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Global coagulation potential was assessed in 59 patients with acquired hemophilia A (PwAHA) by clot waveform analysis (CWA) and/or thrombin and plasmin generation assay. Relationships between factor VIII activity (FVIII:C) and the parameters from CWA and T/P-GA in patients with congenital HA were compared by grading coagulation potential related to FVIII:C: T1 (FVIII:C < 1 IU/dL), T2 (1 ≤ , ≤ 5 IU/dL), T3 (5 < , 12 ≤ IU/dL), and T4 (12 < , ≤ 50 IU/dL). The median FVIII:C and inhibitor titers in PwAHA on admission were 3.3 IU/dL and 63.0 BU/mL, respectively, but global coagulation parameters corresponded to T1 or less. Median FVIII:C levels during follow-up in PwAHA were 1.7-9.6-6.7-40.0-21.7 IU/dL on days 0-14-28-56-93, respectively. CWA-based data corresponded to less than T2 until day 28, but more closely reflected FVIII:C after day 56. Peak thrombin was severely low (near T1) until day 28 and improved modestly after day 56 but remained less than T2. Peak plasmin was lower than T1 until day 56, and returned to T4 on day 93. In conclusion, global coagulation function in PwAHA was impaired to a greater extent than could be anticipated from assays of FVIII:C, until approximately 1 month after immunosuppression and treatment with FVIII-bypassing agents.

Previous studies have reported a positive association between poor health-related quality of life (HRQOL) and disability mainly in relation to the physical component of HRQOL. Given the mental component’s responsivity to interventions, this study aimed to investigate whether the mental component of HRQOL independently predicted functional disability. We targeted all residents aged ≥ 65 years in one municipality and analyzed 3858 men and 4475 women without disability at baseline (November 2016). HRQOL was measured using the physical component summary (PCS) and mental component summary (MCS) of the SF-8 Health Survey. At 3-year follow-up (October 2019), incident functional disability was measured, defined as a new certification according to the Japanese long-term care insurance system. Multivariable Poisson regression models stratified by gender were used to estimate adjusted cumulative incidence ratio (CIR) and 95% confidence interval (CI) for functional disability. Among both genders, there was a significant dose-response relationship between better MCS and lower risk of functional disability, independent of potential confounders including the PCS (P for trend = 0.026 in men and 0.003 in women). Compared with the worst MCS group, the CIRs (95% CIs) for functional disability in the second worst, the middle, the second best, and the best MCS quintile groups were 1.09 (0.80–1.48), 0.58 (0.40–0.85), 0.90 (0.59–1.37), and 0.70 (0.48–1.02) for men, and 0.76 (0.58–1.00), 0.62 (0.46–0.84), 0.73 (0.53–0.99), and 0.63 (0.48–0.85) for women, respectively. The MCS is an independent predictor of functional disability among high-functioning older adults. This suggests that strategies focused on mental HRQOL are important for realizing a healthy, long-lived society.

A combined product of plasma-derived factor (F)VIIa and FX (pd-FVIIa/FX; Byclot®) is currently available for the hemostatic treatment of hemophilia A and B patients with inhibitors in Japan. Limited information is available, however, on its coagulant effect in acquired hemophilia A (AHA). In the present study, we assessed the coagulant effect of pd-FVIIa/FX on impairment of coagulation potentials in AHA. The bypassing agents, pd-FVIIa/FX, recombinant FVIIa (rFVIIa), and activated prothrombin complex concentrates (aPCC) were spiked with normal plasma preincubated with anti-FVIII monoclonal antibody (AHA-model plasma), and added to plasmas from AHA patients. Clot waveform analysis (CWA) triggered by the mixture of tissue factor and ellagic acid was subsequently performed. In the AHA-model, pd-FVIIa/FX improved all of the CWA parameters in a dose-dependent manner, irrespective of epitope specificity, with significant improvements relative to rFVIIa and aPCC. The coagulant effect of pd-FVIIa/FX at 1.6 µg/mL (corresponding to 120 µg/kg infusion) at the maximum therapeutic dose was outside the normal range. Moreover, the addition of pd-FVIIa/FX led to a greater improvement in the coagulant potentials in AHA plasmas than those of rFVIIa and/or aPCC. These data suggest that pd-FVIIa/FX significantly improves the impaired coagulant potentials in AHA and is potentially therapeutic.

