ABTIBODY SCREENING Dr. Mohammed H Saiemaldahr BLOOD BANK MED TECH

Antibody Screening Antibody Detection and Identification Purpose  The purpose of the antibody screen is to detect red blood cell antibodies other than anti-A or anti-B.  These antibodies are called “unexpected” because only 0.3 to 2 % of the general population have positive antibody screen.  Once an unexpected antibody is detected, antibody identification studies are performed to determine the antibodies specificity and clinical significance.

Antibody Screening All red blood cell antibodies are significance if they cause shortened survival of antigen positive red blood cells. For example, anti-D is a clinically significant antibody, because it will bind to D-positive red blood cells, resulting in immune destruction or hemolysis. Proper detection and identification of red blood cell antibodies is important for the selection of appropriate blood for transfusion and in the investigation of hemolytic disease of the new born, and immune hemolytic anemia.

Antibody Screening  Antibody screening test involve testing patient’s serum against two or three reagent red blood cell samples called screening cells  Screening cells are commercially prepared group O cell suspensions obtained from individual donors that are phenotype for the most commonly encountered and clinically important red blood cell antigens.

Antibody Screening  Group O cells are used so that naturally occurring anti-A or anti-B will not interfere with detection of unexpected antibodies.  The cells are selected so that the following antigens are present on at least one of the cell sample; D, C, E, c, e, M N, S, s, P, Le a, Le b, K, k, Fy a, Fy b, and Jk b.

Antibody Screening  An anti-gram listing the antigen makeup of each cell provided with each lot of screening cells issued from a manufacture. It is important that the lot number on the screening cells matches the lot number printed-On the anti-gram because antigen make up will vary with each lot.  The “ideal” screening cells have red cells with homozygous expression of as many antigens as possible.

Antibody Screening  There is no requirement that screening cells contain red cells with homozygous expression of antigens, however, the most workers prefer that such red cells are included in screening cells sets because many antibodies, especially JK and M antibodies, show dosage effect and give stronger reactions when tested against cells with homozygous expression of their corresponding antigen.

Antibody Screening  As a result of dosage, weakly reacting antibody may not be detected if serum samples are not tested against red cells with homozygous expression of the their corresponding antigen.

Antibody Screening Screening Cells.  The screening cells are available in three form 1- a single vial of no more than two donors pooled together in one vial. 2- two vials each with a different donor vials representing three different donors. Two or three cells screening sets are required for detection of antibodies in pre-transfusion testing.

Antibody Screening Detection of very low levels of antibody in a recipient’s serum is important because transfusion of antigen-positive red cells may result in a secondary immune response with rapid production of antibody and subsequent destruction of transfused red blood cells.

Antibody Screening Red blood cells should be re-suspended by gentle shaking or tilting the tube until the cells no longer adhere to the sides. Agglutination is graded once the red blood cells are re-suspended. Agglutination reactions are routinely graded as negative (no agglutination). Agglutination reactions are routinely graded as negative (no agglutination). Weakly positive, and 1+ through 4+. The degree of the positive reaction generally indicates the amount of Ab present not its significance.

Antibody Screening Auto-logous Control.  Autologous control is considered as part of the Ab screening, it can be performed in parallel with the Ab screen and involves testing the patient’s serum against the patient’s red blood cells.  A positive auto-logous control is an abnormal finding and usually means that patient has a positive direct antiglobulin test (DAT).

Antibody Screening Grading Reactions.  Aggregation or hemolysis of test red blood cells is the visible end point of an Ab-Ag interaction.  Test results should be read immediately after centrifugation as delays in reading may cause elution of antibody and false- negative test results  The first step in reading hem-agglutination reactions is inspection of the supernatant for signs of hemolysis (red or pink coloration).

Antibody Screening Interpretation. Evaluation of the antibody screening and auto- logous control results can provide clues and give direction for the identification and resolution of the Ab or Abs. The investigator should consider the following questions;

Interpretation  1- In what phase (s) did the reaction (s) occur?  Low temperature: Abs of the IgM class will react best at low temperatures and are capable of causing agglutination of saline-suspended red blood cells (immediate spin reading (IS). Of the commonly encountered Abs are, anti-N, -I. and -P (IgM)  Abs of the IgG class will react best at the AHG Phase (37°C). Of the commonly encountered Abs are; anti-Rh, kell, Kidd, and Duffy (IgG).  Where as Lewis, M, Abs may be IgG, IgM, or a mixture of both.

Interpretation  2- Is the auto-logous control negative or positive?  A positive Ab screen and a negative auto-logous control indicate an allo-antibodv has been detected.  A positive auto-logus control may indicate the presence of auto- antibodies, antibody to medication or it may idiopathic  If the patient has been recently transfused, the positive auto-logous control may be due to alloantibody coating circulating donor red blood cells.

Interpretation  3- Did more than one screening cell react and if so, did they react at the same strength and phases?  If the patient has multiple Abs, when the Abs corresponding Ag is found on more than one screening cell, or when the patient’s serum contains an autoantibody, more than one screening cell will be positive.  A single Ab specificity should be suspected when all cells react at the same phase and strength.  Multiple Abs are most likely when cells react at different phases and strengths and auto-antibodies are suspected when the auto-logous control is positive.

Interpretation  4-Are the cells truly agglutinated or rouleaux present?  Serum from patients with multiple myeloma or who have received high molecular weight plasma expanders (dextran) may cause non-specific aggregation of red blood cells known as rouleaux.  Rouleaux is not a significant finding in Ab screening tests but it is easily confused with Ab mediated agglutination. To differentiate between rouleaux and agglutination;

Interpretation  1- Rouleaux cells have “a stacked coin” appearance when viewed microscopically.  2- It will not interfere with the AHG phase of testing because the patient’s serum is washed away prior to the addition of the AHG reagent.  3- It is observed in Ab tests containing the patient’s serum including the auto-logous control and the reverse ABO typing.  4- Rouleaux is dispersed by the addition of 1 to 3 drops of saline to the test tube.

Reagent Red Blood Cell Screening Cell Sectional Listing of Antigens Present CCAHG 3737 ISIS P1P1 NMsS LebLeb LeaLea JkbJkb JkaJka FybFyb FyaFya kKecECD No cell I II