Telemedicine pre-screening for blood donor.

INTRODUCTION: The COVID-19 pandemic had an important impact on blood bank services. The onset of the pandemic led to a decrease in the number of blood donors. A remote interview would avoid deferred donors from having to travel to the blood bank. We evaluate the feasibility of using telemedicine as an alternative to a face-to-face interview as a first blood donor screening. METHODS: Our retrospective study included 404 whole blood and platelets donors, who underwent the clinical interview remotely via telemedicine. The deferred donor would not need to go to the blood bank and eligible candidates were required to donate within 7 days. On the day of donation, a mini-interview was held to ensure donor and blood safety. RESULTS: The appointments were made from June 2020 to June 2022, including 263 candidates for whole blood (WB) and 141 for platelets (PLTs). At the end of the telemedicine interview, 285 (70.6 %) candidates were considered eligible. Telemedicine was not performed for 60 (14.8 %) candidates due to technical problems (with audio or video) or absences. The deferral rate among candidates who underwent telemedicine pre-screening was 14.6 % and, among eligible donors after telemedicine, only 7 (2.9 %) were unable to donate blood. CONCLUSION: Telemedicine is a viable alternative and a welcome convenience for potential donors to avoid unnecessary travel.

Novel method for determining when a field-collected donor unit is sufficiently full.

BACKGROUND: Whole blood (WB) collections can occur downrange for immediate administration. An important aspect of these collections is determining when the unit is sufficiently full. This project tested a novel method for determining when a field collection is complete. METHODS: The amount of empty space at the top of WB units, destined to become LTOWB or separated into components, that were collected at blood centers or hospitals was measured by holding a WB unit off the ground and placing the top of a piece of string where the donor tubing entered the bag. The string was marked where it intersected the top of the column of blood in the bag and measured from the top. The WB units were also weighed. RESULTS: A total of 15 different bags, two of which were measured in two different filling volumes, from 15 hospitals or blood centers were measured and weighed. The most commonly used blood bag, Terumo Imuflex SP, had a median string length of 9 mm (range: 2-24 mm) and weighed a median of 565.1 g (range: 524.8-636.7 g). CONCLUSION: Pieces of string can be precut to the appropriate length depending on the type of bag before a mission where field WB collections might be required and a mark placed on the bag before the collection commences to indicate when the unit is full.

Virtual platelet cross-matching as transfusion management for patients with immune platelet refractoriness.

BACKGROUND AND OBJECTIVES: This study describes the use of the Epvix platform for virtual cross-matching (VC) of human leucocyte antigen (HLA)-compatible platelets for patients with immune platelet refractoriness, and demonstrates effectiveness of the selected platelets. MATERIALS AND METHODS: A prospective cohort of haematological patients was evaluated from 2018 to 2022. HLA-typed donor bank profile was previously uploaded to the Epvix platform. Each patient's antibody reactivity panel (PRA) was included in the platform. Then, search, selection and VC were performed, and 24-h-corrected count increment (CCI) platelet transfusion was calculated (reference ≥2500). RESULTS: Six patients were included (four female, two male), with mean age of 61 years. HLA antibodies were detected as the cause of immunity for all patients, whereas four patients also had non-immune causes. High percentage of alloimmunization was detected in all studied patients (mean PRA: 85.7%). Thirty different donors were able to schedule and perform platelet donations. The mean 24-h CCI count was 9882. All platelet transfusions achieved a satisfactory CCI count except for two transfusion events. Presence of non-immune causes identified in these two cases could account for the unsatisfactory CCI. CONCLUSION: Epvix is a free application hosted on the Web and uses the HLAMatchmaker algorithm to generate histocompatibility reports. This study demonstrates the efficiency of VC performed by Epvix. However, physical cross-matching will still be necessary in some instances, as the platform does not support human platelet antigen polymorphism.

Infectious diseases and the impact on transfusion medicine: A historical review and lessons for the future.

Blood transfusion saves millions of lives each year. It is a well-established treatment, and many procedures are applied to avoid transmitted infections. However, throughout the history of transfusion medicine, many infectious diseases arose or were recognised, bringing up an impact on the blood supply, as the difficulties in diagnosing new diseases, the decrease in blood donors, the challenges for the medical team, the risks for the receptor and the related costs. This study aims to review historically the principal infectious diseases transmitted through the blood that circulated worldwide in the 20th and 21st centuries, considering the impact on the blood banks. Despite the current blood bank control of transfusion risks and the hemovigilance improvements, transmitted and emerging infections can still compromise the blood bank supply, as we have witnessed during the first waves of the COVID-19 pandemic. Moreover, new pathogens will continue emerging, and we must be prepared for the future.

Quality assessment of red blood cell concentrates from blood donors at the extremes of the age spectrum: The BEST collaborative study.