Type 2N von Willebrand disease (VWD) is characterized by impaired factor VIII (FVIII) binding to von Willebrand factor (VWF). Type 2N VWD patients generally exhibit mild bleeding tendency, but some exhibit a more severe hemorrhagic pattern. An assay for assessing hemostatic potential and predict clinical severity could significantly improve clinical management in these patients. We examined the relationship between bleeding score (BS) and the potential for thrombus formation in whole blood from type 2N VWD patients with various BS using rotational thromboelastometry (ROTEM) and microchip flow-chamber system (T-TAS®). Collagen-coated PL-chips, or thromboplastin- and collagen-coated AR-chips, were utilized in the T-TAS to assess platelet thrombus formation at high shear flow, or fibrin-rich platelet thrombus formation at low shear flow, respectively. Neither ROTEM nor the T-TAS using PL-chips reflected the BS. The AR-chip parameters in the T-TAS, however, were highly sensitive to different BS levels among these patients, despite similar FVIII/VWF-related measurements including FVIII/VWF binding. Additionally, the results with AR-chip assay were restored to normal after infusions of FVIII/VWF concentrates in the most severe patients. The data indicate that T-TAS using AR-chips may be a useful assay for predicting clinical severity and assessing therapeutic efficiency in type 2N VWD patients.

Emicizumab is a bispecific antibody to factor (F) IX/IXa and FX/FXa, which mimics FVIIIa cofactor function. Emicizumab prophylaxis significantly decreases bleeding events for patients with hemophilia A (PwHA). However, global hemostatic monitoring in emicizumab-treated PwHA remains poorly investigated. Using rotational thromboelastometry (ROTEM), we evaluated coagulation potentials of whole blood samples from seven emicizumab-treated PwHA who participated in ACE001JP/ACE002JP studies. Dose-dependent coagulation-enhancing effects of emicizumab to whole blood from PwHA mixed with an anti-FVIII C2 antibody in vitro were evident by non-activated ROTEM analysis (NATEM). The relationship between FVIII levels and NATEM parameters in PwHA not participating in the clinical trials demonstrated that CT + CFT inversely correlated with FVIII levels. These parameters were defined as NATEM-based grading of coagulation potential; ‘T1’ (FVIII

Rurioctocog alfa (recombinant Factor VIII: AdvateⓇ) is available for the control of bleeding in patients with hemophilia A in Japan. To evaluate the immunogenicity, safety, and efficacy of prophylactic and on-demand use of rurioctocog alfa, postmarketing surveillance was conducted on 114 previously untreated Japanese patients aged 0–82 years with ≤ 3 exposure days under the conditions of routine clinical practice. A post-hoc comparison of mean annualized bleeding rates between patients in the regular prophylaxis group (7.4 bleeds/year) and in the on-demand treatment group (15.7 bleeds/year) using a negative binomial model found a statistically significant difference (P = 0.0164) in the subset of patients with severe hemophilia A. Favorable prophylactic and on-demand hemostatic efficacy (“excellent” or “good”) was shown in 71.4–88.5% across all treatment regimens. A total of 31 events of adverse drug reactions were reported. Of 114 patients, 21 (18.4%) developed de novo FVIII inhibitor; of these, 17 occurred within 50 exposures. One death was reported. A family history of positive inhibitors was significantly associated with inhibitor development (Fisher exact P value = 0.0004); no other risk factors were identified. Rurioctocog alfa was found to be well-tolerated and effective in previously untreated Japanese patients with hemophilia A in this postmarketing surveillance of routine clinical practice.

Rurioctocog alfa (recombinant factor VIII: Advate®) is available for the control of bleeding among patients with hemophilia A in Japan. To evaluate the perioperative safety and hemostatic efficacy of Advate®, a postmarketing surveillance was conducted in Japanese patients undergoing surgery in a real-world setting. A total of 74 surgical procedures performed in 58 subjects aged 0–75 years, including three females, were studied. A hemostatic efficacy rating of “excellent” or “good” was reported in 73/74 surgical procedures (98.6%). Perioperative bleeding was successfully controlled by Advate® in five subjects with positive FVIII inhibitors (2.4-9.1 BU/mL). Advate® was administered at higher initial bolus doses (114-385 IU/kg) and at higher rates by subsequent initial continuous infusion (8.3-15 IU/kg/hour) in the five subjects with inhibitor than in the subjects without inhibitor (n = 47; mean initial bolus dose: 53.4 IU/kg; subsequent mean initial continuous infusion: 3.8 IU/kg/h). Adverse drug reactions were reported in 7/74 (9.5%) procedures, two of which were the development of de novo FVIII inhibitors. Overall, the perioperative use of Advate® in a real-world setting was found to be safe and effective among Japanese patients with hemophilia A.

