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.     

Quinine- and Quinidine-dependent Antiplatelet Antibodies: REQUIREMENT OF FACTOR VIII-RELATED ANTIGEN FOR PLATELET DAMAGE AND FOR IN VITRO TRANSFORMATION OF LYMPHOCYTES FROM PATIENTS WITH DRUG-INDUCED THROMBOCYTOPENIA

The requirement of Factor VIII-related antigen (VIIIR:Ag) for platelet damage by quinine-and quinidine-dependent antibodies was studied in platelet-rich plasma (PRP) of four patients with severe von Willebrand's disease (vWd) (Factor VIII deficiency). Platelet factor 3 availability, platelet aggregation, and release of [¹⁴C]serotonin from labeled vWd-PRP by drug-dependent antibodies were significantly reduced in comparison with PRP from normal controls. Addition of purified VIIIR:Ag restored levels of platelet damage to that of normal PRP. In vWd-PRP, platelet damage by two human antiplatelet sera, not dependent on drugs, and by a rabbit antiplatelet serum did not differ from that in normal PRP. PRP from patients deficient in Factor VIII coagulant activity, Factor IX, or Factors II, VII, IX, and X behaved like normal PRP.     

Standardization of assays of factor VIII and factor IX

Summary The development of international standards over the last 15–20 years has led to improved interlaboratory agreement on assays of factor VIII and factor IX. In the most recent international collaborative study, the coefficient of variation for one-stage assays (26 laboratories) was 5.6%. However, in quality assurance surveys, carried out in the UK and USA, agreement between laboratories is much less good, with coefficients of variation ranging from 30% to over 50%. Improvements in agreement between clinical laboratories could be obtained by increasing the amount of testing on each sample, especially the number of dilutions, and reducing the number of reagent systems used. A large number of laboratories now use immunodepleted plasmas instead of congenitally deficient plasmas as substrates for one-stage assays. These plasmas may give satisfactory assays, but many of them have not been thoroughly evaluated in comparison with congenitally deficient plasma. In assessment of potency of very high purity (VHP) factor VIII concentrates, some immunodepleted plasmas were found to give lower potencies than hemophilic plasma. This is partly due to the fact that VHP concentrates contain little or no von Willebrand factor (vWF), and most immunodepleted plasmas are also deficient in vWF. In recent collaborative studies, assays of VHP factor VIII concentrate were much more variable, both within and between laboratories, than assays of intermediate purity concentrates. Standardization of these new products will require careful attention to methodological detail. Presented at the ‘2nd International Symposium on Standardization and Quality Control of Coagulation Tests: Implications for the Clinical Laboratory’, Rome, September 28–29, 1989.     

Factor VIII preparations of varying purity-examination of physico-chemical parameters (author's transl)

Commerical preparations of cryoprecipitates, factor VIII concentrates of intermediate purity, and factor VIII concentrates of high purity were studied for their factor VIII related clotting- and platelet-aggregating activities as well as their content of factor VIII antigen. The ratios of the values obtained of these three parameters were used to determine the extent of inactivation of the factor in the preparations. By means of two-dimensional immunelectrophoresis and gel chromatography on Sepharose 4B, the preparations were tested for the presence of atypical factor VIII molecules. Investigations were made to qualify and quantify some of the concomitant proteins in the factor VIII preparations.     

Dissociation of the Factor-VIII complex during clotting: role of thrombin and phospholipids

Abstract. To study the dissociation of the two moieties of the factor-VIII complex during clotting, plasma, concentrate and serum were chromatographed on 4% agarose. In plasma and concentrate factor-VIII coagulant activity (VIII C), factor-VIII coagulant antigen (VIII C:Ag), and factor-VIII-related antigen (VIII R:Ag) eluted together in the void volume, but some VIII C: Ag eluted after the void volume. The amount of VIII C:Ag eluting after the void volume was made smaller by adding proteinase inhibitors. In serum nearly all VIII C:Ag eluted after the void volume.
From factor-VIII complex immunoadsorbed by means of an antibody against VIII R:Ag no VIII C:Ag was dissociated by thrombin or by thrombin and physiologic CaCl₂ concentrations. Radiolabelled human thrombin did not bind to the VIII C:Ag of immunoadsorbed factor-VIII complex. VIII C:Ag displaying VIII C was dissociated from immunoadsorbed factor-VIII complex by human brain thromboplastin or by phosphatidyl-serine.
Our results suggest that VIII C:Ag and VIII R:Ag dissociate during clotting. This dissociation seems not to be mediated by thrombin, but may be mediated by phospholipids.     

