Pathogen Reduction Technology Research Articles

Improvement of Blood Processing and Safety by Automation and Pathogen Reduction Technology

Introduction: The objective of the present study was to describe the experience of the Blood and Tissues Bank of Aragon with the Reveos® Automated Blood Processing System and Mirasol® Pathogen Reduction Technology (PRT) System, comparing retrospectively routine quality data obtained in two different observation periods. Methods: Comparing quality data encompassing 6,525 blood components from the period 2007–2012, when the semi-automated buffy coat method was used in routine, with 6,553 quality data from the period 2014–2019, when the Reveos system and subsequently the Mirasol system were implemented in routine. Results: Moving from buffy coat to Reveos led to decreased discard rates of whole blood units (1.2 to 0.1%), increased hemoglobin content (48.1 ± 7.6 to 55.4 ± 6.6 g/unit), and hematocrit (58.9 ± 6.5% to 60.0 ± 4.9%) in red blood cell concentrates. Platelet concentrates (PCs) in both periods had similar yields (3.5 ×10¹¹). Whereas in the earlier period, PCs resulted from pooling 5 buffy coats, in the second period 25% of PCs were prepared from 4 interim platelet units. The mean level of factor VIII in plasma was significantly higher with Reveos (92.8 vs. 97.3 IU). Mirasol PRT treatment of PCs reduced expiry rates to 1.2% in 2019. One septic transmission was reported with a non-PRT treated PCs, but none with PRT-treated PCs. Conclusion: Automation contributed to standardization, efficiency, and improvement of blood processing. Released resources enabled the effortless implementation of PRT. The combination of both technologies guaranteed the self-sufficiency and improvement of blood safety.

Measurement of Tissue Oxygen as a Novel Approach to Optimizing Red Blood Cell Quality Assessment.

The effectiveness of blood transfusions can be impacted by storage and extensive processing techniques that involve treatment of red blood cells (RBCs) with pathogen reduction technologies (e.g., UV-light and chemical treatment), ex vivo stem cell derivation/maturation methods, and bioengineering of RBCs using nanotechnology. Therefore, there is a need to have methods that assess the evaluation of the effectiveness of transfusions to achieve their intended purpose: to increase oxygenation of critical tissues. Consequently, there has been intense interest in the development of techniques targeted at optimizing the assessment of RBC quality in preclinical and clinical settings. We provide a critical assessment of the ability of currently used methods to provide unambiguous information on oxygen levels in tissues and conclude that they cannot do this. This is because they are based on surrogates for the true goal of transfusion, which is to increase oxygenation of critical organs. This does not mean that they are valueless, but it does indicate that other methods are needed to provide direct measurements of oxygen in tissues. We report here on the initial results of a method that can provide direct assessment of the impact of the transfusion on tissue oxygen: EPR oximetry. It has the potential to provide such information in both preclinical and clinical settings for the assessment of blood quality posttransfusion.

Blood transfusion service in Poland in 2019

Background: In this study we evaluated the basic aspects of the activity of the Polish Blood Transfusion Service (hereinafter referred to as Centers) in 2019. Materials and methods: Retrospective analysis of the 2019-data supplied by the Centers. Results: In 2019, blood and blood components were collected in 21 Polish Centers and 129 local collection sites as well as during 13 048 mobile collections. The overall number of blood donors was estimated at 590 893, the majority of which were non-remunerated donors (590 280 — including 40 738 responders to donation appeals), as well as 57 remunerated donors and 556 autologous donors. Most frequent were whole blood collections (1 202 079) and least frequent — granulocyte concentrate collections (94) and RBCs collections by apheresis (48 donations). Whole blood was collected mostly in local collection sites (45.25%), in Centers (28.75%) and mobile collection sites (26%). Most frequently prepared blood components were RBCs — 1 180 333 units) and fresh frozen plasma (FFP — 1 344 092 units, 21.42% dedicated for clinical use). Platelet concentrates (PCs) collected by apheresis amounted to 53 379 units and 81 905 were whole blood-derived. Additional processing methods (leukocyte depletion, irradiation) were more frequently applied to PCs (43.34% leukocyte-depleted, 0.04% irradiated, 54.63% both leukocyte-depleted and irradiated) than to RBCs (18.71% leukocyte-depleted, 0.06% irradiated, 9.17% both leukocytedepleted and irradiated). Pathogen reduction technologies were applied to 20.76% of FFP units issued for clinical use (transfusion) and 11.87% of PCs. In 2019 — for a variety of reasons — 14 013 units of whole blood, 32 798 units of RBCs, 49 285 units of FFP, 1334 units of cryoprecipitate, 4778 units of pooled PCs and 1279 of apheresis PCs were wasted. Conclusions: Our study data may be considered as starting point for assessment of the tendencies observed in the Polish blood transfusion service and may serve practical-benchmarking with benefit to the transfusion community as a whole.