A case of acquired hemophilia A (AHA) that developed in a patient with autoimmune pancreatitis (AIP) is presented. A 64-year-old woman was diagnosed with AIP in 2007. The symptoms resolved with prednisolone (PSL). Although the dose of PSL was tapered to 7.5 mg/day for maintenance, serum IgG4 levels remained high. She suddenly presented with subcutaneous bleeding in 2015. Her activated partial thromboplastin time was prolonged (80.0 s). A mixing test showed an inhibitor pattern, factor VIII (FVIII) activity was less than 1%, and FVIII inhibitor was 290 BU/mL. She was diagnosed with AHA. Her serum IgG4 was elevated to 133 mg/dL. She was treated first with PSL alone, but she developed bladder tamponade. Cyclophosphamide and activated prothrombin complex concentrate were combined with PSL. She then achieved hemostasis, and FVIII inhibitor disappeared. FVIII inhibitor had been detected since PSL was tapered and AHA recurred two months later. An enzyme-linked immunosorbent assay showed that the inhibitor was mainly IgG4 and IgG1. This case suggests that elevation of IgG4 may be associated with the development of both AHA and AIP.

Continuous infusions (CI) of factor (F)VIII are preferable to the conventional bolus injections for the maintenance of consistent FVIII levels during surgery in patients with severe hemophilia A. A third generation, B domain-truncated recombinant FVIII (turoctocog alfa, Novo Nordisk, NovoEight®), was approved for clinical use in 2014. The hemostatic efficacy and safety of bolus injections of turoctocog alfa in patients undergoing surgery have been reported, but no reports on CI therapy have been published. We describe a 43-year-old patient with severe hemophilia A who required arthroscopic synovectomy of the right elbow and arthrodesis of the right ankle. He was treated with a bolus injection of turoctocog alfa (36 IU/kg) immediately before operation, followed by CI (infusion rate; 2.9 IU/kg/h) to maintain FVIII activity > 80 IU/dl throughout the perioperative period. Surgery was completed successfully with uncomplicated hemostatic control. CIs were continued until post-operative day (POD) 4. Further bolus injections were given from POD5. No anti-FVIII inhibitor has been detected post-operation. This case provides important information on CI therapy using turoctocog alfa during surgery for patients with severe hemophilia A.

Patients with mild/moderate hemophilia (H)A, acquired HA (AHA) and lupus anticoagulants (LA), have prolonged aPTTs with low levels of factor (F)VIII activity, but the differentiation of these disorders is complex and time consuming. We established an approach to quickly differentiate these disorders using comprehensive coagulation tests. Patients' plasmas with mild/moderate HA, AHA, LA without anti-phospholipid syndrome [LA-APS(-)], and LA with APS [LA-APS(+)] were examined using clot waveform analysis (CWA) and thrombin generation test (TGT). Activated protein C (APC) sensitivity was assessed by TGT. CWA revealed similarly prolonged clot times in all groups [NP/mild/moderate HA/AHA/LA-APS(-)/LA-APS(+); 33 ± 1/82 ± 12/116 ± 44/90 ± 29/96 ± 15 s] but significantly different decreased maximal coagulation velocity (3.1 ± 0.1/1.3 ± 0.3/0.9 ± 0.5/1.6 ± 0.3/2.2 ± 0.5). In TGT, AHA group demonstrated severely reduced peak-thrombin levels (362 ± 23/170 ± 27/49 ± 21/158 ± 75/158 ± 99 nM), whilst both LA groups markedly prolonged lag times (4.5 ± 0.3/5.0 ± 0.4/4.7 ± 0.8/12.5 ± 7.7/28.8 ± 11.8 min), suggesting that AHA could be readily identified, but the different LA sub-types failed to be classified. An APC sensitivity demonstrated that 'normalized' APC-induced levels of peak thrombin in LA-APS(+) were significantly lower relative to LA-APS(-) (normalized %inhibition; 5 ± 7/42 ± 39 %). Our studies confirmed that %inhibition by APC was significantly decreased in NP preincubated with purified IgGs from LA-APS(+) compared to LA-APS(-), facilitating differentiation between LA groups. A combined approach using CWA and TGT could be a useful means of differentiating coagulation disorders with prolonged aPTT.

