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Background and objectives Pathogen reduction of donor platelets with amotosalen/UVA has been shown to effectively inactivate pathogens and also contaminating white blood cells (WBCs). We wanted to determine whether WBC inactivation could also decrease alloimmune refractoriness to donor platelets. Materials and methods Platelets were prepared from a donor dog's whole blood, and the platelets were either transfused without modification [standard (STD) platelets] or treated with amotosalen/UVA under conditions modelling the amotosalen/UVA Blood System for human platelets (APR) using either 4 or 3 J/cm2 of UVA exposure. Platelets were transfused weekly from a single donor dog for 8 weeks or until the recipient dog became refractory to their donor's platelets. Antibody samples were drawn weekly and tested against the donor dog's platelets and WBCs (CD8 and B cells). Results Only 1/7 (14%) dogs that received STD platelets accepted 8 weeks of donor transfusions. Following APR 4 J/cm2 donor transfusions, 3/9 (33%) recipients accepted their donor's transfusions, but only one recipient remained antibody negative. Following APR 3 J/cm2 donor transfusions, the same dose as used for human platelet transfusions, 7/10 (70%) recipients accepted their donor's transfusions, but only two remained antibody negative. Conclusion As a very high percentage of recipient dogs (70%) accepted APR 3 J/cm2 donor transfusions, these data suggest that preventing alloimmune platelet refractoriness may be another benefit of pathogen reduction using amotosalen/UVA.

Background Leukoreduction (LR) of platelet concentrate (PC) has evolved as the standard to mitigate risks of alloimmunization, clinical refractoriness, acute transfusion reactions (ATRs), and cytomegalovirus infection, but does not prevent transfusion-associated graft-versus-host disease (TA-GVHD). Amotosalen-ultraviolet A pathogen reduction (A-PR) of PC reduces risk of transfusion-transmitted infection and TA-GVHD. In vitro data indicate that A-PR effectively inactivates WBCs and infectious pathogens. Study design and methods A sequential cohort study evaluated A-PR without LR, gamma irradiation, and bacterial screening in hematopoietic stem cell transplant (HSCT) recipients. The first cohort received conventional PC (control) processed without LR, but with gamma irradiation and bacterial screening. The second cohort received A-PR PC (test) processed without: LR, bacterial screening, or gamma irradiation. The primary efficacy outcome was the 1-hour corrected count increment. The primary safety outcome was treatment-emergent ATR. Secondary outcomes included clinical refractoriness, and 100-day status for engraftment, TA-GVHD, HSCT-GVHD, infections, and mortality. Results Mean corrected count increment (× 103 ) of 33 test PC recipients was similar (18.9 ± 8.8 vs. 16.6 ± 8.4; p = 0.296) to that of 31 control PC recipients. Test recipients had a reduced, but nonsignificant, incidence of ATR (test = 9.1%, Control = 19.4%; p = 0.296). The frequencies of clinical refractoriness (0 of 33 vs. 4 of 31 patients) and refractory transfusions (6.6% vs. 19.3%) were lower in the test cohort (p = 0.05 and 0.02), respectively. No patient in either cohort had TA-GVHD. Day 100 engraftment, HSCT-GVHD, mortality, and infectious disease complications were similar between cohorts. Conclusions This study indicated that A-PR PC without LR, gamma irradiation, or bacterial screening is feasible for support of HSCT.

Infection from SARS-CoV-2 elicits an immune response to the nucleocapsid (N) and spike proteins (subunits S1 and S2). In this study, we set out to understand the utility of the multiplexed Quidel Sofia 2 SARS-CoV-2 IgG Antibody Fluorescent Immuno-Assay (FIA) that measures IgG antibodies against these three primary SARS-CoV-2 antigens from a single sample in 15 minutes. Using this assay with samples that were collected prior to the COVID-19 pandemic (n=816) and diseased state samples (n=99), the specificities for the three antigens were 98.4-99.9% and 98.0-100.0%, respectively. A longitudinal study was designed to collect weekly fingerstick, venous whole blood, serum and plasma samples from subjects vaccinated with the Moderna or Pfizer/BioNtech mRNA vaccines. The majority of these enrolled subjects had no known prior infection while a subset was known to have had prior COVID-19 infection. We found that the fingerstick whole blood samples performed as effectively as serum, plasma, and venous whole blood samples with a 95.8-99.5% agreement allowing physicians in a near-patient setting to rapidly provide results to their patients. Additionally, as this assay measures an IgG response against three viral proteins, S1, S2 and N, we were able to characterize immune response between i) naturally infected subjects, ii) vaccinated subjects with no prior infection, iii) vaccinated subjects with known prior infection, and iv) vaccinated subjects with prior asymptomatic exposure/infection. The Quidel Sofia 2 SARS-CoV-2 IgG FIA will aid in providing insights to the protective humoral responses as an increasing number of the world population is vaccinated against SARS-CoV-2.

Background The INTERCEPT Blood System pathogen reduction technology (PRT), which uses amotosalen and ultraviolet A light treatment (amotosalen/UV-PRT), inactivates pathogens in plasma and platelet components (PCs). This review summarizes data describing the inactivation efficacy of amotosalen/UVA-PRT for a broad spectrum of viruses and parasites. Methods Twenty-five enveloped viruses, six nonenveloped viruses (NEVs), and four parasites species were evaluated for sensitivity to amotosalen/UVA-PRT. Pathogens were spiked into plasma and PC at high titers. Samples were collected before and after PRT and assessed for infectivity with cell cultures or animal models. Log reduction factors (LRFs) were defined as the difference in infectious titers before and after amotosalen/UV-PRT. Results LRFs of ≥4.0 log were reported for 19 pathogens in plasma (range, ≥4.0 to ≥7.6), 28 pathogens in PC in platelet additive solution (PC-PAS; ≥4.1-≥7.8), and 14 pathogens in PC in 100% plasma (PC-100%; (≥4.3->8.4). Twenty-five enveloped viruses and two NEVs were sensitive to amotosalen/UV-PRT; LRF ranged from >2.9 to ≥7.6 in plasma, 2.4 or greater to greater than 6.9 in PC-PAS and >3.5 to >6.5 in PC-100%. Infectious titers for four parasites were reduced by >4.0 log in all PC and plasma (≥4.9 to >8.4). Conclusion Amotosalen/UVA-PRT demonstrated effective infectious titer reduction for a broad spectrum of viruses and parasites. This confirms the capacity of this system to reduce the risk of viral and parasitic transfusion-transmitted infections by plasma and PCs in various geographies.

Background Risk of transfusion-transmitted (TT) malaria is mainly associated with whole blood (WB) or red blood cell (RBC) transfusion. Risk mitigation relies mostly on donor deferral while a limited number of countries perform blood testing, both negatively impacting blood availability. This study investigated the efficacy of the pathogen reduction system using amustaline and glutathione (GSH) to inactivate Plasmodium falciparum in WB. Study design and methods WB units were spiked with ring stage P. falciparum infected RBCs. Parasite loads were measured in samples at time of infection, after 24 hours at room temperature (RT), and after a 24-hour incubation at RT post-treatment with 0.2 mM amustaline and 2 mM GSH. Serial 10-fold dilutions of the samples were inoculated to RBC cultures and maintained up to 4 weeks. Parasitemia was quantified by cytometry. Results The P. falciparum viability assay has a limit of detection of a single live parasite per sample. Input parasite titer was >5.7 log10 TCID50 per mL. A 24-hour incubation at RT paused parasite development in controls, but they retained viability and infectivity when tested in culture. In contrast, no infectious parasites were detected in the amustaline/GSH-treated sample after 4 weeks of culture. Conclusion A robust level of P. falciparum inactivation was achieved in WB using amustaline/GSH treatment. Parasite log reduction was >5.7 log10 TCID50 per mL. Development of such a pathogen reduction system may provide an opportunity to reduce the risk of TT malaria and improve blood availability.

Background Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare, often fatal complication of blood transfusion that can occur in immunocompromised or immunocompetent recipients and is the result of viable T cells present in the blood components transfused. Study design and methods We examined the TA-GVHD clinical case literature including numerous original clinical case reports and several comprehensive case series. We also evaluated recent in vitro experimental data on the inhibition of T cell proliferation, comparing the effect of a specific pathogen inactivation (PI) technology to that of the Food and Drug Administration-recommended gamma irradiation dose of 2500 cGy. Results We identified 12 published TA-GVHD cases with atypical/milder or delayed symptom presentations and/or an atypical clinical course; these included cases attributed to leukoreduced or suboptimally irradiated units. We summarize recent in vitro data using a sensitive limiting dilution assay that establish that, compared to irradiation at the recommended 2500 cGy dose, PI using amotosalen/ultraviolet A, or amustaline/glutathione achieves a greater degree of inhibition of T cell proliferation. Conclusion We propose that TA-GVHD has a spectrum of disease severity indicating that additional mild cases may still occur but be undiagnosed and/or underreported, opening up the possibility, supported by in vitro experimental data, that irradiation at the currently established dose may not be fully protective. Furthermore, since many US institutions use component irradiation selectively only for immunocompromised patients, immunocompetent recipients are not fully protected. PI technologies appear to be at least equal to, if not better than, gamma irradiation in abrogating the ability of T cells to proliferate, and if applied to all blood components, protection against TA-GVHD would be an additional benefit that would allow for the elimination of component irradiation.

