Detection of Antiphospholipid Antibodies by Flow Cytometry: Rapid Detection of Antibody Isotype and Phospholipid Specificity

1993 ◽  
Vol 70 (04) ◽  
pp. 603-607 ◽  
Author(s):  
M W Stewart ◽  
W S Etches ◽  
A S Russell ◽  
J S Percy ◽  
C A Johnston ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Antiphospholipid Syndrome ◽  
Antiphospholipid Antibodies ◽  
Fetal Loss ◽  
Laboratory Diagnosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Isotype ◽  
Anionic Phospholipids ◽  
Flow Cytometric Method ◽  
Antibody Isotypes

SummaryLaboratory diagnosis of antiphospholipid antibodies is important in patients with clinical features of the antiphospholipid syndrome, such as thrombosis and fetal loss. We have developed a novel method for the detection of antiphospholipid antibodies using flow cytometry. Anionic phospholipids cardiolipin, phosphatidylserine and phosphatidylinositol are coated onto polystyrene beads of different sizes, allowing detection and semiquantitation of their respective phospholipid antibody isotypes. The results of the flow cytometric method closely correlate those of the standardised anticardiolipin enzyme-linked immunosorbent assay (ELISA), but the method is quicker and is versatile in its ability to detect IgG, IgM and IgA antibody isotypes at the same time. The method promises to be useful in evaluating the significance of phospholipid specificity and antibody isotypes in patients with the antiphospholipid syndrome.

Study of antiphospholipid antibodies by enzyme immunoassay and chemiluminescent methods in patients with antiphospholipid syndrome and systemic lupus erythematosus (preliminary data)

2021 ◽  
Vol 66 (9) ◽  
pp. 546-551
Author(s):  
F. A. Cheldieva ◽  
T. M. Reshetnyak ◽  
M. V. Cherkasova ◽  
A. M. Lila
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Antiphospholipid Syndrome ◽  
Lupus Erythematosus ◽  
Preliminary Data ◽  
Antiphospholipid Antibodies ◽  
Fetal Loss ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serological Markers ◽  
Systemic Lupus

Antiphospholipid antibodies (aPL) are a family of different autoantibodies that lead to recurrent vascular thrombosis of any localization and caliber, and/or obstetric pathology - fetal loss. Serological markers of antiphospholipid syndrome (APS) include only three types of aPL - lupus anticoagulant (VA), antibodies to cardiolipin (aCL) classes IgG and IgM, antibodies to β2-glycoprotein1 (aβ2GP1) classes IgG and IgM. Medium and high levels of aCL and aß2HP1 (IgG and / or IgM) were selected as serological markers of APS in the 2006 classification criteria. However, the threshold of values used from low to moderately high levels has not been standardized. aPL standardization issues are still unresolved, resulting in heterogeneous results of the ongoing studies. The aim of the study was to assess the comparability IgG/IgM-aCL and IgG/IgM-ab2GP1 by enzyme-linked immunosorbent assay and chemiluminescent analysis in patients with APS with and without (systemic lupus erythematosus) SLE. The study included 70 patients (49 women and 21 men) with APS, of which 21 (30%) were with primary APS (pAPS) and 49 (70%) with APS in combination with SLE. All study participants underwent determination of IgG/IgM-aCL and IgG/IgM-aβ2GP1 by enzyme-linked immunosorbent. A study was performed by the chemiluminescent analysis: IgG/IgM-aCL - in 70 patients; IgG/IgM-aβ2GP1 - in 69 patients. Results. According to preliminary data, the determination of IgG-aCL and IgG-aβ2GP1 by the chemiluminescent analysis is informative in assessing positivity according to the manufacturer, compared with the enzyme-linked immunosorbent (p < 0.05). However, when taking into account the levels of antibody positivity determined by enzyme-linked immunosorbent, the level of positive values according to chemiluminescent analysis was much higher than the performance of the manufacturer.

scholarly journals The antiphospholipid syndrome: still an enigma

Hematology ◽  
2015 ◽  
Vol 2015 (1) ◽  
pp. 53-60 ◽  
Author(s):  
Shruti Chaturvedi ◽  
Keith R. McCrae
Keyword(s):  
Antiphospholipid Syndrome ◽  
Antiphospholipid Antibodies ◽  
Oral Anticoagulants ◽  
Direct Oral Anticoagulants ◽  
Clinical Manifestations ◽  
Fetal Loss ◽  
Basic Research ◽  
Laboratory Diagnosis ◽  
Pregnancy Morbidity ◽  
Common Causes

