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biomedicines
Review
Current Promising Biomarkers and Methods in the Diagnostics
of Antiphospholipid Syndrome: A Review
Pavla Bradacova 1,2 , Ludek Slavik 2, *, Jana Ulehlova 2, Adela Skoumalova 3, Jana Ullrychova 1,
Jana Prochazkova 2, Antonin Hlusi 2, Gayane Manukyan 4and Eva Kriegova 4
Citation: Bradacova, P.; Slavik, L.;
Ulehlova, J.; Skoumalova, A.;
Ullrychova, J.; Prochazkova, J.;
Hlusi, A.; Manukyan, G.; Kriegova, E.
Current Promising Biomarkers and
Methods in the Diagnostics of
Antiphospholipid Syndrome:
A Review. Biomedicines 2021,9, 166.
https://doi.org/10.3390/
biomedicines9020166
Academic Editor: Matteo Di Minno
Received: 31 December 2020
Accepted: 2 February 2021
Published: 8 February 2021
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1
Masaryk Hospital Usti nad Labem, Department Clinical Hematology, 40113 Usti nad Labem, Czech Republic;
pavla.bradacova@kzcr.eu (P.B.); jana.ullrychova@kzcr.eu (J.U.)
2Department of Hematology-Oncology, Faculty of Medicine and Dentistry, University Hospital Olomouc,
Palacky University Olomouc, 77900 Olomouc, Czech Republic; jana.ulehlova@fnol.cz (J.U.);
jana.prochazkova@fnol.cz (J.P.); antonin.hlusi@fnol.cz (A.H.)
3Department of Internal Medicine III-Nephrology, Rheumatology and Endocrinology, University Hospital
Olomouc and Faculty of Medicine and Dentistry, Palacky University Olomouc, 77900 Olomouc,
Czech Republic; adela.skoumalova@fnol.cz
4Department of Immunology, Faculty of Medicine and Dentistry, Palacky University Olomouc and Faculty
Hospital, 775 15 Olomouc, Czech Republic; martindihel@seznam.cz (G.M.); eva.kriegova@fnol.cz (E.K.)
*Correspondence: ludek.slavik@fnol.cz; Tel.: +420-585855350
Abstract:
Antiphospholipid syndrome (APS) is a hypercoagulation condition associated with the
incidence of heterogenic antiphospholipid antibodies (aPLs), which non-specifically affect hemostasis
processes. APS is clinically manifested by recurrent arterial and venous thromboses and reproduction
losses. The aPL antibodies, which may induce clinical manifestations of APS, include criteria antibod-
ies anti-cardiolipin, anti-
β
2-glycoprotein-I, and lupus anticoagulant, but also non-criteria antibodies,
for example anti-
β
2-glycoprotein-I domain I, anti-phosphatidylserine/prothrombin, anti-annexin
V, and many others. APS occurs mostly in patients of younger and middle age, most frequently
in females. Laboratory diagnostics of APS are quite difficult, as they include a wide spectrum of
examining methods, which are based on various principles of detection and are performed using
various laboratory techniques. The objective of the review is to describe the current state of po-
tentially examined biomarkers and methods in APS diagnostics. The aforementioned biomarkers
are lupus anticoagulant, anti-
β
2-glycoprotein-I, anti-cardiolipin, anti-
β
2-glycoprotein-I domain I,
anti-phosphatidylserine/prothrombin, anti-
β
2-glycoprotein-I IgA, anti-cardiolipin IgA, anti-annexin
V and II, anti-prothrombin, anti-cardiolipin/vimentin, anti-protein S/protein C, and antibodies
against phospholipid antigens for whose diagnostics we may use some of the methods established
for a long time and some of the modern methods—the coagulation method for the determination of
lupus anticoagulant (LA), enzyme-linked imunosorbent assay (ELISA), chemiluminescence analysis
(CLIA), multiplex fluorescence flow immunoassay (MFFIA), fluorescence enzyme immunoassay
(EliA), line immunoassay (LIA), multiline dot assay (MLDA), and thin-layer chromatography (TLC).
Conclusion: Antibodies against phosphatidylethanolamine, phosphatidic acid, phosphatidylserine,
phosphatidylinositol, cardiolipin/vimentin complex, and annexin V are currently the most studied
new markers. However, these assays have not been standardized until now, both from the laboratory
and clinical point of view. In this review we summarize the evidence of the most studied aPL markers
and their potential clinical significance in seronegative APS (SN-APS).
Keywords:
antiphospholipid syndrome; thrombosis; seronegative APS; lupus anticoagulant; anti-
cardiolipin; anti-
β
2-glycoprotein-I; anti-phosphatidylserine/prothrombin; anti-cardiolipin/vimentin;
anti-annexin; ELISA; chemiluminescence analysis; multiplex fluorescence flow immunoassay; fluo-
rescence enzyme immunoassay; line immunoassay
Biomedicines 2021,9, 166. https://doi.org/10.3390/biomedicines9020166 https://www.mdpi.com/journal/biomedicines
Biomedicines 2021,9, 166 2 of 15
1. Introduction
Antiphospholipid syndrome (APS), also known as Hughes syndrome, was reported for
the first time in 1983 by Dr. Graham Hughes [
1
]. APS is an autoimmune disease associated
with persistent antiphospholipid antibodies (aPLs). The main target of the aPLs is binding
to the phospholipid membranes of platelets with their subsequent activation. However,
they also bind to endothelia, monocytes, and neutrophils with a procoagulation effect [
2
,
3
].
Antiphospholipid antibodies also interfere with the activation of the complement. All this
may subsequently result in the development of thrombosis [
4
]. APS may be primary and
also secondary. Primary APS is a condition in which the patient has no other autoimmune
disease. Secondary APS occurs in relation with another autoimmune disease: systemic
lupus erythematosus (SLE) [5–7].