Background Chikungunya virus, a mosquito-borne arbovirus, often co-circulates with the Zika, dengue, and yellow fever viruses in Aedes mosquito-infested areas where cases of arbovirus transfusion-transmitted infections have been reported. Building on past experience to help maintain the availability of safe components during major outbreaks of chikungunya virus in La Reunion, Italy, and Thailand and of Zika virus in the Pacific, the Caribbean, and the Americas, pathogen inactivation is a mitigation strategy to reduce the risk of transfusion-transmitted infection. Inactivation of chikungunya virus was investigated for platelets in 100% plasma using amotosalen/ultraviolet A light, and in red blood cells using amustaline/glutathione. Study design and methods Platelets in 100% plasma and red blood cells (RBCs) were spiked with chikungunya virus. Infectious chikungunya virus titers were measured in contaminated blood products before and after treatment with amotosalen/ultraviolet A light for platelets in 100% plasma and after treatment with amustaline/glutathione for RBCs. Viral infectivity was quantified by plaque assay. Results The mean chikungunya virus infectivity titers before inactivation were 6.50 log10 plaque-forming units/mL for platelets in 100% plasma and 7.60 log10 plaque-forming units/mL for RBCs. No infectivity was detected after amotosalen/ultraviolet A light or amustaline/glutathione treatment, corresponding to greater than 6.5 log10 plaque-forming units/mL and greater than 7.1 log10 plaque-forming units/mL of inactivation, respectively. Conclusion Robust levels of chikungunya virus inactivation were achieved for platelets in 100% plasma and for RBC components. The licensed amotosalen/ultraviolet A light technology and the amustaline/glutathione pathogen-reduction system under development may provide an opportunity for comprehensive mitigation of the risk of chikungunya virus transfusion-transmitted infection by plasma, platelets, and RBCs.

Physicochemical properties of nanoparticles, such as size, shape, surface charge, density, and porosity play a central role in biological interactions and hence accurate determination of these characteristics is of utmost importance. Here we propose tunable resistive pulse sensing for simultaneous size and surface charge measurements on a particle-by-particle basis, enabling the analysis of a wide spectrum of nanoparticles and their mixtures. Existing methodologies for measuring zeta potential of nanoparticles using resistive pulse sensing are significantly improved by including convection into the theoretical model. The efficacy of this methodology is demonstrated for a range of biological case studies, including measurements of mixed anionic, cationic liposomes, extracellular vesicles in plasma, and in situ time study of DNA immobilisation on the surface of magnetic nanoparticles. The high-resolution single particle size and zeta potential characterisation will provide a better understanding of nano-bio interactions, positively impacting nanomedicine development and their regulatory approval.

BACKGROUND Dengue virus (DENV) is an arbovirus primarily transmitted through mosquito bite; however, DENV transfusion-transmitted infections (TTIs) have been reported and asymptomatic DENV RNA–positive blood donors have been identified in endemic countries. DENV is considered a high-risk pathogen for blood safety. One of the mitigation strategies to prevent arbovirus TTIs is pathogen inactivation. In this study we demonstrate that the amustaline and glutathione (S-303/GSH) treatment previously found effective against Zika virus in red blood cells (RBCs) is also effective in inactivating DENV. STUDY DESIGN AND METHODS Red blood cells were spiked with high levels of DENV. Viral RNA loads and infectious titers were measured in the untreated control and before and after pathogen inactivation treatment of RBC samples. DENV infectivity was also assessed over five successive cell culture passages to detect any potential residual replicative virus. RESULTS The mean ± SD DENV titer in RBCs before inactivation was 6.61 ± 0.19 log 50% tissue culture infectious dose (TCID50)/mL and the mean viral RNA load was 8.42 log genome equivalents/mL. No replicative DENV was detected either immediately after completion of treatment using S-303/GSH or after cell culture passages. CONCLUSION Treatment using S-303/GSH inactivated high levels of DENV in RBCs to the limit of detection. In combination with previous studies showing the effective inactivation of DENV in plasma and platelets using the licensed amotosalen/UVA system, this study demonstrates that high levels of DENV can be inactivated in all blood components.

BACKGROUND Concerned over the risk of Zika virus (ZIKV) transfusion transmission, public health agencies recommended the implementation of mitigation strategies for its prevention. Those strategies included the use of pathogen inactivation for the treatment of plasma and platelets. The efficacy of amotosalen/ultraviolet A to inactivate ZIKV in plasma had been previously demonstrated, and the efficacy of inactivation in platelets with the same technology was assumed. These studies quantify ZIKV inactivation in platelet components using amotosalen/ultraviolet A. STUDY DESIGN AND METHODS Platelet components were spiked with ZIKV, and ZIKV infectious titers and RNA loads were measured by cell culture-based assays and real-time polymerase chain reaction in spiked platelet components before and after photochemical treatment using amotosalen/ultraviolet A. RESULTS The mean ZIKV infectivity titers and RNA loads in platelet components before inactivation were either 4.9 log10 plaque forming units per milliliter, or 4.4 log10 50% tissue culture infective dose per milliliter and 7.5 log10 genome equivalents per milliliter, respectively. No infectivity was detected immediately after amotosalen/ultraviolet A treatment. No replicative virus remained after treatment, as demonstrated by multiple passages on Vero cell cultures; and ZIKV RNA was not detected from the first passage after inactivation. Additional experiments in this study demonstrated efficient inactivation to the limit of detection in platelets manufactured in 65% platelet additive solution, 35% plasma, or 100% plasma. CONCLUSION As previously demonstrated for plasma, robust levels of ZIKV inactivation were achieved in platelet components. With inactivation of higher levels of ZIKV than those reported in asymptomatic, RNA-reactive blood donors, the pathogen-inactivation system using amotosalen/ultraviolet A offers the potential to mitigate the risk of ZIKV transmission by plasma and platelet transfusion.

BACKGROUND The potential for Zika virus (ZIKV) transfusion-transmission (TT) has been demonstrated in French Polynesia and Brazil. Pathogen inactivation (PI) of blood products is a proactive strategy to inactivate TT pathogens including arboviruses. Inactivation of West Nile, dengue, Zika, and chikungunya viruses was previously demonstrated by photochemical treatment with amotosalen and ultraviolet A (UVA) illumination. In this study, we evaluated ZIKV inactivation in red blood cell (RBC) components by a chemical approach that uses amustaline (S-303) and glutathione (GSH). STUDY DESIGN AND METHODS RBC components were spiked with a high titer of ZIKV. Viral titers (infectivity) and ZIKV RNA loads (reverse transcription–polymerase chain reaction) were measured in spiked RBCs before and after S-303 and GSH treatment and confirmed using repetitive passages in cell culture. A mock-treated arm validated the approach by demonstrating stability of the virus (infectivity and RNA load) during the process. RESULTS The mean ZIKV infectivity titer and RNA load in RBCs were 5.99 ± 0.2 log 50% tissue culture infectious dose (TCID50)/mL and 7.75 ± 0.16 log genomic equivalents/mL before inactivation. No infectivity was detected immediately after S-303 and GSH treatment and after five serial passages in cell culture. CONCLUSION Complete ZIKV inactivation of more than 5.99 log TCID50/mL in RBCs was achieved using S-303 and GSH at levels higher than those found in asymptomatic ZIKV-infected blood donors. Therefore, the S-303 and GSH PI system is promising for mitigating the risk of ZIKV TT.

Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare, often fatal complication of blood transfusion that can occur in immunocompromised or immunocompetent recipients and is the result of viable T cells present in the blood components transfused.We examined the TA-GVHD clinical case literature including numerous original clinical case reports and several comprehensive case series. We also evaluated recent in vitro experimental data on the inhibition of T cell proliferation, comparing the effect of a specific pathogen inactivation (PI) technology to that of the Food and Drug Administration-recommended gamma irradiation dose of 2500 cGy.We identified 12 published TA-GVHD cases with atypical/milder or delayed symptom presentations and/or an atypical clinical course; these included cases attributed to leukoreduced or suboptimally irradiated units. We summarize recent in vitro data using a sensitive limiting dilution assay that establish that, compared to irradiation at the recommended 2500 cGy dose, PI using amotosalen/ultraviolet A, or amustaline/glutathione achieves a greater degree of inhibition of T cell proliferation.We propose that TA-GVHD has a spectrum of disease severity indicating that additional mild cases may still occur but be undiagnosed and/or underreported, opening up the possibility, supported by in vitro experimental data, that irradiation at the currently established dose may not be fully protective. Furthermore, since many US institutions use component irradiation selectively only for immunocompromised patients, immunocompetent recipients are not fully protected. PI technologies appear to be at least equal to, if not better than, gamma irradiation in abrogating the ability of T cells to proliferate, and if applied to all blood components, protection against TA-GVHD would be an additional benefit that would allow for the elimination of component irradiation.