On the Quest for In Vitro Platelet Production by Re-Tailoring the Concepts of Megakaryocyte Differentiation.

The demand of platelet transfusions is steadily growing worldwide, inter-donor variation, donor dependency, or storability/viability being the main contributing factors to the current global, donor-dependent platelet concentrate shortage concern. In vitro platelet production has been proposed as a plausible alternative to cover, at least partially, the increasing demand. However, in practice, such a logical production strategy does not lack complexity, and hence, efforts are focused internationally on developing large scale industrial methods and technologies to provide efficient, viable, and functional platelet production. This would allow obtaining not only sufficient numbers of platelets but also functional ones fit for all clinical purposes and civil scenarios. In this review, we cover the evolution around the in vitro culture and differentiation of megakaryocytes into platelets, the progress made thus far to bring the culture concept from basic research towards good manufacturing practices certified production, and subsequent clinical trial studies. However, little is known about how these in vitro products should be stored or whether any safety measure should be implemented (e.g., pathogen reduction technology), as well as their quality assessment (how to isolate platelets from the rest of the culture cells, debris, microvesicles, or what their molecular and functional profile is). Importantly, we highlight how the scientific community has overcome the old dogmas and how the new perspectives influence the future of platelet-based therapy for transfusion purposes.

PMD13 A Health Economic Evaluation of the Impact of Pathogen Reduction Technology on Emerging Pathogens within the Platelet Collection and Transfusion Space

Objectives: Given the recent COVID-19 outbreak our objective was to assess the role a European installed base of pathogen reduction technology (PRT) could have on controlling the potential devastating impact of future bloodborne emerging pathogens In specific we wanted to assess its impact within the platelets collection and transfusion environment Methods: Based on literature review, the characteristics of common bloodborne pathogens (e g the basic reproduction number, mortality and morbidity), current blood testing (BT) capabilities (e g blood test availability and sensitivity), pathogen reduction levels (e g log kill rate) as well as cost information in case of infection, were identified and modelled to assess the potential scenarios from a direct and indirect costs point of view The assessment was performed for platelets collected within the current EU countries Results: Assuming 2 million platelet collections and in case a new pathogen emerges for which no sensitive BT is immediately available PRT could, within the first wave of infections, prevent 3696 infections through blood transfusion and avoid 2634 infections from transfused patients to others Avoiding these infections would reduce health care expenditures by 97 million euro and save 25 000 days of hospitalization Up to 1500 lives would be saved which would have an immediate positive economic impact of 74 million euro Indirectly PRT would save 46189 days of productivity and reduce overall indirect morbidity costs by 19,6 million euro Conclusions: In case of an emerging pathogen for which no highly sensitive DT is directly available, PRT and its known ability to avoid transfusion transmitted infections in platelets, may facilitate substantial economic and societal savings As we only investigated for platelets, future research that considers plasma collections or red blood cell collections may be relevant Country differences as well as differences based on the procedure to collect platelets may occur

Complete Inactivation of Blood Borne Pathogen Trypanosoma cruzi in Stored Human Platelet Concentrates and Plasma Treated With 405 nm Violet-Blue Light.

