label 3 tubes S1, S2 (Saline) and A1 (Albumin).To each tube add 2 drops of fresh serum from recipient.

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

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

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

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

ABO incompatibility may be detected in this phase.Slide56

Incubate the Tube S1 at room temperature for 15 min (Optional).Incubate the Tube S2 and A1 in the water bath for 30 min at 37o C.

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

Read the tube/tubes macroscopically for

Haemolysis

and/or agglutination and record results.

Wash Tube A1 with saline 3 times.

Add drops of Anti Human Globulin serum and mix well.

Centrifuge tube A for 15 second at 3400.

Read for agglutination and record the results. Slide57

Interpretation: If no agglutination of Haemolysis is present in corssmatch procedure, the blood is regarded compatible and reported as crossmatch Negative. Slide58

Cross Match – Minor Compatibility Test: Label a test tube with donor number and recipient's initials.

Add one drop of 2-5% suspension Recipient cells.Add 2 drops of Donor serum and 1 drop of 22% bovine albumin to the tube.

Centrifuge immediately 1 min at 1000 rpm.

Read macroscopically for

Haemolysis

and agglutination.

Incubate at 37o C for 30 minutes.

Centrifuge 1 min at 1000 rpm.

Read macroscopically for

Haemolysis

and agglutination.

Wash the tube 3 times with saline.

Add 2 drops of anti human globulin serum to the dry cell button.

Centrifuge 1 min at 1000 rpm.

Read macroscopically for

Haemolysis

and agglutination.

Add

Check Cells to all negative tests; spin, read and record results.Slide59

Interpretation: If no agglutination of Haemolysis is present in corssmatch procedure, the blood is regarded as serological compatible and reported as crossmatch Negative. Slide60

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.Slide61

The following Questions and Answers will help guide subsequent investigations:

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?

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.

Rh Type:

Recheck recipient and donor, determination of the Rh phenotype may be helpful in some cases.

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.Slide62

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 37

o

C

Irregular Antibody

Autoagglutinin

Rouleaux

Other serum direct Antiglobulin test

Antiglobulin or Enzyme

Irregular Antibody

Autoantibody

Positive Direct Antiglobulin testSlide63

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.

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

.

What was the patient diagnosis?

Is the direct Antiglobulin test of either recipient or donor positive?

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 reactionSlide64

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.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 22

o

C and 37

o

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.Slide65

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.Slide66

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.

Technical

Causes of apparent incompatibility (False Positive):

Dirty Glassware

Bacterial Contamination.

Chemical or other contamination or reagents, including saline.

Fibrin clots.

Over-centrifugation.  Slide67

What to Do With Crossmatch CluesSlide68

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 Rh

o

(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

.Slide69

Selection of Blood for Exchange transfusion