Loss of enzyme-sensitive antigens due to the presence of leukocytes, neomycin sulfate, and LISS.

Previous studies have shown that RBCs with residual WBCs stored in LISS and neomycin sulfate develop characteristics associated with enzyme-treated RBCs. During a mass screening program to antigen type donor RBCs, we observed that the Fya antigens on a RBC sample from an in-house panel became non-detectable with anti-Fya after incubation overnight in Diluent 2 from Micro Typing Systems, Inc. (MTS, Pompano Beach, FL). In response to this observation, we initiated an investigation to determine the cause. Tests were performed according to the manfacturer's instructions in MTS neutral gel cards or gel cards containing anti-IgG. We found that a reduction or loss of the Fya, Fyb, and M antigens occurs when RBCs were prepared from samples containing residual WBCs (as a source of enzymes) and subsequently incubated in media containing neomycin sulfate and LISS. We showed that the effect did not occur in the absence of neomycin sulfate. RBC antigens can be altered in LISS if they have first been exposed to neomycin. We recommend restricting the use of RBCs suspended in MTS Diluent 2 to the day of dilution (as indicated in the package insert) if preparing reagent RBCs from sources that were not leukoreduced and were stored in the presence of neomycin.

Murine monoclonal antibodies can be used to type RBCs with a positive DAT.

RBCs with a positive DAT due to IgG coating require the use of directly agglutinating reagents or treatment with chemicals to remove sufficient IgG to permit typing of the RBCs with antisera that require use of the IAT. In this study we demonstrate that murine IgG MoAbs to human RBC antigens can be used as an alternative if the anti-mouse IgG is neutralized or affinity purified to prevent cross-reaction with cell-bound IgG. We performed DATs on RBC samples coated with IgG in vivo and in vitro, comparing two anti-human IgG reagents (Organon Teknika, Durham, NC, and Ortho-Clinical Diagnostics, Raritan, NJ) with two affinity-purified anti-mouse IgG reagents (The Binding Site, San Diego, CA, and Sigma, St. Louis, MO), and one non-purified anti-mouse IgG reagent. The affinity-purified anti-mouse IgG reagents were nonreactive with the four in vitro sensitized RBC samples and were nonreactive with 8 of 11 in vivo sensitized RBC samples. Non-purified antimouse IgG and both anti-human IgG reagents reacted with every sample. Use of murine MoAbs to antigen type RBCs coated with human IgG is reliable only when the anti-mouse IgG reagents have been affinity purified or neutralized to prevent cross-reactivity. Our results also show the importance of including a saline/RBC control as well as an anti-mouse IgG/RBC control. Murine MoAbs are valuable reagents and we have applied them successfully in typing patients' RBCs that have a positive DAT.

MIMA-9, a valuable antibody for screening for rare donors.

Since monoclonal antibodies (Mabs) are potentially available in an unlimited volume, they can be used to screen numerous donor blood samples to identify antigen-negative donors. We have used a Mab (MIMA-9) with characteristics that allow for the simultaneous screening of RBCs of any ABO group for high-incidence antigen-negativity in the Kell and Gerbich blood group systems. MIMA-9, a murine IgG2a antibody, previously shown to facilitate the identification of K+k-, Kp(a+b-), K0, McLeod, or Ge:-3 red blood cells (RBCs), was used in MTS gel cards containing anti-mouse IgG as the second antibody to test 1134 K- donors. Among the 1134 donors tested, we found one Kp(a+b-) and one Ge:-2,-3,4 donor. If random donor samples had been used instead of preselecting for K-, we would have expected to identify two K+k- donors. One reagent (MIMA-9) can be used to simultaneously screen for K+k-, Kp(a+b-), K0, McLeod, and Ge:-3 RBCs and thereby conserve rare antisera. Inclusion of anti-mouse IgG in gel cards allowed for rapid screening. MIMA-9 is also a useful reagent to type RBCs with a positive direct antiglobulin test. This antibody is available to donor screening laboratories at no cost for this specific use.

Effects on blood group antigens from storage at low ionic strength in the presence of neomycin.

Red blood cells (RBC) stored without plasma in a neomycin, low ionic strength medium at 4 degrees C in excess of 24 h show alterations in antigen reactivity. There is a loss of protease-sensitive RBC antigens and a protease-type increased IgG saline agglutinability of Rh antigens that is associated with increased binding of 125I anti-D. Both the serological findings and the alteration in RBC membrane polypeptides are consistent with protease modification of the membrane due to contamination of the RBC by leukocytes. Neomycin, low ionic strength or leukocytes alone or in dual combination do not produce the observed changes in antigen reactivity. The role of neomycin and low ionic strength in this phenomenon and implication for quality control of reagent RBC used for antibody detection and identification are discussed.