Plasma haemostatic factors and diabetic retinopathy

Plasma coagulation factors were measured in twelve male insulin-dependent diabetics with no retinopathy, ten with background and ten with proliferative retinopathy and ten non-diabetics. Factor VIII pro-coagulant activities (VIII:C), ristocetin cofactor activities and factor VIII-related antigen concentrations (VIIIR:ag) were significantly related to the severity of diabetic retinopathy (P less than 0.025, trend test). The mean ratio of VIII:C/VIIIR:ag was lower in the diabetics with proliferative retinopathy than in the other groups of diabetics (P less than 0.05) or the controls (P less than 0.02). Concentrations of alpha 2 macroglobulin and alpha 1 antitrypsin were highest in diabetics with proliferative retinopathy (0.1 greater than P greater than 0.05, trend test) but mean prothrombin and activated partial thromboplastin times and mean concentrations of alpha 2 antiplasmin, plasminogen activator and antithrombin III were similar in all groups. Concentrations of the platelet-specific protein beta thromboglobulin, though higher in diabetics than controls (P less than 0.005), were not related to retinopathy. The plasma concentrations of coagulation factors did not correlate with creatinine clearance and there were no significant differences between groups in concentrations C-reactive protein; this suggests that the raised concentrations of coagulation factors in diabetics with retinopathy were not a result of associated nephropathy or an 'acute phase protein' response to diabetic tissue damage. Increased coagulation activity in diabetics may contribute to the development of retinopathy.     

Freezing of plasma and recovery of factor VIII

The freezing procedure and its influence on the plasma quality was studied with a new plasma container and freezing bath and compared with the results from those obtained with commonly used equipment. Cryoprecipitation was performed on plasma pools frozen at three different rates, and quality and recovery of the cryoprecipitates were determined by analysis of factor VIII, factor VIII-related antigen (vWF:Ag), and fibrinogen. The plasma freezing time was 95 and 65 percent shorter with a new -40 degrees C freezing bath as compared to -25 and -80 degrees C freezing boxes, respectively. A further 30 percent reduction of the plasma freezing time was gained by the introduction of a new flat 750-ml plasma container. Rapid plasma freezing prevented substantial loss of factor VIII in frozen plasma. Cryoprecipitate purity measured as factor VIII-to-fibrinogen ratio increased from 0.50 to 0.82 (IU/mg) when the freezing time was decreased from 10 hours to 45 minutes, although the recovery of factor VIII increased less. In summary, freezing of plasma in small flat containers in an effective ethanol bath resulted in rapid freezing with high recovery of factor VIII in plasma, and increased purity and recovery of subsequently processed cryoprecipitate. This freezing concept, adapted at Swedish blood banks, has contributed to higher source plasma quality and increased self-sufficiency.     

Familial incidence of precipitating antibodies in von Willebrand's disease: a study of four cases.

Precipitating antibodies directed toward human F.VIII/WF were found in the plasma of four out of 17 multitransfused patients with severe, homozygous-like VWD. The familial incidence was illustrated by the development of these antibodies in three patients from the same kindred. Such antibodies, titrated with newly developed quantitative assays of anti-VIIIR:Ag and anti-VIIIR:RCo, were directed toward only these components of F.VIII/WF. VIII:C was neutralized in a time-independent manner in plasma and was not inactivated when separated from F.VIII/WF by solid-phase PE absorption. Plasma, serum, or immunoglobulin reacted in precipitation systems (immunodiffusion, EID, CIE) with human VIIIR:Ag, with some degree of cross-reactivity toward VIIIR:Ag from other mammalian plasmas. When used in IRMA, these antibodies demonstrated the same abnormalities as heterologous antisera in variant VWD: decreased binding affinity or nonparallelism of the dose-response curves. They are polyclonal IgG with both kappa and lambda light chains. It is suggested that in some patients with severe homozygous-like VWD, the synthesis of the component of F.VIII/WF carrying VIIIR:Ag and VIIIR:RCo is suppressed whereas VIII:C production is not completely abolished.     

