Detection Of Antiphospholipid Antibodies Research Articles

Pulmonary embolism (PE) diagnosed in a 19-year-old lady found to have underlying Antiphospholipid syndrome (APS)

ABSTRACT Background A 19-year-old woman visited a family medicine clinic to obtain her laboratory test results. This case highlights a close call involving a young woman with a potentially serious condition. Pulmonary embolism (PE) is often overlooked during initial assessments in emergency departments or primary care clinics. Case presentation The patient casually mentioned having a cough toward the end of the consultation, revealing she has been using a salbutamol inhaler due to a family history of asthma. Despite appearing well, she had an audible wheezy cough during the examination. Following a chest X-ray (CXR) that indicated a right lung opacity and possible cardiomegaly, an electrocardiogram (ECG) was arranged. The ECG showed an incomplete right bundle branch block (RBBB) and STT changes in leads V3-V6 and lead 3, prompting her transfer to the emergency department for suspected Pulmonary embolism (PE). Further evaluation confirmed acute on chronic PE, with a positive lupus screen leading to the diagnosis of antiphospholipid syndrome (APS) upon detection of anti-phospholipid antibodies in her serology. Discussion The patient’s case was unique, but a basic CXR was performed to investigate their exertional shortness of breath. This led to appropriate action being taken to ensure the patient’s safety. It is important to note that pulmonary embolism can occur in young individuals, highlighting the need for attentive and skilled healthcare professionals. Basic tests such as CXR and ECG can be crucial in cases of ongoing symptoms where uncertainty exists.

Cytokine Profiling in Antiphospholipid Syndrome Demonstrates Persistent Immune Dysregulation and a Procoagulant Phenotype

Background: The antiphospholipid syndrome (APS) is an autoimmune thromboinflammatory disease characterized by thrombosis and the persistent detection of antiphospholipid antibodies (aPL). The rate of recurrent thrombosis in APS is between 3 and 24% despite standard of care anticoagulation with a vitamin K antagonist, suggesting a persistent prothrombotic milieu in APS which may be driven by altered cytokine signaling. Objective: To test the hypothesis that cytokine signaling is dysregulated in APS and contributes to thrombotic risk, we profiled cytokine levels in patients with thrombotic APS and healthy individuals. Methods: We measured a panel of 46 serum cytokines using the Meso Scale Discovery electrochemiluminescent platform, a validated measurement system that provides linear readouts of analytes over several logs of concentration, in 21 APS patients and 12 healthy individuals. All APS patients met the Revised Sapporo Criteria for thrombotic APS. Measurements were performed in triplicate and the log2 transformed averages were analyzed, setting significance at a FDP-adjusted p value of <0.05, while controlling for age and sex. Cytokine values were correlated with clinical history and antibody isotype profile. Results: Our cohort was 67% female and 85% identified as white. All APS patients had thrombotic disease: 57% with history of VTE, 19% with ATE, and 24% with both VTE and ATE. 14% had catastrophic APS. 67% were triple-positive for anti-cardiolipin (aCL), anti-β2 glycoprotein 1 (aβ2GP1), and lupus anticoagulant (LAC). 10% had isolated IgM isotypes of aCL or aβ2GP1. We found significantly elevated levels of TNF-α, IL-6, IL-7, IL-8, IL-10, IL-16, IL-12/IL-23p40, soluble VCAM-1, soluble ICAM-1, PIGF, SAA and CRP and significantly decreased levels of IL-17genB, IL-22 and IL-31 in APS compared with healthy controls (Figure 1). Cytokine levels were measured a median of 5 months (1-240) from the last thrombotic event, demonstrating persistence of the inflammatory response. To determine if cytokine levels were predictive of thrombotic risk, we correlated the number and pattern of positive aPL isotypes and LAC with cytokine levels. We found that TNF-α, IL-8, IL-10, sVCAM-1, sICAM-1, VEGF, IL-7, IL-12/IL-23p40, IL-4, IL-6, IL-16, PIGF, IL-23 and IL-17A each correlated with aPL positivity, with higher levels in triple-positive APS, and the highest levels observed in patients positive for all five measured antibody isotypes (aCL IgG, IgM, IgA; aβ2GP1 IgG, IgM) plus the functional LAC (Figure 2). IL-31 level inversely correlated to the number of positive aPL tests. A sub-analysis of cytokines in patients with isolated IgM aCL or aβ2GP1 antibodies with or without LAC showed significantly increased levels of TNF-α compared to healthy controls, and did not differ from APS patients with other patterns of aPL positivity. This suggests that a similar proinflammatory or procoagulant background is present in patients with isolated antiphospholipid IgM antibodies as those with IgG. Discussion: The sustained differences in cytokine levels measured in APS patients suggests a picture of ongoing immune dysregulation and compensation capable of driving vascular cell activation, and tipping the hemostasis balance into thrombosis. We find upregulation of proinflammatory cytokines, such as TNF-α and IL-6, capable of activating endothelial cells and triggering amplification loops leading to chemokine and acute phase reactant release, cell adhesion molecule expression, recruitment of leukocytes, and accumulation of complement and coagulation proteins, with the net effect of driving inflammation and thrombosis. Decreased levels of IL-17, IL-22 and IL-31 are indicative of dysregulation of the Th17 CD4+ T cell response, which has been implicated in autoimmune pathogenesis. The correlation of cytokine levels with triple or greater aPL positivity suggests a model for advancing risk stratification and has utility as a biomarker of thrombosis. Our study, although limited by size, represents one of the most extensive profiling of cytokines in thrombotic APS.

