Molecular and phenotypic determinants of the response to desmopressin in adult patients with mild hemophilia A

Summary. Background: The relationship of the biologic response to desmopressin with the F8 mutation and physiological characteristics has been poorly investigated in patients with mild hemophilia A. Objectives: We prospectively assessed the molecular and phenotypic determinants of the biologic response to desmopressin in a cohort of 50 patients with mild hemophilia A. Methods: Up to 24 h after desmopressin, blood samples were serially obtained and factor (F)VIII and von Willebrand factor (VWF) measured. The promoter region, all exons and exon–intron boundaries of the F8 gene were screened using denaturing high‐performance liquid chromatography (DHPLC). Direct sequencing was done when DHPLC screening was normal. Genomic DNA was also sequenced for exons 18–21, 24 and 27 of VWF. Results: Mean basal FVIII:C was 19 ± 9 IU dL^?1 (range 6–37) and the median postdesmopressin peak increase was 2.5‐fold (range 1.1–7.1). Eleven patients with a cross‐reacting material positive (CRM⁺) phenotype had similar basal levels and relative increases of FVIII:C to the remaining patients with low FVIII:Ag. Using multivariate regression, FVIII:C half‐life was positively related to basal and peak VWF:Ag levels (P = 0.008) and patient age (P = 0.004). Eleven patients had evidence of reduced FVIII survival. While 27 different gene mutations were identified in 41 patients, nine patients had no detectable mutation. These patients had significantly smaller peaks and smaller relative increase of postdesmopressin FVIII:C (median FVIII:C 26 IU dL^?1 vs. 54 IU dL^?1; P < 0.001; fold 1.8 ± 0.6 vs. 2.9 ± 0.8; P = 0.002). Conclusions: In this cohort of patients with mild hemophilia A, a poor biologic response to desmopressin was frequently associated with the absence of detectable F8 mutations.     

A multicenter pharmacokinetic study of the B-domain deleted recombinant factor VIII concentrate using different assays and standards

Summary. When the one‐stage clotting assay is used in comparison with the chromogenic and immunological assays, plasma levels of factor (F)VIII are underestimated by 40–50% after infusion of B‐domain deleted recombinant FVIII (BDD‐rFVIII) in patients with hemophilia. A possible way to counteract the underestimation of FVIII levels by the one‐stage assay is the adoption of a recombinant FVIII reference standard instead of a plasma standard. To evaluate the usefulness of such a standard [ReFacto^® Laboratory Standard (RLS)], the pharmacokinetic parameters of a single dose of BDD‐rFVIII (25 U kg^?1) were evaluated in a multicenter study carried out in 18 patients with severe hemophilia A. The very low in vivo recovery, obtained with the combination of the one‐stage assay and plasma reference standard, was increased up to the values obtained by the chromogenic assay when the results were expressed in terms of RLS. When the plasma standard was used, the one‐stage/chromogenic ratio was 0.82 ± 0.12 for FVIII levels above 25 U dL^?1 and 1.42 ± 0.99 for FVIII levels below 25 U dL^?1. Using the RLS, the one‐stage/chromogenic ratio increased to 1.01 ± 0.19 at FVIII levels above 25 U dL^?1, as a consequence of a complete overlap of the two decays; however, at FVIII levels below 25 U dL^?1, the one‐stage/chromogenic ratio was still 1.6 ± 0.85. After the twelfth hour, FVIII concentrations obtained by chromogenic assay were always lower than those resulting from the one‐stage clotting assay, independently of the standard used. Results obtained by chromogenic assay were not affected by the type of standard used. Compared with those obtained by the one‐stage assay, higher values of clearance, lower volume of distribution area and shorter plasma half‐life or mean residence time were obtained by chromogenic assay because of a shape change of the decay curve due to a shift to higher values in the first part (time interval 0–12 h) and to lower values in the second part of the decay curve (time interval 12–48 h). As a consequence, the slope of the decay curve obtained by means of chromogenic assay was steeper. In conclusion, the more homogeneous results of in vivo recovery and pharmacokinetic analysis, due to the decrease of discrepancy between the two methods when RLS was used, make the cheaper and more widely used one‐stage assay preferable to the more expensive chromogenic assay, on condition that the ReFacto specific standard has used.     

