REVIEW ARTICLE

Ferrata Storti Foundation Seronegative antiphospholipid syndrome:

refining the value of “non-criteria” antibodies
for diagnosis and clinical management
Pasquale Pignatelli,1,2* Evaristo Ettorre,3* Danilo Menichelli,1 Arianna Pani,4,5
Francesco Violi,1,2** and Daniele Pastori1**
1
I Clinica Medica, Department of Internal Medicine and Medical Specialties, Sapienza
University of Rome, Rome; 2Mediterranea Cardiocentro, Naples; 3Department of
Cardiovascular, Respiratory, Nephrologic, Anesthesiologic and Geriatric Sciences, Division
of Gerontology, Sapienza University, Rome; 4Department of Oncology and Onco-
Haematologica 2020
Hematology, University of Milan, Milan and 5Clinical Pharmacology Unit, ASST Grande
Volume 105(3):562-572
Ospedale Metropolitano Niguarda, Milan, Italy
*PP and EE contributed equally to this work.
**FV and DP contributed equally to this work as co-senior authors.

ABSTRACT

A
ntiphospholipid syndrome (APS) is a systemic autoimmune disease
characterized by arterial and venous thrombotic manifestations
and/or pregnancy-related complications in patients with persistently
high antiphospholipid antibodies (aPL), the most common being represent-
ed by anticardiolipin antibodies (aCL), anti-beta 2 glycoprotein-I (aβ2GPI),
and lupus anticoagulant (LAC). A growing number of studies have showed
that, in some cases, patients may present with clinical features of APS but
with temporary positive or persistently negative titers of aPL. For these
patients, the definition of seronegative APS (SN-APS) has been proposed.
Nevertheless, the negativity to classic aPL criteria does not imply that other
antibodies may be present or involved in the onset of thrombosis. The diag-
Correspondence: nosis of SN-APS is usually made by exclusion, but its recognition is impor-
tant to adopt the most appropriate anti-thrombotic strategy to reduce the
PASQUALE PIGNATELLI rate of recurrences. This research is in continuous development as the clin-
[email protected].
ical relevance of these antibodies is far from being completely clarified. The
most studied antibodies are those against phosphatidylethanolamine, phos-
Received: June 10, 2019. phatidic acid, phosphatidylserine, phosphatidylinositol, vimentin/cardi-
Accepted: December 18, 2019. olipin complex, and annexin A5. Moreover, the assays to measure the levels
Pre-published: January 30, 2020. of these antibodies have not yet been standardized. In this review, we will
summarize the evidence on the most studied non-criteria aPL, their poten-
tial clinical relevance, and the antithrombotic therapeutic strategies avail-
doi:10.3324/haematol.2019.221945 able in the setting of APS and SN-APS.
Check the online version for the most updated
information on this article, online supplements,
and information on authorship & disclosures: Introduction
www.haematologica.org/content/105/3/562
The prevalence of antiphospholipid antibodies (aPL) in the general population is
difficult to estimate due to the lack of population-based studies. The most fre-
©2020 Ferrata Storti Foundation quently detectable aPL are anticardiolipin antibodies (aCL), antiβ2-glycoprotein I
Material published in Haematologica is covered by copyright. antibodies (anti-β2-GPI), and lupus anticoagulant (LAC).1 A large review of the lit-
All rights are reserved to the Ferrata Storti Foundation. Use of erature in 2013 estimated that the prevalence of aPL positivity is 6% among
published material is allowed under the following terms and
conditions:
women with pregnancy complications, 10% among patients with deep venous
https://creativecommons.org/licenses/by-nc/4.0/legalcode. thrombosis (DVT), 11% among patients with myocardial infarction, and 17%
Copies of published material are allowed for personal or inter- among patients with juvenile stroke (<50 years of age). As acknowledged by the
nal use. Sharing published material for non-commercial pur- Authors, this prevalence should be considered with caution, because 60% of the
poses is subject to the following conditions:
papers were published before 2000, all three criteria aPL tests were performed in
https://creativecommons.org/licenses/by-nc/4.0/legalcode,
sect. 3. Reproducing and sharing published material for com- only 11% of the papers, and 36% of papers used a low-titer aCL cut off.2
mercial purposes is not allowed without permission in writing Subjects carrying aPL who develop thrombotic complications are diagnosed with
from the publisher. the antiphospholipid syndrome (APS), which was first described in 1983 by
Hughes, who initially defined it as “anticardiolipin syndrome”.3 This definition was
derived from clinical observation of recurrent miscarriages, central nervous system

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 Seronegative antiphospholipid syndrome

