Anti-Phospholipid Antibodies in
COVID-19 Are Different From Those
Detectable in the Anti-Phospholipid
Syndrome
Maria Orietta Borghi
1,2
, Asmaa Beltagy
1,3
, Emirena Garrafa
4,5
, Daniele Curreli
1
,
Germana Cecchini
6
, Caterina Bodio
1
, Claudia Grossi
1
, Simonetta Blengino
7
,
Angela Tincani
8
, Franco Franceschini
8
, Laura Andreoli
8
, Maria Grazia Lazzaroni
8
,
Silvia Piantoni
8
, Stefania Masneri
8
, Francesca Crisafulli
8
, Duilio Brugnoni
5
,
Maria Lorenza Muiesan
9
, Massimo Salvetti
9
, Gianfranco Parati
7
, Erminio Torresani
6
,
Michael Mahler
10
, Francesca Heilbron
7
, Francesca Pregnolato
1
, Martino Pengo
7
,
Francesco Tedesco
1
, Nicola Pozzi
11
*
and Pier Luigi Meroni
1
*
1
Immunorheumatology Research Laboratory, Istituto Auxologico Italiano, Istituto di Ricovero Cura a Carattere Scientifico (IRCCS),
Milan, Italy,
2
Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy,
3
Rheumatology and Clinical
Immunology Department, Faculty of Medicine, Alexandria University, Alexandria, Egypt,
4
Department of Molecular and Translational
Medicine,UniversityofBrescia,Brescia,Italy,
5
Department of Laboratory Diagnostics, Azienda Socio-Sanitaria Territoriale (ASST)
Spedali Civili, Brescia, Italy,
6
Departme nt of Chemical Chemistry, Istituto Auxologico Italiano , Istituto di Ricovero Cura a Carattere
Scientifico (IRCCS), Milan, Italy,
7
Department of Cardiovascular, Neural and Metabolic Sciences, Istituto Auxologico Italiano, Istituto
di Ricovero Cura a Carattere Scientifico (IRCCS), San Luca Hospital, Milan, Italy,
8
Rheumatology and Clinical Immunology Unit,
Department of Clinical and Experimental Sciences, Azienda Socio-Sanitaria Territoriale (ASST) Spedali Civili and University of Brescia,
Brescia, Italy,
9
Unità Operativa Complessa (UOC) 2° Medicina, Department of Clinical and Experimental Sciences, Azienda Socio-
Sanitaria Territoriale (ASST) Spedali Civili and University of Brescia, Brescia, Italy,
10
Research and Development, Inova Diagnostics,
Inc., San Diego, CA, United States,
11
Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine,
St.Louis,MO,UnitedStates
Background: Critically ill patients with coronavirus disease 2019 (COVID-19) have a
profound hypercoagulable state and often develop coagulopathy which leads to organ
failure and death. Because of a prolonged activated partial-thromboplastin time (aPTT), a
relationship with anti-phospholipid antibodies (aPLs) has been proposed, but results are
controversial. Functional assays for aPL (i.e., lupus anticoagulant) can be influenced by
concomitant anticoagulation and/or high levels of C reactive protein. The presence of anti-
cardiolipin (aCL), anti-beta2- glycoprotein I (anti-b
2
GPI), an d anti-phosphat idylser ine/
prothrombin (aPS/PT) antibodies was not investigated systematically. Epitope
specificity of anti-b
2
GPI antibodies was not reported.
Objective: To evaluate the prevalence and the clinical association of aPL in a large cohort
of COVID-19 patients, and to characterize the epitope specificity of anti-b
2
GPI antibodies.
Methods: ELISA and chemiluminescence assays were used to test 122 sera of patients
suffering from severe COVID-19. Of them, 16 displayed major thrombotic events.