A modified thrombin generation test for the measurement of factor VIII concentrates

Summary. It has been well documented that there is an uncertainty over the true factor (F)VIII level in postinfusion samples due to assay discrepancies. The thrombin generation test (TGT) was used as a potentially more physiological approach to assess and compare FVIII concentrates. FVIII concentrates were added to artificial FVIII‐deficient plasma. Thrombin generation was initiated by the addition of FIXa (14 nm ), phospholipid and CaCl₂. Thrombin was measured by subsampling into fibrinogen, and curves quantified as area under the curve (AUC) and time taken to half‐maximum (t_½max). Addition of one plasma‐derived concentrate to as little as 0.005 IU mL^?1 gave a normal AUC, but prolonged t_½max. Increasing FVIII to 1 IU mL^?1 had little effect on AUC, but did reduce the t_½max to 64 s (normal 114 s). A range of plasma‐derived and recombinant concentrates were tested at 1 IU mL^?1; results were similar, except the B‐domain deleted concentrate, which had the most rapid initial rate of thrombin generation (t_½max 48 s, P < 0.05). Two hemophilic plasmas (< 0.01 IU mL^?1) produced large amounts of thrombin (AUC 65% and 69%), although t_½max was prolonged. Addition of a FVIII antibody abolished thrombin generation, indicating that these plasmas contained low levels of FVIII. Decreasing the FIXa concentration (0.2 nm ) minimized thrombin generation in hemophilic plasma but not in normal plasma. These results indicate that FVIII < 0.01 IU mL^?1 can generate significant quantities of thrombin depending upon the amount of FIXa present. The TGT could prove useful for patient monitoring in gene therapy and prophylaxis.     

Factor VIII requirement to maintain a target plasma level in the prophylactic treatment of severe hemophilia A: influences of variance in pharmacokinetics and treatment regimens

Summary. Background: Prophylactic factor (F)VIII has been shown to reduce bleeds and arthropathy in patients with severe hemophilia A. Objectives: Assuming that the trough FVIII level is an important determinant of the efficacy of prophylaxis, this paper addresses the effect of the inter‐patient variability in pharmacokinetics and different dosing regimens on trough levels. Methods: Simulations used FVIII half‐lives and in vivo recoveries (IVR), observed during clinical trials with Advate [Antihemophilic Factor (Recombinant), Plasma/Albumin‐Free Method], and commonly used prophylactic regimens to calculate their effect on FVIII levels during prophylaxis. Results and conclusions: Half‐life and dose frequency had a larger effect on trough FVIII and time per week with FVIII < 1 IU dL^?1 than IVR and infused dose per kg. The combined effect of these parameters resulted in substantial inter‐patient variability in the amount of FVIII required to sustain a desired trough level. Prophylactic regimens based on Monday, Wednesday, Friday dosing were less cost effective in maintaining a desired trough level throughout the week. Dose escalation on Friday to cover the weekend would require potentially harmful doses of FVIII in many patients, especially in young children where more than 50% would require a Friday dose of over 100 IU kg^?1 and some would require more than 400 IU kg^?1. Knowledge of individual patients’ half‐lives and alteration of frequency of infusions may allow the more cost‐effective use of FVIII and potentially expand access to prophylaxis to a greater number of patients, especially in regions where healthcare resources are scarce.     

Antidote strategies to reverse anticoagulation with TB‐402, a long‐acting partial inhibitor of factor VIII

Summary. Background: TB‐402 is a partially inhibiting antibody of factor VIII that is under development as a long‐acting anticoagulant. Patients and Methods: The reversibility of FVIII inhibition by TB‐402 was evaluated in vitro after spiking with recombinant human FVIII (rhFVIII), human plasma‐derived FVIII (hpdFVIII), recombinant activated human FVII (rhFVIIa), FVIII inhibitor bypassing activity (FEIBA), and prothrombin complex concentrate (PCC). Twelve subjects were randomized to placebo or 35 or 70 IU kg^?1 rhFVIII 48 h after a single dose of 620 μg kg^?1 TB‐402. TB‐402 concentrations, FVIII activity (FVIII:C), activated partial thromboplastin time (APTT) and thrombin generation were measured over a period of 8 weeks. Results: In spiked samples, TB‐402 inhibited FVIII:C by 30%, prolonged APTT by 4.5 s, and reduced the peak height in the thrombin generation assay to 56% ± 13% of the control value. In the presence of 10 μg mL^?1 TB‐402, rhFVIII restored FVIII:C and APTT to the values obtained in the absence of TB‐402. The inhibitory effect of TB‐402 on thrombin generation was entirely reversed by rhFVIII, hpdFVIII, rhFVIIa, FEIBA, and PCC. In men, the mean half‐life (t_1/2) of TB‐402 was 14.2 days. TB‐402 lowered the endogenous thrombin potential by 23% for ～ 35 days. Infusion of 35 IU kg^?1 rhFVIII had a marginal effect, whereas 70 IU kg^?1 rhFVIII restored FVIII:C, reduced APTT back to baseline for 9 h, and restored thrombin generation for ～ 3 h. Conclusions: TB‐402 resulted in a stable long‐term anticoagulant effect. rhFVIII and other procoagulants counteracted the effect of TB‐402 temporarily, and may be effective antidotes for future clinical practice.     

