FVIII Preparations Research Articles

Analysis of Perioperative Factor Replacement Therapy for Hemophilia

Background Hemophilia is a hereditary bleeding disorder, especially in severe hemophilia, that can lead to spontaneous bleeding. For hemophilia patients with inhibitors, even minor acute bleeding events or surgical procedures can have catastrophic consequences. Some hemophiliacs are forced to make surgical choices. There are many risks in perioperative period when hemophiliacs undergo major surgery. Seeking effective and safe perioperative clotting factor replacement therapy or bypass therapy is the basis of successful surgery. Aims To seek effective and safe perioperative coagulation factor replacement therapy or bypass therapy. Methods HA patients were treated with FVIII preparation during perioperative period. To calculate dosage, multiply the patient ' s weight in kilograms by the FVIII level in IU/dL desired, then multiply by 0.5. Example: 50 kg × 40 (IU/dL level desired) × 0.5 = 1000 IU of FVIII. HB patients were treated with PCC or FIX during perioperative period. To calculate dosage, multiply the patient ' s weight in kilograms by the FIX level in IU/dL desired. Example: 50 kg body weight × 40 (IU/dL level desired) = 2000 IU of plasma-derived FIX. For hemophilia patients with low titer inhibitors, estimate the amount of FVIII required as a neutralizing inhibitor loading dose as recommended by WFH. The following formula is used to estimate the amount of FVIII needed as a loading dose to neutralize the inhibitor: [body weight (kg) × 80 × [(1 − hematocrit) × antibody titer (BU)]. An additional 50 IU/kg above the calculated loading dose is added to achieve a measurable FVIII activity. Patients with hemophilia combined with high titer inhibitors were treated with rFVIIa combined with PCC SCBT regimen. Sequential use refers to alternating rFVIIa and aPCC every 3 hours. Results 69 patients with indications for surgery and/or desire for surgery who underwent different types of surgery with clotting factor replacement therapy or bypass therapy were evaluated (Table 1). There were 37 cases of severe hemophilia and 32 cases of non-severe hemophilia. There were 63 cases of HA, including 50 cases of negative inhibitor and 13 cases of positive inhibitor. After meeting the inclusion and exclusion criteria, 69 patients were evaluated for different types of surgery under clotting factor replacement therapy or bypass therapy during perioperative period. 59 cases underwent major operations, including: 49 cases underwent joint replacement, among which 34 cases underwent single joint replacement and 6 cases underwent double joint replacement and so on. 10 cases underwent minor operations. There were 14 cases of hemophilia with inhibitors, of which 13 were major operations with 175.38 (85.00-270.00) minutes of operation and 2 were minor operations with 45.00 minute of operation. In 12 patients with severe hemophilia and high titer inhibitors, the perioperative consumption of factor FVII was as follows: 447.62 (331.04-738.48)ug/kg on the day of surgery, and the total consumption was 2484.21 (1499.40-4500.00)ug/kg. The perioperative consumption of factor PCC was as follows: 126.10 (0-206.45)ug/kg on the day of surgery, and the total consumption was 1332.84 (529.20-1970.00)ug/kg. The mean preoperative FVIII:C level of the HA patients was 1.19 (0.00~6.00)%, the mean preoperative FIX:C level was 0.022 (0.01~0.07)% of the HB patients.The average intraoperative blood loss of the 69 patients was 537.98 (0-3000) ml. 20 patients received intraoperative transfusion of blood products (red blood cells or fresh frozen plasma) and 49 patients did not receive intraoperative transfusion of blood products. In 55 hemophiliacs without inhibitor, no inhibitor was found after surgery. Conclusion(s) It is safe and effective to adopt different schemes for perioperative alternative therapy for different hemophilia types.

