Pretranfusion Compatibility Testing Mr. Mohammed A. Jaber.
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Transcript of Pretranfusion Compatibility Testing Mr. Mohammed A. Jaber.
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.