Factor VIII Assays Research Articles

Damoctocog Alfa Pegol, a PEGylated B-domain Deleted Recombinant Extended Half-life Factor VIII for the Treatment of Hemophilia A: A Product Review.

Damoctocog alfa pegol (BAY 94-9027, Jivi®), is a site-specifically PEGylated, extended half-life recombinant factor VIII (FVIII) that is approved in several European and non-European countries for on-demand treatment and prophylaxis of bleeding in previously treated patients aged ≥ 12 years with hemophilia A. Reliable measurements can be obtained using most one-stage and chromogenic FVIII assays over a wide concentration range. The efficacy, safety and pharmacokinetics (PK) of damoctocog alfa pegol have been studied extensively in the PROTECT VIII clinical trials, and its long-term safety and effectiveness profile is continuing to build through observational and interventional real-world studies. The PK of damoctocog alfa pegol was shown to be improved as compared with that of sucrose-formulated rFVIII (rFVIII-FS, Kogenate®), and was also demonstrated to be non-inferior to and, for some variables, more favorable than rFVIII-Fc fusion protein, efmoroctocog alfa (Elocta®; NCT03364998), rurioctocog alfa pegol (BAX 855, Adynovate®/Adynovi®; NCT04015492), and antihemophilic factor (recombinant) plasma/albumin-free method (rAHF-PFM, Advate®; NCT02483208). Damoctocog alfa pegol was generally well tolerated and none of the patients in any of the clinical trials, including the PROTECT VIII clinical program, HEM-POWR, or ongoing single-center studies, developed FVIII inhibitors. Efficacy for perioperative hemostasis has been demonstrated. Low bleeding rates were achieved across the studies, with twice weekly, every 5-day and every 7-day prophylaxis offering patients ≥ 12 years and their clinicians the chance to tailor treatment to individual needs and lifestyles, while maintaining long-term protection from bleeds and their consequences.

Transplacental Transfer of Emicizumab: Experience with Emicizumab in a Pregnant Female with Severe Hemophilia Α and an Inhibitor

Introduction: Severe hemophilia A (HA) in females is incredibly rare particularly if complicated by a high-titer factor VIII (FVIII) inhibitor. We report, to the best of our knowledge, the first-of-its-kind use of emicizumab (emi) ante/post-partum, its effect on the newborn, and its excretion in breast milk. Baseline details: 29-year-old-female was diagnosed with severe HA at age 1 following prolonged bleeding from a venipuncture site. She had no family history of HA. One stage FVIII activity (OSA) was less than 1%. She was compound heterozygous for F8 type 1 and type 2 intron 22 inversions. At 21 months of age, she developed a high-titer inhibitor (peak: 35 Bethesda unit[BU]). She was not treated with immune tolerance induction and had remained on factor eight inhibitor bypassing activity (FEIBA) as prophylaxis until transferring to our center. She was transitioned to monthly emi prophylaxis and recombinant FVIIa (rFVIIa, NovoSeven) for breakthrough bleeding at age 26. She was medically advised against pregnancy due to unclear safety profile of emi in pregnancy and theoretical concerns for an increased risk of bleeding and thrombotic complications associated with the use of bypassing agents in the peripartum setting. Nevertheless, she desired pregnancy and continued on emi prophylaxis every 4 weeks during her pregnancy. Clinical course in pregnancy: She had an uncomplicated antepartum course and did not require rFVIIa for breakthrough bleeds. Blood emi levels and FVIII inhibitor levels were measured in each trimester of pregnancy (Table 1). Her labor was induced with oxytocin while on rFVIIa and tranexamic acid (TXA) support. She had a spontaneous, unassisted vaginal delivery immediately complicated by post-partum hemorrhage resulting in estimated blood loss of 1100cc. She did not require blood transfusion. She continued on 90 mcg/kg rFVIIa support every 2 hours, gradually prolonging the dosing interval by 2 hours every 24 hours (q2, q4, q6, etc.), and experienced no further in-hospital bleeding. She delivered a healthy female newborn. The infant developed cephalhematoma but transcranial ultrasound was negative for intracranial bleeding. Cord blood was collected at the time of delivery and sent for emi level, FVIII activity and FVIII inhibitor titer (Table 2). FVIII inhibitor assay with bovine reagent (CBU) was performed using chromogenic substrate assay (CSA) after pre-analytical heat-inactivation of patient plasma. The infant's CSA FVIII level was measured using a bovine chromogenic FVIII assay, and FVIII was also assayed using a one-stage APTT-based assay. Mother opted to breast-feed the infant. Emi level was measured in breast milk (Table 1). Conclusion: In this highly unusual case, we demonstrate that emi crossed the placenta and is excreted in breast milk. We also demonstrate that the maternal FVIII inhibitor did not cross the placenta into the newborn. Drawing conclusions on the safety of emi despite placental and breast milk transmission and transmissibility of inhibitor based on a single case would be premature. The conglomeration of several rarities in a single case renders our report unique and insightful and provides a baseline experience for future cases.

