Pathogen inactivation methods to prevent transfusion-transmissible arboviruses: A systematic review and meta-analysis.

OBJECTIVE: Arboviruses are emerging as a relevant threat to transfusion safety. Pathogen inactivation methods (PIMs) may reduce the risk of transmission through transfusion, as long as they meet minimum standards for effectiveness. This study aims to assess the log reduction of viral load achieved with different PIMs, according to the blood product they are used on and the arbovirus targeted. METHODS: Systematic literature review and meta-analysis. Searches were conducted in MEDLINE and Embase. The study protocol was registered in PROSPERO CRD42022312061. We selected records reporting the log reduction of viral load achieved with the main PIMs (amotosalen + UVA light [INTERCEPT], riboflavin + UV light [Mirasol], methylene blue + visible light/UVC light [THERAFLEX], solvent detergent, amustaline [INTERCEPT] and PEN110 [Inactine]), applied to any blood product (plasma, platelets, red blood cells or whole blood) and for any arbovirus. The log reduction of viral loads was assessed by obtaining the mean log reduction factor (LRF). We compared and classified the LRF of different techniques using statistical methods. RESULTS: We included 59 publications reporting LRF results in 17 arboviruses. For 13 arboviruses, including Chikungunya virus, Dengue virus, West Nile virus and Zika virus, at least one of the methods achieves adequate or optimal log reduction of viral load-mean LRF ≥4. The LRF achieved with riboflavin + UV light is inferior to the rest of the techniques, both overall and specifically for plasma, platelets preserved in platelet additive solution (PAS)/plasma, and red blood cells/whole blood. The LRF achieved using Mirasol is also lower for inactivating Chikungunya virus, Dengue virus and Zika virus. For West Nile virus, we found no significant differences. In plasma, the method that achieves the highest LRF is solvent/detergent; in platelets, THERAFLEX and INTERCEPT; and in red blood cells/whole blood, PEN110 (Inactine). CONCLUSION: Not all PIMs achieve the same LRF, nor is this equivalent between the different arboviruses or blood products. Overall, the LRFs achieved using riboflavin + UV light (Mirasol) are inferior to those achieved with the rest of the PIMs. Regarding the others, LRFs vary by arbovirus and blood product. In light of the threat of different arboviruses, blood establishments should have already validated PIMs and be logistically prepared to implement these techniques quickly.

Mortality in acquired thrombotic thrombocytopenic purpura in the pre-caplacizumab era.

Despite the effectiveness of plasma exchange (PEX) and immunosuppressants in the treatment of acquired thrombotic thrombocytopenic purpura (aTTP), a number of patients still die as a result of the disease. Whether caplacizumab could rescue these patients remains still unsettled. The objective of this study was to characterise mortality patterns and prognostic factors in the first episode of aTTP.We queried the Spanish TTP Registry for patients with a diagnosis of aTTP in their presenting episode who fulfilled complete clinical and follow-up data (n = 102). The patients were diagnosed between 2004 and 2018, and all were treated with daily PEX and corticosteroids. Clinical and laboratory data were analysed at diagnosis and during the treatment course.Eight patients (7.7%) died between 12 h and 36 days after presentation, and could be classified into three patterns: death before treatment, early death driven by acute cardiac or neurologic events, and late death due to unremitted aTTP. Stupor or coma at diagnosis and platelet count < 20 × 109 /L by the 6th treatment day were independently associated with increased risk of death.Stupor or coma at diagnosis and lack of response to PEX by the 6th day in patients experiencing the first episode of aTTP are strong predictors of mortality. These patients could be rescued by novel agents aimed at halting the microvascular thrombosis until adequate immunosuppression is achieved.

Contamination of tissue allografts from a multiorgan-multitissue donor colonized by Candida auris.

Standards on tissue banking determine the need of microbiological monitoring during critical steps (recovery, processing, storage, and transplantation). This information will be useful for both discarding contaminated tissues or risk analysis (in case of recipient infection). In this study, we show the case of a multiorgan-multitissue donor colonized by Candida auris. This microorganism is characterized by multidrug resistance, with higher transmissibility and severe outcome. Some of the microbiological cultures from arteries tested positive for this microorganism, but it was not cultured in samples from musculoskeletal tissues and corneas. No recipient case of infection transmission by Candida species was observed (organs and cornea). The implementation of active surveillance protocol for C. auris detection in critical care units (as source of tissue donors) has been suggested as a part of our hospitals' infection control policy.