disease, and recurrent venous thromboembolism (VTE) in plasma, IgM/IgG anti-β2 glycoprotein-I (aβ2GPI) antibod-
patients with systemic lupus erythematosus (SLE) and ies in serum or plasma, and LAC in plasma. Clinical and
serum positivity for anticardiolipin antibodies (aCL) and laboratory criteria are listed in Figure 1.7
lupus anticoagulant (LAC).3 Recently, Duarte-Garcia et al. The persistence of high antibody values should be test-
found an annual incidence of APS of 2.1 per 100,000 per ed at least 12 weeks apart, and, in addition, the antibody
year, with a prevalence of 50 APS patients per 100,000, titers should be dosed at least 12 weeks after the throm-
equally distributed between males and females.4 botic event but no more than five years afterwards.7,8 Of
A more clinically challenging scenario is represented by note, not all patients remain positive over time, and fac-
patients with a clinical history characterized by episodes tors associated with persistence of aPL positivity are not
of thrombosis (especially if recurrent) without cardiovas- well known, but may include inflammation and oxidative
cular risk factors, and more in general, in absence of an stress.9
identifiable cause of thrombosis, suggestive of a throm- Subjects positive for aPL have a low risk of developing
bophilic condition, such as APS, but in absence of any pos- thrombotic events (<1%/year), but after a first episode,
itivity of aPL. For these patients, the definition of seroneg- the risk of recurrence increases by 10-67%.10 This finding
ative APS (SN-APS) was proposed.5 In the context of SN- was supported by a recent study performed by Kearon et
APS, several non-criteria aPL have been investigated with al.,11 in which 307 patients with a first unprovoked VTE
divergent results. were tested for aPL. In this study, the persistence of aPL on
In this review, we will discuss criteria for defining the ≥2 occasions was associated with an increased risk of
SN-APS, the new potential non-criteria antibodies implied recurrent thrombosis, despite negative D-Dimer values
in SN-APS and its clinical management. (HR 4.5: 95%CI: 1.5-13.0; P=0.006).11

Clinical presentation of antiphospholipid syndrome

Antiphospholipid syndrome Antiphospholipid syndrome can be broadly classified in
venous, arterial or obstetric APS, which are, however, not
Diagnosis of antiphospholipid syndrome mutually exclusive. In a retrospective analysis of a cohort
Antiphospholipid syndrome is a systemic autoimmune of 160 patients with a definite APS, VTE was the most
disorder characterized by arterial and venous thrombotic common manifestation (47.5%), followed by arterial
manifestations and/or pregnancy morbidity in patients thromboembolism (43.1%), while obstetrical complica-
with persistently high levels of aPL.6 APS may be classified tions was found in only 9.7% of patients; in this study,
as primary or secondary, the latter being present in 30- catastrophic APS (C-APS) represented 2.5% of the cases.12
40% of patients with SLE.7 The 2006 Sapporo criteria are Stroke and transient ischemic attack often involve APS
those currently recommended to diagnose APS.8 They patients, but also lower limb ischemia and myocardial
include the presence of one clinical criterion and high val- infarction can occur.12 In this context, the relationship
ues of at least one aPL among IgM/IgG aCL in serum or between aPL and myocardial infarction seems to be bidi-

Figure 1. Summary of criteria for antiphospholipid syndrome (APS) diagnosis according to Sapporo criteria. GPL: glycopeptidolipid; MPL: monophosphoryl lipid A.

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 P. Pignatelli et al.

rectional. Therefore, patients carrying aPL have an examinations are mandatory. An MRI and an electroen-
increased risk of ischemic heart disease, and conversely, cephalogram could be useful in recognizing brain atrophy
after a first coronary artery disease event, patients positive associated with dementia and seizures, and could identify
for aPL showed twice the risk of recurrent major adverse more elusive symptoms such as chorea and migraine if
cardiovascular events at 12 and 24 months.13 This risk was associated with an accurate physical examination.
also evident in subjects with juvenile myocardial infarc- Other blood alterations include thrombocytopenia
tion in absence of cardiovascular risk factors.13 (commonly mild with platelet count between 50x109/L
Concerning obstetric complications, fetal loss, especially and 150x109/L, but also severe with platelet counts
after 10th week of gestation and premature birth due to <20x109/L often associated with microangiopathy) and
eclampsia or placental insufficiency are frequent complica- hemolytic anemia with the possible presence of schisto-