The introduction of pathogen reduction technologies (PRTs) to inactivate bacteria, viruses and parasites in donated blood components stored for transfusion adds to the existing arsenal toward reducing the risk of transfusion-transmitted infectious diseases (TTIDs). We have previously demonstrated that 405 nm violet-blue light effectively reduces blood-borne bacteria in stored human plasma and platelet concentrates. In this report, we investigated the microbicidal effect of 405 nm light on one important bloodborne parasite Trypanosoma cruzi that causes Chagas disease in humans. Our results demonstrated that a light irradiance at 15 mWcm−2 for 5 h, equivalent to 270 Jcm−2, effectively inactivated T. cruzi by over 9.0 Log10, in plasma and platelets that were evaluated by a MK2 cell infectivity assay. Giemsa stained T. cruzi infected MK2 cells showed that the light-treated parasites in plasma and platelets were deficient in infecting MK2 cells and did not differentiate further into intracellular amastigotes unlike the untreated parasites. The light-treated and untreated parasite samples were then evaluated for any residual infectivity by injecting the treated parasites into Swiss Webster mice, which did not develop infection even after the animals were immunosuppressed, further demonstrating that the light treatment was completely effective for inactivation of the parasite; the light-treated platelets had similar in vitro metabolic and biochemical indices to that of untreated platelets. Overall, these results provide a proof of concept toward developing 405 nm light treatment as a pathogen reduction technology (PRT) to enhance the safety of stored human plasma and platelet concentrates from bloodborne T. cruzi, which causes Chagas disease.

The Fares Study: A Multicenter, Randomized, Active-Control, Pragmatic, Phase 2 Pilot Study Comparing Prothrombin Complex Concentrate Versus Frozen Plasma in Bleeding Adult Cardiac Surgical Patients

Introduction: Coagulopathic bleeding is a frequent complication of cardiac surgery and can lead to excessive blood loss, blood product transfusion, and bleeding-related complications. Hemostatic management in bleeding surgical patients is evolving towards targeted therapy with purified products undergoing pathogen reduction technology. Unlike frozen plasma (FP), purified prothrombin complex concentrates (PCCs) do not require ABO blood type matching or thawing, are pathogen-reduced, and are associated with a lower risk of transfusion-associated circulatory overload and transfusion-related acute lung injury. The FARES study will compare the efficacy and safety of PCC (Octaplex; Octapharma) versus FP in bleeding cardiac surgical patients with confirmed or suspected coagulopathy. Preliminary non-randomized studies suggest PCC may be superior to plasma for management of bleeding after cardiac surgery. Methods: The randomized, active-control, single-blinded, pragmatic, investigator-initiated, Phase 2 FARES pilot study will enrolled 120 bleeding adult cardiac surgery patients requiring coagulation factor replacement from two hospitals to achieve 100 treated patients. The study outline is shown in Figure 1. Patients were randomized to PCC or FP, with two weight-based doses, for management of bleeding where coagulation factor deficiency is known or suspected (patients over 50 kg were transfused either 2000 IU of PCC or 4 units of plasma; patients under 50 kg were transfused 1500 IU of PCC or 3 units of plasma). Required second doses will be given according to randomization group allocation, but all patients will receive FP for their third and any subsequent doses. The primary endpoint is hemostatic effectiveness and transfusion requirements. Adverse events will be recorded from the beginning of surgery. Results: The study commenced in September of 2019 and completed enrollment of the last patient in June of 2020. Results were monitored and overseen by the Independent Data and Safety Monitoring Committee. The database will be locked in August 2020 after 28 day follow-up is complete for all patients. Statistical analysis plan is complete. The results will inform a definitive, pragmatic, multi-center trial to determine if PCCs can replace plasma for the management of coagulopathy in the bleeding cardiac surgery patient. Conclusions: This study will determine the feasibility, and inform the design and primary outcome parameter, of a definitive Phase 3 trial comparing the efficacy and safety of PCC versus FP in bleeding cardiac surgical patients requiring coagulation factor replacement. The pilot study results will also be used in sample size calculations and to aid detection of any safety issues. Disclosures Karkouti: Octapharma: Research Funding; Canadian blood services: Research Funding. Callum:Canadian Blood Services: Research Funding; Octapharma: Research Funding. OffLabel Disclosure: Coagulation factor replacement with Prothrmobin Complex Concentrates (off-label in the USA and on label in Canada/Europe)

