Raymond P. Goodrich

Riboflavin and UV-Light Based Pathogen Reduction: Extent and Consequence of DNA Damage at the Molecular Level

Abstract We are developing a technology based on the combined application of riboflavin (RB) and light for inactivating pathogens in blood products while retaining the biological functions of the treated cells and proteins. Virus and bacteria reduction measured by tissue culture infectivity or colony formation with UV light alone and in combination with RB yield equivalent results. The effects of RB as a sensitizing agent on DNA in white cells, bacteria and viruses in combination with UV light exposure have been evaluated. UV-mediated DNA degradation in Jurkat T cells and leukocytes in plasma as measured by the FlowTACS assay was significantly increased in the presence of RB. Agarose gel electrophoretic analysis of DNA in Escherichia coli and leukocytes in plasma demonstrated enhanced DNA degradation in the presence of RB. UV light in combination with RB prevented the reactivation of lambda phage compared with samples irradiated in the absence of RB. UV-mediated oxidative damage in calf thymus DNA was also enhanced in the presence of RB. These observations clearly demonstrate that the presence of RB and UV light selectively enhances damage to the guanine bases in DNA. These data also suggest that the type and extent of damage to DNA for virus in the presence of RB and light make it less likely to be repaired by normal repair pathways available in host cells.

A randomized controlled clinical trial evaluating the performance and safety of platelets treated with MIRASOL pathogen reduction technology

BACKGROUND: Pathogen reduction of platelets (PRT‐PLTs) using riboflavin and ultraviolet light treatment has undergone Phase 1 and 2 studies examining efficacy and safety. This randomized controlled clinical trial (RCT) assessed the efficacy and safety of PRT‐PLTs using the 1‐hour corrected count increment (CCI1hour) as the primary outcome.

Correlation of in vitro platelet quality measurements with in vivo platelet viability in human subjects

Background and Objectives  Changes in in vitro platelet quality parameters during platelet storage are associated with a decrease of in vivo platelet viability after platelet transfusion. Many attempts have been made to identify the most predictable in vitro parameters for in vivo performance. We used a riboflavin‐based ultraviolet (UV) light treatment process designed to inactivate pathogens and white blood cell (WBC) contaminants in blood products as a model system in which to study the correlation of in vitro cell quality with in vivo viability.

Effects of a new pathogen-reduction technology (Mirasol PRT) on functional aspects of platelet concentrates.

BACKGROUND: Several strategies are being developed to reduce the risk of pathogen transmission associated with platelet (PLT) transfusion.

Photochemical inactivation of chikungunya virus in plasma and platelets using the Mirasol pathogen reduction technology system

BACKGROUND: Chikungunya virus (CHIKV) is a reemerging mosquito‐borne virus that has been responsible for a number of large‐scale epidemics as well as imported cases covering a wide geographical range. As a blood‐borne virus capable of mounting a high‐titer viremia in infected humans, CHIKV was included on a list of risk agents for transfusion and organ transplant by the AABB Transfusion‐Transmitted Diseases Committee. Therefore, we evaluated the ability of the Mirasol pathogen reduction technology (PRT) system (CaridianBCT Biotechnologies) to inactivate live virus in contaminated plasma and platelet (PLT) samples.

Separation, Identification and Quantification of Riboflavin and its Photoproducts in Blood Products using High-performance Liquid Chromatography with Fluorescence Detection: A Method to Support Pathogen Reduction Technology¶

A medical device using riboflavin (RB) and light is being developed for the reduction of pathogens in platelet concentrates (MIRASOLTM pathogen reduction technology [PRT]). A high‐performance liquid chromatography (HPLC) method for the quantification of RB and its main photoproduct, lumichrome (LC) in blood components has been developed and validated. In addition, the same method has been used to identify and quantify the presence of additional photoproducts‐catabolites of RB. Levels of these agents before and after treatment as well as endogenous levels present in normal donor blood are reported using this analytical technique. The method allows for quantitative and qualitative analysis of RB and LC in blood components using HPLC fluorescence detection, a Zorbax® SB‐CN (stable bond cyano) column and a methanol‐water mobile phase. Quantitation and qualitative analysis of additional photoproducts of RB was also performed, but the method has not been validated for these other components. The method described has passed an 8 day validation and has been found to be adequate for its intended use. The range of the method for RB is 0.016–1.500 μM and for LC is 0.060–1.500 μM. The method detection limit for RB is 0.0006 μM and for LC is 0.012 μM. The acceptance criteria for repeatability were met; the relative standard deviation for RB was 0.64% and for LC was 0.76%. The acceptance criteria for bias were met with a 97% average recovery for RB and a 102% recovery for LC. Samples were centrifuged and diluted 1:50 with 0.9% saline before analysis. No protein precipitation or extraction was required. A mass balance of approximately 93.4–94.4% was achieved after exposure of products to UV light in the intended pathogen reduction treatment method. The method permitted the identification of photoproducts in blood that were both naturally occurring and produced after photolysis of blood samples treated with the PRT process. The identity of these photoproducts has been established using HPLC Tandem Mass Spectrometry (MS/MS) and UV spectroscopic methods and has been correlated with known metabolites and catabolites of RB. HPLC with fluorescence detection using a reverse phase cyano‐column allows for accurate separation, identification and quantification of both RB and LC in blood products without the need for solvent extraction or protein precipitation. Additional photoproducts could also be identified and quantified using this method. The presence of these agents in normal, untreated blood suggests that their presence in blood is ubiquitous.