New opportunities in Haemophilia treatment: Efmoroctocog Alfa for patients with Haemophilia A


 Recently new opportunities are emerging for improving the way patients with Haemophilia A are treated. Among these opportunities, efmoroctocog alfa is a first-in-class recombinant factor VIII-Fc fusion protein (rFVIIIFc) produced by recombinant DNA technology with an extended half-life compared with conventional FVIII preparations. The available evidence coming from an Italian HTA report indicates that efmoroctocog alfa provides an effective alternative to conventional FVIII preparations (including standard rFVIIIs) for the management of Haemophilia A. Moreover, by reducing the frequency of injections required, it has the potential to reduce treatment burden, and hence improve adherence to prophylaxis and patient Quality-of-Life. 

Diagnosis and Treatment of Acquired Hemophilia: One Single-Center Experience from PR. China

Objective: To deepen the understanding of the clinical manifestations of acquired hemophilia A for timely and correctly treatment.Methods: The clinical data of the acquired hemophilia A patients diagnosed in the hospital from Jan 2006 to Mar 2021 were retrospectively analyzed, and the relevant literature was reviewed.Results: 17 patients with acquired hemophilia A, male: female =10: 7, median age 61 years (19 to 78 years), were diagnosed and treated in the hospital with the median time from the onset to diagnosis 21 days (2 days to 6 months). Six patients had comorbidity, including hepatitis B carrying, chronic myelomonocytic leukemia, diabetes, hypertension and positive autoantibodies, pemphigoid and gastric cancer, respectively. Other 11 patients were healthy before the onset. All patients had large large ecchymosis of skin, and one case was combined with hematuria, and one case with retroperitoneal hematoma. All patients had APTT extension (45s-144.7s) and the prolonged APTT could not be corrected with normal mixed plasma with and without incubation at 37℃ for 2 hours. FVIII activity was 1% - 8.9% and inhibitor titer 2 - 128 Bu/ml. All patients with bleeding were with prothrombin complex/recombinant activated coagulation factor VII, some of them with pd-coagulation factor FVIII preparations. Inhibitors were removed with prednisone acetate (1 case) + chemotherapy (1 case), prednisone acetate / + CTX (11 cases) + chemotherapy (1 case), prednisone acetate/prednisolone + mabthera (2 cases) + CTX (1 case), respectively. The removal time of inhibitor was from 8 days to 4 years. During the treatment process, two patients developed lower extremity venous thrombosis, and one patient was complicated with lung infection.Conclusion: Patients with unexplained bleeding and prolonged APTT should be conducted normal mixed plasma correction test, coagulation factor activity and inhibitor titer examination. After correctly diagnosis, bypass agents /coagulation factor VIII preparations should be given timely for hemostasis, protocol based on glucocorticoid + CTX/mabthera to remove the inhibitor and symptomatic treatment for patients with primary comorbidity disease at the same time. DisclosuresNo relevant conflicts of interest to declare.

Gene Expression after Hemarthrosis Differs between FVIII-Deficient Mice Treated with Recombinant FVIII or FVIII-Fc Fusion Protein

