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Technical Information for Further Testing

  1. If all cells in the first panel are reactive and the autocontrol is negative, try chemically treated screen cells and see if this alters the reactivity in any way. Chemical treatment of red cells includes: enzymes, DTT, AET or CDP (chloroquine diphosphate). Methods and information on obtaining any of the products required, can be found in the American Red Cross Immunohematology Methods and Procedures, D. Mallory, ed. Selecting 2 or 3 cells from the panel, or using the screen cells, will save time, save plasma and still provide new information. If the information is that nothing changed, go to step 2.

    [More information on the actions of specific chemicals is listed in step 2, (iii).]

  2. If all cells in the first panel tested are reactive and the auto control is negative, perform a common phenotype on the patient's red cells (patient must not have been transfused in the preceding 90 days). Blood group systems to include Rh, Kell, Duffy, Kidd and MNS. Select a cell from any of the panels or screen cells in your inventory that is negative for the same antigens the patient lacks and test it. This is referred to as running a phenotype matched cell.

    EXAMPLE: PATIENT D+C+E-c+e+, K-, Fy (a+b-), Jk(a-b+), M+N+S-s+.

    POSSIBLE MATCHES: D-C-E-c+e+, K-, Fy(a-b-), Jk(a-b+), M-N+S-s+

    D+C+E-c-e+, K-, Fy(a+b-), Jk(a-b+), M+N-S-s

    1. If the phenotype matched cell does not react with the patient's plasma, select a panel of red cells based on one example of each antigen to be excluded per cell.

      EXAMPLE: For the patient listed above the selection criteria would be:

      Cell Antigens
      1 E+, K-, Fy(b-), Jk(a-), S-
      2 E-, K+, Fy(b-), Jk(a-), S-
      3 E-, K-, Fy(b+), Jk(a-), S-
      4 E-, K-, Fy(b-), Jk(a+), S-
      5 E-, K-, Fy(b-), Jk(a-), S+

      This will confirm or exclude which antibody(ies) to the common antigens the patient has formed and what is left to form in the future.

    2. If the phenotypically matched cell(s) is reactive with the patient's plasma and the autocontrol is negative, the most probable cause is an antibody to a high frequency antigen.

      Use Antigen Plus to select any examples of high frequency antigen-negative red cells in your cell library. I.e… At(a-), k-, Kp(b-), Js(b-) etc.

    3. If you have a limited rare cell library or failed to obtain a negative reaction with the high frequency antigen-negative red cells available, consider that various red cell treatments which modify or destroy certain antigens on red cells can also be useful. DTT/AET treatment will destroy: Kell (not Kx), Lutheran, Knopps/McCoy, Cartwright, Cromer, Dombrock, Auberger, JMH and Lwa. Gerbich is destroyed by DTT but not AET. ENZYMES (papain, ficin, trypsin, bromelin) will remove: Duffy, MNS, Chido, Rogers, JMH, Gerbich, Gilfeather, Xga, Pr, Vw, Cartwright, Mia, Cla, Jea, Nya and most Ena. Chloroquine diphosphate (CDP) will destroy Bg and class I HLA antigens.

  3. If all cells in the first panel tested are reactive and the auto control is positive, do a direct antiglobulin test (DAT) on the patient's cells before doing further serum testing.

    1. If the DAT is positive, a warm autoantibody should be suspected and may hide underlying alloantibodies in the patient's plasma. The autoantibody should be removed by adsorption before further antibody identification can be achieved. Adsorption can only be performed using pre-transfusion patient red cells. The patient must not have been pregnant or transfused in the preceding 90 days. For further information and testing methods, refer to the AABB Technical Manual, Current Edition, or the American Red Cross Immunohematology Methods And Procedures, D. Mallory, ed.

    2. If the DAT is negative, a cold autoantibody should be considered. A cold screen tested from room temperature down to 4o C will indicate if an autologous absorption done at 4o C will be useful to remove the autoantibody. Again, the patient must be untransfused in the preceding 90 days.

    3. If adsorption, warm or cold, is not possible, there are alternatives. Allogeneic warm absorption is possible using R1R1, R2R2, and rr cells. A prewarm method may be useful to remove any cold reactivity. Prewarm methods must be used with caution preferably after the clinically significant antibodies have been tentatively identified. Prewarm methods have been documented to remove anti-H activity in a Bombay phenotype and an example of anti-Vel. References on request.

  4. If all the cells tested are negative, several things must be considered:

    1. Repeat the cell that gave the original positive reaction that initiated the panel, i.e. the antibody screen test or one of the units being crossmatched. Note where the antibody reacts - immediate spin or IAT? Was the panel tested at the same phase the reaction occurred in?

    2. How soon after the original testing, was the panel performed? Was the sample stored properly?

    3. Is the reactive cell double dose (from an apparent homozygous donor) for any common antigen and is there another cell double dose (from an apparent homozygous donor) for the same antigen that can be used to repeat the result?

    4. Will enhancement media help reveal a weak reacting alloantibody pattern, i.e.

      Enz, PEG, LISS with a longer incubation time, or polybrene?

    5. If none of this adds any information about antibody specificity, you may have an antibody directed against a low frequency antigen. While the identity of the antibody is a puzzle, it is rarely a significant problem when looking for compatible units of red cells for transfusion. If you have time to play with it, use Antigen Plus to locate as many low incidence antigen positive cells as you have listed under special antigens. If none of them react, and you still wish to resolve the specificity, send the sample to a reference lab for crosstesting. Most reference labs keep their unidentified lows in hopes of finding another example.

  5. If your routine test method can detect weak reacting autoantibodies at any phase of testing, positive antibody screen results can lead to a common panel result of several weak reactions with no specificity determined. Antigen Plus will perform exclusions based on its policy and may well exclude all common specificities depending on the number of reactions and phenotype of the cells which do not react. The technologist should follow his or her Blood Bank SOP for antibody identification which should address this type of panel testing outcome. Depending on method used, patient population and number of occurrences, the most common choices are likely to include:

    1. Try more sensitive method if an alloantibody may be just forming or was not stimulated in the recent past.

    2. Repeat the original test by a less sensitive method, e.g. from solid phase testing back to tube LISS-IAT (a common approach to dealing with the very sensitive capabilities of solid phase testing.)

    3. Seen only at immediate spin – assess possible clinical significance and drop the IS test if appropriate.

      Use the prewarm method only after significant antibodies (anti-Vel, anti-PP1Pk, anti-H in a Bombay) have been excluded.