ORIGINAL RESEARCH

published: 05 March 2019

doi: 10.3389/fimmu.2019.00376

Reliability of Lupus Anticoagulant

and Anti-phosphatidylserine/
prothrombin Autoantibodies in
Antiphospholipid Syndrome: A
Multicenter Study
Savino Sciascia 1,2*† , Massimo Radin 1,3† , Irene Cecchi 1,3 , Elena Rubini 1,3 , Anna Scotta 1,3,4 ,
Roberta Rolla 4 , Barbara Montaruli 5 , Patrizia Pergolini 4 , Giulio Mengozzi 6 ,
Emanuela Muccini 6 , Simone Baldovino 1,3 , Michela Ferro 2 , Antonella Vaccarino 7 ,
Michael Mahler 8 , Elisa Menegatti 3 and Dario Roccatello 1,2
1
Center of Research of Immunopathology and Rare Diseases- Coordinating Center of Piemonte and Valle d’Aosta Network
Edited by: for Rare Diseases, Department of Clinical and Biological Sciences, University of Turin and S. Giovanni Bosco Hospital, Turin,
Ricard Cervera, Italy, 2 Nephrology and Dialysis Unit, S. Giovanni Bosco Hospital and University of Turin, Turin, Italy, 3 Department of Clinical
Hospital Clínic de Barcelona, Spain and Biological Sciences, School of Specialization of Clinical Pathology, University of Turin, Turin, Italy, 4 AOU Clinical
Chemistry Laboratory, “Maggiore della Carità” University Hospital, Amedeo Avogadro University of Eastern Piedmont,
Reviewed by:
Novara, Italy, 5 AO Ordine Mauriziano, Turin, Italy, 6 Department of Clinical Biochemistry, AOU Cittá della Salute e della
Jason S. Knight,
Scienza, Turin, Italy, 7 Hematology Division, and S. Giovanni Bosco Hospital, Turin, Italy, 8 Inova Diagnostics, Research and
University of Michigan, United States
Development, San Diego, CA, United States
Ian P. Giles,
University College London,
United Kingdom Background: Is it well-known that one of the major drawbacks of Lupus Anticoagulant
*Correspondence: (LA) test is their sensitivity to anticoagulant therapy, due to the coagulation based
Savino Sciascia
[email protected]
principle. In this study we aimed to assess the reproducibility of LA testing and to evaluate
the performance of solid assay phosphatidylserine/prothrombin (aPS/PT) antibodies.
† These authors have contributed
equally to this work Methods: We included 60 patients that fulfilled the following inclusion criteria: (I)
diagnosis of thrombotic antiphospholipid syndrome (APS); (II) patients with thrombosis
Specialty section:
and (a) inconstant previous LA positivity and/or (b) positivity for antiphospholipid
This article was submitted to
Autoimmune and Autoinflammatory antibodies (aPL) at low-medium titers [defined as levels of anti-β2Glycoprotein-I or
Disorders, anticardiolipin (IgG/IgM) 10–30 GPL/MPL] with no previous evidence of LA positivity.
a section of the journal
Frontiers in Immunology aPL testing was performed blindly in 4 centers undertaking periodic external quality
Received: 30 November 2018
assessment.
Accepted: 14 February 2019
Results: The 60 patients enrolled were distributed as follows: 43 (71.7%) with
Published: 05 March 2019
thrombotic APS, 7 (11.7%) with thrombosis and inconstant LA positivity and 10 (16.7%)
Citation:
Sciascia S, Radin M, Cecchi I, with low-medium aPL titers. Categorical agreement for LA among the centers ranged
Rubini E, Scotta A, Rolla R, from 0.41 to 0.60 (Cohen’s kappa coefficient; moderate agreement). The correlation
Montaruli B, Pergolini P, Mengozzi G,
Muccini E, Baldovino S, Ferro M,
determined at the 4 sites for aPS/PT was strong, both quantitatively (Spearman rho
Vaccarino A, Mahler M, Menegatti E 0.84) and when dichotomized (Cohen’s kappa coefficients = 0.81 to 1.0). Discordant (as
and Roccatello D (2019) Reliability of
defined by lack of agreement in ≥3 laboratories) or inconclusive LA results were observed
Lupus Anticoagulant and
Anti-phosphatidylserine/prothrombin in 27/60 (45%) cases; when limiting the analysis to those receiving vitamin K antagonist
Autoantibodies in Antiphospholipid (VKA), the level of discordant LA results was as high as 75%(15/20). Conversely, aPS/PT
Syndrome: A Multicenter Study.
Front. Immunol. 10:376.
testing showed an overall agreement of 83% (up to 90% in patients receiving VKA),
doi: 10.3389/fimmu.2019.00376 providing an overall increase in test reproducibility of +28% when compared to LA,

