Unit 8 Pretransfusion Testing
Part 2
Terry Kotrla, MS, MT(ASCP)BB
Compatibility Testing - History
 Early 1980’s 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
Compatibility Testing - History
 During 1984-85 FDA and AABB allowed the AHG phase of
the CROSSMATCH to be deleted if the patient’s 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.
Compatibility Testing – Coomb’s
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.
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.
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.
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
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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.
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)
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RT incubation
Additives such as albumin or LISS
Enzymes
Polyspecific AHG in IAT
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
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 patient’s 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.
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??
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 recipient’s 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.
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.
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.
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.
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.
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.
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.
General Considerations
 Labeling tubes
 Each tube labeled properly BEFORE use.
 Recipient’s 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.
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.
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.
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.
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.
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 manufacturer’s instructions.
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.
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
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