Prevalence of aPLs in the population is approximately 1–5%, but only a minor part
develops APS [
8
]. However, APS is considered to be the most common cause of acquired
thrombophilia despite this fact. Clinical manifestations of APS are very variable. Ve-
nous thromboses may be manifested by phlebothrombosis of the lower or upper limbs,
or by pulmonary
embolism. Myocardial infarction or cerebrovascular accident is usually a
consequence of arterial thromboses. In the group of pregnancy-related complications, APS
is frequently a cause of preeclampsia, miscarriages, premature labor, growth retardation of
the fetus due to an insufficient placenta, or death of the fetus. Migraine, immune thrombo-
cytopenia, transient ischemic attack, livedo reticularis, autoimmune hemolytic anemia, and
many others were observed as other non-criteria clinical manifestations of APS [
9
]. Pro-
gression of catastrophic antiphospholipid syndrome (CAPS) occurs in approximately 1% of
patients with APS, whereby the patient is affected by thromboses mostly in small vessels,
leading to multiorgan failure. CAPS is a very severe condition with high mortality [
10
,
11
].
Criteria for APS according to the Sydney classification are very strictly defined; at
least one clinical and at least one laboratory criterion must be met. Clinical criteria of APS
include the occurrence of arterial or venous thromboses and reproduction losses [
12
,
13
]. Up
to 10–20% of recurrent reproduction losses and up to 20% of cerebrovascular accidents in
patients below the age of 50 are caused by APS [
14
,
15
]. Laboratory criteria include positivity
of at least one antibody of the anti-cardiolipin (aCL) IgG and IgM, anti-
β
2-glycoprotein-I
(anti-
β
2GPI) IgG and IgM, and the lupus anticoagulant (LA) type [
16
]. In order to meet the
laboratory criteria, the aPLs must be repeatedly positive in an interval of 12 weeks [
17
]. It
is evaluated whether this is single, double, or triple positivity, since patients with triple
positivity have the highest risk of thromboses and recurrent miscarriages [
18
–
20
]. It is
required to avoid laboratory examination of APS during ongoing infection due to false
positivity of the aPLs [21].
2. Antiphospholipid Antibodies
There is a wide range of antiphospholipid antibodies that interact with negatively
charged phospholipid surfaces of many cells and tissues by various mechanisms. These
aPLs, described on Figure 1, include APS criteria antibodies of the lupus anticoagulant,
anti-cardiolipin, anti-
β
2-glycoprotein-I type, and APS non-criteria antibodies of the anti-
β
2-glycoprotein-I domain I (anti-DI), anti-annexin V, anti-annexin II, anti-prothrombin
(anti-PT), anti-phosphatidylserine/prothrombin (anti-PS/PT), anti-cardiolipin/vimentin
(aCL/Vim), anti-protein S/protein C (anti-PS/PC) type, and others.
Biomedicines 2021,9, 166 3 of 15
Biomedicines 2021, 9, x FOR PEER REVIEW 3 of 15
91
Figure 1. The spectrum potential antiphospholipid antibody targets in the diagnostics of antiphos-92
pholipid syndrome (APS). 93
2.1. APS Criteria Antibodies 94
2.1.1. Lupus Anticoagulant 95
Lupus anticoagulants are a heterogenic group of immunoglobulins that specifically 96
aim at epitopes of negatively charged protein binding phospholipids of the cellular mem-97
brane, prothrombin, and beta2-glycoprotein I, which in vitro prolongs the coagulation 98
tests dependent on phospholipids when there is competition with coagulation factors for 99
phospholipids [22]. 100
Positivity of LA is a much more risky factor for the development of thromboembo-101
lism, cerebral ischemia, and recurrent reproduction losses in comparison with aCL and 102
anti-β2GPI and even other non-criteria antibodies [23]. LA was demonstrated in 69% in a 103
group of 192 patients with APS [24]. Choi et al. [25] carried out a retrospective study of 104
833 patients with a persistent presence of aPLs and they found that 46.9% of 96 patients 105
with clinical manifestations of APS had positive LA vs. a group of 737 asymptomatic car-106
riers, where the incidence of LA was only 25.6%. There were no significant differences 107
between the two groups in other aPLs. 108
2.1.2. Anti-β2-Glycoprotein-I 109
β2-glycoprotein-I is anionic glycoprotein with five domains binding to phospholip-110
ids. Four domains have regular, conserved sequences, but the fifth domain is aberrant. 111
This domain contains of the insertion of six residues, C-terminal extension of 19 residues, 112
and another disulphide bond that includes the C-terminal cysteine. These additional 113
amino acids in domain V are responsible for unique characteristics of this CUP domain 114
because they form a large positively charged patch that determines affinity to anionic 115
phospholipids [26]. 116
The anti-β2GPI IgG and IgM antibody plays a major role in the pathogenesis of APS. 117
Its presence is very strongly associated with thromboembolic complications. The β2-gly-118
coprotein-I molecule consists of five homologous domains and occurs in two confor-119
mations, either in a closed circular form or in an open form. In the circular form, there is 120
interaction with anti-β2GPI mainly between domains 1 and 5; in the open form, epitope 121
is uncovered on domain 1, to which anti-β2GPI binds. 122
Detection of anti-β2GPI IgG (Figure 2) and IgM (Figure 3) is performed by the en-123
zyme-linked imunosorbent assay (ELISA) method according to the international guide-124
line of the Society of Thrombosis and Haemostasis Scientific and Standardization Com-125
mittee ISTH SSC. The determined cut-off (99th percentile) in the enzyme-linked imuno-126
sorbent assay (ELISA) for positivity of anti-β2GPI is > 40 IgG antiphospholipid units/mL 127
(GPL), or IgM antiphospholipid units/mL (MPL) [16]. According to Liu et al., anti-β2GPI 128
Commented [M2]: Figure 1 is not behind of first
citation.
Please check if there is Figure 1 citation in this
manuscript
Figure 1.
The spectrum potential antiphospholipid antibody targets in the diagnostics of antiphos-
pholipid syndrome (APS).
2.1. APS Criteria Antibodies
2.1.1. Lupus Anticoagulant
Lupus anticoagulants are a heterogenic group of immunoglobulins that specifically
aim at epitopes of negatively charged protein binding phospholipids of the cellular mem-
brane, prothrombin, and beta2-glycoprotein I, which
in vitro
prolongs the coagulation
tests dependent on phospholipids when there is competition with coagulation factors for
phospholipids [22].