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Sciascia et al. LA and aPS/PT Testing: A Multicenter Study

becoming even more evident (+65%) when analyzing patients on VKA. In patients treated
with VKA, we observed a good correlation for aPS/PT IgG testing (Cohen’s kappa
coefficients = 0.81–1; Spearman rho 0.86).
Conclusion: Despite the progress in the standardization of aPL testing, we observed
up to 45% of overall discrepant results for LA, even higher in patients on VKA. The
introduction of aPS/PT testing might represent a further diagnostic tool, especially when
LA testing is not available or the results are uncertain.

Keywords: antiphosphospholipid syndrome, Antiphospholipid Antibodies, Lupus Anticoagulant, aPS/PT,

thrombosis, laboratory, diagnostic performance, reliability

INTRODUCTION over a time of more than 12 weeks) that presented at San

Giovanni Bosco Hospital in the last 5 years. The study was
Since clinical features of Antiphospholipid Syndrome (APS) performed in compliance with the Declaration of Helsinki;
(thrombosis and pregnancy complications) are common in the approval from the ethic committee was not required according
general population and often related to other underlying factors, to the local and national guidelines. We enrolled 60 patients who
the diagnosis of APS requires next to clinical assessment the met one of the following inclusion criteria:
detection of persistently positive Antiphospholipid Antibodies
(aPL). Thus, reliable laboratory tests with good clinical and 1) Fulfilled the diagnosis of thrombotic APS defined as per
analytical performance reproducibility are required. There is a Sydney criteria (11).
large variety of assays available to assess aPL, but despite progress, 2) Patients with thrombosis and suspected APS not completely
standardization is still not optimal (1–3). fulfilling the laboratory criteria (11), as follows: (a)
Lupus anticoagulant (LA) has been shown to be the strongest inconsistent previous LA positivity; and/or (b) low-medium
risk factor for aPL-related clinical manifestations (4), and the aPL titers [defined as levels of anticardiolipin (aCL) IgG/IgM
correct interpretation of this functional assay is crucial for or anti-β2-glycoprotein I (aβ2GPI) IgG/IgM antibodies 10–30
diagnosis of APS. However, testing patients during treatment GPL/MPL]. Clinical and laboratory characteristics were
with vitamin K antagonists (VKA) or other oral anticoagulants retrospectively collected.
remains a contentious issue and has been discouraged by official
guidelines (5–7) because of interpretational problems affecting Previous Autoantibody Detection
the mixing test. Besides, the clinical significance of low aPL titer The aPL profile, at the diagnosis, included aCL, LA, and
and/or weak LA positivity, especially when detected in patients aβ2GPI antibodies.
receiving anticoagulation [either VKA or direct anticoagulant The aCL and aß2GPI (IgG and IgM) were detected by
agents (DOAC)], remains uncertain and certainly needs a more commercial ELISA (Inova Diagnostics, Inc., San Diego, CA, US).
thorough evaluation. Plasma samples were tested for the presence of LA according
More recently, the family of aPL has been expanded to include to the recommended criteria from the International Society on
a heterogeneous group of autoantibodies whose specificity is Thrombosis and Haemostasis (ISTH) Subcommittee on Lupus
directed to proteins involved in coagulation or to a complex Anticoagulant/Phospholipid-Dependent Antibodies (12, 13).
of these proteins with phospholipids (8). Among others,
autoantibodies that recognize a phosphatidylserine/prothrombin Study Design
(aPS/PT) complex have been reported to be associated with APS LA and aPS/PT testing was performed in a blind fashion
and may have diagnostic relevance in these settings (9, 10). in four centers of the “Antiphospholipid Antibodies Regional
However, since aPS/PT antibodies are not currently included in Consortium” in northwest Italy: San Giovanni Bosco Hospital,
the current APS classification criteria (11), aPS/PT antibodies Turin, Italy, A.O.U. Città della Salute e della Scienza, Turin, Italy,
are not assessed in all patients suspected to suffer from APS. A.O. Ordine Mauriziano, Turin, Italy, and A.O.U. Maggiore della
Given the importance of aPL confirmation to improve the Carità, Novara, Italy (14).
interpretability of laboratory test results for clinical trials and LA was tested with the detection of two different reagents,
research studies, the objective of this study was to assess used as screening and confirmatory tests, Silica Clotting
the reproducibility of LA and aPS/PT antibody testing when Time HemosIL and dRVVT Screen and Confirm HemosIL,
performed in different expert centers and to assess the diagnostic respectively (Instrumentation Laboratory, Bedford, MA, USA).
performance of these tests in different clinical settings of APS. Both tests were automated on ACL TOP 750 LAS instruments
and results were normalized by means of plasma pools obtained
METHODS from healthy donors without any deficit in coagulation factors,
as per the current criteria from the ISTH Subcommittee on
Patients LA-Phospholipid-dependent antibodies (12, 13).
We chart-reviewed patients with thrombotic events who tested Both IgG and IgM aPS/PT were assayed using commercial
persistently positive for at least one aPL (more than two occasions ELISA kits (QUANTA Lite R , Inova Diagnostic), in accordance

