Patients with antiphospholipid syndrome and a first venous or arterial thrombotic event: clinical characteristics, antibody profiles and estimate of the risk of recurrence.

OBJECTIVES: Thrombosis in antiphospholipid syndrome (APS) involves in most cases the venous circulation. Why in some patients thrombotic APS affects the arterial circulation and in particular cerebral circulation is unknown. In previous studies, both patient characteristics and antiphospholipid antibody types and titers have been associated with arterial thrombosis. Aim of this study was to compare the clinical characteristics and laboratory findings of venous and arterial thrombotic APS from a large series of patients. METHODS: Data were retrieved from the Start 2 antiphospholipid, a multicenter prospective register of long-term collected data from Thrombosis Centers in Italy. RESULTS: Of 167 patients with thrombotic APS, 114 (68 %) had a venous and 53 (32 %) had an arterial event as first clinical manifestation. Several clinical characteristics and risk factors were different among groups in univariate analysis. Using logistic regression analysis, reduced creatinine clearance and hyperlipidemia were independent variable for the occurrence of arterial APS. Notably, no difference in antiphospholipid antibody profiles and aß2-Glycoprotein I levels were found between groups. A higher adjusted global antiphospholipid syndrome score (aGAPSS) was found in arterial group indicating a possible high recurrence rate in arterial APS. CONCLUSIONS: These data have pathophysiological and clinical implication since associated conditions might predispose patients to arterial rather than venous events and call to a close monitoring and treatment of arterial APS due to their increased tendency to recurrence.

Close link between antiphosphatidylserine/prothrombin antibodies, lupus anticoagulant, and activated protein C resistance in tetra antiphospholipid antibody-positive subjects.

BACKGROUND: Most of the carriers/patients triple-positive for antiphospholipid antibodies (lupus anticoagulant [LAC], immunoglobulin G [IgG]/immunoglobulin M [IgM] anticardiolipin, and anti-ß2-glycoprotein I antibodies) are tetra-positive, being positive for antiphosphatidylserine/prothrombin (aPS/PT) antibodies. The relationship between aPS/PT titer, LAC potency, and resistance to activated protein C (aPC-R) has not been investigated. OBJECTIVES: The aim of this study was to clarify the mutual interdependence of these parameters in tetra-positive subjects. METHODS: Twenty-three carriers and 30 patients with antiphospholipid syndrome, none of whom were being treated with anticoagulants, and 30 age- and sex-matched controls were studied. Detection of aPS/PT, LAC, and aPC-R in each individual was performed with established methods in our laboratory. Carriers and patients were positive for IgG or IgM aPS/PT or for both isotypes without significant difference. Since both IgG and IgM aPS/PT have anticoagulant activity, we used the sum of their titers (total aPS/PT) for the correlation studies. RESULTS: Total aPS/PT in all individuals studied exceeded that in controls. There was no difference in total aPS/PT titers (P = .72), LAC potency (P = .56), and aPC-R (P = .82) between antiphospholipid antibody-carriers and patients with antiphospholipid syndrome. There was a significant correlation between total aPS/PT and LAC potency (r = 0.78; P < .0001) and between total aPS/PT titers and aPC-R (r = 0.80; P < .0001). LAC potency also was correlated significantly with aPC-R (r = 0.72; P < .0001). CONCLUSION: This study shows that there is interdependence between aPS/PT, LAC potency, and aPC-R.

Fulminant Myocarditis: When One Size Does Not Fit All - A Critical Review of the Literature.

Fulminant myocarditis, rather than being a distinct form of myocarditis, is instead a peculiar clinical presentation of the disease. The definition of fulminant myocarditis has varied greatly in the last 20 years, leading to conflicting reports on prognosis and treatment strategies, mainly because of varied inclusion criteria in different studies. The main conclusion of this review is that fulminant myocarditis may be due to different histotypes and aetiologies that can be diagnosed only by endomyocardial biopsy and managed by aetiology-directed treatment. This life-threatening presentation requires rapid, targeted management both in the short term (mechanical circulatory support, inotropic and antiarrhythmic treatment and endomyocardial biopsy) and in the long term (including prolonged follow-up). Fulminant presentation has also recently been identified as a risk factor for worsened prognosis, even long after the resolution of the acute phase of myocarditis.