tions in APS women.14 In young women with a history of cytes.6 In particular, thrombocytopenia is common in APS,
multiple miscarriage, the immunological study for aPL affecting 20-46% of patients and could paradoxically be
should be considered. associated with an increased risk of thrombosis.18
Finally, catastrophic APS (C-APS) is a severe and life- Thrombocytopenia may be the result of an increased acti-
threatening manifestation characterized by simultaneous vation and destruction of platelets by an immune-mediat-
venous or/and arterial thrombosis, often triggered by ed mechanism involving aPL or by thrombotic microan-
infections and surgical procedures. C-APS involves multi- giopathy.22
ple organs and systems due to excess of proinflammatory After the exclusion of a pseudo-thrombocytopenia, and
cytokines, coagulation cascade, and platelet activation, performing a Coombs test to ascertain the autoimmune
leading to thrombosis and microangiopathic hemolytic nature of thrombocytopenia, corticosteroids, immunosup-
anemia.15 pressive agents, immunoglobulins and new drugs such as
In addition to the above described signs and symptoms, Mammalian target of rapamycin (mTOR) inhibitors and
the clinical presentation of patients with APS may be monoclonal antibodies could be helpful in patients with
more heterogeneous, involving thrombosis of medium autoimmune thrombocytopenia.23
and small vessels (Table 1).6,7,16-18 The relevance in clinical
practice of non-conventional APS criteria was investigated
during the 14th Congress on Antiphospholipid Antibodies, Definition of seronegative antiphospholipid
in which each relevant clinical manifestation was ana- syndrome and non-criteria antiphospholipid
lyzed and each evidence was evaluated by the GRADE
system. This system also considers the balance of patient- antibodies
important outcomes, the overall quality of the evidence
for each outcome, and any uncertainty about values.18 The first definition of SN-APS was given in 2003 by
The most commonly affected sites are the kidney, the Hughes and Khamashta5 who described patients with clin-
skin, and the cardiovascular and nervous systems. In the ical manifestations highly suggestive of APS in absence of
kidney, it is possible to find an acute thrombotic the laboratory criteria such as LAC, aCL and aβ2GPI anti-
microangiopathy or a chronic pattern of vaso-occlusive bodies.
lesions such as cortical ischemic lesions, arterial fibrous Seronegative APS is usually a diagnosis of exclusion and
intimal hyperplasia or interstitial fibrosis.6 APS should be suspected in patients with a clinical history sug-
nephropathy can be identified with a urine test associat- gestive of APS, such as those with recurrent arterial venous
ed with a 24-hour investigation of proteinuria. A biopsy thrombotic events, recurrent miscarriage, or unexplained
is mandatory in cases where the cause is not clearly iden- thrombocytopenia, with persistent negativity of aPL tested
tifiable, as in patients with concomitant diabetes, uncon- on at least two occasions, and when other causes of throm-
trolled arterial hypertension or other autoimmune dis-
eases such as SLE.
Concerning the skin, livedo reticularis can be found, and Table 1. “Extra-criteria” manifestations of antiphospholipid syndrome.
recurrent ulcerations called livedoid vasculopathy have
Nervous system
also been described.8 To evaluate skin abnormalities, a Dementia
clinical examination is often adequate, and there is usually Seizures
no need for skin biopsy. Multiple sclerosis–like illness
Cardiac abnormalities include valve leaflet thickening,6 Chorea
and diastolic dysfunction, especially of the right ventri- Myelitis
cle.19 Heart valve disease and diastolic dysfunction can be Skin
investigated by resting transthoracic echocardiography Livedo reticularis
and by cardiac magnetic resonance imaging (MRI) if a Livedoid vasculopathy
myocardial involvement is suspected (i.e. myocarditis).
Heart
Pericardium may also be involved, especially in patients
Valve vegetations or thickening (Libman-Sacks Endocarditis)
with APS and SLE. Diastolic dysfunction
Finally, APS is associated with an increased risk of
dementia, seizures, multiple sclerosis-like illness, Blood
migraine, myelitis transversa and chorea, due to vascular Thrombocytopenia
Hemolytic anemia
damage and a direct action of antibodies on neurons and
ependymal cells.7,20,21 However, unlike other neurological Kidney
disorders, seizure is not considered a non-conventional Microangiopathy
criterion due to lack of strength of evidence.18 To identify Chronic vaso-occlusive lesions (atherosclerosis, glomerular
critical illness such as neurological disorders, instrumental ischemia, interstitial fibrosis, arterial fibrous intimal hyperplasia)

