Terry Kotrla, MS, MT(ASCP)BB

Unit 8 Pretransfusion TestingPart 3

Testing Techniques - GelBased on principle of controlled centrifugation

of RBCs through a dextran-acrylamide gel.Strip or card of several microtubes of reactants

allows for performance of several tests simultaneously.

Can be used for detection of direct agglutination (ABO, Rh, RBC phenotyping)and for IAT and DAT.

RBCs allowed to interact with antibodies in chambers at TOP of column.

Centrifuged to force RBCs into column to separate agglutinated from nonagglutinated.

Testing Techniques - GelNonagglutinated cells pass freely through

gel and pellet at bottom of microtube.Agglutinated cells are to large to enter the

gel matrix and remain at top of column.A= positive and E=negative

Testing Techniques - Gel Reactions are graded from 0 to 4+. (A) 4+ reaction is indicated by solid band of red cells on the top of the gel; (B) 3+ reaction displays agglutinated red cells in the upper half of the gel

column; (C) 2+ reaction is characterized by red cell agglutinates through length of

column; (D) 1+ reaction indicated by RBC agglutinates mainly in lower half of gel

column with some unagglutinated red cells pelleted at the bottom; and (E) Negative reactions display a pellet at the bottom and no agglutinates in

the matrix of the gel column. A mixed field reaction may be observed.

Testing Techniques - GelMicroliters of plasma added, CANNOT USE

SERUM.Incubate, spin and read, NO washing or

tube shaking.Sesnitivity of 98% compared to LISS tube

method.Cards can be saved for peer review or

documented with photo.Simplifies cross training, improves

productivity and workflow efficiency.

Microplate TestingFor routine processing of DONOR samples.Microtiter plate with 96 wells.Adapted to RBC or serum testing.Add appropriate sample, centrifuge and

dislodge button.

Solid Phase AdherenceRBC antigens or antibodies are immobilized

in microplate wells to detect antibody-antigen interaction.Intact RBCs or RBC membranes of known

phenotype.Add patient serum or plasma.IncubateWashAntibody, if present, will attach.Add IgG coated coated RBCsCentrifuge, agglutinates will coat well.

Solid Phase AdherenceSlide 1 – antibody screenSlide 2 – antibody detection top, ABO

grouping bottom

Solid Phase Adherence

Automated Testing PlatformsPerform multiple tests using

Solid phaseColumn agglutinationMicrotiter plateOther technologies

All process from pipetting to result interpretation performed under computerized control.

Usually interfaces with laboratory information system (LIS).

Examples: Galileo Echo, Biotest Tango

Antiglobulin TechniqueREQUIRED for testing samples from recipients

for presence of unexpected antibodies and, in selected circumstances, verifying serologic compatibility with donors.

Either IgG or polyspecific AHG may be used.Polyspecific rarely used

False positive nuisance antibodies react.Transfusion service medical director should

evaluate time working up nuisance antibodies

Crossmatch Interpretation

Negative screen, Compatible XMVast majority will have this outcome.DOES NOT guarantee the absence of

antibody in patient sample directed against donor antigens OR the donor cells will survive normally.

Negative screen simply means there are no DETECTABLE antibodies in patient sample directed against antigens present on these particular screen cells.

Compatible crossmatch viewed in same manner.

Negative screen, Compatible XM

IS 37C AHG CHECK INTERP

Screen 1 0 0 0 2+ Negative

Screen 2 0 0 0 2+

Screen 3 0 0 0 2+

Donor 1 0 0 0 2+ Compatible

Positive screen, Incompatible XMMay be due to alloantibodies or

autoantibodiesIf ALL tubes positive could be problems

with reagents or, at IS or 37C, rouleauxMUST IDENTIFY PROBLEM PRIOR TO

TRANSFUSION.Need for blood urgent consult with medical

director and patient’s physician.Risk of death transfusing incompatible may

be less than depriving patient of oxygen carrying capacity.

Positive screen, Incompatible XMThe following table would indicate the

presence of an alloantibody directed against screen 1 and donor 1.

