Clinical efficacy of a highly purified SD-treated factor IX concentrate prepared by conventional chromatography

Summary. A highly purified (HP) FIX concentrate was used in 12 patients with haemophilia B in order to prevent bleeding following surgery. The HP FIX concentrate (CRTS, Lille, France) was prepared by ion-exchange chromatograpy and was SD treated. The patients underwent orthopaedic surgery (3), neurosurgery (3) or multiple dental extraction (6). They were treated for 2–18 days and received 8500 to 102000 FIX units. The mean FIX:C recovery was 0·92 ± 0·20%/U/kg. Satisfactory haemostasis was provided in all patients. None of them experienced thrombotic manifestations. There was no change in FDP or soluble fibrin complexes. Prothrombin fragments one plus two (F1 + 2), thrombin-antithrombin III complexes (TAT) and cross-linked fibrin degradation products (D-dimer) remained in the normal range in one patient treated for 2·5 days for dental surgery. In contrast, elevated levels of F1+2, TAT and D-dimer were observed in one patient undergoing a bilateral hip arthroplasty treated for 16 consecutive days with the same lot of HP FIX concentrate as the previous patient. There was no seroconversion for HIV, HBV and HCV. These results indicate that the HP FIX concentrate provides satisfactory haemostasis in surgery while avoiding excessive thrombogenic risks. However, some clinical circumstances, such as orthopaedic surgery, are known to be associated with especially high thrombotic risks that must be taken into account in case of FIX replacement.     

Parvovirus B19 transmission by a high-purity factor VIII concentrate

BACKGROUND: Parvovirus B19 (B19) is known to cause a variety of human diseases in susceptible individuals by close contact via the respiratory route or by transfusion of contaminated blood or blood products. In this study, whether a case of B19 transmission was causally related to the infusion of implicated lots of a solvent/detergent (S/D)-treated, immunoaffinity-purified factor VIII concentrate (antihemophilic factor [human][AHF]) was investigated. STUDY DESIGN AND METHODS: Anti-B19 (both immunoglobulin M [IgM] and immunoglobulin G [IgG]) and B19 DNA (by a nucleic acid testing [NAT] procedure) were assayed in two implicated product lots, a plasma pool, and a recipient's serum sample. Analysis of the partial B19 sequences obtained from sequencing clones or direct sequencing of the samples was performed. RESULTS: Only one of the two implicated lots was B19 DNA-positive. It contained 1.3 x 10(3) genome equivalents (geq or international units [IU]) per mL. The negative lot was derived from plasma screened for B19 DNA by NAT in a minipool format to exclude high-titer donations, whereas the positive lot was mostly from unscreened plasma. This high-purity AHF product had no detectable anti-B19 IgG. A 4-week postinfusion serum sample from a recipient, who received both lots and became ill, was positive for the presence of B19 antibodies (both IgM and IgG) as well as B19 DNA. The B19 sequences from the positive lot, its plasma pool, and the recipient's serum sample were closely related. CONCLUSION: These findings and the recipient's clinical history support a causal relationship between the implicated AHF product and B19 infection in this recipient. The seronegative patient became infected after receiving 2x10(4) IU (or geq) of B19 DNA, which was present in this S/D-treated, high-purity AHF product.     

A factor VIII concentrate of intermediate purity and higher potency

A fractionation method has been modified to produce a factor VIII concentrate of intermediate purity and a potency of about 12 iu/ml, suitable for injection by syringe. The solubility, ease of filtration and purity of the concentrate have been improved by including a stage in which contaminants are precipitated at controlled temperature, pH and ionic strength. A summary of the first 50 production-scale batches is presented. Plasma can be harvested from whole blood and frozen within eight hours of donation, or after overnight storage of whole blood, with no significant difference in overall yield.     

Successful preoperative apheresis of factor VIII antibody using factor VIII concentrate as a replacement fluid

Plasma exchange was performed on a patient with hemophilia A and inhibitor to factor VIIIC prior to bilateral cataract removal. Koate was used as replacement fluid with effective reduction of the inhibitor level. From the technical standpoint we found out that factor VIII is best reconstituted in water and directly infused through the venous line and not the centrifuge bowl.     

