C. Prowse

Application of a Time–Resolved Fluoroimmunoassay for the Analysis of Normal Prion Protein in Human Blood and Its Components

Background and Objectives: To quantify the cellular isoform of prion protein (PrPc) in human blood using a new time‐resolved dissociation‐enhanced fluoroimmunoas‐say (DELFIA®). Materials and Methods: The DELFIA was optimised for human blood samples and applied to isolated cell and plasma fractions from blood donations. The physicochemical properties of PrPc were analysed. Results: 26.5% of blood PrPc was associated with the platelet fraction, 0.8% with polymorphonuclear leucocytes, 2.4% with mononuclear leucocytes, 1.8% with red cells and 68.5% with plasma (mean values from 4 processed donations). Conclusion: The majority of blood PrPc is found in the platelet and plasma compartments.

Human parvovirus B19 and blood products

Background and objectives: Human B19 parvovirus (B19), identified in 1975, was only recognised as the causative agent of fifth disease in 1983. The incidence of viraemia is low, around 1 in 1,000, but is sufficient to ensure that most plasma pools for fractionation contain some virus. While infection usually occurs in childhood and is benign, chronic infection sometimes occurs and may be of concern in certain patient groups. Materials and methods: This review is based on a meeting held in March 1995, and addresses recent concerns regarding the potential transmission of B19 infection by pooled plasma products. Results: Recent data on the pathophysiology and assay of this virus are summarised along with possible approaches to donor screening, product screening, and virus removal. Only five cases of symptomatic infection have been reported in persons with haemophilia, but no technology for virus removal is established, and infection may be of concern in pregnant women, and in patients with enhanced red cell turnover or who are immunosuppressed, including those infected with human immunodeficiency virus, but only rarely in immunocompetent patients. Conclusions: For the future, well‐validated assays relevant to virus infectivity are required if blood donations, plasma pools, or plasma products are to be screened, and an in‐process virus inactivation step for B19 would be highly desirable. In the interim, non‐plasma or recombinant products or a selective transfusion policy might be used in patient groups in which B19 infection is of particular concern. Further clinical data on the prognosis and impact of B19 infection are needed to justify both such policies and the future adoption of new technologies designed to reduce any excess B19 infectivity arising from transfused products.

The in vivo release of human platelet factor 4 by heparin

Intravenous and subcutaneous injection of heparin or the heparin analogue SSHA into normal volunteers induced release of platelet factor 4 (PF4) but not beta-thromboglobulin (beta-TG). At low heparin doses the amount of PF4 released was related to the plasma heparin concentration achieved. The rise in plasma PF4 was coincident with, and appeared to be a response to, the increase in plasma heparin concentration rather than to the absolute heparin level. After the primary response, the system became refractory to further challenge by the same heparin dose; the full initial magnitude of the response was not regained until 144 h. after heparin was first injected. The maximum amount of PF4 released corresponded to only about 5% of that potentially available from platelets. Moreover, heparin did not stimulate PF4 release from whole blood in vitro. We have demonstrated the presence of PF4 on the vascular endothelium, and suggest that this is the immediate source of the PF4 released by heparin, though it is probably initially derived from platelets. The effect of such binding on the antithrombotic potential of the endothelial surface is discussed.

In vitro amplification and detection of variant Creutzfeldt–Jakob disease PrPSc

Variant Creutzfeldt–Jakob disease (vCJD) poses a serious risk of secondary transmission and the need to detect infectivity in asymptomatic individuals is therefore of major importance. Following infection, it is assumed that minute amounts of disease‐associated prion protein (PrPSc) replicate by conversion of the host cellular prion protein (PrPC). Therefore, methods of rapidly reproducing this conversion process in vitro would be valuable tools in the development of such tests. We show that one such technique, protein misfolding cyclic amplification (PMCA), can amplify vCJD PrPSc from human brain tissue, and that the degree of amplification is dependent upon the substrate PRNP codon 129 polymorphism. Both human platelets and transgenic mouse brain are shown to be suitable alternative substrate sources, and amplified PrPSc can be detected using a conformation‐dependent immunoassay (CDI), allowing the detection of putative proteinase K sensitive forms of PrPSc. Copyright