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Sciascia et al. LA and aPS/PT Testing: A Multicenter Study

with manufacturer’s instructions. Samples were considered Overall, categorical agreement for LA among all the four
positive for aPS/PT IgG/IgM if tested >30 U. centers, as expressed by Cohen’s kappa coefficients, ranged
Agreement was defined when all four laboratories had a from 0.41 to 0.60 (corresponding to moderate agreement). The
concordant binomial result (positive/negative), both for LA and correlation among quantitative results for aPS/PT IgG/IgM was
aPS/PT IgG/IgM testing. strong (Spearman rho 0.84; when dichotomizing for positive vs.
negative results, Cohen’s kappa coefficients = 0.81–1.00).
Statistical Analysis Overall categorical agreement is resumed in Figure 1.
Categorical variables are presented as number (%) and We observed 27 (45.0% of the total) cases (15/20, 75% patients
continuous variables are presented as mean (S.D.). Categorical on VKA) in which LA results were discordant (defined by lack
agreement and degree of linear association was analyzed. The of agreement) or inconclusive. Conversely, in those cases, we
significance of baseline differences was determined by the observed a good correlation for aPS/PT IgG/IgM testing (Cohen’s
chi-squared test, Fisher’s exact test or the unpaired t-test, kappa coefficients = 0.81–1.00, Spearman rho 0.86).
as appropriate. A two-sided p-value < 0.05 was statistically When considering previous LA testing, we observed a
significant. All statistical analyses were performed using SPSS statistically significant higher agreement among centers of LA
version 19.0 (IBM, Armonk, NY, USA). testing if LA testing was previously positive [LA previously
positive testing vs. negative: full agreement among centers 74.5%
vs. 30.7% (chi Square test p < 0.05)]. Interestingly, the level
RESULTS of agreement of aPS/PT IgG/IgM among centers was similar
regardless of previous LA testing [LA previously positive vs.
Demographic, clinical and laboratory characteristics of the 60 negative: full agreement among centers 85.1% vs. 92.3% (chi
patients enrolled in the study are summarized in Table 1. Square test p = 0.49)].
Briefly, mean age at data collection was 49.9 years old (SD When stratifying patients according to the inclusion criteria,
± 10.9) (females: males = 71.7%: 28.3%). Forty-three patients we observed that in patients with confirmed diagnosis of APS,
(71.7%) had a confirmed diagnosis of thrombotic APS (arterial LA, and aPS/PT (IgG/IgM) agreements were 24/43 (55.8%)
58.1%; venous 56.3%), and 17 patients presented with thrombosis and 40/43 (93.0%), respectively. Conversely, in patients with
and inconsistent LA positivity [7/17 (41.2%)] and/or with low- thrombosis not completely fulfilling the Sydney laboratory
medium titers [10/17(58.8%)]. In the latest, 10/17 patients with criteria, we found aPL testing agreement among the four centers
suspected APS were tested positive (titer > 30 UI) for aPS/PT, as follows: LA 9/17 (52.9%) and aPS/PT IgG/IgM 11/17 (64.7%).
IgG and/or IgM.