Factor XIII – an under diagnosed deficiency – are we using the right assays?

Summary. Background: The clot solubility test is the most widely used method for detection of factor (F)XIII deficiency. However, it will only detect severe deficiencies; consequently mild deficiencies and heterozygous states are probably under diagnosed. Objective: As an alternative first‐line screening test, we assessed an automated quantitative ammonia release assay (QARA). Patients/methods: Inter‐assay imprecision was evaluated with commercial normal and pathological control plasmas (10 replicates on each of 5 days). Using the QARA and other commercial assays a comparative assessment of congenital (FXIII range < 1–70 u dL^?1, n = 9) and acquired (n = 43) deficiencies was made. We also investigated the prevalence of acquired deficiencies in hospitalized patients using residual samples from adult patients (n = 1004) and from a paediatric intensive care unit (ICU, n = 56). Results: Assay imprecision was acceptably low (normal control: mean 86.6 u dL^?1; cv = 2.0%; pathological control: mean 27.5 u dL^?1; cv = 3.8%). Using an iodoacetamide blanking procedure, the QARA results (FXIII range < 1–70 u dL^?1) exhibited close agreement with those from an immuno‐turbidometric FXIII A‐subunit (FXIII‐A) method. There was also good correlation (R² ≥ 0.89) between the QARA (range 20–180 u dL^?1), a second chromogenic assay, the FXIII‐A and FXIII A+B‐subunit ELISA. We found that 21% of samples from adult patients had FXIII levels < 70 u dL^?1 (mean normal ± 2 SD 73–161 u dL^?1) with 6% < 50 u dL^?1. Within the paediatric ICU samples, 52% were < 70 u dL^?1, with 21% < 50 u dL^?1. Conclusions: Our data demonstrates that the automated assay is sensitive, highly reproducible and the results from clinical samples suggest that acquired FXIII deficiency is a relatively common phenomenon in hospital patients after surgery and in ICU.     

Efficacy and safety of secondary prophylactic vs. on-demand sucrose-formulated recombinant factor VIII treatment in adults with severe hemophilia A: results from a 13-month crossover study

Summary. Background: Hemarthroses in severe hemophilia precipitate physical, psychosocial and financial difficulties. Objective: To compare the effects of secondary prophylaxis with on‐demand sucrose‐formulated recombinant factor VIII (rFVIII‐FS) therapy in severe hemophilia A. Patients and methods: This open‐label study included patients aged 30–45 years with factor VIII (FVIII) coagulant activity < 1 IU dL^?1 who were using on‐demand FVIII treatment. Patients were treated with rFVIII‐FS on demand for 6 months, followed by 7 months prophylaxis (20–40 IU kg^?1, three times per week, with the first month considered a run‐in). The primary endpoint was the number of hemarthroses. Results: Twenty patients were enrolled (n = 19 completed); the mean age was 36.4 years, and 16 had target joints. The median (25–75%) number of joint bleeds decreased significantly with prophylaxis [0 (0–3)] vs. on‐demand [15 (11–26); P < 0.001] therapy. The number of all bleeds was 0 (0–3) vs. 20.5 (14–37; P < 0.001), respectively. Median (range) total Gilbert scores improved after prophylaxis [18 (3–39)] compared with on‐demand [25 (4–46)] therapy, predominantly reflecting the improved bleeding score. Median time from last prophylactic infusion to bleed was 2 days; 82.5% of bleeds occurred 2–3 days after the last infusion. Median 48‐h and 72‐h FVIII trough levels measured during months 10 and 13 were consistently > 6 and > 4 IU dL^?1, respectively. Treatment was well tolerated, and no inhibitor formation was observed. Conclusion: Secondary prophylaxis with rFVIII‐FS significantly reduced the frequency of hemarthroses compared with on‐demand therapy in adult patients with severe hemophilia A.     