Sensitive Measurement of Clinically Relevant Factor VIII Levels in Thrombin Generation Assays Requires Presence of Factor XIa.

Hemophilia A (HA) is characterized by decreased or absent factor VIII (FVIII) activity. Current FVIII assays are based on clotting time and thus only provide information about the initiation of coagulation. In contrast, thrombin generation assays (TGAs) can be used to measure the full coagulation spectrum of initiation, propagation, and termination that provide information on the whole course of thrombin generation and inhibition. However, the commercially available TG kits lack sensitivity for measurements of hemophilia plasma within lower FVIII ranges, which is essential for explaining differences in bleeding phenotypes in hemophiliacs at clinically low levels of FVIII. Optimization of the TGA for measurements of low FVIII levels in severe HA patients. TGA measurements were performed in severe HA pooled plasma (n = 10). Investigations of several preanalytical and analytical variables of the assay were performed in a stepwise process and adjusted based on sensitivity toward intrinsic coagulation activation. TGA initiated by tissue factor (TF) alone at varying concentrations was unable to significantly differentiate between FVIII levels below 20%. In contrast, TGA activation with low concentrations of TF in presence of FXIa appeared to be highly sensitive for FVIII changes both in high and low ranges. In addition, a representative TGA curve at trough levels could only be produced using the dual TF/FXIa TGA. We propose a critical optimization for the setup of the TGA for measurements in severe HA plasma. The dual TF/FXIa TGA shows increased sensitivity, especially in lower FVIII ranges, which allows for better individual characterization at baseline, prediction of interventions, and follow-up.

POSTER ABSTRACTS

POSTER ABSTRACTS

The combination of emicizumab and recombinant factor VIII in plasma: Which assays can we use for accurate measurement?

The bispecific antibody, emicizumab, is a prophylactic therapy used for the treatment of haemophilia A (HA). Patients may require additional replacement factor VIII (FVIII) to ensure adequate haemostasis. This study investigated the laboratory measurement of severe HA (SHA) plasma spiked with 36 combinations of emicizumab plus recombinant (r) FVIII concentrates. FVIII assays were performed by one stage assay (OSA) using eight APTT reagents from three manufacturers and chromogenic assays (CSA) using seven kits. CSA kits comprised a range of bovine FX/FIXa, bovine FX/human FIXa or human FX/FIXa. Thrombin generation (TG) was assessed by CAT and ST-Genesia. Emicizumab-calibrated modified OSA and human FX CSA both overestimated rFVIII in the presence of emicizumab; median FVIII:C of up to 89% higher was observed in plasma spiked with both drugs compared to just rFVIII. In bovine FX CSA assays, there was a FVIII:C increase of up to 11% in plasmas spiked with both drugs compared to rFVIII alone. TG parameters were not all normalized by the presence of emicizumab however addition of rFVIII increased TG. ETP and peak thrombin were normalized at 50 μg/ml emicizumab using ST-Genesia but were still reduced at 75 μg/ml with CAT. Addition of rFVIII further normalized results. Modified OSA and human FX CSA could not distinguish between rFVIII or emicizumab. The presence of both emicizumab and rFVIII increased thrombin generation to normal levels compared to each drug alone. Bovine FX CSA can be used to accurately determine FVIII activity of rFVIII in plasma which also contains emicizumab.