DISCUSSION
TABLE 1 | Characteristics of the patients included in the study.
The diagnosis and consequent management as well as the
APS patients Suspected classification of APS relies on the identification of persistent
(43; 72%) APS (17; 28%)
aPL positivity in patients with thrombosis and/or pregnancy
ANAGRAPHIC
morbidity (11). Among aPL tests, LA has been shown to
Mean age (±S.D.) at 45.7 (±11.9) 51.9 (±7.3)
be the strongest risk factor for thrombotic events (15) and
data collection LA testing should always be performed in parallel with
Females 30 (69.8%) 11 (64.7%) aCL and aβ2GPI (3, 16–18) when a patients is investigated
CLINICAL MANIFESTATIONS for APS.
Arterial thrombosis 21 (48.8%) 5 (29.4%) However, despite significant progress in LA testing thanks to
Venous thrombosis 26 (60.5%) 12 (70.6%) the updated guidelines of the ISTH (12, 13), LA testing still suffers
aPL PROFILE AT DIAGNOSIS from some shortcomings and remain much more labor intensive
LA (positive, n)* 37 (86%) 11 (64.7%)
and complicated to perform compared to immunoassays.
aCL (IgG/M)* 22 (51.2%) 7 (41.2%)
In our study, when testing for LA in a blind fashion in four
aβ2GPI (IgG/M)* 23(53.5%) 6 (35.3%)
centers all undergoing regular external quality assessment (EQA)
ANTICOAGULANT THERAPY AT THE MOMENT OF TESTING
(14), we observed that up to 45% of LA positive samples were not
unanimously identified. When limiting the analysis to patients
VKA (warfarin) 18 (41.9%) 2 (11.8%)
with VKA, the observed level of agreement dropped to 55%.
LMWH 8 (18.6%) 2 (11.8%)
Is it well-known that one of the major drawbacks of LA tests
DOAC 13 (30.2%) 0
is their sensitivity to anticoagulant therapy (such as VKA, and
Anti-platelets therapy 17 (39.5%) 13 (76.5%)
DOAC), due to the coagulation based principle. Preferably, tests
SD, Standard Deviation; APS, Antiphospholipid Syndrome; aPL, Antiphospholipid should be postponed until therapy is stopped; however, in the
Antibodies; LA, Lupus Anticoagulant; aCL, Anticardiolipin Antibodies; anti-β2GPI, Anti- real world, requests during therapy still occur very frequently
β2Glycoprotein I antibodies; VKA, Vitamin K antagonists; LMWH, Low Molecular Weight
Heparins; DOAC, Direct Anticoagulants. *When considering patients with suspected APS:
with potentially false-positive or false-negative results (13, 19).
defined as inconsistent LA positivity and/or low levels of ACA IgG/IgM or anti-β2GPI In addition, it might be logistical inconvenient for the patients to
IgG/IgM antibodies 10–30 GPL/MPL. switch (or stop) anticoagulant therapy for LA testing purpose.

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Sciascia et al. LA and aPS/PT Testing: A Multicenter Study

FIGURE 1 | Results of lupus anticoagulant (LA) and anti-PS/PT antibody obtained in four laboratories. Results are summarized for all the patients included in the study
(Left) and for patients with suspected APS (Right).