Factor VIII concentrate prepared from blood donors stimulated by intranasal administration of a vasopressin analogue

With an interval of eight weeks between collections, blood was drawn twice from 120 blood donors. At one of the donations, 0.25 ml of synthetic vasopressin, (DDAVP, 1300 micrograms/ml), was administered intranasally 60 minutes prior to collection of the blood. No drug was given at the other donation. The yield of factor VIII clotting activity (VIII:C) and factor VIII antigen (VIIIR:Ag) was compared in blood drawn from the treated and untreated donors. To prevent degradation of VIII:C by fibrinolysis, tranexamic acid was added to the plasma from treated donors immediately after separation from the red blood cells. Plasma from treated donors and the derived Cohn fraction I-O contained approximately twice as much VIII:C as plasma and fraction I-O from untreated control donors. The concentration of VIIIR: Ag was also increased in fraction I-O made from plasma from treated donors, however to a lesser degree. The in vivo properties of factor VIII concentrates made from each group of donors were studied. Half-life in plasma and recovery of VIII:C were identical. Thus, intranasal synthetic vasopressin may be used to increase the yield of VIII:C in production of factor VIII concentrates.     

Effect of delayed blood processing on the yield of factor VIII in cryoprecipitate and factor VIII concentrate

Current standards for the preparation of factor VIII (FVIII) concentrates from human plasma recommend separation of plasma from red cells (RBCs) within 6 hours of blood donation, thereby reducing the volume of plasma from donated whole blood available for processing to FVIII concentrate. The decay of FVIII clotting activity (FVIII:C) in whole blood and plasma stored at 22 and 4 degrees C and the recovery of FVIII:C in cryoprecipitate and FVIII concentrate prepared from plasma separated from whole blood stored overnight at 4 degrees C were investigated. In whole blood stored at 22 degrees C and plasma stored at either 4 or 22 degrees C, 90 percent of the original FVIII:C was present at 6 hours, 80 percent at 12 hours, and 65 to 70 percent at 18 hours. At these times lower levels of FVIII:C were recovered from whole blood stored at 4 degrees C, that is, 84, 68, and 56 percent, respectively. In cryoprecipitates prepared from plasma separated from RBCs after 18 hours' storage at 4 degrees C (18-hour plasma), 43 percent of FVIII:C activity was recovered, as compared with 61 percent recovered from standard plasma separated within 6 hours of donation (6-hour plasma), p less than 0.05. With large-scale preparation of FVIII concentrates, however, the yield of FVIII:C was similar whether 18- or 6-hour plasma was used. Thus FVIII concentrates--but not cryoprecipitates--can be prepared from plasma separated from whole blood stored at 4 degrees C for up to 18 hours without undue loss of potency.     

A pharmacokinetic study of an ion-exchange solvent—detergent-treated high-purity factor VIII concentrate

SUMMARY. An ion-exchange chromatography purified factor VIII concentrate (Liberate) that had undergone a solvent—detergent viral inactivation treatment was compared with an intermediate-purity concentrate (Z8), which was terminally heated at 80°C for 72 h, in 15 haemophilia A patients in a blinded crossover pharmacokinetic study. Both products achieved a peak level close to that predicted (100IU/dl for Liberate and 103IU/dl for Z8) and there were no significant differences in the recoveries achieved nor of any of the other pharmacokinetic parameters. We conclude that the pharmacokinetic properties of factor VIII: C, following solvent—detergent treatment and ion-exchange chromatography, are equivalent to those of the lower purity terminally heat-treated product (Z8), and it is therefore likely to be a clinically efficacious concentrate.     

A comparison of factor VIII activity in cryoprecipitates prepared from ACD and CPD plasma

In an attempt to develop a method to produce a cryoprecipitate with a predictable Factor VIII potency, several variables were studied and statistically analyzed. These included the hematocrit, age, blood group and Rh type of the donor; possible epinephrine release; the degree of lipemia, volume, pH and Factor VIII activity of the donor plasma; the volume of cryoprecipitates, its Factor VIII activity and the per cent yield; and the Factor VIII activity of the supernatant plasma. Cryoprecipitates were prepared by the method of Pool and Shannon from the plasmas of 40 random male donors, half the bloods being drawn in ACD and half in CPD. None of the variables had a significant influence on the per cent yield of activity, although the mean values obtained suggest that the per cent yield of Factor VIII activity in the cryoprecipitates prepared from CPD plasmas is higher than in those prepared from ACD plasma. Although the per cent yield of Factor VIII in cryoprecipitates prepared from CPD plasma is not significantly different from that of ACD plasma, the mean total units of Factor VIII activity is significantly higher in CPD cryoprecipitates. It also was confirmed that a higher per cent Factor VIII activity in the donor results in a relatively higher activity in the cryoprecipitate. The data indicate that if CPD plasma collected from donors having above a certain minimum per cent activity were used for cryoprecipitate production, one could be assured of having a minimum of 100 units of Factor VIII activity per bag.     

