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Background: Anticardiolipin antibodies (aCL) and anti-β 2 -glycoprotein I antibodies (aβ 2 GPI) are essential in diagnosing antiphospholipid syndrome (APS) according to the international APS guideline. Five commercial assays for aCL and aβ 2 GPI are available in Japan, but their test results are quite discordant. For harmonization of diagnosing APS, upper reference limit (URL) and diagnostic accuracy of each assay were evaluated and compared by testing common sets of specimens across all assays., Methods: We evaluated two manual and three automated assays for aCL and aβ 2 GPI of IgG- and IgM classes. 99%URL (the upper limit of reference interval: as per guideline) together with 97.5%URL were determined by testing sera from 198 to 400 well-defined healthy subjects. Both URLs were compared with the cutoff values, which were determined based on ROC analysis by testing 50 each of plasma specimens from patients with/without APS. Diagnostic accuracy was evaluated as area under curve (AUC) of the ROC curve., Results: A variable degree of discrepancy between URLs and the cutoff values was observed, which was partly attributable to between-year assay variability. 97.5%URLs were set lower and closer to the cutoff values than 99%URLs. For all assays, diagnostic accuracies of both aβ 2 GPI-IgG and aCL-IgG were generally high (AUC: 0.84-0.93); whereas those for IgM-class assays were low (AUC: 0.57-0.67), implicating its utility is limited to rare IgG negative APS cases., Conclusion: To ensure harmonized APS diagnosis, the diagnostic thresholds of the five assays were evaluated by common procedures. Contrary to the guideline, 97.5%URL is rather recommended for diagnosing APS, which showed a closer match to the cutoff value., (© 2022 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

The causes of thrombosis in antiphospholipid syndrome (APS) remain unknown, though several hypotheses in regard to hypofibrinolysis have been proposed. To clarify the mechanism, we measured plasma levels of thrombin-activatable fibrinolysis inhibitor (TAFI) in APS patients. Both the TAFI antigen (TAFI:Ag) level measured with an ELISA, and thrombin-thrombomodulin-dependent TAFI activity (TAFI:Ac) were elevated in 68 APS patients as compared with those in 66 healthy controls, though they were lower than those in 46 patients with autoimmune diseases. As for the influence of antiphospholipid antibodies (aPL) on TAFI levels, the mean TAFI:Ac level in 39 SLE patients positive for APS was significantly lower than that in 27 SLE patients without APS, whereas there was no difference in TAFI:Ag between those groups. Furthermore, purified IgG from patients positive for aPL, and monoclonal aPL (EY2C9 and 23-1D) inhibited the activation of TAFI in a concentration dependent manner. These results suggest that aPL inhibits TAFI activation by affecting the function of thrombomodulin-thrombin complex through phospholipids. Although TAFI in plasma is elevated in autoimmune diseases including APS, we concluded that an elevated level is not likely a risk factor for thrombosis in APS patients, because of the inhibition of TAFI activation by aPL.

Antiphospholipid syndrome (APS), formerly detected by the association between clinical events (venous/arterial thrombosis or pregnancy morbidity) and positivity in at least one of the laboratory tests used to detect antiphospholipid antibodies (aPL), namely lupus anticoagulant (LA), IgG- or IgM-class anticardiolipin antibodies (aCL), and IgG or IgM-class anti-beta2GPI antibodies (abeta2GPI), is now diagnosed using revised APS classification criteria. However, there are a number of problems with the methods used to detect aPL. IgM-class aCL and abeta2GPI measurements are not covered by the health insurance system of Japan, while ELISA to measure abeta2GPI has not been standardized LA which is strongly associated with thrombosis in APS, can be determined using methods recommended by the Scientific and Standardization Committee of the International Society of Thrombosis and Haemostasis (ISTH-SSC). According to the guidelines for detection of LA recently published by ISTH-SSC, APTT reagents with silica as an activator and double centrifugation-treated samples (including control plasma) are recommended to detect LA, while 50% patient plasma mixed with control plasma is recommended for a cross-mixing test. However, some APTT reagents with ellagic acid are sensitive for LA activity. In the present study, we obtained favorable results for LA detection with cross-mixing tests by measuring APTT in mixtures of control plasma with 0%, 10%, 20%, 50%, and 100% concentrations of patient plasma treated with a 0.2 microm filter. In the future, laboratory examinations for APS will change with diagnostic criteria, as APS has not yet been established as a distinct disease concept.