Coagulation factor activity and clinical bleeding severity in rare bleeding disorders: results from the European Network of Rare Bleeding Disorders

Summary. Background: The European Network of Rare Bleeding Disorders (EN‐RBD) was established to bridge the gap between knowledge and practise in the care of patients with RBDs. Objectives: To explore the relationship between coagulation factor activity level and bleeding severity in patients with RBDs. Patients/Methods: Cross‐sectional study using data from 489 patients registered in the EN‐RBD. Coagulation factor activity levels were retrieved. Clinical bleeding episodes were classified into four categories according to severity. Results: The mean age of patients at data collection was 31 years (range, 7 months to 95 years), with an equal sex distribution. On linear regression analysis, there was a strong association between coagulation factor activity level and clinical bleeding severity for fibrinogen, factor (F) X, FXIII, and combined FV and FVIII deficiencies. A weaker association was present for FV and FVII deficiencies. There was no association between coagulation factor activity level and clinical bleeding severity for FXI. The coagulation factor activity levels that were necessary for patients to remain asymptomatic were: fibrinogen, > 100 mg dL^?1; FV, 12 U dL^?1; combined FV + VIII, 43 U dL^?1; FVII, 25 U dL^?1; FX, 56 U dL^?1; FXI, 26 U dL^?1; FXIII, 31 U dL^?1. Moreover, coagulation factor activity levels that corresponded with Grade III bleeding were: undetectable levels for fibrinogen, FV and FXIII, < 15 U dL^?1 for combined FV + VIII; < 8 U dL^?1 for FVI; < 10 U dL^?1 for FX; and < 25 U dL^?1 for FXI. Conclusions: There is a heterogeneous association between coagulation factor activity level and clinical bleeding severity in different RBDs. A strong association is only observed in fibrinogen, FX and FXIII deficiencies.     

Plasma and albumin-free recombinant factor VIII: pharmacokinetics, efficacy and safety in previously treated pediatric patients

Summary. Background: The pharmacokinetics of factor VIII replacement therapy in preschool previously treated patients (PTPs) with hemophilia A have not been well characterized. Objectives: To assess the pharmacokinetics, efficacy and safety of a plasma‐free recombinant FVIII concentrate, ADVATE [Antihemophilic Factor (Recombinant), Plasma/Albumin‐Free Method, rAHF‐PFM], in children < 6 years of age with severe hemophilia. Patients/methods: Fifty‐two boys, one girl, mean (± SD) age 3.1 ± 1.5 years and ≥ 50 days of prior FVIII exposure, were enrolled in a prospective study of ADVATE rAHF‐PFM at 23 centers. Results: The mean terminal phase half‐life (t_1/2) was 9.88 ± 1.89 h, and the mean adjusted in vivo recovery (IVR) was 1.90 ± 0.43 IU dL^?1 (IU kg^?1)^?1. Over the 1–6‐year age range, t_1/2 of rAHF‐PFM increased by 0.40 h year^?1. IVR increased by 0.095IU dL^?1(IU kg^?1)^?1 (kg m^?2)^?1 in relation to body mass index (BMI). Patients primarily received prophylaxis. Median (range) annual joint bleeds were 0.0 (0.0–5.8), 0.0 (0.0–6.1) and 14.2 (0.0–34.5) for standard prophylaxis, modified prophylaxis and on‐demand treatment, respectively. Bleeds were managed in 90% (319/354) of episodes with one or two rAHF‐PFM infusions; response was rated excellent/good in 93.8% of episodes. Over a median 156 exposure days, no FVIII inhibitors were detected and no related severe adverse events or unusual non‐serious adverse events were seen. Conclusions: Children < 6 years of age appear to have shorter FVIII t_1/2 and lower IVR values than older subjects. However, these parameters increased with age (t_1/2) and BMI (adjusted IVR), respectively. rAHF‐PFM was clinically effective and well tolerated, with no signs of increased immunogenicity in previously treated young children with hemophilia A.     

Comparative immunogenicity of recombinant B domain‐deleted porcine factor VIII and Hyate:C in hemophilia A mice presensitized to human factor VIII

