Evaluation of the von Willebrand factor antigen (vWF-Ag) assay using an immuno-turbidimetric method (STA Liatest vWF) automated on the MDA 180 coagulometer

The standard enzyme-linked immunosorbent assay (ELISA) test for von Willebrand factor antigen (vWF-Ag), though sensitive and specific, does not deliver the flexibility to handle single sample assessments economically or provide rapid, emergency testing capability. The present study examined the performance characteristics of an immuno-turbidimetric assay kit (STA Liatest) modified for automation on the MDA(R)180 coagulation analyser using a supplied protocol. One hundred and sixteen patient samples were assessed by both the standard and the modified method. The correlation coefficient was 0.98 across the range of values 1-487 IU/dl. Above 200 IU/dl, where specimen dilution was indicated, there was greater diversity (r = 0.86) between the techniques. Plotting the difference between methods against the mean of both showed excellent agreement between methods below 100 IU/dl vWF. The intra-assay and interassay coefficients of variation were less than 3% at both low and normal range levels. The MDA(R)180 automated vWF assay merits consideration as an alternative to ELISA testing that provides random access and result availability within 30 min.     

Clinical observation on safety and efficacy of a plasma‐ and albumin‐free recombinant factor VIII for on‐demand treatment of Chinese patients with haemophilia A

Summary. Recombinant FVIII (rFVIII) has become the best choice for treating bleeding of haemophilia A patients. A plasma‐ and albumin‐free recombinant FVIII (rAHF‐PFM, ADVATE^®), as the third generation rFVIII, virtually eliminates the risk of blood‐borne disease transmission by excluding all human blood derived additives throughout cell culture, purification and formulation. In this multicentre prospective clinical study we evaluated the efficacy, safety and immunogenicity of ADVATE^® in Chinese patients with haemophilia A. Fifty‐eight patients enrolled and received ADVATE^® treatment. Of the patients enrolled, eight (13.79%) had severe haemophilia, 45 (77.59%) had moderate haemophilia and five (8.62%) had mild haemophilia. Fifty‐four patients completed 6 months of observation. A total of 781 bleeds occurred in these 58 subjects, all evaluable per‐protocol. A total of 984 infusions were administered with a mean of 17.0 ± 11.1 infusions per patient. On average, each patient received a mean of 15030.2 ± 7972.7 IU ADVATE^® (median 13 625 IU, range 9500–19 750 IU) during 6 months. The majority of bleeding episodes (95.9%) were successfully treated with one or two infusions of ADVATE^®. Overall, response to the first ADVATE^® treatment was rated as either ‘excellent’ (82.8%) or ‘improved’ (17.2%) in all subjects. All patients tolerated ADVATE^® infusions well. One patient (1/58, 1.7%) developed an inhibitor of 4 Betheseda units at day 180 visit. The results of this clinical observational study support that ADVATE^® is efficacious, safe and well tolerated in the treatment of Chinese patients with haemophilia A.     

Field evaluation of GeneXpert® (Cepheid) HCV performance for RNA quantification in a genotype 1 and 6 predominant patient population in Cambodia

GeneXpert^® (Cepheid) is the only WHO prequalified platform for hepatitis C virus (HCV) nucleic acid amplification testing that is suitable for point‐of‐care use in resource‐limited contexts. However, its application is constrained by the lack of evidence on genotype 6 (GT6) HCV. We evaluated its field performance among a patient population in Cambodia predominantly infected with GT6. Between August and September 2017, we tested plasma samples obtained from consenting patients at Médecins Sans Frontières’ HCV clinic at Preah Kossamak Hospital for HCV viral load (VL) using GeneXpert^® and compared its results to those obtained using COBAS^® AmpliPrep/Cobas^® TaqMan^® HCV Quantitative Test, v2.0 (Roche) at the Institut Pasteur du Cambodge. Among 769 patients, 77% of the seropositive patients (n = 454/590) had detectable and quantifiable VL using Roche and 43% (n = 195/454) were GT6. The sensitivity and specificity of GeneXpert^® against Roche were 100% (95% CI 99.2, 100.0) and 98.5% (95% CI 94.8, 99.8). The mean VL difference was ?0.01 (95% CI ?0.05, 0.02) log₁₀ IU/mL for 454 samples quantifiable on Roche and ?0.07 (95% CI ?0.12, ?0.02) log₁₀ IU/mL for GT6 (n = 195). The limit of agreement (LOA) was ?0.76 to 0.73 log₁₀ IU/mL for all GTs and ?0.76 to 0.62 log₁₀ IU/mL for GT6. Twenty‐nine GeneXpert^® results were outside the LOA. Frequency of error and the median turnaround time (TAT) for GeneXpert^® were 1% and 0 days (4 days using Roche). We demonstrated that the GeneXpert^® HCV assay has good sensitivity, specificity, quantitative agreement, and TAT in a real‐world, resource‐limited clinical setting among GT6 HCV patients.     

