Antibody Detection

1. Antibody Detection Mohammed Jaber

2. Antibody Detection • The test used to detect antibodies is called an antibody screen • Antibody screens are used for: • Patients needing a transfusion • Pregnant women • Cases of transfusion reactions • Blood and plasma donors

3. Antibody Screen • Uses patients plasma/serum against reagent red cells to detect unexpected antibodies • Unexpected antibodies are found in addition to the expected anti-A and/or anti-B • Unexpected antibodies are a result of red cell stimulation (transfusion, HDN) • Unexpected antibodies may be: • Clinically significant (IgG) • Not clinically significant (IgM)

4. Clinically significant antibodies • Usually IgG • React best at 37° and AHG phase (IAT) • Clinically significant antibodies are associated with hemolytic transfusion reactions (HTR) and hemolytic disease of the newborn (HDN)

5. Performing an antibody screen • Patients plasma/serum is incubated with screening cells • After incubation, an IAT is performed (indirect antiglobulin test) using AHG reagent • This will detect any IgG antibodies

6. Screening Cells • Screening cells are single or pooled donor group O cells, however, single-donor vials offer increased sensitivity • Why group O? so anti-A and anti-B won’t react • Screening cells come in sets of 2 or 3 vials each • Each vial (donor) has been phenotyped for each antigen • 18 antigens are required on at least one of the vials: D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Jka, Jkb, Fya, Fyb

7. Screening Cells

8. Screening cells • Screening cells come with a sheet of paper called an antigram • Screening cells are an already prepared 2-5% RBC suspension • An antigram (2 or 3 cells) will list the antigens present in each vial • A reaction to one or more cells indicates the presence of an unexpected antibody

9. 2-Cell Antigram Screening Cells I & II AHG IS 37° CC Some manufacturers will use “+” instead of “1” to indicate the presence of the antigen

10. Screening cells • The technologist should be aware that some antigens demonstrate dosage • An attempt should be made to used screening cells that are homozygous for the clinically significant antigens (Rh, Duffy, Kidd). Just be aware that different strengths can occur • Homozygous antigens will react stronger • Heterozygous antigens will react weaker

11. Examples If patient’s serum contains anti-Fya, there will be a stronger reaction because SCI is homozygous for the Duffy antigen In this case, the person has anti-Fyb. The antibody reacts weaker with SCI (heterozygous) and stronger with SCII (homozygous)

12. Screening Cells • Screening cells may also contain low-incidence antigens like V, Cw, and Kpa • The presence of these antigens is not required for screening cells

13. Pretransfusion Screening • Screening for antibodies is normally performed prior to blood transfusion to detect antibodies that react at body temperature (37°) • Colder reacting antibodies (RT and below) are therefore considered insignificant and just cause interference when performing lab testing • The only important thing to remember concerning cold antibodies is that they may bind complement if a persons body temperature becomes low • Open-heart surgery • hypothermia

14. Autocontrol • Tests patient serum with their own red cells • Some labs may or may not perform an autocontrol (AC) with the screen…depends on the hospital • However, the AC should be run with the antibody panel…we’ll discuss this later • AC is incubated with the antibody screen (or antibody panel) • If a lab uses an AC with the screen and it is positive, they may run a DAT (patient cells + AHG) to detect in vivo coating

15. Autocontrol • The AC and DAT can help in determining whether the antibodies are directed against the patient’s cells or transfused cells (allo-or autoantibody) • In this hospital: Screen If positive Antibody Panel (w/AC) If positive DAT

16. Potentiators • Used in antibody detection and identification to enhance antigen-antibody reaction • Saline (may only enhance if incubated long time) • Low-ionic strength solution (LISS)…common • Bovine serum albumin (BSA) • Polyethylene glycol (PEG) • Proteolytic enzymes (can destroy some antigens)

17. Potentiators

18. 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.

19. 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.

20. 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.

21. 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.

22. 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

23. Procedure • Antibody screening tests using a test tube method are performed in a variety of ways. American Association of Blood Banks Standards requires that these tests detect clinically significant antibodies and that they include a 37°C incubation and an AHG test. Generally, testing includes the following steps: • Appropriately label each tube. • Add 2 drops of patient serum to each tube. • Add 1 drop of appropriate screening cells to each tube. • Centrifuge, then gently resuspend the cell button and read for agglutination or hemolysis. Record results. It should be noted that this step is optional because most significant antibodies are IgG and do not cause agglutination of saline-suspended RBCs. • Add 2 drops of enhancement reagent to each tube (may vary with enhancement reagent used). • Incubate at 37°C for 15 to 30 minutes, according to the manufacturer's recommendation for the

24. Procedure • Incubate at 37°C for 15 to 30 minutes, according to the manufacturer's recommendation for the enhancement reagent being used. During the incubation, antibody in the patient serum will bind to antigens on the reagent RBC. This is called the sensitization phase. • Centrifuge, then gently resuspend the cell button and read for agglutination or hemolysis. Record results. • Fill all tubes with saline, centrifuge, and discard supernatant. This is called washing, and it removes unbound IgG that neutralizes the AHG reagent. • Repeat step 8 two or three times to remove unbound antibody completely. • Add 2 drops of AHG to each tube (polyspecific or anti-IgG).