The Presence of Antineutrophil Cytoplasmic Antibodies and Antiphospholipid Antibodies in Patients with Severe Acute Respiratory Syndrome Coronavirus 2: A Case-Control Study among Sudanese Patients.

Patients infected with COVID-19 are at an increased risk for thrombosis, suggesting a possible role of COVID-19 in the induction of coagulopathy. This study aimed to investigate the presence of prothrombotic antineutrophil cytoplasmic antibodies (ANCA) and antiphospholipid antibodies (aPLs) in the course of COVID-19 infection and to correlate these markers with severity and fatality, suggesting that COVID-19-induced autoimmune thrombosis is a possible axis in the inflammatory circuit of this infection. To investigate this, we conducted a case-control study which included patients with a positive reverse transcription-polymerase chain reaction (RT-PCR) test of COVID-19 and a control group with negative COVID-19 PCR and antibody (IgG-IgM and IgA nucleoprotein) ELISA results. An indirect immunofluorescence assay using granulocyte biochips (Aesku slides from AESKU DIAGNOSTICS, Germany) was used to detect ANCA (IgG), as well as multiplex ELISA for the detection of antiphospholipid antibodies for all patients with COVID-19 and for the control group. The results revealed the detection of antiphospholipid antibodies (IgG) in one patient out of the 45 patients in the case group. 1/45(2.2%) and 7/45(15.6%) tested positive for ANCA. Five were men and two were females, with one case revealed to be positive for both aPL and ANCA. A cytoplasmic reaction on the eosinophil granulocytes was observed in 2 cases; both were positive for ANCA. Other markers (CRP, APTT, PT, INR, ESR, and neutrophil and lymphocyte counts) were included in the study, along with demographic data. No aPL or ANCA reactions were detected for any of the control groups. These findings suggest that aPL and ANCA may be induced during the course of inflammation in COVID-19 and possibly contribute to the disease's severity and mortality.

Pathogenetic significance of antiphospholipid antibodies in the development of placenta-associated complications in pregnant women with thrombocytopenia