The International Immune Tolerance Induction (I-ITI) Study in hemophilia A patients with inhibitors included 16 Japanese patients among a total of 115 test subjects. The results within this group of Japanese patients were 11 cases of I-ITI off-study, three cases of I-ITI on-study, and two cases of tolerance on prophylaxis. There was no significant difference in success rate between the low-dose and high-dose groups (Study I). Successively, independent follow-up survey in Japan was conducted in 14 cases, with consent (Study II). Ten cases were off-study at the end of the I-ITI Study. Of these 10 cases, seven of seven successful cases remained clinical successes at the end of the follow-up study, one partial success became a full success while a second relapsed, and one failure was subsequently evaluated as a partial success. Four cases that were on-study at the end of I-ITI Study were classified as three successes and one failure at the end of the follow-up study. As a result, the status at the end of follow-up study was: 11 ITI successes (78.6 %); one partial success; one failure; and one relapse. Thus, the ITI follow-up study was helpful in providing a long-term prognostic determination of inhibitors.

Measurements of von Willebrand factor ristocetin cofactor activity (VWF:RCo) are necessary for the diagnosis of von Willebrand disease (VWD). However, the conventional manual glass-plate method is technically demanding, and exhibits high intra-/inter-laboratory variation. An automated technique has been recently described utilizing the CS-2000i™ with specific VWF:RCo reagents, but measurements at lower levels (

A group of international physicians committed to the unique care of pediatric hemophilia patients form the International Network for Pediatric Hemophilia (INPH). The main objectives of the group center on scientific studies, educational presentations and networking. By joining together on an international level, large hemophilia centers with extensive experience in the clinical care of children with hemophilia perform and publish scientifically valid studies. Annual meetings of the group include invited speakers who present hot topics or new developments in this therapeutic area for the purpose of educating the group on cutting edge research. Furthermore, since members have experience in pediatric treatment from different parts of the world, current practices as well as new trends can be discussed and shared among the group. The Fifth Annual INPH meeting was held in Buenos Aires, Argentina, on July 8–9, 2010 and focused on the topics of prophylaxis treatment and immune tolerance induction in the clinical care of children with hemophilia. The meeting is structured around roundtable discussions on the members’ current research and clinical activities, project reports of INPH study initiatives, followed by invited educational presentations with interactive discussions.

Although the efficacy of recombinant factor VIII (rFVIII) in the treatment of type 3 von Willebrand disease (VWD) has been reported, the mechanisms by which FVIII concentrates devoid of von Willebrand factor (VWF) induce improvements in hemostasis are poorly understood. To address the role of FVIII or intrinsic coagulation in the absence of VWF, we performed a hemostatic analysis. Blood samples were obtained before and after the administration of rFVIII to 2 patients with type 3 VWD. A rotating thromboelastometry assay was performed to examine global interactions in hemostasis. Studies of thrombin-and shear-induced platelet aggregation were also conducted to elucidate the effect on platelet activation. Furthermore, we assessed the rise in the thrombin-induced intracellular concentration of free calcium ([Ca2+]i). Addition of rFVIII to preinfusion blood in vitro corrected thromboelastometric parameters and thrombin-induced aggregation. In ex vivo studies, thromboelastometry analysis showed that rFVIII shortened the onset and progression of the coagulation process. Furthermore, rFVIII corrected low shear-induced and thrombin-induced platelet aggregation in platelet-rich plasma. In addition, rFVIII improved thrombin-induced [Ca2+]i flux in washed platelets. Our observations suggested that FVIII is incorporated into platelets to activate them, as well as to act directly in intrinsic coagulation in the absence of VWF. FVIII may play a critical role even in the absence of VWF.