Results: Anti-b
2
GPI IgG/IgA/IgM was the most frequent in 15.6/6.6/9.0% of patients,
while aCL IgG/IgM was detected in 5.7/6.6% by ELISA. Comparable values were found by
Frontiers in Immunology | www.frontiersin.org October 2020 | Volume 11 | Article 5842411
Edited by:
Rolando Cimaz,
University of Milan, Italy
Reviewed by:
Andras Perl,
Upstate Medical University,
United States
Tadej Avcin,
University Medical Centre Ljubljana,
Slovenia
*Correspondence:
Nicola Pozzi
nicola.pozzi@health.slu.edu
Pier Luigi Meroni
pierluigi.meroni@unimi.it
Specialty section:
This article was submitted to
Autoimmune and
Autoinflammatory Disorders,
a section of the journal
Frontiers in Immunology
Received: 16 July 2020
Accepted: 25 September 2020
Published: 15 October 2020
Citation:
Borghi MO, Beltagy A, Garrafa E,
Curreli D, Cecchini G, Bodio C,
Grossi C, Blengino S, Tincani A,
Franceschini F, Andreoli L,
Lazzaroni MG, Piantoni S, Masneri S,
Crisafulli F, Brugnoni D, Muiesan ML,
Salvetti M, Parati G, Torresani E,
Mahler M, Heilbron F, Pregnolato F,
Pengo M, Tedesco F, Pozzi N and
Meroni PL (2020) Anti-Phospholipid
Antibodies in COVID-19 Are Different
From Those Detectable in the Anti-
Phospholipid Syndrome.
Front. Immunol. 11:584241.
doi: 10.3389/fimmu.2020.584241
ORIGINAL RESEARCH
published: 15 October 2020
doi: 10.3389/fimmu.2020.584241
chemiluminescence. aPS/PT IgG/IgM were detectable in 2.5 and 9.8% by ELISA. No
association between thrombosis and aPL was found. Reactivity against domain 1 and 4-5
of b
2
GPI was limited to 3/58 (5.2%) tested sera for each domain and did not correlate with
aCL/anti-b
2
GPI nor with thrombosis.
Conclusions: aPL show a low prevalence in COVID-19 patients and are not associated
with major thrombotic events. aPL in COVID-19 patients are mainly directed against b
2
GPI
but display an epitope specificity different from antibodies in antiphospholipid syndrome.
Keywords: anti-phospholipid antibodies, b
2
-glycoprotein I, prothrombin, autoimmunity, COVID-19, thrombosis
INTRODUCTION
Critically ill patients with coronavirus disease 2019 (COVID-19)
have a profound hypercoagulable state and often develop
thrombosis in veins, arteries and in the microcirculation (1, 2).
A recent analysis showed several coagulation abnormalities in
these patients, including prominent elevation of fibrin/fibrinogen
degradation products (i.e., D-dimer) and a prolonged activated
partial-thromboplastin time (aPTT). While high levels of D-dimer
are consistent with sustained activation of the clotting and
fibrinolytic cascades, the combination of prolonged aPTT and
both arterial and venous thrombosis was, however, surprising, and
it is reminiscent of a clinical scenario known as antiphospholipid
syndrome (APS) (3).
Looking at the causes of aPTT prolongation, recent studies
have shown that lupus anticoagulant (LA) can be detected in a
significant percentage of COVID-19 samples (4–6). Since LA is
often caused by anti-phospholipid antibody (aPL), these findings
support the idea that aPL may play a role in COVID-19 (7).
However, it is important to point out that LA is a very sensitive
assay and its outcome can be influenced by several factors, most
notably heparin administration (8) and a profound inflammatory
state characterized by high levels of C reactive protein (CRP) (9,
10). Both of them are present in COVID-19 patients (11).
Another method to detect aPL that is in principle insensitive to
anticoagulation and other con founding agents re lies on the
detection and quantification of autoantibodies using solid-phase
assays (3). Using this method, the presence of aPL was recently
reported in a handful of case reports and small cohorts of patients
(4, 6, 7, 12, 13). While encouraging, this data is limited and its
interpretation remains controversial, with some investigators
proposing an important role of aPL in COVID-19 patients (7)
while others suggesting a very poor correlation between aPL and
thrombotic events (14). There is no information on the antigen
specificity of COVID-19 aPL in comparison with APS antibodies.