Frozen Platelets-Development and Future Directions.

Storage requirements and outdating of platelets represent a continued challenge for blood banks. These hurdles are confounded for rural area hospitals or in military deployments. Over 60 years of research and development into frozen platelets have generated a stable and reproducible product. Valeri's method to freeze platelets in 6% dimethyl sulfoxide (DMSO) and storage at -80°C allows for long-term storage alleviating burdens placed on blood banks. Clinical studies show that frozen platelet transfusions are safe with no related thrombotic or other serious adverse events. There are ongoing efforts to demonstrate cryopreserved platelet (CPP) superiority in efficacy studies designed in trauma or cardiac surgery patients. Technical advances in CPP manufacturing including closed system manufacturing, applications of pathogen reduction technology and potency standard characterization add to the appeal of CPP as an alternative to traditional liquid-stored platelets (LP) in settings of supply shortages, mass casualty, active bleeding, rapid provision of HLA-compatible platelets, and remote care.

Analysis of the mechanism of damage produced by thiazole orange photoinactivation in apheresis platelets.

Pathogen Reduction Technologies (PRTs) are broad spectrum nucleic acid replication-blocking antimicrobial treatments designed to mitigate risk of infection from blood product transfusions. Thiazole Orange (TO), a photosensitizing nucleic acid dye, was previously shown to photoinactivate several types of bacterial and viral pathogens in RBC suspensions without adverse effects on function. In this report we extended TO treatment to platelet concentrates (PCs) to see whether it is compatible with in vitro platelet functions also, and thus, could serve as a candidate technology for further evaluation. PCs were treated with TO, and an effective treatment dose for inactivation of Staphylococci was identified. Platelet function and physiology were then evaluated by various assays in vitro. Phototreatment of PCs yielded significant reduction (≥4-log) in Staphylococci at TO concentrations ≥20 μM. However, treatment with TO reduced aggregation response to collagen over time, and platelets became unresponsive by 24 hours post-treatment (from >80% at 1 h to 0% at 24 h). TO treatment also significantly increased CD62P expression (<1% CD62P+ for untreated and >50% for TO treated at 1 h) and induced apoptosis in platelets (<1% Annexin V+ for untreated and >50% for TO treated at 1 h) and damaged mitochondrial DNA. A mitochondria-targeted antioxidant and reactive oxygen species (ROS) scavenger Mito-Tempo mitigated these adverse effects. The results demonstrate that TO compromises mitochondria and perturbs internal signaling that activates platelets and triggers apoptosis. This study illustrates that protecting platelet mitochondria and its functions should be a fundamental consideration in selecting a PRT for transfusion units containing platelets, such as PCs.