IntroductionLocal and systemic molecular responses to hemarthroses in hemophilia are not well understood. Emerging clinical evidence suggests that treatment with FVIII-Fc Fusion protein (FcFVIII), using the Fc-portion of immunoglobulin for half-life extension, may mitigate joint inflammation and modulate immune stimulation. We analyzed gene expression profiles in synovium and spleen (a major immune regulatory organ) in FVIII-deficient mice at baseline and after hemarthrosis treated with recombinant human FVIII (rhFVIII) or murine (m)FcFVIII to characterize the respective differences and molecular pathways for each FVIII preparation.MethodsHemarthrosis was induced by sub-patellar needle puncture in FVIII-deficient mice treated with saline (vehicle), 100 IU/kg mFcFVIII (Fc murine due to species specificity) or 120 IU/kg rhFVIII (to account for differences in t 1/2) administered intravenously 2 hours before and 6 hours after injury. Spleen and synovium were harvested on day 3 or day 14 post-injury (n=3-5 per group). Spleen and synovium from uninjured mice served as controls. RNA libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit and sequenced on an Illumina NextSeq500 platform (single-end; 75bp reads). The limma-voom method (R Bioconductor) was used for differential expression analyses. The criteria for differential expression were: i) a log fold-change (logFC) >1 or <-1, and ii) an adjusted p value <0.05. Functional enrichment was performed using Signaling Pathway Impact Analysis (SPIA). Molecular pathways were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathway databases.ResultsKnee injury in FVIII-deficient mice resulted in hemarthrosis and a drop in mean hematocrit from 44.7% to 27.5% (saline), which was prevented by rhFVIII and mFcFVIII prophylaxis (mean day 2 hematocrit ~45%).In the spleen, differential gene expression (DGE) in saline treated mice was pronounced but fleeting with 5295 and 9 differentially expressed genes (DEG) on day 3 and 14. DGE was abrogated by either rhFVIII or mFcFVIII, without significant expression differences between rhFVIII and mFcFVIII at the single gene level. However, differences were noted between rhFVIII and mFcFVIII during KEGG pathway analyses, where rhFVIII predominantly perturbed pathways related to inflammation, T-cell signaling and innate immune regulation, while mFcFVIII perturbed pathways related to B-cell signaling and cellular homeostasis.In the synovium, DGE in saline treated mice was also pronounced on day 3 (3388 DEG) but, in contrast to the spleen, continued on day 14 (1030 DEG). Unlike in the spleen, DGE was not fully corrected with rhFVIII or mFcFVIII; moreover, gene expression differed between rhFVIII and mFcFVIII. This was particularly evident on day 14, with a partial reduction to 603 and 294 differentially expressed genes with rhFVIII and mFcFVIII, respectively. Only 163 genes overlapped, whereas 440 were unique to rhFVIII and 131 to mFcFVIII. A comparison between gene expression with rhFVIII and mFcFVIII yielded that ≥ 2 log-fold expression difference was present for 74 genes, with 72 genes upregulated with rhFVIII relative to mFcFVIII. Many of these genes are involved in pathways related to innate and adaptive immune responses, cell cycle regulation and extracellular matrix organization in the Reactome database. This response was dampened by mFcFVIII.ConclusionsJoint bleeding in hemophilic mice induced pronounced systemic and synovial gene expression, which was diminished by rhFVIII or mFcFVIII. However, while there were many similarities with regard to regulation of gene expression and/or molecular pathways, there were also notable differences, especially involving immune regulatory, inflammatory and tissue repair functions. These observations provide new molecular insights regarding Fc-driven effects of FVIII after hemarthrosis, suggesting that the type of FVIII preparation used for bleed control may have implications beyond hemostasis. Disclosuresvon Drygalski: Hematherix, Inc: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Super FVa; uniQure: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CSL Behring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Biomarin: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Polysialic Acid-Mediated Activity Measurement of Polysialylated Recombinant Coagulation Factor VIII

Measurement of modified biologic including coagulation factors with extended half-life obtained, for example, by polysialylation pose an analytical challenge especially if both biological activity and presence of modification have to be determined. Analytical methods applied so far address only 1 of the 2 quality attributes of modified biologics. Here, we describe the development and bioanalytical validation of a polysialic acid-mediated factor VIII activity assay: Polysialic acid-specific capture of polysialylated recombinant factor VIII is combined with a chromogenic FVIII activity test using commercially available reagents. This assay principle enabled measurement of FVIII activity down to the pico mole-range without any interference by nonmodified factor VIII. To the best of our knowledge, this is the first method to selectively, accurately, and precisely measure simultaneously activity and modification integrity of a polysialylated biologic in complex matrices, as shown by the bioanalytical validation data. The convenience, robustness, and reliability of using this method has been demonstrated by its application for the nonclinical development of the polysialylated recombinant FVIII preparation. The method principle could be applied to protein modifications other than polysialylation and to activity tests other than the chromogenic FVIII assay.