Such information and a larger study, possibly multicenter, may be
instrumental to clarify the real clinical value of these autoantibodies.
MATERIALS AND METHODS
Patients
A total of 122 patients were enrolled from two COVID-19
referral centers in Lombardia. All patients tested positive to
SARS-CoV-2, and classified as severe or c ritical COVID-1 9
(11). The mean age was 68.5 (± SD 16.4) years; 77 were men
and 45 women. No diagnosis of previous autoimmune diseases
was made; six patients had a thrombotic event (three arterial and
three venous) in the past clinical history. The presence of
antinuclear antibodies (ANAs) was investigated in 58 patients
at Istituto Auxologico Italiano by HEp2-IIF and solid phase CTD
screening following the guidelines described in Agmon-Levin
et al. (15). Of the 58 samples, none was positive for ANA.
Eighty-seven patients suf fering from APS were also tested for
anti-cardiolipin (aCL) and anti-b
2
GPI IgG/IgM (16).The study was
approved by the Ethics Committees (IstitutoAuxologicoItaliano3-
04-2020 - Milan and ASST Spedali Civili NP4187 - Brescia).
Detection of aPL
aCL and anti-b
2
GPI IgG/IgA/IgM were detected by
chemiluminescence immunoassay (CIA; Quanta Flash, Inova,
San Diego, CA, US) and a home-made ELISA as described ( 16,
17). Anti-b
2
GPI domain 1 IgG (anti-D1) were detected by CIA
(16, 17), IgG anti-D4-5 by a home-made ELISA, as described ( 16,
17). Detailed methods are reported in the Supplementary
Material. Anti-phosphatidylserine/prothrombin (aPS/PT)
IgG/IgM were detected by a commercial ELISA as reported
(18). Blood samples were collected in the first week after
hospital admission.
Statistical Analysis
Data were analyzed using R v3.4.0. Descriptive statistics was used
to summarize dat a. Associ ations and differences between
categorical or continuous variables were tested by Fisher’sexact
test and non-parametric Mann-Whitney test, respectively. A
p-value < 0.05 was considered statistically significant.
RESULTS
Patients
Table 1 reports the median with minimum and maximum values
for different coagulation and inflammation parameters in 122
severe or critical COVID-19 patients. In particular, prolonged
aPTT (>30 s) was found in 57.6% while PT INR values were
above the cut-off in 24.8% of the cases. Most of the patients (120/
122) were on anticoagulation with low molecular weight heparin
(70% on therapeutic and the remaining on prophylactic dosage).
Borghi et al. aPL Antibodies and COVID-19
Frontiers in Immunology | www.frontiersin.org October 2020 | Volume 11 | Article 5842412
Despite anticoagulation, we observed sixteen thrombotic events
(13.1%, 8 in veins and 8 in arteries). These statistics are in
agreement with previous reports (2, 19–24) and document a
systemic inflammation and a coagulopathy in our patients.
Anti-Cardiolipin and Anti-b
2
GPI
Antibody Testing
In the APS field, testing for LA is not recommended when
patients are on heparin, since the presence of heparin, even if
neutralized, may lead to false-positive results (8). Likewise, high
levels CRP, such as those found in our cohort of patients, have
been shown to prolong aPTT independently from the presence of
aPL (9, 10). On these bases, the presence of aPL was researched
using solid-phase assays, and not LA. First, we investigated the
presence of aCL and anti-b
2
GPI, two APS classification criteria
(3). Testing was independently performed in Milan and Brescia,
using harmonized methodologies (25). Th e prevale nce of
COVID-19 patients positive for aCL and anti-b
2
GPI IgG/IgA/
IgM detected by ELISA and CIA is summarized in Table 2. The
ELISA raw data are shown in Figure 1. We found IgG/IgM aCL
in 5.7/6.6% of patients, whereas anti-b
2
GPI IgG/IgA/IgM were
found in 15.6/6.6/9.0% of patients. Similar values were obtained
for aCL antibodies using CIA (Table 2), whereas a slightly lower
sensitivity was obtained for anti-b
2
GPI antibodies (26). The
positivity for aCL and anti-b
2
GPI antibodies was at medium/
low titer in contrast with the medium/high titers found in the
control group of primary APS (Figure 1). There is no association
between aPL positivity and thrombotic events.