Factor VIII (FVIII) inhibitors develop as either alloantibodies against exogenous FVIII in patients with congenital hemophilia A after FVIII-replacement therapy or as autoantibodies against endogenous FVIII in previously healthy, nonhemophilic individuals. The predominant immunoglobulin G (IgG) subclass of FVIII inhibitors is IgG(4). The main epitopic regions are known to be located, however, in the A2, A3, and C2 domains. The A2 and A3 epitopes have been identified between amino acid residues 484 and 509 and residues 558 and 565, respectively. Both of these regions are close to the binding sites for activated FIX (FIXa). Two regions have been identified in the C2 domain, one in the amino-terminal portion of the domain (residues 2181-2243) and the other in the carboxy-terminal portion of the domain (residues 2248-2312 and residues 2315-2330). In addition, a crystallographic analysis of a complex of the C2 domain and a human monoclonal IgG(4)(K) Fab revealed that this type of antibody is in direct contact with hydrophobic and basic residues of the membrane-binding surface. Inactivated FVIII is rapidly cleared from the circulation in the presence of inhibitors. The inhibitors also bind to essential FVIII ligand proteins, including von Willebrand factor, FIXa, FXa, and thrombin, and to surface membrane phospholipid. Some type 2 inhibitors interfere with binding to activated protein C.

To elucidate differences in the antigenic structure of factor VIII (FVIII) among mammals, we evaluated cross-reactivities of well-defined antihuman FVIII antibodies with canine and other mammalian FVIII proteins. Monoclonal antibodies against human FVIII recognizing the A1 domain in the heavy chain and the A3 domain in the light chain showed 2.7% and 0% cross-reactivities, respectively, with canine FVIII. The cross-reactivities of 2 alloantibodies and a monoclonal antibody that recognized the A2 domain in the heavy chain were 10%, 38%, and 0%, respectively. On the other hand, 2 kinds of alloantibodies and a monoclonal antibody recognizing the C2 domain in the light chain showed 160%, 390%, and 130% cross-reactivity, respectively, with canine FVIII. The anti-C2 monoclonal antibody (NMC-VIII/5) showed a type 2 inactivating property when tested with canine and human plasma. Moreover, cross-reactivities of NMC-VIII/5 with simian and feline FVIII were 54.5% and 82.8%, respectively, while the cross-reactivities of the anti-A2 monoclonal antibody (JR8) with simian and feline FVIII were 1.3% and 0%, respectively. These findings suggest that the antigenic structure of the C2 epitope has remained relatively conserved throughout mammalian evolution in contrast to the A2 epitope and that a canine model of hemophilia A is useful for FVIII inhibitor experiments.

Hemophilia A is genetically very heterogeneous because disease-causing mutations involving deletions, point mutations, insertions, and inversions are scattered throughout the factor VIII gene. Of these mutations, inversions, which are intrachromosomal recombinations between int22h-1 (intron 22 homologous region 1) and 1 of 2 other extragenic copies located 500 kilobases upstream, are the more frequently found defects, especially in patients with severe hemophilia A. Reportedly, approximately half of all severe hemophilia A patients have inversions in intron 22. A group of unrelated patients from the middle of Japan with severe hemophilia A were screened by Southern blot analysis for gene inversions. Forty-two of 100 severely affected patients presented factor VIII gene rearrangements. Of these patients, 36 exhibited the distal type of inversion, and 6 exhibited the proximal type. No other variant type of recombination was observed. In this study, neither the prevalence of inhibitor development against factor VIII nor the frequency of sporadic cases in the group presenting gene inversions was significantly different from that in the group without chromosomal inversions. Southern blot analysis successfully detected a carrier in a hemophilia family for which no patient was available. Genetic counseling of patients with severe hemophilia A and their families will be considerably improved, because the inversions occur in 42% of the Japanese patients with severe hemophilia.

A multicenter and open-labeled clinical trial of human recombinant factor VIIa (rFVIIa) was conducted in Japanese patients with severe hemophilia A or B with inhibitors. The trial consisted of 2 parts. In study 1, the pharmacokinetics, pharmacodynamics, and safety of a single dose of 120 microg/kg of rFVIIa were investigated in 8 patients. In the subsequent study 2, the hemostatic effect and safety of rFVIIa were evaluated during a 24-week period in 10 patients. In study 1, the mean maximum FVII-coagulant activity (FVII:C) was found to occur after 10 minutes; activity then decreased rapidly and returned to the baseline within 24 hours after a single intravenous infusion of rFVIIa. The mean half-life of FVII:C was 3.5 hours. The activated partial thromboplastin time and prothrombin time in the patients were immediately shortened but returned to the baseline within 24 hours after dosing. In study 2, 86 microg/kg to 120 microg/kg of rFVIIa (mean, 97 microg/kg) was administered 1 to 85 times to 10 patients. A total of 58.0% (91/157) of bleeding episodes were treated excellently or effectively, with 5 (3.2%) ineffective episodes. There was no apparent trend in the relationship of the hemostatic effect with bleeding sites, mean dose, or number of injections. The efficacy rate, however, was significantly higher (90.0%) in bleeding episodes treated within 3 hours than in those treated at longer intervals (31.0%). No treatment-related adverse events were observed, and there was no evidence of antibody formation to rFVIIa. In conclusion. rFVIIa is an effective and well-tolerated option for treatment of bleeding episodes in hemophilia patients with inhibitors.