The use of the new ReFacto AF Laboratory Standard allows reliable measurement of FVIII:C levels in ReFacto AF mock plasma samples by a one‐stage clotting assay

Summary. Factor VIII coagulant (FVIII:C) levels measured in patients receiving ReFacto^® (B‐domain‐deleted recombinant FVIII) using chromogenic substrate assay (CSA) and one‐stage clotting assay (OSA) have frequently shown discrepancies, and the use of the ReFacto Laboratory Standard (RLS) has therefore been recommended to minimize these differences. The potency of ReFacto AF^®, the albumin‐free successor of ReFacto^®, is determined using CSA for the titration of vials, and a new standard (RLS‐AF) was developed to measure its biological efficacy using OSA. This multicentre study therefore evaluated the efficacy of this new RLS in minimizing differences between OSA and CSA when measuring FVIII:C levels in plasma. Mock plasma samples were prepared by diluting ReFacto AF^® in FVIII‐deficient plasma to obtain four concentrations ranging from 15 to 90 IU dL^?1. FVIII:C levels were then measured in six laboratories on four separate days using three different procedures, i.e. OSA with a plasma standard (PS) as reference, OSA with RLS‐AF and CSA with PS. The inter‐centre standard deviation ranged from 1.4 to 5.5 IU dL^?1. However, FVIII:C levels measured with OSA were closer to the expected values when RLS‐AF was used. In addition, the uncertainty of measurement, reflecting the inter‐method discrepancy was greatly reduced when RLS‐AF was employed in OSA (15%) in place of PS (33%). This study demonstrates that the OSA performed with RLS‐AF to establish calibration curves provides a valuable alternative to CSA to measure FVIII:C in ReFacto‐AF‐treated patients.     

International comparative field study of N8 evaluating factor VIII assay performance

Summary. Discrepancies between the one‐stage clotting assay and the chromogenic method, and also among different variations of each method, have been a significant challenge for one B‐domain deleted FVIII product. N8 is a B‐domain truncated FVIII product developed by Novo Nordisk. The comparison of N8 and Advate^® was performed in an international, multicentre, randomized and blinded field study of simulated postinfusion samples. Overall, Advate^® and N8 performed similarly in the one‐stage assay. In the one‐stage clotting assay, the measured mean FVIII levels of Advate^® vs. N8 were 0.046/0.047, 0.24/0.24, 0.58/0.60 and 0.82/0.83 IU mL^?1 for the target values of 0.03, 0.2, 0.6 and 0.9 IU mL^?1, respectively. In the chromogenic assays, the concentration estimates showed a tendency towards higher N8 values as compared with Advate^®; the measured FVIII levels of Advate^® vs. N8 were 0.030/0.032, 0.22/0.24, 0.65/0.74 and 0.98/1.08 IU mL^?1 for the target values of 0.03, 0.2, 0.6 and 0.9 IU mL^?1, respectively. In the one‐stage assays, the measured values were above 150% of target at the lowest concentration, decreasing to around 90% of target at the highest concentration. In contrast, the chromogenic assays were close to target at the lowest concentration and consistently above target at the three highest concentrations. Therefore, the ratio of chromogenic/one‐stage potencies was concentration dependent, ranging from 0.66 to 1.30. The SSC plasma standard was similar in both. Assay variability was similar for both compounds. The results show that N8 can be reliably measured in plasma without the need for a separate N8 standard.     

