Practical Blood Bank

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Practical Blood Bank
Compatibility Testing
Lab 11
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Blood Transfusion Process

• Pre-transfusion
• Transfusion
• Post-transfusion

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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

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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

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Compatibility testing

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

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Pre-analytical phases

• Patient identification
• Specimen collection
• Review of patient history

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Patient Identification

• Must confirm recipients ID from bracelet ON the
  patient
• Full patient name and hospital number
• Name of physician

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Sample Identification

• The sample should also have the full patient
  name, hospital number, and physician
• Date and time of collection, phlebotomists
  initials
• All of this should be on the request form and the
  sample

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Specimen Tubes
Red Top no additives
Pink Top - EDTA
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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

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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)

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Getting the history

• Look at recipients records for any prior
  unexpected antibodies
• Previous transfusion reactions

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Serological Testing

• 3 tests
• ABO/Rh
• Antibody detection/identification
• Crossmatch

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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

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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

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Crossmatching

• Purpose
• Prevent transfusion reactions
• Increase in vivo survival of red cells
• Double checks for ABO errors
• Another method of detecting antibodies

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Crossmatch

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

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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

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Crossmatches

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

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Crossmatch
No agglutination compatible
Donor RBCs (washed)
Patient serum
Agglutination incompatible
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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

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Units of whole blood with segments attached
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Procedure

• ABO/Rh typing is FIRST performed
• Antibody Screen is performed next.

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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

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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

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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 blood
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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)

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Manual (IS and IAT)

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

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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.

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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

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Computer crossmatch (contd)

• 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

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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.

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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.

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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

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SPAA
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Post-analytical phase

• Involves labeling, inspecting, and issuing the
  blood unit
• Labeling form includes patients 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

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Issuing blood

• When its 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

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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 10C for RBCs)
• If not at correct temp, unit must be returned
  within 30 minutes of issue

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Special Circumstances
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Emergency Release

• In an emergency, there may not be enough time to
  test the recipients 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..)

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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

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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

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Massive transfusion

• Defined as a transfusion approaching or exceeding
  the recipients 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
  patients condition
• Complete Crossmatch not necessary (if no
  antibodies were detected originally)
• Give ABO identical units
• If antibodies were originally IDs, continue to
  give antigen negative units

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Donor Selection Appropriate donor units to give

• 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).
  

Patients Type 1st 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
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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.

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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.

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Major Crossmatch Tests

• It is done both for IgM and IgG antibodies
• Requirement
• Recipients serum.
• Donors red cells taken from the tube attached to
  the bag.

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A-Saline technique

• Saline technique is designed to detect
  compatibility of IgM antibody(ies) in patients
  serum against antigens on donors red cells.
• Method
• Label 1 tube for each donor sample to be tested.
• Put 2 drop of patients 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.

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• 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.

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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 recipients serum and
  donors cells. The majority of incomplete
  antibodies are IgG and are detected by AHG test.

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Method

1. Put 2 drops of patients 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.

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• Perform IAT test
• Add 2 drops of polyspecific AHG serum to washed
  cells
• Centrifuge at 1000 rpm for 1 minute
• See for agglutination
• Add IgG coated red cells to negative AHG test.
• Centrifuge and check for agglutination - if there
  is no agglutination test is invalid.

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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 patients serum after step 5 in saline
  technique is incubated at 37C 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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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.

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• 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. 

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Interpretation

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

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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.

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Interpretation

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

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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.

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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.

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• 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
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• 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 reaction

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• 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 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.

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• 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.

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• 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.  

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What to Do With Crossmatch Clues
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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.

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Selection of Blood for Exchange transfusion