Antiphospholipid syndrome (APS) is an autoimmune disorder in which vascular thrombosis and recurrent pregnancy loss occur in patients with antiphospholipid antibodies(aPL). Measurements of the beta2-glycoprotein I-dependent anticardiolipin antibody(aCL) and lupus anticoagulant(LAC) are the only laboratory tests available for the diagnosis of APS. Recently, phosphatidylserine-dependent antiprothrombin antibody(aPS/PT) has been detected. aPS/PT was measured by ELISA using the phosphatidylserine-prothrombin complex as an antigen immobilized on ELISA plates in the presence of CaCl2. In our study of 219 patients with APS and autoimmune diseases, the prevalence of aPS/PT-IgG in those with APS was 42.2%, which was significantly higher than that(4.6%) in patients with autoimmune diseases. Furthermore, aPS/PT was closely associated with APS manifestations with an odds ratio (OR) of 2.92 (95% confidence interval (95% CI): 1.33 to approximately 6.40), whereas the OR for aCL was 2.06 (95% CI: 0.91 to approximately 4.66). In addition, aPS/PT-IgG was strongly correlated with the presence of LAC as detected with a diluted Russell viper venom time test (dRVVT) (OR: 38.2, 95% CI: 13.4 to approximately 109.1). The monoclonal antibody (23-1D) of aPS/PT also prolonged the clotting time in LAC tests (aPTT, dRVVT, and kaolin clotting time) in a concentration-dependent manner. In conclusion, aPS/PT is more closely associated with manifestations of APS and LAC, and positive results from an aPS/PT test can mark thrombotic events in APS patients. The determination of aPS/PT in clinical practice, in conjunction with that of other aPL, may improve the likelihood of recognizing APS.

A 61-year-old woman experienced a high fever with anemia and APTT prolongation after suffering a herpes zoster virus infection. Physical examination revealed a large splenomegaly without lymphadenopathy. Laboratory evaluations were positive for lupus anticoagulant (LA) and monoclonal IgM-kappa protein. LA was associated with the presence of anti-beta2GPI antibody, anti-cardiolipin antibody, and anti-prothrombin antibody. Moreover, the results of factors IX, XI, and XII assays and CRP and FDP-E were disturbed. A splenectomy was performed, and a splenic marginal zone lymphoma (SMZL) was diagnosed. All hematological findings rapidly recovered after the splenectomy. No thrombotic events occurred after the splenectomy even though thrombosis prophylaxis was not performed. The clinical course suggested that the SMZL-producing antibody induced immunological abnormalities in the labolatory tests. Since the patient suffered disease progression soon after the splenectomy, an autologous peripheral stem cell transplantation with rituximab administration was performed.

Beta2-glycoprotein-I (beta2GPI) is a phospholipid-binding plasma protein that consists of five homologous domains. Domain V is distinguished from others by bearing a positively charged lysine cluster and hydrophobic extra C-terminal loop. Beta2GPI has been known as a natural anticoagulant regulator. Beta2GPI exerts anticoagulant activity by inhibition of phospholipid-dependent coagulation reactions such as prothrombinase, tenase, and factor XII activation. It also binds factor XI and inhibits its activation. On the other hand, beta2GPI inhibits anticoagulant activity of activated protein C. According to the data from knockout mice, beta2GPI may contribute to thrombin generation in vivo. Phospholipid-bound beta2GPI is one of the major target antigens for antiphospholipid antibodies present in patients with antiphospholipid syndrome (APS). Binding of pathogenic anti-beta2GPI antibodies increases the affinity of beta2GPI to the cell surface and disrupts the coagulation/fibrinolysis balance on the cell surface. These pathogenic antibodies activate endothelial cells via signal transduction events in the presence of beta2GPI. Impaired fibrinolysis has been reported in patients with APS. Using a newly developed chromogenic assay, we demonstrated lower activity of intrinsic fibrinolysis in euglobulin fractions from APS patients. Addition of monoclonal anti-beta2GPI antibodies with beta2GPI also decreased fibrinolytic activity in this assay system. beta2GPI is proteolytically cleaved by plasmin in domain V (nicked beta2GPI) and becomes unable to bind to phospholipids, reducing antigenicity against antiphospholipid antibodies. This cleavage occurs in patients with increased fibrinolysis turnover. Nicked beta2GPI binds to plasminogen and suppresses plasmin generation in the presence of fibrin, plasminogen, and tissue plasminogen activator (tPA). Thus, nicked beta2GPI plays a role in the extrinsic fibrinolysis via a negative feedback pathway loop.