Adoptive transfer of ex vivo expanded SARS-CoV-2-specific cytotoxic lymphocytes: A viable strategy for COVID-19 immunosuppressed patients?

Cellular and humoral response to acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is on focus of research. We evaluate herein the feasibility of expanding virus-specific T cells (VST) against SARS-CoV-2 ex vivo through a standard protocol proven effective for other viruses. The experiment was performed in three different donors' scenarios: (a) SARS-CoV-2 asymptomatic infection/negative serology, (b) SARS-CoV-2 symptomatic infection/positive serology, and (c) no history of SARS-CoV-2 infection/negative serology. We were able to obtain an expanded VST product from donors 1 and 2 (1.6x and 1.8x increase of baseline VST count, respectively) consisting in CD3 + cells (80.3% and 62.7%, respectively) with CD4 + dominance (60% in both donors). Higher numbers of VST were obtained from the donor 2 as compared to donor 1. T-cell clonality test showed oligoclonal reproducible peaks on a polyclonal background for both donors. In contrast, VST could be neither expanded nor primed in a donor without evidence of prior infection. This proof-of-concept study supports the feasibility of expanding ex vivo SARS-CoV-2-specific VST from blood of convalescent donors. The results raise the question of whether the selection of seropositive donors may be a strategy to obtain cell lines enriched in their SARS-CoV-2-specificity for future adoptive transfer to immunosuppressed patients.

Hydroxyethyl starch is an alternative washing solution for peripheral bloodstem cells products.

Cryopreservation of hematopoietic stem cells (HSC) involves slow rate cooling in the presence of a cryoprotectant (DMSO) to avoid the damaging effects of intracellular ice formation. The infusion of DMSO with the thawed product has been related to adverse events. Reduction of DMSO content by washing the HSCs after thawing has been suggested as a method to avoid infusion-related side-effects. Albumin-dextran washing methods have proved useful in thawing HSC products. Dextran40 shortages prompted us to search for suitable alternatives. We report the results of a comparative study of the use of hydroxyethyl starch (HES) as an alternative to dextran40 for washing thawed HSCs products. A total of 10 HSC bags cryopreserved with 10 % DMSO were used. We conducted a paired study; one of the bags was thawed and washed with our standard washing solution (Dextran 40) and the paired bag with HES solution with a final HES and Human Serum Albumin (HSA) concentration of 2.4 % and 4.2 % respectively. Each final product was tested immediately after washing (sample 0') and after 90 min (sample 90') for total nucleated cells (TNC) recovery, acridine orange viability, viable CD34+ enumeration, and clonogenicity. No significant difference was found for any of the cell counts, viability tests, cell recovery, or potency. We can state that the washing solution based on 2.4 % HES and 4.2 % HSA is equivalent to that used in our routine practice. Therefore, we could use the solution with HES, paying special attention to the renal function of the recipient.

Biomarker profile predicts clinical efficacy of extracorporeal photopheresis in steroid-resistant acute and chronic graft-vs-host disease after allogenic hematopoietic stem cell transplant.

We conducted a multicenter interventional study to assess the efficacy of Therakos ECP to treat steroid-resistant graft-vs-host disease (SRes-GVHD) after allogeneic HSCT and to identify biomarkers of GVHD response. A total of 62 patients were treated for acute SRes-GVHD (n = 37) or chronic SRes-GVHD (n = 25). Median time to best response was 35 days (range, 28-85) and 90 days (range, 27-240) in acute and chronic SRes-GVHD, respectively. Overall, 27 patients (72.9%) with SRes-aGVHD responded to treatment (40.5% CR and 32.4% PR). The response rate was significantly higher in grade I-II than in grade III-IV aGVHD (100% vs 50.0%, respectively, P-value = .001). In chronic SRes-GVHD, 22 patients (88%) achieved a clinical response (24.0% CR and 64% PR). Response was higher in moderate than in severe SRes-cGVHD (100% vs 75%, P = .096). In both acute and chronic SRes-GVHD patients, the percentage of peripheral blood CD3+ CD4+ was higher and CD3+ CD8+ lower in responding than nonresponding patients. Acute SRes-GVHD responding patients presented a higher number of Treg cells (CD4+ CD25+ CD127low/- ) at day 0 (P = .028) than nonresponding patients, differences that were maintained over the observation period. Phenotypic analysis of T-cell maturation showed a trend toward reduction in TCD8 naive cells, along with an increased percentage of TCD8 Mem Efect T cells after starting ECP in responding patients. None of the studied serum cytokines displayed statistically significant changes in either acute or chronic SRes-GVHD. ECP is an effective treatment for patients with SRes-GVHD. Biomarkers could help guide decision-making on ECP treatment initiation and duration.