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 Seronegative antiphospholipid syndrome

bosis are excluded, such as genetic thrombophilia (factor V To better characterize the entity of SN-APS, antibodies
and II mutations), active cancer, trauma, major surgery, or against different phospholipids or protein co-factors have
prolonged bed rest. This is particularly evident in young been investigated in patients negative to conventional aPL
patients without established cardiovascular risk factors (i.e. (Table 2).
obesity, diabetes, hypertension, dyslipidemia). Most The antibodies that have been most studied so far are
importantly, other forms of coagulopathy should be those directed against: 1) a zwitterionic phospholipid,
excluded first, including Protein C and S and anti-thrombin namely phosphatidylethanolamine (PE); 2) negatively
deficiency. In addition, the patient's personal medical his- charged phospholipids other than cardiolipin, including
tory should be carefully investigated to exclude previous phosphatidic acid (PA), phosphatidylserine (PS), phos-
positivity to aPL. phatidylinositol (PI); 3) vimentin (forming a complex with

Table 2. Summary of positivity for extra-criteria antibodies in each study of seronegative antiphospholipid syndrome (SN-APS).
Authors Study typology Population aPL positivity in SN-APS (n) Main findings
(year) P=prospective; studied
R=retrospective
CS=cross-sectional APS SN-APS IgA aPL aPE NCP AVA/CL aPS/PT aANX
CaS= case series (n) (n)
Sanmarco39 CS 67 18 - 18 - - - -
(2001)
Sanmarco40 CS - 25 - 25 - - - - 25 of the 40 aPE-positive patients (63%) were
(2007) negative for the APS laboratory criteria
Kumar83 CaS - 5 5 - - - - -
(2009)
Ortona47 CS 40 29 - - - 27 - - Vimentin seems to be positive in a large
(2010) number of mainly SN-APS patients.
Conti 49 CS 25 24 - - - 11 1 1 SN-APS were positive for 11/24 (45.8%) for
(2013) anti-vimentin/cardiolipin antibodies, 3/24 (12.5%)
for anti-prothrombin antibodies, and 1/24 (4.2%) for
anti-annexin V antibodies.
Ruiz-García35 CS 22 35 35 - - - - - Isolated IgA aβ2GPI antibodies were found in 22%
(2014) of patients. Patients with arterial thrombosis were
positive only for IgA aβ2GPI.
Cousins32 CS 40 40 1 - - - - - IgA aCL or IgA aβ2GPI antibodies, were present in
(2015) a significant proportion of patients with APS,
and in a small proportion of SN-APS.
Mekinian27 CS 83 96 - 47 - 5 57 68% of patients with obstetrical SN-APS have
(2016) non-conventional aPL
Zohoury24 CS 107 68 - 8 - 11 8 - 1/3 of SN-APS patients showed reactivity to 1
(2017) or more non-criteria markers
Tortosa36 R - 38 38 - - - - - The presence of IgA aβ2GPI in people with no history
(2017) of APS-events is the main independent risk factor
for the development of these types of events,
mainly arterial thrombosis
Litvinova25 CS 41 17 - - 5 - 4 4 87 patients: 41 APS, 11 aPL carriers, 17 SN-APS.
(2018) <1% of patients with thrombotic/obstetrical
SN-APS had non-conventional aPL
Anti-PS/PT antibodies were correlated with LA.
APS triple patients were also positive for
anti-PS/PT antibodies
Truglia50 CS - 61 - - - 33 - - Non-conventional tests, mainly aCL/Vim and aCL
(2018) seem to be the most sensitive approaches
for identifying aPL in patients with obstetric SN-APS
Billoir84 R - 23 - 23 - - - - aPE persists in 23 patients (10%): 15 with
(2019) a thrombotic event, 6 with obstetrical morbidity
and 2 with a combined event
Ganapati61 R 58 12 - - - - 7 - Addition of aPS/PT to current APS criteria to SN-APS
(2019) patients led to reclassification of additional 12.1%
patients as APS overall and 42.8% in obstetric
APS category
aANX: Annexin A5 antibody; aPE: phosphatidylethanolamine; aPL: antiphospholipid antibodies; APS: antiphospholipid syndrome; aPS/PT antiphosphatidylserine/prothrombin; AVA/CL:
anti vimentin/cardiolipin complex; LA: lupus anticoagulant; NCP: negatively charged phospholipids (phosphatidic acid, phosphatidylserine and phosphatidylinositol); SN-APS: seronega-
tive APS.

