Potency measurement of factor VIII in recombinant concentrates.

In preliminary studies on the stability of recombinant factor VIII (rFVIII) concentrates post reconstitution, a rise in potency to 200% of labelled values was observed in concentrates stored at 22 °C over 24 h. This was observed in potency estimates by the one-stage clotting, but not the two-stage clotting or chromogenic assays, and was not observed for intermediate-purity product derived from plasma (IPVIII). Use of human serum albumin (HSA), rather than the usual bovine material (BSA), to dilute product for the stability study abolished the rise in potency.

Platelet size analysis in the quality assurance of platelet concentrates for transfusion.

The platelet distribution width (PDW) as analysed on standard haematology cell counters is an indicator of size dispersion in the platelet population. Using a Sysmex E-2500 analyser platelet concentrates prepared for transfusion showed an increase in PDW over storage. This increase correlated strongly with in vitro indicators of platelet viability (pH and response to osmotic stress).

Platelet concentrates for transfusion-metabolic and storage aspects.

Transfusion of platelets concentrated from donated blood is an established therapeutic modality in clinical medicine. Over the past 25 years much effort has gone into optimising the conditions for the collection, preparation and storage of platelets for transfusion. Despite significant advances, platelet production is still a costly process requiring a dedicated environment and the use of specially formulated plastic storage containers.

Relative efficiency of leucocyte removal procedures for the production of leucocyte-poor red cell concentrates assessed by flow cytometry.

Flow cytometry was used to: (1) determine residual leucocyte numbers in red cell suspensions following the range of leucocyte depletion procedures used in our organisation, and (2) to characterize phenotypically the leucocytes using direct immunofluorescence with monoclonal antibodies to cell surface receptors. Under the conditions used, a lower limit of detection of 2.5 leucocytes per microliter (equivalent to 3.

Storage of platelet concentrates harvested from blood collected into dextrose-free preservative without agitation.

This study addresses the possibility of platelet quality being maintained during storage by an endogenous metabolic fuel, while avoiding dextrose-induced lactate accumulation. This was achieved by harvesting platelet concentrates from blood donations collected into a dextrose-free anticoagulant. Adequate maintenance of all metabolic and functional parameters was observed in platelets from blood collected into 4% citrate.

Cryopreservation of red blood cells: effect of freezing on red cell quality and residual lymphocyte immunogenicity.

Aims: To investigate treatment with glycerol/washing as a potential substitute for freeze-thawing in the production of leucocyte depleted red cell concentrates for patients with a history of non-haemolytic reactions following transfusion.

Methods: The standard procedure of treatment with glycerol/-80 degrees C freezing/thawing/washing was compared with a similar procedure in which freezing was omitted. The quality of the resulting red cell products was assessed in relation to: (1) standard red cell biochemical parameters; (2) leucocyte and lymphocyte subset composition using flow cytometry with fluorescent labelled monoclonal antibodies; and (3) immunogenicity of the residual lymphocytes in mixed lymphocyte culture.

Stabilization of von Willebrand factor in banked blood by leucocyte depletion.

The von Willebrand factor (vWf) activity, as measured by the ristocetin co-factor (vWf:RCo) and collagen binding (vWf:CBA) assays, declined progressively in standard blood units stored at 4 degrees C after a 2-day storage period. This loss of activity was accompanied by a loss and degradation of high molecular weight (HMW) vWf multimers. In studies using a paired design, filtration of blood with a high efficiency leucocyte-removal filter, prior to storage at 4 degrees C, led to significantly improved maintenance of vWf:RCo and vWf:CBA compared with unfiltered units (P < 0.

Biotechnology and the plasma fractionation industry--the impact of advances in the production of coagulation factor VIII.

Several excellent reviews have described the role of biotechnological procedures in the production of pharmaceuticals derived from human blood plasma (Meulien and Tuddenham 1990, Brodniewicz-Proba 1991). The system of blood collection and fractionation is driven by the need to manufacture concentrates of coagulation Factor VIII. This review will analyse the effect of biotechnology on the manufacture of this product.

Modulation of fibrinogen content in cryoprecipitate by temperature manipulation during plasma processing.

In routine blood bank production of single-donation cryoprecipitate, the introduction of a 16-hour hold at 4 degrees C, with the frozen plasma units packed into polystyrene containers, resulted in plasma prethaw temperatures of -4 degrees C to -8 degrees C. This in turn resulted in cryoprecipitate fibrinogen levels that were 214 percent of those obtained when units were thawed immediately after removal from -30 degrees C storage. In scale-model production of factor VIII concentrate, plasma warmed from -30 to -10 to -15 degrees C over 18 hours before pooling and thawing yielded cryoprecipitate fibrinogen levels that were 66 percent of those found in plasma warmed to -2 to -5 degrees C over the same period.

Some characteristics of blood shed into the Solcotrans postoperative orthopaedic drainage/reinfusion system.

Objective: To assess the suitability of blood shed into the Solcotrans orthopaedic autotransfusion system as a source of autologous blood for transfusion.