The storage of skull bone flaps for autologous cranioplasty: literature review.

The use of autologous bone flap for cranioplasty after decompressive craniectomy is a widely used strategy that allows alleviating health expenses. When the patient has recovered from the primary insult, the cranioplasty restores protection and cosmesis, recovering fluid dynamics and improving neurological status. During this time, the bone flap must be stored, but there is a lack of standardization of tissue banking practices for this aim. In this work, we have reviewed the literature on tissue processing and storage practices. Most of the published articles are focused from a strictly clinical and surgical point of view, paying less attention to issues related to tissue manipulation. When bone resorption is avoided and the risk of infection is controlled, the autograft represents the most efficient choice, with the lowest risk of complication. Otherwise, depending on the degree of involvement, the patient may have to undergo new surgery, assuming further risks and higher healthcare costs. Therefore, tissue banks must implement protocols to provide products with the highest possible clinical effectiveness, without compromising safety. With a centralised management of tissue banking practices there may be a more uniform approach, thus facilitating the standardization of procedures and guidelines.

Effect of freezing and storage temperature on stability and antimicrobial activity of an antibiotic mixture used for decontamination of tissue allografts.

One of the most important risks to be controlled in tissue banking is the infection associated with the clinical use of auto- and allografts. Thus, tissue disinfection protocols are used, in addition to processing in controlled environments. For this purpose, combinations of antibiotics are designed to ensure a broad spectrum of antimicrobial activity. This type of protocol is usually validated by testing its antimicrobial efficacy. In this work, we have studied the effect of several factors on the potential of an antibiotic mixture: container, freezing, storage at 4 °C, storage at - 30 °C and storage at - 80 °C. The molecular stability of the compounds has also been tested, additionally to their efficacy. Our findings show that storage conditions affect the molecular stability of Fungizone and Tobramycin (only in case of frozen storage for the last one). Nevertheless, the solution retains its antimicrobial activity for several weeks. The availability of stored aliquots of disinfectant solution and defining expiry dates for different storage conditions can help to schedule tissue bank tasks.

Detection of hepatitis B virus in bone allografts from donors with occult hepatitis B infection.

The implementation of nucleic acid testing in donor screening has improved the safety of tissue allografts. Although infectious disease transmission can be considered a rare event, the detection of occult hepatitis B infection remains challenging. The studies concerning this risk are mainly based on testing blood specimens. This work shows the correlation between results of samples obtained from donor blood and the corresponding tissue washing solution. Hepatitis B virus deoxyribonucleic acid was detected both in bone allografts from donors with serological profiles associated to active hepatitis B infection and occult hepatitis B infection. These results suggest that hepatitis B virus seems to concentrate in bone marrow even when a low viral load is present in peripheral blood. Even detection at molecular level is not enough to avoid the risk of hepatitis B virus transmission and a multiparametrical evaluation is required in tissue donor screening. The role of clinicians in recognition and reporting of allograft-associated infections is a major concern for the acquisition of experience to be applied in risk control of disease transmission.

Autologous cell therapy with CD133+ bone marrow-derived stem cells for refractory Asherman's syndrome and endometrial atrophy: a pilot cohort study.

