MLS 4240 AB ID Panals PDF

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Immunohematology Lab Manual Page 10...

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ANTIBODY IDENTIFICATION USING RED BLOOD CELL PANELS

Summary and Explanation When an unexpected antibody has been detected in serum it must be identified to determine its clinical significance. Blood group antibodies are not all equally dangerous in transfusion therapy or in pregnancy. Once the identity of the antibody (or antibodies) has been established, standard texts may be consulted for guidance in specific situations. Antibody identification is accomplished by testing the serum against a set of group O red blood cells typed for known blood group antigens, observing the presence or absence of hemolysis or agglutination, and comparing the pattern of reactivity with the antigen profile of the cells. If the reaction pattern corresponds to an antigen profile, identification of the unknown antibody is presumed. Identification of antibodies is largely dependent upon the methods, media and temperature used. Principle of the Procedure The serum under test is combined with the panel cells. Several techniques may be required to detect unexpected blood group antibodies. For this laboratory these include the use of room temperature, 37o C, LISS/PEG/ALB, and anti-human globulin. The presence of agglutination and/or hemolysis in any test phase indicates a positive reaction and the presence of unexpected antibody(ies). A negative result (no agglutination and/or hemolysis) indicates the absence of detectable unexpected antibody(ies) capable of reacting with antigens present on the red cells provided. By comparing the agglutination patterns obtained with the antigen profiles of the panel cells printed on the worksheet, identity of the unknown antibody(ies) may be determined. .

Rule Out Procedure for Interpretation 1. Look at the phase of reactivity. RT or IS reactions are due to IgM antibodies. 370 C reactions may be either IgM carryover or IgG. AHG reactions are IgG antibodies (or may also detect complement bound to RBCs if polyspecific AHG is used). 2. Look at reaction strength. Do all positive cells react equally? Dosage may cause stronger reactions with homozygous cells, or there may be more than one antibody present with each reacting antibody reacting differently. 3. “Rule out” antibodies that are definitely not present. 4. Beginning with the first test cell having all negative reactions on the worksheet, cross out the antigens present on that cell. Cross out cells at this step in accordance with the following criteria. a. The following antibodies are ruled out without regard to the zygosity of the rule out cells on the screen or panel: D, K, P1, Lua, Lub, Kpa, Kpb

b. The following antibodies are ruled out using homozygous rule out cells only: C, E, c, e, M, N, S, s, Le a, Leb, k, Fya, Fyb, Jka, Jkb. You cross out homozygous cells at this time in case your antibody is demonstrating dosage (i.e. only reacting with cells having a “double dose” of the antigen). 5. Repeat this procedure with each panel cell which did not react with the serum. 6. List the antigens that have not been crossed out. Compare the reaction pattern with the remaining antigen patterns printed on the worksheet. A reaction pattern matching an antigen pattern presumes the identification of the corresponding antibody. This is typically observed when there is a single antibody present. 7. From the list of antigens that cannot be ruled out, determine whether the corresponding antibodies are likely to be present in the serum or not. a. Generally low-incidence antigens are crossed out unless crossmatches with antigen negative units (for known antibodies present) are still incompatible. b. For our laboratory protocol and to solve the case studies, if the following antigens are not crossed out using the routine panel and screen cells, you may cross them out and do NOT need to select additional cells as outlined below: Cw, Lua, Lub, Kpa, Kpb, Jsa, and Js b. 8. Apply the Rule of Three. To obtain at least a 95% probability that the antibody or antibodies are indeed causing the reactions observed, there must be at least 3 antigen positive cells that react and at least 3 antigen negative cells that do not react. If your panel sheet does not have at least 3 antigen (+) cells and 3 antigen (-) cells then additional cells from another panel (or screening cells) need to be selected for further testing to achieve the 95% confidence that your results are valid. 9. Selected cells (rule out cells) are tested to obtain 95% probability and to determine if multiple antibodies are present in the serum. Choose panel cells that are negative for the antigen corresponding to the antibody most likely present in the patient’s serum and positive (preferably double dose) for antigens corresponding to antibodies that have not been ruled out. 10. Phenotype the patient. Except in cases of autoantibodies, patients do not make antibodies to antigens that they possess. If the patient is antigen positive, you can conclude that the corresponding antibody is not present. Therefore, the patient’s RBCs should be phenotyped to ensure they lack the antigen corresponding to the antibody(ies) identified. Phenotyping should only be done if the patient has not received a transfusion within the last 3 months. Transfused RBCs may cause erroneous phenotyping results since any specimen drawn after a transfusion would contain both the donor unit(s) RBCs as well as the patient’s RBCs. 11. Agglutination in the auto-control tube may indicate an autoagglutinin. If the AC and all panel cells are (+) in the IS or RT phases of testing, a cold-reactive autoantibody should be suspected. Anti-I, the most common autoagglutinin, requires cord cells or adult I negative cells for identification. Anti-H is also a common cold autoagglutinin. Rouleaux may cause a similar pattern of reactivity. But, is easily eliminated by saline addition/replacement methods. If the AC and all panel cells are (+) at the AHG phase, a warm-reactive autoantibody should be suspected. 12. If the AC is (-) and all panel cells are (+) with the same strength, consider the possibility of an antibody to a high incidence antigen or multiple alloantibodies of

similar strength. Retesting with panel cells pretreated with enzymes (which destroy certain antigens) may help differentiate these and offer clues to antibody specificity. Phenotyping the patient’s RBCs may also offer clues in this situation. 13. When the results with the panel cells do not permit clear identification, consider: a. More than one Ab may be present. Differences in reactivity among the panel cells under different test conditions (i.e. temperature, suspending medium, etc) may indicate the identify of one or more antibodies present. b. Type the patient’s cells to determine their antigenic composition. This is not effective if the patient has been recently transfused. c. The antibody may react in an unexpected manner (i.e. a typically coldreactive alloantibody such as an anti-M may be showing reactivity at 370 C and AHG) d. Weak antibodies may be reacting only with red cells that exhibit a double dose of the antigen (e.g. anti-M, anti-c, anti-Jka) or only with those cells possessing the strongest amount of antigen (e.g. anti-P1). e. IMPORTANT!! If the identified antibody(ies) are clinically significant, phenotype the potential donor units for the antigen corresponding to the patient’s antibody, using commercially prepared antisera. A mathematical calculation may be done to determine how many units probably need to be screened in order to find the number of units of blood ordered that would be compatible with the patient. Phenotype that number of units to find units that lack the antigen corresponding to the patient’s antibody. Limitations of Procedure 1.

Caution should be taken not to rely completely on reaction patterns to identify an antibody. An antibody may not react with all cells possessing the corresponding antigen (an antibody showing `dosage'). In some cases, a mixture of two or more antibodies may give a reaction pattern similar to a single antibody. Noting differences in strengths of reactions may aid in the identification of multiple antibodies.

2.

Additional testing to confirm antibody specificity should include: a) Testing of the patient cells for the antigen corresponding to the suspected antibody b) Use of specific cells from other panels which may allow differentiation of antibodies not accomplished with panel in use c) Use of additional techniques such as absorption and elution for separation of multiple antibodies.

3.

There are nearly 700 recognized antigens associated with red blood cells. No panel of reagent red cells will contain all the known antigens. Therefore, negative results only indicate the test serum lacks antibodies of a detectable level to those antigens present on that panel....