IS 37C AHG CHECK INTERP

Screen 1 0 0 2+ Positive

Screen 2 0 0 0 2+

Screen 3 0 0 0 2+

Donor 1 0 0 2+ Incompatible

Positive screen, Incompatible XMPositive usually indicates alloantibody present.

Perform panel study identify antibody specificity.Estimate liklihood of finding antigen negative blood

in inventory.Use appropriate antiserum to verify units are

antigen negative.NOT necessary to confirm antigen negative for M, N, P1 or

Le antigens.Must perform if required at your facility.

If multiple antibody specificities present must identify all, may have to send to reference lab.

Antibody to high incidence antigen most promising source will be siblings or AABB rare donor blood file.

Positive screen, Incompatible XMIf autocontrol positive (required for

antibody identification studies) obtain patient history.Transfused within last 3 months alloantibody

may be present reacting with transfused donor cells.

MF agglutination is usually noted as only donor cells are positive for antigen and coated with antibody.

Perform elution procedure to remove bound antibody from cells and identify specificity.

Positive screen, Incompatible XMPotent cold reactive antibodies may cause problem with

ABO, D typing as well as antibody detection and crossmatch.

Most common specificity is anti-I which may show high thermal amplitude, reacts with adult but NOT cord cells.

Important to determine if cold is masking warm antibody.AFTER IDENTIFICATION perform prewarm technique

Prewarm serum/plasma and add to prewarmed screen cells.After incubation wash with WARM saline.Add AHG and evaluate for positive and negative reactions.

Cold auto-absorption may be necessary – covered later.

Positive screen, Incompatible XMRouleaux is property of serum that causes ALL

cells tested to appear agglutinated at RT and 37C, DOES NOT affect AHG test as serum is removed.

Stacked coin appearance of RBCs.To confirm perform saline replacement.

Spin tubes down, remove serum.Add 1-3 drops saline, mix, respin.Rouleaux formation will disperse, TRUE

agglutination will NOT.Spin, remove saline, add serum and proceed to

37C incubation.

Positive screen, Incompatible XMReagent Related Problems

May be due to a variety of drugs or additives present in reagents which cause false POSITIVE results.

If reagent RBCs positive but XMs appear compatible suspect patient reacting with additive in reagent RBCs, wash reagent RBCs and retest.

If ALL screen and XM tubes positive, suspect patient antibody reacting with substance in enhancement media, repeat with different enhancement or do saline test.

Positive Screen, Compatible XMPatient has antibody directed against

antigen on screen cell which absent on donor cell.

Patient has a weak antibody reacting with homozygous screen cell, donor is either heterozygous or negative for antigen.

Perform antibody identification (panel study) and test donors for antigen.

Crossmatch units negative for antigen.

Positive screen, Compatible XMThe following table would indicate the

presence of an alloantibody directed against screen 1 which is not present in donor 1 OR

Antibody reacting with homozygous screen cell, donor 1 is heterozygous or negatve.

IS 37C AHG CHECK INTERP

Screen 1 0 0 2+ Positive

Screen 2 0 0 0 2+

Screen 3 0 0 0 2+

Donor 1 0 0 0 2+ Compatible

Negative Screen, Incompatible XMMOST COMMON CAUSE IS DONOR UNIT

HAS POSITIVE DAT.Perform DAT on DONOR UNIT.If positive return to blood provider

RARELY, patient may have antibody to low incidence antigen present on donor cells but absent from screen cells.Perform DAT on donor unit.If negative perform antibody ID

Repeat ABO grouping on DONOR sample.

Negative screen, Incompatible XMThe following table would indicate the

presence of an alloantibody directed against donor 1 OR donor has positive DAT.

Perform DAT FIRST, if negative, panel study.

IS 37C AHG CHECK INTERP

Screen 1 0 0 0 Negative

Screen 2 0 0 0 2+

Screen 3 0 0 0 2+

Donor 1 0 0 2+ Incompatible

Negative Screen, Incompatible XMIf at RT only this may be due to:

Donor RBCs are incompatible with patient, retype donor.Anti-A1 in serum of A2 or A2B patients.Other alloantibodies reactive at RT, perform antibody ID

Crossmatch incompatible at AHG phase ONLY.Donor RBCs have positive DAT.Antibody reactive only with cells homozygous for a

particular antigen, screen cells may not have homozygous cell – perform antibody identification (panel) study.