Analysis of individualized primary prophylactic treatment of 19 cases of children with severe hemophilia A

Objective: To study the current situation of primary prophylaxis in severe hemophilia A children and to explore rational regimen in order to provide evidence for the development of primary prophylaxis in China. Method: A retrospective clinical data collection and analysis was conducted for 19 severe hemophilia A children who received primary prophylaxis in Beijing Children's Hospital outpatient clinic between February 2011 and September 2015 and evaluated the regimen and efficacy. Result: (1) Primary prophylaxis regimen: the median beginning age 1.8 (range 0.5-2.9) years, the median FⅧ preparation using dosage 16.7 (8.0-23.5) U/(kg·time), the median using frequency was 1.0 (1.0-3.0) time/week. Eight cases among the patients received escalation of treatment intensity because of the poor bleeding control. (2) Efficacy: the median annual bleeding rate (ABR) was 1.9 (0-6.0) times/year, the median annual joint bleeding rate (AJBR) was 0 (0-3.3) times/year, without life threatening bleeding. All of them kept in 4th scale of Beijing Children Hospital daily activity level. The median annual factor consumption was 1 844 (840-5 040) U/kg. Conclusion: Low-dose primary prophylaxis regimen which were in low-dose /low frequencies and adjusted by bleeding frequency could decrease bleeding and joint bleeding frequency significantly, maintained the normal daily activity capacity and saved the factor consumption compared to standard regimen in severe hemophilia A children.

Native analysis of plasma-derived clotting factor VIII concentrates: “Sponge effect” and contaminants

In the present study, we analysed two commercially available plasma-derived FVIII preparations, Beriate and Emoclot, through native gel-based approaches (CN-PAGE). The rationale behind this study was to assess whether protein complexes from plasma resisted the aggressive manufacturing processes. A preliminary analysis of plasma complexes was performed focusing on the molecular weight range between 45 kDa and 1 MDa. As a result, we could evidence in both preparations the presence of complexes, which resisted the cryoprecipitation, chromatographic and heat treatment processing steps. These complexes behaved in a "sponge-effect"-like fashion through the enrichment of otherwise less abundant contaminants. The use of an alternative electrophoretic approach such as CN-PAGE allowed us to confirm 2DE-based observations and expand the list of non-FVIII proteins which contaminate the preparations.

Factor VIII Lacking the C2 Domain Retains Cofactor Activity in Vitro

Factor (F) VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains). The activated form of FVIII, FVIIIa, functions as a cofactor for FIXa in catalyzing the membrane-dependent activation of FX. Whereas the FVIII C2 domain is believed to anchor FVIIIa to the phospholipid surface, recent x-ray crystal structures of FVIII suggest that the C1 domain may also contribute to this function. We constructed a FVIII variant lacking the C2 domain (designated DeltaC2) to characterize the contributions of the C1 domain to function. Binding affinity of the DeltaC2 variant to phospholipid vesicles as measured by energy transfer was reduced approximately 14-fold. However, the activity of DeltaC2 as measured by FXa generation and one-stage clotting assays retained 76 and 36%, respectively, of the WT FVIII value. Modest reductions ( approximately 4-fold) were observed in the functional affinity of DeltaC2 FVIII for FIXa and rates of thrombin activation. On the other hand, deletion of C2 resulted in significant reductions in FVIIIa stability ( approximately 3.6-fold). Thrombin generation assays showed peak thrombin and endogenous thrombin potential were reduced as much as approximately 60-fold. These effects likely result from a combination of the intermolecular functional defects plus reduced protein stability. Together, these results indicate that FVIII domains other than C2, likely C1, make significant contributions to membrane-binding and membrane-dependent function.