BACKGROUND Pathogen inactivation methods are increasingly used to reduce the risk of infections after transfusion of blood products. Photochemical treatment (PCT) of platelets (PLTs) and plasma with amotosalen and ultraviolet A (UVA) light inactivates pathogens and white blood cells through formation of adducts between amotosalen and nucleic acid that block replication, transcription, and translation. The same adducts block the amplification of nucleic acids using polymerase chain reaction (PCR) in a manner that correlates with the number of adducts formed, providing a direct quality control (QC). Current QC measures for PCT rely on indirect methods that measure the delivered UVA dose or percent residual amotosalen after illumination, rather than directly measuring nucleic acid modification. STUDY DESIGN AND METHODS Endogenous mitochondrial DNA (mtDNA), which is detectable in PLT and plasma units, was chosen as a target for the quantification of photochemically induced modifications. DNA was extracted from untreated or amotosalen and UVA–treated PLTs or plasma, and mtDNA fragments of variable lengths were quantified using a real-time PCR inhibition assay. RESULTS PCT induced increasing real-time PCR inhibition of mtDNA amplification for larger amplicon sizes. Amplification was unaffected by treatment with amotosalen or UVA alone, whereas up to 3 log inhibition was observed after PCT. Blinded PCR testing of a panel of 110 samples each, from PLT or plasma components prepared for routine use within a blood center, allowed 100% discrimination between untreated and treated units. CONCLUSION Our initial findings indicate that an adequately sensitive, quantitative real-time PCR inhibition assay targeting mtDNA could provide a valuable tool to confirm and monitor PCT.

BACKGROUND Red blood cell (RBC) transfusion risks could be reduced if a robust technology for pathogen inactivation of RBC (PI-RBCs) were to be approved. MATERIALS AND METHODS Estimates of per-unit and per-patient aggregate infectious risks for conventional RBCs were calculated; the latter used patient diagnosis as a determinant of estimated lifetime exposure to RBC units. Existing in vitro data for the two technologies under development for producing PI-RBCs and the status of current clinical trials are reviewed. RESULTS Minimum and maximum per-unit risk were calculated as 0.0003% (1 in 323,000) and 0.12% (1 in 831), respectively. The minimum estimate is for known lower-risk pathogens while the maximal estimate also includes an emerging infectious agent (EIA) and endemic area Babesia risk. Minimum and maximum per-patient lifetime risks by diagnosis grouping were estimated as 1.5 and 3.3%, respectively, for stem cell transplantation (which includes additional risk for cytomegalovirus transmission); 1.2 and 3.7%, respectively, for myelodysplastic syndrome; and 0.2 and 44%, respectively, for hemoglobinopathy. DISCUSSION There is potential for PI technologies to reduce infectious RBC risk and to provide additional benefits (e.g., prevention of transfusion-associated graft-versus-host disease and possible reduction of alloimmunization) due to white blood cell inactivation. PI-RBCs should be viewed in the context of having a fully PI-treated blood supply, enabling a blood safety paradigm shift from reactive to proactive. Providing insurance against new EIAs. Further, when approved, the use of PI for all components may catalyze operational changes in blood donor screening, laboratory testing, and component manufacturing.

Background and Objectives A photochemical treatment process (PCT) utilizing amotosalen and UVA light (INTERCEPT™ Blood System) has been developed for inactivation of viruses, bacteria, parasites and leucocytes that can contaminate blood components intended for transfusion. The objective of this study was to further characterize the safety profile of INTERCEPT-treated platelet components (PCT-PLT) administered across a broad patient population. Materials and Methods This open-label, observational haemovigilance programme of PCT-PLT transfusions was conducted in 21 centres in 11 countries. All transfusions were monitored for adverse events within 24 h post-transfusion and for serious adverse events (SAEs) up to 7 days post-transfusion. All adverse events were assessed for severity (Grade 0–4), and causal relationship to PCT-PLT transfusion. Results Over the course of 7 years in the study centres, 4067 patients received 19 175 PCT-PLT transfusions. Adverse events were infrequent, and most were of Grade 1 severity. On a per-transfusion basis, 123 (0·6%) were classified an acute transfusion reaction (ATR) defined as an adverse event related to the transfusion. Among these ATRs, the most common were chills (77, 0·4%) and urticaria (41, 0·2%). Fourteen SAEs were reported, of which 2 were attributed to platelet transfusion (

Background and objectives Chikungunya virus (CHIKV) infections have been reported in all continents, and the potential risk for CHIKV transfusion-transmitted infections (TTIs) was demonstrated by the detection of CHIKV RNA-positive donations in several countries. TTIs can be reduced by pathogen inactivation (PI) of blood products. In this study, we evaluated the efficacy of amustaline and glutathione (S-303/GSH) to inactivate CHIKV in red-blood-cell concentrates (RBCs). Material and methods Red-blood-cells were spiked with high level of CHIKV. Infectious titres and RNA loads were measured before and after PI treatment. Residual CHIKV infectivity was also assessed after five successive cell culture passages. Results The mean CHIKV titres in RBCs before inactivation was 5·81 ± 0·18 log10 50% tissue culture infectious dose (TCID50 )/mL, and the mean viral RNA load was 10·49 ± 0·15 log10 genome equivalent (GEq)/mL. No CHIKV TCID was detected after S-303 treatment nor was replicative CHIKV particles and viral RNA present after five cell culture passages of samples obtained immediately after S-303 treatment. Conclusion Chikungunya virus was previously shown to be inactivated by the PI technology using amotosalen and ultraviolet A light for the treatment of plasma and platelets. This new study demonstrates that S-303/GSH can inactivate high titres of CHIKV in RBCs.

INTRODUCTION Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare complication after transfusion of components containing viable donor T cells. Gamma irradiation with doses that stop T-cell proliferation is the predominant method to prevent TA-GVHD. Treatment with pathogen inactivation methodologies has been found to also be effective against proliferating white blood cells, including T cells. In this study, T-cell inactivation was compared, between amotosalen/ultraviolet A (UVA) treatment and gamma-irradiation (2500 cGy), using a sensitive limiting dilution assay (LDA) with an enhanced dynamic range. METHODS AND MATERIALS Matched plasma units (N = 8), contaminated with 1 × 106 peripheral blood mononuclear cells (PBMCs) per mL, were either treated with amotosalen/UVA or gamma irradiation, or retained as untreated control. Posttreatment, cells were cultured under standardized conditions. T-cell proliferation was determined by the incorporation of 3 H-thymidine and correlated with microscopic detection. RESULTS Range-finding experiments showed that after gamma irradiation (2500 cGy), significant T-cell proliferation could be observed at a 1 × 107 cell culture density, some proliferation at 1 × 106 , and none at 1 × 105 cells/well. Based on these facts, a quantitative comparison was carried out between amotosalen/UVA at the highest challenge of 1 × 107 PBMCs/well, and gamma irradiation at 1 × 106 and 1 × 105 PBMCs/well. Complete inactivation of the T cells after amotosalen/UVA treatment was observed, equivalent to greater than 6.2 log inactivation. Complete inactivation of the T cells was also observed after gamma irradiation when 1 × 105 PBMCs/well were cultured (>4.2 log inactivation). Proliferation was observed when 1 × 106 PBMCs/well were cultured (≤5.2 log inactivation) after gamma irradiation. CONCLUSION Amotosalen/UVA treatment more effectively inactivates T cells than the current standard of gamma irradiation (2500 cGy) for the prevention of TA-GVHD.

BACKGROUND With an increasing number of recognized transfusion-transmitted (TT) babesiosis cases, Babesia microti is the most frequently TT parasite in the United States. We evaluated the inactivation of B. microti in red blood cells (RBCs) prepared in Optisol (AS-5) using amustaline and glutathione (GSH) and in platelet components (PCs) in 100% plasma using amotosalen and low-energy ultraviolet A (UVA) light. STUDY DESIGN AND METHODS Individual RBCs and apheresis PCs were spiked with B. microti–infected hamster RBCs (iRBCs) to a final concentration of 106 iRBCs/mL and treated with the respective inactivation systems according to the manufacturer's instruction. Samples were collected before (control) and after (test) each treatment. Dilutions of the control samples to 10−6 were inoculated into hamsters, while the test samples were inoculated neat or at 10−1 dilution. At 3 and 5 weeks postinoculation, hamsters were evaluated for B. microti infection by microscopic observation of blood smears and 50% infectivity titers (ID50) were determined. Log reduction was calculated as control log ID50 minus test log ID50. RESULTS Parasitemia was detected in hamsters injected with as low as 100,000-fold diluted control samples, while no parasites were detectable in the blood smears of any hamsters receiving neat test samples. Mean log reduction was more than 5 log/mL by amustaline/GSH for RBCs and more than 4.5 log/mL by amotosalen/UVA for PCs. CONCLUSION B. microti was inactivated to the limit of detection in RBCs and PCs after the respective inactivation treatment. Complete inactivation of B. microti was achieved in this animal infectivity model, and pathogen reduction treatment inhibited transmission of infection.