25. Procedure • Centrifuge, then gently resuspend the cell button and read for agglutination or hemolysis. Tests that are macroscopically negative are usually checked for microscopic agglutination. Record results. • Add 1 drop of Coombs control cells (or "check cells") to all negative tests. • Centrifuge and read for agglutination. Repeat test if agglutination is not observed.

26. Grading Reactions

27. Interpretation • Agglutination or hemolysis at any stage of testing is a positive test result, indicating the need for antibody identification studies. However, evaluation of the antibody screen and autologous control results can provide clues and give direction for the identification and resolution of the antibody or antibodies. • The investigator should consider the following questions: • In what phase(s) did the reaction(s) occur? • Is the autologous control negative or positive? • Did more than one screening cell sample react, and, if so, did they react at the same strength and phase? • Is hemolysis or mixed-field agglutination present? • Are the cells truly agglutinated, or is rouleaux present?

28. Interpretation • In what phase(s) did the reaction(s) occur? Antibodies of the IgM class react best at low temperatures and are capable of causing agglutination of saline-suspended RBCs (immediate spin reading). Antibodies of the IgG class react best at the AHG phase. Of the commonly encountered antibodies, • anti-N. anti-I, and anti-PI are frequently IgM, • whereas those directed against Rh. Kell. Kidd, and Duffy antigens are usually IgG. • Lewis and M antibodies may be IgG, IgM, or a mixture of both.

29. Interpretation • Is the autologous control negative or positive? A positive antibody screen and a negative autologous control indicate that an alloantibody has been de­tected. A positive autologous control may indicate the presence of autoantibodies or antibodies to medica­tions. If the patient has been recently transfused, the positive autologous control may be caused by alloanti­body coating circulating donor RBCs. Evaluation of samples with positive autologous control or DAT re­sults is often complex and may require a lot of time and experience on the part of the investigator.

30. Interpretation • Did more than one screening cell sample react, and, if so, did they react at the same strength and phase? More than one screening cell sample is positive when the patient has multiple antibodies, when the antibodies' corresponding antigen is found on more than one screening cell, or when the patient's serum contains an autoantibody. A single antibody specificity should be suspected when all cells react at the same phase and strength. Multiple antibodies are most likely when cells react at different phases and strengths, and autoantibodies are suspected when the autologous control is positive. Figure provides several examples of antibody screen results with possible causes.

31. Interpretation • Is hemolysis or mixed-field agglutination present? Certain antibodies-such as anti-Lea, anti-Leb, anti P+P1+Pk, and anti-Vel-areknown to cause in vitro hemolysis. Mixed-field agglutination is associated with anti-Sda and Lutheran antibodies.

32. Interpretation • Are the cells truly agglutinated, or is rouleaux present? Serum from patients with altered albumin-to-globulin ratios (e.g., patients with multiple myeloma) or who have received high-molecular-weight plasma expanders (e.g.. dextran) may cause nonspecific aggregation of RBCs, known as rouleaux. • Rouleaux is not a significant finding in antibody screening tests, but it is easily confused with antibody-mediated agglutination.

33. Interpretation • Knowledge of the following characteristics of rouleaux helps in differentiation between rouleaux and agglutination: • Cells have a "stacked coin" appearance when viewed microscopically (see Color Plate ). • Rouleaux is observed in all tests containing the patient's serum, including the autologous control and the reverse ABO typing. • Rouleaux does not interfere with the AHG phase of testing because the patient's serum is washed away prior to the addition of the AHG reagent. • Unlike agglutination, rouleaux is dispersed by the addition of 1 to 3 drops of saline to the test tube.

35. Limitations • Very effective in detecting antibodies • If negative, then the crossmatch should be compatible

36. Limitations • Antibody screening tests are designed to detect significant RBC antibodies, but they cannot detect all such antibodies. Antigens with frequencies of less than 10 percent (e.g., Cw. Lu-, Kpa) are not usually represented on screening cells, and, as a result, their corresponding antibodies are not detected in routine screening tests. • Antibody screening tests may also yield negative results when the titer or concentration of antibody drops below detectable limits. • antibody levels decrease over time when the individual is no longer exposed to the corresponding antigen. If the level of an RBC antibody drops too low, results of antibody screening tests and crossmatches will appear negative and may lead to transfusion of donor units that carry the corresponding antigen.

37. Limitations • Re exposure to the RBC antigen will elicit a secondary immune response, resulting in a dramatic increase in the antibody titer and possible immunologic destruction of the transfused RBCs. this is called a delayed hemolytic transfusion reaction (DHTR) because it occurs days or weeks after the transfusion. • The student should keep in mind that proper performance and interpretation of antibody detection tests minimize the risk of DHTRs,

38. Patient History • GET THE HISTORY!! • Mixed red cell populations from a previous transfusion can remain for up to 3 months • Patient may have come from another hospital • Some diseases are associated with antibodies • Some antibodies occur at a higher frequency in some races عرق • Get diagnosis, age, race, etc…

39. Example 1 Not Done • IgG antibody • Single specificity *Coombs Control Red Blood Cells (CC)

40. Example 2 • IgG antibody • Multiple specificities

41. Example 3 Neg AHG, add CC • IgM antibody • Single specificity showing dosage

42. Example 2 • IgG antibody • Allo- or autoantibody? (don’t know without further testing)