Von Willebrand Factor-Specific Antibodies Developing upon Treatment of FVIII-Deficient Mice with Different FVIII Preparations

Von Willebrand Factor-Specific Antibodies Developing upon Treatment of FVIII-Deficient Mice with Different FVIII Preparations

The role of VWF in the immunogenicity of FVIII

Up to 33% of patients with severe haemophilia A develop inhibitory antibodies to factor VIM (FVIII) that can significantly impair treatment with FVIII. The plasma protein von Willebrand factor (VWF) binds to FVIII and is known to be important for the functioning of FVIII. Accumulating data suggest that VWF may also be important for reducing the immunogenicity of therapeutically administered FVIII in patients with haemophilia A. Although contradictory results have been reported for studies in patients, studies in mice have shown that the levels of FVIII-binding antibodies induced following treatment with recombinant FVIII (rFVIII) are higher than those following treatment with plasma-derived FVIII preparations containing VWF, and that the addition of VWF to rFVIII reduces the levels of FVIII-binding antibodies induced. In in vitro studies, VWF has been shown to inhibit both the uptake of FVIII by immature dendritic cells and the activation of FVIII-specificT-cells in a dose-dependent manner. However, recombinant VWF (rVWF) lacking the FVIII-binding domain did not inhibit T-cell activation. These data suggest that VWF may reduce the immunogenicity of FVIII by inhibiting the uptake of FVIII by antigen presenting cells, the first step in the development of an immune response against a foreign antigen. Further studies are required to confirm the applicability of these results to patients with haemophilia. If confirmed, these data would encourage the use of VWF in conjunction with FVIII in the management of patients with haemophilia A.

Clearance mechanisms of von Willebrand factor and factor VIII.

Clearance mechanisms of von Willebrand factor and factor VIII.

B‐domain deleted recombinant factor VIII preparations are bioequivalent to a monoclonal antibody purified plasma‐derived factor VIII concentrate: a randomized, three‐way crossover study

Deletion of the B-domain of recombinant blood coagulation factor VIII (BDDrFVIII) increases the manufacturing yield of the product but does not impair in vitro or in vivo functionality. BDDrFVIII (ReFacto) has been developed with the additional benefit of being formulated without human albumin. The primary objective of this three-way crossover-design study was to compare the pharmacokinetic (PK) parameters of two BDDrFVIII formulations (one reconstituted with 5 mL of sterile water, the other reconstituted with 4 mL sodium chloride 0.9% USP) with those of a plasma-derived, full-length FVIII preparation (Hemofil M) in patients with haemophilia A to determine bioequivalence. A series of blood samples were collected over a period of 48 h after i.v. administration of each of the FVIII preparations. Plasma FVIII activity was determined using a validated chromogenic substrate assay. Plasma FVIII activity vs. time curves was characterized for a standard set of PK parameter estimates. Two parameter estimates, the maximum plasma concentration (Cmax) and the area under plasma concentration vs. time curves (AUCs), were used to evaluate bioequivalence. The two preparations were considered bioequivalent if the 90% confidence intervals for the ratio of geometric means for Cmax and AUCs fell within the bioequivalence window of 80% to 125%. Results show that each BDDrFVIII formulation is bioequivalent to Hemofil M and the two formulations of BDDrFVIII are bioequivalent to each other.

Evaluation of Stability of a Sucrose-Formulated, Full-Length rFVIII for Use in Continuous Infusion Therapy.

Introduction: Stability of a FVIII preparation in mini-pumps is an important consideration for its use in a continuous infusion manner. Our goal was to evaluate the stability of a specific recombinant FVIII (Kogenate®FS; KOGENATE®Bayer) in two commonly used infusion mini-pumps, with and without anticoagulant additives that may be used in clinical practice to prevent local thrombophlebitis.Methods: Kogenate® FS (250 IU/vial and 1000 IU/vial) was reconstituted using aseptic technique according to the manufacturer's instructions with normal water for injection (100 IU/ml and 400 IU/ml, respectively); an additional dilution of 100 IU/ml was made from the 1000 IU/vial size. Reconstituted material was spiked with unfractionated heparin (UFH) or low molecular weight heparin (LMWH; Enoxaparin/Clexane) to a final concentration ~5 U/ml, or left untreated and then transferred to reservoirs of 2 mini-pumps (Walkmed, Medfusion Inc., Duluth, GA and CADD, Deltec, Inc., St. Paul, MN). All bags were stored in a light-protected environment at room temperature (22°C) for 7 days. Samples were drawn at baseline (immediately post reconstitution), 3h, 6h, 12h, 24h, and days 2–7, frozen at −30°C, and assayed for FVIII activity by one-stage assay. On Day 7, the residual volume of the mini-pump containers was cultured for bacteria.Results: All samples except one* had FVIII activity >90% of baseline.Conclusions: Kogenate® FS appears to retain excellent stability at room temperature during 7 days in both CADD and Walkmed infusion pumps with and without addition of UFH or LMWFH. No bacterial growth was observed. These results indicate that Kogenate® FS may be useful in continuous infusion therapy. [Display omitted]