The potential for Zika virus (ZIKV) transfusion-transmission (TT) has been demonstrated in French Polynesia and Brazil. Pathogen inactivation (PI) of blood products is a proactive strategy to inactivate TT pathogens including arboviruses. Inactivation of West Nile, dengue, Zika, and chikungunya viruses was previously demonstrated by photochemical treatment with amotosalen and ultraviolet A (UVA) illumination. In this study, we evaluated ZIKV inactivation in red blood cell (RBC) components by a chemical approach that uses amustaline (S-303) and glutathione (GSH).RBC components were spiked with a high titer of ZIKV. Viral titers (infectivity) and ZIKV RNA loads (reverse transcription-polymerase chain reaction) were measured in spiked RBCs before and after S-303 and GSH treatment and confirmed using repetitive passages in cell culture. A mock-treated arm validated the approach by demonstrating stability of the virus (infectivity and RNA load) during the process.The mean ZIKV infectivity titer and RNA load in RBCs were 5.99 ± 0.2 log 50% tissue culture infectious dose (TCIDComplete ZIKV inactivation of more than 5.99 log TCID

We performed a risk-benefit analysis for implementation of pathogen-inactivated (PI) apheresis platelets (APs) in the United States, focusing on the amotosalen/ultraviolet-A system. Risks and benefits were quantified per patient assuming a mean of 6 AP units per treatment cycle and using available clinical data, mathematical modeling, and observational studies. Current risks associated with AP transfusion can be divided into known partially addressed risks, known well-addressed risks, and unknown risks associated with acute or chronic emerging infectious agents (EIAs). Bacterial contamination dominates the first category, at a per-patient rate of 1:250, which correlates with an estimated septic transfusion reaction rate of 1:1000. Quantitation of per-patient EIA risk was modeled to be between 1:370 (acute) and 1:667 (chronic). Due to its broad range of action PI is expected to reduce or eliminate these infectious risks and also to reduce the rate of febrile transfusion reactions and possibly alloimmunization. These benefits are weighed against 1) concerns for excess bleeding, 2) an apparent increase in acute respiratory distress syndrome in the initial report of the SPRINT clinical trial, and 3) the possible toxicity associated with the introduction of a new chemical into platelet (PLT) units. However, transfusion of an estimated 100,000 patients with PI PLTs worldwide has occurred without reported serious adverse effects. We conclude that evidence indicates a favorable risk-benefit profile for the implementation of PLT PI and argues for a path forward toward US regulatory approval.

Contexte Les procedes d’inactivation des agents pathogenes (IP) ont pour but l’inactivation des agents infectieux (bacteriens, viraux et parasitaires) dans les produits sanguins labiles (PSL). En ce qui concerne les arbovirus, l’IP par procede photochimique avec amotosalen (S-59) et ultraviolets A (UVA) a ete demontree, entre autres, pour les virus de la dengue (DENV) et du chikungunya (CHIKV) dans le plasma et les plaquettes et pour le virus Zika (ZIKV) dans le plasma ; l’IP par traitement chimique utilisant l’amustaline et le glutathion (S-303/GSH) a ete demontree pour le ZIKV dans les concentres de globules rouges (CGR). Objectifs Pour completer ces travaux, nous avons etudie l’inactivation du ZIKV dans les plaquettes par S-59/UVA ; et celles du DENV et du CHIKV dans les CGR par S-303/GSH. Methodes Les unites plaquettaires ont ete inoculees avec des titres eleves de ZIKV et les CGR avec des titres eleves de DENV et CHIKV. Les niveaux d’infectiosite (log 10 TCID 50 /mL) et les quantites d’ARN (log 10 Geq/mL) ont ete mesures avant et apres IP. Resultats S-59/UVA inactive > 4,4 log 10 ZIKV TCID 50 /mL (7,5 log 10 Geq/mL) a la limite de detection dans les plaquettes ; S-303/GSH inactive > 6,61 log 10 DENV TCID 50 /mL (8,42 log 10 Geq/mL) et > 5,81 log 10 CHIKV TCID 50 /mL (10,49 log 10 Geq/mL) a la limite de detection dans les CGR. Conclusion Ces resultats combines a ceux d’etudes anterieures montrent que les procedes d’inactivation utilisant S-59/UVA et S-303/GSH inactivent ZIKV, DENV et CHIKV dans le plasma, les plaquettes et les CGR. Ces donnees sont particulierement importantes car, la plupart du temps, ces arbovirus co-circulent.

Introduction: HSCT patients require platelet components (PC) for support of thrombocytopenia. Donors are tested for selected viruses to prevent transfusion-transmission infection (TTI), and PC are screened for bacteria and irradiated to prevent transfusion associated graft versus host disease (TA-GVHD). Leukocyte reduction (LR) is commonly, but not universally, used to mitigate the risks of acute transfusion reactions (ATR), allo-immunization, clinical refractoriness, and CMV TTI. However, donor testing, PC bacterial screening, and LR do not provide complete protection from ATR, transfusion related sepsis (TRS), refractoriness, CMV TTI, or residual risks of window period and emerging pathogen TTI. Amotosalen-UVA pathogen reduction treatment (A-PRT) inactivates a broad spectrum of pathogens (e.g. Zika), inhibits leukocyte antigen presentation and cytokine synthesis, and inhibits T-cell proliferation. A-PRT with LR has replaced bacterial screening, CMV serology, and gamma irradiation. Objective: To control cost, the Hong Kong Red Cross Blood Transfusion Service (HKRCBTS) has used whole blood platelet-rich-plasma (PRP) PC without LR. Based on data showing effective leukocyte inactivation by A-PRT, the HKRCBTS evaluated use of A-PRT buffy coat PC (BC PC) in platelet additive solution (PAS) without LR, bacterial screening, and gamma irradiation to manage costs, salvage more donor plasma, reduce TTI risk, and simplify production. Study Design and Methods : An open-label, prospective, sequential, two cohort study at Queen Mary Hospital, Hong Kong enrolled HSCT patients for up to 30 days of PC support with 100-day follow up. Cohorts were comparable for primary disease and type of HSCT. The first cohort received PRP PC prepared from CPD whole blood, a current HKRCBTS standard PC (Control-C). Five ABO-matched PRP PCs in 100% plasma were pooled without LR to yield ≥ 3 x 1011 platelets. C-PCs were screened for bacteria, gamma irradiated (2,500 cGy), stored up to 5 days at 22- 24 °C; and contained ~ 5 x 109 leukocytes. For the 2nd cohort, buffy coat PCs (BC PC) were prepared from CPD whole blood in PAS (SSP+, Macopharma, France). Five ABO-matched BC PC in 35% plasma-65% PAS were pooled, and prepared with A-UVA PRT to yield ≥ 3 x 1011 platelets (Test-T). T-PCs were not LR, not screened for bacteria, not gamma irradiated, stored for up to 5 days at 22- 24 °C; and contained ~ 5 x 108 leukocytes. HSCT patients ≥ 18 years expected to require PC were included. Exclusion criteria were: history of clinical refractoriness to platelet transfusion (2 successive 1-HR corrected count increments (CCI) < 5.0 x 103), immune thrombocytopenia, or disorders confounding 1-HR CCI determination. For both cohorts, a transfusion threshold of 10 x 109/L was specified for stable patients unless adjusted to 20 x 109/L for sepsis. Patients were not screened for HLA antibody at study entry. The efficacy outcome was the 1-hour (HR) count increment (CI), and the safety outcome was ATR incidence. Secondary (post-hoc) outcomes were the 1-HR CCI and the incidence of clinical refractoriness (two successive CCI < 5x103 per patient and transfusions with CCI < 5x103). Results: 64 patients were transfused (31 Control and 33 Test). Demographics were similar between cohorts (Table). The majority of patients had allogeneic HSCT from related donors. Mean pre-transfusion platelet counts were < 16 x 109/L. The mean PC dose and storage duration were similar between cohorts (Table). The 1-HR count increments (CI) and 1-HR CCI responses were within therapeutic ranges for both cohorts (Table). The proportion of transfusions with CCI responses < 5.0 x 103 and the proportion of patients with clinical refractoriness were less in the Test cohort (Table). ATRs trended lower with A-PRT (Test = 9.1%, Control = 19.4%, p=0.296).No patients had TRS or TA-GVHD reported during the active transfusion period. Day-100 engraftment (Table), HSCT-GVHD, mortality, and infectious disease complications were similar between cohorts. Conclusions: This exploratory study of A-PRT PC without LR demonstrated therapeutic 1-HR CCI responses, safety, and conserved HSCT engraftment. The proportions of transfusions with CCI < 5.0x103 and the proportion of patients with clinical refractoriness were less for A-PRT PC than conventional PC. A-PRT of pooled whole blood BC PC without LR offers the potential to improve PC supply, and to reduce the net cost of PRT PC by replacing other interventions. Table Table. Disclosures Corash: Cerus Corporation: Employment, Equity Ownership. Liu:Cerus Corporation: Employment, Equity Ownership. Huang:Cerus Corporation: Employment, Equity Ownership. Vermeij:Cerus Corporation: Employment, Equity Ownership. Stassinopoulos:Cerus Corporation: Employment, Equity Ownership. Benjamin:Cerus Corporation: Employment, Equity Ownership.

A series of binuclear iron compounds has been synthesized using diamide, bis-phenolate ligands in which the carbon-linker between the amide nitrogen atoms has been varied. Two diferrous compounds in the series, Fe 2 II ( H 2 Hbach ) 2 ( N-MeIM ) 2 and Fe 2 II ( H 2 Hbame ) 2 ( N-MeIM ) 2 along with their two-electron oxidized, di-μ-methoxy-bridged counterparts, Fe III ( H 2 Hbach ) 2 ( OMe ) 2 and Fe III ( H 2 Hbame ) 2 ( OMe ) 2 have been crystallographically characterized, as have the di-μ-methoxy compounds Fe 2 III ( H 2 Hbap ) 2 ( OMe ) 2 , Fe 2 III ( H 2 Hbabn ) 2 ( OMe ) 2 and Fe 2 III ( H 2 Hbapen ) 2 ( OMe ) 2 (H2Hbab = 1,2-bis(2-hydroxybenzamido) benzene, H2Hbach = trans-1,2-bis(2-hydroxybenzamido) cyclohexane, H2Hbame = 1,2-bis(2-hydroxybenzamido) ethane, H2Hbap = 1,3-bis(2-hydroxybenzamido) propane, H2Hbabn = 1,4-bis(2-hydroxybenzamido) butane, H2Hbapen = 1,5-bis(2-hydroxybenzamido) pentane, N-MeIM = N-methylimidazole and OMe = methoxide). Fe 2 II ( H 2 Hbach ) 2 ( N-MeIM ) 2 and Fe 2 II ( H 2 Hbame ) 2 ( N-MeIM ) 2 are structurally very similar to previously reported diferrous compounds of this family of ligands that have been shown to be active as oxygen atom transfer catalysts. Flexibility in the carbon-linker allows some variability in the orientation of the phenolate arms of the ligands in the diferric di-μ-methoxy compounds, but the Fe2O2 core remains largely unchanged across the series. Two-electron oxidation of the ferrous compounds in methanol shows a substantial ligand rearrangement that is consistent with other spectroscopic, electrochemical and kinetic investigations. The loss of both phenolate bridges upon oxidation is reminiscent of the “carboxylate shift” observed in binuclear non-heme enzymes and could provide insight into the driving force behind this family of compounds’ function as a catalyst.