An ultrapure plasma‐derived monoclonal antibody‐purified factor IX concentrate (Nonafact®), results of phase III and IV clinical studies

Summary. Nonafact^®, an ultrapure, monoclonal antibody‐purified factor IX concentrate (FIX) was developed to minimize risk of thrombotic complications and viral transmission. To investigate the pharmacokinetics, efficacy and safety, phase III/IV studies were performed in the Netherlands and Poland from 1996 to 2007. The mean half‐life, in vivo response and recovery of Nonafact^® were 18.7 (SD 2.0) h, 1.1 (SD 0.2) IU dL^?1 per IU kg^?1 b.w. of FIX infused and 49% (SD 10%), respectively. Eleven surgical procedures were performed in eight patients. During two surgeries, both high‐risk, blood loss was observed. No postoperative bleeding occurred. The in vivo recovery of FIX was higher than expected. In the phase III follow‐up study, 26 previously treated patients (PTP) were included with a median follow‐up of 1130 days. From the 1617 minor bleedings, 80.5% was stopped after a single infusion. In the phase IV study thirteen patients were treated for a median study period of 737 days. In the two follow‐up studies the investigators rated the effect of Nonafact^® as excellent/good in 95% of major bleedings. Surgeries for which Nonafact^® was given prophylactically were without bleeding problems. In total more than 10 million units of Nonafact^® were used during almost 120 person‐years. Only one minor adverse event was reported. No inhibitors, viral transmissions and thrombogenic events occurred. In conclusion, Nonafact^® is safe and provides excellent haemostasis in haemophilia B patients treated for spontaneous bleeding or undergoing surgical procedures. Due to the excellent in vivo recovery characteristic, treatment with Nonafact^® is cost saving compared to other FIX products.     

The PFA‐100® does not predict delta‐granule platelet storage pool deficiencies

Summary.  There are no published reports investigating the ability of the platelet function analyzer (PFA‐100^®) to detect the presence of delta‐granule platelet storage pool deficiencies (δ‐PSPD), a common mild bleeding disorder. Prior studies of the PFA‐100^® and congenital platelet disorders have been limited by small numbers of patients with a variety of disorders. We examined PFA‐100^® results in a large paediatric patient population diagnosed specifically with δ‐PSPD, and determined the relationship between PFA‐100^® and platelet electron microscopy (the gold standard for diagnosis). This study is a retrospective review of patients <19 years of age diagnosed with δ‐PSPD at Nationwide Children’s Hospital from 2008 to 2010. To examine the correlation between PFA‐100^® and average number of granules per platelet we used Spearman’s Rho as a non‐parametric measure of dependence. A total of 105 patients diagnosed with δ‐PSPD were included, of which 99 patients underwent PFA‐100^® testing. Of those tested 46% had at least one abnormal closure time, whereas 16% had abnormal results for both cartridges. We found no statistical correlation between C‐EPI closure time and average number of granules per platelet (ρ= ?0.0095, P‐value = 0.9328), nor between C‐ADP closure time and the average number of granules (ρ = 0.0315, P‐value = 0.7798). The PFA‐100^®, a widely used screening test for suspected bleeding disorders, did not correlate with presence or severity of δ‐PSPD as determined by platelet electron microscopy. When evaluating patients with suspected bleeding disorders, PFA‐100^® alone cannot be used to rule out the presence of a δ‐PSPD.     

Prospective evaluation of a rapid nanoparticle‐based lateral flow immunoassay (STic Expert® HIT) for the diagnosis of heparin‐induced thrombocytopenia

A rapid lateral flow immunoassay (LFIA) (STic Expert^® HIT), recently developed for the diagnosis of heparin‐induced thrombocytopenia (HIT), was evaluated in a prospective multicentre cohort of 334 consecutive patients. The risk of HIT was estimated by the 4Ts score as low, intermediate and high in 28·7%, 61·7% and 9·6% of patients, respectively. Definite HIT was diagnosed in 40 patients (12·0%) with positive results on both enzyme‐linked immunosorbent assay (Asserachrom^® HPIA IgG) and serotonin release assay. The inter‐reader reproducibility of results obtained was excellent (kappa ratio > 0·9). The negative predictive value of LFIA with plasma samples was 99·6% with a negative likelihood ratio (LR) of 0·03, and was comparable to those of the particle gel immunoassay (H/PF4‐PaGIA^®) performed in 124 cases. Positive predictive value and positive LR were 44·4% and 5·87, respectively, and the results were similar for serum samples. The probability of HIT in intermediate risk patients decreased from 11·2% to 0·4% when the LFIA result was negative and increased to 42·5% when it was positive. In conclusion, the STic Expert^® HIT combined with the 4Ts score is a reliable tool to rule out the diagnosis of HIT.     