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 P. Pignatelli et al.

cardiolipin); 4) prothrombin (forming complex with PS – ciated to APS as compared to IgG aβ2GPI (HR 33.4,
anti-PS/PT); and 5) the anticoagulant protein Annexin A5 95%CI: 13.0-86.1), but showed a higher association com-
(Table 2). In addition, the IgA isotype aβ2GPI is under pared to IgM aβ2GPI (HR 9.2, 95%CI: 4.6-18.4) and was
investigation in APS and SN-APS patients. associated with thrombotic but not obstetric complica-
A collaborative USA/UK study analyzed a comprehen- tions in patients with APS.34
sive panel of ‘non-criteria’ aPL tests in a series of 175 con- Indeed, IgA aβ2GPI levels seem to be associated with
secutive patients matching the criteria for APS and 68 SN- thrombotic events in patients without other aPL.33,35 Thus,
APS patients with clinical manifestations suggestive of a case-control study including 244 asymptomatic patients
APS but having negative serology. The Authors found that screened for aPL and positive only for IgA aβ2GPI and 221
one-third of the ‘seronegative’ sera gave positive results.24 negative patients followed for five years showed that the
The study concluded that patients with clinical features of presence of IgA aβ2GPI was associated with an increased
APS, but negative for conventional criteria markers, risk for developing clinical thrombotic APS events (OR
should undergo additional testing for non-criteria bio- 5.15; P<0.001).36
markers.24 Although attractive, these data were not confirmed by
A recent study evidenced that positivity for the extra- another study evaluating the presence of IgA aβ2GPI anti-
criteria aPL was <1% in SN-APS (thrombotic or obstetric); bodies in SN-APS.25
however, the lack of clear inclusion and exclusion criteria Based on this evidence, it is not clear whether testing for
does not allow a precise estimation of the prevalence to be IgA aCL and IgA aβ2GPI antibodies in addition to the rou-
made.25 tine tests may improve thrombotic risk stratification.
Similarly, non-criteria antibodies were detected in Thus, the use of IgA antibodies to identify a SN-APS
18.8% of SN-APS patients also in a Chinese cohort com- needs to be further investigated.
posed of APS and patients with only clinical criteria for
APS.26 This is also confirmed in obstetric SN-APS patients, Antibodies to phosphatidylethanolamine
in whom 68% were positive for non-conventional aPL.27 Phosphatidylethanolamine is mainly found in the inner
Here we will provide an overview of current evidence leaflets of plasma membranes and contributes to 20-50%
on the most studied non-criteria aPL that, although not of total phospholipids. It works as an anticoagulant by
validated in large cohort studies, may have a potential role enhancing activated protein C (APC) activity. Other
in the pathogenesis of APS. investigators have demonstrated that PE inhibits coagula-
tion activity interfering with the factor Xa-prothrombin
IgA antibody isotype anti-β 2 glycoprotein-I system.37
and anticardiolipin antibodies Several studies reported that antibodies against PE (aPE)
There is a growing body of evidence to suggest a poten- are significantly associated with major clinical events
tial usefulness of IgA in the context of APS.28 Very recent such as fetal loss and/or thrombosis, and are mainly pres-
evidence suggested that, while IgG/M isotypes recognize ent in the absence of the laboratory criteria of APS. Bérard
an epitope in domain 1, the epitopes recognized by IgA et al. showed that aPE were the only aPL found in 6 of 34
are the domains 3, 4 and 5.29 However, as reported by the patients suffering from thrombotic events and with a neg-
13th International Congress on Antiphospholipid ative screening for antibodies to anionic phospholipids,
Antibodies, testing for IgA-aβ2GPI should be considered including LA.38 A second study focused on patients with
only in patients negative for IgG and IgM isotypes with unexplained thrombosis and no criteria for APS. Thus, in
APS symptoms.30 98 patients with unexplained thrombosis, 142 with
Studies investigating the prevalence of IgA aPL reported thrombophilia, 67 with APS, 75 with hereditary hemo-
a variable prevalence ranging from 14% to 72% according static defects and 110 without thrombosis, the authors
to different reports;28 however, these studies have a retro- found that aPE prevalence was significantly higher both
spective design, used different assays to measure IgA aPL, in patients with APS (43%; P<0.0001) and in those with
and used different cut-off values to define aPL positivi- unexplained thrombosis (18%; P=0.001) compared to
ty.31,32 patients without thrombosis.39 Subsequently, in a large
A large study including 5,892 patients (803 with SLE and multicenter study including 317 patients with deep
5,089 from the Antiphospholipid Standardization venous thrombosis and 52 with arterial events, aPE were
Laboratory sent for evaluation for APS) found that IgA found in 15% of the thrombotic patients, most of whom
aβ2GPI isotype was positive in 255 (4.3%) patients, in 198 were only positive for aPL.40 Some interesting data were
cases in association with other aPL, while only aPL was also reported regarding the association between aPE and
detectable in 57.33 Isolated IgA aβ2GPI positivity was asso- obstetric complications. Gris et al. measured various aPL
ciated with an increased risk of arterial thrombosis in a large cohort of 518 women with unexplained or
(P<0.001), venous thrombosis (P=0.015), and all thrombo- explained early fetal losses and a control group of healthy
sis (P<0.001).33 mothers. IgM-aPE were found to be independent risk fac-
A second study evaluated, in addition to IgM and IgG, tors for unexplained early fetal loss.41
the positivity and predictivity of IgA aCL and IgA aβ2GPI A retrospective study on 228 SN-APS demonstrated a
in 430 patients: 111 with APS, 119 with SLE, and 200 positivity for aPE in 10% of the patients.42 In contrast, a
healthy controls.34 Positivity for IgA aCL was 38%; IgA recent study on a Chinese population composed of APS
aβ2GPI was 46% in patients with APS. All three antibody and patients with only clinical criteria for APS failed to
isotypes (IgM, IgG and IgA) were significantly associated demonstrate any aPE positivity in SN-APS.26
with a diagnosis of APS, with high specificity but not The above reported results suggest that aPE could be
good sensitivity, based on receiver operating characteristic considered as markers of a variant of APS when they are
(ROC) analysis. Looking at likely hazard ratios, the IgA associated with thrombosis and a potential tool to define
aβ2GPI (HR 33.9, 95%CI: 10.5-109.5) was similarly asso- the SN-APS subjects.