EVALUATION OF CLOTTING FACTORS IN HOSPITALIZED CRITICALLY-ILL INDIVIDUALS WITH SUSPECTED COVID-19

The Coronavirus Disease 2019 (COVID-19), that results of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, manifests with dysfunction of hemostasis and thrombosis. This study aims to evaluate laboratory parameters of hemostasis in hospitalized individuals with suspected COVID-19. Individuals aged 18 years or older with suspected COVID-19 admitted to two hospitals were invited to participate in this study. The study inclusion period was from April 2020 to March 2021. The study was approved by the institutional ethics committees. The diagnosis of COVID-19 was confirmed by positive SARS-CoV-2 real-time reverse-transcription polymerase chain reaction. Antithrombin, factor V, factor VII, factor XI, factor XII, factor XIII and prothrombin assays were performed using the Multiplex immunoassay technique (ThermoFisher Scientific, Vienna, Austria). Propensity score matching for sex and age were estimated by a logistic regression model for COVID-19 and non-COVID-19 individuals in the R software. The proportions found in each group were compared by Fisher's exact test, when the variable was categorical, and by the Mann-Whitney test, when it was continuous. Linear regression was performed adjusting the levels of clotting factors and antithrombin for the severity score (Sequential Organ Failure Assessment - SOFA). Correlation between SOFA, clotting factors and antithrombin in individuals with COVID-19 were performed by using the Spearman test. Only very strong correlations (≥0.9) were considered; p-value<0.05 was considered statistically significant. A total of 151 individuals were included in the study, of whom 138 (91.4%) with the diagnosis of COVID-19 and 13 (8.6%) non-COVID-19. After 2:1 matching, 26 individuals with COVID-19 and 13 non-COVID-19 participated in the study. In the univariate analysis, the group of COVID-19 had higher levels of antithrombin, factor V, factor VII, factor XI and prothrombin compared to the non-COVID-19 group. However, after adjusting for SOFA, only the levels of factor XI and prothrombin remained different between the groups (higher in the COVID-19) (p=0.04 and p=0.04, respectively). We found no association between factor XI and prothrombin with mortality. However, we found a very strong correlation between coagulation factors V and VII (r=0.923, p<0.0001). Our results show that plasma levels of antithrombin, factor V, factor VII, factor XI and prothrombin were higher in the COVID-19 when compared with non-COVID-19 group of critically-ill patients, but the difference was lost after adjusting the analysis for SOFA. Only the levels of factor XI and prothrombin remained significant in the COVID-19 group after adjustment. This finding suggests that the severity of the disease rather than viral etiology was the main determinant of the difference in the plasma levels of these proteins. We also showed a strong correlation between factor V and VII in our study. Indeed, factor VII is the major trigger of coagulation in vivo . Therefore, it is possible that factor V and VII could act together to boost coagulation and promote thrombus formation in patients with COVID-19. Our study suggests that increased levels of procoagulant factors in hospitalized critically-ill individuals with suspected COVID-19 are rather related to disease severity than to its cause.

Selective human factor VIII activity measurement after analytical in‐line purification

BackgroundIt is essential to measure the activity of factor VIII (FVIII) throughout the life cycle of a coagulation FVIII concentrate. Such measurement in nonclinical pharmacokinetic studies is potentially biased by the presence of endogenous nonhuman FVIII, and certain manufacturing process–related additives can also impact the assay performance. Finally, the presence of FVIII activity–mimicking antibodies poses challenges when measuring FVIII in samples. Therefore, we developed an antibody‐based chromogenic FVIII assay, which facilitates the selective and sensitive activity measurement of human FVIII in the presence of animal plasma and interfering agents. MethodsPlate‐bound monoclonal anti‐FVIII antibody specifically captured human FVIII, which was then measured with a chromogenic activity assay. A human reference plasma preparation was used to construct the calibration curve. Spike recovery was carried out in a citrated cynomolgus monkey plasma–solvent/detergent mixture and in the presence of the bispecific antibody emicizumab. ResultsThe calibration curve ranged from 3.03 to 97.0 mIU FVIII/ml and was obtained repeatedly with good accuracy. B domain–deleted and full‐length FVIII did not differ in their responses. Recovery of spiked human FVIII in citrated cynomolgus monkey plasma was 102.7%, while neither native monkey plasma nor the other animal specimen tested showed any activity. Solvent/detergent solution and the bispecific antibody emicizumab had no influence on the assay. ConclusionCombining antibody‐mediated specific capture of human FVIII and a chromogenic activity assay resulted in a selective and sensitive measurement of human FVIII with no interference by endogenous, nonhuman FVIII, manufacturing process additives, or an FVIII activity–mimicking antibody.

Assessment of Internal Quality Control of Blood Products; Experience at a Regional Transfusion Centre from Northern Pakistan