Heating Lyophilised Factor VIII Does Not Alter Its Recognition by Specific Antibodies

Background and objectives: Alterations of factor Vili (FVIII) during preparation procedures can potentially affect its immunogenicity. One method evaluating such alterations could be by determining the reactivity of FVIII with specific antibodies. Materials and methods: Since heat treatment is currently used to reduce the risk of viral transmission, we evaluated the immunoreactivity of plasma-derived FVIII before and after heating at different temperatures and for different periods. Freeze-dried FVIII was used for these experiments as part of the validation procedure of a novel FVIII preparation. Results: Ideating FVIII for up to 72 hat 80 °C does not alter its reactivity with specific rabbit antibodies or mouse monoclonal antibodies, although some loss of FVIII activity occurred after 72 h. After heating for 2 h at 100°C, a procedure that reduced FVIII activity by about 50%, there were still no significant effects on FVIII reactivity with monoclonal antibodies. Conclusions: Freeze-dried preparations of plasma-derived FVIII seem to be resistant to heat-induced structural dénaturation.

Strategy for pre-clinical evaluation of factor VIII concentrates.

A series of methods and assay systems was designed, using both mouse monoclonal antibodies and purified human polyclonal antibodies, by which alterations in the antigenic properties, and potentially therefore in the immunogenic properties, of FVIII concentrates could be identified. Those methods could be applied to the pre-clinical evaluation of FVIII concentrates. It has become evident that very subtle alterations in FVIII can have dramatic effects on its antigenicity and immunogenicity and it is suggested that an analysis of FVIII preparations, using reagents such as mouse monoclonal antibodies, should be carried out as a pre-clinical evaluation. It is also thought that any haemophiliac patient is at risk of developing anti-FVIII antibodies, even though he had been considered as tolerant to the infusion of FVIII. The diversity of FVIII concentrates now available on the market has multiplied the chances of encountering a product that would give an immune response in some patients and this further stresses the need for a careful check of FVIII concentrates.

Pharmacokinetic Properties of Recombinant Factor VIII Compared with a Monoclonally Purified Concentrate (Hemofil® M)

A recombinant FVIII preparation, Recombinate, was compared with a high-purity plasma-derived concentrate, Hemofil M, in 47 hemophilia A patients in a cross-over evaluation of pharmacokinetic properties. The recombinant material showed a significantly lower clearance, volume of distribution, and higher in vivo recovery, but a similar half-life to the plasma-based product. In a comparison with reported data from other standard concentrates, the recombinant preparation exhibited potentially better pharmacokinetic properties in that its clearance was slower and its half-life was longer. We conclude that the recombinant DNA method of preparation does not adversely affect the biological and pharmacological characteristics of the factor VIII molecule.

Two affinity immunoelectrophoretic methods for studying collagen interaction with von Willebrand factor antigen.