Head‐to‐head comparison of the pharmacokinetic profiles of a high‐purity factor IX concentrate (AlphaNine®) and a recombinant factor IX (BeneFIX®) in patients with severe haemophilia B

Head‐on comparative studies of factor IX (FIX) concentrates performed under standardized conditions are rarely conducted regardless of being a valuable instrument guiding health care providers towards better informed and cost‐effective decisions. This study is an extension of a multicentre study that assessed the efficacy, safety and pharmacokinetics (PK) of AlphaNine^® in 25 previously treated patients with severe haemophilia B (FIX:C ≤ 2%). After a washout period ≥7 days following the last PK performed with AlphaNine^® after a dose of 65–75 IU kg^?1, an identical PK study was performed with BeneFIX^® on 22 of the same patients. Venous blood samples for analysis were taken at baseline and at 0.25, 0.5, 1, 3, 6, 9, 24, 48, 72 and 74 h post infusion. The outcomes of the comparison of the PK parameters were as follows: Mean (±SD) in vivo recovery (IVR) was 1.3 ± 0.4 IU dL^?1 per IU kg^?1 for AlphaNine^® and 1.0 ± 0.3 IU dL^?1 per IU kg^?1 for BeneFIX^® (P < 0.01). Mean terminal half‐life, mean residence time, area under the curve, clearance and volume of distribution of BeneFIX^® were 36.0 ± 12.8 h, 39.3 ± 13.9 h, 1631 ± 467 IU h dL^?1, 0.046 ± 0.01 dL kg^?1 min^?1 and 1.75 ± 0.52 mL kg^?1 respectively. These values were not significantly different to those observed in AlphaNine^®, although BeneFIX^® displayed higher than expected IVR values and lower than expected clearance values. In conclusion, AlphaNine^® showed a comparable half‐life, but an IVR significantly higher than that of BeneFIX^®. This dissimilarity may have implications on dosing requirements for on‐demand treatment regimes affecting optimal resource allocation.     

Evaluation of a new venom‐based clotting assay of protein C

Congenital protein C deficiency significantly increases the risk of venous thromboembolism, a serious and potentially lethal condition. Protein C levels can be determined by chromogenic, clotting and antigenic assays, each type of assay has differences in specificity and sensitivity to protein C deficiency. In principle, clotting‐based assays of protein C are preferred over chromogenic assays, as they can detect some rare mutations that are missed by the chromogenic assay, however, clotting‐based assays may be prone to inaccuracy because of poor specificity. We have evaluated a new venom‐based clotting assay of protein C, and optimized it for use on Sysmex CA‐1500 analyser. The assay was linear from 0 to 130 U/dl, a normal plasma demonstrated good inter‐assay precision, with a coefficient of variation of 4.8%. The assay compared well with antigenic‐ and venom‐based chromogenic protein C assay in normal individuals, subjects with lupus anticoagulant, and subjects with FV Leiden. Median protein C levels by clotting, chromogenic and antigen for the three subject groups were 108 U/dl, 108 IU/dl and 109 IU/dl for normal subjects, 94 U/dl, 106 IU/dl and 103 IU/dl for subjects with lupus anticoagulant, and 102 U/dl, 104 IU/dl and 100 IU/dl for subjects heterozygous for FV Leiden. Comparing levels of clotting protein C with protein C antigen by ratio (clotting/antigen), the three groups showed small differences that did not quite reach statistical significance, (mean ratios ranged from 0.95 to 1.01, anova P = 0.0561), the lowest ratio was with the lupus anticoagulant group. Comparing clotting assay with chromogenic assay by ratio (clotting/chromogenic), the three groups did show a statistically significant difference (P = 0.0033) which was due to a difference in mean ratios between normal and lupus anticoagulant groups (ratios 1.00 and 0.91, respectively, P < 0.01). There was no statistical difference in any of the groups when comparing chromogenic protein C with protein C antigen (mean ratios ranged from 1.02 to 1.05, P = 0.3925). In a normal sample, the clotting‐based protein C level was unaffected by increasing FVIII level by up to 1000 IU/dl, using intermediate purity FVIII concentrate. The new assay is considered to be a suitable assay for the routine diagnosis of protein C deficiency.     