BACKGROUND: Thrombocytopenia is one of the most common cytopenic syndromes that complicate pregnancy. It is considered a typical hematological criterion and the second most common manifestation of antiphospholipid syndrome. In recent years, the relationship between the development of thrombotic and placenta-associated pregnancy complications and the circulation of antiphospholipid antibodies has been actively studied, which makes it necessary to search for new methods for hemostasis correction and for prevention of the development of obstetric complications.
 AIM: The aim of this study was to assess the incidence of antiphospholipid antibodies in pregnant women with thrombocytopenia in order to determine the risk of obstetric complications and adverse pregnancy outcomes depending on the presence of antiphospholipid antibodies in the examined women.
 MATERIALS AND METHODS: 299 pregnant women were included in this multicenter prospective study: the main study group (n = 249) consisted of patients with thrombocytopenia, and the control group (n = 50) comprised women with normal platelet counts during a physiological pregnancy. All participants underwent a complete clinical and anamnestic examination and a study of the hemostasis system. To determine antibodies to cardiolipin, beta-2 glycoprotein 1, annexin V, and prothrombin (IgG/IgM/IgA), we performed enzyme-linked immunosorbent assay in 84 pregnant women with thrombocytopenia and in 25 women in the control group. Lupus anticoagulant was determined in 123 pregnant women of the main group and 25 women in the control group using an ACL-200 coagulometer (Instrumentation Laboratory, Spain).
 RESULTS: A comparative analysis of antiphospholipid antibodies detection has shown that in pregnant women with thrombocytopenia, lupus anticoagulant was detected in every third case (35.8% of cases, 44/123 women), which is more common than in the control group (8.0% of cases, 2/25 women; p = 0.008). Antibodies to beta-2 glycoprotein 1 were detected in pregnant women with thrombocytopenia in every fourth case (27.4% of cases, 23/84 women), and in the control group only in 4% of cases (1/25 women; p = 0.013). Antibodies to cardiolipin were also more often detected in pregnant women with thrombocytopenia (23.8% of cases, 20/84 women) compared to the control group (4% of cases, 1/25 women; p = 0.040). Based on the data obtained with V Cramer, we established medium-strength direct correlations between criterial antiphospholipid antibodies circulation and the development of placenta-associated complications of pregnancy (p 0.05; 0.2 V 0.4). We also identified statistically significant risks of developing placenta-associated complications in pregnant women with circulating antiphospholipid antibodies (95 % confidence interval excludes 1).
 CONCLUSIONS: Thrombocytopenia during pregnancy develops more often in women with antiphospholipid antibodies. Antiphospholipid antibodies circulation elevates coagulation potential and activates intravascular coagulation, resulting in an increased risk of placenta-associated complications of pregnancy, which requires hemostasis correction.

Anticardiolipin Autoantibodies as Useful Biomarkers for the Prediction of Mortality in Septic Patients.

Background The detection of antiphospholipid antibodies (aPL) is of interest because of their importance in the pathogenesis of arterial or venous thrombosis. They could be a “second hit” of an inflammatory event such as infection. The aim of our study was to assess the performance of antiphospholipid antibody biomarker to predict in-hospital mortality in intensive care unit (ICU) septic patients. Methods We conducted a prospective single-center observational study including consecutive critically ill septic adults admitted to the intensive care unit. Clinical and laboratory data including enzyme-linked immunosorbent assay for antiphospholipid antibodies (anticardiolipin (aCL), antiphosphatidylserine (aPS)) were obtained. Blood samples were collected on days 1, 3, 5, 8, and 10 of hospitalization. The primary study endpoint was ICU mortality defined as death before ICU discharge. Secondary end points included correlation between SOFA score and biological parameters. Results A total of 53 patients were enrolled. 18.8% of patients were aPL positive. In-hospital mortality rate was 60%. Multivariate analysis showed that age and aCL at days 3 and 5 along with SOFA at day 3 were independent outcome predictors. A significant positive correlation existed between SOFA at days 3, 5, and 8 and antiphospholipid antibody concentrations. Conclusions Our data showed that antiphospholipid was useful biomarkers for the prediction of mortality in critically ill septic patients. We found a positive correlation between SOFA score and antiphospholipid antibodies.

Concordance and discordance among antiphospholipid syndrome screening assay

Abstract Introduction/Objective The lupus anticoagulant (LA) panel utilizing dilute Russell’s viper venom and LA-responsive APTT remains the front-line screen for detecting the presence of clinically significant antibodies in the diagnosis of antiphospholipid syndrome. Receiving anticoagulant therapy limits the utility of clotting-based methods due to increased risk of false positive results. Anti-cardiolipin (aCL) and anti-ß2-glycoprotein I (a2GßPI) antibody titers have also been employed for the detection of anti-phospholipid antibodies; however, they are not influenced by anticoagulation therapy. The International Society on Thrombosis and Haemostasis recommends that all three tests should be performed to appropriately assess the risk of lupus anticoagulant/antiphospholipid antibodies. In this study, the concordance and discordance of rates of among these three assays were examined. Methods/Case Report The electronic medical record was mined according to lab tests codes for the results of the LA panel, aCL and a2GßPI antibody titers for all patients having at least one resulted study at the University of Cincinnati Medical Center in Cincinnati, Ohio, over a one-year period (2020). Results were compared to determine rates of concordance and discordance among the assays as well as Spearman’s correlation. Non-parametric binary regression analysis was applied to explore the relationship connecting aCL and a2GßPI titers to LA panel findings. Results (if a Case Study enter NA) Strong concordance was seen between the results of LA panels and antibody titer assays. A weak but significant positive correlation was found among the three assays. A robust logarithmic relationship was developed from which the LA panel result could be predicted utilizing outcomes from aCL and a2GßPI antibody titer findings. Conclusion These data further support the reliability of aCL and a2GßPI titers in the evaluation of antiphospholipid syndrome. Furthermore, the robust relationship among these three assays suggests that antibody titers may provide a reliable diagnostic avenue for patients actively undergoing anticoagulation therapy.