Two new immunoelectrophoretic methods are described for studying the interaction of collagen fibrils with von Willebrand factor antigen (vWF: Ag). In the first, the sample was electrophoresed through a collagen-agarose wedge into an antibody-agarose area, and immunoprecipitin lines was detected by staining. Different immunoprecipitin patterns were obtained with the vWF: Ag of normal plasma, commercial FVIII preparations, and von Willebrand disease (vWD) type IIa plasma as the result of collagen binding of vWF: Ag. In the other method, the sample was electrophoresed into agarose for preliminary separation of forms, followed by migration in the second dimension through a collagen spacer gel into an antibody-agarose area. This method demonstrated preferential binding of high molecular weight forms of vWF: Ag in normal plasma and slight binding of the lower molecular weight forms of antigen found in vWD type IIa plasma. The affinity wedge method is a convenient general method for finding quickly a useful concentration of affinity reagent.

Small-pool high-yield factor VIII production.

Hemophilia care depends on several factors for the production of purified FVIII: plasma procurement, plasma logistics, production method, and efficacy. The latter two are restrictive factors, both for the supply and the safety of FVIII preparations. Conventional production methodology unavoidably recovers only 10 to 20% of usable protein, therefore requiring large pools of source plasma. Related to pool size is the transmission of diseases, which poses unnecessary risks for patients. The development of new technologies to better recover FVIII allows reduction of the pool size: crush-thaw, controlled pore-glass chromatography, and heparin double-cold precipitation techniques. This review will reflect on current production methods, pool size concept, small-pool approaches in FVIII production, and future developments.

Inactivation of Hepatitis B and Non-A, Non-B Viruses by Combined Use of Tween 80®, β-Propiolactone, and Ultraviolet Irradiation

To assess the sterilization efficacy of a combined Tween 80, beta-propiolactone and ultraviolet irradiation procedure applied to a F VIII preparation to which an estimated 10(5.9) chimpanzee infectious doses (CID50) of hepatitis B virus had been added per ml, two chimpanzees were inoculated with 10 ml each of treated and untreated preparations. The untreated preparation, which was obtained from donors with normal alanine aminotransferase (ALT) levels, induced non-A, non-B hepatitis in both recipient animals, and delayed hepatitis B infection in one of these. Neither animal receiving the treated preparation developed either type of hepatitis. When subsequently challenged with the untreated material, both of the latter animals developed non-A, non-B and hepatitis B infection, proving their susceptibility to both types of infection. It was concluded that the combined procedure inactivated an estimated 10(6.9) CID50 of hepatitis B virus and an unknown quantity of a non-A, non-B virus. The finding of non-A, non-B virus infectivity in a pooled F VIII preparation despite careful ALT screening of plasma donors emphasizes the necessity of subjecting such preparations to sterilization procedures.

802 VARIANT VON WILLEBRAND'S DISEASE (vWD): ABNORMALITY IN MULTIMERIC ASSEMBLY OF FACTOR VIII RELATED ANTIGEN (VIIIRAg)

Factor VIII in normal plasma is a complex of at least two molecules, VIIIRAg and VIIIC, each having distinct functional and antigenic properties. VIIIRAg normally assembles as a series of multimeric forms ranging from 1 to >20×106 daltons. We show, using a new crossed immuno-electrophoresis procedure, the importance of identifying alterations in the distribution of these multimeric forms. A normal distribution for at least 5 distinct forms of VIIIRAg was established using both pooled and individual plasmas. The slowest migrating form contained the most of the antigen while the four faster forms had progressively decreasing concentrations. Two patients presented as possible vWD variants because of prolonged bleeding times and low ristocetin cofactor activity despite normal to elevated levels of VIIIRAg. Both plasmas showed essentially complete absence of the larger multimers and an absolute increase in the concentration of the anodal forms that corresponded to the faster migrating forms in normal plasma. An additional faster migrating form was found in one of the plasmas. Patterns of cryo-supernatant and commercial FVIII preparations were similar to the variant patterns; cryoprecipitate resembled more closely the normal plasma pattern. Results suggest that some types of vWD can result from an abnormality in the ability of VIIIRAg to assemble into the larger multimeric forms and supports the hypothesis that the largest forms of VIIIRAg are necessary for normal hemostasis through participation in platelet-vessel wall interactions.