Epitope Characterization of
Anti-b
2
GPI Antibodies
Fifty-eight sera were also tested with D1 and D4-5-coated plates
in order to characterize their epitope specificity. Figure 2B shows
that three out of 58 samples reacted with D1, while in Figure 2C,
TABLE 2 | Prevalence of COVID-19 patients positive for aPL.
ELISA CIA
aCL ab
2
GPI aCL CIA ab
2
GPI CIA
IgG 5.7 (7)* 15.6 (19) 9.8 (12) 5.0 (6)
IgM 6.6 (8) 9.0 (11) 6.6 (8) 5.0 (6)
IgA nd 6.6 (8) 2.5 (3) 0.8 (1)
*Values are expressed as percentage (n) of positive patients. aCL, anti-cardiolipin
antibodies; ab
2
GPI, anti-b
2
glycoprotein I antibodies; ELISA, enzyme linked
immunosorbent assay; CIA, chemiluminescence immunoassay; nd, not done.
A
B
FIGURE 1 | Titers of aCL and ab
2
GPI antibodies detected by ELISA in COVID-19 patients (black, n = 122) and comparison with APS patients (green, n = 86).
Values are expressed as median levels [first and third quartile]. (A) aCL. From the left to the right: COVID-19 IgG: 15 [8–15]; APS IgG: 65 [22–103]; COVID-19 IgM:
6.2 [2.6–10.8]; and APS IgM: 4.0 [1–11]. (B) ab
2
GPI. From the left to the right: COVID-19 IgG: 0.06 [0.04–0.10]; APS IgG: 1.14 [0.52–1.55]; COVID-19 IgM: 0.065
[0.02–0.142]; APS IgM: 0.23 [0.105–0.741]; and COVID-19 IgA: 0.04 [0.02–0.09]. Cutoff values are aCL IgG/IgM 20 phospholipid units (GPL/MPL); ab
2
GPI IgG/IgM/
IgA ELISA 0.13, 0.27, and 0.16 optical units (OD), respectively.
TABLE 1 | Coagulation and inflammation parameters expressed as median with minimum and maximum in severe or critical COVID-19 patients.
D-dimer
mg/L
CRP
mg/dl
Ferritin
mg/L
IL-6
ng/L
White cells
n/ml
Neuthrophils
n/ml
Plateletsn × 10
3
/ml PTratio aPTTs Fibrinogen
mg/dl
COVID-19 984.47 126.99 1,024 25.1 8000 6600 350 1.196 30.13 521
200–40,234 0.1–470.3 55–9,002 3–496 2,500–12,900 1,560–12,510 60–800 0.9–6.9 21–75.4 202–840
Normal range <500 0.00–0.05 30–400 <10 4,300–10,500 1,800–8,100 140–450 ≤1.2 <30 s 200–400
Borghi et al. aPL Antibodies and COVID-19
Frontiers in Immunology | www.frontiersin.org October 2020 | Volume 11 | Article 5842413
three samples tested positive for D4-5. None of the sera was
positive for both domains and all displayed a weak reactivity with
no association with thrombosis.
Anti-Phosphatidylserine/Prothrombin
Antibody Testing
Prolonged aPTT (>30 s) was found in 57.6% of the patients.
Although aPS/PT are not included in the APS classification
laboratory tests, they can be associated with a prolonged aPTT
and with the presence of LA (18). Consequently, we looked at the
presence of aPS/PT antibodies in our cohort and we found fifteen
out of 122 sera positive for aPS/PT (12.3%), mostly of the IgM
isotype (12 out 15) and at a low titer (Figure 3). There was no
association between prolonged aPTT and the presence of aPS/PT
antibodies nor with thrombotic events in our COVID-19 cohort.