Rapid response platform to manufacture human immunoglobulins

Abstract A rapidly deployable drug product for use in the early stages of a disease outbreak could prevent further spread of the pathogen. Emergent BioSolutions is developing a rapid response platform (RRP) to manufacture human immunoglobulins (HIG) for use as a passive immunotherapy during public health emergencies. Passive immunotherapies derived from either convalescent plasma or purified immunoglobulins (Ig) have a long history of success in treating a range of viral, bacterial and toxin diseases, as they provide the immediate benefit of a protective response. Emergent's RRP is being developed to address the need for front-line therapy/prophylaxis with immediate benefit with the potential to overcome the current limitations of convalescent plasma. Emergent's proposed RRP begins with identification of plasma donors with measurable levels of pathogen-specific antibodies using a field deployable real-time donor screening assay. Plasma collected from eligible donors is treated using the Mirasol® Pathogen Reduction Technology, pooled, purified and concentrated into HIG using Emergent's rapid HIG manufacturing process to produce product formulated for intramuscular administration. Our rapid HIG process is chromatography based and each 30 L run aims to purify, formulate, and fill ∼65 doses (1 g of total IgG) with testing and release onsite for immediate use. Multiple innovations to streamline operations, including single-use technologies and minimal human interfaces, allow for the manufacture of HIG product in ∼30 hours. To house the process, Emergent has designed and constructed a working modular manufacturing unit (MMU) prototype. The MMU consists of a modified 53-foot shipping container designed for multiple modes of transport, to meet ISO 8 environment requirements with self-contained utilities, including HVAC, and can run off a diesel generator or local power. Product from multiple development runs within the MMU have been characterized and validation work is ongoing. Key messages A modular manufacturing unit is being developed with the potential to provide a local capability to produce health solutions for endemic diseases. A modular manufacturing unit is being developed with the potential to rapidly manufacture human immunoglobulins during public health emergencies and pandemics.

Effects of riboflavin and ultraviolet light treatment on pathogen reduction and platelets.

Pathogen reduction technology has become an accepted method for limiting transfusion-transmitted infections (TTIs). Riboflavin and ultraviolet light (RUV) treatment of platelets (PLTs) is an optional means of pathogen inactivation owing to its safety, effectiveness, and ease of use. However, the literature on effects of ultraviolet (UV) light spectra and doses on pathogen reduction is still contradictory. We tested the effectiveness of killing Escherichia coli following RUV exposure with one broad-spectrum and two narrow-spectrum light sources centered at 311 and 365 nm and at successively higher doses by limited dilution survival assays. After comparing the effectiveness of E coli and phage inactivation (n = 6) and the changes in PLT count and metabolism caused by RUV treatment with optimized UV light at increasing doses, we confirmed our results by using four model virus systems that represent common TTIs, as well as PLT function and activation assays at an optimized light dose. The narrow-spectrum UV, centered at 311 nm, optimally reduced the E coli titer with a light dose ≥8.11 J/mL, resulting in the same trend of E coli and phage reduction at different light doses. At 8.11 J/mL, 311-nm narrow-spectrum UV had a good inactivation effect on E coli and phages, eliminating many viruses, and resulted in acceptable PLT quality after RUV treatment and during storage for 4 days. Our data suggest restricting exposure to narrow-spectrum UV centered at 311 nm can increase E coli elimination and potentially optimize virus titer reduction without significantly compromising PLT quality.

Convalescent Plasma Therapy for COVID-19: State of the Art

Convalescent plasma (CP) therapy has been used since the early 1900s to treat emerging infectious diseases; its efficacy was later associated with the evidence that polyclonal neutralizing antibodies can reduce the duration of viremia. Recent large outbreaks of viral diseases for which effective antivirals or vaccines are still lacking has renewed the interest in CP as a life-saving treatment. The ongoing COVID-19 pandemic has led to the scaling up of CP therapy to unprecedented levels. Compared with historical usage, pathogen reduction technologies have now added an extra layer of safety to the use of CP, and new manufacturing approaches are being explored. This review summarizes historical settings of application, with a focus on betacoronaviruses, and surveys current approaches for donor selection and CP collection, pooling technologies, pathogen inactivation systems, and banking of CP. We additionally list the ongoing registered clinical trials for CP throughout the world and discuss the trial results published thus far.

