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Mabel was kind enough to share this sample with our lab as we had not had the pleasure of seeing one of these. The sample was limited and it technically was not a referral, so my work was centered on getting to know anti-CD47. The patient forward typed as an A with no issues (I've heard the forward type can at times be affected), but reverse typed as an O, with 2+ reactivity in the A1 and A2 reverse cells. The patient typed Rh positive; no reactivity noted in the Rh control. The DAT was micro positive with poly specific AHG, heavy-chain IgG (HC) and C3, but was not interpret-able due to a micro positive saline control. Warm washing x4 with 37C saline resolved the positive saline control and still demonstrated micro reactions in the previously mentioned AHG sources. Interestingly, the reactivity was actually slightly stronger than the initial testing. Initial antibody testing was 2+ reactive in saline at initial spin, 3+ in saline at 37C spin (post 30 minute incubation) and 3-4+ reactive in saline at IAT with HC IgG, 3-4+ reactive in PEG at IAT (4+ reactions with the Rh negative cells tested as compared to 3+ reactivity with Rh positive cells; consistent with reports in literature), and 4+ reactive with papain treated cells (carryover reactivity was noted as a "no spin read" was 3+ and agglutination was noted prior to the addition of HC AHG). Testing with Immucor's murine monoclonal IgG Gamma Clone in PEG at IAT was macro negative, micro positive. Alloadsorption x2 (using 2 volumes of cells to one volume of plasma) onto papain-treated R1R1 and rr cells of known phenotypes (we usually do R1, R2 and r cells, but with the limited sample I dropped the R2 adsorption) removed the initial spin reactivity and confirmed the patient was group A. Alloadsorptions x3 were 1+ reactive in saline at 37C, and 3+ reactive at IAT using the HC IgG. x4 adsorptions yielded the same results. Out of curiosity, I did run a D-- cell with the x4 and it was nonreactive at 37C, but 2+ reactive at IAT. For fun, I performed titration studies on both the R1 and rr x4 adsorptions (saline, 60 minutes 37C incubation). Macro reactivity at 37C was noted at 1:32 in both samples, but technically they had a titer of 4 (1st observed 1+ reaction). At IAT, both samples had a titer of 16,384.... AFTER 4 adsorptions! A recent report in IMMUNOHEMATOLOGY stated that using equal volume (cells:plasma) adsorptions onto papain-treated cells x3 and x4 resulted in "the majority" of the samples being nonreactive in saline at initial spin and in PEG at IAT. Lastly, I did perform EGA treatment of the patient's red cells to see its affect on the positive DAT. The micro reactivity was removed following a 1min. 30sec treatment of the patient's cells, but since the sample was way beyond the manufacturer's specimen recommendation, I'm not totally confident in the observed results. Net result: It looks like alloadsorptions will be needed to resolve any observed ABO discrepancies due to reverse cell issues and PEG testing with Immucor's murine monoclonal Gamma Clone, with a macro only read, would resolve any the issue of detecting underlying alloantibodies. Thanks much Mabel for sharing this sample with us!

Right then. The first thing I would say is that I am simply amazed that these cells were found to be negative, and yet these types were NOT represented in any way in the original screening cells or the 18 panel cells. The first thing I would do, therefore, is to change my reagent supplier as a matter of urgency. It would be equally amazing for all four of these red cell samples to be uniquely negative for the same antigen. I noticed is that the reactions are all 2+, which suggests that there is a single specificity in the plasma, directed against a high prevalence antigen. 2+ reactions are pretty weak, which suggests the presence of what used to be called high titre, low avidity (HTLA) antibodies - I am STILL confused as to what we are supposed to call them these days, but hey! - so the first thing I would do is to titre the antibody to see if the end point is unexpectedly high, compared with what is expected for the reaction strength. The other thing I would do is to see if the antibody can be inhibited 1:2 with pooled human plasma, using the patient's own plasma diluted 1;2 in saline as the negative control. If the antibody is inhibitable as above, the probability is that the antibody is either anti-Ch or anti-Rg. Neither specificity is clinically significant, and so many people would go no further, but one thing that could be easily done these days is to try to inhibit the plasma with recombinant red cell protein (rRCP) that is specific for the C4d molecule where these antigens are expressed. Inhibition with this will prove the specificity. I, however, being a purist, would try to inhibit the plasma with plasma from a Ch-, Rg+ individual, and a Ch+, Rg- individual to prove the more exact specificity. If none of this works, I would have to have another think!

Off to reference lab...LOL We are very limited with what we can do. 2 panels...Enhancement only

LOL! We would send it to our reference lab! We have other things to do here... Scott