Detection and Identification of alloantibodies to Red Cell Antigens

1. Detection and Identification of alloantibodies to Red Cell Antigens

2. Unexpected Alloantibodies: Any Red Cell Alloantibodies other than naturally occurring anti-A or anti-B Detected by: Performing an antibody screen test Found in: 0.3-38% of the population - Depending on group of patient or Donor Studied - Sensitivity of the test methods used Reaction: Only with allogenic red cells VS. Auto- antibodies that reacts with red cells from other individuals

3. Immunization to red cell Ag: Results from pregnancy-Transfusion- Transplantation- Injection with immunogenic material-No specific- Immunizing event Initial detection of alloantibodies In tests that use serum of Plasma including: • ABO Test • Antibody Screen Test • Cross-match Test • Eluate

4. Once an antibody is detected: • Determine Specificity • Asses Clinical significance Clinically Significant antibodies defined as: • One that shortens the survival of transfused red cells • Cause Hemolytic disease of the newborn (HDN) • Hemolytic Transfusion reaction * Reported experience with other examples of antibody with the same specificity can be used in assessing clinical significance

5. NOTE:If no data exists on certain antibody ,decision must be based on the temp. that one antibody is active • 37C And Or • Reactive by the Indirect Antiglobulin Test (IAT) Preparing Compatible blood for a recipient : Goals • Detect as many clinically significant Antibodies • Detect as few clinically insignificant Antibodies • Complete the procedure in a timely manner • Patients with clinically significant antibodies should, receive red cells that are negative for the corresponding antigen. • In prenatal testing, the specificity and immunoglobulin class of an antibody influence the likelihood of HDN

6. Techniques: Techniques for Antibody detection Antibody identification are similar, but methods for: • Antibody detection is broad to detect antibodies with differing patterns of reactivity • Antibody Identification is more focused based on reactivity patterns identified in the antibody detection • Use of flowcharts as a guide to - expediting process - minimizing unnecessary tests.

7. Specimen Requirements: Either 1- Serum Or 2- Plasma 10-ml aliquot is enough for identifying simple antibody specificities Medical History: When performing an antibody identification it is useful to know: 1- Patient’s clinical diagnosis 2- History of Transfusion 3- History of pregnancies

8. 4- Recent drug therapy 5- Ethnic background 6- Sex M.F. 7- Age Reagents: Screening Cells: - Group O red cells commercially available - Available as sets of two or three vials of single-donor red cells - Pooled antibody screening cells are used for donor serum and not for recipients’ specimens

9. Reagents: - Reagent cells licensed by the Food and Drug administration (FDA) must Express the following antigens D,C,E, c, e, M,N,S, s, P1,Lea ,Leb,K,K, Fya, Fyb, Jka, Jkb -weakly reactive antibodies react only with screening red cells from donors who are homozygous , a serologic phenomenon called dosage effect . - When not in use reagent should be refrigerated at 2-8ºС

10. Red cell Panels: - Use panel of selected red cell with known antigen composition - Obtained commercially or institutionally (Horne made) from local - Group O cells except in special circumstances - Each cell from different individual - The pattern of reactivity should not overlap with any other, Example 0 Not all K+ should also be E+ - Expiration for commercially prepared cells is every 2-4 weeks

11. - Use only reagent phenotype listing sheet for the specific kit - Suspension of 2-5% Red Cells in a Preservative medium Saline – Suspended Red cells Technique: • Simplest serologic • Incubation of saline suspended cell with serum at at: • Immediate spin • Room Temperature • 37oC

12. Antibodies reacting below 37 oC are Anti-M, N, P1, Lea, Leb • This phase reading can be omitted to avoid finding little clinically significant antibodies. • 37 oC antibody detection anti – D, K, E also some • Some antibodies like anti- Lea, Jka can be detected in this phase by Lysing antigen- incompatible red cells

13. Antiglobulin Reagents • Antiglobulin phase to detect clinically significant antibodies • Use antiglobulin reagent as • Polyspecific to detect antibodies that bind complement kidd antibodies • IgG- specific

14. Enhancement Media & Enhancement Techniques • Techniques • Temperature reduction • Increased serum to cell ratio • Increased incubation time • Alteration of PH • Inhibition tests • Lewis substances (saliva) • P1 substance (pigeon egg whites) • Sola substance (body fluids-urine) • Pooled plasma (Chido & Rodgers)

15. Enhancement Media & Enhancement Techniques • Include incubating Serum/Plasma and reagent red cells in such media as: • Albumin 22% or 30% • LISS (low-ionic-strength saline) • PEG (polyethylene glycol) • Enzyme techniques • Polybrene

16. Autologous Control • To determine alloantibody from Autoantibody • Not performed in antibody screening • Used with panel cells

17. Basic Antibody Identification Techniques • Traditional serologic method in the united states are based agglutination performed in tubes or micro-plates • Other methods are not dependent on agglutination • Solid- Phase • Flocytometry • Agglutination test (gel tests) column techniques • Automated systems • Initial observation

18. Basic Antibody Identification Techniques • Interpreting results • Positive and Negative • Exclusion or “crossing out” • Antibodies to High- Incidence Antigens anti-H, I, P, LW , Sda, Vel , Ch, Rg, U, Jsb • Antibodies to low-Incidence Antigens anti-Wra

19. Selecting Blood for Transfusion For a patient with clinically significant Antibodies (37oC and IAT) • Red cell should be tested and be negative for the appropriate antigen • Even if Ab. Is no longer detectable to prevent a secondary immune response • An antiglobulin cross-match is required • The absence of Ag should be confirmed with a potent commercial antisera • FDA requires use of licensed (commercial) reagents

20. Selecting Blood for Transfusion • When rare type is needed • High- Incidence • Multiple antibodies frequency of random donors negative for each antigen should be used, Example: serum contains anti-C, Fya and s among random donors 18% C Neg 34% Fya Neg 45% S Neg

21. Selecting Blood for Transfusion The Frequency of compatible units would be 0.18×0.34 ×0.45= 0.028 If patient is group O then 45% of random donors are group O then 0.028 ×0.45=1.3% of random donors would be compatible with the patient serum.