Apoptosis as an underlying mechanism in lymphocytes induced by riboflavin and ultraviolet light

Riboflavin plus UV light pathogen reduction technology (RF-PRT) is an effective method for inactivating donor-derived leukocytes (DDLs) in blood components. Literature data have shown that reactive oxygen species (ROS) increased in lymphocytes after RF-PRT treatment. Sustained high levels of ROS may abolish the endogenous antioxidant system, leading to damage to proteins, lipids, and nucleic acids, resulting in cell apoptosis. Nevertheless, whether riboflavin plus UV light can trigger leukocyte apoptosis remains obscure. In this study, a pool-and-split design, ABO/D-matched lymphocytes treated with RF-PRT or UV light or left untreated. After treatment, the level of ROS and intracellular calcium were measured in samples. Changes in the protein expression of cleaved PARP, Bax, and Bcl-2 and the activities of caspase-3 and caspase-9 were determined by immunoblot analysis or luminometer, respectively. Cell apoptosis was evaluated by flow cytometry. The effect of ROS on apoptosis was assessed. The RF-PRT treatment significantly augmented ROS production, intracellular calcium concentration. The pro-apoptotic proteins expression levels of Bax, but did not the anti-apoptotic protein Bcl-2, were markedly increased after the RF-PRT treatment.Furthermore, the percentage of apoptotic cells was increased in RF-PRT-treated lymphocytes compared to UV-treated cells or untreated cells. Moreover, the inhibition of ROS generation partially neutralized the apoptosis effects of riboflavin plus UV treatment. These findings revealed that RF-PRT-treated lymphocytes significantly increase the proportion of apoptotic cells by promoting ROS generation delineation of the biochemical processes influenced by RF-PRT are a necessary step to provide novel insights into the riboflavin pathogen inactivation technology.

3062 – ASSESSING COMPATIBILITY OF ANTIMICROBIAL VIOLET-BLUE LIGHT FOR PATHOGEN REDUCTION OF RED BLOOD CELL CONCENTRATES

Transfusion-related sepsis is the most frequent infectious complication of blood transfusion. With millions of units transfused per year, product safety is key to public health but currently relies upon donor deferral and product testing. Pathogen reduction technologies can also be used, with several systems CE marked or FDA approved for platelet and plasma use, but only one CE marked for whole blood. 405 nm violet-blue light has recently demonstrated potential for in situ bacterial reduction of ex vivo stored plasma and platelet products, along with preliminary antiviral potential in plasma. This study assesses the potential compatibility of 405 nm antimicrobial light with red blood cell (RBC) components. Sheep RBC were analysed, showing light transmissibility and absorption characteristics over wavelengths of 220-1100 nm using neat–1000X dilutions. Irradiances of 10-100 mW/cm² 405 nm light were then applied to red cell samples with ∼1.3 & 7.8 mm depths over 10-60 minutes, giving doses of 9-360 J/cm². Treatment effects on cell integrity were demonstrated by microscopy and photometric detection of leaked haemoglobin using the Harboe method. RBC transmission analysis highlighted the high opacity of RBC suspension at 18% haematocrit with 1 cm depths, with peak absorption found to be at 417 nm. Results suggested the requirement of sample depths <1 cm to provide potential for penetration. Photometric analysis demonstrated RBC damage was influenced by the irradiance levels and exposure times used. Haemolysis increased by 0.37 & 1.26% over 15-60 minutes with 20 & 50 mW/cm² irradiances respectively. A greater increase of 5.06% was observed by 100 mW/cm² exposures over 15-30 minutes. Haemolysis rose sharply during 100 mW/cm² exposures and was supported by a visual decrease in stained viable cells. Low treatment doses ≤90 J/cm² with irradiances ≤50 mW/cm², showed potential compatibility, producing haemolysis below the 0.8% European limit. Future work will build on these findings and investigate the potential for antimicrobial efficacy of a 405 nm light treatment system at levels compatible with RBC physiology.