In the last few years, the transfusion medicine community has been paying special attention to emerging vector-borne diseases transmitted by arboviruses. Zika virus is the latest of these pathogens and is responsible for major outbreaks in Africa, Asia and, more recently, in previously infection-naïve territories of the Pacific area. Many issues regarding this emerging pathogen remain unclear and require further investigation. National health authorities have adopted different prevention strategies. The aim of this review article is to discuss the currently available, though limited, information and the potential impact of this virus on transfusion medicine.

BACKGROUND: The human erythrovirus B19 (B19) is a small (18- to 26-nm) nonenveloped virus with a single-stranded DNA genome of 5.6 kb. B19 is clinically significant and is also generally resistant to pathogen inactivation methods. Photochemical treatment (PCT) with amotosalen and ultraviolet A (UVA) inactivates viruses, bacteria, and protozoa in platelets (PLTs) and plasma prepared for transfusion. In this study, the capacity of PCT to inactivate B19 in human PLT concentrates was evaluated. STUDY DESIGN AND METHODS: B19 inactivation was measured by a novel enzyme-linked immunosorbent spot (ELISPOT) erythroid progenitor cell infectivity assay and by inhibition of long-range (up to 4.3 kb) polymerase chain reaction (PCR), under conditions where the whole coding region of the viral genome was amplified. B19-infected plasma was used to test whether incubation of amotosalen with virus before PCT enhanced inactivation compared to immediate PCT. RESULTS: Inactivation of up to 5.8 log of B19 as measured by the infectivity assay, or up to 6 logs as measured by PCR inhibition can be achieved under non-limiting conditions. Inactivation efficacy was found to increase with incubation prior to UVA illumination. Without incubation prior to illumination 2.1 +0.4 log was inactivated as determined by infectivity assay. When measured by PCR inhibition, inactivation varied inversely with amplicon size. When primers that spanned the entire coding region of the B19 genome were used, maximum inhibtion of PCR amplification was demonstrated. CONCLUSION: Under defined conditions, PCT with amotosalen combined with UVA light can be used to inactivate B19, a clinically significant virus that can be transmitted through blood transfusion, and heretofore has been demonstrated to be refractory to inactivation.

Background. In evaluating a photochemical treatment process for inactivating parvovirus B19, there lacked simple culture methods to measure infectivity. The recently developed enzyme‐linked immunospot (ELISpot) infectivity assay uses late‐stage erythropoietic progenitor cells and is labor intensive and time consuming. We evaluated a novel, efficient polymerase chain reaction (PCR) inhibition assay and examined correlations with reductions in infectivity. Methods. Contaminated plasma was treated with 150 μmol/L amotosalen and 3 J/cm2 ultraviolet A light and then tested for DNA modification using conventional PCR inhibition and a novel preamplification approach. The novel assay subjected the samples to preamplification cycles using long‐template PCR, followed by quantitative PCR (QPCR) inhibition detection. Both approaches were tested for correlations with reductions in viral infectivity by comparing ELISpot assay results of identical samples. Results. The B19 preamplification inhibition assay showed detection ranges of 2–2.5 log and demonstrated quantitative correlation with up to a 5.8‐log reduction in viral infectivity in ELISpot results. Conventional PCR detected a >5 log reduction in amplification, correlated with a 4.4‐log reduction in viral infectivity. A range of 4‐log inhibition of hepatitis B virus DNA amplification was also achieved. Conclusions. The results demonstrated that a novel preamplification QPCR assay is a useful tool for predicting reductions in infectivity after photochemical treatment. This assay was extended to show utility in circumstances where practical in vitro assays are unavailable for the determination of the efficacy of pathogen inactivation.

Introduction Depuis 2015, l’epidemie de virus ZIKA (ZIKV) s’est etendue. Les infections asymptomatiques frequentes ainsi que le risque de transmission sexuelle soulevent la possibilite de transmission par transfusion. Ce risque peut etre limite par la reduction des agents pathogenes (RP) avec le procede Intercept™ Blood System. La technologie a ete demontree efficace sur d’autres arbovirus ainsi que capable d’inactiver > 6,57 log ZIKV dans le plasma humain (Aubry M et al.). Le mode d’action (MDA) est base sur la formation d’adduits covalents irreversibles avec les acides nucleiques. Une technologie avec le meme MDA utilisant l’Amustaline et le Glutathion (GSH) est en developpement pour les concentres de globules rouges (CGR). Nous rapportons ici l’inactivation de ZIKV dans les CGR et les concentres de plaquettes (CP) quel que soit le milieu de suspension. Methodes Des CP soit dans 65 % de solution additive plaquettaire (PAS) ou 100 % de plasma, ou des CGR prepares en AS-5 (n = 3), ont ete inocules avec ZIKV (souche CDC PRVABC59 ; ∼ 107pfu/mL). Les CP ont ete traites avec Amotosalen et UVA (3j/cm2). Les CGR ont ete traites avec Amustaline/GSH (0,2 mM/20 mM) a temperature ambiante pendant 18–24 h. L’inactivation a ete determinee en comparant les titres infectieux (log10) avant et apres le traitement. Resultats Les titres initiaux dans les CP en 65 % PAS ou 100 % de plasma etaient de 4,0 et 4,3 log pfu/mL respectivement. Apres inactivation, aucune infectivite n’a ete observee, soit une inactivation moyenne > 4,6 log pfu, ou > 4,1 log pfu/mL. Les titres moyens initiaux dans les hematies AS-5 etaient de 4,3 log pfu/mL, sans infectivite apres, soit une inactivation moyenne de > 5,0 log pfu, ou > 4,3 log pfu/mL. Dans tous les cas, l’etendue de l’inactivation a ete limitee par l’inoculum initial. Conclusions Les technologies de reduction des pathogenes avec Amotosalen/UVA et Amustaline/GSH inactivent efficacement ZIKV dans les plaquettes, le plasma et les CGR.

Introduction Intercept™ Blood System pour les globules rouges (non approuve commercialement) utilise une petite molecule d’amustaline pour former des adduits covalents au sein des acides nucleiques des leucocytes et des agents pathogenes contaminants pour empecher leur replication. Avant incubation de 18–24 h a temperature ambiante, 0,2 mM d’amustaline et 20 mM de glutathion (GSH) sont ajoutes. L’inactivation est complete au bout de 3 h. Le temps supplementaire assure une decomposition complete de l’amustaline. Une centrifugation et l’echange du surnageant avec une solution additive (SAG-M) fournissent un concentre de globules rouges (CGR) inactive. L’objectif de cette etude etait d’evaluer l’inactivation du Calicivirus felin (CVF) pour des CGR en Optisol (AS-5). CVF est un virus a ARN simple brin sens positif, genre Vesivirus , famille Caliciviridae et sert de modele pour les petits virus non enveloppes tels que le virus de l’hepatite E. Methodes Pour chaque experience, un CGR a ete contamine avec du CVF a une concentration finale de ∼10 8,0 pfu/mL. Les CGR contamines ont ete melanges avec le GSH et des echantillons de controle (T = 0) pris pour determiner les titres pre-amustaline. L’amustaline a ete melangee aux CGR et des echantillons tests (T = 3) ont ete preleves pour determiner les niveaux d’inactivation. Les echantillons controles et tests ont ete dilues en serie et inocules sur des cellules CrFK. Les plaques ont ete incubees 3 jours a 37 °C, colorees avec du cristal violet avant numeration. La reduction Log a ete calculee sur base de la difference entre le titre moyen dans des echantillons pre et post-amustaline a 3 h. Resultats Une inactivation robuste de CVF a ete obtenue comme resume dans le Tableau 1 . Conclusion Le Calicivirus felin non enveloppe a ete inactive a la limite de detection dans les CGR apres traitement avec le GSH et l’amustaline. Une inactivation de > 5,8 log de CVF a ete obtenue dans le modele d’infectivite CrFK.