Positivity of LA is a much more risky factor for the development of thromboembolism,
cerebral ischemia, and recurrent reproduction losses in comparison with aCL and anti-
β
2GPI and even other non-criteria antibodies [
23
]. LA was demonstrated in 69% in a
group of 192 patients with APS [
24
]. Choi et al. [
25
] carried out a retrospective study of
833 patients
with a persistent presence of aPLs and they found that 46.9% of 96 patients
with clinical manifestations of APS had positive LA vs. a group of 737 asymptomatic
carriers, where the incidence of LA was only 25.6%. There were no significant differences
between the two groups in other aPLs.
2.1.2. Anti-β2-Glycoprotein-I
β
2-glycoprotein-I is anionic glycoprotein with five domains binding to phospholipids.
Four domains have regular, conserved sequences, but the fifth domain is aberrant. This
domain contains of the insertion of six residues, C-terminal extension of 19 residues,
and another disulphide bond that includes the C-terminal cysteine. These additional
amino acids in domain V are responsible for unique characteristics of this CUP domain
because they form a large positively charged patch that determines affinity to anionic
phospholipids [26].
The anti-
β
2GPI IgG and IgM antibody plays a major role in the pathogenesis of
APS. Its presence is very strongly associated with thromboembolic complications. The
β
2-glycoprotein-I molecule consists of five homologous domains and occurs in two confor-
mations, either in a closed circular form or in an open form. In the circular form, there is
interaction with anti-
β
2GPI mainly between domains 1 and 5; in the open form, epitope is
uncovered on domain 1, to which anti-β2GPI binds.
Detection of anti-
β
2GPI IgG (Figure 2) and IgM (Figure 3) is performed by the enzyme-
linked imunosorbent assay (ELISA) method according to the international guideline of the
Society of Thrombosis and Haemostasis Scientific and Standardization Committee ISTH
SSC. The determined cut-off (99th percentile) in the enzyme-linked imunosorbent assay
(ELISA) for positivity of anti-
β
2GPI is >40 IgG antiphospholipid units/mL (GPL), or IgM
antiphospholipid units/mL (MPL) [
16
]. According to Liu et al., anti-
β
2GPI IgG is the
best predictor of arterial thrombosis, with an odds ratio (OR) = 6.5 [
24
]. Demonstration of
anti-
β
2GPI IgG has higher specificity for APS than aCL IgG, but lower sensitivity for APS
than demonstration of aCL IgG at the same time [
27
]. However, the results of anti-
β
2GPI
do not always significantly correlate with clinical manifestations of APS, which may be
due to insufficient standardization of the ELISA method [
28
–
32
]. The modern method of
anti-
β
2GPI detection is the chemiluminescence analysis (CLIA), in which the cut-off for
Biomedicines 2021,9, 166 4 of 15
positivity is >20 chemiluminescence unit (CU) (99th percentile) [
33
]. Multiline dot assay
(MLDA) is also an available method.
Biomedicines 2021, 9, x FOR PEER REVIEW 4 of 15
IgG is the best predictor of arterial thrombosis, with an odds ratio (OR) = 6.5 [24]. Demon-
stration of anti-β2GPI IgG has higher specificity for APS than aCL IgG, but lower sensi-
tivity for APS than demonstration of aCL IgG at the same time [27]. However, the results
of anti-β2GPI do not always significantly correlate with clinical manifestations of APS,
which may be due to insufficient standardization of the ELISA method [28–32]. The mod-
ern method of anti-β2GPI detection is the chemiluminescence analysis (CLIA), in which
the cut-off for positivity is >20 chemiluminescence unit (CU) (99th percentile) [33]. Multi-
line dot assay (MLDA) is also an available method.
Figure 2. The IgG monomer structure.
Figure 3. The IgM pentamer structure.
2.1.3. Anti-Cardiolipin
Anti-cardiolipin antibodies include a group of antibodies against the cardiolipin part
of the VDRL (venereal disease research laboratory) antigen, which are the antibodies that
react with phospholipids of the prothrombin activator complex and antibodies that can
react with cardiolipin in the fixed phase [34].
aCL IgG is much more associated with cerebral thromboses and myocardial infarc-
tions than aCL IgM. Detection of aCL may be performed by ELISA, CLIA, and MLDA.
The determined cut-off (99th percentile) in ELISA for positivity of aCL is >40 GPL/MPL
[16]. The cut-off recommended by the manufacturer in CLIA for positivity of aCL is >20
CU (99th percentile) [24].
2.2. APS Non-Criteria Antibodies
2.2.1. Anti-β2-Glycoprotein-I Domain I
The presence of APS anti-DI antibodies correlates more significantly with the inci-
dence of thromboses and reproduction losses against other aPLs [35]. The occurrence of
anti-DI together with LA is significantly associated with patients with APS and venous
thrombosis [27]. Sensitivity of anti-DI after APS of 85% and specificity of 99.5% point to
quite great usefulness of anti-DI for APS diagnostics, however, more studies are still
needed [36]. Radin et al. [37] analyzed 11 studies involving 1218 patients with APS, where
positivity of anti-DI was demonstrated in 45.4%. Tonello et al. [38] carried out a study of
105 patients with APS and persistent presence of the aPL criteria and they demonstrated
anti-DI in 41.9%. Positivity of anti-DI was significantly associated with triple positivity.
On the contrary, anti-DI negativity was significant in patients with an isolated presence
Figure 2. The IgG monomer structure.
Biomedicines 2021, 9, x FOR PEER REVIEW 4 of 15
IgG is the best predictor of arterial thrombosis, with an odds ratio (OR) = 6.5 [24]. Demon-
stration of anti-β2GPI IgG has higher specificity for APS than aCL IgG, but lower sensi-
tivity for APS than demonstration of aCL IgG at the same time [27]. However, the results
of anti-β2GPI do not always significantly correlate with clinical manifestations of APS,
which may be due to insufficient standardization of the ELISA method [28–32]. The mod-
ern method of anti-β2GPI detection is the chemiluminescence analysis (CLIA), in which
the cut-off for positivity is >20 chemiluminescence unit (CU) (99th percentile) [33]. Multi-
line dot assay (MLDA) is also an available method.