DRAMATIC IMPROVEMENTS IN VIRAL INACTIVATION WITH BROMINATED PSORALENS, NAPHTHALENES AND ANTHRACENES

Amino or polyamino derivatives of naphthalene (N‐H), anthracene (A‐H) and 8‐alkoxypsoralen (PSR‐H) were prepared along with their monobrominatcd analogs (N‐Br, A‐Br and PSR‐Br). The ammonium salts of these compounds are all water soluble and bind strongly to calf thymus DNA and to λ phage, a double‐helical DNA, protein‐coated virus. Binding of the sensitizer to DNA occurs, presumably by a mixture of hydrophobic, intercalative and electrostatic interactions. Relative binding constants to calf thymus DNA and to λ phage were measured by the cthidium bromide fluorescence quenching assay. In general the brominated analogs bind more tightly to calf thymus DNA and to the virus than to the nonhalogenated analogs. It is demonstrated that the brominated aromatics are much more effective at inactivating λ phage upon photoactivation (λ 310 or 350 nm) than are their nonbrominated analogs. At identical sensitizer concentrations (by weight) and light flux N‐Br, A‐Br, and PSR‐Br produce 5–6 more logs of viral inactivation than their nonbrominated counterparts (N‐H, A‐H and PSR‐H, respectively). The bromine effect may originate from light‐induced electron transfer and subsequent cleavage of the C‐Br bond of the sensitizer radical anion bonds to form aryl radicals. Singlet oxygen cannot be responsible for the viral inactivation because the brominated sensitizers are equally effective in the presence and absence of oxygen. Dithiothreitol does not protect λ phage from light‐induced inactivation by the brominated sensitizer thereby demonstrating that the photogenerated reactive intermediates responsible for the effect are complcxed to the virus and are not generated free in solution.

Lyophilized and reconstituted blood platelet compositions

A process and medium are disclosed for the lyophilization of cells, specifically platelets, and cell-like matter, which comprises the use of solutions including monosaccharide hexoses and pentoses, and biocompatible amphipathic polymers to permit the reconstitution of transfusably useful cells, specifically platelets, and cell-like matter.

An Action Spectrum of the Riboflavin-photosensitized Inactivation of Lambda Phage¶

Abstract The Action Spectrum of riboflavin (RB) sensitized inactivation of lambda phage was determined between 266 and 575 nm. Below 304 nm, RB depresses the phage reduction by screening phage from radiation that it would otherwise absorb directly. Between 308 and 525 nm, RB sensitizes the inactivation of phage. Enhanced phage reduction is observed at 320 and 500 nm because of binding of RB to the phage and the shifting of the absorption curve of the phage-bound flavin relative to free flavin in phosphate-buffered saline. Enhanced inactivation at 320 and 500 nm and depressed phage inactivation between 360 and 410 nm is also influenced by the inner filter effect.

Reduction of Prion Infectivity in Packed Red Blood Cells

The link between a new variant form of Creutzfeldt-Jakob disease (vCJD) and the consumption of prion contaminated cattle meat as well as recent findings showing that vCJD can be transmitted by blood transfusion have raised public health concerns. Currently, a reliable test to identify prions in blood samples is not available. The purpose of this study was to evaluate the possibility to remove scrapie prion protein (PrP(Sc)) and infectivity from red blood cell (RBC) suspensions by a simple washing procedure using a cell separation and washing device. The extent of prion removal was assessed by Western blot, PMCA and infectivity bioassays. Our results revealed a substantial removal of infectious prions (3 logs of infectivity) by all techniques used. These data suggest that a significant amount of infectivity present in RBC preparations can be removed by a simple washing procedure. This technology may lead to increased safety of blood products and reduce the risk of further propagation of prion diseases.