Unit 8 Pretransfusion Testing Part 3

1. Unit 8 Pretransfusion TestingPart 3 Terry Kotrla, MS, MT(ASCP)BB

2. Testing Techniques - Gel • Based 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.

3. Testing Techniques - Gel • Nonagglutinated 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

5. 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.

6. Testing Techniques - Gel • Microliters 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.

7. Microplate Testing • For routine processing of DONOR samples. • Microtiter plate with 96 wells. • Adapted to RBC or serum testing. • Add appropriate sample, centrifuge and dislodge button.

8. Solid Phase Adherence • RBC 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. • Incubate • Wash • Antibody, if present, will attach. • Add IgG coated coated RBCs • Centrifuge, agglutinates will coat well.

9. Solid Phase Adherence • Slide 1 – antibody screen • Slide 2 – antibody detection top, ABO grouping bottom

10. Solid Phase Adherence

11. Automated Testing Platforms • Perform multiple tests using • Solid phase • Column agglutination • Microtiter plate • Other technologies • All process from pipetting to result interpretation performed under computerized control. • Usually interfaces with laboratory information system (LIS). • Examples: Galileo Echo, Biotest Tango

12. Antiglobulin Technique • REQUIRED 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

13. Crossmatch Interpretation

14. Negative screen, Compatible XM • Vast 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.

15. Negative screen, Compatible XM

16. Positive screen, Incompatible XM • May be due to alloantibodies or autoantibodies • If ALL tubes positive could be problems with reagents or, at IS or 37C, rouleaux • MUST 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.

17. Positive screen, Incompatible XM • The following table would indicate the presence of an alloantibody directed against screen 1 and donor 1.

18. Positive screen, Incompatible XM • Positive 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.

19. Positive screen, Incompatible XM • If 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.

20. Positive screen, Incompatible XM • Potent 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.

21. Positive screen, Incompatible XM • Rouleaux 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.

22. Positive screen, Incompatible XM • Reagent 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.

23. Positive Screen, Compatible XM • Patient 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.

24. Positive screen, Compatible XM • The 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.

25. Negative Screen, Incompatible XM • MOST 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.

26. Negative screen, Incompatible XM • The 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.

27. Negative Screen, Incompatible XM • If 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.

28. Labeling and Release of Blood • A tag or label must be attached to blood container indicating • Recipient’s two independent identifiers • Donor unit number • Interpretation 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.

29. Labeling and Release of Blood • Prior 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.

30. Labeling and Release of Blood • A process must exist to confirm that all of the following are in agreement: • Identifying information • Request • Records • Blood or components • All discrepancies must be resolved before issue.

31. Labeling and Release of Blood • Final 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.

32. Release of Blood in Urgent Situations • Desperate 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.

33. Release of Blood in Urgent Situations • IMMEDIATELY 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.

34. Release of Blood in Urgent Situations • Begin compatibility testing PROMPTLY, i.e., STAT. • If incompatibility is detected at ANY stage of testing notify patient’s physician and transfusion service physician IMMEDIATELY.

35. Massive Transfusion • Several 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 to • Unexpected surgical or medical emergencies. • Planned circumstances in cardiac or vascular surgery. • Exchange transfusion in and infant or adult.

36. Massive Transfusion • Such 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 crossmatch • Computer crossmatch • If unexpected alloantibody present abbreviating crossmatch acceptable so long as units are antigen negative and ABO compatible.

37. Massive Transfusion Protocol • Studies 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.

38. Transfusion After Non-Group Specific Transfusion • Switch 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. • D • D positive switch immediately. • D negative continue if at all possible. • If any question as to true D type, transfuse D negative.

39. 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