Design: Blood samples were taken from patients after surgery and from shed blood within the Solcotrans units.

Setting: Surgery was performed at a public hospital.

von Willebrand factor characterization of a severe dry-heat treated factor VIII concentrate, AHF (high purity).

Eight batches of a severe dry-heat treated (80 degrees C for 72 hours) Factor VIII concentrate manufactured by the Commonwealth Serum Laboratories (CSL Ltd.) were analysed for the following von Willebrand factor-related (vWf) activities: ristocetin cofactor activity (vWf:RCof), collagen binding activity (CBA), vWf antigen levels (vWf:Ag), vWf multimeric analysis and 2-stage FVIII clotting activity (VIII:C). The average potency per vial of vWf:Ag was 440 +/- 80 units, vWf:RCof 500 +/- 60 units, CBA 350 +/- 50 units and VIII:C 242 +/- 36 International Units.

Red cell and platelet concentrates from blood collected into half-strength citrate anticoagulant: improved maintenance of red cell 2,3-diphosphoglycerate in half-citrate red cells.

This study confirms previous work suggesting equivalent in vitro properties in blood components prepared from donations collected into half-citrate preservative (HCPD) compared to components derived from donations collected into standard citrate-phosphate-dextrose (CPD) preservatives. In addition, red cell products harvested from HCPD donations showed significantly improved maintenance of pH over storage, and this was reflected in improved maintenance of intracellular 2,3-diphosphoglycerate (2,3-DPG). This effect was observed in whole blood and in red cells suspended in a phosphate-containing additive solution (Tuta AAS).

Improved in-vitro quality of platelet concentrates stored in a dextrose-free synthetic medium.

The licensed balanced salt solution Plasma-Lyte, buffered with a clinical solution of sodium bicarbonate, was evaluated as a suspending fluid for platelet concentrates. Platelets suspended in this medium showed better pH maintenance over 5 days of storage compared to platelets stored in plasma (7.0 vs 6.

Use of plasma with high levels of ionised calcium in the production of model scale coagulation factor concentrates.

We have attempted to exploit the Ca2(+)-dependent stability of factor VIII in producing factor VIII concentrates of higher yield. Plasma levels of ionised calcium were increased in two ways: (a) whole blood collection into half-strength citrate CPD anticoagulant, leading to free Ca2+ levels of ca 120 microM and (b) apheresis collection of plasma which was then recalcified to free Ca2+ levels of ca 300 microM under heparin cover. Coagulation factor concentrates were prepared using model versions of our industrial scale manufacturing methods.

Microtitre plate measurement of platelet response to hypotonic stress.

The conventional method of assessing the platelet response to hypotonic stress (HSR) was adapted to allow microtitre plate technology to be used. After water is added to a platelet suspension two sequential readings are taken at 414 nM on a vertical microplate reader. The difference between the second (three minutes) and the first (one minute) was defined as the HSR.

Effects of plasma collection systems and processing parameters on the quality of factor IX concentrate.

Using pilot-scale production of our present factor IX (II and X) concentrate, we have studied the effects of starting plasma source and processing parameter on two in-vitro indicators of product quality - yield and thrombogenic potential. Plasma source did not affect factor IX yield but had a marked effect on thrombogenic potential. Factor IX concentrates produced from plasma derived through centrifugation-based technology showed significantly higher thrombogenic potential than products derived from plasma derived through a filtration-based system.

Studies on the procurement of blood coagulation factor VIII in vitro studies on blood components prepared in half-strength citrate anticoagulant.

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.

Studies on the procurement of blood coagulation factor VIII: effects of plasma freezing rate and storage conditions on cryoprecipitate quality.

Plasma was frozen and stored in different ways before processing to cryoprecipitate by a standard thawing technique. Freezing rate was found to be important with slow freezing having a deleterious effect on cryoprecipitate quality. Storage of frozen plasma at constant temperatures for periods up to six months had no effect on the quality of cryoprecipitate, with no difference being found for plasma stored at -20 degrees C or -40 degrees C.

Studies on the procurement of coagulation factor VIII: selective precipitation of factor VIII with hydrophilic polymers.

Early work on the purification of factor VIII using polyethylene glycol (PEG) indicated that other polymers might also be used to precipitate factor VIII leaving fibrinogen in solution. Recently polyvinylpyrrolidone (PVP) has also been advocated for this purpose. In this study, different concentrations and molecular weights of hydroxyethyl starch, dextran, PEG, PVP, Ficoll, Percoll and albumin were examined for their ability to precipitate the factor VIII complex from cooled (not frozen) fresh CPD plasma.

A comparative study using immunological and biological assay of the haemostatic responses to DDAVP infusion venous occlusion and exercise in normal men.

In a group of six normal male volunteers, infusion of DDAVP, venous occlusion and exercise were shown to increase plasma levels of factor VIII and plasminogen activator, activity and antigen, to different extents and at differing rates. Any mechanisms suggested to explain release of these proteins by various stimuli should account for such differences. All three stimuli could also increase plasma levels of prostacyclin metabolites, although this was only significant for high doses of DDAVP.