Background Membranous nephropathy is one of the most common causes of nephrotic syndrome in adults. In contrast, acquired factor V inhibitor is a rare bleeding disorder. Case presentation A 62-year-old Asian man with a history of cerebral hemorrhage, purpura, eosinophilia and hyper immunoglobulin E syndrome developed proteinuria. The bleeding disorder was diagnosed with acquired factor V inhibitors. A renal biopsy revealed that he suffered from membranous nephropathy with glomerular endothelial damage which is reported to be involved in another factor disorder. After the steroid administration, the coagulation test and proteinuria were improved. Conclusions The presence of factor V inhibitors may have been involved in the development of membranous nephropathy.

Background Acquired hemophilia A (AHA) is a rare bleeding disorder caused by an autoantibody to coagulation factor (F) VIII. It is characterized by soft tissue bleeding in patients without a personal or family history of bleeding. Bleeding is variable, ranging from acute, life-threatening hemorrhage, with 9-22% mortality, to mild bleeding that requires no treatment. AHA usually presents to clinicians without prior experience of the disease, therefore diagnosis is frequently delayed and bleeds under treated. Methods Structured literature searches were used to support expert opinion in the development of recommendations for the management of patients with AHA. Results Immediate consultation with a hemophilia center experienced in the management of inhibitors is essential to ensure accurate diagnosis and appropriate treatment. The laboratory finding of prolonged activated partial thromboplastin time with normal prothrombin time is typical of AHA, and the diagnosis should be considered even in the absence of bleeding. The FVIII level and autoantibody titer are not reliable predictors of bleeding risk or response to treatment. Most patients with AHA are elderly; comorbidities and underlying conditions found in 50% of patients often influence the clinical picture. Initial treatment involves the control of acute bleeding with bypassing agents. Immunosuppressive treatment to eradicate the FVIII inhibitor should be started as soon as the diagnosis is confirmed to reduce the time the patient is at risk of bleeding. Conclusions These recommendations aim to increase awareness of this disorder among clinicians in a wide range of specialties and provide practical advice on diagnosis and treatment.

Dear Editor, Approximately 2 % of multiple myeloma (MM) patients present with hemorrhage at diagnosis. However, hemorrhage due to abnormalities in the coagulation system is a rare complication [1, 2]. Although lupus anticoagulant (LA), which is infrequently reported to be in association with MM, is commonly a risk factor for arterial or venous thrombosis, bleeding tendencies in patients with LA are strongly related to a low prothrombin activity [3–6]. Acquired hypoprothrombinemia with LA, also called LA hypoprothrombinemia syndrome (LAHPS), is a rare disease which appears mostly in young females with systemic lupus erythematosus or in healthy children after viral infection and is usually associated with the presence of antiprothrombin antibodies [7]. We herein report the case of an 86-year-old male with Bence-Jones protein (BJP) κ-type MM who presented with hypoprothrombinemia and LA associated with antibodies directed to the phosphatidylserine–prothrombin complex (or phosphatidylserine-dependent antiprothrombin antibodies, aPS/PT). The patient was admitted with anemia and had no past history of bleeding disorders or thrombotic events. A urinalysis showed massive proteinuria (5.3 g/day), which was determined to be κ-type BJP using immunoelectrophoresis. Bone marrow aspiration showed proliferation of abnormal plasma cells. Computerized tomography showed hematomas in the bilateral gluteus maximus muscle and the supraclavicular area. Initial coagulation tests showed prolonged prothrombin time and activated partial thromboplastin time (aPTT) (Table 1). Reduced clotting activity of factors II (FII), VIII (FVIII), and IX (FIX) was noted in a pattern typical of that observed in previously reported cases of LAHPS [8, 9]. FVIII and FIX inhibitors were not detected. The prolonged aPTT with LA-sensitive aPTT reagent (PTT-LA Roche Diagnostics, Tokyo, Japan), which could not be corrected by mixing with normal plasma, suggested the presence of LA. The results were confirmed using the Staclot LA® assay, and the dilute Russell viper venom time test was used to confirm the presence of LA with the phospholipid-neutralizing LA test (Gradipore, Frenchs Forest, Australia). IgG/M anticardiolipin antibodies and IgG aPS/PT were negative, while strong positive IgM aPS/PT was detected, which was measured with ELISA using the phosphatidylserine–prothrombin complex as antigen immobilized on ELISA plates in the presence of CaCl2 [10]. Based on these findings, the patient was diagnosed as MM with LAHPS associated with aPS/PT and treated with melphalan and prednisolone (MP) therapy. The FII levels were observed to normalize after one cycle of MP therapy and the patient has remained in remission without any hemorrhage for 10 months. Table 1 Laboratory findings In our case, aPTT continued to be prolonged with reduced levels of FVIII and FIX in spite of normalizing the FII level after therapy. LA and IgM aPS/PT remained positive, although these values were improved, suggesting that the presence of LA might have an influence on coagulation tests after treatment. There are very rare reports showing the presence of aPS/PT in patients with LAHPS [9]. These reports describe the patients as having bleeding tendencies with mildly reduced FII levels, similar to that observed in our patient. However, in previously reported child cases of LAHPS, severe hemorrhage usually occurs when the FII levels are very low (under 10∼15 %). It is possible that other coagulation factors associated with aPS/PT in LAHPS might be present. A diagnosis of LAHPS should always be considered in MM patients with bleeding tendencies associated with LA, and aPS/PT detection should be performed in conjunction with LA tests.