Figure 2. The IgG monomer structure.
Figure 3. The IgM pentamer structure.
2.1.3. Anti-Cardiolipin
Anti-cardiolipin antibodies include a group of antibodies against the cardiolipin part
of the VDRL (venereal disease research laboratory) antigen, which are the antibodies that
react with phospholipids of the prothrombin activator complex and antibodies that can
react with cardiolipin in the fixed phase [34].
aCL IgG is much more associated with cerebral thromboses and myocardial infarc-
tions than aCL IgM. Detection of aCL may be performed by ELISA, CLIA, and MLDA.
The determined cut-off (99th percentile) in ELISA for positivity of aCL is >40 GPL/MPL
[16]. The cut-off recommended by the manufacturer in CLIA for positivity of aCL is >20
CU (99th percentile) [24].
2.2. APS Non-Criteria Antibodies
2.2.1. Anti-β2-Glycoprotein-I Domain I
The presence of APS anti-DI antibodies correlates more significantly with the inci-
dence of thromboses and reproduction losses against other aPLs [35]. The occurrence of
anti-DI together with LA is significantly associated with patients with APS and venous
thrombosis [27]. Sensitivity of anti-DI after APS of 85% and specificity of 99.5% point to
quite great usefulness of anti-DI for APS diagnostics, however, more studies are still
needed [36]. Radin et al. [37] analyzed 11 studies involving 1218 patients with APS, where
positivity of anti-DI was demonstrated in 45.4%. Tonello et al. [38] carried out a study of
105 patients with APS and persistent presence of the aPL criteria and they demonstrated
anti-DI in 41.9%. Positivity of anti-DI was significantly associated with triple positivity.
On the contrary, anti-DI negativity was significant in patients with an isolated presence
Figure 3. The IgM pentamer structure.
2.1.3. Anti-Cardiolipin
Anti-cardiolipin antibodies include a group of antibodies against the cardiolipin part
of the VDRL (venereal disease research laboratory) antigen, which are the antibodies that
react with phospholipids of the prothrombin activator complex and antibodies that can
react with cardiolipin in the fixed phase [34].
aCL IgG is much more associated with cerebral thromboses and myocardial infarctions
than aCL IgM. Detection of aCL may be performed by ELISA, CLIA, and MLDA. The
determined cut-off (99th percentile) in ELISA for positivity of aCL is >40 GPL/MPL [
16
].
The cut-off recommended by the manufacturer in CLIA for positivity of aCL is >20 CU
(99th percentile) [24].
2.2. APS Non-Criteria Antibodies
2.2.1. Anti-β2-Glycoprotein-I Domain I
The presence of APS anti-DI antibodies correlates more significantly with the inci-
dence of thromboses and reproduction losses against other aPLs [
35
]. The occurrence of
anti-DI together with LA is significantly associated with patients with APS and venous
thrombosis [
27
]. Sensitivity of anti-DI after APS of 85% and specificity of 99.5% point
to quite great usefulness of anti-DI for APS diagnostics, however, more studies are still
needed [
36
]. Radin et al. [
37
] analyzed 11 studies involving 1218 patients with APS, where
positivity of anti-DI was demonstrated in 45.4%. Tonello et al. [
38
] carried out a study of
105 patients with APS and persistent presence of the aPL criteria and they demonstrated
anti-DI in 41.9%. Positivity of anti-DI was significantly associated with triple positivity. On
the contrary, anti-DI negativity was significant in patients with an isolated presence of other
aPL criteria. The cut-off recommended by the manufacturer for positivity of anti-DI in
CLIA is >20 CU (99th percentile) [
24
,
33
]. Serrano et al. specified their own cut-off of >23.8
units (99th percentile) in ELISA for anti-DI in a measurement of 321 healthy volunteers [
39
].
Slavík et al. [
40
] examined 74 patients with APS who had positivity at least in one aCL and
anti-
β
2GPI class at the same time. They demonstrated positivity of anti-DI in 21 samples,
of which 57% had clinical manifestations of APS. They increased the predictive value for
thrombosis from 25% to 68% in anti-DI positive patients by an examination of anti-DI.
Biomedicines 2021,9, 166 5 of 15
2.2.2. Anti-β2-Glycoprotein-I IgA
Antibodies of the IgA class are produced by B-lymphocytes, which may be found
in the mucosae, therefore, IgA are also called mucosal antibodies; they are the most
common antibodies in the body. IgA antibodies are structurally similar to IgF, but IgA
more frequently occur as dimers (Figure 4). The basic function of IgA is to block bacterial
adhesion molecules and their opsonization. IgA do not active the complement.
Biomedicines 2021, 9, x FOR PEER REVIEW 5 of 15
of other aPL criteria. The cut-off recommended by the manufacturer for positivity of anti-
DI in CLIA is >20 CU (99th percentile) [24,33]. Serrano et al. specified their own cut-off of
>23.8 units (99th percentile) in ELISA for anti-DI in a measurement of 321 healthy volun-
teers [39]. Slavík et al. [40] examined 74 patients with APS who had positivity at least in
one aCL and anti-β2GPI class at the same time. They demonstrated positivity of anti-DI
in 21 samples, of which 57% had clinical manifestations of APS. They increased the pre-
dictive value for thrombosis from 25% to 68% in anti-DI positive patients by an examina-
tion of anti-DI.
2.2.2. Anti-β2-Glycoprotein-I IgA
Antibodies of the IgA class are produced by B-lymphocytes, which may be found in
the mucosae, therefore, IgA are also called mucosal antibodies; they are the most common
antibodies in the body. IgA antibodies are structurally similar to IgF, but IgA more fre-
quently occur as dimers (Figure 4). The basic function of IgA is to block bacterial adhesion
molecules and their opsonization. IgA do not active the complement.
Figure 4. The IgA dimer structure.