A Machine-Learning Model for the Prognostic Role of C-Reactive Protein in Myocarditis.

Aims: The role of inflammation markers in myocarditis is unclear. We assessed the diagnostic and prognostic correlates of C-reactive protein (CRP) at diagnosis in patients with myocarditis. Methods and results: We retrospectively enrolled patients with clinically suspected (CS) or biopsy-proven (BP) myocarditis, with available CRP at diagnosis. Clinical, laboratory and imaging data were collected at diagnosis and at follow-up visits. To evaluate predictors of death/heart transplant (Htx), a machine-learning approach based on random forest for survival data was employed. We included 409 patients (74% males, aged 37 ± 15, median follow-up 2.9 years). Abnormal CRP was reported in 288 patients, mainly with CS myocarditis (p < 0.001), recent viral infection, shorter symptoms duration (p = 0.001), chest pain (p < 0.001), better functional class at diagnosis (p = 0.018) and higher troponin I values (p < 0.001). Death/Htx was reported in 13 patients, of whom 10 had BP myocarditis (overall 10-year survival 94%). Survival rates did not differ according to CRP levels (p = 0.23). The strongest survival predictor was LVEF, followed by anti-nuclear auto-antibodies (ANA) and BP status. Conclusions: Raised CRP at diagnosis identifies patients with CS myocarditis and less severe clinical features, but does not contribute to predicting survival. Main death/Htx predictors are reduced LVEF, BP diagnosis and positive ANA.

Antiphospholipid syndrome: Reversal of antiphosphatidylserine/prothrombin-induced activated protein C resistance.

BACKGROUND: Anti-phosphatidylserine/prothrombin (aPS/PT) antibodies are the major contributor to activated Protein C resistance (APC-R) in tetra-positive thrombotic high-risk patients with Antiphospholipid Syndrome (APS). OBJECTIVES: To evaluate the role of phospholipids (PL) on aPS/PT mediated APC-R. PATIENTS/METHODS: Total IgG were purified from plasma of 6 tetra-positive patients and IgG aPS/PT were affinity-purified from 3 of these patients. Purified material was spiked into Normal Pooled Plasma (NPP) and tested for APC-R in thrombin generation assay and in Factor Va inactivation assay using increasing amounts of phospholipids. RESULTS AND CONCLUSIONS: Total IgG showed APC-R at low PL concentration (1.5 µg/mL) that disappeared at increasing PL concentrations (5.8, 11.6 and 46.6 µg/mL). In the same way, affinity purified aPS/PT showed a robust (59 %, 52 %, 36 %) APC-R in patients #4, #5 and #6, respectively at low PL concentration (1.5 µg/mL) that was completely reversed at higher concentration (11.6 µg/mL). The inactivation of FVa by activated Protein C (aPC) was impaired in the presence of aPS/PT at low aPL concentration and reversed by increasing amounts of PL. These data point out the relevance of PL in reversing APC-R in this 'in vitro' system. The mechanism for reversal might be explained by loss of PL availability for aPC. These results may give some insight into the pathogenesis of thrombosis or suggestions for alternative treatments.

Platelet- and endothelial-derived microparticles in the context of different antiphospholipid antibody profiles.