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 Seronegative antiphospholipid syndrome

Antibodies against phosphatidic acid, tic marker. Hence, the observation by Ortona et al.47 needs
phosphatidylserine and phosphatidylinositol to be confirmed by larger prospective clinical studies in
In an effort to expand the panel of aPL to other negative- order to better define the role of AVA/CL in SN-APS.
ly-charged phospholipids, antibodies against phospha-
tidic acid (PA), phosphatidylserine (PS), and phos- Anti-prothrombin and
phatidylinositol (PI), which fall under the category of
anionic phospholipids, were proposed.43 Anti-PS antibod- antiphosphatidylserine/prothrombin antibodies
ies inhibited the development and invasion of the tro- Prothrombin is a plasma glycoprotein involved in the
phoblast, decreased hCG levels, and retarded the forma- coagulation cascade converted to thrombin by extrinsic
tion of syncytiotrophoblast in in vitro models.44 Few clinical thromboplastin during the second stage of blood clotting.51
studies have investigated this issue. In a first study on 866 A large amount of data, obtained from various, mainly ret-
women with recurrent pregnancy loss (RPL), the authors rospective, studies gave contrasting evidence concerning
found that 87 of 866 women who were negative for aCL the clinical significance of anti-prothrombin antibody
had a positivity for one of the other aPL.42 In a second (aPT). Thus, in a comparison between 106 subjects who
study on 872 women with RPL, 49 (3.6%) were negative experienced either a non-fatal myocardial infarction or
for both aCL and LA but positive for aPS.46 In this second cardiac death and 106 subjects without coronary disease,
study, the presence of aPS had a positive correlation with Vaarala et al. found that a high level of aPT (highest tertile
the number of consecutive pregnancy losses.46 This result of distribution) predicted a 2.5-fold increase in the risk of
was not confirmed when the same author analyzed a larg- cardiovascular events.52 Conversely, Atsumi et al. did not
er population of 1,020 woman with RPL.46 Moreover, find any correlation between clinical manifestation of aPT
Zhang et al. did not find any positivity for aPE, aPS or aPI and APS in an evaluation of 265 APS patients.53 More
in an evaluation study of 288 subjects (86 patients with recently, two prospective studies validated the role of aPT
APS, 30 patients with non-APS thrombosis, 32 patients in predicting the first or recurrent risk of thrombosis in
with non-APS pregnancy-related morbidity, 42 patients patients with APS.54,55 Considering a group of 142 LA pos-
with SLE, and 39 healthy controls).26 itive patients, Forastiero et al. found that a higher rate of
Based on the current evidence, testing for aPA, aPI and thrombosis in patients with positive anti-PT compared
aPS is not recommended, as these antibodies appeared to with patients without anti-PT (8.6% vs. 3.5% per patient
overlap with the accepted diagnostic markers of APS. year). The highest incidence of thrombosis was detected
Nonetheless, the results obtained on RPL with a seroneg- in patients positive for both aβ2GPI and aPT (8.4% per
ative profile suggest a potential role for aPA, aPI and aPS patient year).54 Moreover, a 15-year longitudinal prospec-
in defining SN-APS in this particular setting. tive study by Bizzaro et al. identified IgG aPT antibody as
the most useful thrombosis predictor in SLE patients.55
Another intriguing issue is represented by the different
Anti-vimentin/cardiolipin complex potential role of IgG/IgM antiphosphatidylserine/pro-
thrombin (aPS/PT) compared to aPT. Indeed, a high corre-
Vimentin is the most abundant type III intermediate fil- lation between APS classical antibody panel and aPS/PT
ament of the cytoskeletal system and it was recently local- IgG/IgM suggests that this marker may be useful in the
ized on the surface of apoptotic neutrophils and T cells, evaluation of APS.56 The clinical significance of aPT and
activated macrophages, platelets, and vascular endothelial aPS-PT was evaluated by testing for the presence of these
cells. After becoming antigenic with a still unexplained antibodies in 212 SLE patients and in 100 healthy individ-
mechanism, Vimentin is exposed and could be bound by uals. Results show that aPT and aPS-PT were found in
anti-vimentin antibodies (AVA).47,48 Vimentin could also 47% of the patients (aPT in 31% and aPS-PT in 31%).
electrically interact with cardiolipin on the surface of Their presence did not correlate with that of aCL, aβ2GPI,
apoptotic cells generating the vimentin/cardiolipin com- LA and/or anti-protein S. IgG but not IgM aPT were more
plex. Antibodies against this complex (vimentin/cardi- frequently found in patients with thrombosis than in
olipin antibodies, AVA/CL) show a prothrombotic effect. those without. IgG and IgM aPS-PT were also more fre-
Thus, Ortona et al. demonstrated an AVA/CL-mediated quent in patients with thrombosis (venous and/or arterial)
activation of the TLR4/IRAK/Nf-kB molecular pathway than in those without. Levels of IgG aPT and IgG and IgM
that leads to the release of pro-inflammatory and procoag- aPS-PT were higher in patients with thrombosis than in
ulant factors by endothelial cells.47 Hence, AVA/CL could those without. More significantly, 48% of the patients
play a role in arterial thrombosis by inducing platelet and with aPL-related clinical features who were negative for
coagulation cascade activation. standard tests had aPT.57 Recently, the clinical significance
The role of AVA/CL in SN-APS has been investigated of aPS/PT antibodies was prospectively evaluated in a
only in a few clinical studies. Thus, Ortona et al.47 ana- cohort of 191 aPL carriers:58 IgG aPS/PT antibodies were
lyzed serum IgG AVA/CL antibodies detected by ELISA in detected in 40 (20.9%) and IgM aPS/PT in 102 (53.4%) of
29 SN-APS, 40 APS, 30 patients with SLE, 30 with the carriers. The cumulative incidence rate of thrombotic
rheumatoid arthritis, and 32 healthy controls. They found events was significantly higher in the IgG aPS/PT positive
a positivity for AVA/CL in almost all APS patients (92%), (P=0.035) but not in the IgM aPS/PT positive carriers.
and also in a large proportion of SN-APS (55%); interest- Similar results were obtained in a second study evaluating
ingly, this positivity was persistent in almost all cases. 152 patients with a previous thrombosis of whom 90
Similarly, Conti et al. found AVA/CL positivity in 24 SN- were SN-APS; 10% of SN-APS patients in this study were
APS patients.49 Moreover, in a retrospective analysis of 61 positive for aPS/PT.59 Of note, aPS/PT are associated with
obstetric SN-APS, 76% resulted positive for AVA/CL.50 recurrent early or late abortions and with premature deliv-
However, the overlapping presence of AVA/CL antibodies ery irrespective of other aPL.60
in SLE and APS weakens the specificity of such a diagnos- Based on the above studies, aPT and aPS/PT can be

