Terry Kotrla, MS, MT(ASCP)BB

Unit 8 Pretransfusion TestingPart 2

Compatibility Testing - HistoryEarly 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 unitsWeak D testingRepeat antibody screen on donor unitsDAT testingPerformance of elutionsSignificance of antibodies reactive at RT or below.Usefulness of albumin in antibody detection testsUse of AHG in both antibody screen AND

crossmatch

Compatibility Testing - HistoryDuring 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 CrossmatchWho 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 cellsRead 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 - ISWhen 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 - ISDecision 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 - ComputerAntibody 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.

Optional Pretransfusion TestingABO Grouping

Testing RBCs with anti-A,BSerum/plasma tested against A2 cells

D typingWeak D test Rh control with chemically modified reagents

unless AB posAntibody Screen (IAT)

RT incubationAdditives such as albumin or LISSEnzymesPolyspecific AHG in IAT

Optional Pretransfusion TestingAutocontrol or DAT

Published data indicate performance is of limited value even in recently transfused patients.

Standards does not require autocontrol or DATMicroscopic reading of tests, magnifier viewing

lamp adequate.Crossmatch

37C and AHG when antibody screen and history is negative.

RT incubationEnzyme testsPolyspecific AHGMinor crossmatch

Selection of Donor GroupWhen 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 TransfusionDetermine 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 GroupsUnnecessary 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 TechniquesUse 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/ISAfter 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 TechniquesAntibody 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 TechniquesMethod 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 TechniquesLab 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 agglutinationDetect mixed field agglutination seen in anti-Sda.

Antibody Detection TechniquesReactions 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 ConsiderationsLabeling 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 ConsiderationsVolume 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 ConsiderationsCell 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 TubeSimplest 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 TubeUtilized 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 TubeLow 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 TubePolyethylene 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

37CWash and add AHG.

Testing Techniques – Enzymes TubeMore 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 includePapainBromelainTrypsinFicin – MOST POPULAR

ReferencesAABB Technical Manual, 16th edition, 2008CAP Today http://tinyurl.com/4cd4qgd Basic & Applied Concepts in

Immunohematology, 2nd edition, 2009Ortho WIRE, http://www.ortho-wire.com