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GinaL

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GinaL last won the day on November 9 2018

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  • Birthday 11/03/1953

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  1. Both ficin and papain work well for adsorptions. We have routinely used ficin in our reference lab for decades, both for antibody ID and adsorptions. And both enzymes can be used as constituents of ZZAP. A couple of years ago I compared cysteine-activated papain and ficin as constituents of ZZAP, and the volume of 1%ficin used in ZZAP. In the AABB Technical Manual, the ZZAP recipe calls for twice as much 1% ficin as 1% papain without explanation. I found there is no need to use 2x the volume of ficin. I suspect there may have been some issues with the activity of ficin some years ago. We prepare and standardize our enzymes. Both the ficin and papain for this study were purchased from Sigman-Aldrich. ref: Leger & Garratty. Comparison of papaina and ficin as constituents of ZZAP for adsorption using allogeneic RBCs to remove warm autoantibodies for detection of alloantibodies (abstract). Transfusion 2011;51(Suppl):173A. Gina Leger
  2. Drug-induced immnune hemolytic anemia is uncommon, but piperacillin is now the top contender of all the drug antibody investigations we perform in Dr. George Garratty's research lab at the American Red Cross, Southern California Region. Ceftriaxone and cefotetan are also high on our list. Piperacillin, although it is a semi-synthetic penicillin is unlike penicillin G in both the clinical presentation and in the serology. Piperacillin is often given in combination with tazobactam, a beta lactamase inhibitor, and is commonly given to patients with cystic fibrosis for lung infections. Patients who develop anti-piperacillin often have prior exposure to the drug. Also unlike with penicillin, intravascular hemolysis can be seen and the hemoglobin can drop dramatically. Fatalities have been reported. Unfortunately, we cannot predict which patients will or will not develop anti-piperacillin. Monitoring the hemobglobin is the best initial indicator, then following up with the other typical indicators of hemolysis (indirect bili, LDH, haptoglobin, evaluating peripheral blood smear). A direct antiglobulin test should then follow suspected hemolysis to determine if it is immune-mediated. A significant finding with piperacillin antibodies is that while the patient is still receiving the drug (it is intravenous), the serology can mimic warm autoantibodies, and often with anti-e specificity. One-to two days after the drug is stopped, however, this plasma reactivity disappears. We believe this reactivity is caused by circulating drug (i.e., no in vitro drug is needed). And to baffle this serologist, we have even seen reactive eluates is some patients with this phenomenom. We have even seen some patients who have mistakenly been treated for warm autoimmune hemolytic anemia (e.g. with steroids) based primarily on the serology. Once the drug is stopped the hemolysis subsides. Testing for antibodies to piperacillin is performed by adding a solution of the drug to serum and untreated RBCs (we also test enzyme-treated RBCs). Piperacillin does bind readily to RBCs so testing can be performed with piperacillin-treated RBCs, but we have found that plasma from blood donors and random patients can directly agglutinate piperacillin-treated RBCs, so we do not recommend this method in order to avoid misinterpretation (false positive result). Piperacillin antibodies do not have high titers so testing a dilution of the serum to get around this problem is not advised. So, in addition to a dropping hemoglobin, a serologic clue is a new warm autoantibody in a patient whose initial clinical presentation is not warm autoimmune hemolytic anemia! If the eluate is reactive, it may react weaker than the plasma. Gina Leger
  3. How I would interpret these results is dependent upon what reagents were used! Different reagents are in use all over the world. We cannot expect that all reagents of a given specificity will react in the same way! In the US, we have one manufacturer's monoclonal anti-A, Ortho's BioClone anti-A which contains MHO4, that reacts with B(A) RBCs. While Most B(A) react weakly with this anti-A, some react quite well (e.g. 2+). The plasma is expected to contain anti-A (reacting with both A1 and A2 RBCs) whereas plasma from AsubB may occasionally contain weak anti-A1. Testing other monoclonal anti-A reagents (i.e. not containing the MHO4 clone) would be used to differentiate a B(A) from AsubB. The weak reactivity of the plasma with the A1 and A2 RBCs could just be this donor's anti-A, reacting weaker than expected. I would test this donor's RBCs with other monoclonal anti-A and the plasma with other A1 and A2 RBCs.
  4. Sorry for the delay, but I've been on vacation! The TSEN antigen results from the formation of a hybrid glycophorin A-B or GPB-A-B. In the case of the probable TSEN homozygote I mentioned above, the immunoblotting studies indicated hybrid GPA-B, but no normal GPA or GPB, so it was believed that the patient inherited genes for a hybrid GP from both parents (hence probable homozygote). So the En(a-) status of this patient is not the same as the MkMk type or En(a-)Fin. Her antibody was anti-EnaFR so there was no compatible blood available for her. This case was included in the report by Storry JR et al. Vox Sang 2000;798:175-9. As Peter stated, some anti-S react with the TSEN antigen, but others do not. Gina