Summary. Hyate is a commercial plasma‐derived porcine factor (F)VIII concentrate that is used in the treatment of patients with inhibitory antibodies to FVIII. OBI‐1 is a recombinant B domain‐deleted form of porcine FVIII that is in clinical development for the same indication. Hemophilia A mice were presensitized with human FVIII to simulate clinical inhibitory antibody formation and then were randomized to receive OBI‐1 or Hyate:C in a comparative immunogenicity trial. OBI‐1 or Hyate:C were given in a series of four intravenous injections at weekly intervals at doses of 1, 10, or 100 U kg^?1. Inhibitory antibodies to porcine FVIII were not detected by Bethesda assay in most of the mice given OBI‐1 or Hyate:C at doses of 1 or 10 U kg^?1, but were identified in 81% and 94% of mice given 100 U kg^?1 of OBI‐1 or Hyate:C, respectively. There was no significant difference between OBI‐1 and Hyate:C in inhibitory antibody formation at any dose, although there was a trend toward a lower Bethesda titer in OBI‐1‐treated mice at 10 U kg^?1 (P = 0.09). Total anti‐FVIII antibodies to Hyate:C and OBI‐1 were also measured by ELISA using immobilized purified plasma‐derived porcine FVIII and OBI‐1, respectively, as antigens. At the 10 and 100 U kg^?1 doses, the mean anti‐FVIII response was higher in Hyate:C‐treated‐mice than in OBI‐1‐treated mice (P = 0.02 and P = 0.004, respectively). The results using this model suggest that OBI‐1 may be less immunogenic and safer than Hyate:C in FVIII inhibitor patients.     

Population pharmacokinetic modeling for dose setting of nonacog beta pegol (N9‐GP), a glycoPEGylated recombinant factor IX

Summary. Background: nonacog beta pegol (N9‐GP) is a glycoPEGylated recombinant factor IX (rFIX) molecule with a prolonged half‐life. Objectives: To provide information on potential dose regimens for N9‐GP for phase 3 pivotal and surgery trials. Methods: A population pharmacokinetic model was developed from single‐dose data derived from the first human‐dose trial with N9‐GP in hemophilia B patients, and was used to extrapolate to steady‐state conditions for different N9‐GP dose regimens for prophylaxis. The model was also used to compare prophylaxis using N9‐GP with standard prophylactic regimens using rFIX or plasma‐derived (pd) FIX (40 IU kg^?1 every third day). Plasma activity following dosing with N9‐GP, rFIX and pdFIX for surgery and on‐demand treatment of bleeds was also simulated. Results: A linear two‐compartmental model best described the pharmacokinetic profiles of N9‐GP, rFIX and pdFIX. A prophylactic regimen of 10 U kg^?1 N9‐GP once weekly predicted mean peak and trough levels of 18 and 4.2 U dL^?1, while 40 U kg^?1 once weekly predicted values of 72 and 17 U dL^?1, respectively. Standard prophylactic regimens with rFIX and pdFIX predicted mean peak and trough levels of 34 and 3.9 IU dL^?1 for rFIX, and mean values of 43 and 2.1 IU dL^?1 for pdFIX. Additional simulations predicted significantly reduced dosing frequency and factor concentrate consumption for N9‐GP vs. rFIX and pdFIX for surgery and the treatment of bleeds. Conclusions: N9‐GP may allow prophylaxis, surgical dosing regimens and on‐demand treatment of bleeding episodes with less frequent injections and lower factor concentrate consumption; this possibility is being investigated in prospective clinical trials.     

Apolipoprotein C‐III predicts cardiovascular mortality in severe coronary artery disease and is associated with an enhanced plasma thrombin generation

Summary. Background: Apolipopoprotein C‐III (apo C‐III) plays a pivotal role in controlling plasma triglyceride (TG) and contributes to the atherogenic properties of TG‐rich lipoproteins. Objectives: (i) To examine the predictive value of serum apo C‐III for cardiovascular mortality in the setting of secondary prevention of coronary artery disease (CAD); and (ii) to evaluate possible associations between apolipoprotein levels and the thrombin generation assay, a global test to estimate plasma thrombogenic potential. Methods and results: A cohort of 633 patients with angiographically proven CAD was prospectively followed for a median follow‐up of 57 months. The large majority of them (92%) underwent coronary (endovascular or surgical) revascularization. During the follow‐up, 91 (14.3%) out of 633 patients died, with 64 events (10.1%) attributed to cardiovascular causes. After adjustment for all the other predictors of mortality during univariate analysis (i.e. age, statin therapy, myocardial infarction history, diabetes, hs‐CRP and creatinine), elevated apo C‐III levels (≥ 10.5 mg dL^?1– the median value) significantly predicted both total and cardiovascular mortality (HR for total mortality 2.22 with 95% CI 1.16–4.24; HR for cardiovascular mortality 2.35 with 95% CI 1.19–4.62). In a subgroup of 225 subjects, apo C‐III levels were significantly associated with endogenous thrombin potential in regression models (standardized β coefficient = 0.207, P = 0.002). Conclusions: Basal concentrations of apo C‐III levels ≥ 10.5 mg dL^?1 in CAD patients independently predicted cardiovascular mortality during the subsequent 5‐year period. Such concentrations were associated with an enhanced plasma endogenous thrombin generation, suggesting a complex interplay between TG‐rich particles and the coagulation cascade as well as a new ‘thrombogenetic’ role for apo C‐III.     