A clinical study assessing the pharmacokinetics,efficacy and safety of AlphaNine®, a high‐purity factor IX concentrate,in patients with severe haemophilia B

Summary. Effective treatment with factor IX (FIX) requires a thorough consideration of the properties of the concentrate to be used as replacement therapy, to date, the only available treatment for haemophilia B. The aim of the study was to determine the pharmacokinetics, clinical efficacy and safety in routine clinical use of AlphaNine^®, a high‐purity human FIX concentrate. This open, single‐arm, multicentre, non‐randomized trial included 25 subjects (age ≥ 12) with moderate/severe haemophilia B. Pharmacokinetics was assessed at baseline and after a 6‐month follow‐up. The degree of haemostasis control achieved was evaluated during a 12‐month follow‐up. Safety was evaluated in terms of tolerance, thrombogenicity, immunogenicity and viral safety. Mean recovery was 1.01 ± 0.19 IU dL^?1 per IU kg^?1 at baseline and 1.23 ± 0.34 IU dL^?1 per IU kg^?1 6 months later. Terminal half‐life was 34.5 ± 6.2 h and 33.7 ± 5.4 h, respectively. Ratios of each parameter between the two pharmacokinetic studies were all close to 1. A total of 1,576,890 IU AlphaNine^® were administered in 889 infusions (mean dose per infusion: 1774 IU; 3.2 infusions per month per patient). The main reasons for infusion were mild/moderate bleeding (62.3%) and prophylaxis (20.5% continuous, 15.6% intermittent). Overall, 93.0% of the efficacy assessments were rated as excellent/good and 88.8% of bleedings resolved after the first infusion. Twenty‐one adverse events were reported in eight patients, none of which was considered related to the study medication. AlphaNine^® showed a pharmacokinetic profile in agreement with that of other plasma‐derived FIX concentrates and provides safe and clinically effective substitution therapy for patients with haemophilia B.     

Clinical experience with Optivate®, high‐purity factor VIII (FVIII) product with von Willebrand factor (VWF) in young children with haemophilia A

Summary. Optivate^® is a high‐purity FVIII/VWF product. Its safety, tolerability and efficacy in subjects ≥12 years have been demonstrated. This study was undertaken to assess Optivate^® in children with haemophilia A. Twenty‐five children, including one PUP (previously untreated patient), aged 1–6 years (mean 4.67 years) were treated with Optivate^® for 26 weeks. Inhibitors were assessed every 3 months and viral status at the study start and end. Prophylaxis was used by five boys and on demand by twenty. The mean number of bleeds in the study was lower compared to the same period pre‐study (12.0/child vs. 16.2/child), with fewer bleeds (P < 0.05) in the prophylactic subgroup (8.0/child) compared with the on‐demand sub‐group (13.4/child). Fourteen major bleeds were reported, all by the on‐demand sub‐group. Children on prophylaxis were administered a mean of 59.4 infusions; on‐demand group 35.1 infusions. A total of 998 infusions were used with a mean dose of 29.1 IU kg^?1, and a mean of 38.6 exposure days (ED). Children <4 years used higher doses, and reported fewer bleeds than older children. Children’s Parents/Guardians rated Optivate^® as helpful or very helpful in controlling 97.5% of bleeds by the prophylactic group, and in 98.5% of the bleeds in the on‐demand group. Only 5 of 101 ADRs were treatment‐related events (5%), all were mild and non‐serious. There were no clinically significant changes in vital signs, viral transmissions or inhibitors. In young children Optivate^® was well tolerated, safe and efficacious.     

Pharmacokinetics and pharmacodynamics of a new recombinant FVIII (N8) in haemophilia A mice

Summary. N8 is a new recombinant factor VIII (rFVIII) compound produced and formulated without human‐ or animal‐derived protein. The aims of the present studies were to evaluate the pharmacokinetics and pharmacodynamics properties of N8 and to compare with a commercially available rFVIII product (Advate^®) in haemophilia A mice. The pharmacokinetics were evaluated after single i.v. administration of 80, 120 and 280 IU kg^?1 of N8 and Advate^® and measurements of FVIII blood concentrations as a function of time. The efficacy and dose response curves of N8 and Advate^® (1–200 IU kg^?1) were evaluated in a tail bleeding model. Furthermore, the effects in a newly developed haemophilia knee joint haemarthrosis model were investigated. No significant differences were found in the pharmacokinetic parameters between N8 and Advate^®. The clearances were 11 ± 1 vs. 10 ± 2 mL h^?1 kg^?1 (P = 0.14) and the half‐lives 7.2 ± 0.9 vs. 7.7 ± 1.4 h (P = 0.31) after administration of N8 and Advate^® respectively. Dose‐independent pharmacokinetics was shown, and comparable efficacy and potency were shown between N8 and Advate^® in the tail bleeding model. Both compounds normalized the bleeding at the dose of 200 IU kg^?1, and for blood loss ED₅₀ values of 27 IU kg^?1 (N8) and 28 IU/kg (Advate^®) were found (P = 0.97). In the haemarthrosis model, treatment with N8 and Advate^® at 200 IU kg^?1 reduced the mean increase in the joint diameter significantly from 1.23 ± 0.19 to 0.32 ± 0.08 mm (P < 0.01) and 0.25 ± 0.08 mm (P < 0.001) respectively. Pharmacokinetics and pharmacodynamics of N8 and Advate^® were comparable after i.v. administration to haemophilia A mice.     