  5. I have seen both auto anti-Ena and alloanti-Ena. Anti-Ena can be IgM or IgG. Alloanti-Ena is an exceptionally rare antibody. The few examples I have seen have been associated with hybrid glycophorins, not with the extremely rare Mk type. A clue for a hybrid in such a case is usually only M or N plus S or s are present, but we had a TSEN homozygote (probable) come our way and her RBCs typed M+N-S-s-U+ and the antibody was anti-EnaFR. The antibody was reactive with all RBCs, except the autologous RBCs, in the indirect antiglobulin test with PEG-IgG (nonreactive in a short room temp incubation) and with ficin-treated RBCs at 37C and AHG. The antibody also reacted with DTT-treated RBCs in LISS-IgG. Rare RBCs, including null phenotypes, for specificities that are not denatured by ficin and DTT were tested to attempt to determine the specificity. The MNS typing, combined with an Hispanic ethnicity was a clue in this case that we might be dealing with an hybrid (we don't expect S-s- for individuals other that Blacks, but learn to expect the unexpected!). The patient's serum was nonreactive with two examples of RBCs from individuals with hybrids. Adsorptions were used to complete exclusion of common alloantibodies. The New York Blood Center completed the investigation into the hybrid part with immunoblots for us. One of the difficulties with proving anti-EnaFR is that anti-Wrb may also be present since Wrb requires the presence of GPA. I am only aware of one example of RBCs that are Ena+;Wr(b-). The few examples of anti-Ena associated with hybrids that I have seen had anti-EnaFR/-Wrb in the serum. By the way, there is no donor blood available I have also seen autoanti-Ena, both autoanti-EnaFR and autoanti-EnaFS, also reactive only at the antiglobulin test. Again, all RBCs are reactive, including the patient's RBCs. Here, since this is not because of a missing normal GPA, the MNS typing is noninformative. I suspect that autoanti-EnaFR is most often not recognized because it looks just like an idiopathic IgG warm autoantibody. Autoanti-EnaFS, however, does not react with ficin-treated RBCs. The serologic problem here is to distinguish anti-EnaFS from autoanti-Pr, which is also nonreactive with ficin-treated RBCs. (Autoanti-Ge also need to be excluded). While I was taught that anti-Pr is a cold reactive antibody, there are examples of warm reactive anti-Pr. Jumping through some hoops, these two specificities can be readily distinguished by testing with neuraminidase-treated RBCs. Check Issitt and Anstee, Applied Blood Group Serology (or Issitt's earlier editions) for details - remeber neuraminidase-treated RBCs react with anti-T in most sera! Defining an autoantibody specificity, however, is purely of academic interest. Nonreactivity with ficin-treated RBCs is not typical of warm autoantibodies, so it gets our attention. Sorry for the long post. Anti-Ena is not a simple antibody!
  6. Continuing on this thread, both column agglutination (eg, gel) and solid phase methods are more sensitive for detecting warm autoantibodies, just as PEG and enzyme methods. Repeating the test by tube with LISS or even saline suspended RBCs is an acceptable and often used approach. But I do need to comment on the dilution approach to detect alloantibodies underlying warm autoantibodies. We (Dr. Garratty and myself) reported on the comparison of adsorbing autoantibodies with ZZAP-treated RBCs, adsorbing in the presence of PEG, and the dilution technique in Transfusion 1999;39:11-16. The New York Blood Center had previously reported using a 1 in 5 dilution of patient's serum to detect alloantibodies underlying autoantibodies. With the volume of warm autoantibody workups that our reference lab performs, we had wanted to see if we could similarly reduce turn around time on these cases. The bottom line is that 27 of 119 sera with alloantibodies (plus autoantibodies) did not react by tube test with LISS-suspended RBCs after diluting 1 in 5. In other words, we would have missed these potentially clinically significant anitobides. Our caveat, however, is that if adsorptions are not possible (eg, insufficient time prior to transfusion) that dilution is better than just giving least incompatible units. It is established that warm autoantibodies plus alloantibodies do not necessarily react more stronly than warm autoantibody alone, even for 1+ reactivity of the auto. Also, a retrospective adsorption under these circumstances could be used to ensure alloantibodies were not missed by the dilution method. I do have to add that these were the results in our hands; another laboratory may have established their own criteria and approach. The dilution technique described by Petz and Garratty did not have a predetermined dilution, eg 1 in 5, as used in the NYBC method. Rather, a titration of the patient's sample was performed against a pool of screening RBCs and the dilution where the serum reacted 1+ in the IAT against the pool was selected for testing agianst a panel of RBCs. In the 1980 edition of their text they stated that this method is efficient when (my emphasis/underline) the alloantibody is of higher titer than the autoantibody, but it cannot be used to confidently exldue the presence of alloantibody. Gina Leger
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