Objective: To analyze internal quality control of blood components, including red cell concentrates, fresh frozen plasma, cryoprecipitate and random donor platelets, to measure our blood bank performance.&#x0D; Study Design: Cross-sectional study.&#x0D; Place and Duration of Study: Armed Forces Institute of Transfusion (AFIT), Rawalpindi Pakistan from Jul to Dec 2021.&#x0D; Methodology: Whole blood units were separated into red cell concentrates, fresh frozen plasma and random platelets by the platelet-rich plasma method. Cryoprecipitates were prepared from FFPs. Quality control was done on representative components. For red cell concentrates, hematocrit was measured. For platelet concentrates, pH and platelet yield were measured. For fresh frozen plasma, Factor-VIII assay and for cryoprecipitate, Factor VIII and fibrinogen assays were done. The blood components were also tested for bacterial cultures.&#x0D; Results: A total of 1130 units were analyzed for quality control, including 360 red cell concentrates, fresh frozen plasma, random donor platelets each and 50 cryoprecipitates. Red cell concentrates had a mean hematocrit of 68.5±3.7%. Random donor platelets had a yield of 10.1±1.4× 1010 / unit and a mean pH of 6.7±0.2. Fresh frozen plasma had a Factor VIII level of 2.2±0.98 IU/ml. Cryoprecipitate had a mean fibrinogen level of 202.8±27.8 mg/unit and Factor VIII132.5±54.1IU/unit. All blood components met internal quality control standards. Blood cultures were negative in 99.2% of random donor platelets tested.&#x0D; Conclusion: The internal quality control of blood products was in concordance with the national and international standards for quality control in blood banks.

Applicability of the Thrombin Generation Test to Evaluate the Hemostatic Status of Hemophilia A Patients in Daily Clinical Practice.

Hemophilia A (HA) is a rare bleeding disorder caused by factor VIII (FVIII) deficiency due to various genetic mutations in the F8 gene. The disease severity inversely correlates with the plasma levels of functional FVIII. The treatment of HA patients is based on FVIII replacement therapy, either following a prophylactic or on-demand regime, depending on the severity of the disease at diagnosis and the patient’s clinical manifestations. The hemorrhagic manifestations are widely variable amongst HA patients, who may require monitoring and treatment re-adjustment to minimize bleeding symptoms. Notably, laboratory monitoring of the FVIII activity is difficult due to a lack of sensitivity to various FVIII-related molecules, including non-factor replacement therapies. Hence, patient management is determined mainly based on clinical manifestations and patient–clinician history. Our goal was to validate the ST Genesia® automated thrombin generation analyzer to quantify the relative hemostatic status in HA patients. We recruited a cohort of HA patients from the Principality of Asturias (Spain), following treatment and at a stable non-bleeding phase. The entire cohort (57 patients) had been comprehensively studied at diagnosis, including FVIII and VWF activity assays and F8 genetic screening, and then clinically monitored until the Thrombin Generation Test (TGT) was performed. All patients were recruited prior to treatment administration, at the maximum time-window following the previous dose. Interestingly, the severe/moderate patients had a similar TGT compared to the mild patients, reflecting the non-bleeding phase of our patient cohort, regardless of the initial diagnosis (i.e., the severity of the disease), treatment regime, and FVIII activity measured at the time of the TGT. Thus, TGT parameters, especially the peak height (Peak), may reflect the actual hemostatic status of a patient more accurately compared to FVIII activity assays, which may be compromised by non-factor replacement therapies. Furthermore, our data supports the utilization of combined TGT variables, together with the severity of patient symptoms, along with the F8 mutation type to augment the prognostic capacity of TGT. The results from this observational study suggest that TGT parameters measured with ST Genesia® may represent a suitable tool to monitor the hemostatic status of patients requiring a closer follow-up and a tailored therapeutic adjustment, including other hemophilia subtypes or bleeding disorders.

Laboratory coagulation tests and recombinant porcine factor VIII: A United Kingdom Haemophilia Centre Doctors' Organisation guideline.

Acquired haemophilia A (AHA) is a rare bleeding disorder caused by development of auto-antibodies to endogenous coagulation factor VIII (FVIII). Recombinant porcine factor VIII (rpFVIII) is currently licensed only for the management of bleeding in patients with AHA. Regular monitoring of rpFVIII is recommended to assess treatment effectiveness. This guideline from the United Kingdom Haemophilia Centre Doctors' Organisation (UKHCDO) examines the current publications in the area and aims to offer advice for the laboratory monitoring of rpFVIII in patients with AHA. A review of the current literature was undertaken followed by critical review by the authors. A validated one-stage clotting FVIII assay is recommended for the measurement and regular monitoring of rpFVIII. Assessment of cross-reacting rpFVIII inhibitors by one-stage porcine Bethesda assay should be performed as part of the initial diagnosis of AHA or prior to treatment with rpFVIII. Available data show that chromogenic FVIII assays underestimate rpFVIII and this should be considered if measurement of rpFVIII is required in patients receiving Emicizumab.

Multicenter performance evaluation and reference range determination of a new one-stage factor VIII assay.

