Unit 8 Pretransfusion Testing Part 2

1. Unit 8 Pretransfusion TestingPart 2 Terry Kotrla, MS, MT(ASCP)BB

2. 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

3. 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.

4. 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.

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 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.

11. Selection of Donor Group

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 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.

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. • 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.

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 manufacturer’s 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