Coffee consumption is associated with a reduced risk of venous thrombosis that is mediated through hemostatic factor levels

Summary. Background: Coffee consumption is associated with a lower risk of venous thrombosis, but the role of confounding and the pathophysiology behind these findings are unclear. Objective: To assess the role of hemostatic factors in the relationship between coffee consumption and venous thrombosis. Methods: From a large case–control study, 1803 patients with a first venous thrombosis and 1803 partner controls were included. With conditional logistic regression, odds ratios (ORs) and 95% confidence intervals (CIs) for venous thrombosis were calculated for coffee consumption vs. no coffee consumption. In addition, mean differences in hemostatic factor levels between these groups were calculated in the controls. Results: Coffee consumption yielded a 30% lower risk of venous thrombosis than no coffee consumption (OR 0.7, 95% CI 0.5–0.9). Adjustment for several putative confounders (age, sex, body mass index, smoking, hormonal factors, statin, aspirin, alcohol, malignancy, and chronic disease) yielded an OR of 0.8 (95% CI 0.6–1.1). Results were similar for provoked and unprovoked events, and for deep vein thrombosis and pulmonary embolism. In controls, von Willebrand factor levels were 11 (3–19) IU dL^?1 lower and factor (F) VIII levels were 11 (1–21) IU dL^?1 lower in coffee consumers than in non‐consumers. After adjustment of the risk estimates for these hemostatic factors, the inverse association between coffee consumption and venous thrombosis diminished (OR 1.0, 95% CI 0.7–1.4). There was no association between coffee consumption and anticoagulant proteins, fibrinogen levels, or fibrinolytic markers. Conclusions: Coffee consumption is associated with a lower risk of venous thrombosis, which seems to be mediated through von Willebrand factor and FVIII.     

Comparative pharmacokinetics of plasma‐ and albumin‐free recombinant factor VIII in children and adults: the influence of blood sampling schedule on observed age‐related differences and implications for dose tailoring

Summary. Background: Dose tailoring of coagulation factors requires reliably estimated and reproducible pharmacokinetics (PK) in the individual patient. Objectives: To investigate the contribution of both biological and methodological factors to the observed variability of factor VIII (FVIII) PK, with the focus on differences between children and adults, and to examine the implications for dosing. Patients: Data from 52 1–6‐year‐old and 100 10–65‐year‐old patients with hemophilia A (FVIII ≤ 2 IU dL^?1) in three clinical studies were included. Results: In vivo recovery was lower, weight‐adjusted clearance was higher and FVIII half‐life was on average shorter in children than in adults. However, a reduced blood sampling schedule for children was estimated to account for up to one half of the total observed differences. Intrapatient variance in PK was smaller than interpatient variance in 10–65‐year‐olds. Age and ratio of actual to ideal weight only showed weak relationships with PK parameters. Variance in PK caused large variance in the calculated dose required to maintain a target FVIII trough level during prophylactic treatment. Conclusion: Differences in blood sampling schedules should be taken into account when results from different PK studies are compared. However, even with this consideration, PK cannot be predicted from observable patient characteristics but must be determined for the individual. Because the influence of reducing the blood sampling was minor in comparison to the true variance between patients, a reduced blood sampling protocol can be used. Low intrapatient variability supports the use of PK measurements for dose tailoring of FVIII.     

Coagulation procofactor activation by factor XIa

Summary. Background: In the extrinsic pathway, the essential procofactors factor (F) V and FVIII are activated to FVa and FVIIIa by thrombin. In the contact pathway and its clinical diagnostic test, the activated partial thromboplastin time (APTT) assay, the sources of procofactor activation are unknown. In the APTT assay, FXII is activated on a negatively charged surface and proceeds to activate FXI, which activates FIX upon the addition of Ca²⁺. FIXa feeds thrombin generation through activation of FX. FIXa is an extremely poor catalyst in the absence of its FVIIIa cofactor, which, in the intrinsic FXase complex, increases FXa generation by ～ 10⁷. One potential APTT procofactor activator in this setting is FXIa. Objective: To test the hypothesis that FXIa can activate FVIII and FV. Methods: Recombinant FVIII and plasma FV were treated with FXIa, and the activities and integrities of each procofactor were measured using commercial clotting assays and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Results: Kinetic analyses of FXIa‐catalyzed activation and inactivation of FV and FVIII are reported, and the the timing and sites of cleavage are defined. Conclusions: FXIa activates both procofactors at plasma protein concentrations, and computational modeling suggests that procofactor activation during the preincubation phase of the APTT assay is critical to the performance of the assay. As the APTT assay is the primary tool for the diagnosis and management of hemophilias A and B, as well as in the determination of FVIII inhibitors, these findings have potential implications in the clinical setting.     