STUDY QUESTION: Could cell therapy using autologous peripheral blood CD133+ bone marrow-derived stem cells (BMDSCs) offer a safe and efficient therapeutic approach for patients with refractory Asherman's syndrome (AS) and/or endometrial atrophy (EA) and a wish to conceive? SUMMARY ANSWER: In the first 3 months, autologous cell therapy, using CD133+ BMDSCs in conjunction with hormonal replacement therapy, increased the volume and duration of menses as well as the thickness and angiogenesis processes of the endometrium while decreasing intrauterine adhesion scores. WHAT IS KNOWN ALREADY: AS is characterized by the presence of intrauterine adhesions and EA prevents the endometrium from growing thicker than 5 mm, resulting in menstruation disorders and infertility. Many therapies have been attempted for these conditions, but none have proved effective. STUDY DESIGN, SIZE, DURATION: This was a prospective, experimental, non-controlled study. There were 18 patients aged 30-45 years with refractory AS or EA were recruited, and 16 of these completed the study. Medical history, physical examination, endometrial thickness, intrauterine adhesion score and neoangiogenesis were assessed before and 3 and 6 months after cell therapy. PARTICIPANTS/MATERIALS, SETTING, METHODS: After the initial hysteroscopic diagnosis, BMDSC mobilization was performed by granulocyte-CSF injection, then CD133+ cells were isolated through peripheral blood aphaeresis to obtain a mean of 124.39 million cells (range 42-236), which were immediately delivered into the spiral arterioles by catheterization. Subsequently, endometrial treatment after stem cell therapy was assessed in terms of restoration of menses, endometrial thickness (by vaginal ultrasound), adhesion score (by hysteroscopy), neoangiogenesis and ongoing pregnancy rate. The study was conducted at Hospital Clínico Universitario of Valencia and IVI Valencia (Spain). MAIN RESULTS AND THE ROLE OF CHANCE: All 11 AS patients exhibited an improved uterine cavity 2 months after stem cell therapy. Endometrial thickness increased from an average of 4.3 mm (range 2.7-5) to 6.7 mm (range 3.1-12) ( ITALIC! P = 0.004). Similarly, four of the five EA patients experienced an improved endometrial cavity, and endometrial thickness increased from 4.2 mm (range 2.7-5) to 5.7 mm (range 5-12) ( ITALIC! P = 0.03). The beneficial effects of the cell therapy increased the mature vessel density and the duration and intensity of menses in the first 3 months, with a return to the initial levels 6 months after the treatment. Three patients became pregnant spontaneously, resulting in one baby boy born, one ongoing pregnancy and a miscarriage. Furthermore, seven pregnancies were obtained after fourteen embryo transfers, resulting in three biochemical pregnancies, one miscarriage, one ectopic pregnancy, one baby born and one ongoing pregnancy. LIMITATIONS, REASONS FOR CAUTION: Limitations of this pilot study include the small sample size and the lack of control group. WIDER IMPLICATIONS OF THE FINDINGS: This novel autologous cell therapy is a promising therapeutic option for patients with these incurable pathologies and a wish to conceive. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the Spanish Ministry of Science and Innovation (SAF 2012-31017, Principal Investigator C.S.), Spanish Ministry of Health (EC11-299, Principal Investigator C.S.) and Regional Valencian Ministry of Education (PROMETEOII/2013/018, Principal Investigator C.S.). Four authors (X.S., I.C., A.P. and C.S.) are co-inventors of the patent resulting from this work (Application number: 62/013,121). S.C., C.A., F.R., J.F., J.P. and J.R. have no conflict of interest in relation to this work. TRIAL REGISTRATION NUMBER: This study was registered with ClinicalTrials.gov (NCT02144987).

Development and characterization of a cell donor registry for virus-specific T cell manufacture in a blood bank.

Adoptive cell therapy using virus-specific T cells (VST) is a strategy for treating common opportunistic viral infections after transplantation, particularly when these infections do not resolve through antiviral drug therapy. The availability of third-party healthy donors allows for the immediate use of cells for allogeneic therapy in cases where patients lack an appropriate donor. Here, we present the creation of a cell donor registry of human leukocyte antigen (HLA)-typed blood donors, REDOCEL, a strategic initiative to ensure the availability of compatible cells for donation when needed. Currently, the registry consists of 597 healthy donors with a median age of 29 years, 54% of whom are women. The most represented blood groups were A positive and O positive, with 36.52% and 34.51%, respectively. Also, donors were screened for cytomegalovirus (CMV) and Epstein-Barr virus (EBV). Almost 65% of donors were CMV-seropositive, while less than 5% were EBV-seronegative. Of the CMV-seropositive donors, 98% were also EBV-seropositive. High-resolution HLA-A, -B, -C, -DRB1 and -DQB1 allele and haplotype frequencies were determined in the registry. Prevalent HLA alleles and haplotypes were well represented to ensure donor-recipient HLA-matching, including alleles reported to present viral immunodominant epitopes. Since the functional establishment of REDOCEL, in May 2019, 87 effective donations have been collected, and the effective availability of donors with the first call has been greater than 75%. Thus, almost 89% of patients receiving an effective donation had available at least 5/10 HLA-matched cell donors (HLA-A, -B, -C, -DRB1, and -DQB1). To summarize, based on our experience, a cell donor registry from previously HLA-typed blood donors is a useful tool for facilitating access to VST therapy.