When a thrombotic event occurs in patients suspected for these antibodies in APS (9, 23, 25, 26). Recent evidence support
APS with inconsistent LA positivity and/or with low-medium that while aPS/PT are frequently found in patients with LA,
aPL titers, clinical management can be challenging, as no their association with thrombosis seems to be independent of the
consensus exists on the choice and, more critically, the duration presence of LA (27).
of anticoagulation in this setting. In this study, when analyzing Among the so-called extra-criteria aPL tests, besides aPS/PT,
patients not completely fulfilling the criteria for APS, we observed antiβ2GPI-domain1 antibodies have been also proposed to
a level of LA agreement of only 53%, supporting the need of potentially improve the diagnostic accuracy in patients with
further diagnostic tool to help physicians in the management of suspected APS (28, 29), especially when assessing the risk for
these patients. both thrombosis and pregnancy morbidity. Other antibody
Autoantibodies directed toward PS/PT complexes have been specificities, such as anti-annexin A5 and anti-vimentin
extensively studied for their diagnostic and prognostic utility in antibodies, might be considered for thrombotic risk assessment
patients with suspected APS (9). Due to the observation that anti- only in selected patients, particularly when other aPL tests are
prothrombin antibodies associate significantly with LA (20–24), negative and in the presence of clinical signs and/or symptoms
several studies have sought to define the diagnostic relevance of strongly suggestive for APS (26, 30).

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Sciascia et al. LA and aPS/PT Testing: A Multicenter Study

In our cohort, aPS/PT testing showed an overall agreement of In conclusion, despite the progress in the standardization of
83% (up to 90% in patients receiving VKA), providing an overall aPL testing, we observed up to 45% of overall discrepant results
increase in test reproducibility of +28% when compared to LA, for LA, even higher in patients on VKA. Our findings showed that
becoming even more evident (+65%) when analyzing patients the persistence of significant discordance in the reliability of LA
on VKA. These observations have important implications. On testing. The introduction of aPS/PT antibodies in the diagnostic
the one hand, LA testing remains a cornerstone for APS process of APS might represent a further valuable diagnostic
diagnosis. On the other, ongoing efforts to reduce the LA tool, especially when LA is not available or reliable. In addition,
testing interlaboratory/interassay variations remain important. detection of aPS/PT antibodies provides another tool which can
Taking into account the methodological shortcomings of LA, complement and support current testing with aCL and aβ2GPI
aPS/PT might represent a reliable and reproducible test, even assays, and further help guiding clinical management.
during VKA or when APS diagnosis in uncertain. Besides the
diagnosis, these findings also might have significant implications DATA AVAILABILITY
for classification criteria and therefore for clinical trials of
new treatments. This author takes responsibility for all aspects of the reliability
Besides, albeit investigating the impact of aPS/PT testing and freedom from bias of the data presented and their
on the management of patients with suspected APS was discussed interpretation.
out of the scope of this study, one might note that up to
nearly 60% of the patients with suspected APS were found ETHICS STATEMENT
positive for aPS/PT. From a speculative point of view, this
observation might support a role for aPS/PT testing when APS This study was carried out in accordance with the
is suspected but currently classification criteria aPL are not recommendations for rare diseases in Piedmont Region,
fully informative/reliable. Northwest Italy with written informed consent from all subjects.
Although our investigation suffers for some limitations (cross- All subjects gave written informed consent in accordance with
sectional approach limiting the analysis of the longitudinal the Declaration of Helsinki.
fluctuation in aPL positivity; limited sample size; no further
analysis on the level of agreement for aCL and aβ2GPI), the AUTHOR CONTRIBUTIONS
strengths of this study relies on the blind approach of aPL
testing, performed in four different centers all undergoing SS, MR, IC, ER, MM, and DR drafted the manuscript, figures,
periodic EQA. Besides, in this investigation, we evaluated the and tables and critically reviewed the manuscript. AS, RR, BM,
robustness of aPS/PT ELISA testing in different clinical settings, PP, GM, EMu, SB, MF, AV, and EMe participated in laboratory
including patients suspected for APS but tested negative/low- testing and critically reviewed the manuscript.
titers for aCL and aβ2GPI antibodies. In such cases, a
further diagnostic tool for APS with reliable performances ACKNOWLEDGMENTS
might be crucial to guide the diagnostic process and to
avoid under/over treatment (31). Finally, testing for aPS/PT This study was performed under the support of the School
by a commercial kit was proven to be a reproducible and of Specialization of Clinical Pathology, Department of Clinical
accurate test for the detection of aPS/PT, bringing the added and Biological Sciences, University of Turin, Italy. The study
advantage of shorter running times when compared to in-house was presented in abstract form during the American College
assays (32). of Rheumatology.

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Frontiers in Immunology | www.frontiersin.org 6 March 2019 | Volume 10 | Article 376