Antiprothrombin antibodies: a comparative analysis of homemade and commercial methods. A collaborative study by the Forum Interdisciplinare per la Ricerca nelle Malattie Autoimmuni (FIRMA).
OBJECTIVE
Prothrombin (PT) is a target for antibodies with lupus anticoagulant (LA) activity, suggesting the possible application of anti-prothrombin antibody (aPT) assays in patients with antiphospholipid syndrome (APS). Different methods - both homemade and commercial - for the detection of aPT are available, but they seem to produce conflicting results. The purpose of this study was to compare the performance of different assays on a set of well-characterized serum samples.


METHODS
Sera were gathered from 4 FIRMA institutions, and distributed to 15 participating centres. Forty-five samples were from patients positive for LA and/or anticardiolipin antibodies (aCL) with or without APS, and 15 were from rheumatoid arthritis (RA) patients negative for antiphospholipid antibodies. The samples were evaluated for IgG and IgM antibodies using a homemade direct aPT assay (method 1), a homemade phosphatidylserine-dependent aPT assay (aPS/PT, method 2), and two different commercial kits (methods 3 and 4). In addition, a commercial kit for the detection of IgG-A-M aPT (method 5) was used.


RESULTS
Inter-laboratory results for the 5 methods were not always comparable when different methods were used. Good inter-assay concordance was found for IgG antibodies evaluated using methods 1, 3, and 4 (Cohen k > 0.4), while the IgM results were discordant between assays. In patients with thrombosis and pregnancy losses, method 5 performed better than the others.


CONCLUSIONS
While aPT and aPS/PT assays could be of interest from a clinical perspective, their routine performance cannot yet be recommended because of problems connected with the reproducibility and interpretation of the results.
Tincani
A
A
Reumatologia e Immunologia Clinica, Ospedale Civile e Università di Brescia, Italy. Tincani@bresciareumatologia.it
Morozzi
G
G
Afeltra
A
A
Alessandri
C
C
Allegri
F
F
Bistoni
O
O
Bizzaro
N
N
Caccavo
D
D
Galeazzi
M
M
Gerli
R
R
Giovannelli
L
L
Longobardo
G
G
Lotzniker
M
M
Malacarne
F
F
Migliorini
P
P
Parodi
A
A
Pregnolato
F
F
Radice
A
A
Riccieri
V
V
Ruffelli
M
M
Sinico
R A
RA
Tozzoli
R
R
Villalta
D
D
Marcolongo
R
R
Meroni
P
P
D000888:Antibodies, Anti-Idiotypic / Q000097:blood*; D016736:Antiphospholipid Syndrome / Q000097:blood / Q000276:immunology*; D001172:Arthritis, Rheumatoid / Q000097:blood / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D007075:Immunoglobulin M / Q000097:blood; D016682:Lupus Coagulation Inhibitor / Q000276:immunology; D011516:Prothrombin / Q000276:immunology*; D015203:Reproducibility of Results
D000888:Antibodies, Anti-Idiotypic; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D016682:Lupus Coagulation Inhibitor; D011516:Prothrombin