Positivity of the anti-β2GPI IgA class, but with LA negativity at the same time, may
be a cause of recurrent unexplainable reproduction losses in females [27,41]. Positivity is
put in relation with thrombocytopenia, livedo reticularis, and pulmonary hypertension,
and it increases the risk of fatal graft rejection in patients after kidney transplantation [42].
Anti-β2GPI IgA antibodies are more associated with APS than with anti-β2GPI IgM [43].
Ruiz-Garcia et al. performed ELISA measurement of anti-β2GPI IgA in 156 patients with
clinical criteria of APS and they demonstrated isolated positivity of anti-β2GPI IgA in
22.4% [44]. Vlagea et al. [45] carried out a study for the presence of anti-β2GPI IgA (cut-
off >20 U/mL 99th percentile, 100 healthy follow-ups) in 314 patients with APS and SLE.
The presence of isolated positivity of anti-β2GPI IgA in the group of APS was detected
only in 7.2%, whereas the presence was detected in 76.2% in the SLE group. Chayoua et
al. [46] analyzed a multicentric study of aPL detection in 1068 patients from 8 sites by 4
various methods (CLIA, ELISA, multiplex fluorescence flow immunoassay (MFFIA), flu-
orescence enzyme immunoassay (EliA)) and they determined isolated positivity of anti-
β2GPI IgA in patients with clinical manifestations of APS in 0.3–5% dependent on the
device used.
2.2.3. Anti-Cardiolipin IgA
The significance of aCL IgA for the development of thrombotic complications has
also been of much interest recently [47]. Using CLIA (cut-off recommended by the manu-
facturer >20 CU), Liu et al. detected aCL IgA in 192 samples of APS in 42%, in 90 samples
of seronegative APS (SN-APS) in 12%, and in healthy donors in 0% [24].
2.2.4. Anti-Prothrombin and Anti-Phosphatidylserine/Prothrombin Complex
The anti-PT IgG antibody may be a very useful predictive factor for the development
of thrombosis in patients with SLE [48]. Anti-PT is capable of a bond even to the PS/PT
complex. Positivity of anti-PS/PT IgG, IgM with positivity of LA at the same time is very
Figure 4. The IgA dimer structure.
Positivity of the anti-
β
2GPI IgA class, but with LA negativity at the same time, may
be a cause of recurrent unexplainable reproduction losses in females [
27
,
41
]. Positivity is
put in relation with thrombocytopenia, livedo reticularis, and pulmonary hypertension,
and it increases the risk of fatal graft rejection in patients after kidney transplantation [
42
].
Anti-
β
2GPI IgA antibodies are more associated with APS than with anti-
β
2GPI IgM [
43
].
Ruiz-Garcia et al. performed ELISA measurement of anti-
β
2GPI IgA in 156 patients with
clinical criteria of APS and they demonstrated isolated positivity of anti-
β
2GPI IgA in
22.4% [
44
]. Vlagea et al. [
45
] carried out a study for the presence of anti-
β
2GPI IgA
(
cut-off >20 U/mL
99th percentile, 100 healthy follow-ups) in 314 patients with APS and
SLE. The presence of isolated positivity of anti-
β
2GPI IgA in the group of APS was detected
only in 7.2%, whereas the presence was detected in 76.2% in the SLE group. Chayoua
et al. [
46
] analyzed a multicentric study of aPL detection in 1068 patients from 8 sites by
4 various methods (CLIA, ELISA, multiplex fluorescence flow immunoassay (MFFIA),
fluorescence enzyme immunoassay (EliA)) and they determined isolated positivity of
anti-
β
2GPI IgA in patients with clinical manifestations of APS in 0.3–5% dependent on the
device used.
2.2.3. Anti-Cardiolipin IgA
The significance of aCL IgA for the development of thrombotic complications has also
been of much interest recently [
47
]. Using CLIA (cut-off recommended by the manufacturer
>20 CU), Liu et al. detected aCL IgA in 192 samples of APS in 42%, in 90 samples of
seronegative APS (SN-APS) in 12%, and in healthy donors in 0% [24].
2.2.4. Anti-Prothrombin and Anti-Phosphatidylserine/Prothrombin Complex
The anti-PT IgG antibody may be a very useful predictive factor for the development
of thrombosis in patients with SLE [
48
]. Anti-PT is capable of a bond even to the PS/PT
complex. Positivity of anti-PS/PT IgG, IgM with positivity of LA at the same time is
very significantly associated with arterial and also venous thromboses and pregnancy
complaints [
49
–
51
] and sensitivity, and specificity for APS is also higher than during
positivity of aCL [
52
]. Using ELISA (cut-off >30 [
53
]), Liu et al. detected anti-PS/PT IgG,
IgM in samples of APS in 72%, in SN-APS in 36%, and in healthy donors in 0%. Anti-PS/PT
was more commonly detected in the group of APS and SN-APS than aCL IgG and IgM and
anti-
β
2GPI IgG and IgM. They further found out that particularly anti-PS/PT IgG is the
best predictor for deep vein thrombosis, OR = 9.2 [
24
]. Hui shi et al. found in a study of
186 samples with APS + SN-APS that if LA is positive together with anti-PS/PT, then the
OR for the development of thrombosis is 101.6 [54].
Biomedicines 2021,9, 166 6 of 15
2.2.5. Anti-Annexin V and Anti-Annexin II
Annexins are in the group of Ca2
+
-dependent proteins binding phospholipids. An-
nexin V is the main part of trophoblast and vascular endothelia. Annexin V binds phospho-
lipids with anticoagulation activity; it serves as a so-called protective shield. This shield
may be impaired in case of the interaction of annexin V with antibodies, causing thrombosis
and reproduction losses [
55
]. However, the correlation of anti-annexin V with pregnancy
complications is not completely significant and more studies are needed [
56
]. Annexin II is
important for the bonding of
β
2GPI to endothelium and to monocytes. Using the ELISA
method, Canas et al. [
57
] found that demonstration of anti-annexin II is significantly higher
in patients with APS than in healthy donors and patients with SLE without thrombosis.