Contexte Le traitement photochimique (PCT) de concentres de plaquettes avec amotosalen/UVA (INTERCEPT™ Blood System) remplace l’irradiation gamma (IRRG) dans des pays Europeens depuis > 10 ans pour reduire le risque de maladie du greffon contre l’hote liee a la transfusion. Objectif L’inactivation des cellules T par PCT et IRRG a ete comparee a l’aide d’un dosage par dilution limite sensible (DDL). Methode Des cellules mononuclees de sang peripherique (CMN) ont ete inoculees (10 6 /mL) dans des unites de plasma et inactivees en utilisant soit PCT ou IRRG, soit laissees non traitees. Les cellules traitees par PCT ou IRRG ont ete incubees (10 7 /mL) pendant 14 jours avec des CMN allo-stimulatrices melangees de 10 donneurs (5 × 10 6 traitees avec 7500 cGy) et des facteurs de croissance (PHA, IL-2). L’inactivation des cellules T par PCT (10 7 CMN/puits) a ete comparee a celle par IRRG (10 5 ou 10 6 CMN/puits) et a un controle non traite. La proliferation a ete evaluee par incorporation de 3H-thymidine (3H-Thy ; 6,7 Ci/mmol) dans les CMN (score sous microscope). Douze puits ont ete utilises pour chaque dilution et le protocole a ete effectue avec 6 replicats. Resultats Aucune croissance de cellules T pour les CMN inactivees par PCT n’a ete detectee avec 10 7 cellules cultivees/puits. Cependant, l’incorporation de 3H-Thy au-dessus du seuil de detection a ete observee avec 10 6 CMN IRRG/puits. Aucune incorporation n’a ete observee avec10 5 CMN IRRG/puits alors que 4/6 replicats ont montre une proliferation avec 10 6 CMN IRRG/puits. Conclusion L’inactivation des cellules T avec PCT (traitement amotosalen/UVA) est plus robuste (> 6,2 log 10 ) qu’avec 2500 cGy IRRG (> 4,2 log 10 – ≤ 5,2 log 10 ).

Pathogen inactivation (PI) is an alternative approach to blood safety that is gaining widespread use.1 PI for platelets (PLTs) has been in use in Europe for more than 10 years and one of these systems, the INTERCEPT Blood System, was recently approved by the Food and Drug Administration (FDA).2 There are costs for implementing PI, but it is also likely that there will be offsetting savings. The availability of PI PLTs may have substantial economic impact both in terms of operating costs for the facilities which collect and prepare them and in the acquisition costs to hospitals. The purpose of this study was to carry out a detailed cost analysis of the operational activities that may be affected by the adoption of PI PLTs and to project those that will apply to either blood centers or hospitals. The costs will affect hospitals in different ways depending on whether they purchase all their PLTs from an outside supplier or if they produce some fraction of the PLTs. While PI PLTs may reduce the costs associated with transfusion-transmitted disease treatment those costs were not considered in this project. This study focuses on PLTs collected by apheresis because in the most recent survey in the United States, 91% of the total of 2,516,000 PLT units was collected by apheresis.3 In the United States, blood centers collected approximately 89% of the apheresis PLT supply while hospitals collected 11%.3 Collection can be done with different devices and the PLTs can be stored for up to 5 days in either donor plasma or PLT additive solution and plasma mix at room temperature. When PLTs are collected at a blood center, testing is done along with standardized whole blood testing; however, when PLTs are collected at a hospital, samples are often sent to a different facility for testing. Bacterial screening of each unit of PLTs is performed at the place of collection but when performed, point-of-release (PoR) testing is usually done at the hospital.

Le traitement photochimique avec amotosalen et UVA (INTERCEPT Blood System) permet d’inactiver des agents pathogenes dans les plaquettes et le plasma. La compatibilite du procede avec divers types de plasma a ete testee. Methodes Sept publications etudiant la fonction coagulante de 6 types de plasma ont ete identifiees : plasma congele dans les 8 ou dans les 24 heures, collecte par apherese ou a partir de sang total, fabrique a partir de plasma frais ou prealablement congele, liquide, lyophilise ou appauvri en cryoprecipite. Les mesures d’une selection de parametres representant la qualite du plasma ont ete rassemblees : facteur VIII, fibrinogene, proteine C, proteine S, alpha 2-antiplasmine et generation de thrombine (ETP) a 5 pM de facteur tissulaire (FT). Resultats Le Tableau 1 montre les valeurs rapportees pour les 6 parametres de qualite du plasma choisis (quand testes). Le facteur VIII et le fibrinogene sont maintenus dans les valeurs de reference pour tous les types de plasma sauf le plasma appauvri en cryo et le plasma stocke liquide (FVIII) comme attendu. Les valeurs de proteine C, proteine S et d’alpha 2-antiplasmine sont dans les references. La capacite de generation de thrombine des differents produits est conservee ou peu modifiee apres traitement. Conclusion Le procede d’inactivation des agents pathogenes INTERCEPT peut etre applique avec succes aux produits plasmatiques de differentes sources ou formes, provenant de sang total ou d’apherese, congele, liquide, lyophilise ou appauvri en cryoprecipite. Les marqueurs de la fonction de coagulation du plasma sont conserves a des niveaux satisfaisants et la capacite de generation de thrombine n’est pas affectee.

Nucleic acid bulges have been implicated in a number of biological processes and are specific cleavage targets for the enediyne antitumor antibiotic neocarzinostatin chromophore in a base-catalyzed, radical-mediated reaction. The solution structure of the complex between an analog of the bulge-specific cleaving species and an oligodeoxynucleotide containing a two-base bulge was elucidated by nuclear magnetic resonance. An unusual binding mode involves major groove recognition by the drug carbohydrate unit and tight fitting of the wedge-shaped drug in the triangular prism pocket formed by the two looped-out bulge bases and the neighboring base pairs. The two drug rings mimic helical DNA bases, complementing the bent DNA structure. The putative abstracting drug radical is 2.2 +/- 0.1 angstroms from the pro-S H5' of the target bulge nucleotide. This structure clarifies the mechanism of bulge recognition and cleavage by a drug and provides insight into the design of bulge-specific nucleic acid binding molecules.

The base-catalyzed (bc) thiol-independent cleavage reaction of neocarzinostatin chromophore (NCS chrom) has been characterized with long single-stranded (ss) DNA in order to use this reaction as a selective probe for the tertiary structure of naturally occurring ss nucleic acids. The ss circular phi chi 174 phage and M13mp18 phage DNAs (approximately 5000 and 7500 bases, respectively) were shown to be bc NCS chrom reaction substrates, exhibiting the expected pH dependence. The ss DNA fragments (150-450 bases) were cleaved at six major sites; the lesions occurred at T-rich non-double-stranded sequences, as predicted from comparison with the minimal energy secondary structures. These sites exhibited the expected pH and drug: DNA ratio dependence shown to be required for this reaction. Optimization of the shortest sequence, which gave the highest cleavage yield, identified the minimal sequence requirements for the site (19-mer of the sequence 3'TACTGAGTCTCCTTTTGTA5', attacked residue in bold). Folding pattern analysis predicted that the oligonucleotide contained a two-base bulge at the cleavage site; this result was consistent with the observation that removing features which destabilize the bulged structure increased the cleavage yield. Furthermore, the derived 19-mer was shown to generate maximal amounts of the final drug product of the bc DNA cleavage reaction. Reaction of an RNA 339-mer containing the same sequence as one of the long ss DNA fragments showed it not to be a substrate for the bc reaction, while similar results were obtained for the RNA analog of shorter oligodeoxyribonucleotides identified in this and earlier studies. Through a combination of thermodynamic and kinetic assays, the observed difference in reactivity was shown to be the result of the low binding of the cleaving species to RNA.

Introduction : Depending on the conditioning intensity and graft source, mosthematopoietic stem cell transplant(HSCT) patients invariably experience a cytopenic phase of at least 2-3 weeks and require transfusion support until engraftment. All transfused blood components post-transplant need to be gamma irradiated to prevent transfusion associated graft versus host disease (TA-GVHD). Platelet component (PC) manufacturing methodologies differ around the globe. Currently, Hong Kong HSCT patients are supported with individual platelet rich plasma (PRP) PCs without leukocyte filtration (NLF), tested by short-term aerobic cultures for bacterial contamination (STABC), and gamma irradiated. When new PC manufacturing methodologies are considered for adoption, it is important to assess the immediate and longer term effects on transfused patients. Amotosalen+UVA light (A-UVA) pathogen reduction technology (INTERCEPT™ Blood System, Cerus Corporation, Concord, CA) inactivates bacteria, viruses, parasites and leukocytes by efficient covalent adduct modification of DNA and RNA. Treatment with A-UVA prevents TA- GVHD in an animal model, inhibits clonal T cell proliferation, prevents allogeneic antigen stimulation in mixed lymphocyte reactions, and inhibits transcription mediated cytokine production and early activation antigen expression (Corash et al, BMT 2004). The common strategy of prescribing gamma irradiated PC only for suspected "high risk" patients is sub-optimal, as failure to identify recipients at risk for TA-GVHD was responsible for 50% of reported cases (Kopolovic et al. Blood 2016). Treatment of PC with A-UVA addresses two key issues for HSCT patients: bacterial contamination, and universal prevention of TA-GVHD. Here, we compared the clinical support of HSCT patients with conventional PCs (C-PC) vs. an equivalent HSCT group supported with A-UVA PCs (I-PC) using a sequential patient cohort design within a single HSCT clinical center. Methods : I-PCs were prepared from 5 pooled ABO-matched, NLF buffy coat PCs in platelet additive solution and treated with A-UVA replacing gamma irradiation and bacterial detection. C-PCs were prepared from 5 ABO-matched, STABC-negative, NLF PRP PCs, and treated with gamma irradiation. Each patient could receive up to 5 PC transfusions. The primary efficacy endpoint was the 1-hour corrected count increment (CCI) and the primary safety endpoint was the proportion of patients (P) with acute transfusion reactions (ATR). Follow-up data on mortality, hematopoiesis engraftment, and immune status were collected up to 100 days post HSCT. Results: Patient demographics and type of HSCT were similar between patient cohorts. 33 patients received 76 A-UVA PCs and 31 patients received 89 C-PCs. The mean days of platelet support (p=0.618) and mean 1-hour CCIs per patient averaged for all transfusions were comparable (p=0.296) between the I- PC and conventional C-PC cohorts (Table). The proportion of patients with AEs was lower (p=0.021) for the I-PC group (Table), and no related SAEs were observed during the entirety of trial. Survival at 100 days post HSCT and rates of remission were similar between the cohorts (Table). The ATR rate trended lower, although not significantly different (p=0.296), in the I- PC group (Table). Follow-up data showed that the patients had comparable neutrophil and platelet engraftment (Table) with comparable immune system reconstitution by 100 days post HSCT. No cases of TA-GVHD were observed in either cohort. Conclusions: A-UVA-treated PCs prepared without LF, gamma irradiation, and bacterial detection can replace C-PCs for support of HSCT patients resulting in comparable post transfusion CCI responses and short and intermediate term clinical outcomes, while offering additional protection against transfusion transmitted bacteria and emerging or untested pathogens. Table Table. Disclosures Sim Pui Yin: Cerus Corporation: Other: Investigator sponsored trial. Leung Yuk Yan:Cerus Corporation: Other: Investigator sponsored trial. Lam Chun Kit:Cerus Corporation: Other: Investigator sponsored trial. Tsoi Wai:Cerus Corporation: Other: Investigator sponsored trial. Lee Cheuk:Cerus Corporation: Other: Investigator sponsored trial. Lie Kwok Wai:Cerus Corporation: Other: Investigator sponsored trial. Huang:Cerus Corporation: Employment. Rico:Cerus Corporation: Employment. Lin:Cerus corp: Employment. Corash:Cerus Corporation: Employment. Stassinopoulos:Cerus Corporation: Employment.