An overview of turoctocog alfa pegol (N8‐GP; ESPEROCT®) assay performance: Implications for postadministration monitoring

Factor replacement therapy with factor VIII (FVIII) concentrates is the current standard of care for patients with haemophilia A. Postadministration monitoring of FVIII activity during on‐demand or prophylactic treatment is important, for example to guide a suitable dosing regimen. While the use of two‐stage chromogenic substrate (CS) assays is increasing, activated partial thromboplastin time (APTT)‐based one‐stage clotting (OSC) assays are most commonly used to measure FVIII activity in clinical laboratories. Substantial variations in activity measurements have been observed in association with some OSC assay reagents when assessing extended half‐life FVIII molecules. Certain silica‐based APTT reagents have previously been shown to underestimate FVIII activity with the polyethylene glycol (PEG)‐conjugated product turoctocog alfa pegol (N8‐GP [ESPEROCT^®]; Novo Nordisk A/S). As a wide range of assay reagents are used in clinical laboratories worldwide, it is essential to establish which can be used to accurately measure activity with modified FVIII concentrates. Here, we describe the approach taken by Novo Nordisk to determine the suitability and accuracy of assays and reagents to measure FVIII activity in samples that contain N8‐GP. While accurate activity measurements were possible with all tested CS assays and most of the OSC APTT reagents tested, three APTT reagents that contain silica as a contact activator were found to underestimate N8‐GP recovery (APTT‐SP, TriniCLOT?, STA^® PTT‐Automate). The data demonstrate the importance of characterizing the accuracy of each FVIII activity assay. Any limitations should be communicated to treating physicians and the clinical laboratories that test samples containing N8‐GP.     

Pharmacokinetics of Optivate®, a high‐purity concentrate of factor VIII with von Willebrand factor,in patients with severe haemophilia A

Summary. Optivate^® is a high purity factor VIII/von Willebrand factor (FVIII/VWF) concentrate, which is manufactured using two antiviral processes: solvent/detergent and terminal dry heating (80°C for 72 h). A multicentre, non‐randomized open‐label study in 15 patients was conducted to test the pharmacokinetics (PK) of Optivate^®. PK variables were analysed for the patients’ prior FVIII product (PK1), their first dose of Optivate^® (PK2) and at 3 months therapy (PK3). Mean non‐compartmental half‐lives (h) were 14.1, 12.4 and 12.1, respectively (P = 0.45), mean clearances (mL h^?1 kg^?1) were 3.6, 3.2 and 3.1, respectively (P = 0.051), MRTs (h) were 19.0, 17.3 and 17.4, respectively (P = 0.39) and mean AUC_0–48h (h IU mL^?1) were 14.3, 15.4 and 16.6, respectively (P = 0.051) and mean AUC_0–∞ (h IU mL^?1) were 15.9, 16.4 and 17.9, respectively (P = 0.18). The recovery data from this PK study was aggregated with recovery data collected from another study, with similar design but devoid of the other PK measurements. A total of 309 recoveries were conducted in 70 patients. The overall mean recovery per subject across 27 Optivate^® batches was 2.7 IU dL^?1 per IU kg^?1. There were no clinical differences between Optivate^® and other FVIII products, and except for volume of distribution (Vd), no statistically significant differences were seen with respect to any of the other PK variables, or in recovery between weeks 0 and 12. Therefore, the PK of FVIII is not affected by the processes used to manufacture Optivate^®, which can be expected to be effective in the management of patients with haemophilia A.     