The Placental Vascularization Indices Value and Placental Volume in Pregnancies with Antiphospholipid Syndrome for Pregnancy Outcome Prediction

Background: The detection of antiphospholipid antibodies in the setting of arterial and venous thrombus and/or pregnancy loss involves antiphospholipid syndrome. APS may be primary in 40 % of instances where no proof of autoimmune disease is present, or secondary to autoimmune processes such as systemic erythematous lupus. Objectives: To explore the value of placental volume and placental vascularization indexes for predicting pregnancy results in pregnant women with antiphospholipid syndrome. Patient and Methods: One hundred pregnant women have been classified into 2 groups: Group A) group of controls (50 cases), (Group B) group of anti-phospholipids (50 cases). The perinatal results were associated with placental volume findings and indexes of placental vascularization. The precision in predicting the negative result was determined. Results: The indexes of placental vascularization, VI, FI and VFI in group B became lower in 1st and 2nd US scans with statistically significant P values (<0.05). Also placental volume was lower in antiphospholipid group in 1st and 2nd US scans with statistically significant P values (<0.001). A significant correlation was found among the two groups concerning the gestational age at termination of pregnancy, Apgar score and birth weight (with P-value < 0.05). Moreover, IUGR in group B was significantly associated with placental volume and placental vascularization indexes. Conclusion: In women who are pregnant with antiphospholipid syndrome, placental volume and placental vascularization indexes are good methods for evaluating fetal well-being and predicting neonatal outcomes.

Thromboembolic complications in antiphospholipid syndrome and ankylosing spondylitis (two clinical cases)

We presented two clinical cases with clinical manifestations of antiphospholipid syndrome (APS) and ankylosing spondylitis (AS). The peculiarity of these cases is the onset of diseases in childhood, as well as the presence of not only extra-skeletal manifestations, but also complications or manifestations of other pathology. In the first case, it was thrombosis of the superficial veins of the lower limbs with the development of postthrombotic syndrome. In the second case, aortic valve defect, as a result of aortitis with a dilatation of the ascending aorta, which led to aortic valve replacement and its subsequent dysfunction because of thrombosis of the valve prosthesis. The frequency of detection of antiphospholipid antibodies (aPL), APS and thrombosis in AS is discussed. The role of tumor necrosis factor α (TNFα) inhibitors in the induction of aPL synthesis and the development of APS in patients with AS is considered either. Separately, we discussed the role of TNFα inhibitors, which are the main drugs in the treatment of ankylosing spondylitis, in the induction of aPL synthesis and the development of APS. Data on the occurrence of aPL, the reasons for the development of thrombosis in APS and the role of TNFα inhibitors remains incomplete. Perhaps the combination of APS and AS is an underestimated problem, and the information available in the literature does not reflect the real numbers. It is obvious that further research is needed to improve the treatment of patients with AS with thrombosis.