IntroductionWe conducted a multicenter evaluation of a new one‐stage factor VIII (FVIII) assay (Roche Diagnostics), intended for the quantitative assessment of FVIII activity. We evaluated the analytical performance of the FVIII assay on the cobas t 711 analyzer.MethodsExperiments performed at three laboratories used 3.2% citrated residual or commercially purchased plasma samples. Five human plasma pools and two controls were used to determine assay within‐run and within‐laboratory precision, and total reproducibility; coefficients of variation (CVs) and/or standard deviations (SDs) were calculated. Lot‐to‐lot variability and method comparison (vs Coagulation FVIII Deficient Plasma/Dade Actin FS Activated PTT reagent/Standard Human Plasma Calibrator on the Sysmex CS‐5100 analyzer; Siemens Healthineers) were evaluated by Passing–Bablok and Deming regression, respectively, and Pearson's r calculated. Assay‐specific reference range was determined using 199 fresh plasma samples from healthy adults, not receiving anticoagulants.ResultsAcross sites, SDs for repeatability were 0.016–0.046 for samples with ≤1.0 international units (IU)/dL FVIII activity; CVs were 0.9%–3.8% for samples with >1.0 IU/dl activity. Among samples with mean FVIII activity 0.344–133 IU/dl, good intermediate precision (SD 0.020 for samples with 0.344 IU/dl activity; CV 1.8%–4.7%) and good total reproducibility (CV 2.0%–13.3%) were observed. The FVIII assay showed excellent lot‐to‐lot variability (Pearson's r = .999) and good correlation with the comparator assay (Pearson's r = .993–.996). The reference range for FVIII activity was 82.2−218.0 IU/dl.ConclusionThe one‐stage FVIII assay demonstrated robust analytical performance on the cobas t 711 analyzer, supporting its use in routine laboratory practice.

Factor V11 deficiency: A rare cause of nasal bleeding

Factor VII deficiency is a rare inherited disorder. Clinically the patient presents with bleeding tendencies. Diagnosis is made by prolonged prothrombin time, normal activated partial thromboplastin time and low functions factor VII assay or factor VII antigen. Therapy involves factor VII concentrates, recombinant factor VII, fresh frozen plasma and fibrinolytic inhibitors. We present a 6 years old boy with nose bleed for six months of whom prothrombin time was prolonged with functional factor VII assay of less than 1% confirming factor VII deficiency. He was managed with fresh frozen plasma, blood transfusion, tranexamic acid. Factor VII deficiency even though rare should be sought out in children presenting with bleeding. Keywords: Factor VII deficiency; coagulation; hemorrhage.

Anticoagulation Therapy Considerations in Left Ventricular Assist Devices in the Setting of Factor VII Deficiency