OBJECTIVES: Studies on microparticles (MPs) in patients with antiphospholipid antibodies (aPL) are sparse and inconclusive. The relation between MPs and different aPL antibody profiles has never been tested. We evaluated the presence of platelet and endothelial microparticles in patients positive for IgG anti-ß2-glycoprotein I (aß2GPI) antibodies according to triple, double and single positive aPL profiles. METHODS: Megamix (Biocytex) was used to set up the MPs gating according to the datasheet. Markers of Platelet Microparticles (PMPs) were CD41a-PE and annexin-V-FITC that was used to determine phosphatidylserine (PS) exposure. CD144-FITC was used as a marker of Endothelial Microparticles (EMPs). RESULTS: The number of total MPs and EMPs was significantly higher in triple positive groups with respect to single positive group and showed a significant correlation with IgG aß2GPI titers. The number PMPs was the lowest in triple positive group and inversely correlated with IgG aß2GPI titers. CONCLUSIONS: Elevated levels of total MPs and EMPs suggest a state of vascular activation in IgG aß2GPI positive individuals according to the number of positive tests. PMPs may be fast cleared from circulation in high risk triple positive patients.

Predictors of relapse, death or heart transplantation in myocarditis before the introduction of immunosuppression: negative prognostic impact of female gender, fulminant onset, lower ejection fraction and serum autoantibodies.

AIMS: Outcome predictors in myocarditis are not well defined; we aimed at identifying predictors of death, heart transplantation (HTx) and relapse before the introduction of immunosuppression. METHODS AND RESULTS: From 1992 to 2012, 466 consecutive patients (68% male, mean age 37 ± 17 years, single centre recruitment, median follow-up 50 months) were included, of whom 216 had clinically suspected and 250 biopsy-proven myocarditis. Serum anti-heart (AHA) and anti-intercalated disk (AIDA) autoantibodies were measured by indirect immunofluorescence. Univariable and multivariable analyses of clinical and diagnostic features at diagnosis were performed. Survival free from death or HTx at 10 years was 83% in the whole study population and was lower in biopsy-proven versus clinically suspected myocarditis (76% vs. 94%, p < 0.001). Female gender (hazard ratio [HR] 2.7, 95% confidence interval [CI] 1.1-6.5), fulminant presentation (HR 13.77, 95% CI 9.7-261.73), high-titre organ-specific AHA (HR 4.2, 95% CI 1.2-14.7) and anti-nuclear antibodies (ANA) (HR 5.2, 95% CI 2.1-12.8) were independent predictors of death or HTx; higher echocardiographic left ventricular ejection fraction (LVEF) at diagnosis was protective, with a 0.93-fold risk reduction for each 1% LVEF increase (95% CI 0.89-0.96). History of myocarditis at diagnosis (HR 8.5, 95% CI 3.5-20.7) was an independent predictor of myocarditis relapse at follow-up; older age was protective (HR 0.95, 95% CI 0.91-0.99). Predictors of death, HTx and relapse did not differ in biopsy-proven versus clinically suspected myocarditis. CONCLUSIONS: Young age and a previous myocarditis were independent relapse predictors; female gender, fulminant onset, lower LVEF at presentation and high-titre organ-specific AHA and ANA were independent predictors of death and HTx, suggesting that autoimmune features predict worse prognosis.

Serum Organ-Specific Anti-Heart and Anti-Intercalated Disk Autoantibodies as New Autoimmune Markers of Cardiac Involvement in Systemic Sclerosis: Frequency, Clinical and Prognostic Correlates.