Introduction Le procede Intercept™ Blood System (IBS) plaquettes est utilise pour la preparation ex vivo de concentres plaquettaires d’apherese (CP) inactives pour un large spectre de virus enveloppes/non enveloppes, bacteries, spirochetes, parasites et leucocytes. Le mecanisme d’action se fait par generation d’adduits aux acides nucleiques qui inhibent la replication, la traduction et la transcription. Base sur ce mecanisme, nous emettons l’hypothese que l’inactivation des bacteries ne depend pas du profil de sensibilite aux antibiotiques, determine par des variations genomiques. Objectif Comparer l’inactivation de 2 souches de Klebsiella pneumoniae, genetiquement differentes, isolees chez des donneurs, avec des profils de sensibilite differents pour au moins 10 antibiotiques, dans des CP en solution PAS-3. Methodes Les dispositifs Intercept™ petit volume ont ete utilises, 150 μM d’amotosalen et illumination UVA (3 J/cm2). Deux souches de K. pneumoniae ont ete selectionnees basees sur des profils de sensibilite differents aux antibiotiques. La PCR ADNr 16s et l’analyse de la membrane de Klebsiella pneumoniae confirment le genre et l’espece. Les tests de sensibilite aux antibiotiques montrent une difference de phenotype entre les souches. Les souches ont ete cultivees jusqu’a des titres tres eleves (9–10 log10/mL) et diluees au 1/100 dans les CP. Un echantillon de controle a ete preleve avant l’illumination et la viabilite bacterienne a ete determinee sur plaque de culture en milieu agar en parallele avec differentes dilutions des echantillons tests obtenus apres traitement. L’inactivation a ete calculee en comparant les titres des controles et des tests. Resultats Les profils de sensibilite aux antibiotiques de K. pneumoniae sont presentes dans le Tableau 1 , les resultats d’inactivation dans le Tableau 2 . Conclusions L’inactivation de K. pneumoniae par Intercept™ Blood System pour plaquettes est robuste et ne depend pas du profil de sensibilite aux antibiotiques.

Introduction Le virus du chikungunya (CHIKV) est un arbovirus a ARN, genre Alphavirus , famille Togaviridae . La transmission d’une infection a CHIKV par transfusion est un probleme de sante dans les regions endemiques bien que non encore demontree. L’objectif de l’etude etait d’evaluer l’inactivation du CHIKV par le procede Intercept TM Blood System (IBS) pour les concentres de globules rouges (CGR) prepares en Optisol (AS-5). IBS pour CGR (en developpement) utilise une molecule d’amustaline pour former des liaisons covalentes au sein des acides nucleiques des leucocytes et agents pathogenes contaminants pour empecher leur replication. Avant incubation 18–24 h a temperature ambiante, 0,2 mM amustaline/20 mM glutathion (GSH) sont ajoutes, centrifugation et echange du surnageant avec une solution additive (SAG-M). L’inactivation est complete au bout de 3 h. Le temps additionnel assure une decomposition complete de l’amustaline. Methodes Pour chaque experience, un CGR a ete contamine par le CHIKV a une concentration finale de ∼10 7,0 pfu/mL et traite avec l’amustaline. L’echantillon controle etait preleve avant addition de l’amustaline et dilue en serie jusqu’a 10 −7 . Les dilutions ont ete inoculees sur des cellules Vero76 pour determiner le titre initial. L’amustaline a ete ensuite ajoutee et un echantillon preleve apres 3 heures pour determiner le niveau d’inactivation. Les echantillons tests ont ete dilues de 10 −1 a 10 −3 et inocules sur cellules Vero76. Les plaques ont ete incubees 5 jours a 37 °C, colorees avec du cristal violet avant numeration. La reduction Log a ete calculee soit la difference entre le titre moyen dans les echantillons pre- et post-amustaline a 3 heures. Resultats Une inhibition robuste de l’infectivite au CHIKV a ete atteinte ( Tableau 1 ). Conclusions Le CHIKV a ete inactive a la limite de detection dans les CGR apres traitement avec l’amustaline et le GSH. L’inactivation > 7,1 log du CHIKV a ete obtenue dans le modele d’infectivite Vero76.

Introduction Le procede par traitement photochimique utilisant l’amotosalen et les rayons ultraviolets (UVA), developpe pour inactiver les pathogenes et les leucocytes residuels dans les plasmas et les concentres plaquettaires (CP), est le premier systeme de reduction des pathogenes approuve par la FDA aux Etats-Unis. Initialement, ce procede etait approuve pour des CP prepares dans 65 % de solution additive (PAS). L’objectif de cette etude etait d’evaluer l’inactivation du Plasmodium falciparum (Pf) dans les CP suspendus dans 100 % de plasma pour etendre l’approbation. Pf est une des especes du parasite intracellulaire Plasmodium qui affecte specifiquement les globules rouges (GR) et provoque le paludisme. Methodes Pour chaque experience, un CP d’apherese dans 100 % plasma (∼ 365 mL) a ete inocule avec des GR humains infectes par du Pf (GRi), stade en anneau, a une concentration finale de ∼10 6 GRi/mL. Un echantillon controle de 80 mL a ete preleve, puis l’amotosalen ajoute, l’unite illuminee et un echantillon post-traitement a ete preleve. Pour les echantillons de controle et de test, les GRi ont ete isoles par separation Ficoll, resuspendus et dilues en serie, a 10 −8 pour le controle et a 10 −3 pour le test, dans des flacons contenant un milieu avec 5 % de GR frais. Les echantillons dilues etaient utilises pour inoculer les flacons en quatre exemplaires. La parasitemie etait controlee par comptage des GRi sur frottis sanguins et cytometrie de flux. Les cultures etaient terminees quand parasitemie > 1 %. La reduction en log correspond a la difference entre le titre moyen des echantillons avant et apres UVA. Resultats Le niveau d’inactivation du P. falciparum obtenu dans les plaquettes prelevees dans 100 % de plasma ( n = 4) est robuste ( Tableau 1 ). Conclusion Le traitement photochimique de CP dans 100 % plasma avec amotosalen et UVA inactive > 7,9 log de P. falciparum . Ces resultats sont comparables avec les ≥ 6,0 log obtenus dans les plaquettes avec 65 % de PAS.

Introduction La reaction du greffon contre l’hote transfusionnelle (TA GVHD) est causee par les lymphocytes T contaminants presents dans les produits sanguins. L’irradiation gamma (IRRG) avec 2500 cGy est utilisee pour prevenir la TA GVHD. Pour les concentres de plaquettes (CP) et le plasma, une methode alternative a IRRG est le traitement photochimique (PCT) avec amotosalen/UVA (Intercept™ Blood System [IBS]) utilise en Europe depuis plus de 10 ans. Une autre methode en developpement pour les concentres de globules (CGR) est le procede IBS avec amustaline et GSH. Dans cette etude, nous avons evalue la capacite de l’amustaline/GSH d’inactiver les lymphocytes T dans les CGR pour prevenir la TA GVHD. Methodes L’inactivation de lymphocytes T isoles de CGR traites par amustaline/GSH (0,2 mM/20 mM) avec incubation a temperature ambiante pendant 18–24 h suivie d’une centrifugation et de l’echange du surnageant avec la solution additive AS-5 a ete testee. Une methode de dilution limite (LDA) comportant l’incorporation d’un analogue ethinyl desoxyuridine (EDU) fluorescent de thymidine en presence de cellules allostimulatrices (Click-iT ® ) et identification des cellules viables en cytometrie de flux a ete utilisee. Resultats L’analyse a montre un signal fort (1–8 × 10 4 cellules) associe a l’analyse des cellules viables non traitees qui ne pouvait etre observe que lorsque les cellules allostimulatrices, l’EDU et le marquage fluorescent etaient utilises ; tandis que le signal obtenu etait negligeable lorsque l’un des reactifs manquait. Pas de signal (1–7 cellules) n’a ete observe avec les echantillons tests (traites amustaline/GSH), ce qui indique que les cellules ont ete incapables de proliferer. Il est prevu que si l’incubation des cellules est prolongee pendant 14 jours, une plage dynamique plus grande sera atteinte. Conclusions Le traitement des cellules T avec amustaline/GSH resulte dans l’inactivation de ≥ 4 log 10 de cellules T vs controle GSH seul.

