title: Detection of red blood cell antibodies in mitogen-stimulated cultures from patients with hereditary spherocytosis.
abstract: BACKGROUND
  Hereditary spherocytosis (HS) is a congenital hemolytic anemia caused by defects in red blood cell (RBC) membrane proteins leading to premature RBC clearance in the spleen. The presence of RBC autoantibodies has never been extensively investigated in HS.
  
  
  METHODS
  RBC antibody-bound immunoglobulin (Ig)G was investigated in 91 consecutive HS patients by mitogen-stimulated direct antiglobulin test (MS-DAT), a sensitive method able to magnify latent RBC antibody autoimmunity and related with hemolytic variables, previous splenectomy, and type of membrane defect.
  
  
  RESULTS
  A total of 61% of HS cases had RBC antibodies by MS-DAT (29 Band 3, 17 spectrin deficiency, and nine no defined defect). The amount of RBC-bound IgG was greater in HS compared with controls (236 ± 192 ng/mL vs. 52 ± 29 ng/mL, p < 0.0001), although lower than that observed in autoimmune hemolytic anemia (AIHA; 634 ± 371 ng/mL vs. 236 ± 192 ng/mL, p < 0.0001). Western blot experiments showed that purified IgG fraction from MS-DAT-positive patients bind to α- and β-spectrin, Band 3, and Band 4.9. Positive cases displayed increased reticulocytosis and slightly reduced hemoglobin (Hb) values compared to negative ones. Patients displaying RBC-bound IgG of more than 250 ng/mL (the positive threshold of AIHA) showed increased number of spherocytes and mainly had spectrin deficiency. RBC-bound IgG and free Hb increased over time after storage at 4°C, a surrogate of ex vivo aging, more evidently in HS than controls, and particularly in Band 3 deficiency.
  
  
  CONCLUSIONS
  RBC autoantibodies were detected by MS-DAT in more than a half of HS patients. Positive cases showed a more evident hemolytic pattern suggesting a pathogenic role of these autoantibodies in RBC opsonization and splenic removal.
authors.affiliation: UO Oncoematologia, UOS Fisiopatologia delle Anemie.
authors.forename: Anna
authors.initials: A
authors.lastname: Zaninoni
authors.affiliation: UO Oncoematologia, UOS Fisiopatologia delle Anemie.
authors.forename: Cristina
authors.initials: C
authors.lastname: Vercellati
authors.affiliation: UO Oncoematologia, UOS Fisiopatologia delle Anemie.
authors.forename: Francesca G
authors.initials: FG
authors.lastname: Imperiali
authors.affiliation: UO Oncoematologia, UOS Fisiopatologia delle Anemie.
authors.forename: Anna P
authors.initials: AP
authors.lastname: Marcello
authors.affiliation: UO Oncoematologia, UOS Fisiopatologia delle Anemie.
authors.forename: Bruno
authors.initials: B
authors.lastname: Fattizzo
authors.affiliation: UO Oncoematologia, UOS Fisiopatologia delle Anemie.
authors.forename: Elisa
authors.initials: E
authors.lastname: Fermo
authors.affiliation: UO Oncoematologia, UOS Fisiopatologia delle Anemie.
authors.forename: Paola
authors.initials: P
authors.lastname: Bianchi
authors.affiliation: Experimental Laboratory of Immunological and Rheumatologic Researches, IRCCS Istituto Auxologico Italiano.
authors.forename: Claudia
authors.initials: C
authors.lastname: Grossi
authors.affiliation: UO Laboratorio Centrale di Analisi Chimico Cliniche e Microbiologiche Dipartimento Dei Servizi, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Cattaneo
authors.affiliation: UO Oncoematologia, UOS Fisiopatologia delle Anemie.|Università Degli Studi di Milano, Milan, Italy.
authors.forename: Agostino
authors.initials: A
authors.lastname: Cortelezzi
authors.affiliation: UO Oncoematologia, UOS Fisiopatologia delle Anemie.
authors.forename: Alberto
authors.initials: A
authors.lastname: Zanella
authors.affiliation: UO Oncoematologia, UOS Fisiopatologia delle Anemie.
authors.forename: Wilma
authors.initials: W
authors.lastname: Barcellini
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001323:Autoantibodies / Q000097:blood*; D016922:Cellular Senescence; D002648:Child; D002675:Child, Preschool; D003298:Coombs Test; D004912:Erythrocytes / Q000276:immunology*; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D008297:Male; D008875:Middle Aged; D008934:Mitogens / Q000494:pharmacology; D013103:Spherocytosis, Hereditary / Q000276:immunology*
chemical_list: D001323:Autoantibodies; D007074:Immunoglobulin G; D008934:Mitogens