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.

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. STUDY DESIGN AND METHODS: Twenty fresh plasma units (230 +/- 30 mL) were treated by the Mirasol process (CaridianBCT Biotechnologies) and frozen within 8 hours of donation. Plasma units were combined with 35 mL of a 500 micromol/L riboflavin solution in an illumination bag to achieve a final concentration of approximately 60 micromol/L riboflavin. The bag was placed in the Mirasol illuminator and exposed to UV light (6.24 J/mL). Samples were frozen before and after treatment. RESULTS: Recoveries observed were 67.7 +/- 3.9% Factor (F)XI, 68.5 +/- 3.3% FVIII:C, 78.8 +/- 4.5% fibrinogen, 78.9 +/- 4.1% FV, 79.0 +/- 4.2% FVII, 79.0 +/- 8.6% F IX, 79.7 +/- 2.6% FX, and 85.0 +/- 3.7% FII. Von Willebrand factor (VWF) antigen, VWF:ristocetin cofactor, and ADAMTS13 recoveries were 87.0 +/- 7.1, 85.5 +/- 6.6, and 73.3 +/- 15.2%, respectively, while that of protein C was 83.6 +/- 2.6%. A loss of high-molecular-weight VWF multimers was observed in most units. Recoveries for protein S, antithrombin, and plasmin inhibitor were greater than 90%. The mean FVIII:C concentration, after treatment, was 0.76 +/- 0.17 IU/mL. CONCLUSIONS: As with other pathogen reduction technologies, the Mirasol process resulted in some loss of coagulation factor activity. For most Mirasol-treated units and for most of the tested factors this is unlikely to have clinical impact, but trials are required to demonstrate this.

Freezing of buffy coat-derived, leukoreduced platelet concentrates in 6 percent dimethyl sulfoxide.

BACKGROUND: There has recently been renewed interest in freezing platelets (PLTs) in dimethyl sulfoxide (DMSO) for the treatment of major traumatic injuries, especially in military situations. This study examined PLTs that were frozen in small volumes of 6 percent DMSO at -80 degrees C. STUDY DESIGN AND METHODS: Buffy coat-derived pooled leukoreduced PLT concentrates were frozen in 6 percent DMSO and stored at -80 degrees C. Assays included hypotonic shock response (HSR); aggregation; glycoprotein (GP)Ibalpha and P-selectin binding sites; annexin V binding to phosphatidylserine, glycocalicin, and lactate dehydrogenase (LDH). Cone and plate technology (DiaMed Impact-R, DiaMed) was used to test PLT function under near physiologic conditions. RESULTS: The freeze-thaw loss of PLTs was 23 percent. HSR was 17 +/- 7 percent. Cytometry demonstrated two populations of PLTs: one with normal levels of GPIbalpha binding sites (27 x 10(3) +/- 3 x 10(3)/PLT) and one with reduced levels (5.5 x 10(3) +/- 1.2 x 10(3)/PLT). There were 1.4 x 10(3) +/- 0.2 x 10(3) P-selectin binding sites per PLT. Annexin V binding to phosphatidylserine was 50 +/- 9 percent and LDH was 496 +/- 207 IU per 10(12) PLTs. Surface coverage and aggregate size, as measured by the DiaMed Impact-R, were similar to those observed with PLTs stored for 2 days at 22 degrees C. CONCLUSION: Some degree of activation was demonstrated by the proportion of PLTs with reduced levels of GPIbalpha binding sites, increased P-selectin expression, and increased Annexin V binding. LDH concentrations indicated a degree of lysis. The DiaMed Impact-R results showed that the PLTs were still capable of adhering to surfaces and forming aggregates under shear force.

Purification of normal cellular prion protein from human platelets and the formation of a high molecular weight prion protein complex following platelet activation.

A method for the extraction and purification of PrP(C), in its native monomeric form, from outdated human platelet concentrates is described. Both calcium ionophore platelet activation and lysis in Triton X-100 were evaluated as methods for the extraction of soluble platelet PrP(C) in its monomeric form. Following platelet activation, the majority of released PrP(C) was detected as a disulphide linked high molecular weight complex, which under reducing conditions could be separated into what appear to be stable non-disulphide linked PrP dimers or PrP covalently linked to another as yet unidentified protein. This phenomenon appears to be unique to activation since only monomeric PrP(C) was detected following lysis of resting platelets. Subsequently, PrP(C) was purified from the Triton X-100 lysate by sequential cation ion exchange and Cu2+ affinity chromatography. From 10 L of outdated platelet concentrate, we were able to recover 1.29 mg PrP(C) at a purity of 92%.

The effect of methylene blue photoinactivation and methylene blue removal on the quality of fresh-frozen plasma.

BACKGROUND: T: he effects of using fresh or frozen-thawed plasma, WBC reduction of plasma before freezing, and the use of two different methylene blue (MB) removal filters on the quality of MB-treated plasma were compared. STUDY DESIGN AND METHODS: In a paired study (n = 11/arm) plasma was frozen within 8 hours of collection, thawed, MB photoinactivated, and then filtered using one of two MB removal filters. Fresh plasma (n = 16) and plasma WBC reduced before freezing (n = 19) were MB inactivated. RESULTS: Freeze-thawing resulted in loss of activity of FXII and VWF of 0.06 and 0.04 units per mL, respectively, but no significant loss of activity of factors II through XI or fibrinogen. Further loss of activity occurred after MB treatment: FII (0.07 IU/mL), FV (0.11 U/mL), FVII (0.08 IU/mL), FVIII (0.28 IU/mL), F IX (0.12 IU/mL), FX (0.16 IU/mL), FXI (0.28 U/mL), FXII (0.15 U/mL), VWF antigen (0.05 IU/mL), VWF activity (0.06 U/mL), and fibrinogen (0.79 g/L). Losses due to this step were significantly (5-10%) lower in fresh plasma compared to frozen-thawed plasma. Neither MB removal filter resulted in significant loss of activity of any factor studied. CONCLUSION: MB removal, by either of the available filters, has little impact on the coagulation factor content of plasma, but freezing of plasma before MB treatment results in a small additional loss.