Virus inactivation by addition of neutralizing antibodies.

This study has shown that the principle of virus neutralization by specific antibodies is feasible at least in the case of hepatitis B. Virus neutralization is our preferred method since it entails no loss of functional activity and no risk of induction of neo-antigens in the plasma derivatives. Although double-blind clinical trials have not been performed, this study--in combination with some other studies - brought the conclusive evidence that virus neutralization in the case of hepatitis B transmission is efficacious. The suggested occurrence of side effects related to the formation of immune complexes during or after administration is not borne out by more than 7 years of experience with neutralized plasma derivatives with no reported side effects. Whether this method is useful for other viruses will depend on the availability of specific neutralizing antibodies.

Immunogenicity and safety of heat-inactivated hepatitis B vaccine (CLB) in low risk human volunteers and in patients treated with chronic haemodialysis in the Netherlands.

The immunogenicity and safety of a heat-inactivated hepatitis B vaccine (HB-vaccine) were studied in healthy human volunteers (n = 471) at a low risk to be infected with hepatitis B virus (HBV) and in patients on chronic haemodialysis (n = 227), who were treated in hepatitis B free centres. After vaccination with 3 injections of 3 micrograms heat-inactivated HBsAg adsorbed to A1PO4, given at monthly intervals, 93% of the healthy low risk volunteers had formed anti-HBs. The haemodialysis patients were randomly divided into 2 groups, which were vaccinated with 3 monthly injections of 3 micrograms and 27 micrograms heat-inactivated HBsAg, respectively. It appeared, that this 9-fold increase of the vaccine-dosage enhanced the anti-HBs response from 77% in the 3 micrograms vaccinees to 94% in the 27 micrograms recipients. Side-effects of the vaccination were negligible, formation of autoantibodies in consequence of the vaccine treatment was not demonstrated in any of the vaccinees.

Contributions to the optimal use of human blood. IX. Elimination of hepatitis B transmission by (potentially) infectious plasma derivatives.

Investigations were performed concerning the elimination of the risk of hepatitis B transmission of potentially infectious plasma derivatives by the addition of a low dose of hepatitis B immunoglobulin (HBIg). To this end, clotting factor VIII concentrate, prothrombin complex, C1 esterase inhibitor concentrate, plasminogen and antithrombin III were prepared from plasma strongly positive for hepatitis B surface antigen (HBsAg). To one half of every preparation, HBIg was added up to a final concentration of 0.4 IU anti-HBs/ml (test preparations), the other half was not treated (control preparations). Furthermore, to 10(-3) diluted infectious reference plasma (Bureau of Biologics, FDA, USA), an overdose HBIg was added to a final concentration of about 0.4 IU anti-HBs/ml. 6 chimpanzees, injected either with the control plasma derivatives or with the untreated infectious reference plasma, were infected with hepatitis B virus, whereas 5 chimpanzees, injected either with the test plasma derivatives or the infectious reference plasma to which the HBIg had been added, did not show any evidence of hepatitis B infection during the follow-up of 1 year. Addition of a low dose of HBIg to potentially infectious plasma derivatives appears to be a reliable measure to eliminate the hepatitis B transmission and is preferred to other methods for labile plasma derivatives.

Prothrombin complex concentrates for clinical use.

Prothrombin complex concentrates prepared for clinical use are reviewed with emphasis on frequently observed adverse reactions such as viral hepatitis and thromboembolic complications. Preparation procedures and quality control are described briefly, and the use of these concentrates in the treatment of hemophilia A patients who develop factor VIII inhibitors is also described. The causes of and methods for the detection of potential thrombogenicity are discussed and suggestions which may result in a safer product are made.

Prevention of chronic HBsAg carrier state in infants of HBsAg-positive mothers by hepatitis B immunoglobulin.

21 children of HBsAg-carrier mothers were given 0.5 ml/kg hepatitis B immunoglobulin (HBIg) within 48 h after birth and subsequently 0.16 ml/kg every month for 6 months; 20 children were not treated. None of the HBIg-treated children became HBsAg positive, compared with 5 of the untreated children (p less than 0.02). 2 of 3 children who were not started on HBIg until the fourth or fifth day after birth also became HBsAg positive. 4 children of mothers who had acute hepatitis B in the third trimester of pregnancy were treated with HBIg and remained HBsAg negative, whereas another, untreated, child became HBsAg positive.

Report of a working group for the European Pharmacopoeia. Collaborative assay on human albumin of different origin.

A collaborative assay was conducted by 9 laboratories on 31 samples of human albumin which were in clinical use. It was the object of the study to establish test systems which would differentiate between albumins of venous or placental origin. The properties examined for this purpose were: appearance, total protein, haem, polymers, alkaline phosphatase and blood group substances. Additional tests such as for beta-thromboglobulin and citrate were included; pyrogenicity, however, was excluded because this was under study for all plasma proteins at that time. Results obtained were in satisfactory agreement both between laboratories and between samples. They, therefore, enabled the verification of a number of correlations in the test systems. The evaluation did not allow, however, the differentiation of the samples in relation to their origin. The results were, therefore, regarded as a tool to define the upper limits of acceptance for human albumins corresponding to the quality prescribed by the European Pharmacopoeia.

Antigenic properties of Bioplast.