Role of antiphospholipid antibodies in the diagnosis of antiphospholipid syndrome

The diagnosis of antiphospholipid syndrome (APS) relies on the detection of antiphospholipid antibodies (aPL). Currently, lupus anticoagulant (LA), anticardiolipin (aCL), and antibeta2-glycoprotein I antibodies (aβ2GPI) IgG or IgM are included as laboratory criteria, if persistently present. LAC measurement remains a complicated procedure with many pitfalls and interfered by anticoagulant therapy. Solid-phase assays for aCL and aβ2GPI show interassay differences. These methodological issues make the laboratory diagnosis of APS challenging. In the interpretation of aPL results, antibody profiles help in identifying patients at risk. Other aPL, such as antibodies against the domain I of beta2-glycoprotein (aDI) and antiphosphatidylserine-prothrombin (aPS/PT) antibodies have been studied in the last years and may be useful in risk stratification of APS patients. Because of the methodological shortcomings of immunological and clotting assays, these non-criteria aPL may be useful in patients with incomplete antibody profiles to confirm or exclude the increased risk profile. This manuscript will focus on the laboratory aspects, the clinical relevance of assays and interpretation of aPL results in the diagnosis of APS.

Consultative Interpretation for Lupus Anticoagulant by Expert Pathologist Reduces False-Positive Rates in the Era of Direct Oral Anticoagulants.

The diagnosis of antiphospholipid syndrome requires detection of antiphospholipid antibodies (aPL). A retrospective review of our testing practices revealed that societal recommendations for lupus anticoagulant (LA) testing as part of aPL testing are largely not followed by clinicians, and there was a high proportion of positive LA results. Increasing direct oral anticoagulant (DOAC) usage creates additional challenges in identifying LA. This prompted us to establish an order set with pathologist consultation ("LA panel") and testing algorithm to reduce false-positive LA and to ensure optimal LA identification and best practices for interpretation and follow-up. The laboratory database was reviewed to determine the number of LA tests ordered and rate of LA positivity before and after the LA panel was instituted. We assessed the impact of pathologist consultation to minimize false-positive findings and on following diagnostic guidelines. LA panels were ordered for 1146 patients. LA was detected in 10% (111 of 1146) by dilute Russel viper venom time (dRVVT) normalized ratio [includes dRVVT screen (dRVVTs) positive/lupus-sensitive partial thromboplastin time (PTT-LA) positive and dRVVTs positive/PTT-LA negative] and 20% (228 of 1146) by Staclot-LA (includes dRVVTs negative/PTT-LA positive and dRVVTs positive/confirm negative). There was a reduction of false-positive LA by Staclot-LA; previously, 48% positive. We saw increased cancellation of LA testing for interfering anticoagulants [6.8% (16 of 236) vs 14.4% (55 of 383); P = 0.0061]. There was also increased adherence to follow-up LA testing [3% (8 of 236) vs 13.8% (53 of 383); P ≤ 0.001]. Creating a predetermined order set and testing algorithm with pathologist consultation improved LA testing interpretation and diagnostic follow-up testing.

Experts consensus on clinical application of antiphospholipid antibodies detection

Antiphospholipid antibodies (aPLs) are a group of autoantibodies that target phospholipids and/or phospholipid-binding proteins. aPLs are important serum markers of anti phospholipid syndrome and are also risk factors for thrombosis and pathological pregnancy. Standardization of aPLs detection is critical to its clinical application.

Contribution from the laboratory in the diagnosis of heparin-induced thrombocytopenia. A case study

The diagnosis of antiphospholipid syndrome (APS) relies on the detection of antiphospholipid antibodies (aPL). Currently, lupus anticoagulant (LA), anticardiolipin (aCL), and antibeta2-glycoprotein I antibodies (aβ2GPI) IgG or IgM are included as laboratory criteria, if persistently present. LAC measurement remains a complicated procedure with many pitfalls and interfered by anticoagulant therapy. Solid-phase assays for aCL and aβ2GPI show interassay differences. These methodological issues make the laboratory diagnosis of APS challenging. In the interpretation of aPL results, antibody profiles help in identifying patients at risk. Other aPL, such as antibodies against the domain I of beta2-glycoprotein (aDI) and antiphosphatidylserine-prothrombin (aPS/PT) antibodies have been studied in the last years and may be useful in risk stratification of APS patients. Because of the methodological shortcomings of immunological and clotting assays, these non-criteria aPL may be useful in patients with incomplete antibody profiles to confirm or exclude the increased risk profile. This manuscript will focus on the laboratory aspects, the clinical relevance of assays and interpretation of aPL results in the diagnosis of APS.