BACKGROUND: Heart involvement (HInv) in systemic sclerosis (SSc) may relate to myocarditis and is associated with poor prognosis. Serum anti-heart (AHA) and anti-intercalated disk autoantibodies (AIDA) are organ and disease-specific markers of isolated autoimmune myocarditis. We assessed frequencies, clinical correlates, and prognostic impacts of AHA and AIDA in SSc. METHODS: The study included consecutive SSc patients (n = 116, aged 53 ± 13 years, 83.6% females, median disease duration 7 years) with clinically suspected heart involvement (symptoms, abnormal ECG, abnormal troponin I or natriuretic peptides, and abnormal echocardiography). All SSc patients underwent CMR. Serum AHA and AIDA were measured by indirect immunofluorescence in SSc and in control groups of non-inflammatory cardiac disease (NICD) (n = 160), ischemic heart failure (IHF) (n = 141), and normal blood donors (NBD) (n = 270). AHA and AIDA status in SSc was correlated with baseline clinical, diagnostic features, and outcome. RESULTS: The frequency of AHA was higher in SSc (57/116, 49%, p < 0.00001) than in NICD (2/160, 1%), IHF (2/141, 1%), or NBD (7/270, 2.5%). The frequency of AIDA was higher (65/116, 56%, p < 0.00001) in SSc than in NICD (6/160, 3.75%), IHF (3/141, 2%), or NBD (1/270, 0.37%). AHAs were associated with interstitial lung disease (p = 0.04), history of chest pain (p = 0.026), abnormal troponin (p = 0.006), AIDA (p = 0.000), and current immunosuppression (p = 0.01). AHAs were associated with death (p = 0.02) and overall cardiac events during follow-up (p = 0.017). CONCLUSIONS: The high frequencies of AHA and AIDA suggest a high burden of underdiagnosed autoimmune HInv in SSc. In keeping with the negative prognostic impact of HInv in SSc, AHAs were associated with dismal prognosis.

Anti-phosphatidyl-serine/prothrombin antibodies (aPS/PT) in isolated lupus anticoagulant (LA): is their presence linked to dual test positivity?

OBJECTIVES: Anti phosphatidylserine/prothrombin antibodies (aPS/PT) are often present in patients with antiphospholipid syndrome (APS) and might be relevant in the pathogenesis of this condition. They are major determinant of lupus anticoagulant (LA) in triple-positive antiphospholipid (aPL) profile. Whether they are present and pathogenic in patients with isolated LA [negative anticardiolipin (aCL) and anti ß2-glycoprotein I (aß2GPI) antibodies] is a matter of debate. METHODS: We measured aPS/PT in a large number of isolated LA with the aim to ascertain whether there is a link between the way isolated LA is assessed and the presence of these antibodies. APS/PT were measured in 86 patients with isolated LA (aCL- and abeta2GPI-). LA was assessed by two test systems, the dilute Russell Viper Venom Time (dRVVT) and the Silica Clotting Time (SCT). RESULTS: Sixty-six (77%) individuals with isolated LA were positive for aPS/PT (IgM 44, IgG and IgM 15, IgG in 7). Diagnosis of LA was made based on positive results in both dRVVT and SCT in 40 patients (Group 1) and based on only one positive test in the remaining 46 patients (Group 2). The rate of positive aPS/PT antibodies was significantly higher in Group 1 (OR=7.2, 95% CI 1.9-27.0, p<0.002). Moreover, the titre of IgM aPS/PT was significantly increased in Group 1 as compared to Group 2 (137 U, IQR 64-179 vs. 43 U, IQR 11-120, p=0.008). CONCLUSIONS: These data indicate an association between LA based on two positive coagulation tests and the presence of aPS/PT antibodies, especially of IgM isotype.

Serum Anti-Heart and Anti-Intercalated Disk Autoantibodies: Novel Autoimmune Markers in Cardiac Sarcoidosis.

