In vitro Quality of Platelets with Low Plasma Carryover Treated with Ultraviolet C Light for Pathogen Inactivation.

BACKGROUND: The THERAFLEX UV-Platelets system uses shortwave ultraviolet C light (UVC, 254 nm) to inactivate pathogens in platelet components. Plasma carryover influences pathogen inactivation and platelet quality following treatment. The plasma carryover in the standard platelets produced by our institution are below the intended specification (<30%). METHODS: A pool and split study was carried out comparing untreated and UVC-treated platelets with <30% plasma carryover (n = 10 pairs). This data was compared to components that met specifications (>30% plasma). The platelets were tested over storage for in vitro quality. RESULTS: Platelet metabolism was accelerated following UVC treatment, as demonstrated by increased glucose consumption and lactate production. UVC treatment caused increased externalization of phosphatidylserine on platelets and microparticles, activation of the GPIIb/IIIa receptor (PAC-1 binding), and reduced hypotonic shock response. Platelet function, as measured with thrombelastogram, was not affected by UVC treatment. Components with <30% plasma were similar to those meeting specification with the exception of enhanced glycolytic metabolism. CONCLUSION: This in vitro analysis demonstrates that treatment of platelets with <30% plasma carryover with the THERAFLEX UV-Platelets system affects some aspects of platelet metabolism and activation, although in vitro platelet function was not negatively impacted. This study also provides evidence that the treatment specifications of plasma carryover could be extended to below 30%.

In vitro comparison of cryopreserved and liquid platelets: potential clinical implications.

BACKGROUND: Platelet (PLT) concentrates can be cryopreserved in dimethyl sulfoxide (DMSO) and stored at -80°C for 2 years. These storage conditions improve availability in both rural and military environments. Previous phenotypic and in vitro studies of cryopreserved PLTs are limited by comparison to fresh liquid-stored PLTs, rather than PLTs stored over their clinically relevant shelf life. Further, nothing is known of the effect of reconstituting cryopreserved PLTs in plasma stored at a variety of clinically relevant temperatures. STUDY DESIGN AND METHODS: Apheresis PLTs were either stored at room temperature for 5 days or cryopreserved at -80°C with 5% DMSO. Cryopreserved PLTs were thawed at 37°C and reconstituted in plasma (stored at different temperatures) and compared to fresh and expired liquid-stored PLTs. In vitro assays were performed to assess glycoprotein expression, PLT activity, microparticle content, and function. RESULTS: Compared to liquid PLTs over storage, cryopreserved PLTs had reduced expression of the key glycoprotein receptors GPIbα and GPIIb. However, the proportion of PLTs expressing activation markers CD62P and CD63 was similar between cryopreserved and liquid-stored PLTs at expiry. Cryopreserved PLT components contained significantly higher numbers of phosphatidylserine- and tissue factor-positive microparticles than liquid-stored PLTs, and these microparticles reduced the time to clot formation and increased thrombin generation. CONCLUSION: There are distinct differences between cryopreserved and liquid-stored PLTs. Cryopreserved PLTs also have an enhanced hemostatic activity. Knowledge of these in vitro differences will be essential to understanding the outcomes of a clinical trial comparing cryopreserved PLTs and liquid PLTs stored for various durations.

Increased lipid droplet accumulation associated with a peripheral sensory neuropathy.

Hereditary sensory neuropathy type 1 (HSN-1) is an autosomal dominant neurodegenerative disease caused by missense mutations in the SPTLC1 gene. The SPTLC1 protein is part of the SPT enzyme which is a ubiquitously expressed, critical and thus highly regulated endoplasmic reticulum bound membrane enzyme that maintains sphingolipid concentrations and thus contributes to lipid metabolism, signalling, and membrane structural functions. Lipid droplets are dynamic organelles containing sphingolipids and membrane bound proteins surrounding a core of neutral lipids, and thus mediate the intracellular transport of these specific molecules. Current literature suggests that there are increased numbers of lipid droplets and alterations of lipid metabolism in a variety of other autosomal dominant neurodegenerative diseases, including Alzheimer's and Parkinson's disease. This study establishes for the first time, a significant increase in the presence of lipid droplets in HSN-1 patient-derived lymphoblasts, indicating a potential connection between lipid droplets and the pathomechanism of HSN-1. However, the expression of adipophilin (ADFP), which has been implicated in the regulation of lipid metabolism, was not altered in lipid droplets from the HSN-1 patient-derived lymphoblasts. This appears to be the first report of increased lipid body accumulation in a peripheral neuropathy, suggesting a fundamental molecular linkage between a number of neurodegenerative diseases.

