IAT elution by MAHG IgA(+) IAT elution by MAHG IgG(+) Performing elution test with monospecific antihuman globulin unmasks multiple immunoglobulin coating; implications on improving multiple immunoglobulin detection sensitivity in AIHA screening. Z.Bezirgiannidou1,2, E.Kontekaki2, S.Papamichos1, A.Christoforidou1 , S.Zisaki2, K.Chouchos3, J.Kotsiannidis1, D.Margaritis1, E. Mantadakis4 1 Department of Haematology, Democritus University of Thrace Medical School, Alexandroupolis, Thrace, Greece. 2 Blood Transfusion Center, University Hospital of Alexandroupolis, Thrace, Greece. 3Biomedical engineer, Democritus University of Thrace Medical School Alexandroupolis, Thrace, Greece. 4Department of Paediatrics, Democritus University of Thrace Medical School, Alexandroupolis, Thrace, Greece BACKGROUND Picture 1: Examples of additional Igs unmasked by MAHG in IAT elution versus use of MAHG in DAT Mixed type AIHA Blood donor Evans Syndrome IAT elution by MAHG IgA(+) IAT elution by MAHG IgG(+) DAT by MAHG IgG(-) DAT by MAHG IgA(-) Table 1: Comparison of results by DAT& IAT elution with MAHGs Autoimmune Haemolytic Anemia (AIHA) basic screening includes: (i) Direct Antiglobulin Test (DAT) using monospecific antihuman globulin (MAHG), namely anti-IgG, anti-IgA, anti-IgM, anti-C3c and anti-C3d, to detect the autoantibody immunoglobulin (Ig) class(es) or complement fragments on erythrocyte membrane; (ii) Indirect Antiglobulin Test (IAT) using polyspecific antihuman globulin (PAHG), comprised of anti-IgG and anti-C3d, to identify autoantibody(ies) in serum or red blood cells (RBCs) elution. Current elution techniques are valuable for allowing the identification of antibody specificity and the elimination of false positive DAT cases. The use of only anti-IgG in IAT implies that: (a) Non-IgG classes causing AIHA will be missed in elution test, rendering false negative results, (b) Elution test in AIHA caused by multiple Ig classes would allow non-IgG classes to be missed, generating misleading results. Patients with masked Igs DAT IAT elution 6 IgM(-) IgM(+) 3 IgG(-) IgG(+) 5 IgA(-) IgA(+) Total 14 Ιn 14 out of 52 cases (26.92%), the DAT missed to identify multiple Igs coating AIM RESULTS CONCLUSIONS To highlight that in DAT(+) RBCs coated with multiple Igs, DAT is not sufficient to unmask all the putative Ig classes and to suggest a method that potently unmasks multiple Ig coating. Initial screening of blood samples by DAT identified 49 warm and 2 cold autoantibodies, as well as one mixed type autoantibody. When AIHA caused by multiple Ig classes, the use of MAHG in elution IAT allowed the identification of Ig classes that were not detected by the standard DAT. In detail, six DAT(+) and IgM(- ) cases by DAT were identified as IgM(+) by elution IAT; three IgG(-) DAT(+) cases were found to be IgG(+) by elution IAT; five DAT(+) IgA(-) cases by DAT were identified as IgA(+) by elution IAT. Overall, in 14 out of 52 cases (26.92%), the DAT missed to identify multiple Igs coating (Table 1). Even though elution IAT with PAHG would identify the three IgG(-) DAT(+) cases, still 11 cases with multiple Igs (21.15%) would have been missed, a statistically significant difference (p=0.012). In our study AIHA was present more frequently when multiple(missed) Igs were implicated [11 out of 14 (78.57%)] versus single Ig [10 out of 38(26,31)], a statistically significant difference (p=0,0003). In cases of AIHA caused by multiple Ig classes, we assume that monospecific antiserums in DAT bind mainly with the dominant Ig class due to high concentration of bound antibodies on RBC membrane (like the prozone phenomenon). This constraint is bypassed when Igs are discarded from the RBC membrane in elution, since they are available to react with PAHG by IAT; importantly the latter applies only for IgGs, since non-IgGs would be missed by PAHG. In conclusion, we report an efficient method for unmasking non-IgGs participating in multiple Ig coating, i.e., the use of MAHG in IAT elution (picture 1). This method may have clinical implications, because AIHA is frequently severe when multiple Ig coating occurs. MATERIALS & METHODS Acid elution (DiaCidel/BIO-RAD) was performed on 59 patients’ DAT(+) RBCs. The eluates were screened by IAT against a commercially available panel of RBCs (ID-DiaPanel) using PAHG Liss/Coombs gel cards (Biorad, Switzerland). An additional method using MAHG gel cards (DC-Screening I/BIO- RAD) in elution IAT was also applied. Seven patients were excluded from the study since their DAT(+) was due to alloantibodies following recent transfusion or non-specific reactions. Of the remaining 52 patients, only 21 (40.38%) fulfilled the diagnostic criteria for autoimmune haemolysis; 17 presented with AIHA and 4 with Evans syndrome. DAT positivity without haemolysis was observed in 31(59.61%) patients, representing a random finding during pretransfusion testing. REFERENCES Virginia Vengelen-Tyler, Vengelen-Tyler. Investigation of a positive Direct Antiglobulin Test. In: Technical Manual of American Association of Blood Banks 13th ed. Bethesda, Maryland, USA: ABBB, 1999 (p690-4, 699). L.Petz-G.Garratty. Hemolytic Transfusion Reactions. In: Immune Hemolytic Anemias. 2nd ed. Philadelphia, USA: Churchill Livingstone, 2004 (p 552). L.Petz-G.Garratty. The serologic Investigation of Autoimmune Hemolytic Anemia. 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