BACKGROUND: Sarcoidosis is an immune-mediated disease. Cardiac involvement, a granulomatous form of myocarditis, is under-recognized and prognostically relevant. Anti-heart autoantibodies (AHAs) and anti-intercalated disk autoantibodies (AIDAs) are autoimmune markers in nonsarcoidosis myocarditis forms. OBJECTIVE: The aim was to assess serum AHAs and AIDAs as autoimmune markers in cardiac sarcoidosis. METHODS: This is a cross-sectional study on AHA and AIDA frequency in: 29 patients (aged 46 ± 12, 20 male) with biopsy-proven extracardiac sarcoidosis and biopsy-proven or clinically suspected and confirmed by 18-fluorodeoxyglucose positron emission tomography and/or cardiovascular magnetic resonance (CMR) cardiac involvement; 30 patients (aged 44 ± 11, 12 male) with biopsy-proven extracardiac sarcoidosis without cardiac involvement (no cardiac symptoms, normal 12-lead electrocardiogram, echocardiography and CMR), and control patients with noninflammatory cardiac disease (NICD) (n = 160), ischemic heart failure (IHF) (n = 141) and normal blood donors (NBDs) (n = 270). Sarcoidosis patients were recruited in two recruiting tertiary centers in the USA and Italy. AHAs and AIDAs were detected by indirect immunofluorescence on the human myocardium and skeletal muscle. RESULTS: AHA and AIDA frequencies were higher in sarcoidosis with cardiac involvement (86%; 62%) than in sarcoidosis without cardiac involvement (0%; 0%), NICD (8%; 4%), IHF (7%; 2%) and NBD (9%; 0%) (p = 0.0001; p = 0.0001, respectively). Sensitivity and specificity for cardiac sarcoidosis were 86% and 92% for positive AHAs and 62% and 98% for positive AIDAs, respectively. AIDAs in cardiac sarcoidosis were associated with a higher number of involved organs (p = 0.04). CONCLUSIONS: Serum AHAs and AIDAs provide novel noninvasive diagnostic autoimmune markers for cardiac sarcoidosis.

Insight into the hypercoagulable state of high-risk thrombotic APS patients: Contribution of aß2GPI and aPS/PT antibodies.

OBJECTIVE: Most high-risk thrombotic antiphospholipid syndrome (APS) patients test positive for anti-ß2-glycoprotein I (aß2GPI) and anti-phosphatidylserine/prothrombin (aPS/PT) antibodies. Information on the influence of these antibodies on thrombin generation and activated protein C resistance (aPCr) is still sparse and contradictory. METHODS: Plasma of 16 patients poured into a ß2GPI affinity column allowed the perfect separation of aß2GPI and aPS/PT antibodies. aPS/PT antibodies were further purified through a prothrombin affinity column. Obtained material was spiked into normal pooled plasma (NPP) and tested in the thrombin generation assay in the absence or presence of aPC. RESULTS: aPS/PT antibodies showed a marked anticoagulant effect. Affinity purified aPS/PT and aß2GPI antibodies from five patients were compared. aPS/PT antibodies showed significantly prolonged lag time and time to peak (5.0 minutes [interquartile range (IQR)3.5-6.1] versus 2.7 minutes [IQR2.2-3.5], P = .03 and 8.7 minutes [IQR6.7-10.3] versus 5.7 minutes [IQR4.5-6.2], P = .05, respectively) and significantly lower peak and velocity index (143 nmol/L [IQR131-163] versus 171 nmol/L [IQR157-182], P = .03 and 35 nmol/L/min [IQR32-59] versus 72 nmol/L/min [IQR54-77], P = .03, respectively). When aPC was added to the system, aPCr was significantly increased compared to controls for both aß2GPI and aPS/PT antibodies. However, it was significantly stronger using aPS/PT antibodies. Median inhibition of endogenous thrombin potential was 22% (IQR16-33) with aPS/PT compared to 52% (IQR46-56) with aß2GPI antibodies (P = .002). CONCLUSIONS: Aß2GPI antibodies show a mild anticoagulant and moderate procoagulant effect in thrombin generation and moderate aPC resistance. Conversely, aPS/PT antibodies show a strong anticoagulant effect and a strong aPCr.

Antibody profiles comprising anti phosphatidylserine/prothrombin differently affect thrombin generation and protein C resistance in antiphospholipid antibody carriers.