3135 – VISIBLE VIOLET-BLUE LIGHT FOR PATHOGEN REDUCTION OF BLOOD PLASMA: ASSESSMENT OF BROAD SPECTRUM ANTIBACTERIAL EFFICACY AND PRELIMINARY COMPATIBILITY STUDIES

Bacterial contamination of blood products is a major concern in transfusion medicine. Continued efforts to improve blood safety include the use of pathogen reduction technologies (PRTs), which for plasma includes solvent-detergent treatment, or the use of ultraviolet or visible light in combination with photosensitizers. Non-ionizing violet-blue light, in the region of 405nm, has recently demonstrated potential for in situ treatment of ex vivo stored plasma and platelet products, without the need for additional photosensitizers. This study investigates the broad spectrum antibacterial efficacy of 405 nm light for pathogen reduction of plasma samples. Human plasma was seeded with a range of bacteria implicated in transfusion transmitted infections: Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae and Yersinia enterocolitica. The organisms were seeded at 10², 10⁵ and 10⁷ CFU mL⁻¹ densities and exposed to a 405 nm light dose of 360 Jcm⁻² (1-hr at 100 mWcm⁻²). Broad spectrum antibacterial efficacy was observed, with significant bacterial inactivation achieved for all species (P≤0.05). 98.9 – 100 % inactivation was achieved across all seeding densities for all organisms except E. coli, which achieved 95.1 – 100.0 % inactivation. Preliminary protein analysis, conducted using SDS-PAGE, assessed the compatibility of an effective antibacterial dose with plasma proteins. Protein stability tests provide the first evidence for the compatibility of 405 nm light for the decontamination of plasma, with no visible signs of protein degradation detected for doses ≤ 1.8 kJcm⁻². Future studies are required to determine the treatment regime that provides the optimal balance of antibacterial efficacy and product stability.

Vox Sanguinis International Forum on Hospital Transfusion Services' Response to COVID‐19: Responses

Question 1Demographics:a. Beer Yaakov, Israel.b. Large academic medical center.c. 956 beds.d. 9034 RBC units issued in 2019, approximately 9000.e. 4831 confirmed SARS‐CoV‐2 cases and 17 deaths (March 31, 2020, 12:40 pm, data from the Israeli Ministry of Health).f. 9,136,000 inhabitants.g. The spread of SARS‐CoV‐2 in Israel is still increasing, but as of yesterday the rate of increase is declining sharply (from almost 30% of samples tested per day at the peak to 12% yesterday).

COVID-19 insights from transfusion medicine.

The emergence of CoVID‐19 infection in the first months of 2020 resulted in a massive surge in admissions to hospitals and intensive care units due to the nature of the respiratory pathophysiology. In some countries, health care systems were rapidly overwhelmed, while in others their hospitals coped with the first wave and all patients received the level of care needed, depending upon both the containment measured and the level of development of the heath care systems.

Digital droplet polymerase chain reaction to monitor ultraviolet C treatment of single-donor and buffy coat platelet units.

UVC illumination of agitated platelet concentrates (PCs) inactivates pathogens and white blood cells by modifications of their nucleic acids. Related effects on mitochondrial DNA (mtDNA) in platelets serve as a basis for an efficient monitoring suited for routine quality control (QC) of this purely physical pathogen reduction technology. Samples from PCs (n = 530) were tested with an established LightCycler PCR (LC PCR) for QC of the UVC procedure. RNR2 and TRNK/ATP8 genes were sequenced in the PCs (n = 21) with out-of-specification results in the LC PCR. A digital droplet PCR (ddPCR) was developed to minimize the outliers and cross-validated by testing the 530 PCs. The ddPCR was further evaluated in a subgroup of 300 PCs without mtDNA extraction and in samples from systematic variations of UVC dose and agitation speed. Apheresis PCs (n = 380) resulted in 5.3% outliers in LC PCR versus only 0.7% in buffy coat pool PCs (n = 150). Sequencing of these outliers revealed single-nucleotide polymorphisms in the primer- and probe-binding sites of LC PCR. The development of a ddPCR assay with modified probe sequences reduced the outliers to 0.4%. The ddPCR analysis of PCs both with and without mtDNA extraction demonstrated low intra- and interassay variabilities and congruent results also compared to LC PCR. Experiments varying the UVC dose and the agitation speed demonstrated that the ddPCR results closely reflect functional effects of the UVC treatment. The ddPCR assay offers a valid and reliable tool for QC of routine production of the UVC-treated PCs as well as for monitoring treatment conditions during optimization of the UVC procedure.

