Measurement of immunoglobulins anti VWF in patients with probable AVWS with an enzyme-linked immunosorbent assay (ELISA) using platelet VWF as an antigen.

KEMPFER AC, WOODS AI, PAIVA J, CASINELLI MM, SANCHEZ-LUCEROS A, LAZZARI MA

Dublin

Congreso; Congreso de ISTH; 2018

ISTH

Title: Measurement of immunoglobulins anti VWF in patients with probable AVWS with an enzyme-linked immunosorbent assay (ELISA) using platelet VWF as an antigen. Normal 0 21 false false false ES-AR X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;} Topic: Laboratory Tests Author(s): A.C. Kempfer1, A.I. Woods1, J. Paiva2, A. Sanchez-Luceros1,2, M.A. Lazzari1 Institute(s): 1IMEX-CONICET-Academia Nacional de Medicina, Laboratorio de Hemostasia y Trombosis, Ciudad Autonoma de Buenos Aires, Argentina, 2Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Hemostasia y Trombosis, Ciudad Autonoma de Buenos Aires, Argentina Travel Grant: I want to apply for Reach the World 849-cv-1517492665.pdf Disclosure: I have no potential conflict of interest. Text: Background: Autoantibodies play a role in the pathogenesis of some patients with AVWS. Inhibitory antibodies (Ab) can be detected in mixing studies with normal plasma (KPTT; VWF:RCo) and presence of non-neutralizing Ab can be detected by ELISA. Aims: The detection of immunoglobulin (IgG, IgA or IgM) in plasma or anti-platelet (pt) or anti-VWF, the elevated VWFpp ratio and the absence of responsible mutations are useful tools to discriminate inherited VWD from AVWS. We showed here our results in detecting anti VWF antibodies by ELISA in 6 patients (P) with probable AVWS and one P VWD 2M. Methods: ELISA measurement of anti-VWF antibodies. We developed an ELISA technique to detect anti-VWF Ab as described by Siaka et al (2003), but with modifications: Covalink plates were coated overnight at 4ºC with 100 µL of pt VWF (obtained from 20 ml blood, pt washed three times and frozen and thawed 30 times) used at a final concentration of 4 IU/dL VWF:RCo. The 99th percentile of the distribution of values obtained in 20 healthy controls (normalized OD units) was assumed as cut-off for normal levels of: median IgG 0.34 normalized OD units, median IgA 0.72 normalized OD units and median IgM 1.00 normalized OD units anti-VWF. Results: When we used VWF from the Hemate-P as Ag, the P 7 had a median IgM anti-VWF of 2.2 (cut off 0.98) different value that observed in table, median IgM anti-VWF=1.00 (cut off 1.00). The same behavior had a P VWD type 3 median IgG anti-VWF=1.2 (cut-off 0.98), while with pt VWF as Ag, median IgG anti-VWF was 0.34 (cut-off 0.34). Patients Sex-Age (years old) 1 F-58 2 F-45 3 F-18 4 M-47 5 M-51 6 F-41 7 M-47 KPTT (s) P+N 66 No correction 196 No correction 42 correction 66 correction 53 No correction 53 correction 46 VWF:Ag (IU/dL) 18 126 25 9 20 9.5 73 VWF:RCo (IU/dL) P+N No detection No correction <10 No correction <10 No detected <10 No correction <10 correction 10 VWFpp/VWF:Ag ratio 13.3 Not done 2.9 13.2 8.20 12.2 1.96 VWF multimers ultralarge Not done absence large normal ultralarge absence large+ intermediate normal molecular biology Normal exons 27-28 Not done Normal exon 28 Normal exons 26-27-28 Normal exons 27-28 Normal exons 13,14, 26-28 p.T1468I ANTI VWF cut-off=0.34-0.72-1.00 (IgG-IgA-IgM) 0.40-0.27-0.33 0.86-2.38-2.14 0.46-0.78-0.74 0.25-0.32-0.35 0.26-0.44-1.00 0.23-1.00-0.44 0.23-0.70-1.00 Other methods Not done Not done Not done Increased titles of IgG kappa IgM asociated to Pt (Pt count: 39 109/L) Not done Not done Probable diagnostic AVWS (neutralizing Ab) AVWS Lupus anticoagulant) AVWS non-neutralizing Ab) AVWS (monoclonal gammopathy) AVWS (Ab associated to pt) AVWS (gammopathy monoclonal) VWD2M [Table 1. Different tests made to prove or rule out the diagnosis of AVWS in 6 P from IIHema.] Conclusions: Plasma-derived VWF contains ABO blood group Ag and should not be used as an Ag for ELISA to detect Ab because the presence of isoagglutinins may cause false positive results (Tiede, 2011). We believe that our election of pt-VWF as a reagent (Ag) has resolved this issue because A or B blood group antigens were not detected in pt (Sarode, 2010).