Abstract Antiphospholipid syndrome (APS) is defined by clinical manifestations that include thrombosis and/or fetal loss or pregnancy morbidity in patients with antiphospholipid antibodies (aPL). Antiphospholipid antibodies are among the most common causes of acquired thrombophilia, but unlike most of the genetic thrombophilias are associated with both venous and arterial thrombosis. Despite an abundance of clinical and basic research on aPL, a unified mechanism that explains their prothrombotic activity has not been defined; this may reflect the heterogeneity of aPL and/or the fact that they may influence multiple pro- and/or antithrombotic pathways. Antiphospholipid antibodies are directed primarily toward phospholipid binding proteins rather than phospholipid per se, with the most common antigenic target being β2-glycoprotein 1 (β2GPI) although antibodies against other targets such as prothrombin are well described. Laboratory diagnosis of aPL depends upon the detection of a lupus anticoagulant (LA), which prolongs phospholipid-dependent anticoagulation tests, and/or anticardiolipin and anti-β2-glycoprotein 1 antibodies. Indefinite anticoagulation remains the mainstay of therapy for thrombotic APS, although new strategies that may improve outcomes are emerging. Preliminary reports suggest caution in the use of direct oral anticoagulants in patients with APS-associated thrombosis. Based on somewhat limited evidence, aspirin and low molecular weight heparin are recommended for obstetrical APS. There remains a pressing need for better understanding of the pathogenesis of APS in humans, for identification of clinical and laboratory parameters that define patients at greatest risk for APS-related events, and for targeted treatment of this common yet enigmatic disorder.

scholarly journals Autoantibodies against the fibrinolytic receptor, annexin 2, in antiphospholipid syndrome

Blood ◽  
2006 ◽  
Vol 107 (11) ◽  
pp. 4375-4382 ◽  
Author(s):  
Gabriela Cesarman-Maus ◽  
Nina P. Ríos-Luna ◽  
Arunkumar B. Deora ◽  
Bihui Huang ◽  
Rosario Villa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Antiphospholipid Syndrome ◽  
Lupus Erythematosus ◽  
Antiphospholipid Antibodies ◽  
Cell Surface Receptor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Target Antigen ◽  
Healthy Individuals ◽  
Annexin 2

AbstractThe association of thrombosis and gestational morbidity with antiphospholipid antibodies is termed antiphospholipid syndrome (APS). Annexin 2 (A2) is a profibrinolytic endothelial cell surface receptor that binds plasminogen, its tissue activator (tPA), and β2-glycoprotein I (β2GPI), the main antigen for antiphospholipid antibodies. Here, we evaluate A2 as a target antigen in APS. Serum samples from 434 individuals (206 patients with systemic lupus erythematosus without thrombosis, 62 with APS, 21 with nonautoimmune thrombosis, and 145 healthy individuals) were analyzed by enzyme-linked immunosorbent assay (ELISA) and immunoblot for antiphospholipid and A2 antibodies. Anti-A2 antibodies (titer > 3 SDs) were significantly more prevalent in patients with APS (22.6%; venous, 17.5%; arterial, 34.3%; and mixed thrombosis, 40.4%) than in healthy individuals (2.1%, P < .001), patients with nonautoimmune thrombosis (0%, P = .017), or patients with lupus without thrombosis (6.3%, P < .001). Anti–A2 IgG enhanced the expression of tissue factor on endothelial cells (6.4-fold ± 0.13-fold SE), blocked A2-supported plasmin generation in a tPAdependent generation assay (19%-71%) independently of β2GPI, and inhibited cell surface plasmin generation on human umbilical vein endothelial cells (HUVECs) by 34% to 83%. We propose that anti-A2 antibodies contribute to the prothrombotic diathesis in antiphospholipid syndrome.

scholarly journals Antiphospholipid Antibodies and Fertility: No Impact on Ovarian Reserve in Premenopausal Women

2020 ◽  
Vol 20 (01) ◽  
pp. 4-8
Author(s):  
Sciascia Savino ◽  
Radin Massimo ◽  
Menegatti Elisa ◽  
Barinotti Alice ◽  
Sini Federica ◽  
...  
Keyword(s):  
Risk Assessment ◽  
Antiphospholipid Syndrome ◽  
Immunosorbent Assay ◽  
Ovarian Reserve ◽  
Antiphospholipid Antibodies ◽  
Premenopausal Women ◽  
Classification Criteria ◽  
Enzyme Linked Immunosorbent Assay ◽  
Asymptomatic Carriers ◽  
Healthy Donors