Background: Factor VII (FVII) deficiency is a rare bleeding disorder that affects approximately 1 out of every 500,000 people. As Warfarin inhibits the synthesis of vitamin K dependent clotting factors, it is generally contraindicated in patients with bleeding tendencies or blood dyscrasias; therefore, the literature regarding the use of warfarin in FVII deficiency is very limited. We report a successful re-challenge of warfarin therapy in a patient with FVII deficiency who needed left ventricular assist device (LVAD).Clinical case: Hematology was consulted to aid in the anti-coagulant management of a 57-year old female with coronary artery disease, ischemic cardiomyopathy (ejection fraction 15%), end stage heart failure (stage D) on home milrinone, elevated baseline prothrombin time (PT), and INR who was admitted for placement of LVAD, as a bridge to transplant. Of note, she had spontaneous oral bleeding when warfarin was given 7 years ago, after myocardial infarction after which warfarin was discontinued. She has been on aspirin 81 mg and clopidogrel 75 mg once daily. Moreover, 6 months ago, she had rectal bleeding after colonic polypectomies and needed 2 units of packed red blood cell transfusion. She always had heavy menstrual cycles and now she is post-menopausal. Family history was negative for any bleeding disorder. Social history was positive for 30 pack years smoking, which she stopped 5 years ago. Vital signs were significant for low systolic blood pressures in 90s-100s mm Hg on milrinone and heart rate in low 60s and saturating 96% on 4 liter of oxygen. Physical exam was remarkable for jugular venous distention, systolic murmur, bibasilar crackles, and grade 2 pitting edema. Laboratory tests were significant for hemoglobin (Hgb) 8.7g/dL (12-15.5 g/dl), hematocrit 28.2 (normal: 38-46%), platelet count 204 (150-450 x 103/microliter), PT 15.3 to 18.8 (normal: 11-13.5 seconds), INR between 1.4 to 1.7, PTT 21.1 sec. Liver function was normal except for mild decrease in albumin levels 3.3 g/dL (normal: 3.5-5.5 g/dl), vitamin K level 1 (0.1-2.2 ng/dl). Mixing study (1:1) has corrected the PT to 13.1 sec (ruling out the presence of inhibitory antibodies). Meanwhile, FVII assay was back with lower values– 35% (65-180%). A repeat testing also revealed a similar value (37%).As the acquired causes of FVII deficiency were ruled out (negative anti-phospholipid antibodies, appropriate reticulocyte index in the setting of anemia, no malignancy, no history of stem cell transplant) mild FVII deficiency, likely congenital, was diagnosed. Of note, sequential analysis genetic testing for F7 gene was positive for missense mutation 11128delC in exon 8. Despite lack of data of warfarin use in the setting of FVII deficiency, and LVAD, she consented to LVAD after fully understanding of risk of death from bleeding. HeartMate II LVAD was implanted. Intravenous heparin was started post-operatively with goal anti-Xa initially at 0.2 to 0.3 IU/ml that was safely titrated between 0.3 to 0.7 IU/mL with careful monitoring of bleeding. Warfarin at 2mg/day was started. Factors II, VII and X, PT and INR were closely monitored with slow titration of warfarin. The INR between 3.0 to 3.6 correlated with Factor II level of 20-40%, FVII level of 15% and Factor X level of 15-25%. Aspirin 81mg once daily was added eventually. Around 10 months after LVAD, she presented with melena, anemia (Hgb 4.8 g/dL) for which she received 3 units of packed red blood cells. Detailed evaluation showed angioectasias of the stomach and jejunum, which were cauterized. Warfarin was safely re-initiated 1 month after with goal INR lowered between 2.5 to 3.0. No more bleeding episodes occurred to date.Discussion and conclusion: Both bleeding and thrombosis occur at very high rates in the patients on LVAD, present difficult management issues, and can be life threatening. This was more challenging in the setting of FVII deficiency. We used the goals of factors II, factor VII and X levels between 20-40%, 10-15% and 15-25%, respectively, for the therapeutic window with warfarin use. For our patient, these levels correlated with INR 3-3.6. With recent GI bleeding, goal INR was lowered between 2.5-3.0 with no further bleeding episode on 6 month follow up. Our case describes a successful re-challenge of warfarin therapy in a patient with FVII deficiency. However, we must recognize the varying degrees of FVII and the corresponding bleeding risk among patients with FVII deficiency. DisclosuresNo relevant conflicts of interest to declare.

512 A CASE OF AN IDIOPATHIC ACQUIRED HEMOPHILIA A IN AN ELDERLY WOMAN

Abstract Introduction Acquired Hemophilia is a bleeding diathesis caused by autoantibodies that interfere with factor VIII (FVIII). Reasons for autoantibodies production are not clear but may be related to gene polymorphisms and/or CD4+ T lymphocytes. 1.3 to 1.5 cases per million population per year are reported in the UK. Half of the cases are secondary to malignancy, pregnancy related conditions, connective tissue disorders or drug reactions while the rest are idiopathic. Case Report We report a case of an acquired hemophilia A in an 86-year-old lady with underlying type 2 diabetes, hypertension, and cognitive impairment, being treated as the left lower limb cellulitis with antibiotics. She was found to have a sudden hemoglobin drop and her CT (Abdomen) confirmed a spontaneous intra-abdominal hematoma. Clotting profile showed prolonged APTT to 168.5 seconds, being not corrected at mixing study, with normal PT and INR. The FVIII assay was reduced to 18.4 iU/dL with FVIII inhibitor concentration of 0.7 Bu. Viral and autoimmune screenings were negative. The idiopathic acquired hemophilia A was diagnosed. Red blood cell transfusions, bypassing agents (FEIBA) and oral tranexamic acid were given for acute bleeding episode. Concomitantly, oral prednisolone was used to reduce the inhibitor levels. Repeated FVIII assay showed 121 iU/dL and 199iU/dL on day 6 and 12, respectively. Steroid was continued for the next 4 weeks and then gradually tapered. No further bleeding episode was noted. Conclusion The diagnosis of acquired hemophilia should be considered in any elderly patient with prolonged APTT. Mixing study is to measure the presence of inhibitors of coagulation or to detect coagulation factor deficiency. Quantitative coagulation factor assays and Bethesda Assays are performed for definitive diagnosis. Immunosuppressive regimens are the mainstay treatment. However, premorbid conditions and co-morbidities should be taken into consideration before initiating the aggressive immunosuppressive therapy in the elderly patients.