Blood component separation of pathogen-reduced whole blood by the PRP method produces acceptable red cells but platelet yields and function are diminished.

This study evaluated blood components processed by the platelet rich plasma (PRP) method from fresh whole blood (FWB) treated with a pathogen reduction technology (PRT). The effects of storage temperature on PRT treated platelet concentrates (PCs) were also examined. PRT was performed using riboflavin and ultraviolet light on FWB in citrate phosphate dextrose anticoagulant. Following PRT, red blood cells (RBCs), PCs, and plasma for fresh frozen plasma (FFP), were isolated by sequential centrifugation. RBCs were stored at 4°C, FFP at -80°C, and PC at 22°C or at 4°C. Components were assayed throughout their storage times for blood gases, chemistry and CBC, hemostatic function as well as platelet (PLT) and RBC integrity. Component processing following PRT resulted in a significant drop in platelet recovery. Most PRT-PC bags fell below AABB guidelines for platelet count. PRT-PC also showed a decrease in clot strength and decreased aggregometry response. Platelet caspases were activated by PRT. Storage at 4°C improved platelet function. In PRT-FFP, prothrombin time and partial thromboplastin time (PT and aPTT) were prolonged; factors V, VII, VIII, and XI, protein C, and fibrinogen were significantly decreased. Free hemoglobin was elevated two-fold in PRT-RBC. Blood components isolated by the PRP method from PRT-treated WB result in a high percentage of PC that fail to meet AABB guidelines. FFP also shows diminished coagulation capacity. However, PRT-RBC are comparable to control-RBC. PRT-WB retains acceptable hemostatic function but alternatives to the PRP method of component separation may be more suitable.

Pathogen reduction of SARS-CoV-2 virus in plasma and whole blood using riboflavin and UV light.

BackgroundSevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has recently been identified as the causative agent for Coronavirus Disease 2019 (COVID-19). The ability of this agent to be transmitted by blood transfusion has not been documented, although viral RNA has been detected in serum. Exposure to treatment with riboflavin and ultraviolet light (R + UV) reduces blood-borne pathogens while maintaining blood product quality. Here, we report on the efficacy of R + UV in reducing SARS-CoV-2 infectivity when tested in human plasma and whole blood products.Study design and methodsSARS-CoV-2 (isolate USA-WA1/2020) was used to inoculate plasma and whole blood units that then underwent treatment with riboflavin and UV light (Mirasol Pathogen Reduction Technology System, Terumo BCT, Lakewood, CO). The infectious titers of SARS-CoV-2 in the samples before and after R + UV treatment were determined by plaque assay on Vero E6 cells. Each plasma pool (n = 9) underwent R + UV treatment performed in triplicate using individual units of plasma and then repeated using individual whole blood donations (n = 3).ResultsRiboflavin and UV light reduced the infectious titer of SARS-CoV-2 below the limit of detection for plasma products at 60–100% of the recommended energy dose. At the UV light dose recommended by the manufacturer, the mean log reductions in the viral titers were ≥ 4.79 ± 0.15 Logs in plasma and 3.30 ± 0.26 in whole blood units.ConclusionRiboflavin and UV light effectively reduced the titer of SARS-CoV-2 to the limit of detection in human plasma and by 3.30 ± 0.26 on average in whole blood. Two clades of SARS-CoV-2 have been described and questions remain about whether exposure to one strain confers strong immunity to the other. Pathogen-reduced blood products may be a safer option for critically ill patients with COVID-19, particularly those in high-risk categories.