Objective: To investigate possible differences in levels of ovarian reserve between antiphospholipid antibodies (aPL) asymptomatic carriers and antiphospholipid syndrome (APS) patients, by measuring the levels of anti-Müllerian hormone (AMH). Methods: We enrolled 69 premenopausal women divided in 2 groups: a) patients with APS, either primary (PAPS) or secondary (SAPS), according to the Sydney classification criteria; b) asymptomatic aPL carriers. Aged-matched premenopausal healthy donors (HDs) were also recruited. Complete aPL testing was performed and AMH levels were measured using enzyme-linked immunosorbent assay. Results: Among the 69 patients included in the study, 22 were diagnosed with PAPS, 13 with SAPS, and 14 patients were asymptomatic aPL carriers. No differences in AMH levels were observed among the three groups [mean AMH: PAPS 3.09 ng/ml ± 1.9 (range 1.02 − 7.1); SAPS 3.1 ng/ml ± 2.2 (range 1.1 − 7.6); aPL carriers 2.2 ng/ml ± 5.4 (range 1 − 6.3)] and between patients/aPL carriers and HDs [mean AMH 2.82 ng/ml ± 2.9 (range 1 − 6.9)]. Any correlation between the global APS score (GAPSS) and AMH levels failed to be found (rho = 0.31; p = 0.073). Conclusion: With the limitations of the current study, as observed in women with APS, we confirm that ovarian reserve, assessed with AMH levels, is not reduced in premenopausal women with isolated aPL positivity. Moreover, when granulating the aPL profile in terms of risk assessment, using the GAPSS, no impact on fertility was observed.

Manifestations of the Antiphospholipid Syndrome in Patients with Malignancies.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4039-4039 ◽  
Author(s):  
Wolfgang A. Miesbach ◽  
Geneth Asmelash ◽  
Birgit Puetz ◽  
Martina Boehm ◽  
Inge Scharrer
Keyword(s):  
Antiphospholipid Syndrome ◽  
Antiphospholipid Antibodies ◽  
Clinical Manifestations ◽  
Anticardiolipin Antibodies ◽  
Solid Tumours ◽  
Enzyme Linked Immunosorbent Assay ◽  
Thromboembolic Complications ◽  
Non Hodgkin Lymphoma ◽  
Thrombotic Complications ◽  
Very High

Abstract The presence of antiphospholipid antibodies has been reported in a large variety of malignancies. It is not clear, however, if the antiphospholipid antibodies are related to thrombotic associations of the antiphospholipid syndrome (APS) in these patients. We investigated the frequency of thrombotic manifestations in 58 patients with the presence of antiphospholipid antibodies and a history of neoplasia, including haematologic and lymphoproliferative malignancies. Methods Antiphospholipid antibodies were detected by clotting assay (lupus anticoagulant, LA) or by enzyme-linked immunosorbent assay (anticardiolipin antibodies). LA were tested by more than 2 different methods according to the proposed criteria of the SSC of the ISTH. Results 39/58 patients suffered from solid tumours mostly from carcinoma of the breast, prostate, and colon and 19/58 patients from malignant haematologic or lymphoproliferative diseases mostly from Non-Hodgkin lymphoma. One patient was suffering simultaneously from two carcinomas of the prostate and the testicle and a Non-Hodgkin’s lymphoma. Among the patients with solid tumours 18/39 (46 %) patients had thromboembolic complications of the antiphospholipid syndrome. Among the patients with haematologic and lymphoproliferative malignancies only 6/19 (32 %) suffered from thromboembolic complications. Thrombotic manifestations were more common on the arterial than the venous site. There was no relation between the titres of aCL antibodies and the rate of clinical manifestations. In two patients aPL disappeared after the effective treatment of the tumor. Especially patients with very high titres did not present any thromboembolic manifestation. Conclusion The presence of antiphospholipid antibodies may identify a subset of cancer patients with high risk of developing thrombotic complications but the frequency of thrombosis is lower in aPL positive patients with lymphoproliferative and haematological malignancies. Especially in these patients very high titres of aCL antibodies do not seem to be associated with clinical manifestations of the APS.

The role of antiphospholipid antibodies in reproductive failure

1997 ◽  
Vol 6 (3) ◽  
pp. 133-143
Author(s):  
D Ware Branch ◽  
Harry H Hatasaka
Keyword(s):  
Pregnancy Outcome ◽  
Clinical Features ◽  
Antiphospholipid Syndrome ◽  
Antiphospholipid Antibodies ◽  
Lupus Anticoagulant ◽  
Fetal Loss ◽  
Placental Insufficiency ◽  
Antiphospholipid Antibody ◽  
The Relationship

