Exploratory analysis to identify the best antigen and the best immune biomarkers to study SARS-CoV-2 infection.

BACKGROUND: Recent studies proposed the whole-blood based IFN-γ-release assay to study the antigen-specific SARS-CoV-2 response. Since the early prediction of disease progression could help to assess the optimal treatment strategies, an integrated knowledge of T-cell and antibody response lays the foundation to develop biomarkers monitoring the COVID-19. Whole-blood-platform tests based on the immune response detection to SARS-CoV2 peptides is a new approach to discriminate COVID-19-patients from uninfected-individuals and to evaluate the immunogenicity of vaccine candidates, monitoring the immune response in vaccine trial and supporting the serological diagnostics results. Here, we aimed to identify in the whole-blood-platform the best immunogenic viral antigen and the best immune biomarker to identify COVID-19-patients. METHODS: Whole-blood was overnight-stimulated with SARS-CoV-2 peptide pools of nucleoprotein-(NP) Membrane-, ORF3a- and Spike-protein. We evaluated: IL-1ß, IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL- 15, IL-17A, eotaxin, FGF, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1ß, PDGF, RANTES, TNF-α, VEGF. By a sparse partial least squares discriminant analysis we identified the most important soluble factors discriminating COVID-19- from NO-COVID-19-individuals. RESULTS: We identified a COVID-19 signature based on six immune factors: IFN-γ, IP-10 and IL-2 induced by Spike; RANTES and IP-10 induced by NP and IL-2 induced by ORF3a. We demonstrated that the test based on IP-10 induced by Spike had the highest AUC (0.85, p < 0.0001) and that the clinical characteristics of the COVID-19-patients did not affect IP-10 production. Finally, we validated the use of IP-10 as biomarker for SARS-CoV2 infection in two additional COVID-19-patients cohorts. CONCLUSIONS: We set-up a whole-blood assay identifying the best antigen to induce a T-cell response and the best biomarkers for SARS-CoV-2 infection evaluating patients with acute COVID-19 and recovered patients. We focused on IP-10, already described as a potential biomarker for other infectious disease such as tuberculosis and HCV. An additional application of this test is the evaluation of immune response in SARS-CoV-2 vaccine trials: the IP-10 detection may define the immunogenicity of a Spike-based vaccine, whereas the immune response to the virus may be evaluated detecting other soluble factors induced by other viral-antigens.

Two new RHD alleles with deletions spanning multiple exons.

BACKGROUND: The most common large-deletion RHD allele (RHD*01N.01) includes the entire coding sequence, intervening regions and untranslated regions. The rest of large-deletion RHD alleles reported to-date consist of single-exon deletions, such as RHD*01N.67 which includes exon 1. MATERIALS AND METHODS: Samples from two donors with RhD-negative serology yielded unclear or inconclusive results when subject to confirmatory testing on RHD genotyping arrays. To determine their RHD genotypes, genomic DNA was analyzed with a combination of allele-specific PCR, long-range PCR, Sanger sequencing, and next-generation sequencing assays. RESULTS: Allele-specific PCR failed to detect products for RHD exons 1 to 3 in one sample and RHD exons 1 to 5 in the other. A quantitative next-generation sequencing assay confirmed deletion of exons 1 to 3 and 1 to 5 respectively, and detected the absence of an RHD gene in trans in both samples. Long-range PCR and Sanger sequencing enabled identification of the breakpoints for both alleles. Both deletions start within the 5' Rhesus box (upstream of the identity region for the 1-to-3 deletion, downstream of it for the 1-to-5 deletion), and end within introns. CONCLUSIONS: Resolution of unclear or inconclusive results from targeted genotyping arrays often leads to the discovery of new alleles. The 5' Rhesus box may be a hot spot for genetic recombination events, such as the large deletions described in this report.

Pain control and functional improvement in patients treated by autologous conditioned serum after failure of platelet rich plasma treatments in knee osteoarthritis.

