Session Information
Title: Antiphospholipid Syndrome: Clinical Manifestations and New Biomarkers in Antiphospholipid Syndrome
Session Type: Abstract Submissions (ACR)
Background/Purpose:
Recurrent IgG or IgM anti-β2glycoprotein (ab2GPI) antibody positivity is a key laboratory indicator for classification of antiphospholipid syndrome (APS). Considerable inter-laboratory variation still exists for both isotyopes hindering efforts at standardization. At the 13th International Congress on Antiphospholipid Antibodies in 2010, the “Criteria aPL Testing” task force recommended both the development of international units (IU) and of suitable polyclonal and monoclonal reference material (RM) for aβ2GPI measurement. As such, we sought to prepare polyclonal RM and establish IU for the measurement for IgM aβ2GPI antibodies.
Methods:
IgM aβ2GPI fractions were affinity-purified (AP) from the sera of 3 APS patients using a combination of ion exchange and affinity chromatography. Purity was confirmed using SDS-PAGE; high-activity fractions were pooled, concentrated and sterile-filtered. The Bradford assay was used to assign IU values (1 IU/ml = 1µg/ml AP- aβ2GPI). The RM serum was assigned an IU value using the AP- ab2GPI material and sent with 30 samples to six commercial companies (INOVA, Bio-Rad, Corgenix, Phadia, Human and Instrumentation Laboratory) for testing in their kits (eight total) according to an approved protocol to evaluate linearity, unit equivalency and commutability (CLSI guidelines EP14-A2 and C53-A).
Results:
The pooled AP material had a protein concentration of 15.125 µg/ml and was assigned a value of 15 IgM aβ2GPI IU/ml. The RM had a value of 220.3 IgM aβ2GPI IU/ml. The linearity (R2) of the RP curve for the various assays ranged from 0.9649 to 0.9996. The value of the RM in the various arbitrary kit units ranged from 71.6 to 568 units. Commutability among the different assays was confirmed as demonstrated in figure 1 (representative of all assays) in which dilutions of the IgM RM fell within the 95% prediction interval limits of the regression curve created by the 30 native serum samples. Commutability was also demonstrated using principal components analysis.
Conclusion: The results of linearity and commutability studies suggest that this material can be used as a calibrant/reference in a wide array of assays of different formats and new international consensus are now available for IgM-aβ2GPI measurement. These studies contribute significantly to the much-needed standardization of aβ2GPI immunoassays and further characterization by international bodies (IRMM/IFCC) will be done Performance of IgM RM in various IgM ab2GPI assays
|
Kit |
Method |
Cut-off |
Linearity |
RM in kit units |
|
Bio-Rad Bio-plex |
ELISA |
<20 M units |
R2 :0.99 |
93.8 M Units |
|
Bio-Rad ELISA |
Multiplex |
<20 M units |
R2: 0.96 |
96.4 M Units |
|
Corgenix ELISA |
ELISA |
< 20 M units |
R2: 0. 98 |
77.4 M Units |
|
IL ACUSTAR |
Chemiluminescence |
< 10 U/ml |
R2: 0.99 |
69.1 U/ml |
|
INOVA ELISA |
ELISA |
<20 SMU |
R2 0.99 |
98.4 SMU |
|
INOVA Bio-Flash |
Chemiluminescence |
<10 U/ml |
R2 :0.99 |
72.5 CU |
|
Phadia |
Immunofluoroescence |
<10 U/ml |
R2 :0.99 |
568 U/ml |
|
Human GmbH |
ELISA |
< 7 U/ml |
R2 :0.99 |
71.6 U/ml |
Table. Performance of IgM RM in various IgM aβ2GPI assays
Disclosure:
R. Willis,
None;
C. Grossi,
None;
M. Borghi,
None;
P. L. Meroni,
None;
G. Lakos,
Inova Diagnostics, Inc.,
3;
T. Buckner,
Corgenix,
3;
F. S. Cavalcanti,
Biokit,
3;
M. Crisostomo,
Bio-Rad,
3;
C. Dima,
TheraTest Laboratories,
3;
K. Jaskal,
Instrumentation Laboratories ,
3;
M. Kast,
Phadia ,
3;
L. R. Lopez,
None;
N. Olschowka,
Phadia ,
3;
S. Paul,
Bio-Iad,
3;
T. Prestigiacomo,
Bio-Rad,
3;
J. Puig,
Bio-Kit,
3;
W. Vandam,
Bio-Rad,
3;
A. Villarreal,
Bio-Rad,
3;
R. Walker,
Bio-Rad,
3;
M. Watkins,
Bio-Rad,
3;
S. S. Pierangeli,
None.
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