Development, optimization and use of an enzyme linked immunosorbent assay (ELISA) to measure factor VIII antigen utilizing monoclonal antibodies

An enzyme linked immunosorbent assay (ELISA) has been developed to measure VIII:Ag in plasma and concentrates. The assay utilizes two commercially available monoclonal antibodies to VIII:Ag and provides an alternative to the established immunoradiometric assay (IRMA). It has the advantage of not requiring the use of radioactive material and human antibodies. The assay sensitivity is 0.006 u/ml and the interassay coefficient of variation is 6.3%. Forty-eight samples with VIII:Ag levels ranging from 0.006 to 1.5 u/ml were assayed by both ELISA and IRMA. The coefficient of correlation between the two assays was 0.89. In addition to measuring human VIII:Ag, it is also possible to detect antigen in several animal plasma and sera.     

Heterogeneity of human factor VIII. I. Characterization of factor VIII present in the supernatant of cryoprecipitate

Recent observations suggest that plasma F VIII consists of a series of molecules with different molecular weights. The data described in this paper suggest that sup F VIII represents the molecules with relatively low molecular weights whereas the molecules with the highest molecular weights appear in cryo F VIII. Sup F VIII was associated with VIII:C and VIIIR:Ag, but ristocetin cofactor activity was lacking. Although the immunoprecipitation characteristics of sup F VIII with rabbit antifactor VIII were different from those of cryo F VIII, immunological identity was observed in immunodiffusion and crossed immunoelectrophoresis. In 0.8M NaCl sup F VIII dissociated into VIIIR:Ag of relatively high molecular weight and VIII:C of low molecular weight. No indications were obtained that the presence of sup F VIII was the result of proteolytic degradation of factor VIII. VIII:C of sup F VIII was more labile in vitro than VIII:C in plasma. It could be activated by traces of thrombin in a way similar to plasma F VIII. In patients with classic von Willebrand's disease relatively more VIII:C remained in the supernatant after cryoprecipitation of plasma.     

The Effect of Several Variables on Cryoprecipitated Factor VIII (AHG) Concentrates

Because of current interest in the new CPD anticoagulant and in acidified platelet-rich plasma for the production of platelet concentrates, the effect of these changes on cryoprecipitation of Factor VIII was investigated. It was found that use of CPD anticoagulant results in Factor VIII concentrates at least as good as those prepared from corresponding ACD plasma. It was also found that cryoprecipitation of Factor VIII is strikingly affected by pH, with the yield being very low at pH 6.0 and rising steadily to a plateau at about pH 6.8, with no further change up to pH 8.0. Acidified plasma can be restored to normal for production of cryoprecipitates by neutralization of the added acid with NaOH. A survey of five possible diluents for the cryoprecipitates showed that normal saline and retained supernatant plasma both function as well as citrated saline; good stability at room temperature was demonstrated for the AHG in all diluents examined.     

Orthotopic liver transplantation in a patient with severe hemophilia A: a life-saving treatment for the first Italian case

Summary Clinical cure of hemophilia A by orthotopic liver transplantation has been reported in 11 cases. We describe the first successful Italian case. A 27-year-old man had cirrhosis caused by previous infections with the hepatitis B, C and D viruses following life-long treatment with factor VIII concentrates made from large plasma pools. He was, however, seronegative for the human immunodeficiency virus. In the year before transplantation, life-threatening gastrointestinal bleeding due to severe esophageal varices required a large transfusion regimen (on average, 13 bags of red cell concentrates and 35,000 U of factor VIII/week). To perform orthotopic liver transplantation 8,000 U of factor VIII were given during surgery together with 10 bags of red cells and 11 of fresh-frozen plasma. Intraoperative bleeding was not different from that of non-hemophilic patients undergoing orthotopic liver transplantation. No additional factor VIII was used after transplantation and factor VIII levels in plasma were always above 50 U/dl, reaching the highest value of 184 U/dl on day 4 post transplantation. He was discharged from hospital 10 weeks after trans-plantation with factor VIII levels of 68 U/dl. All virological markers are currently negative, except anti-hepatitis C virus antibodies. In this patient orthotopic liver trans-plantation was a life-saving treatment for end-stage cirrhosis and a cure for hemophilia A.     