DISCUSSION
Taken together, our data show a low prevalence of classification
criteria aPL in COVID-19 patients. In this regard, our study
confirms recent studies obtained with smaller cohorts of patients
(4, 14, 24). Importantly, our data also shows that aPL are slightly
more reactive towards b
2
GPI-coated plates as compared to CL-
coated ones and that, regardless of the nature of aPL, there is no
association between aPL positivity and thrombotic events (p = 1).
A striking difference between the autoantibody profile in
COVID-19 patients as compared to the one in APS concerned
the titers of aPL. Medium/low aPL titers were consistently found in
patients with COVID-19. By contrast, medium/high titers are
usually found in APS patients (Figure 1). This dif ference suggests
that aPL found in COVID-19 may be dif ferent from aPL found in
APS and led us to further investigate the epitope specificity of anti-
b
2
GPI antibodies. We focused on autoantibodies directed against
the N-terminal domain 1 (anti-D1) or the C-terminal domains 4-5
(anti-D4-5) of the molecule (17)(Figure 2A). This is because anti-
D1 antibodies are associated with an increased risk of thrombosis
and pregnancy complications in APS (16, 17, 28). By contrast, anti
D4-5 antibodies are associated neither with vascular nor obstetric
APS manifestations (16, 29). Furthermore, anti D4-5 antibodies are
also reported at high levels in the so called asymptomatic aPL
carriers and are frequently found in non-APS (e.g., patients with
leprosy, atopic dermatitis, atherosclerosis, and in children born to
mothers with systemic autoimmune diseases) (29). We found that
three out of 58 samples reacted with D1, and three samples tested
positive for D4-5. None of the sera was positive for both domains
and all displayed a weak reactivity. Although the number of the
investigated sera is relatively small, this finding is quite dif ferent
from the results found in APS in which almost all the sera positive
for the whole b
2
GPI molecule also reacted with domain D1 at high
titer (16, 28). Furthermore, at variance with APS patients, none of
the anti-D1 positive patients displayed thrombotic events (28).
Approximately, 57% of COVID-19 patients have prolonged
aPTT. Yet, only a small proportion of COVID-19 patients carry
aCL and anti-b
2
GPI antibodies. This suggests that other factors
must be responsible for the prolonged aPTT phenomenon and
likely for the LA activity. LA may be affected by the concomitant
heparin treatment and the high CRP levels. Although more
sensitive and specificdiagnosticalgorithmshavebeen
suggested (30), we followed the ISTH guidelines available at
the beginning of the study (31). Since aPS/PT can be associated
with a prolonged aPTT and with the presence of LA (18), we
tested our cohort for aPS/PT antibodies. We found a small
percentage (12.3%) of positive sera, mostly of the IgM isotype
(12 out 15) and at a low titer. Again, there was no association
between prolonged aPTT and the presence of aPS/PT antibodies
nor with thrombotic events in our COVID-19 cohort. This
indicates that aPS/PT are not responsible for the prolongation
of aPTT nor are predictors of adverse clinical outcomes.
Furthermore, in contrast to what we would have expected in
A
BC
FIGURE 2 | Epitope specificity of anti-b
2
GPI antibodies in COVID-19 patients. (A) Three-dimensional structure of b
2
GPI solved at 2.4 Å by X-ray crystallography [PDI
ID: 6V06 (27)] displaying the positioning of the fragments used in this study. The N-terminal D1 is shown in red. The C-terminal D4-5 fragment is shown in blue.
N-linked glycosylations are shown as magenta stick. Titers of anti-D1 (B) and anti-D4-5 antibodies (C) in 58 COVID-19 patients detected by chemiluminescence and
ELISA, respectively. Values are expressed as median levels [first and third quartile]. Anti-D1(aD1): 3.6 [3.6–4.7]. Anti-D4-D5 (aD4-D5): 0.10 [0.068–0.199]. Cutoff
values are >20 chemiluminescent units (CU) and >0.405 optical units (OD) for aD1 and aD4-D5, respectively.