Variability in factor VIII concentrate measurement: results from SSC field collaborative studies

Summary.  Seven 'field' collaborative studies on factor (F)VIII concentrate potency measurements were carried out, using local routine methodology, standards and calculation of results. Data from five of the 12 different concentrates studied are described in detail. These studies revealed that, for the intermediate-purity and the recombinant FVIII concentrates, one-stage potencies were significantly lower than chromogenic potencies, whilst for the two high-purity FVIII concentrates one-stage potencies were significantly greater than chromogenic potencies. On comparing predilution methods for the intermediate-purity concentrate, equivalent potencies were obtained using either buffer or FVIII-deficient plasma as prediluent. For the two high-purity and the recombinant concentrates, potencies obtained using buffer as prediluent were significantly greater and lower, respectively, than potencies obtained using FVIII-deficient plasma as prediluent. Interlaboratory variabilities were compared over all 12 concentrates studied and coefficients of variation (CVs) for one-stage assays were found to be much greater than for chromogenic assays. This was true for all concentrates except for the intermediate-purity concentrate and samples A and B from the first study, where the reverse was true. Furthermore, much better CVs were obtained when using FVIII-deficient plasma than when using buffer as prediluent, for all FVIII concentrates except for the intermediate-purity concentrate where the reverse was true, and sample B where CVs were equivalent. Overall, CVs were far worse than those obtained in controlled collaborative studies. Generally, however, CVs were better with chromogenic assays and predilution in FVIII-deficient plasma, as is recommended by the International Society on Thrombosis and Haemostasis/Scientific and Standardization Committee, particularly for higher purity and recombinant concentrates.     

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.     

Virus-inactivated factor VIII concentrate prevents postoperative bleeding in a patient with von Willebrand''s disease

A patient with von Willebrand's disease underwent cholecystectomy after replacement therapy with a factor VIII concentrate that had been sterilized by treatment with tri(n-butyl)phosphate and Tween 80. The patient received 53 units of factor VIII per kg of body weight prior to operation. In addition, a total of 280 units of factor VIII per kg was infused within 10 days after operation. This replacement regimen prevented excessive bleeding during surgery and supported normal hemostasis during the postoperative period. Analysis of the multimeric pattern and the functional assay of von Willebrand factor in factor VIII concentrates indicated that the procedures utilized for virus inactivation had no significant deleterious effect upon the quality of von Willebrand factor molecules.     

Parvovirus B19 transmission by heat-treated clotting factor concentrates

BACKGROUND: Human parvovirus B19 (B19) DNA can be frequently detected in plasma-derived coagulation factor concentrates. The production of some clotting factor products includes heat treatment steps for virus inactivation, but the effectiveness of such steps for B19 inactivation is unclear. Moreover, detailed transmission case reports including DNA sequence analysis and quantification of B19 DNA from contaminated heat-treated blood components have not been provided so far. Therefore, the correlation between B19 DNA in blood components and infectivity remains unclear. STUDY DESIGN AND METHODS: Asymptomatic B19 infections of two patients with hemophilia A were detected by anti-B19 seroconversion after administration of B19-contaminated heat-treated clotting factors. The suitability of nucleic acid sequence analysis for confirmation of B19 transmission was investigated. Furthermore, the B19 DNA level in blood components was determined and the drug administration was reviewed to calculate the amount of inoculated B19 DNA. RESULTS: Both B19 transmissions from clotting factor products could be confirmed by identical nucleic acid sequences of virus DNA from patients and blood components while sequences from unrelated controls could be differentiated. One patient received, for 4 days, a total of 180 mL vapor heat-treated prothrombin complex concentrate containing 8.6 x 10(6) genome equivalents per mL of B19 DNA. The other patient received 966 mL of low-contamination (4.0 x 10(3) genome equivalents/mL) dry heat-treated FVIII concentrate over a period of 52 days. CONCLUSION: B19 transmissions can be confirmed by nucleic acid sequencing. However, due to the low variability of the B19 genome, a large part of the B19 genome must be analyzed. The transmissions show that the applied heat treatment procedures were not sufficient to inactivate B19 completely.     