However, sensitivity is quite low despite this fact, since anti-annexin II was demonstrated
only in 25% of patients with APS.
2.2.6. Anti-Cardiolipin/Vimentin
Vimentin is a part of endothelial cells and may be present even on the surface of
apoptotic neutrophils, T-lymphocytes, activated macrophages, and platelets. Vimentin
and cardiolipin act on the surface of apoptotic cells as immunogens and may induce the
production of antibodies. The presence aCL/Vim is strongly associated with recurrent
thrombosis and pregnancy morbidity [
52
,
58
]. Ortona et al. demonstrated the presence of
aCL/Vim by the ELISA method in patients with APS in 92.5%, in patients with SN-APS in
55.2%, and in patients with SLE in 43.3%. Positivity of aCL/Vim was not demonstrated in
any case in a group of healthy donors [59].
2.2.7. Anti-Protein S/Protein C
The mechanism of action of anti-PS/PC is their bond to complexes of phospholipids
with coagulation inhibitors protein S and protein C; this results in blocking their activity
and subsequently the development of thrombosis. Anti-PS/PC is usually a frequent cause
of pregnancy complications and preeclampsia. However, positivity of anti-PC/PS has
lower sensitivity and also specificity for APS in comparison with aCL IgG [59].
2.2.8. Antibodies Against Phospholipid Antigens
This group of antiphospholipid antibodies includes antibodies against phosphatidic
acid (anti-PA), phosphatidylserine (anti-PS), phosphatidyletanolamine (anti-PE), phos-
phatidylinositol (anti-PI), phosphatidylcholine (anti-PC), phosphatidylglycerol (anti-PG),
lyso-bis-phosphatidic acid (anti-LBPA), and a mixture of phospholipids (APhL). Natural
IgG antibodies to the above-mentioned types of lipids are ubiquitously distributed in
sera of healthy humans and are believed to serve beneficial functions. Although natural
antibodies to lipids generally exhibit germ line or near germ line binding specificities, the
antibodies commonly increase transiently in the acute phases of most, if not all, infectious
diseases and may serve as a first line of defense [
60
]. Some studies show that anti-PE may
be a cause of fetal loss. Even anti-PS, which inhibits production of choriogonadotropin
hormone (HCG), may act similarly [
27
,
61
]. Korematsu et al. [
62
] reported increased levels
of anti-PC and anti-PE in three children with cerebral infarction. The anti-LBPA antibodies
were demonstrated in a significant number of patients with APS, however, sensitivity and
specificity were lower than in aCL and anti-
β
2GPI [
63
]. Castanon et al. [
64
] examined
various aPL IgMs and IgGs in 548 serum samples using the ELISA method. Comparison
of two groups of APS/healthy donors demonstrated the presence of APhL in 89.7/0%,
anti-PI in 89.7/32.1%, anti-PS in 86.2/7.1%, aCL in 93.1/32.1%, and anti-
β
2GPI in 86.2/0%.
Park et al. [
65
] demonstrated by line immunoassay (LIA) detection that single positivity
of anti-PS (OR 16.5) and anti-PA (OR 9.6) is a better predictive factor for thrombosis than
positivity of anti-β2GPI (OR 5.5).
Biomedicines 2021,9, 166 7 of 15
3. Methods
Table 1summarizes the available methods for detecting antibodies in the diagnostis
of APS based on the principle and technique of the procedure.
Table 1. Overview of the methods available for the examination of biomarkers.
Methods Assay Determination
Dilute Russell’s viper venom time (DRVVT)
Activated partial thromboplastin time (aPTT) Liquid-phase Quantitative
Enzyme-linked immunosorbent assay (ELISA)
Solid-phase
Quantitative
Fluorescence enzyme immunoassay (EliA) Quantitative
Chemiluminescence immunoassay (CLIA) Quantitative
Multiplex flow fluorescence immunoassay (MFFIA) Quantitative
Multiline dot assay (MLDA) Semi-quantitative
Line immunoassay (LIA) Qualitative
Thin-layer chromatography (TLC) Qualitative
3.1. Liquid-Phase Assay
Lupus Anticoagulant
LA examination should be performed based on the international guideline ISTH
SSC for detection of lupus anticoagulant [
66
]. Detection is based on the ability of present
antiphospholipid antibodies in the plasma of the patient to extend the coagulation time in
a test dependent on phospholipids. The following basic tests are recommended: di-
lute Russell’s viper venom time (DRVVT) and activated partial thromboplastin time
(aPTT) [
67
]. The traditional procedure of the LA examination is performed in three basic
steps:
1—screening
, 2—mixed tests, and 3—confirmation [
68
]. The LA results are inter-
preted according to ISTH SSC as positive/negative based on the normalized ratio (NR)
calculation (
NR = patient/polled normal plasma (PNP)
) [
69
]. With regard to the use of
different analyzers and reagents, each laboratory should determine its own cut-off for
LA (99th percentile) by measuring ideally 120 (minimally 40) healthy controls [
47
,
70
,
71
].
Cohen et al
. [
72
] carried out a survey in 575 laboratories by means of a “Lupus Program,”
the External quality Control of diagnostic Assay and Test Foundation (ECAT). Despite
the ISTH SSC guidelines, only 55% of laboratories performed the tests in the screen-mix-
confirm order, 50% of laboratories used their own cut-off determined at the 99th percentile,
and 46% for interpretation of the results as an NR. Many different laboratories used a
“universal” NR >1.2 for interpretation of their DRVVT results. Pradella et al. [
73
] carried
out a DRVVT examination in 200 healthy donors and determined a cut-off NR > 1.22 for
positive LA.
3.2. Solid-Phase Assay
3.2.1. Enzyme-Linked Imunosorbent Assay
ELISA is the gold standard for detection of many aPLs. The bond of the aPL antibodies
in examined plasma/serum to the surface of a microtiter plate hole coated with a fixed
phase is the principle of this sandwich method, when a complex antigen/antibody is
formed. Human Ig and peroxidase conjugate is bound to this complex. Peroxidase enzyme
cleaves a specific chromogenic substrate, producing a color change, the intensity of which
is detected through photometry by a reader at a wavelength of 450 nm [
74
–
77
]. The aPL
results are obtained by reading the measured optic density from the calibration curve
and they are usually indicated in arbitrary units IU/mL or in GPL/MPL units. The
cut-off differs for the individual aPLs. Serrano et al. determined a cut-off >20 units in
anti-β2GPI IgA using ELISA (99th percentile) by measuring 321 healthy volunteers [39].
