title: Western immunoblotting as a new tool for investigating direct antiglobulin test-negative autoimmune hemolytic anemias.
abstract: 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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors: Department of Laboratory Medicine and Pathobiology, University of Toronto.|Centre for Innovation, Canadian Blood Services.
authors: Evgenia M
authors: EM
authors: Bloch
authors: Centre for Innovation, Canadian Blood Services.
authors: Darinka
authors: D
authors: Sakac
authors: Department of Medicine, University of Toronto.
authors: Haley A
authors: HA
authors: Branch
authors: Department of Laboratory Medicine and Pathobiology, University of Toronto.|Centre for Innovation, Canadian Blood Services.|Department of Medicine, University of Toronto.|Toronto General Hospital, University Health Network.
authors: Christine
authors: C
authors: Cserti-Gazdewich
authors: Department of Laboratory Medicine and Pathobiology, University of Toronto.|Department of Medicine, University of Toronto.|Toronto General Hospital, University Health Network.
authors: Jacob
authors: J
authors: Pendergrast
authors: Department of Laboratory Medicine and Pathobiology, University of Toronto.|St. Michael's Hospital, Toronto, Ontario, Canada.
authors: Katerina
authors: K
authors: Pavenski
authors: Department of Laboratory Medicine and Pathobiology, University of Toronto.|Centre for Innovation, Canadian Blood Services.|Department of Medicine, University of Toronto.|Toronto General Hospital, University Health Network.
authors: Donald R
authors: DR
authors: Branch
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000743:Anemia, Hemolytic / Q000175:diagnosis / Q000276:immunology; D000744:Anemia, Hemolytic, Autoimmune / Q000175:diagnosis* / Q000276:immunology*; D001323:Autoantibodies / Q000032:analysis / Q000097:blood*; D015153:Blotting, Western; D003298:Coombs Test; D003937:Diagnosis, Differential; D004912:Erythrocytes / Q000276:immunology; D005188:False Negative Reactions; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D008297:Male; D008875:Middle Aged; D012680:Sensitivity and Specificity
chemical_list: D001323:Autoantibodies; D007074:Immunoglobulin G