Detection and Identification of alloantibodies to Red Cell Antigens

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

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

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

Preparing Compatible blood for a recipient : Goals 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

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.

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

commercially available - Available as sets of two or three 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

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

- Use panel of selected red cell with known antigen composition 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

- 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

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

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

Enhancement Media & Enhancement 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)

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

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

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

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

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

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

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.