Human Anelloviruses: Influence of Demographic Factors, Recombination, and Worldwide Diversity.

Anelloviruses represent the major and most diverse component of the healthy human virome, referred to as the anellome. In this study, we determined the anellome of 50 blood donors, forming two sex- and age-matched groups. Anelloviruses were detected in 86% of the donors. The number of detected anelloviruses increased with age and was approximately twice as high in men as in women. A total of 349 complete or nearly complete genomes were classified as belonging to torque teno virus (TTV), torque teno mini virus (TTMV), and torque teno midi virus (TTMDV) anellovirus genera (197, 88, and 64 sequences, respectively). Most donors had intergenus (69.8%) or intragenus (72.1%) coinfections. Despite the limited number of sequences, intradonor recombination analysis showed 6 intragenus recombination events in ORF1. As thousands of anellovirus sequences have been described recently, we finally analyzed the global diversity of human anelloviruses. Species richness and diversity were close to saturation in each anellovirus genus. Recombination was found to be the main factor promoting diversity, although its effect was significantly lower in TTV than in TTMV and TTMDV. Overall, our results suggest that differences in diversity between genera may be caused by variations in the relative contribution of recombination. IMPORTANCE Anelloviruses are the most common human infectious viruses and are considered essentially harmless. Compared to other human viruses, they are characterized by enormous diversity, and recombination is suggested to play an important role in their diversification and evolution. Here, by analyzing the composition of the plasma anellome of 50 blood donors, we find that recombination is also a determinant of viral evolution at the intradonor level. On a larger scale, analysis of anellovirus sequences currently available in databases shows that their diversity is close to saturation and differs among the three human anellovirus genera and that recombination is the main factor explaining this intergenus variability. Global characterization of anellovirus diversity could provide clues about possible associations between certain virus variants and pathologies, as well as facilitate the implementation of unbiased PCR-based detection protocols, which may be relevant for using anelloviruses as endogenous markers of immune status.

Rationale for combined therapies in severe-to-critical COVID-19 patients.

An unprecedented global social and economic impact as well as a significant number of fatalities have been brought on by the coronavirus disease 2019 (COVID-19), produced by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Acute SARS-CoV-2 infection can, in certain situations, cause immunological abnormalities, leading to an anomalous innate and adaptive immune response. While most patients only experience mild symptoms and recover without the need for mechanical ventilation, a substantial percentage of those who are affected develop severe respiratory illness, which can be fatal. The absence of effective therapies when disease progresses to a very severe condition coupled with the incomplete understanding of COVID-19's pathogenesis triggers the need to develop innovative therapeutic approaches for patients at high risk of mortality. As a result, we investigate the potential contribution of promising combinatorial cell therapy to prevent death in critical patients.

Prevalence of Chikungunya, Dengue and Zika viruses in blood donors: a systematic literature review and meta-analysis.