The test results of various kits in various laboratories show quite large variability.
Due to this reason, the results of aPL tests often do not provide a sufficient benefit for the
clinical use; the method needs to be more standardized [28–31].
Biomedicines 2021,9, 166 8 of 15
3.2.2. Fluorescence Enzyme Immunoassay
The EliA method is based on a similar principle as ELISA, except that the conjugate
contains mouse Ig and
β
-galactosidase. Detection is based on fluorescence intensity,
which is optically demonstrated in the detector. The cut-off for positivity of aCL and
anti-
β
2GPI recommended by the manufacturer is >10 U/mL [
78
]. Bor et al. determined
their own cut-off (99th percentile) in 377 samples of patients with APS for the individually
determined aPLs and compared this with the cut-off recommended by the manufacturer.
They subsequently found that based on their own cut-off they evaluated 40 positive samples
fewer than in the cut-off determined by the manufacturer [79].
3.2.3. Chemiluminescence Immunoassay
CLIA is a method of quantitative detection of aCL IgG, IgM, anti-
β
2GPI IgG, IgM,
and anti-DI. CLIA is a very well-standardizable method, performed using an automatic
analyzer, and it is suitable for a higher number of samples [
80
]. The bond of the aPLs in
the examined serum/plasma sample to paramagnetic particles coated by an appropriate
surface is the principle of CLIA. Isoluminol-labeled compatible human Ig is bound to
this formed complex. A chemiluminescence reaction is initiated after the addition of a
triggering reagent [
81
]. The emission of light occurs during the chemiluminescence reaction;
this is detected by an optic module in the device in relative light units (RLU). Measured
RLU are directly proportional to the concentration of the individual aPLs in the sample.
Measured RLU are converted to chemiluminescence units (CU) by means of a logistic curve
4PLC. The cut-off recommended by the manufacturer is >20 U/mL [
82
].
Chayoua et a
l. [
83
]
carried out a multicentric study in 1168 samples. They compared the results of aCL IgM
and IgG and anti-
β
2GPI IgG and IgM in three solid-phase assays (MFFIA, EliA, ELISA)
and found that the best correlation (0.900) in anti-
β
2GPI IgG was between MFFIA and
CLIA. On the contrary, the worst correlation (0.514) in aCL IgM was between MFFIA and
EliA. Salma et al. [
84
] compared CLIA and ELISA in 370 samples and demonstrated a
similar sensitivity of both methods for aCL IgG and IgM and anti-
β
2GPI IgM, but CLIA
had higher sensitivity for anti-β2GPI IgG than ELISA.
3.2.4. Multiplex Flow Fluorescence Immunoassay
MFFIA analysis for the detection of aCL IgG and IgM and anti-
β
2GPI IgG and IgM is
based on the use of paramagnetic particles coated with an appropriate antigen to which
the aPLs are bound in the sample. A conjugate of human Ig with fluorescein phycoerythrin
is subsequently added. Fluorescence is identified in relative fluorescence units (RFI) as the
particles pass through the detector. The method is performed using an automatic analyzer
and is suitable for performing on a larger number of samples [
85
]. The cut-off for positivity
of aCL and anti-
β
2GPI recommended by the manufacturer is >20 U/mL.
Grossi et al
. [
86
]
compared the results of 134 patients on MFFIA and CLIA and demonstrated a very good
compliance between both methods. Compliance for aCL IgG was 88.1%, and for anti-
β
2GPI
IgG was 97.8%.
3.2.5. Multiline Dot Assay
MLDA is a semi-quantitative method for detection of multiple aPLs at the same
time, performed on polyvinylidene difluoride (PVDF) membranes. Various immobilized
phospholipids are piled up on PVDF in strips, to which the respective aPLs from the
serum sample are bound. Detection is performed using densitometry, and the results are
indicated as positive/negative [
87
]. Compared to ELISA, hydrophobic PVDF membranes
imitate the bond of the aPLs
in vivo
, they are more porous, and may hide a large portion
of the phospholipid hydrophobic part, which may result in denser expression of the
phospholipid hydrophilic part on the PVDF surface and intensified interaction with the
examined aPLs. Misasi et al. [
27
] and Egerer et al. [
88
] performed a comparative MLDA
and ELISA study, and in the measurement results they demonstrated a good to very good
compliance of aCL and anti-
β
2GPI between both methods. Using MLDA, aCL and anti-
Biomedicines 2021,9, 166 9 of 15
β
2GPI and the presence of other aPLs may be determined in the sample. The method is
not certainly suitable for an analysis of a larger number of samples due to characteristics of
its implementation, and standardization of MLDA is not completely sufficient either.
3.2.6. Line Immunoassay
LIA is a novel multiline assay for the determination of up to 10 different aPLs at
the same time. Various phospholipids are immobilized on the PVDF membrane with
no addition of a cofactor, and binding of the aPLs is dependent only on
β
2GPI present
in the examined sample. Ig and peroxidase conjugate cleave the substrate. Individual
strips are analyzed qualitatively using positive/negative densitometry. The optical den-
sity (oD) cut-off for positivity is
≥
50 of oD (determined in 150 healthy donors, 99th
percentile).
Thaler et al
. compared an examination of 10 different aPLs by the LIA method
in 53 APS and 34 healthy controls with CLIA and ELISA technologies. The sensitivity
of LIA for aCL and anti-
β
2GPI IgG was significantly higher than in other methods [
89
].
Roggenbuck et al. [90]
and
Nalli et al
. [
91
] independently compared the detection of dif-
ferent aPLs by LIA and ELISA in two files of patients with APS and healthy controls and
demonstrated a very good compliance between the results in patients with APS. In addition
to ELISA, the LIA method could differentiate patients with APS from patients with infec-
tious diseases or asymptomatic carriers probably by exposure of domain I.