Approximation of emicizumab plasma levels in emergency situations. A practical approach.

A dedicated emicizumab assay based on the modified one-stage factor VIII (FVIII) assay (mOSA) is mainly available in haemophilia treatment centres (HTC). A method to estimate emicizumab plasma levels based on a widely available assay would be desirable, especially for emergency situations. A method for emicizumab plasma level approximation (ELA) using a routine FVIII activity measurement with standard one-stage assay (sOSA) was developed and evaluated. Within this pilot study, 59 samples from patients with severe haemophilia A with (n=8) and without (n=8) inhibitors under emicizumab treatment were analysed using sOSA following a manual 1:8 sample pre-test dilution with saline. The sOSA was determined in two different laboratories, using two different analyser platforms each. The results demonstrated an excellent correlation of approximated emicizumab plasma levels (ELA) with the emicizumab plasma concentration determined with mOSA (r>.9; p<.05). The ELA showed a sensitivity of 93.3% and a specificity of 89.6% to predict a pre-defined cut-off-value of ≤30µg/ml for the discrimination between subtherapeutic and therapeutic emicizumab plasma levels. Approximation of emicizumab levels by standard one-stage FVIII assay discriminates between subtherapeutic and therapeutic emicizumab levels and might facilitate clinical decision-making in emergency situations, such as bleeding, trauma or urgent surgery in case that dedicated emicizumab assays are not available.

Extended Half-Life Factor VIII/Factor IX Products: Assay Discrepancies and Implications for Hemophilia Management.

Due to structural differences between extended half-life (EHL) factor VIII (FVIII) or FIX products and equivalent plasma wild-type molecules used for assay calibration, reagent-dependent discrepancies during monitoring of FVIII- and FIX-replacement therapies with EHL products have been described. To assess the performance of available one-stage clotting and chromogenic substrate assays on the Siemens Atellica COAG 360 analyzer, an in vitro study using spiked plasma samples was performed. The described results confirm previously described findings and allowed allocation of each EHL product to an appropriate assay. In addition, corresponding EHL product-specific analytes were defined within the order entry system of the University Hospital Bonn. The requirement of product-specific FVIII and FIX assays complicates patient monitoring and demonstrates the need for both continuous education and communication between treating physicians and the coagulation laboratory.

Comparism Of Cryoprecipitate Production Methods Using Fibrinogen And Factor VIII Assay

Abstract Introduction/Objective cryopecipiitate is of huge importance in the management of some Bleeding disorders especially in a low resource settings like ours where it is still the mainstay of management of disseminated Intravsacular coagulopathy, Haemophilia and Von willebrand Disease. In this study we aimed to compared the quality of cryoprecipitate produced using the Thaw-siphon method (no centrifugation) and the ones produced using the Slow- thawing (with centrifugation) methods Methods Ten units of newly prepared Fresh Frozen plasmas were randomly selected, five of were used to prepare cryoprecipitate using the Thaw-siphon method while cryoprecipitate were prepared using the slow-thawing plus centrifugation method. The Fibrinogen concentration in all the ten units were determined using the modified Claus tehcnique while the Factor Viii assay of each unit was done using the one-stage APTT based Assay method Results using the Thaw-siphon method, the mean fibrinogen concentration (152mg/bag) was found to be lower than that of units prepared with slow-thawing plus centrifugation methods (256mg/bag). In a similar manner Factor Viii concentrations were higher in the Slow-thawing with centrifugation methods, Mean Factor Viii concentration was 172Iu/bag when compared with 82 IU/bag in Thaw-siphon methods Conclusion We concluded that Cryoprecipiate produced usng the Thaw- centrifugation methods have higher Fibrinogen and factor Viii units and as such, smaller quantity will be required for correction of coagulopathy when compared with the Thaw-siphon methods. Despite this glaring and expected findings, it is worthy of note that the cryoprecipitate units produced by Thaw-siphon methods meets the required criteria for quality cryoprecipitate and will be very useful in a low resource settings.

Genetics and Hemostatic Potential in Persons with Mild to Moderate Hemophilia A with a Discrepancy between One-Stage and Chromogenic FVIII Assays.