BACKGROUND: Blood transfusion centres should understand the epidemiology of emerging diseases that are transmissible through the transfusion of blood components. The risk of transmission of arboviruses through this route has become apparent in recent years. The aim of our study is to summarise the reported prevalence (viraemic rate, seroprevalence and/or antigen detection) of Chikungunya (CHIKV), Dengue (DENV) and Zika (ZIKV) viruses in blood donors according to screening test used and world region. MATERIALS AND METHODS: We conducted a systematic literature review and meta-analysis having searched for information in the main bibliographic databases (MEDLINE, Embase, and Scopus). The prevalence for each of the viruses was calculated according to the screening test used and geographic location. RESULTS: We included 18 records on CHIKV, 71 on DENV, and 27 on ZIKV. The highest prevalences of RNA for CHIKV were 1.9% in Puerto Rico (2014), 1.0% in Thailand (2009), and 1.0% in French Polynesia (2014-15). The highest prevalences of RNA for DENV were 5.5% in Saudi Arabia (2015-16), 2.3% in Madeira, Portugal (2012-13), and 0.6% in Brazil (2012). The highest prevalences of RNA for ZIKV were 2.8% in French Polynesia (2013-14), 2.7% in Brazil (2015-16), and 1.8% in Martinique (2016). Overall seroprevalence, as assessed by IgG antibodies, was 21.6% for CHIKV, 24.0% for DENV, and 5.1% for ZIKV. DISCUSSION: Our study shows a high proportion of donors who are viraemic and asymptomatic, especially during outbreaks, with prevalences surpassing 5% for DENV, 1% for CHIKV, and 2% for ZIKV. These data confirm a clear threat to blood transfusion safety. The elevated seroprevalence for these three arboviruses is also indicative of their wide circulation in populations, correlating with an increased risk of infected but asymptomatic donors. Health centres and institutions must address this threat, especially in tropical regions where the biggest outbreaks occur.

Transfusion-transmitted arboviruses: Update and systematic review.

BACKGROUND: The detection of the first cases of transfusion-transmitted West Nile virus in 2002 posed a new challenge for transfusion safety. Institutions like the World Health Organization have stated that blood transfusion centers need to know the epidemiology of the different emerging infectious agents and their impact on blood transfusion. The aim of the study is to review the published cases of arbovirus transmission through transfusion of blood or blood components and to analyze their main clinical and epidemiological characteristics. MATERIAL AND METHODS: Systematic literature searches were conducted in MEDLINE, Embase and Scopus. Pairs of review authors selected a variety of scientific publications reporting cases of transfusion-transmitted arboviruses. Main clinical and epidemiological characteristics were reviewed of the cases described. The study protocol was registered in PROSPERO CRD42021270355. RESULTS: A total of 74 cases of transfusion-transmitted infections were identified from 10 arboviruses: West Nile virus (n = 42), dengue virus (n = 18), Zika virus (n = 3), yellow fever vaccine virus (n = 3), tick-borne encephalitis virus (n = 2), Japanese encephalitis virus (n = 2), Powassan virus (n = 1), St. Louis encephalitis virus (n = 1), Ross River virus (n = 1) and Colorado tick fever virus (n = 1). The blood component most commonly involved was red blood cells (N = 35, 47.3%; 95% confidence interval [CI] 35.9% to 58.7%). In 54.1% (N = 40; 95% CI: 42.7%-65.47%) of the cases, the recipient was immunosuppressed. Transmission resulted in death in 18.9% (N = 14; 95% CI: 10.0%-27.8%) of the recipients. In addition, 18 additional arboviruses were identified with a potential threat to transfusion safety. DISCUSSION: In the last 20 years, the number of published cases of transfusion-transmitted arboviruses increased notably, implicating new arboviruses. In addition, a significant number of arboviruses that may pose a threat to transfusion safety were detected. In the coming years, it is expected that transmission of arboviruses will continue to expand globally. It is therefore essential that all responsible agencies prepare for this potential threat to transfusion safety.

Public Cord Blood Banks as a source of starting material for clinical grade HLA-homozygous induced pluripotent stem cells.

BACKGROUND: The increasing number of clinical trials for induced pluripotent stem cell (iPSC)-derived cell therapy products makes the production on clinical grade iPSC more and more relevant and necessary. Cord blood banks are an ideal source of young, HLA-typed and virus screened starting material to produce HLA-homozygous iPSC lines for wide immune-compatibility allogenic cell therapy approaches. The production of such clinical grade iPSC lines (haplolines) involves particular attention to all steps since donor informed consent, cell procurement and a GMP-compliant cell isolation process. METHODS: Homozygous cord blood units were identified and quality verified before recontacting donors for informed consent. CD34+ cells were purified from the mononuclear fraction isolated in a cell processor, by magnetic microbeads labelling and separation columns. RESULTS: We obtained a median recovery of 20.0% of the collected pre-freezing CD34+, with a final product median viability of 99.1% and median purity of 83.5% of the post-thawed purified CD34+ population. CONCLUSIONS: Here we describe our own experience, from unit selection and donor reconsenting, in generating a CD34+ cell product as a starting material to produce HLA-homozygous iPSC following a cost-effective and clinical grade-compliant procedure. These CD34+ cells are the basis for the Spanish bank of haplolines envisioned to serve as a source of cell products for clinical research and therapy.

