Compatibility-testing

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					Pretranfusion Compatibility
          Testing



      Mr. Mohammed A. Jaber
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…
                    Crossmatching

 Purpose:
   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




                             No agglutination ~ compatible



                                Agglutination ~ incompatible



Donor RBCs   Patient serum
 (washed)
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
    Guarantee normal survival of RBCs
Prevent patient from developing an antibody
           Detect all antibodies
   Prevent delayed transfusion reactions
          Detect ABO/Rh errors
      Incompatible crossmatches

Antibody Crossmatch        Cause               Resolution
 screen
Pos     Neg           Antibody directed      ID antibody, select
                      against antigen on     antigen negative
                      screening cell         blood
Neg     Pos           Antibody directed      ID antibody, select
                      against antigen on     antigen negative
                      donor cell which may   blood OR perform
                      not be on screening    DAT on donor unit
                      cell OR donor unit
                      may have IgG
                      previously attached
Pos     Pos           Antibodies directed    Antibody ID, select
                      against both           antigen negative
                      screening and donor    blood
                      cells
Additional Information on Types of
        Compatibility Tests

 Manual (IS and IAT)
 Gel Technology
 Electronic (Computerized) Cross match
 Red cell Affinity Column Technology (ReACT)
 Solid Phase Adherence Assays (SPAA)
            Manual (IS and IAT)
                       Antibody




Naturally occuring                              Acquired

(Cold agglutinin)

                     Alloantibody                                    Autoantibody


   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
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       dispersed cells
 Negative     indicator cells forming distinct ring
           Major Crossmatch Tests

 It is done both for IgM and IgG antibodies
 Requirement:
 1. Recipient’s serum.
 2. 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
     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.

				
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posted:2/16/2013
language:English
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