The relationship between antiphospholipid antibodies and the clinical features of placental insufficiency, pre-eclampsia, and fetal loss has emerged as one of the most exciting new observations in obstetrics in the last 15 years. Antiphospholipid syndrome is the only convincing ‘immunologic’ disturbance of pregnancy affecting the fetus other than anti-erythrocyte or antiplatelet alloimmunization disorders, and it is now routine to test patients with fetal loss for the two best characterized antiphospholipid antibodies, lupus anticoagulant and anticardiolipin. Although there is no proven mechanism for fetal loss, treatment of antiphospholipid antibody-positive mothers during pregnancy with heparin improves pregnancy outcome.

scholarly journals Urine Proteomics Differentiate Primary Thrombotic Antiphospholipid Syndrome From Obstetric Antiphospholipid Syndrome

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhuochao Zhou ◽  
Yijun You ◽  
Fan Wang ◽  
Yue Sun ◽  
Jialin Teng ◽  
...  
Keyword(s):  
Antiphospholipid Syndrome ◽  
Fetal Loss ◽  
Absolute Quantification ◽  
Mass Spectrometry Analysis ◽  
Response To Treatment ◽  
Enzyme Linked Immunosorbent Assay ◽  
Multisystem Disorder ◽  
Spectrometry Analysis ◽  
Urine Proteomics ◽  
Non Invasive

Antiphospholipid syndrome (APS) is a multisystem disorder characterized by thrombosis and/or recurrent fetal loss. This clinical phenotype heterogeneity may result in differences in response to treatment and prognosis. In this study, we aimed to identify primary thrombotic APS (TAPS) from primary obstetric APS (OAPS) using urine proteomics as a non-invasive method. Only patients with primary APS were enrolled in this study from 2016 to 2018 at a single clinical center in Shanghai. Urine samples from 15 patients with TAPS, 9 patients with OAPS, and 15 healthy controls (HCs) were collected and analyzed using isobaric tags for relative and absolute quantification (iTRAQ) labeling combined with liquid chromatography-tandem mass spectrometry analysis to identify differentially expressed proteins. Cluster analysis of urine proteomics identified differentiated proteins among the TAPS, OAPS, and HC groups. Urinary proteins were enriched in cytokine and cytokine receptor pathways. Representative secreted cytokines screened out (fold change &gt;1.20, or &lt;0.83, p&lt;0.05) in these differentiated proteins were measured by enzyme-linked immunosorbent assay in a validation cohort. The results showed that the levels of C-X-C motif chemokine ligand 12 (CXCL12) were higher in the urine of patients with TAPS than in those with OAPS (p=0.035), while the levels of platelet-derived growth factor subunit B (PDGFB) were lower in patients with TAPS than in those with OAPS (p=0.041). In addition, correlation analysis showed that CXCL12 levels were positively correlated with immunoglobulin G anti-β2-glycoprotein I antibody (r=0.617, p=0.016). Our results demonstrated that urinary CXCL12 and PDGFB might serve as potential non-invasive markers to differentiate primary TAPS from primary OAPS.

The significance of autoantibodies against β2-glycoprotein I

Blood ◽  
2012 ◽  
Vol 120 (2) ◽  
pp. 266-274 ◽  
Author(s):  
Philip G. de Groot ◽  
Rolf T. Urbanus
Keyword(s):  
Autoimmune Disease ◽  
Infectious Diseases ◽  
Antiphospholipid Syndrome ◽  
Plasma Protein ◽  
Antiphospholipid Antibodies ◽  
Fetal Loss ◽  
Thrombotic Risk ◽  
Pregnancy Morbidity ◽  
Glycoprotein I ◽  
History Of

AbstractThe antiphospholipid syndrome (APS) is defined by the persistent presence of antiphospholipid antibodies in patients with a history of thrombosis and/or pregnancy morbidity, including fetal loss. APS is an autoimmune disease with a confusing name because the pathologic auto-antibodies are shown to be directed against the plasma protein β2-glycoprotein I and not against phospholipids. In fact, auto-antibodies that recognize phospholipids themselves are not associated with thrombosis but with infectious diseases. One of the intriguing questions is why autoantibodies against β2-glycoprotein I are so commonly found in both patients and the healthy. Several potential mechanisms have been suggested to explain the increased thrombotic risk in patients with these autoantibodies. In this overview, we will summarize our knowledge on the etiology of the autoantibodies, and we will discuss the evidence that identify autoantibodies against β2-glycoprotein I as the culprit of APS.