Prevalence of immunoglobulin A deficiency in Chinese blood donors and evaluation of anaphylactic transfusion reaction risk

Objectives/Aims: We investigated the incidence of immunoglobulin A (IgA) deficiency in Chinese population. Background: The frequency of IgA deficiency, defined as a serum IgA level of <0·05 mg dL^?1, has been broadly studied in different ethnic groups. Individuals with IgA deficiency may form specific antibodies against IgA, which can cause an anaphylactic response when the patient receives an IgA‐containing blood transfusion. Methods: A sandwich enzyme‐linked immunosorbent assay was performed to screen for IgA deficiency and particle gel immunoassay used for confirmation. IgA antibodies were further detected by the DiaMed anti‐IgA test in IgA‐deficient blood donors. Results: Of the total 22 609 healthy blood donors screened, only seven cases were confirmed as having IgA deficiency (<0·05 mg dL^?1). Another seven cases displayed relative IgA deficiencies, with mean IgA concentrations ranging from 0·39 to 3·70 mg dL^?1. Anti‐IgA was identified in 2 of the 14 IgA‐deficient blood donors whose IgA levels were <5 mg dL^?1. Estimation of the theoretical risk for IgA anaphylactic transfusion reaction was 0·009%. Conclusion: The prevalence of IgA deficiency in Chinese is low. However, potential risks exist in performing blood transfusion to IgA‐deficient persons, and measures should be taken to reduce IgA anaphylaxis.     

Severe hemophilia with mild bleeding phenotype: molecular characterization and global coagulation profile

Summary. Background: Patients with severe hemophilia may show very varied bleeding tendencies, and the reasons for this heterogeneous clinical expression are unclear. The factor VIII/FIX genotype is the main determinant of the residual factor activity; however, different bleeding phenotypes have also been reported in patients sharing the same mutation. Such global coagulation tests as thrombin generation assays are tools with which to investigate different coagulation profiles among severe hemophiliacs. Objectives, patients and methods: This case–control study was aimed at comprehensively evaluating the role of genotype and endogenous thrombin potential (ETP) as predictors of the clinical phenotype in severe hemophiliacs with an extremely mild bleeding tendency (cases, n = 22), in comparison with those showing a typical bleeding tendency (controls, n = 50). Results: Cases were more frequently affected by hemophilia B than by hemophilia A, and showed a lower incidence of severe FVIII/FIX gene defects (referred to as null mutations), higher FVIII and FIX antigen levels and higher ETP values in platelet‐rich plasma than controls (P < 0.05). By multivariate logistic regression, only non‐null mutations were confirmed as an independent predictor of a mild clinical phenotype. Conclusions: These results indicate that non‐null mutations represent the main determinant of the bleeding tendency, and that ETP measurement in platelet‐rich plasma is able to identify severe hemophiliacs with a mild clinical phenotype.     

Collaborative field study on the utility of a BDD factor VIII concentrate standard in the estimation of BDDr Factor VIII:C activity in hemophilic plasma using one‐stage clotting assays

Summary. Advances in production technologies of factor (F)VIII concentrates during the last two decades has resulted in very pure and safe products. In assessment of recombinant FVIII:C, inconsistent assay values are found comparing one‐stage assays with two‐stage (e.g. amidolytic) methods. Such discrepancies have been quite prominent in the case of a B‐domain deleted recombinant FVIII (BDDrFVIII, ReFacto^®). In order to alleviate this assay variance, a product‐specific reference standard [the ReFacto Laboratory Standard? (RLS)], was established for laboratory use with either one‐stage clotting or chromogenic substrate assays for the measurement of FVIII:C in ReFacto‐containing patient samples. The primary objective of the current study was to assess, under field laboratory conditions, the accuracy and precision of the one‐stage clotting assay for the determination of FVIII:C in ReFacto‐containing samples employing the new concentrate standard. A secondary goal was to assess whether use of the RLS would minimize the discrepancy between one‐stage clotting and chromogenic substrate assays. Thirty‐one clinical laboratories worldwide participated in the study of severe‐hemophilic plasma (SHP) samples that had been spiked with ReFacto to target levels of 0.9, 0.6 and 0.2 IU mL^?1. FVIII:C levels were determined against both the RLS and the local in‐house plasma standard (IHS). The results showed good agreement between laboratories in FVIII:C levels obtained by one‐stage clotting assays utilizing the RLS, and a good degree of accuracy was found compared with the intended target values. Consistent with previously published data, a discrepancy of approximately 30% was observed between one‐stage clotting and chromogenic potencies when the IHS was used as the calibrator. The discrepancy between one‐stage and chromogenic assay methodologies was significantly reduced when the RLS was employed as calibrator in the one‐stage assay. In conclusion, the study demonstrates that accurate and precise FVIII:C results can be obtained for ReFacto‐containing SHP samples by clinical laboratories using a product‐specific standard in one‐stage clotting assays. In addition, the product‐specific reference standard significantly reduced the discrepancy between the one‐stage clotting and the chromogenic substrate assay for ReFacto.     