Storage of platelets in additive solutions: a multicentre study of the in vitro effects of potassium and magnesium

Background and Objectives  In a preliminary study, the presence of potassium and magnesium in a modified synthetic medium (PAS‐III) was found to have a significant influence on platelet metabolism (using apheresis‐derived, as well as buffy‐coat‐derived platelets) when compared with standard PAS‐III. The differences included reduced glycolysis, as evidenced by lower consumption of glucose and lower production of lactate, but also better preservation of pH and hypotonic shock response reactivity. The results suggested that storage in modified PAS‐III containing 20% plasma was comparable to storage in standard PAS‐III containing 30% plasma. To confirm the preliminary results and to evaluate the effects of different preparation protocols, an international multicentre study, which included 11 different sites, was conducted.

All Clinically-Relevant Blood Components Transmit Prion Disease following a Single Blood Transfusion: A Sheep Model of vCJD

Variant CJD (vCJD) is an incurable, infectious human disease, likely arising from the consumption of BSE-contaminated meat products. Whilst the epidemic appears to be waning, there is much concern that vCJD infection may be perpetuated in humans by the transfusion of contaminated blood products. Since 2004, several cases of transfusion-associated vCJD transmission have been reported and linked to blood collected from pre-clinically affected donors. Using an animal model in which the disease manifested resembles that of humans affected with vCJD, we examined which blood components used in human medicine are likely to pose the greatest risk of transmitting vCJD via transfusion. We collected two full units of blood from BSE-infected donor animals during the pre-clinical phase of infection. Using methods employed by transfusion services we prepared red cell concentrates, plasma and platelets units (including leucoreduced equivalents). Following transfusion, we showed that all components contain sufficient levels of infectivity to cause disease following only a single transfusion and also that leucoreduction did not prevent disease transmission. These data suggest that all blood components are vectors for prion disease transmission, and highlight the importance of multiple control measures to minimise the risk of human to human transmission of vCJD by blood transfusion.

Studies on the Procurement of Blood Coagulation Factor VIII in vitro Studies on Blood Components Prepared in Half-Strength Citrate Anticoagulant

Abstract. The effect of replacing a standard citrate anticoagulant with one containing half the amount of citrate on the in vitro properties of components prepared from blood donations was investigated. This resulted in a significant improvement in factor VIII stability such that there was little loss during overnight storage, and this was reflected in the factor VIII yield in cryoprecipitate. The quality of cellular components in red cell units stored up to 35 days or platelet concentrates stored up to 7 days was not adversely affected. Although initial levels were similar to those in standard anticoagulant, the extent of fibrinopeptide A generation and complement C3 breakdown in red cell units stored for 35 days in half‐strength citrate was somewhat increased.

Properties of Pathogen-Inactivated Plasma Components

There are now 4 licensed technologies available for the pathogen inactivation of fresh frozen plasma in Europe. None of these are currently available in the United States, whereas in other geographic regions some are licensed others are not. This review addresses the different technologies available for the pathogen inactivation of plasma, their impact on the potency of the product, their efficacy in addressing microbiological contamination, as well as summarizing potential immunologic and toxicologic concerns. Published evidence of clinical efficacy is also reviewed as are various approaches to determining cost-effectiveness.

Prevention of the platelet alpha-granule release reaction by membrane-active drugs

A range of membrane-active drugs were tested for their ability to prevent beta-thromboglobulin and platelet factor 4 release from freshly collected blood platelets. While all the drugs tested could inhibit collagen-induced platelet aggregation, only a few, notably procaine and the anti-malarial drugs chloroquine, hydroxychloroquine, camoquine and quinacrine (mepacrine), effectively prevented the alpha-granule release reaction.

A potentially improved approach to methylene blue virus inactivation of plasma: the Maco Pharma Maco-Tronic system

. Plasma was subjected to methylene blue (MB) photochemical virus inactivation using the Maco Pharma Maco‐Tronic system which allows three units to be illuminated together, thus reducing processing time. The plasma bag system used incorporates an integral membrane plasma filter and a dry MB pill which dissolves in the plasma to give a 1‐µm concentration. There is computer‐controlled processing and datalogging.