OBJECTIVE: To assess the efficacy of autologous conditioned serum (ACS) for the treatment of patients with knee osteoarthritis after failure of medical treatments and platelet rich plasma (PRP) injections. BACKGROUND: Knee osteoarthritis is the most common form of arthritis. Prior to prosthetic surgery these patients might benefit from medical treatments, physiotherapy, and in case of their ineffectiveness, from autologous blood component injections. METHODOLOGY: We have treated 30 patients with Kellgren-Lawrence I-III knee osteoarthritis with ACS after failure of standard medical treatments/physiotherapy and platelet rich plasma (PRP) injections for a full cycle, within the previous year from enrollment. RESULTS: ACS administration was performed in all patients with mild side effects and produced prompt (1 month) improvements of VAS and Lequesne scales in 67% of patients and this result persisted at 6 and 12 months. No relationship between the rate of response and Kellgren-Lawrence scale at enrollment was observed whilst responders had a significantly higher amount of interleukin-1 receptor antagonist (IL1-RA) in ACS as compared to nonresponders. CONCLUSION: The present study confirms the efficacy of ACS in pain control and functional recovery of patients with knee osteoarthritis resistant to medical and PRP treatment. These results were obtained in a well-defined cohort of resistant patients and seem to be related with IL1-RA content in injected ACS.

Preoperative Sucrosomial Iron Supplementation Increases Haemoglobin and Reduces Transfusion Requirements in Elective Heart Surgery Patients: A Prospective Randomized Study.

BACKGROUND: Low preoperative haemoglobin is frequently observed in heart surgery patients and is associated with a significant decrease in haemoglobin between post-operative days 2 and 3, known as haemoglobin drift. Overall, these patients tend to receive many RBC transfusions. Since iron homeostasis is often impaired in these patients, restoration of iron availability might override iron-restricted erythropoiesis. However, reduced tolerance to oral iron salts has limited this strategy to intravenous iron administration. STUDY DESIGN AND METHODS: The purpose of this study was to assess whether preoperative supplementation with oral sucrosomial iron, a new iron-delivery technology with improved tolerance and bioavailability, might be an effective strategy for this patient population. One thousand consecutive patients were randomized and received either a one-month course of sucrosomial iron (60 mg/day) or no treatment prior to elective heart surgery at a single high-volume centre (ClinicalTrials.gov NCT03560687). Primary end-points were haemoglobin concentration on the day of hospital admittance and number of blood transfusions. Secondary end-points were haemoglobin drift, tolerance of treatment and cost-effectiveness of sucrosomial iron administration. RESULTS: Baseline haemoglobin in the treatment group was higher (by 0.67 g/dL; p<0.001) than that in the control group. The percentage of patients in the treatment group who required transfusion (35.4%) was half that in the control group (64.6%). The average number of transfused units per operation was 0.95 vs. 2.03 in the treatment and control groups, respectively. Haemoglobin drift was substantially similar in the two groups, and the tolerability of treatment was excellent (98%). The overall cost of treatment was 156 Euros less in the treatment group, expressed as a raw cost of transfusion. CONCLUSION: In elective heart surgery, routine preoperative sucrosomial iron administration seems to be a safe, well-tolerated and cost-effective strategy to increase preoperative haemoglobin and reduce the need for allogeneic blood transfusions.

A proposal for sectorial organizing and quality standards in therapeutic apheresis: The therapeutic apheresis unit (TAU) standards.

Therapeutic apheresis (TA) includes a wide range of therapeutic procedures based on the separation of blood components and the collection of cells with therapeutic activity or the removal of unwanted plasma or cellular components involved in the etiology of various hematologic, renal, neurological, and medical diseases. The complexity of these interventions requires an organizing model to assure a proper clinical environment, technology, quality requirements, and personnel as well as an active system for outcome monitoring for safety and efficacy. Finally, a structured organizing model may favor the efficiency of the TA unit and economic affordability. Here, we describe the more relevant characteristics of a model of TA standards, named TA unit (TAU) standards, that may help to establish a quality program in units working in the field of TA (shown as supplementary material and available at http://www.ifeit.org/pdf/TAU_Standards_3.0.pdf.

CAR-T with License to Kill Solid Tumors in Search of a Winning Strategy.

Artificial receptors designed for adoptive immune therapies need to absolve dual functions: antigen recognition and abilities to trigger the lytic machinery of reprogrammed effector T lymphocytes. In this way, CAR-T cells deliver their cytotoxic hit to cancer cells expressing targeted tumor antigens, bypassing the limitation of HLA-restricted antigen recognition. Expanding technologies have proposed a wide repertoire of soluble and cellular "immunological weapons" to kill tumor cells; they include monoclonal antibodies recognizing tumor associated antigens on tumor cells and immune cell checkpoint inhibition receptors expressed on tumor specific T cells. Moreover, a wide range of formidable chimeric antigen receptors diversely conceived to sustain quality, strength and duration of signals delivered by engineered T cells have been designed to specifically target tumor cells while minimize off-target toxicities. The latter immunological weapons have shown distinct efficacy and outstanding palmarès in curing leukemia, but limited and durable effects for solid tumors. General experience with checkpoint inhibitors and CAR-T cell immunotherapy has identified a series of variables, weaknesses and strengths, influencing the clinical outcome of the oncologic illness. These aspects will be shortly outlined with the intent of identifying the still "missing strategy" to combat epithelial cancers.