Melkersson\u2013Rosenthal syndrome: a case report of a rare disease with overlapping features

BackgroundMelkersson–Rosenthal syndrome (MRS) is a rare, neuro-mucocutaneous disease which presents as orofacial swelling, facial palsy and fissured tongue. These symptoms may occur simultaneously or, more frequently, with a oligosymptomatic or monosymptomatic pattern. Swelling, that is the most common initial finding, may mimic hereditary or acquired angioedema, a disorder caused by histamine or bradykinin-mediated plasma-leakage affecting subcutaneous and/or submucosal tissue. The differential diagnosis of MRS includes also chronic inflammatory and infective diseases characterized by granulomatous infiltration, as well as rosacea, contact dermatitis, allergic reactions and Bell’s palsy.Case presentationA 71-year old, non-allergic female patient with no familial and personal history of angioedema presented, a few days after a possible herpes simplex or varicella-zoster virus infection, with monolateral facial paraesthesia and lower lip edema. After temporary remission of symptoms on oral steroids and antihistamines, she showed swelling recurrence refractory to valaciclovir therapy and a subsequent course of antihistamines. The clinical picture and a previous history of non-Hodgkin lymphoma prompted us to rule out an acquired form of paraneoplastic, C1-inhibitor (C1-INH) deficiency: C1q and both antigen and functional C1-INH tested normal, whilst we found low plasma levels of C3 and C4 possibly related to the parallel detection of antiphospholipid antibodies. Thus, we hypothesized a non-histaminergic, idiopathic form of angioedema and planned further therapy with tranexamic acid and the leukotriene receptor antagonist montelukast. Treatment failure with both drugs finally suggested a Melkersson–Rosenthal syndrome, which was confirmed by histologic findings of non caseating granulomas on lip biopsy.ConclusionMelkersson–Rosenthal syndrome may occur with rather non-specific symptoms and overlap with alternative conditions, including recurrent angioedema. No specific biomarkers for MRS exist and clinical diagnosis is often of exclusion. The finding of complement or immune alterations, as in our patient, may be further confounding and justify the need for skin or mucosal biopsy to establish a correct diagnosis and prescribe targeted therapy.

RANGING OF ANTIPHOSPOLIPID ANTIBODIES IN THE PATIENTS WITH THROMBOPHILIA AND RECURRENT MISCARRIAGE

Laboratory diagnosis of antiphospholipid syndrome (APS) is based on detection of antiphospholipid antibodies (aPLs). E.g., aPLs are directed against conformational epitopes of the so-called “co-factor” proteins: β2-gycoprotein 1 (β2-GP1), annexin V (An V) and prothrombin (Pt) that are formed during interaction with phospholipids – cardiolipin (CL), phosphatic acid (Pha), phosphatidylcholine (Pch), phosphatidylethanolamine (Pe), phosphatidylglycerol (Pg), phosphatidylinositol (Pi), phosphatidylserine (Ps). A routine methodology of detection based on ELISA testing is challenged by new tests when the antigen is absorbed on another kind of support like microbeads or membranes that can influence density of conformational epitopes for aPL’s binding. The aim of our study was to compare the results of aPLs detection by ELISA and multi-line immunodot assay (MLD). We collected blood serum samples from 45 patients with noncardioembolic ischemic strokes, 19 patients with recurrent deep vein thrombosis of lower limbs, 44 females with recurrent miscarriages, and 50 clinically healthy donors. To compare the results of aPL detection by ELISA and MLD kits, the test systems from different manufacturers were evaluated. We used an ELISA kits for detection of antibodies to CL IgG, aCL IgM, β2-GP1 produced by Euroimmun AG (Mr1) and Orgentec Diagnostica GmbH (Mr2) and MLD – for detection of antibodies to CL, β2-GP1, Pch, Pe, Pg, Pi, Ps, AnV and Pt (Medipan GmbH, Mr3). When a cut-off titer was used as the main index, 30.5% of patients were aPLs-positive with ELISA method by Mr1 and 38%, wiht Mr2. By MLD aPls were detected in 30% of patients. In the same cohort, medium and high aPLs titers (&gt; 40 U/mL) were determined in 12% of patients using ELISA kits. Positive and highly positive aPLs titers were determined in 16% when using a new method by Mr3. Medium and high titer were detected only for antibodies to β2-GP1, CL, An V, Pha and Phs. The use of ELISA approach for detection of aPLs in patients with thrombosis and obstetric pathology is associated with relatively high number of low-positive ELISA results. Due to higher sensitivity for medium and high aPLs titers, MLD testing may be used as a confirming method for APS diagnosis.

