Duncan S. Pepper

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.

Gas sparging to enhance permeate flux in ultrafiltration using hollow fibre membranes

Abstract This study focuses on the use of gas-liquid two-phase crossflow to overcome concentration polarisation in the ultrafiltration of macromolecular solutions as applied to hollow fibre membrane systems. The experimental work was conducted on a purpose built pilot-plant scale rig with albumin and dextran as the test media. The effect of gas injection on the permeate flux and membrane sieving coefficient was examined experimentally at different transmembrane pressures, feed concentrations and gas to liquid flow ratios. The results were encouraging, with flux enhancements of 20–50% obtained for dextrans and 10–60% for albumin, when air was injected into the system over the range of process variables examined. The sieving coefficient of albumin was considerably reduced when gas-liquid two-phase cross-flow was used. These results were compared to those obtained with tubular membrane systems, and an additional mechanism, based on physical displacement of the concentration polarisation boundary layer is proposed. The operational difficulty related to protein foaming is also discussed.

Three Approaches to the Radioimmunoassay of Human β-Thromboglobulin

Summary. Three radioimmunoassays for the measurement of β‐thromboglobulin are described. The standard method, using antiserum in solution, could be used to measure plasma concentrations of β‐thromboglobulin with the results available after 2–3 d. The use of a greater dilution of antiserum and tracer, with delayed addition of tracer, resulted in a more sensitive assay suitable for measuring β‐thromboglobulin in urine. The use of a solid‐coupled antiserum under non‐equilibrium conditions allowed the measurement of plasma levels of β‐thromboglobulin after an assay incubation time of 1 h. These three radioimmunoassay systems for β‐thromboglobulin cover the likely clinical requirements for the measurement of this platelet specific protein.

Catabolism of low-dose heparin in man.

Abstract An iodinated heparin derivative was injected with carrier heparin into healthy volunteers. The iodinated material was of similar molecular weight to the underivatised heparin, highly sulphated, and biologically active. It bound to plasma proteins including antithrombin III in vivo , and more than 85% was firmly bound to platelet factor 4 or protamine in vitro . After injection of 1–100 units, 80–90% was removed from the plasma within 40 minutes, most of it being sequestered by the liver and some by the spleen, lungs, and kidneys. Iodinated material was then again released into the plasma. The molecular weight of this product was unchanged, but it was no longer biologically active. It was not bound to antithrombin III in vivo , or to platelet factor 4 and protamine in vitro , and it was markedly desulphated. Except at doses greater than 1,000 units, the labelled material was degraded to small fragments before excretion in the urine.

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.

A radioimmunoassay for platelet factor 4

Abstract A radioimmunoassay for the measurement of human platelet factor 4 is described which is sufficiently sensitive to measure the concentration of this protein in plasma. This assay can detect the release of platelet factor 4 from platelets in vitro . Evidence is presented which suggests this assay may be potentially useful as a clinical tool for the detection of platelet aggregation in vivo as raised plasma concentrations have been found in some patients with prosthetic heart valves.

A radioimmunoassay for thrombospondin, used in a comparative study of thrombospondin, β-thromboglobulin and platelet factor 4 in healthy volunteers

A radioimmunoassay was developed for the platelet alpha-granule protein thrombospondin; concentrations of thrombospondin as low as 3 ng ml-1 could be measured. There was no interference from other components of human biological fluids and no crossreactivity with beta-thromboglobulin (beta-TG) or platelet factor 4 (PF4). Plasma samples were stable when stored at -20 degrees C. Normal human plasma contained 105.0 +/- 31.0 ng thrombospondin ml-1 compared with beta-TG concentrations of 37.2 +/- 10.9 ng ml-1 and PF4 concentrations of 14.7 +/- 10.1 ng ml-1 when samples were carefully taken into a platelet inhibitor cocktail and processed at 0-4 degrees C. Release of thrombospondin during clotting of blood occurred at the same time as that of beta-TG and PF4 and resulted in a serum concentration of 17.5 +/- 5.5 micrograms ml-1. Assay of whole blood gave a platelet thrombospondin content of 89.1 +/- 28.3 ng/10(6) platelets. The concentration in normal urine fluctuated widely from 3 to 22.5 ng ml-1, and was unrelated to urine flow. The half-life of thrombospondin in vivo was about 9 h, much longer than that of either beta-TG or PF4. Unlike PF4, it was not released into the blood following an intravenous heparin injection. Bovine, ovine, canine and porcine sera contained thrombospondin which crossreacted immunologically with the human molecule; these species would be suitable animal models for the study of thrombospondin and its value as a platelet release marker.

Effect of bubble size and frequency on the permeate flux of gas sparged ultrafiltration with tubular membranes

Abstract Gas sparged ultrafiltration experiments are performed using a tubular membrane module with solutions of dextran and human serum albumin (HSA) as the test media. Air is injected, in a controlled manner with the ability to adjust bubble size and frequency independently, into the membrane module to create a gas–liquid two-phase crossflow operation. The effects of bubble size and frequency on the permeate flux of the sparged ultrafiltration are studied experimentally. It is found that the permeate flux increases with the bubbling frequency in the examined range. The effect of bubble size on flux can be divided into two regions, an increasing region for smaller bubbles and a plateau region for larger slugs. The results are discussed on the basis of bubble wake hydrodynamics.

Investigations on Antithrombin III in Normal Plasma and Serum

Summary. Studies on antithrombin III (AT‐III) were made by a modification of the two dimensional crossed immunoelectrophoresis technique and gel filtration. Mixing various quantities of heparin with agarose in the first phase of electro‐phoresis, AT‐III from normal human plasma and serum revealed a heterogeneity which depended on the heparin concentration in the agarose gel. At heparin concentrations higher than 16 u/ml, AT‐III displayed three components with different electrophoretic mobilities. The component with the highest mobility (designated immunoantithrombin III1: IAT‐III1) dominated in plasma. In normal serum, however, the quantity of this component was decreased and the two other peaks with a slower electrophoretic mobility (IAT‐III2 and IAT‐III3) became more evident.

Haemoglobin-based blood substitutes and sepsis

An important concern that has received little attention is the possible increased susceptibility to bacterial infections of patients infused with cell-free haemoglobin-based blood substitutes. We show that pyridoxalated polymerised human haemoglobin promotes fulminating Escherichia coli septicaemia in mice, which draws attention to the potential danger of such products in the clinic.