"Best practice" for extracorporeal photopheresis in acute and chronic graft-versus-host disease by Societa' Italiana di Emaferesi and Manipolazione Cellulare and Gruppo Italiano Trapianto Midollo Osseo: a national survey to ascertain its degree of application in Italian transplant centers.

BACKGROUND: A recent "best practice" promoted and published by Gruppo Italiano Trapianto Midollo Osseo (GITMO) and Societa' Italiana di Emaferesi and Manipolazione Cellulare (SIDEM) on the use of extracorporeal photopheresis (ECP) in acute and chronic graft-versus-host disease (GVHD) has been tested for the degree of agreement and application among Italian transplant centers. STUDY DESIGN AND METHODS: Twenty-four Italian centers coordinating a pair number of transplant programs completed the Web-based survey within the fixed deadline, representing approximately 52% of those centers that have a GITMO-accredited allogeneic transplant program that routinely uses ECP in GVHD. RESULTS: The level of full agreement was more than 85% for most of the answers given by the best practice (14 of 16) to the various issues. For the answers of two questions dealing with indications of ECP in acute GVHD and with safety and quality measures we observed a full agreement of 62 and 50%, respectively. CONCLUSION: This report shows a very good success of best practice among Italian centers, indicating that, after 3 years, it seems to be still updated and useful. More discussion has been observed for safety and quality measures and, likely, this issue deserves a dedicated focus on a future consensus report.

Emergency response of four transfusion centers during the last Chikungunya outbreak in Italy.

Between September 15 and November 11, 2017, the area of Rome was subjected to cessation of donation in an area of approximately 1,300,000 inhabitants and with a 5-day quarantine for the rest of metropolitan areas due to Chikungunya virus (CHIKV) outbreak. Quarantine was applied to red blood cell (RBC) and plasma units while platelet concentrates (PCs) were subjected to pathogen inactivation methods (PIMs). Quarantine was based on an active recall of all donors and, in case of declared absence of any symptom or illness, on release of RBC and plasma units after 5 days. Four regional centers in which PIMs were already performed were charged for PIMs for the rest of Rome's area. These centers increased by 236% their previous PIM procedures, producing additional 1425 pathogen-reduced (PR) PCs in 57 days, beside their production (996 PR PCs) for local needs. The emergency support was close to the maximal production capacity of the four centers and was successful only thanks to the limited length of emergency. No adverse events were reported by all centers through a passive hemovigilance approach and by a follow-up of approximately 4 months. None of the donors referred symptoms or illness at recall and none of the blood products were discarded after quarantine. To date, no cases of CHIKV-transmitted infection were reported in transfused patients. This experience has taught some relevant strategic and organizing aspects that should be taken into account when an infectious outbreak must be faced in a large metropolitan area.

Lenograstim 5 µg/kg is not superior to biosimilar filgrastim 10 µg/kg in lymphoma patients undergoing peripheral blood stem cell mobilization after chemotherapy: preliminary results from a prospective randomized study.

BACKGROUND: Randomized trials comparing chemomobilization efficiency between lenograstim and biosimilar filgrastim are lacking. Our previous retrospective study suggested that lenograstim could be more effective than biosimilar filgrastim when used at the same conventional dosage (5 µg/kg) only in lymphoma patients undergoing peripheral blood stem cell mobilization. We planned a prospective randomized study comparing lenograstim 5 µg/kg with biosimilar filgrastim 10 µg/kg to verify the hypothesis of lenograstim superiority even at half the dosage (stress test). Herein we report data after enrolling 60% of planned patients. STUDY DESIGN AND METHODS: From October 2014 to November 2017, a total of 42 of 70 planned patients with lymphoma were randomly assigned to receive lenograstim 5 µg/kg (21) or biosimilar filgrastim 10 µg/kg (21). Patients were stratified according to treatment line at the time of mobilization (1 or ≥2). Primary endpoint was the rate of achievement of the CD34+ cell collection target dose (≥ 4 × 106 /kg). An improvement by 23% was expected to validate the hypothesis of lenograstim superiority. RESULTS: The two cohorts were balanced for all the baseline features. We observed an identical rate of patients able to reach the targeted CD34+ cell dose and of mobilization failures (90.4 and 4.8% in both cohorts) and a perfect equivalence in any of the secondary collection outcomes. The hypothesis of lenograstim superiority was not corroborated at interim analysis. CONCLUSION: Lenograstim at conventional dosage has failed to demonstrate its superiority over biosimilar filgrastim at double the dosage at interim analysis in their first head-to-head trial.

