Terry Kotrla, MS, MT(ASCP)BB

1
Unit 8 Pretransfusion TestingPart 2

• Terry Kotrla, MS, MT(ASCP)BB

2
Compatibility Testing - History

• Early 1980s started to question utility of
• Routine use of anti-A,B and A2 cells in ABO
  grouping
• Repeat D typing of D positive donor units
• Weak D testing
• Repeat antibody screen on donor units
• DAT testing
• Performance of elutions
• Significance of antibodies reactive at RT or
  below.
• Usefulness of albumin in antibody detection tests
• Use of AHG in both antibody screen AND crossmatch

3
Compatibility Testing - History

• During 1984-85 FDA and AABB allowed the AHG phase
  of the CROSSMATCH to be deleted if the patients
  antibody screen was negative.
• In 1984 Judd recommended deleting the autocontrol
  as part of routine pretransfusion testing.
• By 1986 the minor crossmatch was of historic
  interest only.

4
Compatibility Testing Coombs Crossmatch

• Who needs a crossmatch? Patients who are
• experiencing clinical signs and symptoms of
  anemia.
• actively bleeding.
• having a surgical procedure where possibility of
  excessive bleeding is high.
• What is the major crossmatch
• Patient serum/plasma added to donor cells
• Read at three phases IS, 37C and AHG.
• Set up and read as part of antibody screen
  procedure
• Agglutination and/or hemolysis are positive.
• Donor cells reacting with patient sample at 37C,
  AHG or causing hemolysis are incompatible
  CANNOT be transfused.

5
Compatibility Testing - IS

• When NO CLINICALLY SIGNIFICANT antibodies are
  detected in the antibody screen AND there is no
  history of antibodies, the AHG phase of testing
  is NOT required.
• Rarely are AHG incompatible crossmatches obtained
  when antibody screen is negative.
• MUST demonstrate ABO incompatibility by
  performing IS crossmatch.
• Policy change requires medical director approval.

6
Compatibility Testing - IS

• Decision to omit AHG phase based on the
  following
• Incidence of incompatible crossmatches when
  antibody screen is negative and the reason.
• Sensitivity of antibody detection procedure used.
• Benefits of omitting AHG phase in the laboratory.
• Expertise of individuals working in the
  transfusion service.
• If clinically significant antibodies are present
  the AHG phase of the crossmatch is required.

7
Compatibility Testing - Computer

• Antibody screen negative and computer validated
  on site to prevent release of ABO-incompatible
  blood it may be used to detect ABO compatibility
  instead of serologic testing.
• Following conditions MUST be met
• TWO determinations of patients ABO group by
  second type on same sample OR second current
  sample, or comparing to previous records.
• Donor ABO/D type, unit number, component name and
  confirmatory type.
• Patient ABO/D type, antibody screen result and
  two unique patient identifiers.
• Method to ensure correct data entry.
• Computer logic to alert to ABO/D discrepancies on
  unit label and testing and ABO incompatibility
  between recipient and donor.

8
Optional Pretransfusion Testing

• ABO Grouping
• Testing RBCs with anti-A,B
• Serum/plasma tested against A2 cells
• D typing
• Weak D test
• Rh control with chemically modified reagents
  unless AB pos
• Antibody Screen (IAT)
• RT incubation
• Additives such as albumin or LISS
• Enzymes
• Polyspecific AHG in IAT

9
Optional Pretransfusion Testing

• Autocontrol or DAT
• Published data indicate performance is of limited
  value even in recently transfused patients.
• Standards does not require autocontrol or DAT
• Microscopic reading of tests, magnifier viewing
  lamp adequate.
• Crossmatch
• 37C and AHG when antibody screen and history is
  negative.
• RT incubation
• Enzyme tests
• Polyspecific AHG
• Minor crossmatch

10
Selection of Donor Group

• When possible ABO identical.
• D positive should be selected for D pos, although
  D neg is acceptable but should be reserved for D
  neg except
• D neg short date unit can be given to D pos sure
  give.
• Multiple antibodies present and D neg more likely
  to lack.
• D negative should be selected for D neg to avoid
  immunization to D antigen.
• Consult with medical director and patients
  physician if need is urgent.
• Use D negative first.
• Weigh risk of patient death versus immunization
  to D.
• May be appropriate to administer Rh immune
  globulin especially after platelet transfusion.

11
Selection of Donor Group
Recipient ABO Compatible Donor ABO In Order of Selection
O O
B B, O
A A, O
AB AB, A, O, B why O before B??
12
Blood Administered after Non-Type Specific
Transfusion

• Determine presence of anti-A and/or anti-B in
  patient.
• When serum from freshly drawn sample is
  compatible at AHG with recipients own blood
  group may return to group specific.
• If AHG incompatible must continue with
  alternative ABO group.
• If change involved D only return to D type
  specific.

13
Other Blood Groups

• Unnecessary to select units based on other blood
  groups UNLESS patient has clinically significant
  unexpected antibody.
• If antibody strongly reactive use patient serum
  to screen then confirm with specific typing sera.
• Weakly reactive screen units with specific typing
  sera.
• If commercially prepared typing sera is not
  available use patient sample or plasma from donor
  with antibody.

14
Antibody Detection Techniques

• Use 2 to 3 commercially prepared group O cells.
• Relatively short shelf life, two weeks.
• Antigen profile (antigram) provided with analysis
  of antigens present on each cell.
• MAKE SURE lot number of screen cell matches
  antigram.
• Add patient serum/plasma to screen cells and
  observe at
• RT/IS
• After incubation at 37C with enhancement media.
• After washing and addition of AHG reagent.
• Agglutination and/or hemolysis is POSITIVE.
• Phase of reactivity of positive reaction
  extremely helpful.