Mechanisms of factor VIIa‐catalyzed activation of factor VIII

Summary. Background: Factor (F)VIIa, complexed with tissue factor (TF), is a primary trigger of blood coagulation, and has extremely restricted substrate specificity. The complex catalyzes limited proteolysis of FVIII, but these mechanisms are poorly understood. Objectives: In the present study, we investigated the precise mechanisms of FVIIa/TF‐catalyzed FVIII activation. Results: FVIII activity increased ～4‐fold within 30 s in the presence of FVIIa/TF, and then decreased to initial levels within 20 min. FVIIa (0.1 nm ), at concentrations present physiologically in plasma, activated FVIII in the presence of TF, and this activation was more rapid than that induced by thrombin. The heavy chain (HCh) of FVIII was proteolyzed at Arg⁷⁴⁰ and Arg³⁷² more rapidly by FVIIa/TF than by thrombin, consistent with the enhanced activation of FVIII. Cleavage at Arg³³⁶ was evident at ～1 min, whilst little cleavage of the light chain (LCh) was observed. Cleavage of the HCh by FVIIa/TF was governed by the presence of the LCh. FVIII bound to Glu‐Gly‐Arg‐active‐site‐modified FVIIa (K_d, ～0.8 nm ) with a higher affinity for the HCh than for the LCh (K_d, 5.9 and 18.9 nm ). Binding to the A2 domain was particularly evident. Von Willebrand factor (VWF) modestly inhibited FVIIa/TF‐catalyzed FVIII activation, in keeping with the concept that VWF could moderate FVIIa/TF‐mediated reactions. Conclusions: The results demonstrated that this activation mechanism was distinct from those mediated by thrombin, and indicated that FVIIa/TF functions through a ‘priming’ mechanism for the activation of FVIII in the initiation phase of coagulation.     

Validation of Nijmegen–Bethesda assay modifications to allow inhibitor measurement during replacement therapy and facilitate inhibitor surveillance

Summary. Background: As part of a pilot U.S. inhibitor surveillance project initiated at the Centers for Disease Control and Prevention (CDC) in 2006, a centralized inhibitor measurement was instituted. Objective: To validate a modified method for inhibitor measurement suitable for surveillance of treated and untreated patients. Methods/Results: In all, 710 subjects with hemophilia A were enrolled; 122 had a history of inhibitor (HI). Nijmegen–Bethesda assay (NBA) results on 50 split specimens shipped on cold packs and frozen were equivalent (r = 0.998). Because 55% of 228 initial specimens had factor (F)VIII activity (VIII:C) present, a heat treatment step was added. Heating specimens to 56 °C for 30 min and centrifuging removed FVIII, as demonstrated by a reduction of VIII:C and FVIII antigen to < 1 U dL^?1 in recently treated patients. Among specimens inhibitor‐negative before heating, one of 159 with negative HI and five of 30 with positive HI rose to ≥ 0.5 Nijmegen–Bethesda units (NBU) after heating. Correlation of heated and unheated inhibitor‐positive specimens was 0.94 (P = 0.0001). The modified method had a coefficient of variation (CV) for a 1 NBU positive control of 10.3% and for the negative control of 9.8%. Based on results on 710 enrollment specimens, a positive CDC inhibitor was defined as ≥ 0.5 NBU. Results were similar when 643 post‐enrollment specimens were included. Of 160 enrolled hemophilia B patients, two had HI. All others had NBU ≤ 0.2 at enrollment. Conclusion: The CDC experience demonstrates that this modified NBA can be standardized to be within acceptable limits for clinical tests and can be used for national surveillance.