Anti-factor VIII (FVIII) inhibitor alloantibodies from 11 patients with hemophilia A, along with five anti-FVIII neutralizing monoclonal antibodies, were examined for differences in their reactivities with the A2 and C2 domains of human and porcine FVIII. None of the patients had been previously treated with porcine FVIII. Six inhibitors which specifically recognized the human FVIII C2 domain bound to both the 76-kDa porcine FVIII light chain and its 69-kDa proteolyzed fragments, showing cross-reactivity against porcine FVIII between 33 and 100%. Two A2-specific inhibitors did not react with porcine FVIII. The cross-reactivity was low (0-0.5%). The inhibitors recognizing both C2 and A2 reacted with the 76- and 69-kDa bands of porcine FVIII light chain, with cross-reactivity of between 11 and 33%. Monoclonal antibodies recognizing A1 (C-5) and A2 (JR8) did not react with the porcine FVIII. No anti-porcine FVIII neutralizing activity was detected in these antibodies. Monoclonal antibodies to the amino-terminal portion of A3 (NMC-VIII/10 and C-2) poorly reacted with the 76-kDa band, the cross-reactivities being 0 and 0.5%, respectively. NMC-VIII/5 recognizing C2 which competes with the C2-specific inhibitor, reacted with both the 76- and 69-kDa fragments showing cross-reactivity of 13%. These findings suggest that porcine A2 is antigenically different from human A2. The A2-specific inhibitor is a useful indicator for therapy with porcine FVIII.

Using enzyme-linked immunosorbent assay (ELISA), we measured anti-factor VIII immunoglobulin G (IgG), IgG4 and IgM in serum samples obtained from hemophilia A patients treated with a recombinant factor VIII (rFVIII) preparation (Kogenate). Twelve pre- and post-treatment serum samples from 3 previously untreated patients who developed an inhibitor (alloantibody) were evaluated. Both rFVIII and plasma-derived factor VIII (pdFVIII) were used as antigen. Serum samples were diluted 37.5-fold. For the measurement of IgM antibody, protein A Sephadex suspension was added to the patients' serum samples and the supernatant was assayed. Anti-human IgG, IgG4, and IgM monoclonal antibodies labelled with peroxidase were added and absorbance at 450nm was determined. The reactivity of the IgG antibody with rFVIII and pdFVIII was extremely low. Samples containing the IgG4 inhibitor with a neutralizing activity of approximately 7.5 Bethesda units (BU)/ml resulted in absorbance values of 0.451-0.551, thus demonstrating a considerably high degree of sensitivity. The correlation between the neutralizing activity and reactions of the IgG4 antibody with rFVIII and pdFVIII antigens was high, with correlation coefficients of 0.912 and 0.966, respectively. Furthermore, the correlation coefficient between the measured absorbance values for the antibody reacted with pdFVIII and rFVIII was 0.961. No correlation was found between the reactivity of IgM to rFVIII and pdFVIII.