Pharmacokinetic study of a high-purity factor IX concentrate (Factor IX Grifols®) with a 6-month follow up in previously treated patients with severe haemophilia B

Summary. Optimal replacement treatment in haemophilia B patients requires a good understanding of the pharmacokinetics of factor IX (FIX). The aim of this study was to compare the pharmacokinetic profile of Factor IX Grífols^®, a highly purified human FIX concentrate with two specific pathogen inactivation/removal steps, to that of available FIX preparations. The study was an open, non‐randomized trial including 25 male subjects older than 12 years of age with severe haemophilia B. Pharmacokinetic profile of the FIX preparation regularly used by the subjects was determined as control. Pharmacokinetic profile of Factor IX Grifols^® was determined twice, one 7–15 days after control assessment and second after a 6 months period had elapsed. Results showed that all products had peak plasma levels of FIX:C within 30 min. Mean recovery was 1.3 ± 0.3 IU dL⁻¹ per IU kg⁻¹ for Factor IX Grifols^® and 1.0 ± 0.3 IU dL⁻¹ per IU kg⁻¹ for control products (P < 0.001). The mean terminal half‐life (t_1/2) for Factor IX Grifols^® was 26.7 h and 26.8 h for control product. Pharmacokinetic parameters after 6 months of treatment with Factor IX Grifols^® did not statistically differ from the parameters obtained with the first infusion. There were no adverse events related to Factor IX Grifols^® for the duration of the study. In conclusion, Factor IX Grifols^® has adequated pharmacokinetic properties comparable to the control plasma‐derived FIX and these parameters remain stable after 6 months of treatment. Factor IX Grifols^® can be an effective and safe plasma‐derived FIX concentrate for replacement therapy in haemophilia B patients.     

Evaluation of an automated platelet‐based assay of ristocetin cofactor activity

Summary. von Willebrand’s disease (VWD) is regarded as the most common congenital bleeding disorder, and although not available in all laboratories von Willebrand factor (VWF) activity is most frequently assessed as ristocetin cofactor (VWF:RCo). This test can be technically challenging, is subject to poor sensitivity (～20 IU dL^?1 VWF:RCo) and has a high degree of inter‐ and intra‐assay imprecision [coefficient of variation (cv) > 25%]. We studied an automated assay using a combined fixed platelet/ristocetin reagent (BC von Willebrand reagent, Siemens Healthcare Diagnostics) on the CS‐2000i analyser (Sysmex UK Ltd). Initially inter‐ and intra‐assay imprecision was assessed. The automated method showed good day‐to‐day reproducibility and linearity of standard curves. This technique, also gave low intra‐ and inter‐assay imprecision using commercial normal (cv < 4.5%) and pathological (cv < 8.1%) control plasmas. We then compared automated technique results from 30 healthy normal subjects and 39 VWD patients to those obtained using standard aggregometry (Bio/Data, PAP4) with lyophilised fixed platelets (Helena BioSciences) and ristocetin (American Biochemical and Pharmaceutical Ltd). The automated method had a sensitivity limit of approximately 10 IU dL^?1 vs. 20 IU dL^?1 for aggregometry. Samples giving results within the aggregometry measurable range (n = 50) exhibited good correlation with the automated technique (median 70 IU dL^?1, range 7–184 IU dL^?1; and 64 IU dL^?1, 6–138 IU dL^?1 respectively; R² = 0.85). We subsequently compared 3 different batches of BC von Willebrand reagent, using a second group of normal subjects and VWD patients (n = 35, 55–139 IU dL^?1 and n = 30, <10–50 IU dL^?1). The CS‐2000i results exhibited no clinically significant variation between batches (mean cv = 7%). The automated VWF:RCo assay offers a more sensitive, reproducible, robust and less laborious alternative to standard aggregometry.     

A simple and inexpensive point‐of‐care test for hepatitis B surface antigen detection: serological and molecular evaluation