von Willebrand factor: evidence for variable clearance in vivo according to Y/C1584 phenotype and ABO blood group.

BACKGROUND: The plasma von Willebrand factor (VWF) level (VWF:Ag) is known to correlate with the VWF Y/C1584 variation and with ABO blood group. The ratio of the VWF propeptide (VWFpp) to VWF:Ag and the ratio of coagulation factor VIII (FVIII:C) to VWF:Ag have previously been used as indicators of VWF clearance and/or secretion. OBJECTIVES AND METHODS: To investigate the mechanism underlying the relationship between VWF phenotype and VWF:Ag, the VWFpp/VWF:Ag ratio and FVIII:C/VWF:Ag ratio were determined for plasmas of phenotype Y/C1584, Y/Y1584, blood group O and blood group A (n = 50 for each set). The blood group O plasmas comprised two sets of 25 with low and high mean VWF levels (Low-O and High-O), respectively; similarly for group A (Low-A and High-A). RESULTS AND CONCLUSIONS: The VWFpp/VWF:Ag ratio was greater than 1 (unity) for Y/C1584 plasmas and significantly higher than for Y/Y1584 plasmas; however, the FVIII:C/VWF:Ag ratio was near unity for both and was not significantly different. These results are consistent with increased clearance for Y/C1584 VWF. Similarly, the VWFpp/VWF:Ag ratio and FVIII:C/VWF:Ag ratio in combination were consistent with increased VWF clearance in blood group O compared with blood group A, and in Low-O and Low-A, respectively, compared with High-O and High-A. The data indicate that in vivo C1584 and blood group O are associated with increased VWF clearance, and that clearance contributes to differing VWF level within a given blood group.     

Plasma factor VIII, variables affecting stability under standard blood bank conditions and correlation with recovery in concentrates

Some of the variables affecting factor VIII during storage of plasma at 4 C prior to preparing factor VIII concentrates and cryoprecipitates were investigated. No significant difference in factor VIII levels could be demonstrated between whole blood, platelet-rich plasma or plasma depleted of platelets when stored at 4 C. However, in frozen plasma platelets were shown to have a deleterious effect on factor VIII. No significant difference in factor VIII levels was found in whole blood or plasma stored at 4 C for 6 hours as compared with blood or plasma stored for 18 hours (p = greater than 0.1). There was, however, a highly significant difference (p = less than 0.001) between plasma and whole blood stored at 4 C for 4 hours as compared with storage for 18 hours. There was a significant correlation (correlation coefficient 0.714) between factor VIII levels in the starting material and the factor VIII recovered in cryoprecipitates, but in a small pilot study no correlation between factor VIII levels in the starting material and that recovered in freeze-dried concentrates, could be found. There was no significant difference in factor VIII levels between the Group O and Group A donations used in this study (p = greater than 0.1), and no correlation was found between the drop in pH during storage and the factor VIII decay pattern.     

Hepatitis C treatment with triple therapy in a patient with hemophilia A

We report a case of successful treatment of chronic hepatitis C infection with telaprevir-based triple therapy in a patient with hemophilia A complicated by factor VIII inhibitor. A twenty-two years old male with hereditary hemophilia A and high-titer factor VIII inhibitor was taking maintenance doses of recombinant factor VIII. He visited our clinic for treatment of his chronic hepatitis C with the newly instituted protease inhibitor based therapy. He was diagnosed with hepatitis C genotype 1a at one year of age. He was initiated on telaprevir, ribavirin and peg-interferon for treatment of hepatitis C and qualified for response-guided therapy. He completed treatment at 24 wk with minimal adverse effects. Notably, after 4 wk of hepatitis C treatment, his factor VIII inhibitor screen was negative and the dose for recombinant factor VIII decreased by half of the initial dosing before he was treated for hepatitis C. We suspect that suppressing hepatitis C may help decrease factor VIII inhibitor level and the need for recombinant factor VIII.     