Neutralization of antifactor VIII inhibitors by recombinant porcine factor VIII

BACKGROUND: Inhibitory antibodies to factor (F) VIII (FVIII inhibitors) present a major clinical challenge as a complication of hemophilia A and as acquired autoantibodies in non-hemophiliacs. Porcine FVIII is a potentially useful therapeutic agent because of its low crossreactivity with many inhibitors. Recombinant porcine FVIII (rpFVIII) is undergoing clinical trials in inhibitor patients. OBJECTIVES: The goals of this study were to neutralize human FVIII inhibitors in vitro with rpFVIII and to characterize the relationship between recovery of FVIII activity and the antiporcine FVIII inhibitor titer. METHODS: rpFVIII was added to 28 antihuman FVIII inhibitor plasmas and assayed either immediately or after a 2 h preincubation at 37 degrees C. The concentration of rpFVIII required for recovery of FVIII activity to 50% of normal (EC(50)) was determined by polynomial regression analysis and compared with the antiporcine FVIII inhibitor titer measured by Bethesda assay. RESULTS: Six of nine plasmas classified as non-crossreactive by Bethesda assay had detectable inhibitory activity in the FVIII recovery assay. Recovery was decreased following a 2 h preincubation compared with immediate assay for the 19 plasmas with detectable antiporcine FVIII inhibitors. There was a linear relationship between EC(50) and inhibitor titer for plasmas with antiporcine FVIII titers ranging from 0.6 to 10 BU per milliliter in both the immediate and the 2 h preincubation assays. CONCLUSION: A quantitative method for analysis of inhibitor neutralization in vitro has been developed, which may be useful for comparison with pharmacodynamic studies of rpFVIII in FVIII inhibitor patients and subsequent rational dosing in this patient population.     

Study of viral safety of Scottish National Blood Transfusion Service factor VIII/IX concentrate

Summary. To assess the viral safety of the Scottish National Blood Transfusion Service (SNBTS) intermediate purity factor VIII and IX concentrates, the liver function and viral status were assessed prospectively in 13 recipients. None developed hepatitis or seroconverted to HIV or HCV. This study provides additional evidence for the efficacy of dry heat treatment at 80°C for 72 h in preventing virus transmission by coagulation factor concentrates.     

Investigation of porcine parvovirus among persons with hemophilia receiving Hyate:C porcine factor VIII concentrate

BACKGROUND: Porcine clotting factor has been used for more than 15 years to treat severe bleeding episodes in persons with hemophilia who have antibodies to human clotting factor. In 1996, QC procedures revealed for the first time the presence of porcine parvovirus (PPV) in the product. This report describes an investigation to determine the extent of product contamination and to evaluate past recipients of porcine clotting factor (Hyate:C, Speywood Biopharm) for evidence of PPV infection. STUDY DESIGN AND METHODS: Stored specimens from 22 lots of previously released Hyate:C were tested for the presence of PPV DNA by PCR and nested PCR assays. Serum specimens from 98 recipients of Hyate:C and 24 controls who did not receive Hyate:C were tested for PPV antibodies by an immunofluorescence assay. RESULTS: PPV DNA was detected in product from 21 of the 22 lots of Hyate:C, primarily by nested PCR testing. In contrast, none of the serum specimens from the 98 Hyate:C recipients tested positive for PPV IgG antibodies. CONCLUSION: The risk of human disease from percutaneous exposure to low levels of PPV seems to be low. Nevertheless, the theoretical risk of human infection with PPV has led to manufacturing changes, including PCR screening of all porcine plasma, which are designed to eliminate this risk.     

Factor VIII/von Willebrand factor concentrate therapy for ventricular assist device-associated acquired von Willebrand disease

BACKGROUND: Acquired von Willebrand disease (aVWD) can lead to a propensity to bleed, and many different modalities have been used to treat this condition. The efficacy of these agents in patients with aVWD secondary to cardiac assist devices is not fully understood. STUDY DESIGN AND METHODS: A case is reported of a patient with two risk factors for aVWD, multiple myeloma and ventricular assist device (VAD), who was successfully treated with von Willebrand factor (VWF)/Factor VIII concentrate (Humate‐P, CSL Behring) during the bridge to VAD replacement. RESULTS: Although continued absence of high‐molecular‐weight VWF persisted after VWF replacement with Humate‐P, the patient's VWF antigen and activity increased and clinical hemostasis was achieved. The VAD clotted on a few occasions, despite a continuous heparin infusion; however, these events were resolved with temporarily holding the Humate‐P. A VAD exchange was performed and the patient was successfully bridged to heart transplant. CONCLUSION: The treatment of VAD‐associated aVWD may be augmented with Humate‐P, and successful treatment can allow a bridge to heart transplantation. However, careful monitoring for thrombosis in the VAD circuit must be undertaken.