Preservation of anti-SARS-CoV-2 neutralising antibodies in convalescent plasma after pathogen reduction with methylene blue and visible light.

BACKGROUND: COVID-19 convalescent plasma (CCP) is an experimental treatment against SARS-CoV-2. Although there has so far been no evidence of transmission through transfusion, pathogen reduction technologies (PRT) have been applied to CCP to mitigate risk of infectious disease. This study aims to assess the impact of methylene blue (MB) plus visible light PRT on the virus-neutralising activity of the specific antibodies against SARS-CoV-2. MATERIAL AND METHODS: Thirty-five plasma doses collected by plasmapheresis from COVID-19 convalescent donors were subjected to MB plus visible light PRT. Anti-SARS-CoV-2 RBD S1 epitope IgGs antibodies were quantified by ELISA. Titres of SARS-CoV-2 neutralising antibodies (NtAbs) were measured before and after the PRT process. A Spearman's correlation was run to determine the relationship between antibody neutralisation ability and SARS-CoV-2 IgG ELISA ratio. Pre- and post-inactivation neutralising antibody titres were evaluated using a Wilcoxon test. RESULTS: The plasma pathogen reduction procedure did not diminish NtAbS titres and so did not cause a change in the viral neutralisation capacity of CCP. There was a strong correlation between pre-and post-PRT NtAbs and anti-SARS-CoV-2 IgGs titres. DISCUSSION: Our results showed PRT with MB did not impair the CCP passive immunity preserving its potential therapeutic potency. Therefore, PRT of CCP should be recommended to mitigate the risk for transmission of transfusion-associated infectious disease. There is a good correlation between SARS-CoV-2 IgG titres determined by ELISA and the neutralising capacity. This allows blood centres to select CCP donors based on IgG ELISA titres avoiding the much more labour-intensive laboratory processes for determining neutralising antibodies.

Deep viral blood metagenomics reveals extensive anellovirus diversity in healthy humans.

Human blood metagenomics has revealed the presence of different types of viruses in apparently healthy subjects. By far, anelloviruses constitute the viral family that is more frequently found in human blood, although amplification biases and contaminations pose a major challenge in this field. To investigate this further, we subjected pooled plasma samples from 120 healthy donors in Spain to high-speed centrifugation, RNA and DNA extraction, random amplification, and massive parallel sequencing. Our results confirm the extensive presence of anelloviruses in such samples, which represented nearly 97% of the total viral sequence reads obtained. We assembled 114 different viral genomes belonging to this family, revealing remarkable diversity. Phylogenetic analysis of ORF1 suggested 28 potentially novel anellovirus species, 24 of which were validated by Sanger sequencing to discard artifacts. These findings underscore the importance of implementing more efficient purification procedures that enrich the viral fraction as an essential step in virome studies and question the suggested pathological role of anelloviruses.

Exploring the Diversity of the Human Blood Virome.

Metagenomics is greatly improving our ability to discover new viruses, as well as their possible associations with disease. However, metagenomics has also changed our understanding of viruses in general. The vast expansion of currently known viral diversity has revealed a large fraction of non-pathogenic viruses, and offers a new perspective in which viruses function as important components of many ecosystems. In this vein, studies of the human blood virome are often motivated by the search for new viral diseases, especially those associated with blood transfusions. However, these studies have revealed the common presence of apparently non-pathogenic viruses in blood, particularly human anelloviruses and, to a lower extent, human pegiviruses (HPgV). To shed light on the diversity of the human blood virome, we subjected pooled plasma samples from 587 healthy donors in Spain to a viral enrichment protocol, followed by massive parallel sequencing. This showed that anelloviruses were clearly the major component of the blood virome and showed remarkable diversity. In total, we assembled 332 complete or near-complete anellovirus genomes, 50 of which could be considered new species. HPgV was much less frequent, but we, nevertheless, recovered 17 different isolates that we subsequently used for characterizing the diversity of this virus. In-depth investigation of the human blood virome should help to elucidate the ecology of these viruses, and to unveil potentially associated diseases.