Antibody reactive with low frequency antigen present on donor RBCs, absent on screen cells – perform panel study.

Labeling and Release of BloodA tag or label must be attached to blood

container indicating Recipient’s two independent identifiersDonor unit numberInterpretation of compatibility testing

Two independent identifiers, of which is the patient’s name.Recipient’s ABO/D types.Donor unit or pool ID number.Donors ABO group and, if required, D type.Interpretation of the crossmatch if performed.The date and time of issue.Special transfusion requirements (CMV reduced risk,

irradiated or antigen negative.

Labeling and Release of BloodPrior to issuing a unit of blood, blood bank

personnel must:Check the expiration date of the blood to

avoid issuing an outdated component. Inspect the unit for abnormal appearance.

Document:Name of the individual issuing the blood.Date and time of issue.Name of person to whom blood was issued or

destination.

Labeling and Release of BloodA process must exist to confirm that all of

the following are in agreement:Identifying informationRequestRecordsBlood or components

All discrepancies must be resolved before issue.

Labeling and Release of BloodFinal identification of the recipient prior

to transfusion rests with the transfusionist, who must identify the patient and donor unit and certify that identifying forms, tags and labels are in agreement.

Release of Blood in Urgent SituationsDesperate need for blood physician must

weigh hazard of transfusing uncrossmatched blood against risk of waiting.

If blood is released before crossmatch is completed must contain statement, usually a form, signed by physician indicating reason.

Does not absolve transfusion service from responsibility to issue properly labeled donor blood of an ABO compatible group.

Compatibility testing must be started as soon as sample reaches transfusion service.

Release of Blood in Urgent SituationsIMMEDIATELY begin compatibility testing.

If possible give ABO/D specific, only takes a few minutes to accurately type an individual.

CANNOT rely on previous records.If ABO/D type unknown release O negative

RBCs.MUST indicate in CONSPICOUS fashion that

blood is UNCROSSMATCHED.

Release of Blood in Urgent SituationsBegin compatibility testing PROMPTLY, i.e.,

STAT.If incompatibility is detected at ANY stage

of testing notify patient’s physician and transfusion service physician IMMEDIATELY.

Massive TransfusionSeveral different definitions:

Infusion of 8 to 10 within 24 hours of a blood volume exceeding the recipients total blood volume OR

Transfusion of 4 to 5 RBCs units in 1 hour.Exchange transfusion.

May occur due toUnexpected surgical or medical emergencies.Planned circumstances in cardiac or vascular

surgery.Exchange transfusion in and infant or adult.

Massive TransfusionSuch a small volume of patient’s blood

remains that complete crossmatching has limited benefit.Pretransfusion sample no longer represents

currently circulating blood.Only important to confirm ABO compatibility of

donors.IS crossmatchComputer crossmatch

If unexpected alloantibody present abbreviating crossmatch acceptable so long as units are antigen negative and ABO compatible.

Massive Transfusion ProtocolStudies have been done to document that in

addition to RBCs transfusion of other components during this time are equally critical.

Must take into consideration “dilutional coagulopathy.

Giving RBCs plus IV solutions will NOT replace clotting factors or platelets.

Trauma protocol for massively bleeding patients may involve: 4 units group O negative RBCs, 4 AB FFP and 1 AB

platelet.Repeat until bleeding is controlled.

Transfusion After Non-Group Specific TransfusionSwitch ASAP to minimize transfusion of ABO

antibodies.Sample should be provided when uncrossmatched

blood picked up.Type STAT and provide future ABO identical.

Massive transfusion with Group O may cause passively acquired anti-A or anti-B to cause problems which require transfusing blood that lacks the ABO antibodies.

DD positive switch immediately.D negative continue if at all possible.If any question as to true D type, transfuse D negative.

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