haematologica | 2020; 105(3) 567

 P. Pignatelli et al.

potentially used as confirmatory diagnostic markers and patients with double or triple positivity (OR >9).67
as indicators of the risk of thrombosis. Recently, the pres- Some scores have also been proposed to stratify the risk
ence of IgG and IgM aPS/PT was also detected in 9 of 17 of events in APS patients; the APL Score has no clinical
SN-APS.25 Similar, and even stronger evidence was provid- items and is based exclusively on the antibody titers
ed by two retrospective studies on SN-APS patients that (Table 3).68 An aPL score of ≥30 was an independent risk
found approximately 50% of subjects were positive for factor for thrombosis (hazard ratio 3.144, 95%CI: 1.383-
aPS/PT.61,62 Nonetheless, further studies must be undertak- 7.150; P=0.006) in patients with autoimmune diseases.69
en before these antibodies can be included in the diagnos- Another score is the Global Anti-Phospholipid
tic criteria of SN-APS. Syndrome Score (GAPSS), which was developed in a
cross-sectional study on a cohort of 211 patients with SLE.
The score includes traditional cardiovascular risk factors
Annexin A5 antibody such as hypertension and hyperlipidemia and the presence
/ absence of aPL70 (Table 3). Of note, both scores also
Annexin A5 is a glycoprotein that binds to negative include one non-criteria aPL, such as aPS/PT.
phospholipids such as PS. It has been proposed that These scores, although potentially useful in clinical
annexin A5 forms a protective anticoagulant shield on vas- practice, require further prognostic validation.
cular endothelial cells and that aβ2GPI antibodies in com- The thrombotic risk stratification is more challenging in
plex with ß2GPI may disturb the shield and hence predis- patients with SN-APS. It is important to identify and char-
pose to thrombosis. Due to this marked heterogeneity, it acterize the presence of non-criteria aPL, as they seem to
remains controversial whether anti-annexin A5 antibodies be associated with different thrombotic complications
(aANX) are associated with clinical manifestations. In a (Table 4). Thus, aPS/PT and antibodies to vimentin/CL
comparison of 112 APS patients with 40 healthy controls, complex increase the risk of arterial thrombosis, while
Singh et al. found aANX positivity in 69 APS and in only 3 pregnancy-related complications are associated with the
controls.63 On the contrary, de Laat et al. found no associ- presence of PE, PA, PS and PI antibodies (Table 4).
ation between aANX and history of thrombosis in 198
patients with primary APS, SLE or lupus-like disease.64
aANX was also found to be predictive for fetal loss in a
study of three groups (total 518 women) of patients with
unexplained primary recurrent early fetal loss, patients Table 3. Scores for risk stratification in antiphospholipid syndrome.
with explained episodes, and mothers with no previous
obstetric incident, respectively.41 APL Score - ITEMS CUT-OFF POINTS
Annexin A5 resistance was proposed as a mechanism APTT mixing >49 sec 5
for APS. The annexin A5 resistance (A5R) assay identifies CONFIRMATION TEST, ratio >1.3 2
patients with an antibody-mediated disruption of annexin >1.1 1
A5 on endothelial surfaces. This is demonstrated by the KCT mixing >29 sec. 8
resistance to the annexin A5 anticoagulant effects (i.e.
annexin A5 resistance) in vitro. This test was validated in DRVVT mixing >45 sec. 4
750 patients with a history of thrombosis, pregnancy CONFIRMATION TEST, ratio >1.3 2
complications, and controls.65 The authors found a reduc- >1.1 1
tion in A5 anticoagulant ratios in aPL antibody-positive IgG ACL, GPL.
patients with thrombosis and/or pregnancy complications High titers >30 20
compared with aPL antibody-negative patients and con- Medium/low titers >18.5 4
trols. This suggests that reduced A5R could identify IgM ACL, MPL >7 2
patients with a propensity for thrombosis or pregnancy IgG Anti-β2GPI
complications.65 High titers >15 U 20
Very recently, a case report described the presence of Medium/low titers >2.2 U 6
multiple annexin autoantibodies in a patient with recur-
IgM anti-β2GPI >6 U 1
rent miscarriages, fulminant stroke, and SN-APS.66 Hence,
although attractive, the evaluation of aANX or A5R in IgG aPS/PT
clinical practice for the management of SN-APS requires High titers >10 U 20
Medium/low titers >2 U 13
larger prospective studies.
IgM aPS/PT >9.2 U 8

From risk assessment to antithrombotic GAPSS Item Points

treatment CLINICAL Hyperlipidemia 3
The first step in the assessment of thrombotic risk in Arterial hypertension 1
APS and SN-APS patients is represented by antibody char- LABORATORY aCL IgG/IgM 5
acterization and evaluation of cardiovascular risk factors. Anti β2GPI IgG/IgM 4
Thus, the thrombotic risk varies according to aPL positiv- aPS/PT IgG/IgM 3
ity and antibody titers. For example, a retrospective study LA 4
on 3,088 APS patients demonstrated that single positivity Total 20
APTT: activated partial thromboplastin time; KCT: kaolin clotting time; dRVVT: dilute
for aCL or aβ2GPI was associated with low risk of event Russell’s viper venom time; aCL: anticardiolipin antibodies; β2GPI: β2-glicoprotein I;
[odds ratio (OR) <5], while LA positivity alone conferred a aPS/PT: phosphatidylserine prothrombin complex; aPL: antiphospholipid antibody;
medium risk of event (OR 5-9); this risk increased in GPL: IgG phospholipid units; MPL: IgM phospholipid units; LA: lupus anticoagulant.

568 haematologica | 2020; 105(3)

 Seronegative antiphospholipid syndrome

Primary and secondary prevention strategies In women with a high-risk aPL profile but no history of
thrombosis or pregnancy complications, treatment with
Antiphospholipid antibody carriers LDA (75-100 mg daily) during pregnancy should be con-
In subjects with positivity for aPL in the absence of clin- sidered according to 2019 European League Against
ical thrombotic events, primary prevention strategy Rheumatism (EULAR) recommendations.
includes cardiovascular risk factors such as arterial hyper-
tension, diabetes, dyslipidemia and cigarette smoking Secondary antiphospholipid syndrome
(Figure 2). Treatment with low-dose aspirin (LDA, 75-100 In APS patients with previous arterial or venous throm-
mg/die) is still controversial6 and could be considered in boembolism, use of unfractionated or low molecular
patients at high risk, such as those with triple positivity or weight heparins (LMWH) is recommended in the acute
persistent positivity with medium-high titer of aCL.71,72 phase6 followed by long-term treatment with warfarin,
Recently, the positivity for IgG aPS/PT has been suggested with an international normalized ratio (INR) range
as a marker of thrombotic risk in aPL carriers in addition between 2-3.8,74
to triple positivity (Figure 2).58 Regarding oral anticoagula- Warfarin therapy in APS has several critical points.
tion, with or without LDA, the quality of evidence is too Indeed, a recent study10 showed that, in the APS popula-
low to demonstrate benefit or harm of anticoagulant use tion, the management of anticoagulant therapy is more
in aPL carriers.73 problematic compared to a population of patients with

Table 4. Suggested extra-criteria antibodies in seronegative antiphospholipid syndrome and its clinical manifestations.
Extra-critera antibodies Clinical manifestations
Anti-prothrombin/phosphatidylserine antibodies Thrombosis
Anti-annexin V antibodies/annexin A5 resistance Thrombosis and/or pregnancy complications
Antibodies to vimentin/CL complex Arterial thrombosis
Phosphatidylethanolamine Fetal loss and/or thrombosis
Phosphatidic acid Fetal loss
Phosphatidylserine Fetal loss
Phosphatidylinositol Fetal loss
IgA aCL and aβ2GPI antibodies Thrombosis
CL:cardiolipin; aβ2GPI: anti- β2 Glycoprotein I.