Anti phosphatidylserine/prothrombin antibodies (aPS/PT) are currently not included in the laboratory work-up of antiphospholipid symdrome (APS). However, several studies indicate that aPS/PT confer additional risk for thromboembolic events when added to classical antiphospholipid (aPL) antibody panel. We aimed to study thrombin generation (TG), a test that describes hyper or hypo-coagulability, in a cohort of antiphospholipid antibody (aPL) carriers with or without aPS/PT. As oral anticoagulants interfere with TG, we performed the study in carriers of aPL antibodies not on oral anticoagulants treatment. TG in tissue factor-triggered platelet-poor plasma and its inhibition by thrombomodulin was measured with a calibrated automated thrombogram method. Data are expressed as minutes (Interquartile Range). Of 55 aPL carriers, 37 were positive and 18 were negative for aPS/PT. Lag Time 5.4 min (4.1; 7.3) vs 3.4 min (3.0;4.5) is significant longer (p < 0.0001) and time to peak 9.6 min (8.1;11) vs 7.7 min (6.8;8.8) is significantly delayed (p = 0.0011) in aPS/PT positive as compared to aPS/PT negative carriers. Endogenous Thrombin Potential (ETP), peak thrombin formation and the velocity index are lower in aPS/PT positive carriers but did not reach statistical significance. Inhibition of ETP by thrombomodulin was significantly lower (protein C resistance) in aPS/PT positive vs aPS/PT negative group (22.8%±11.5 vs 34.9%±20.4, p = 0.01). In conclusion, aPS/PT positive carriers show an anticoagulant effect in TG while they exert a procoagulant effect in response to thrombomodulin-activated protein C.

Prothrombin Is Responsible for the Lupus Cofactor Phenomenon in a Patient with Lupus Anticoagulant/Hypoprothrombinemia Syndrome.

Lupus anticoagulant is a misnomer as it is commonly associated with thromboembolic events. In few cases, the name retains its literal meaning when it characterizes patients with a bleeding disorder. We describe a patient with lupus anticoagulant, hypoprothrombinemia, and major bleeding (lupus anticoagulant/hypoprothrombinemia syndrome). Immunological studies revealed a huge amount of circulating monoclonal immunoglobulin M lambda (IgMλ) antiphosphatidylserine/prothrombin antibodies (14,400 U/mL). Affinity purified monoclonal antibodies (440 U/mL) prolonged the coagulation time of normal plasma by 12.2 seconds (diluted Russell viper venom time) and 25.5 seconds (silica clotting time). The original patient's plasma mixed 1:1 with normal plasma showed a marked prolongation of coagulation times (lupus cofactor) from a ratio of 2.94 to 5.23 in diluted Russel viper venom time and from 2.30 to 3.00 using the silica clotting time. Human prothrombin added to original patient's plasma caused a marked prolongation of coagulation times in diluted Russell viper venom test thus unequivocally explaining the lupus cofactor phenomenon. In conclusion, we have shown that lupus anticoagulant/hypoprothrombinemia syndrome is attributable to monoclonal IgMλ antibodies directed to phosphatidylserine/prothrombin and that prothrombin is the protein responsible for the observed lupus cofactor phenomenon.

Tetra positive thrombotic antiphospholipid syndrome: Major contribution of anti-phosphatidyl-serine/prothrombin antibodies to lupus anticoagulant activity.

BACKGROUND: The concurrent presence of lupus anticoagulant, anticardiolipin, and anti ß2-glycoprotein I antibodies (triple positive profile) identifies patients at high risk of thromboembolic events. These patients are also positive for anti-phosphatidyl-serine/prothrombin antibodies (tetra-positive profile). OBJECTIVE: Understand which antibody among anti-ß2-glycoprotein I and anti-phosphatidyl-serine/prothrombin is responsible for lupus anticoagulant activity. PATIENTS/METHODS: Affinity purified anti-ß2-glycoprotein I antibodies from plasma of 14 tetra-positive patients spiked into normal pooled plasma were tested. RESULTS AND CONCLUSIONS: Anti-ß2-glycoprotein I antibodies did not prolong the diluted Russell viper venom time and silica clotting time (median ratio 0.98, interquartile ratio [IQR] 0.9-1.06; and 1.0, IQR 0.91-1.03, respectively). Anticoagulant activity remained in the flow-through that was deprived of anti-ß2 glycoprotein I antibodies (median ratio 1.88, IQR 1.58-2.77; and 1.75, IQR 1.17-2.9, respectively). This material was loaded on size-exclusion chromatography Sephacryl S-300 column and showed that anticoagulant activity and anti-phosphatidyl-serine/prothrombin antibodies coeluted in the same fractions. Besides, the flow through was poured into a prothrombin affinity column. Protein yield in three patients ranged from 54 to 91 µg/mL and showed strong positivity in phosphatidyl-serine/prothrombin ELISA. The affinity purified material prolonged the coagulation time of normal pooled plasma: the diluted Russell viper venom ratio in the three patients was 2.09, 1.21, and 1.35; that of silica clotting time was 2.05, 1.5, and 2.13. In conclusion, under the assay conditions used, anticoagulant activity in tetra-positive antiphospholipid syndrome patients may largely be attributable to anti-phosphatidyl-serine/prothrombin antibodies.