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.     

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.     

Prevalence,biological phenotype and genotype in moderate/mild hemophilia A with discrepancy between one‐stage and chromogenic factor VIII activity

Summary. Background: In most laboratories, the severity of hemophilia A is assessed by the factor VIII activity (FVIII:C) one‐stage assay. However, comparisons of these results with those of two‐stage assays can reveal discrepancies and suggest misdiagnosis. Patients/Methods: In this monocentric study, we measured FVIII:C with two methods (one‐stage chronometric and chromogenic assays) in 307 (173 families) patients with moderate/mild hemophilia A. To compare results, we used a chronometric/chromogenic ratio. Discrepancy was defined as a ratio < 0.5 or > 1.5. We studied their putative involvement at known FVIII functional sites, their interspecies conservation status, and their spatial position within the FVIII structure. Results: Thirty‐six patients from 17 families exhibited a discrepancy between the two assays: 12 (6.9%) families had a low ratio (< 0.5), and five (2.9%) families had a high ratio (> 1.5). Qualitative deficiency was diagnosed in about 16% of the families. Molecular studies were performed in 15 of these 17 families, resulting in each case in the identification of missense mutations, including three novel mutations. We were further able to propose a pathophysiologic explanation. Conclusions: In this monocentric study, we have demonstrated a discrepancy between FVIII:C assay results in 10% of families with moderate/mild hemophilia A. The prevalence of ‘inverse’ discrepancy (i.e. low chronometric/chromogenic ratio) is high as compared with previous reports. We suggest that both FVIII:C assays are recommended in patients with moderate/mild hemophilia A for a complete biological phenotype. This could also improve our knowledge of the FVIII structure–function relationships.     

The Effect of Cryoprecipitate Volume on Factor VIII Content

Two hundred and thirty-five cryoprecipitates were sampled and assayed for Factor VIII activity. Two hundred and twenty-five of these were given to 11 patients with hemophilia on 53 occasions. The patients' Factor VIII levels were measured just before and 15 minutes after infusion. The rise in Factor VIII activity averaged 97 per cent of the expected rise calculated from the cryoprecipitate assays and plasma volume estimates. The mean Factor VIII content per bag of cryoprecipitate was 61 units. There is a highly significant probability that the smallest volume cryoprecipitates had significantly less Factor VIII than average and that the largest volume cryoprecipitates had significantly more Factor VIII than the average.     

Factor VIII/von Willebrand factor complex in methylene blue-treated fresh plasma

BACKGROUND: The effect of virus inactivation of fresh plasma with methylene blue has been studied on the factor VIII/von Willebrand factor molecular complex (FVIII/vWF), factor XIII, and fibrinogen. STUDY DESIGN AND METHODS: FVIII function or activity, vWF activity, vWF antigen, vWF:Ag, vWF multimeric structure, fibrinogen, and factor XIII were analyzed in paired samples of control fresh plasma (untreated) and the same fresh plasma treated with methylene blue. Treated plasma was filtered (0.8-1.2 microm), mixed with a methylene blue solution (300 microg/L), and illuminated at 50,000 lux for 30 minutes on both sides. RESULTS: Average loss of biologic activity of coagulation factors studied was 25 percent (FVIII function, 29%; fibrinogen, 39%; factor XIII, 16%; and vWF activity, 18%). Reduction in vWF activity was significantly lower than that in FVIII function (p<0.05), and the vWF multimeric structure did not show alterations. CONCLUSION: Methylene blue-treated plasma and the cryoprecipitates obtained from it may be effective for replacement therapy in cases of von Willebrand disease and deficiencies of factor XIII and fibrinogen, but the clinical studies are needed to verify that possibility.