Analytical and clinical comparison of different immunoassay systems for the detection of antiphospholipid antibodies.

We evaluated analytical and clinical performances of IgG and IgM anticardiolipin (aCL) antibodies and anti-β2-glycoprotein I (a-β2GpI) antibodies and upper limit reference ranges (99th percentiles) in comparison with manufacturer's cutoff values with different commercial methods. We assayed aCL and a-β2GpI in serum samples from 30 healthy individuals, 77 patients with antiphospholipid syndrome (APS) diagnosed according to the Sydney criteria, 51 patients with autoimmune diseases, eight patients with previous thrombotic events, six patients with other diseases, and 18 patients with infectious diseases, using ELISA Inova Diagnostics; EliA Phadia Laboratory Systems; CliA Zenit-RA; and CliA Bio-Flash. Anticardiolipin and a-β2GpI IgG and IgM immunoassays showed good analytic performances with both 99th percentile and manufacturer's cutoff reference values. Our results showed fair to moderate agreement among assays. In-house cutoff values gave significantly better performances only for a-β2GpI IgG with all the immunoassays analyzed with the exception of Inova CliA Bio-Flash where we obtained the same performances with in-house and manufacturer's cutoffs. By guidelines, all laboratories are strongly advised to validate/verify the manufacturer's cutoff values. We recommend establishing low-positive, medium-/high-positive, and high-positive CliA IgG aCL and a-β2GpI ranges in order to help clinicians in the diagnosis and treatment of APS.

Detection of multiple annexin autoantibodies in a patient with recurrent miscarriages, fulminant stroke and seronegative antiphospholipid syndrome.

Anti-phospholipid syndrome (APS) is one of the main causes for recurrent miscarriages. The diagnosis of APS is based on the occurrence of clinical symptoms such as thrombotic events or obstetric complications as well as the detection of antiphospholipid antibodies directed against β2-glycoprotein I and cardiolipin, or a positive lupus anticoagulant assay. However, there is a subpopulation of patients with clinical symptoms of APS, but the lack of serological markers (seronegative APS). In addition, a large proportion of patients with unexplained recurrent miscarriages exist. These cases may be attributed, at least in part, to a seronegative APS. The presence of autoantibodies against annexins is potentially associated with APS. Here we used immunoassays and immunoblots to detect autoantibodies directed against annexin A1-5, and A8, respectively, in a patient with a seronegative APS and a history of six recurrent pregnancy losses and fulminant stroke. We found strong IgM isotype antibody reactivity directed against annexin A2 and annexin A8, and moderate to weak IgM isotype antibody reactivity directed against annexin A1, A3, and A5. Further studies will evaluate the diagnostic value of IgM isotype antibodies against annexin A1-A5, and A8 for seronegative APS and recurrent miscarriages.

Diagnostic performance study of an antigen microarray for the detection of antiphospholipid antibodies in human serum.

The parallelization of clinically relevant antigens in a microarray format is of growing importance due to the ability to measure multiple antigen-antibody interactions. With the development of a microarray for the detection of antiphospholipid antibodies we focussed on one important autoimmune disease that is still diagnostically challenging. Reasons are the heterogeneity of the autoantibodies and the unspecific clinical symptoms. For the covalent immobilization of antigenic structures, glass transducers were coated with 11-aminoundecyltrimethoxysilane (11-AUTMS). In total 35 antiphospholipid syndrome (APS) patients, six patients with lupus erythematosus and 24 healthy controls were investigated on a microarray format using polarized imaging reflectometric interference spectroscopy. The novel surface modification based on the short derivative 11-AUTMS resulted in a selective biosensor allowing a clear differentiation of patient and control samples. It combined proteinogenic as well as phospholipid-derived antigens, namely β2-glycoprotein I (β2-GPI), prothrombin, cardiolipin (CL) and a β2-GPI/CL complex. With optimized regeneration conditions, up to 20 consecutive measurements could be performed on one chip. Sensitivity was determined to be 0.800-0.929, specificity was between 0.733 and 0.969, depending on the respective antigen. Multiplexed determination of serological parameters has a great potential. We have shown that our biosensor is capable of detecting four different APS relevant antibodies in parallel exhibiting a sensitivity and specificity comparable to existing ELISA methods.