β2-Glycoprotein I can exist in 2 conformations: implications for our understanding of the antiphospholipid syndrome

Blood ◽  
2010 ◽  
Vol 116 (8) ◽  
pp. 1336-1343 ◽  
Author(s):  
Çetin Ağar ◽  
Gwendolyn M. A. van Os ◽  
Matthias Mörgelin ◽  
Richard R. Sprenger ◽  
J. Arnoud Marquart ◽  
...  
Keyword(s):  
Antiphospholipid Syndrome ◽  
Partial Thromboplastin Time ◽  
Antiphospholipid Antibodies ◽  
Fetal Loss ◽  
Activated Partial Thromboplastin Time ◽  
Ample Evidence ◽  
Glycoprotein I ◽  
Open Conformation ◽  
Cryptic Epitope ◽  
Major Antigen

Abstract The antiphospholipid syndrome is defined by the presence of antiphospholipid antibodies in blood of patients with thrombosis or fetal loss. There is ample evidence that β2-glycoprotein I (β2GPI) is the major antigen for antiphospholipid antibodies. The autoantibodies recognize β2GPI when bound to anionic surfaces and not in solution. We showed that β2GPI can exist in at least 2 different conformations: a circular plasma conformation and an “activated” open conformation. We also showed that the closed, circular conformation is maintained by interaction between the first and fifth domain of β2GPI. By changing pH and salt concentration, we were able to convert the conformation of β2GPI from the closed to the open conformation and back. In the activated open conformation, a cryptic epitope in the first domain becomes exposed that enables patient antibodies to bind and form an antibody-β2GPI complex. We also demonstrate that the open conformation of β2GPI prolonged the activated partial thromboplastin time when added to normal plasma, whereas the activated partial thromboplastin time is further prolonged by addition of anti-β2GPI antibodies. The conformational change of β2GPI, and the influence of the autoantibodies may have important consequences for our understanding of the antiphospholipid syndrome.

Functional Analyses of Patient-derived IgG Monoclonal Anticardiolipin Antibodies Using In Vivo Thrombosis and In Vivo Microcirculation Models

2000 ◽  
Vol 84 (09) ◽  
pp. 388-395
Author(s):  
Xiaowei Liu ◽  
Ricardo Espinola ◽  
Tsawei Olee ◽  
Min Zhu ◽  
Nigel Harris ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Antiphospholipid Syndrome ◽  
Adhesion Molecule ◽  
Antiphospholipid Antibodies ◽  
Cell Function ◽  
Leukocyte Adhesion ◽  
Anticardiolipin Antibodies ◽  
Intercellular Adhesion Molecule ◽  
Enzyme Linked Immunosorbent Assay

SummaryAntiphospholipid antibodies (aPL) have been associated with thrombosis and pregnancy losses in patients diagnosed with antiphospholipid syndrome (APS) and enhance thrombus formation in vivo in mice, but the mechanism of thrombosis by aPL is not completely understood. It has been proposed that aPL may affect endothelial cell (EC) function and/or induce their activation, transforming their anticoagulant surface into procoagulant, thus predisposing to thrombosis. It has been proposed that aPL may affect EC cell function and/or induce their activation, transforming their anticoagulant surface into procoagulant, thus predisposing to thrombosis. This study proposes to test the hypotheses that some IgG anticardiolipins (IgG aCL) with thrombogenic properties in mice, exert their effects through activation of endothelium. We studied seven patient-derived monoclonal aCL for their thrombogenic properties in an in vivo pinch-induced thrombosis model, and their functional activities in activating EC by analyzing in vivo leukocyte adhesion to endothelium in microcirculation in venules in exposed murine cremaster muscle and in vitro adhesion molecule expression in cultured EC. The binding of the monoclonal aCL to EC was also tested. In addition to the previous identified thrombogenic IS2, four of the five new more IgG monoclonal aCL (from two patients) were found to be thrombogenic. Of these five thrombogenic aCL, three caused more in vivo leukocyte adhesion to EC in microcirculation, as compared to that induced by the H2 control human monoclonal IgG, and enhanced expression of adhesion molecules (particularly VCAM-1) on cultured EC. These data show that about 2/3 patientderived IgG monoclonal aCL are thrombogenic and suggest that some thrombogenic IgG aCL exert their effects through activating EC. Abbreviations: aCL, anticardiolipin antibodies; aPL, antiphospholipid antibodies; APS, antiphospholipid syndrome; CL, cardiolipin; dRVVT, dilute Russell’s viper venom time; EC, endothelial cells; ELISA, enzyme-linked immunosorbent assay; GP, glycoprotein; h, hour; HUVEC, human umbilicalvein endothelial cells; ICAM, intercellular adhesion molecule; KCT, kaolin clotting time; LA, lupus anticoagulant; PBS, phosphate-buffered saline; PL, phospholipid; sq, square; TBS, Tris-buffered saline; VCAM, vascular cell adhesion molecule; PMBC, peripheral mononuclear blood cells

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