Early identification of chronic hepatitis B is important for optimal disease management and prevention of transmission. Cost and lack of access to commercial hepatitis B surface antigen (HBsAg) immunoassays can compromise the effectiveness of HBV screening in resource‐limited settings and among marginalized populations. High‐quality point‐of‐care (POC) testing may improve HBV diagnosis in these situations. Currently available POC HBsAg assays are often limited in sensitivity. We evaluated the NanoSign^® HBs POC chromatographic immunoassay for its ability to detect HBsAg of different genotypes and with substitutions in the ‘a’ determinant. Thirty‐seven serum samples from patients with HBV infection, covering HBV genotypes A–G, were assessed for HBsAg titre with the Roche Elecsys HBsAg II quantification assay and with the POC assay. The POC assay reliably detected HBsAg at a concentration of at least 50 IU/mL for all genotypes, and at lower concentrations for some genotypes. Eight samples with substitutions in the HBV ‘a’ determinant were reliably detected after a 1/100 dilution. The POC strips were used to screen serum samples from 297 individuals at risk for HBV in local clinical settings (health fairs and outreach events) in parallel with commercial laboratory HBsAg testing (Quest Diagnostics EIA). POC testing was 73.7% sensitive and 97.8% specific for detection of HBsAg. Although the POC test demonstrated high sensitivity over a range of genotypes, false negatives were frequent in a clinical setting. Nevertheless, the POC assay offers advantages for testing in both developed and resource‐limited countries due to its low cost (0.50$) and immediately available results.     

A Phase 2 study of L‐asparaginase encapsulated in erythrocytes in elderly patients with Philadelphia chromosome negative acute lymphoblastic leukemia: The GRASPALL/GRAALL‐SA2‐2008 study

Purpose : The GRASPALL/GRAALL‐SA2‐2008 Phase II trial evaluated the safety and efficacy of L‐asparaginase encapsulated within erythrocytes (GRASPA®) in patients ≥ 55 years with Philadelphia chromosome‐negative acute lymphoblastic leukemia. Findings : Thirty patients received escalating doses of GRASPA^® on Day 3 and 6 of induction Phases 1 and 2. The primary efficacy endpoint was asparagine depletion < 2 µmol/L for at least 7 days. This was reached in 85 and 71% of patients with 100 and 150 IU/kg respectively but not with 50 IU/kg. Grade 3/4 infection, hypertransaminasemia, hyperbilirubinemia and deep vein thrombosis occurred in 77, 20, 7, and 7% of patients, respectively. No allergic reaction or clinical pancreatitis was observed despite 17% of Grade 3/4 lipase elevation. Anti‐asparaginase antibodies were detected in 50% of patients and related to a reduction in the duration of asparagine depletion during induction Phase 2 without decrease of encapsulated L‐asparaginase activity. Complete remission rate was 70%. With a median follow‐up of 42 months, median overall survival was 15.8 and 9.7 months, in the 100 and 150 IU/kg cohorts respectively. Conclusions : The addition of GRASPA^®, especially at the 100 IU/kg dose level, is feasible in elderly patients without excessive toxicity and associated with durable asparagine depletion. ( clinicaltrials.gov identifier NCT01523782). Am. J. Hematol. 90:811–818, 2015. © 2015 Wiley Periodicals, Inc.     

Bioequivalence between two serum‐free recombinant factor VIII preparations (N8 and ADVATE®) – an open‐label,sequential dosing pharmacokinetic study in patients with severe haemophilia A

Summary. Recombinant coagulation factor VIII (rFVIII) concentrates provide a safe and efficacious replacement therapy for treatment and prevention of bleeding in patients with severe haemophilia A. The aim of this study was to compare the pharmacokinetic (PK) and safety profiles of two serum‐free rFVIII products: N8, a new rFVIII manufactured by Novo Nordisk and Advate^®, a marketed product. Patients with severe haemophilia A with >150 exposure days to FVIII, without current or past inhibitors, were enrolled in an open‐label, first human dose (FHD), multicentre trial. Twenty‐three patients first received a single dose of 50 IU kg^?1 body weight Advate^® followed by 50 IU kg^?1 body weight N8 at the next visit. A 4‐day washout period was required prior to each dosing. Blood samples for PK and safety analyses were drawn prior to dosing and at intervals up until 48 h postdosing. The PK parameters were based on FVIII clotting activity (FVIII:C) measurements. Occurrence of adverse events was closely monitored. The mean profiles of FVIII:C and all primary and secondary parameters for Advate^® and N8 were comparable. The 90% CI for the treatment ratio (Advate^®/N8) for all primary endpoints (incremental recovery, t_1/2, AUC and Cl), and the secondary endpoints (AUC_last and C_max) were within the bioequivalence interval of 0.8–1.25. There were no safety concerns in the study and no reports of inhibitor formation in the 72‐h period following exposure to a single N8 dose. In conclusion, N8 is bioequivalent to Advate^®. Furthermore, N8 is well tolerated in the FHD trial.