Comparison of real world and core laboratory lupus anticoagulant results from the Antiphospholipid Syndrome Alliance for Clinical Trials and International Networking (APS ACTION) clinical database and repository.

BACKGROUND: Variability remains a challenge in lupus anticoagulant (LA) testing. OBJECTIVE: To validate LA test performance between Antiphospholipid Syndrome Alliance for Clinical Trials and International Networking (APS ACTION) Core laboratories and examine agreement in LA status between Core and local/hospital laboratories contributing patients to this prospective registry. METHODS: Five Core laboratories used the same reagents, analyzer type, protocols, and characterized samples for LA validation. Non-anticoagulated registry samples were retested at the corresponding regional Core laboratories and anticoagulated samples at a single Core laboratory. Categorical agreement and discrepancies in LA status between Core and local/hospital laboratories were analyzed. RESULTS: Clotting times for the reference/characterized plasmas used for normalized ratios were similar between Core laboratories (CV <4%); precision and agreement for LA positive/negative plasma were similar (all CV ≤5%) in the four laboratories that completed both parts of the validation exercise; 418 registry samples underwent LA testing. Agreement for LA positive/negative status between Core and local/hospital laboratories was observed in 87% (115/132) non-anticoagulated and 77% (183/237) anticoagulated samples. However, 28.7% (120/418) of samples showed discordance between the Core and local/hospital laboratories or equivocal LA results. Some of the results of the local/hospital laboratories might have been unreliable in 24.7% (41/166) and 23% (58/252) of the total non-anticoagulated and anticoagulated samples, respectively. Equivocal results by the Core laboratory might have also contributed to discordance. CONCLUSIONS: Laboratories can achieve good agreement in LA performance by use of the same reagents, analyzer type, and protocols. The standardized Core laboratory results underpin accurate interpretation of APS ACTION clinical data.

Laboratory Diagnostics of Antiphospholipid Syndrome.

Diagnosis of antiphospholipid syndrome (APS) lies in the recognition of antiphospholipid antibodies (aPL). As standardization of tests for the detection of aPL is far from being optimal and reference material is not available, inappropriate diagnoses of APS are not unusual. In the last few years, the concept of triple test positivity has emerged as a useful tool to identify patients with APS. Clinical studies on patients and carriers of triple positivity clearly show that these individuals are at high risk of thromboembolic events and pregnancy loss. Moreover, triple positivity arises from a single (probably pathogenic) antibody directed to domain 1 of ß2-glycoprotein I, a protein whose function is still unknown. Studies on homogenous group of patients with single or double positivity are scant, and uncertainties arise on their association with clinical events. Promising but undetermined results come also from the determination of antibodies directed to phosphatidylserine/prothrombin complex. Interpretation of laboratory profile in APS is challenging, and the collaboration between clinical pathologists and clinicians is highly desirable.

Lupus Anticoagulant Testing: Diluted Russell Viper Venom Time (dRVVT).