A new standardized clinical-grade protocol for banking human umbilical cord tissue cells.

BACKGROUND: Mesenchymal stromal cells (MSCs) isolated from human umbilical cord tissue (UCT) can be considered the perfect candidates for cell-based therapies and regenerative medicine. UCT-derived MSCs can be cryogenically stored in cell banks and expanded as needed for therapeutic uses. STUDY DESIGN AND METHODS: We developed a new method for UCT-MSC isolation, cryopreservation, and expansion, following all criteria required by a stem cell bank. UCT-MSCs were isolated either by manual dissociation (MM) or by a semiautomatic dissociation system (SAM). In both protocols UCTs were treated enzymatically using Type IV collagenase good manufacturing practices (GMP) graded and hyaluronidase (medicinal product). Isolated UCT-MSCs were cryopreserved and analyzed after thawing for phenotype; for proliferation rate; and for their osteogenic, adipogenic, and chondrogenic differentiation capabilities. RESULTS: We found that SAM reduced the time of tissue enzyme exposure and enabled us to obtain a homogeneous single-cell suspension deprived of tissue fragments. The isolated cells in both groups showed high expression of MSC markers CD105, CD73, and CD90 and similar differentiation capabilities, phenotype, and proliferation potential. Moreover, the final yield of MSCs was comparable between the two techniques. CONCLUSION: In this study, we have established a reliable and standardized protocol to isolate UCT-MSCs from UCT for cell banking purposes. Processing the whole umbilical tissue with GMP-graded enzymes using a semiautomatic dissociator allowed us to obtain a single-cell suspension product with a known number of isolated cells that can be cryopreserved right after isolation and thawed as needed for expansion and clinical use.

A single dose of erythropoietin reduces perioperative transfusions in cardiac surgery: results of a prospective single-blind randomized controlled trial.

BACKGROUND: We conducted a prospective single-blind randomized study to assess whether a single 80,000 IU dose of human recombinant erythropoietin (HRE), given just 2 days before cardiac surgery, could be effective in reducing perioperative allogeneic red blood cell transfusion (aRBCt). STUDY DESIGN AND METHODS: Six-hundred patients presenting with preoperative hemoglobin (Hb) level of not more than 14.5 g/dL were randomly assigned to either HRE or control. The primary endpoint was the incidence of perioperative aRBCt. The secondary endpoints were mortality and the incidence of adverse events in the first 45 days after surgery, Hb level on Postoperative Day 4, and number of units of RBC transfusions in the first 4 days after surgery. RESULTS: A total of 17% (HRE) versus 39% (control) required transfusion (relative risk, 0.436; p<0.0005). After baseline Hb was controlled for, there was no difference in the incidence of aRBCt between HRE (0%) and control (3.5%) among the patients with baseline Hb of 13.0 g/dL or more, which included the nonanemic fraction of the study population. The mean (range) Hb level on Postoperative Day 4 was 10.2 (9.9-10.6) g/dL (HRE) versus 8.7 (8.5-9.2) g/dL (control; p<0.0005). The distribution of number of units transfused was shifted toward fewer units in HRE (p<0.0005). The all-cause mortality at 45 days was 3.00% (HRE) versus 3.33% (control). The 45-day adverse event rate was 4.33% (HRE) versus 5.67% (control; both p=NS). CONCLUSION: In anemic patients (Hb<13 g/dL), a single high dose of HRE administered 2 days before cardiac surgery is effective in reducing the incidence of aRBCt without increasing adverse events.

Stress, burnout, and job satisfaction in 470 health professionals in 98 apheresis units in Italy: A SIdEM collaborative study.

AIMS: In Italian and international background, there are no studies focusing on stress, burnout indicators, and job satisfaction in health professionals working in the apheresis units. This study aims to fill this void both for scientific and clinical reasons. METHODS: The participants were 470 health professionals (220 physicians, 250 nurses), mostly female (73.4%), with an average age of 48.09 (with the 5° percentile under 32 years and the 95° percentile over 60), working in the Apheresis Units in the North (228), in the Center (131) and in the Southern-islands of Italy (111). The health professionals' years on the job were principally between one and 10 years (40.2%) or from 11 to 20 years (33.2%). The prevalent activity was therapeutic apheresis (48.5%). The self-report questionnaires were proposed electronically by a protected online site. RESULTS: Important stress levels were identified in the health professionals. Physicians principally showed medium (47.5%) and high (35.8%) stress levels. Stress levels of nurses were mostly low (57.7%) or medium (25.7%). Female gender in nurses [t(268) = -3.29; P = 0.001] and in physician professions [t(217) = -3.01; P = 0.03] was a risk factor for stress. Both job categories were placed at a high risk level for burnout syndrome comparing with normative scales, especially the health professionals working in the center of Italy for the scales "Emotional exhaustion" [F(2) = 4.39; P = 0.013] and "Professional inefficacy" [F(2) = 4.38; P = 0.013]. CONCLUSIONS: Health professionals working in the apheresis unit show high stress levels and burnout risk. New preventive programs and specific clinical interventions should be constructed.