Figure 2. Summary of antithrombotic treatment options in patients with antiphospholipid syndrome and seronegative antiphospholipid syndrome.72 APS: antiphos-
pholipid syndrome; aPL: antiphospholipid antibodies; aCL: anticardiolipin antibodies; VTE: venous thromboembolism; LDA: low-dose aspirin, LMWH: low molecular
weight heparin, UFH: unfractionated heparin; INR: international normalized ratio; EULAR: European League Against Rheumatism.

haematologica | 2020; 105(3) 569

 P. Pignatelli et al.

atrial fibrillation (AF). Thus, APS patients had a shorter be treated with antepartum administration of prophylac-
time within the therapeutic range than those with AF tic or intermediate dose of unfractionated heparin or pro-
(53.5% vs. 68%; P=0.001) and needed a higher mean phylactic LMWH combined with LDA (75-100 mg/day),
weekly dose of warfarin to reach the therapeutic range.10 according to the 2012 American College of Cardiology
In the case of low-quality therapy with warfarin or recur- (ACC) guidelines.74
rent thrombosis, two possible therapeutic approaches could Recent 2019 EULAR recommendations suggest that
be considered. The first is to adopt a higher intensity war- women: 1) with a history of obstetric APS, such as a his-
farin therapy with target INR 3-4, which is, however, not tory of ≥3 recurrent spontaneous miscarriages <10th week
current practice given its association with a reduced risk of of gestation and in those with a history of fetal loss (≥10th
thrombosis in the majority of patients.6,72,75 A second week of gestation); and 2) with a history of delivery <34
approach is represented by the addition of LDA to antico- weeks of gestation due to eclampsia/severe pre-eclampsia
agulation, which should, however, be reserved for high- or due to placental insufficiency, should be started on a
risk patients, particularly after an arterial thrombotic combined therapy including LDA and prophylactic
event.6,76 heparin during pregnancy.72
More recently, non-vitamin K antagonist oral anticoagu- Heparin at prophylactic dose should be maintained for
lants (NOAC) have been investigated in patients with APS six weeks after delivery to avoid maternal thrombosis.
with divergent results.77 Following the results from the Trial Finally, heparin should be increased to therapeutic doses,
on Rivaroxaban in AntiPhospholipid Syndrome (TRAPS),78 in addition to LDA, in women with a history of throm-
which included triple positive thrombotic APS, rivaroxaban botic APS.
is contraindicated in APS patients with triple aPL positivi-
ty.72 An analysis from the RE-COVER/RE-COVER II and
RE-MEDY trials showed similar safety and efficacy of dabi- Conclusions
gatran in patients with thrombophilia and previous venous
thromboembolic events, in whom APS represented the sec- The diagnosis of SN-APS should be formulated only
ond most common inherited disorders, accounting for 20% after the exclusion of other causes of inherited and
of all patients.79 These results need to be confirmed in real- acquired thrombophilic conditions. Although several dif-
world studies. A randomized trial investigating the efficacy ferent antibodies to a number of antigens are involved in
and safety of apixaban in APS patients is currently ongo- SN-APS, the routine testing of these non-criteria antibod-
ing;80 this study will include patients with both venous and ies is not recommended, but may be considered in
arterial thrombosis. Laboratory testing of NOAC may be patients with a high clinical suspicion of APS, such as
useful in patients with APS as no pre-clinical data in this those presenting with recurrent unexplained thrombosis,
patient population are available. thrombosis at unusual sites, or women with recurrent
Recently, new drugs have been administered in APS pregnancy-related complications. The assessment and
patients with thrombotic events. A first example is repre- interpretation of these non-conventional antibodies
sented by mTOR inhibitors; these were found to reduce the should be performed by specialized centers of hemostasis
onset of new vascular lesions after transplantation in and thrombosis to reduce laboratory variability.
patients with APS nephropathy.81 Monoclonal antibodies The detection of non-criteria aPL may help guide
such as rituximab82 (anti-CD20 agent) and eculizumab23 antithrombotic strategies in SN-APS patients with arterial
(anti-C5 agent) are currently administrated to manage non- or venous thrombosis. As an example, patients treated
criteria symptoms refractory to standard therapy and to with NOAC for recurrent VTE events, who become posi-
add-on in catastrophic APS and kidney transplantation in tive for non-criteria aPL, may be switched to VKA or
APS patients, respectively. Despite these findings, the use of LMWH, especially in cases of a recurrent thrombotic
these drugs should be avoided due to lack of strong evidence events. Moreover, in case of an unprovoked DVT, and
in APS patients; their use could be considered in patients among patients who could be withdrawn from anticoagu-
with refractory C-APS, as suggested by EULAR guidelines.72 lation, the positivity for a non-criteria aPL may help decide
whether or not to continue long-term anticoagulation.
Obstetric antiphospholipid syndrome In conclusion, there is growing evidence to suggest a
role for non-criteria aPL in those patients defined as
Women who are diagnosed with confirmed APS should “seronegative”.

disease, and the lupus anticoagulant. Br Med Diagnosing and treating antiphospholipid
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