Park et al
. [
65
]
detected
9 different aPLs
in 180 patients with APS by LIA and ELISA, and by a comparison
of both methods they demonstrated compliance in the results of aCL IgG (68.2%), aCL
IgM (82.6%), anti-
β
2GPI IgG (71.7%), and anti-
β
2GPI (93.2%). Park et al. demonstrated by
LIA detection that single positivity and anti-PS (OR 16.5) and anti-PA (OR 9.6) are better
predictive factors for thrombosis than anti-β2GPI (OR 5.5).
3.2.7. Thin-Layer Chromatography TLC
TLC is a non-quantitative screening method performed on phospholipid-coated alu-
minum plates. TLC is performed in several basic steps: Antigen separation occurs at first,
followed by immunostaining with the examined aPLs, and finally immunoreactivity is de-
tected using a chemiluminescence reaction [
92
,
93
]. In case of detection of immunoreactivity
(positivity) of aPL by TLC, it is subsequently appropriate to perform a targeted examination
of the individual aPLs using ELISA. Based on a comparative ELISA and TLC study of
120 samples, Sorice et al. found that TLC shows higher specificity, but lower sensitivity
than ELISA [
94
]. As with MLDA, TLC is not suitable for an analysis of a larger number of
samples, and in TLC it is true that there is insufficient standardization of this method.
4. Conclusions
Seronegative APS
In practice, we often find patients with clinical manifestations of APS, but they are
repeatedly negative for all of the criteria for an antiphospholipid antibody. They are
so-called seronegative APS [
52
,
95
,
96
]. A part of patients with SN-APS show repeated
positivity of non-criteria antibodies of the anti-DI, anti-PS/PT IgG and IgM, anti-annexin
V IgG and IgM, anti-PS, anti-PA type, and others [
58
,
97
]. Trugliia et al. [
98
] analyzed
61 samples
of SN-APS in females with reproduction complications. The aCL antibodies
were analyzed using TLC; aCL/Vim antibodies, anti-PS/PT, anti-
β
2GPI IgA, and aCL
IgA were analyzed using the ELISA method. At least one positive aPL was demonstrated
in 81.9%. Repeated testing 12 weeks later demonstrated persistent positivity of at least
one aPL in 57.4% of females.
Patients with SN-APS are at risk for recurrent thrombotic and pregnancy complica-
tions; long-term prophylactic treatment is therefore required [
27
]. Due to this reason, it
has been currently proving increasingly beneficial to revise the original laboratory criteria
of APS [
16
] and to include specifications of other non-criteria antibodies summarized in
Table 2
[
99
]. The introduction of additional aPLs into routine laboratory practice will cer-
tainly represent a useful tool for more precise and accelerated APS diagnostics [
54
,
100
,
101
].
Biomedicines 2021,9, 166 10 of 15
Table 2. Overview of biomarkers and methods used to study them.
Biomarkers Methods Reference
Lupus anticoagulant (LA) DRVVT
aPTT Liu [24], Choi [25], Pengo [66], Linnemann [68]
Anti-β2-glycoprotein-I (anti-β2GPI) IgG, IgM
ELISA
EliA
CLIA
MFFIA
MLDA
LIA
Miykis [16], Liu [24], Misasi [27], Serrano [39]
Vanouverchelde [78], Bor [79], Chayoua [83]
Janek [33], Chayoua [83], Salma [84]
Chayoua [83], Chayoua [85], Grossi [86]
Misasi [27], Bevers [87], Egerer [88]
Park [65], Egerer [88], Thaler [89], Roggenbuck [90], Nalli [91]
Anti-cardiolipin (aCL) IgG, IgM
ELISA
EliA
CLIA
MFFIA
MLDA
LIA
Miykis [16], Liu [24]
Vanouverchelde [78], Bor [79], Chayoua [83]
Janek [33], Chayoua [83], Salma [84]
Chayoua [83], Chayoua [85], Grossi [86]
[Misasi [27], Bevers [87], Egerer [88]
Park [65], Egerer [88], Thaler [89], Roggenbuck [90], Nalli [91]
Anti-β2-glycoprotein-I domain I (anti-DI) ELISA
CLIA
Serrano [39]
Slavik [40]
Anti-β2-glycoprotein-I IgA
ELISA
EliA
CLIA
MFFIA
Ruiz-Garcia [44], Vlagea [45]
Chayoua [46]
Chayoua [46]
Chayoua [46]
Anti-cardiolipin IgA CLIA Liu [24]
Anti-prothrombin (anti-PT)
Anti-phosphatidylserine/prothrombin
(anti-PS/PT)
ELISA Liu [24], Shi [54]
Anti-annexin V
Anti-annexin II ELISA Canas [57]
Anti-cardiolipin/vimentin (aCL/Vim) ELISA Ortona [58]
Anti-protein S/protein C (anti-PS/PC) LIA Arachchillage [59]
Anti-phosphatidic acid (anti-PA)
Anti-phosphatidylserine (anti-PS)
Anti-phosphatidyletanolamine (anti-PE)
Anti-phosphatidylinositol (anti-PI)
Anti-phosphatidylcholine (aPC)
Anti-phosphatidylglycerol (aPG)
Anti-lyso-bis-phosphatidic acid (anti-LBPA)
Anti-mixture of phospholipids (APhL)
ELISA
LIA
Castanon [64]
Park [65]
Author Contributions:
Conceptualization, P.B. and L.S.; writing—original draft preparation, P.B.;
writing—review and editing, L.S.; visualization, L.S.; supervision, P.B., L.S., J.U. (Jana Ulehlova), A.S.,
J.P., A.H., J.U. (Jana Ullrychova), G.M. and E.K. All authors have read and agreed to the published
version of the manuscript.
Funding:
This work was supported by the internal grant agency of Palacky University (IGA
UP_2021_001) and in part by the Ministry of Health of the Czech Republic (MH CZ-DRO) (FNOL,
00098892).
Conflicts of Interest: The authors declare no conflict of interest.
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