The influence of heat treatment (at 150 +/- 2 degrees C for 2 hours) on the antigenicity of human Bioplast fibrin powder and human fibrin Bioplast plates was tested in an experimental study. Untreated and heat-treated fibrin powder with incomplete Freund's adjuvant was subcutaneously injected in rabbits. In addition, untreated and heat-treated plates, to which incomplete Freund's adjuvant was added, were subcutaneously implanted. A booster dose was given 8 weeks after the start of the experiment. Blood samples were collected up to 4 weeks after the second injection or implantation, and the sera were tested for precipitating antibodies by the double diffusion technique (OUCHTERLONY'S method). Unlike previous investigators, we found antibodies in sera from rabbits injected or implanted with heat-treated Bioplast products. Thus, the claim that the antigenic properties of all kinds of fibrin can be fully eliminated by heat treatment does not appear to be tenable. In order to avoid the risk of sensitization in man, it is probably advisable to use only human fibrin for the production of Bioplast fibrin powder and Fibrin Bioplast plates for clinical use in human beings.

Contribution to the optimal use of human blood. VII. Increase of the yield of factor VIII in four-donor cryoprecipitate by an improved processing of blood and plasma.

The influence of different variables on the yield of factor VIII in cryoprecipitate as prepared in the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, was studied. The following conclusions may be drawn: (1) In case blood should be stored, the use of the anticoagulant solution acid citrate dextrose (ACD) is preferable to citrate phosphate dextrose (CPD) or trisodium citrate (TSC). (2) The temperature of stored whole blood should not decrease below 8 degrees C because of a spontaneous precipitation of factor VIII from blood (and plasma) below this temperature. (3) Cryoprecipitate derived from rapidly frozen plasma (1 min) contains a decreased amount of proteins in comparison with cryoprecipitate prepared from slowly frozen plasma (45 min to 4 h). On the other hand, equal amounts of factor VIII activity were obtained in the precipitate after freezing of plasma at varying rates. (4) Rapid thawing of plasma results in both higher yields of factor VIII procoagulant activity and a higher specific activity of this factor in the resulting cryoprecipitate. (5) The sedimentation of cryoprecipitate is completed after 5 min centrifugation at 1,500 g. (6) At temperatures higher than 8 degrees C, cryoprecipitated factor VIII starts dissolving into the supernatant plasma or in buffer. (7) Factor VIII in lyophilized cryoprecipitate is stable at room temperature. At elevated temperatures it rapidly looses its activity. (8) Evidence was obtained that the improvements which are introduced in the preparation of factor VIII do not lead to a product which is less stable in vitro as well as in vivo.

Contributions to the optimal use of human blood. VIII. Stability of blood coagulation factor VII during collection and storage of whole blood and plasma.

Investigations were performed concerning the influence of the pH on the stability of factor VIII during the collection of blood and during the storage of blood and plasma for varying periods and at varying temperatures. It was found that the low pH of ACD anticoagulant solution (pH 4.9) caused a loss of factor VIII procoagulant activity of 10-15% during the collection of blood. However, when less acidic anticoagulant solutions were used, substantial losses of factor VIII occurred during the storage of blood. We concluded that the optimal pH of both the anticoagulant solution and the stored blood, should be between 6.7 and 7.0. However, no anticoagulant solution is known that meets these requirements. In practice ACD ensures the highest recovery of factor VIII in cryoprecipitate, at least in those cases where the blood donations are stored for several hours before separation and freezing of the plasma.

The use of hepatitis B immunoglobulin in the Netherlands.

Hepatitis B immunoglobulin (HBIg) was administered to 241 patients contaminated with HBAg positive material; 116 persons were followed up for 7 months after the HBIg injection. Only 4 (3.4%) cases of hepatitis B with jaundice and demonstrable HBAg occurred and 15 (12.9%) cases of subclinical hepatitis B were observed. HBIg is well tolerated. Passively transferred HBAb were demonstrable for 2-3 months and no chronic carriers of HBAg were seen after administration of HBIg.

Contributions to the optimal use of human blood. VI. Modification of the method to prepare prothrombin complex on a large scale.

The relatively low yield of factor II activity in prothrombin complex preparations gave rise to an investigation in which factor II was determined on the basis of its coagulation activity and on that of its antigenic properties during the routine preparation procedure of the complex. It appeared that factor II, measured as activity, suffered a greater loss than factor II, measured as antigen, during this procedure. This greater loss had to be attributed to changes in the conformation of the factor II protein molecule induced by the freezing step of the preparation method hitherto in use. Omission of this step led to a higher yield of factor II activity in the prothrombin complex preparation. Therefore, freezing and thawing during the preparation of the prothrombin complex should be avoided.

A new solid phase radioimmunoassay (CLB-RIA) for the detection of hepatitis-B antigen and antibody.

A new competitive solid phase radioimmunoassay (CLB-RIA) has been developed for the detection of HBAg and HBAb in human serum and plasma. In the assay, sheep antibodies to HBAg, covalently linked to an insoluble carrier, highly purified 125-I labelled HBAg and the serum or plasma sample are incubated for 20 h at room temperature. After incubation, the bound and the free fraction of the tracer are separated by centrifugation. Both the presence of HBAg and HBAb, result in a decrease of the amount of bound tracer, when compared with a negative control serum. Differentiation between HBAg and HBAb requires the use of another type of radioimmunoassay. For this purpose a sandwich solid phase radioimmunoassay, for the detection of HBAb only, has been developed (CLB-AURIA). In this, assay-purified HBAg is covalently linked to an insoluble carrier. Using a mixture of both immunosorbents (insolubilized HBAg and HBAb), it is possible to detect and to distinguish HBAg and HBAb in one single solid phase radioimmunoassay (CLB-MIRIA). The influence of three parameters on the CLB-RIA, the incubation time, the amount of tracer and the effect of Tween-20 has been studied. The sensitivity of the described solid phase CLB-RIA for the detection of HBAg is comparable to that of other radioimmunoassays reported in literature; its specificity is very high.