15
Antibody Detection Techniques

• Antibody detection procedure used determined by
  what is considered significant antibody.
• Carefully considered if AHG crossmatch not
  performed.
• Once adopted method written in SOP and must be
  followed exactly by all staff members.
• Detection method chosen should
• Detect as many clinically significant antibodies
  as possible.
• NOT detect clinically insignificant antibodies.
• Allow prompt delivery of blood to the patient.

16
Antibody Detection Techniques

• Method should be sufficiently sensitive to detect
  low level of antibody in patient serum or plasma.
• Undetected low levels of patient antibody may
  result in rapid production of antibody if antigen
  positive cells transfused.
• Antibody present in donor plasma will not harm
  recipient.
• Methods for antibody screen and crossmatch may be
  the same or different.
• RT tests such as IS crossmatch required to detect
  ABO incompatibilities, may not be required for
  antibody screen.
• Antibody screen MUST include AHG to detect
  clinically significant antibodies, crossmatch may
  be IS only.

17
Antibody Detection Techniques

• Lab personnel should use same interpretations,
  notations and consistency in grading reactions.
• Consistency in grading reactions crucial.
• Hemolysis and/or agglutination constitutes
  visible endpoint of antigen-antibody reaction and
  must be observed accurately and consistently.
• Use light source or optical aid to enhance
  sensitivity and consistency.
• Microscopic observation is not required but is
  useful to
• Distinguish rouleaux from true agglutination
• Detect mixed field agglutination seen in anti-Sda.

18
Antibody Detection Techniques

• Reactions must be observed for hemolysis then
  agglutination IMMEDIATELY after centrifugation.
• Manner in which RBCs are dislodged is crucial.
• Hold tube at angle so fluid cuts across cell
  button as tube is tilted.
• Reaction is not interpreted until ALL cells
  resuspended.
• Over shaking will result in weak or negative
  reactions.
• Reactions are recorded IMMEDIATELY with tube held
  in hand in front of column to record in.

19
General Considerations

• Labeling tubes
• Each tube labeled properly BEFORE use.
• Recipients initials (or other identifying
  information) and donor unit number or reagent RBC
  identification.
• System must allow for accurate, rapid labeling.
• NEVER rely on the position of a tube in a rack or
  centrifuge head to identify the contents of the
  tube.
• ALWAYS place tubes in the serofuge head in the
  order they will be read.
• Use the SAME organizational techniques when
  labeling and arranging tubes in rack to improve
  organization and speed.

20
General Considerations

• Volume of serum or plasma.
• Most procedures call for 2 drops.
• Research has shown 2 drops provide optimal
  antibody to antigen ratio.
• Some alloantibodies detected only when 3 to 4
  drops used.
• High variability in delivery in transfer
  pipettes.
• Standardize volumes based on equipment used in
  your lab.
• Low ionic reagents require ration of 2 drops
  serum/plasma to 2 drops LISS, cannot vary.

21
General Considerations

• Cell suspension
• RBCs used for crossmatching obtained from sealed
  segment of original tubing attached to blood
  container.
• Wash once and prepare 2-4 suspension, some
  workers prefer 2 as it increases sensitivity of
  the test system.
• Best to use weakest suspension that can be
  observed for agglutination.
• Too heavy of a cell suspension can cause weak
  antibodies to be missed.

22
Testing Techniques Saline Tube

• Simplest to perform.
• Mix serum or plasma with saline suspended RBCs,
  centrifuge and read, incubate at RT or 37C.
• Used in crossmatching to detect ABO
  incompatibility.
• In antibody tests used to detect IgM antibodies
  which react preferentially at RT anti-M, -N,
  -P1, -Le and I.
• Rare examples of antibodies of other
  specificities may be observed at RT but more
  often will be reactive at 37C and/or AHG as well.

23
Testing Techniques Bovine Albumin Tube

• Utilized to enhance agglutination of IgG
  antibodies since 1945.
• Decreases amount of time required for incubation.
• Controversy Decrease zeta potential (affects
  second stage of agglutination) or due to function
  of ionic strength of albumin diluent does it
  increase uptake of antibody onto cells?
• Many antibodies have enhanced reactivity when
  albumin is added to test system.

24
Testing Techniques LISS Tube

• Low Ionic Strength Saline shortens incubation
  time.
• Increases antibody uptake onto cell, enhancing
  agglutination.
• Several important factors to consider
• Incubation time and sensitivity subsequent to AHG
  depends upon desired ionic conditions.
• Adding additional serum will increase ionic
  strength, must not be done.
• MUST adhere to manufacturers instructions.

25
Testing Techniques PEG Tube

• Polyethylene Glycol (PEG) is a water soluble,
  neutral polymer which is an effective potentiator
  of antigen-antibody reactions.
• Advantages over albumin include
• Increases rate of detection of clinically
  significant antibodies.
• Decreases detection of clinically insignificant
  antibodies.
• May decrease need for other enhancement
  techniques.
• Procedure
• Serum or plasma added to RBCs, perform IS.
• Add PEG and incubate at 37C IS NOT READ AFTER
  37C
• Wash and add AHG.

26
Testing Techniques Enzymes Tube

• More appropriate for antibody ID than routine
  testing.
• GREATLY enhance reactivity of Rh antibodies.
• CANNOT be only method used as M, N, S, Fy and
  other antigens are destroyed, those antibody
  specificities would not be detected.
• Enzymes used include
• Papain
• Bromelain
• Trypsin
• Ficin MOST POPULAR

27
References

• AABB Technical Manual, 16th edition, 2008
• CAP Today http//tinyurl.com/4cd4qgd
• Basic Applied Concepts in Immunohematology, 2nd
  edition, 2009
• Ortho WIRE, http//www.ortho-wire.com