Borghi et al. aPL Antibodies and COVID-19
Frontiers in Immunology | www.frontiersin.org October 2020 | Volume 11 | Article 5842414
APS (32), we found no associations between the presence of aPS/
PT, aCL, and anti-b
2
GPI antibodies. This data is in line with the
unusual epitope specificity of anti-b
2
GPI antibodies documented
in Figure 2, supporting the hypothesis that aPL found in
COVID-19 patients are different from aPL found in APS
patients. W hether COVID-19 aPL are similar to the ones
found in other infectious diseases such as HCV, HBV and HIV
(33) remains to be determined.
Despite heparin treatment, 13.1% of our patients
displayed thrombotic event s. Although we cannot e xclude
that treatment co uld be prot ecti ve, the prevalence of v ascular
events was in line with that reported by other studies as recently
reviewed (34).
In conclusion, while the medium/high aPL titers with D1
specificity are associated with vascular events in APS, low
antibody titers with reactiv ity against b
2
GPI epit ope(s)
different from D1 or D4,5 can be found in COVID-19. This
may explain the lack of association with thrombotic events in
COVID-19. In addition, our data do not support the hypothesis
that a PL can be the main cause of prolonged aPTT in these
patients. Although low titer aPL are not predictive of vascular
events in the APS, it i s importan t to keep in mind that COVID-
19 patients suffer from an acute form of systemic in flammation
with complement activation (35), which may be responsible
for endothelial perturbation. In this context, since b
2
GPI can
accumulate on the activated endot helium at high densi ty, even
low titers of aPL may bec ome pathoge nic thus poten tia ting or
even triggering thrombus formation, especially when
anticoagulation is suspended. A comparable condition in
which low titers of aPL can cause substantial damage is seen
in obstetric APS, where high levels of b
2
GPI can be fou nd in the
placenta (36). Hence, while transitory aPL are likely to be
clinically irrelevant in COVID-19 patient s as in other
infections (33), detection of aPL may be useful for identifying
patients potentially at risk of thrombosis after the hospital
discharge. Accordingly, anticoag ulant prophylaxis or therapies
affecting cell signaling involved in inflammatory and
coagulation responses could be justified before a confirmatory
assay (3, 37).
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.
ETHICS STATEMENT
The studies involving human participants were reviewed and
approved by Istituto Auxologico Italiano 3-04-2020 - Milan and
ASST Spedali Civili NP4187 - Brescia. The ethics committee
waived the requirement of written informed consent
for participation.
AUTHOR CONTRIBUTIONS
MB, MP, AT, FF, FT, NP, and PM designed the study. SB, GP,
FH, MP, ML, MLM, and MS collected clinical samples. EG, DC,
GC, CB, CG, SP, SM, FC, DB, ET, MM, and LA performed
research. MB, AB, FP, FT, NP, and PM analyzed data. MB, FT,
NP, and PM wrote the manuscript. All authors contributed to the
article and approved the submitted version.
FUNDING
The study was in part supported by IRCCS Istituto Auxologico
Italiano - Ricerca Corrente 2019 (PM), a grant from the Italian
Ministry of Foreign Affairs and International Cooperation
(MAECI) for foreign citizens and Italian citizens living abroad
(AB) and a National Institutes of Health Research Grant
HL150146 (NP).
ACKNOWLEDGMENTS
The authors would like to thank N. Carabellese and G. Martini
(Department of Laboratory Diagnostics; ASST Spedali Civili,
Brescia, Italy) for their valuable collaboration; all the physicians
of the COVID-19 Units of the IRCCS Istituto Auxologico
FIGURE 3 | Titers of aPS/PT antibodies detected by ELISA in COVID-19
patients. Values are expressed as median levels [first and third quartile]. aPS/
PT IgG:13.6 [8 to 15.2]; aPS/PT IgM:11.4 [8 to 16.5] IgM. Cut-off of the
assays was 30 units/ml.
Borghi et al. aPL Antibodies and COVID-19
Frontiers in Immunology | www.frontiersin.org October 2020 | Volume 11 | Article 5842415
