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Western immunoblotting as a new tool for investigating direct antiglobulin test–negative autoimmune hemolytic anemias
Author(s) -
Bloch Evgenia M.,
Sakac Darinka,
Branch Haley A.,
CsertiGazdewich Christine,
Pendergrast Jacob,
Pavenski Katerina,
Branch Donald R.
Publication year - 2015
Publication title -
transfusion
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.045
H-Index - 132
eISSN - 1537-2995
pISSN - 0041-1132
DOI - 10.1111/trf.13082
Subject(s) - autoimmune hemolytic anemia , hemolysis , autoantibody , antibody , coombs test , hemolytic anemia , immunology , immune system , medicine , haptoglobin , autoimmune disease , anemia
BACKGROUND Direct antiglobulin test–negative (DAT(–)) autoimmune hemolytic anemia, characterized by hemolysis without detectable immunoglobulin or complement on patient red blood cells (RBCs), poses a diagnostic challenge. To select therapy, classification of the hemolysis as immune‐ or non–immune‐mediated is important. We developed a method using Western immunoblot (WB) to classify DAT(–) patients by measuring and comparing levels of RBC immunoglobulin (Ig)G to normal donors. STUDY DESIGN AND METHODS RBC samples from 42 normal donors were made into ghosts and analyzed by WB and densitometry to establish a normal mean relative quantity of IgG (RQIgG) on the RBCs. RQIgG on eight DAT(–) and eluate‐negative patients with hemolytic anemia was determined and compared to RQIgG on normal RBCs. RESULTS RQIgG of 42 normal donors indicated a calculated mean ± SD of 0.0016 ± 0.0015 and we used a cutoff of 0.0047, the mean + 2SD. This was compared with a receiver operating curve cutoff of 0.0041 with 100% sensitivity and 93% specificity. Of the eight patients tested, three were classified as non–immune‐mediated (one had pyruvate kinase deficiency) and five as immune‐mediated. Two of the patients in the latter group underwent splenectomy, followed by remission. CONCLUSION WB analysis is more sensitive than conventional test tube DAT or elution analysis. Our assay confirms: 1) previous studies showing normal RBCs are sensitized with IgG, perhaps due to natural autoantibody to senescence; 2) that some normal RBCs have increased levels of IgG without signs of disease; and 3) that WB distinguishes between non–immune‐ and immune‐mediated hemolytic anemia in DAT(–) patients and may be useful for clinical diagnosis.