Pathogen-free, plasma-poor platelet lysate and expansion of human mesenchymal stem cells.

BACKGROUND: Supplements to support clinical-grade cultures of mesenchymal stem cells (MSC) are required to promote growth and expansion of these cells. Platelet lysate (PL) is a human blood component which may replace animal serum in MSC cultures being rich in various growth factors. Here, we describe a plasma poor pathogen-free platelet lysate obtained by pooling 12 platelet (PLT) units, to produce a standardized and safe supplement for clinical-grade expansion of MSC. METHODS: PL lots were obtained by combining 2 6-unit PLT pools in additive solution (AS) following a transfusional-based procedure including pathogen inactivation (PI) by Intercept technology and 3 cycles of freezing/thawing, followed by membrane removal. Three PI-PL and 3 control PL lots were produced to compare their ability to sustain bone marrow derived MSC selection and expansion. Moreover, two further PL, subjected to PI or not, were also produced starting from the same initial PLT pools to evaluate the impact of PI on growth factor concentration and capacity to sustain cell growth. Additional PI-PL lots were used for comparison with fetal bovine serum (FBS) on MSC expansion. Immunoregulatory properties of PI-PL-generated MSC were documented in vitro by mixed lymphocyte culture (MLC) and peripheral blood mononuclear cells (PBMC) mitogen induced proliferation. RESULTS: PI-PL and PL control lots had similar concentrations of 4 well-described growth factors endowed with MSC stimulating ability. Initial growth and MSC expansion by PI-PL and PL controls were comparable either using different MSC populations or in head to head experiments. Moreover, PI-PL and PL control sustained similar MSC growth of frozen/thawed MSC. Multilineage differentiation of PI-derived and PI-PL-derived MSC were maintained in any MSC cultures as well as their immunoregulatory properties. Finally, no direct impact of PI on growth factor concentration and MSC growth support was observed, whereas the capacity of FBS to sustain MSC expansion in basic medium was irrelevant as compared to PL and PI-PL. CONCLUSION: The replacement of animal additives with human supplements is a basic issue in MSC ex vivo production. PI-PL represents a standardized, plasma-poor, human preparation which appears as a safe and good candidate to stimulate MSC growth in clinical-scale cultures.

Kinetics of the use of cryopreserved autologous stem cell grafts: a GITMO-SIDEM survey.

BACKGROUND AIMS: Hematopoietic stem cell cryopreservation significantly contributed to autologous stem cell transplantation (ASCT). Cryopreserved stem cell units (SCU) are expected to be used soon after harvesting for most purposes, but, in a number of cases, they remain stored for some time, creating an increasing load for SCU depositories. Disposal policies vary widely in each center, and the existing guidelines are insufficient. METHODS: We conducted a survey of seven Gruppo Italiano Trapianto di Midollo Osseo centers to investigate the outcome of SCU harvested from January 2005 to December 2009 for ASCT. The data from 1603 collections were gathered, for a total of 5822 SCU. RESULTS: In our cohort, 79% of patients collected >5 × 106 CD34+ cells/kg, and 3.4% collected <2 × 106 CD34+ cells/kg. Up to 21% of all the patients and 42% of those with acute leukemia did not undergo reinfusion, and 37% of the cryopreserved SCU were excess, resulting from patients not reinfusing or partially reinfusing. Less than one-third of the excess SCU was disposed, and the major causes of disposal were death and, in a minority of cases, withdrawal of the indication for ASCT. In our analysis, very few first reinfusions occurred after 2 years, and those after 5 years were exceptional. Through the use of a multivariate analysis, we sought to identify the risk factors for collection non-use, independent of the centers' policies. Non-use of SCU was significantly associated with patients with acute leukemia, collections of <2 × 106 CD34/kg and lower age groups. CONCLUSIONS: These data serve as a valid basis to support rational recommendations for cost-effective storage and disposal of SCU.