Factor VIII (FVIII) activity (FVIII:C) can be measured by different methods including one-stage clotting assays (OSAs) and chromogenic assays (CSAs). Discrepancy between FVIII:C assays is known and associated with genetic variations causing mild and moderate hemophilia A (HA). We aimed to study the discrepancy phenomenon and to identify associated genetic alterations. Further, we investigated if hemostatic global assays could discriminate the group with discrepant FVIII:C from them. The study contained plasma samples from 45 patients with HA (PwHA) from Hemophilia Centers in Stockholm, Sweden, and Belgrade, Serbia. We measured FVIII:C with OSA and CSA, sequenced the F8 gene, and performed two global hemostatic assays; endogenous thrombin potential and overall hemostatic potential. Nineteen of 45 PwHA had a more than twofold higher FVIII:C using OSA compared to CSA and were considered discrepant. Thirty-four causal mutations were detected, where of five had not previously been associated with assay discrepancy. These novel mutations were p.Tyr25Cys, p.Phe698Leu, p.Met699Leu, p.Ile1698Thr, and Ala2070Val. We found no difference between discrepant and nondiscrepant cases with either of the global assays. There was a discrepancy between FVIII:C assays in almost half of the PwHA, which for some could lead to missed HA diagnoses or misclassification of severity. Genotyping confirmed that mutations associated with FVIII:C discrepancy cluster in the A domains of F8, and five mutations not previously associated with FVIII:C discrepancy was identified. Global hemostatic assays did not contribute to distinguish assay discrepancy in PwHA.

Congenital Deficiency in Factor VII Revealed by Menorrhagia: Case Report

Factor VII (FVII) deficiency is the most common among rare inherited autosomal recessive bleeding disorders. It is a multifaceted disease because of the lack of a direct correlation between plasma levels of coagulation FVII and bleeding manifestations. Clinical phenotypes range from asymptomatic condition—even in homozygous subjects—to severe, life-threatening bleedings (e.g., central nervous system and gastrointestinal bleeding). Menorrhagia is a frequent type of bleeding in FVII deficiency, with a prevalence rate of two in three women aged 10 to 50 years and with a peak prevalence in teenagers. When menorrhagia is observed and once the gynecological causes are excluded, it is important to carry out a hemostasis assessment because, if an anomaly is found, specific treatment can be administered and preventive measures taken. Basic diagnostic work-up includes routine assays, prothrombin level, activated partial thromboplastin time and platelet count, followed by FVII coagulant activity measurement for isolated decreased prothrombin level. To confirm the diagnosis, FVII assay should be repeated at least once. Several treatment options are currently available for FVII deficiency: Recombinant activated Factor VII (rFVIIa), plasma-derived Factor VII, fresh frozen plasma and prothrombin complex concentrates. rFVIIa is the most used replacement therapy. Other medical therapies of menorrhagia includes hemostatic agents and hormonal treatments (combined oral contraceptives, levonorgestrel intrauterine devices), in combination or not with rFVIIa.&#x0D; We report the case of a fourteen-and-a-half-year-old girl who presented menorrhagia of great abundance at the age of thirteen, the exploration of which revealed a congenital deficit in FVII.

A rat model of severe VWD by elimination of the VWF gene using CRISPR/Cas9

BackgroundVon Willebrand Disease (VWD) is the most common inherited bleeding disorder, caused by quantitative and qualitative changes in von Willebrand factor (VWF). The biology of VWD, studied in canine, porcine, and murine models, differ in species‐specific biology of VWF and the amenability to experimental manipulations such as phlebotomy. The factor VIII (FVIII) levels in these models are higher than in humans with type 3 VWD, suggesting functional differences between FVIII and VWF.ObjectivesTo develop a VWF knock out (VWF–/–) rat by excision of all 52 exons of the VWF locus. MethodsThe entire VWF gene was eliminated in Sprague‐Dawley (Crl:SD) rats via CRISPR/Cas9‐mediated gene editing. VWF antigen (VWF:Ag), VWF propeptide, and VWF collagen IV binding (VWF:CB4) levels were determined by ELISA assays and FVIII chromogenic activity (FVIII:C) levels by chromogenic FVIII assays. Lateral tail veins were transected to measure bleeding time. VWF–/– rats were infused with FVIII–/– rat platelet poor plasma (PPP) to determine response of plasma FVIII. ResultsBreeding of VWF ± rats yielded VWF–/– offspring at normal Mendelian ratios. VWF:Ag, VWF propeptide, VWF:CB4, and FVIII:C plasma levels were undetectable in VWF–/– rats. VWF–/– rats bled longer and more than VWF+/– and VWF+/+ rats when challenged. Transfusion of FVIII‐deficient platelet‐poor plasma induced a rapid rise in endogenous FVIII:C in VWF–/– rats. ConclusionThis rat model of severe VWD due to elimination of the entire VWF gene recapitulates the severe secondary deficiency of FVIII seen in human type 3 VWD and facilitates the study of VWF and FVIII and their interactions.