Ian MacGregor

Plasminogen activator inhibitor (PAI-1) in plasma and platelets

The distribution of PAI‐1 in the plasma and platelets of normal individuals and of patients with platelet abnormalities was studied. An ELISA, capable of measuring PAI‐1 in plasma at 1.5 ng/ml, and a functional assay of t‐PA inhibition were used to assay platelet‐free plasma (PFP), platelet‐rich plasma in which the platelets were lysed (PRP) and serum. The PAI‐1 concentration of normal PFP was 21.0 ± 7.2 ng/ml (mean ± SD) and those of PRP and serum were 282.6 ± 68.0 and 270.3 ± 71.9 ng/ml. The concentration of PAI‐1 in PRP was proportional to the platelet count with 0.67 ± 0.18 ng/106 platelets. Patients with thrombocy‐topenia had approximately normal PAI‐1 concentrations in PFP; the extremely low concentrations in serum or PRP reflected the platelet count. A patient with grey platelet syndrome showed a comparable pattern, confirming that PAI‐1 occurs in the platelet α‐granules and indicating that the plasma concentration of PAI‐1 is independent of the platelet pool of PAI‐1. The median inhibitory activities towards t‐PA were 1.6, 8.7 and 8.3 units/ml in normal PFP. PRP and serum respectively. PAI‐1 in PFP had a median specific activity (units/mg PAI‐1) about 5‐fold higher than platelet PAI‐1. Plasma and platelets represent two distinct pools of PAI‐1, both of which should be considered in studies on the relationship between circulating PAI‐1 and thrombotic disease.

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.

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

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.

Prion protein and developments in its detection.

The theoretical risk of transmission of variant Creutzfeldt–Jakob disease (vCJD) via blood transfusions has led to replacement of UK‐derived plasma for fractionation by plasma sourced outwith the UK and the introduction of leucodepletion of donated blood and its components. Prion protein in an abnormal conformation (PrPsc) has been identified as inextricably linked with the infectivity of transmissible spongiform encephalopathies such as vCJD and in this review some of its properties relevant to its detection are considered. In particular its insolubility in nonionic detergents and its partial resistance to proteinase K digestion have provided methods for separating it from the normal abundant isoform PrPc prior to subsequent detection, usually by means of antibody probes. As yet no antibodies which discriminate between the abnormal and normal isoforms have been identified with the sole exception of one which is not suitable for generally used immunodetection systems. Most detection systems, such as immunohistochemistry or Western blotting, have been optimized for brain or lympho‐reticular tissues. Much less information is available for assays applied to blood or plasma, although some recent publications provide indications of their feasibility. However, a number of obstacles remain to be overcome. These include the issues of assay validation, detection of extremely low levels of PrPsc in the presence of large excesses of PrPc, lack of available standardized reagents, assay specificity and practicality for large‐scale screening use. Progress towards these goals is reviewed.

Studies on the removal of a bovine spongiform encephalopathy-derived agent by processes used in the manufacture of human immunoglobulin

Background and Objectives There is still uncertainty over how the agent of variant Creutzfeld‐Jakob disease (vCJD) would partition during the manufacture of plasma derivatives. In this study, a BSE‐derived agent was used as a vCJD model to determine the extent to which infectivity could be removed by selected steps used in the manufacture of intravenous immunoglobulin (IVIG).

Human platelets as a substrate source for the in vitro amplification of the abnormal prion protein (PrPSc) associated with variant Creutzfeldt-Jakob disease

BACKGROUND: Four recent cases of transfusion‐related transmission of variant Creutzfeldt‐Jakob disease (vCJD) highlight the need to develop a highly sensitive and specific screening test to detect infectivity in the blood of asymptomatic infected individuals. Protein misfolding cyclic amplification (PMCA), a method for the amplification of minute amounts of disease‐associated abnormal prion protein (PrPSc) to readily detectable levels, could be incorporated into such a test provided that a suitable substrate source for routine use in human PMCA reactions can be found.

The effects of leukodepletion on the generation and removal of microvesicles and prion protein in blood components.

BACKGROUND:  Universal leukodepletion (LD) has been implemented in the United Kingdom to reduce the risk of transfusion‐transmitted variant Creutzfeldt‐Jakob disease. If LD causes microvesiculation of blood cells, however, potentially infectious membrane‐associated prion could reach the final products.

Pathogen reduction of fresh plasma using riboflavin and ultraviolet light: effects on plasma coagulation proteins.

BACKGROUND: Treatment with riboflavin and ultraviolet (UV) light reduces the pathogens present in blood components. This study assessed changes to the coagulation proteins that had occurred during this treatment of fresh plasma units before freezing.

Distribution of a bovine spongiform encephalopathy‐derived agent over ion‐exchange chromatography used in the preparation of concentrates of fibrinogen and factor VIII

Background and Objectives  The risk of haemophiliacs contracting variant Creutzfeldt‐Jakob disease (vCJD) via treatment with factor VIII concentrates is not known. Therefore, in order to determine the extent to which the vCJD agent might be removed during the preparation of factor VIII concentrate, the partitioning of a bovine spongiform encephalopathy (BSE)‐derived agent was measured over the main purification step used to prepare the Scottish National Blood Transfusion Service high‐purity factor VIII concentrate (Liberate®).