Autoantibody profiling in APS.

The international consensus for the classification of antiphospholipid syndrome (APS) requires clinical and laboratory criteria to be considered at an equal level for diagnosing APS. Thus, detection of antiphospholipid antibodies (aPL) being a hallmark of APS has been the object of intensive investigation over the past 40 years. However, appropriate detection of aPL still remains a laboratory challenge due to their heterogeneity comprising autoantibodies reactive to different phospholipid-binding plasma proteins, such as beta-2 glycoprotein I (β2GPI) and prothrombin. The relevance of aPL interacting with phospholipids other than cardiolipin (CL, diphosphatidylglycerol), such as phosphatidylserine (PS), remains elusive with regard to the diagnosis of APS. Recently, the concept of aPL profiling has been introduced to assess the risk of thrombotic complications in patients with APS. New assay techniques, apart from enzyme-linked immunosorbent assays (ELISAs) recommended by the international consensus for the classification of APS, have been proposed for multiplexing of aPL testing. Line immunoassays (LIAs) employing a novel hydrophobic solid phase for the simultaneous detection of different aPL seem to be an intriguing alternative. We evaluated a novel multiplex LIA employing a hydrophobic membrane coated with different phospholipid (PL)-binding proteins or PLs. The performance characteristics of this new multiplexing assay technique demonstrated its usefulness for aPL profiling.

Evaluation of different immunoassays for the detection of antiphospholipid antibodies: Report of a wet workshop during the 13th International Congress on Antiphospholipid Antibodies

BackgroundThe performance and standardization of anticardiolipin (aCL) and anti-β2 glycoprotein I antibodies (aβ2GPI) tests for the confirmation of diagnosis of antiphospholipid syndrome (APS) remain a matter of debate and concern. We evaluated the performance of different ELISAs and other new immunoassays for the detection of aCL and aβ2GPI in a wet workshop at the 13th International Congress on Antiphospholipid Antibodies in Galveston, TX (April 13th, 2010, APLA 2010). MethodsAliquots of 26 un-identified APS or persistently aPL positive serum samples and 21 controls (9 from healthy individuals and 5 from patients with infectious diseases and 7 with various autoimmune diseases) were distributed to all participants/groups. All serum samples were evaluated in various aCL and aβ2GPI ELISAs, a chemiluminescent immunoassay, a fluoro-enzyme immunoassay, and in a multiplexed immunoassay system. Monoclonal and polyclonal calibrators were also evaluated. ResultsAlthough not all the assays reported the titers of aCL and aβ2GPI in the same units, the correlation of positive titers among the assays was good. All aCL and aβ2GPI tests showed excellent clinical sensitivities, specificities and positive predictive values and good agreement with respect to the levels of the IgG and IgM antibodies, regardless of assay type, or whether tests were done using automated or “manual” systems. ConclusionsNew methodologies for the detection of aPL look promising and comparable to currently approved ELISA tests. This study provides evidence of progress of efforts of harmonization of tests used to detect aPL.

From antibody to clinical phenotype, the black box of the antiphospholipid syndrome: Pathogenic mechanisms of the antiphospholipid syndrome

The antiphospholipid syndrome (APS) is diagnosed by the combination of vascular thrombosis and/or pregnancy morbidity and the detection of antiphospholipid antibodies (aPLs) in plasma. In the last few years, a great effort has been made to unravel the mechanism by which aPLs cause thrombosis and a vast amount of mechanisms have been proposed. aPLs were proposed to induce a prothrombotic state by influencing the cellular blood compartment, the plasma compartment, the vascular wall and even metabolic pathways beyond the hemostatic system. However, due to the diversity in the mechanisms and the differences in the methodology, the focus of the mechanistical studies in this field seems to be largely diffused. It is hard to imagine that aPLs can exert such a diversity of effects, resulting in either thrombosis and/or pregnancy morbidity and the relationship between aPLs and the clinical manifestations remains to be a mysterious "black box". In an attempt to get insight in what takes place inside the black box, we have analyzed 126 mechanistical studies on aPLs and discussed differences in the type of antibodies that were used, the involvement of beta2-glycoprotein I (β2GPI), and the criteria used to diagnose APS patients.