The glutathione post-activated neocarzinostatin chromophore (NCSi-glu)-DNA complex was studied in detail by 2-D NMR spectroscopy. The complex is a model for understanding the sequence specific cleavage of DNA by the native neocarzinostatin chromophore (NCS chrom), a highly potent enediyne antitumor agent. NMR spectral analysis is presented for the free NCSi-glu, the free DNA duplex and the NCSi-glu-DNA complex. In addition to the previously reported structural details of the complex (Gao, X.; Stassinopoulos, A.; Rice, J. S.; Goldberg, I. H. Biochemistry 1995, 34, 40), we demonstrate that the binding of NCSi-glu in minor groove results in a patch of negatively charged surface covering the otherwise relatively neutral minor groove. The formation of the complex is largely driven by hydrophobic forces and the solvation of the polar surface of the complex. Comparison of the conformations of NCSi-glu and DNA duplex in their free and bound form reveals an induced mutual fit of DNA and NCSi-glu upon complex formation. The reduced NCS chrom represents a DNA binding motif for sequence specific recognition of DNA via intercalation and minor groove interactions.

It is shown by fluorescence spectroscopy that the post-activated form of neocarzinostatin chromophore (NCSi-glu) can form stable complexes with single-site oligonucleotides (SSOs) featuring sequences known to be involved in double stranded (AGC·GCT, AGT·ACT, AGA·TCT, ACA·TGT) or single stranded (AGG·CCT) cleavage (attacked residues in bold). Furthermore, the same SSOs form cleavage productive complexes with native neocarzinostatin chromophore (NCS chrom) over a similar concentration range. The productive complexes yield damage similar to that observed if the same sequence is part of a longer DNA piece. Previously identified double stranded site sequences ATT·AAT and TAT·ATA are shown to contain overlapping attack sites. Binding order preference derived from fluorescence quenching experiments for NCSi-glu is consistent with constants derived by quantitative cleavage affinity binding experiments with NCS chrom. This confirms the similarity in interactions between the NCSi-glu and NCS chrom and justifies the use of NCSi-glu as a stable analog of NCS chrom.

We performed a risk-benefit analysis for implementation of pathogen-inactivated (PI) apheresis platelets (APs) in the United States, focusing on the amotosalen/ultraviolet-A system. Risks and benefits were quantified per patient assuming a mean of 6 AP units per treatment cycle and using available clinical data, mathematical modeling, and observational studies. Current risks associated with AP transfusion can be divided into known partially addressed risks, known well-addressed risks, and unknown risks associated with acute or chronic emerging infectious agents (EIAs). Bacterial contamination dominates the first category, at a per-patient rate of 1:250, which correlates with an estimated septic transfusion reaction rate of 1:1000. Quantitation of per-patient EIA risk was modeled to be between 1:370 (acute) and 1:667 (chronic). Due to its broad range of action PI is expected to reduce or eliminate these infectious risks and also to reduce the rate of febrile transfusion reactions and possibly alloimmunization. These benefits are weighed against 1) concerns for excess bleeding, 2) an apparent increase in acute respiratory distress syndrome in the initial report of the SPRINT clinical trial, and 3) the possible toxicity associated with the introduction of a new chemical into platelet (PLT) units. However, transfusion of an estimated 100,000 patients with PI PLTs worldwide has occurred without reported serious adverse effects. We conclude that evidence indicates a favorable risk-benefit profile for the implementation of PLT PI and argues for a path forward toward US regulatory approval.

Detailed structure determination of the major and minor base-catalyzed degradation products of the chromophore of the enediyne anticancer antibiotic neocarzinostatin in the absence of DNA demonstrates that the enolate Michael addition reaction leading to a spirolactone cumulene intermediate is a spontaneous, stereoselective process. The implications of these findings for the mechanism of the thiol-independent, site-specific cleavage by the so-generated radical species of the drug at a DNA bulge are described.

The objective of this study was to examine the effects of polyethylene glycol (PEG) treated red blood cells (RBCs) on the microcirculation in a hamster back skin window chamber model. Donor hamster RBCs were PEGylated through an incubation with an activated PEG solution, washed, resuspended, and infused through a 10% volume top loading procedure into the carotid artery in an awake Syrian Golden hamster. Eight hamster groups were treated with activated PEG different sizes and concentrations: 0.05 mM-5 kDa PEG, 0.5 mM-5 kDa PEG, 1.1 mM-5 kDa PEG, 2.2 mM-5 kDa PEG, 22 mM-5 kDa PEG, 0.05 mM-20 kDa PEG, 0.5 mM-20 kDa PEG, and 5 mM-20 kDa PEG. Non-treated RBCs were used as control. The microvascular bed under observation was videotaped 30 min before the infusion and followed for 30 min post infusion. The diameter of individual blood vessels and blood flow velocities in selected vessels was measured. Hematocrit and hemoglobin concentration were recorded before infusion and at the end of experiment. Tissue pO(2) was also monitored. Results showed the hamsters tolerated the PEGylated RBCs without apparent ill effects. No significant changes were recorded for the hematocrit, the hemoglobin concentration, the blood vessel diameters, blood flow velocities, and the interstitial partial oxygen pressure (pO(2)) before, during, and after the injections of PEG-RBCs (P0.05). Unlike most hemoglobin-based oxygen carrying compounds, which can cause vasoconstriction, the PEGylated RBCs did not produce any measurable vasoactivity. Together with the absence of rouleaux formation and the fact that PEG molecules can mask the surface antigens on RBCs, PEGylation appeared promising as a circulation enhancement treatment.

The subject invention provides a binuclear metal complex having structure (I) wherein M1, and M2 are independently selected from the group consisting of Fe, Co, Mn and Ru; wherein m and n are independently +2 or +3; wherein R1, R2, R3, R4, R5 and R6 are independently a linear C1-C6 alkyl, C5-C6 cycloalkyl, phenyl, etc.; wherein (i) R1 and R2, (ii) R3 and R4, or (iii) R5 and R6 independently and optionally are linked covalently and together with the respective adjoining C atom comprise a spirocyclic ring; wherein i, j and k are integers such that 2≤i + j + k≤4; wherein p is 1 or 2, and q is 0, 1 or 2 such that m + n-4=p x q; wherein (i) R1 or R2 and R3 or R4, (ii) R3 or R4 and R4 or R5, or (iii) R1 or R2 and R5 or R6 independently and optionally are linked covalently and together with the respective adjoining C atoms comprise a fused ring; wherein Ar is 1,2-phenylene, 1,2- or 2,3-naphthylene, etc., wherein said Ar is optionally substitued by C1-C6 alkyl or alkoxy; wherein L is N-methylimidazole, N-ethylimidazole, etc.; and wherein X is fluorine, chlorine, bromine, etc. Also provided are methods of oxidation of alkanes, arenes, and sulfides using the binuclear metal complex as a catalyst and a method of preparing said complex.

Our previous structure elucidation of the complexes of DNA and postactivated neocarzinostatin chromophore (NCS-chrom) compounds revealed two distinctly different binding modes of this antitumor molecule. A thorough understanding of these results will provide the molecular basis for the binding and DNA chain cleavage properties of NCS-chrom. NCSi-gb is one of the postactivated mimics of NCS-chrom which is formed under thiol-free conditions and is able to bind to DNA. This report describes the structure refinement of the NCSi-gb-bulge-DNA complex [Stassinopoulos, A., Jie, J., Gao, X., and Goldberg, I. H. (1996) Science 272, 1943-1946] and the NMR characterization of the free bulge-DNA and free NCSi-gb. These results reveal that the formation of the complex involves conformational changes in both the DNA and the ligand molecule. Of mechanistic importance for the NCS-chrom-DNA interaction, the two ring systems of the drug are brought closer to each other in the complex. This conformation correlates well with the previously observed marked enhancement of the formation of a DNA bulge cleaving species in the presence of bulge-DNA sequences, due to the promotion of the intramolecular radical quenching of the activated NCS-chrom. Interestingly, the binding of NCSi-gb promotes the formation of a bulge binding pocket; this was not found in the unbound DNA. NCS-chrom is unique among the enediyne antibiotics in its ability to undergo two different mechanisms of activation to form two different DNA binding and cleaving species. The two corresponding DNA complexes are compared. One, the bulge-DNA binder NCSi-gb, involves the major groove, and the second, the duplex binder NCSi-glu which is generated by glutathione-induced activation, involves the minor groove. Since the two NCS-chrom-related ligand molecules contain some common chemical structural elements, such as the carbohydrate ring, the striking differences in their DNA recognition and chain cleavage specificity provide insights into the fundamental principles of DNA recognition and ligand design.