Diluted Russell Viper Venom Time (dRVVT) has become the most popular test to detect Lupus Anticoagulant (LA). dRVVT is more sensitive than other global tests employed to detect LA and is not affected by inhibitors of factor VIII or IX. The test is most successfully implemented if you observe three steps in its execution: screening, mixing, and confirmatory studies. Interference due to the presence of heparin in tested plasma must be excluded by means of thrombin time (TT). The prior use of Vitamin K Antagonists (VKAs) or Non-vitamin K Oral Anticoagulants (NOACs) must also be evaluated by means of International Normalized Ratio, or specific tests, respectively.

Diagnosis and therapy of antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is a clinical condition that has not been well defined yet. Although the clinical component is well established, the laboratory part is a mood issue. According to current guidelines, 3 tests (lupus anticoagulant, anticardiolipin, and anti ß2-glycoprotein I antibodies) are officially recommended to assess the presence of antiphospholipid antibodies. According to test positivity, patients are classified into categories in clinical studies. However, it is now clear that classification categories have a different impact on the clinical course of APS. Indeed, patients and healthy carriers with a full positive antibody profile (triple positivity) are those at the highest risk of events. Patients with a single test positivity are those at a lower risk. In this review, on the basis of a laboratory profile, we grade the diagnosis of APS into definite, probable/possible, and uncertain. We also discuss secondary prevention of thrombotic APS, prevention of pregnancy morbidity, and treatment of catastrophic APS. Finally, new tools in laboratory diagnosis and treatment are highlighted.

Incidence of a first thromboembolic event in carriers of isolated lupus anticoagulant.

Among the so called antiphospholipid (aPL) antibodies Lupus Anticoagulant (LAC) is considered the strongest risk factor for thromboembolic events. In individuals without a previous thromboembolic event (carriers), LAC is a risk factor when associated with the presence of anticardiolipin (aCL) and aß2-Glycoprotein I (aß2GPI) antibodies. On the other hand, data on carriers of isolated LAC positivity are sparse and inconclusive. The aim of this study was to prospectively determine the incidence of thrombosis in a cohort of carriers of isolated LAC positivity. One-hundred seventy-nine carriers of LAC confirmed twelve weeks apart and in a reference laboratory were studied. During a total follow up of 552 person-years, there were seven thromboembolic events (1.3% person-y). All the seven patients had at least one adjunctive major risk factor for thrombosis. The cumulative incidence of thromboembolic events was 3.1% (95% CI 0.6-5.6) after 2years, and 5.9% (95% CI 1.2-10.6) after 5 and 10years. On a multivariate regression analysis considering age, sex, autoimmune disease, risk factors for arterial and venous thrombosis, use of aspirin, only age was found to be an independent predictor of thromboembolic events (HR=1.1, 95% CI 1.0-1.2, p=0.02). These data might be relevant in clinical practice and underline the importance of differentiating LAC carriers in terms of isolated positivity or positivity associated with the presence of antibodies to aCL and ß2-glycoprotein I.

The paradox of the lupus anticoagulant: history and perspectives.

A unique coagulation inhibitor prolonging whole-blood clotting time was described more than 50 years ago in two patients with systemic lupus erythematosus (SLE). The immunoglobulin nature of the inhibitor and its interaction with antiphospholipid antibodies was later demonstrated and the term "lupus anticoagulant (LA)" was coined to describe this laboratory finding. It soon became apparent that LA was a misnomer as it is often found in plasma from patients with clinical conditions other than SLE and is associated with thromboembolic events that may occur in otherwise healthy individuals. Individuals with LA have circulating autoantibodies that inhibits blood coagulation. These are mostly of IgG or IgM class and mainly directed against a phospholipid (PL)-binding plasma protein, ß2-glycoprotein I (ß2GPI). The presence of ß2GPI-dependent LA represents a well-recognized risk factor for venous and arterial thromboembolism, as well as pregnancy loss and morbidity. ß2GPI-dependent LA in the presence of documented previous thromboembolism, or history of pregnancy loss/morbidity, identifies definite anti-PL syndrome. Laboratory diagnosis of LA is thus of particular importance, as it may assign patients with a common event (thrombosis) to a group with a high risk for recurrence, which is a prerequisite for long-term oral antithrombotic treatment.