Mutations in the SPTLC1 protein cause mitochondrial structural abnormalities and endoplasmic reticulum stress in lymphoblasts.

Mutations in serine palmitoyltransferase long chain subunit 1 (SPTLC1) cause the typical length-dependent axonal degeneration hereditary sensory neuropathy type 1 (HSN1). Transmission electron microscopy studies on SPTLC1 mutant lymphoblasts derived from patients revealed specific structural abnormalities of mitochondria. Swollen mitochondria with abnormal cristae were clustered around the nucleus, with some mitochondria being wrapped in rough endoplasmic reticulum (ER) membranes. Total mitochondrial counts revealed a significant change in mitochondrial numbers between healthy and diseased lymphocytes but did not reveal any change in length to width ratios nor were there any changes to cellular function. However, there was a notable change in ER homeostasis, as assessed using key ER stress markers, BiP and ERO1-Lα, displaying reduced protein expression. The observations suggest that SPTLC1 mutations cause mitochondrial abnormalities and ER stress in HSN1 cells.

Red blood cell in vitro quality and function is maintained after S-303 pathogen inactivation treatment.

BACKGROUND: Over the past decade there has been a growth in the development of pathogen reduction technologies to protect the blood supply from emerging pathogens. This development has proven to be difficult for red blood cells (RBCs). However the S-303 system has been shown to effectively inactivate a broad spectrum of pathogens, while maintaining RBC quality. STUDY DESIGN AND METHODS: A paired three-arm study was performed to compare the in vitro quality of S-303-treated RBCs with RBCs stored at room temperature (RT) for the duration of the treatment (18-20 hr) and control RBCs stored at 2 to 6°C. Products were sampled weekly over 42 days of storage (n = 10) and tested using an array of in vitro assays to measure quality, metabolism, and functional variables. RESULTS: During S-303 treatment there was a slight loss of RBCs and hemoglobin (Hb < 5 g). Hemolysis, glucose consumption, and potassium release were similar in all groups during the 42 days of storage. S-303-treated RBCs had a significantly lower lactate concentration and pH compared to the paired controls. The S-303-treated RBCs had significantly higher adenosine triphosphate than the RT and control RBCs. There was a significant loss of 2,3-diphosphoglycerate in the S-303-treated products, which was also observed in the RT RBCs. Flow cytometry analysis demonstrated similar RBC size, morphology, expression of CD47, and glycophorin A in all groups. CONCLUSION: RBCs treated with S-303 for pathogen reduction had similar in vitro properties to the paired controls and were within transfusion guidelines.

Measuring hydrogen peroxide reduction using a robust, inexpensive, and sensitive method.

Here, we report an improved method to analyze antioxidant activity using the europium tetracycline assay developed by Duerkop and Wolfbeis (J Fluor 15(5):755-761, 2005). The europium tetracycline hydrogen peroxide reduction assay (EHRA) accurately measures antioxidant activity based on hydrogen peroxide scavenging. Several known antioxidant compounds were assessed with the EHRA and a stoichiometric relationship between the number of oxidant molecules trapped per molecule of antioxidant was identified. Various extracts of hops were also tested to validate this method for use with natural extracts; water extraction yielded the highest level of antioxidant activity. Hop leaves were shown to be a better source of antioxidants relative to the traditional hop cones. The data also indicate that the EHRA may serve to breach the hydrophilic/lipophilic gap in antioxidant screening as the europium tetracycline probe is effective in many solvents. The EHRA thus provides a robust and inexpensive measure of antioxidant activity.