BACKGROUND: Blood donors at the extremes of the age spectrum (16-19 years vs. ≥75 years) are characterized by increased risks of iron deficiency and anemia, and are often underrepresented in studies evaluating the effects of donor characteristics on red blood cells (RBC) transfusion effectiveness. The aim of this study was to conduct quality assessments of RBC concentrates from these unique age groups. STUDY DESIGN: We characterized 150 leukocyte-reduced (LR)-RBCs units from 75 teenage donors, who were matched by sex, and ethnicity with 75 older donors. LR-RBC units were manufactured at three large blood collection centers in the USA and Canada. Quality assessments included storage hemolysis, osmotic hemolysis, oxidative hemolysis, osmotic gradient ektacytometry, hematological indices, and RBC bioactivity. RESULTS: RBC concentrates from teenage donors had smaller (9%) mean corpuscular volume and higher (5%) RBC concentration compared with older donors counterparts. Stored RBCs from teenage donors exhibited increased susceptibility to oxidative hemolysis (>2-fold) compared with RBCs from older donors. This was observed at all testing centers independent of sex, storage duration, or the type of additive solution. RBCs from teenage male donors had increased cytoplasmatic viscosity and lower hydration compared with older donor RBCs. Evaluations of RBC supernatant bioactivity suggested that donor age was not associated with altered expression of inflammatory markers (CD31, CD54, and IL-6) on endothelial cells. CONCLUSIONS: The reported findings are likely intrinsic to RBCs and reflect age-specific changes in RBC antioxidant capacity and physical characteristics that may impact RBC survival during cold storage and after transfusion.

Neutrophil extracellular traps (NETs), transfusion requirements and clinical outcomes in orthotopic liver transplantation.

Inflammatory phenomena have a direct impact on the prognosis of orthotopic liver transplantation (OLT). Neutrophil extracellular traps (NETs) contribute to OLT inflammation and hemostasis imbalance in OLT. The association between NETosis, clinical outcomes and transfusion requirements is not determined. To evaluate NETs release during OLT and the effect of NETosis ontransfusion requirements and adverse outcomes in a prospective cohort of patients submitted to OLT. We quantified citrullinated histones (cit-H3) and circulating-free-DNA (cf-DNA) in ninety-three patients submitted to OLT in three periods: pre-transplant, after graft reperfusion and before discharge. NETs markers were compared between these periods using ANOVA test. The association of NETosis and adverse outcomes was evaluated using regression models adjusted for age, sex and corrected MELD. We observed a peak of circulating NETs following reperfusion, evidenced by a 2.4-fold increase in cit-H3 levels in the post-graft reperfusion period (median levels of cit-H3 pre transplant: 0.5 ng/mL, after reperfusion: 1.2 ng/mL and at discharge 0.5 ng/mL, p < 0.0001). We observed an association between increased levels of cit-H3 and in-hospital death (OR = 1.168, 95% CI 1.021-1.336, p = 0.024). No association was found between NETs markers and transfusion requirements. There is a prompt release of NETs after reperfusion that is associated with poorer outcomes and death. Intraoperative NETs release seems to be independent of transfusion requirements. These findings highlight the relevance of inflammation promoted by NETS and its impact on OLT adverse clinical outcomes.

Nucleated red blood cell: a feasible quality parameter of cord blood units.

INTRODUCTION: Umbilical cord blood is an alternative source of hematopoietic progenitor cells for bone marrow transplantation; however, it is associated with a higher graft failure rate. The presence of a high rate of nucleated red blood cells (NRBCs) seems to be related to a greater capacity for engraftment, although is also associated with fetal distress conditions. We analyzed the correlation of the NRBC with quality parameters and its association with the utilization score of a cord blood unit. STUDY DESIGN AND METHOD: Data of 3346 units collected in a public cord blood bank from May 2010 to December 2017 were analyzed, retrospectively, to identify factors associated with an increased number of nucleated red blood cells and its correlation with the engraftment capacity measured through total nucleated cells (TNCs) and CD34 positive cells. We also evaluated the utilization score of these units and identified an NRBC cutoff associated with a higher score. RESULTS: The median volume collected was 104 mL (42-255), the pre-processing TNC count was 144.77 × 107 (95.46-477.18), the post-processing TNC count was 119.44 × 107 (42.7-477.18), the CD34 count was 4.67 × 106 (0.31-48.01), the NRBC count was 5 (0-202) and the utilization score was 0.0228 (0.00143-0.9740). The NRBC showed a correlation with the collected volume, TNC and CD34 positive cells and a higher utilization score and the receiver operating characteristic (ROC) curve analysis identified the five NRBC/100 leukocytes cutoff that correlates better with the probability of use. No association with pathological conditions and the NRBC rate was observed. CONCLUSIONS: The NRBC is a feasible parameter for the screening of the cord blood unit (CBU) and the minimum cutoff of five NRBC/100 leukocytes can be a strategy in conjunction with the TNC to identify better units for cord blood bank sustainability.

A common variant close to the "tripwire" linker region of NLRP1 contributes to severe COVID-19.

OBJECTIVE AND DESIGN: The heterogeneity of response to SARS-CoV-2 infection is directly linked to the individual genetic background. Genetic variants of inflammasome-related genes have been pointed as risk factors for several inflammatory sterile and infectious disease. In the group of inflammasome receptors, NLRP1 stands out as a good novel candidate as severity factor for COVID-19 disease. METHODS: To address this question, we performed an association study of NLRP1, DPP9, CARD8, IL1B, and IL18 single nucleotide variants (SNVs) in a cohort of 945 COVID-19 patients. RESULTS: The NLRP1 p.Leu155His in the linker region, target of viral protease, was significantly associated to COVID-19 severity, which could contribute to the excessive cytokine release reported in severe cases. CONCLUSION: Inflammasome genetic background contributes to individual response to SARS-CoV-2.

Zika virus RNA detection in blood donors in São Paulo, Brazil

ABSTRACT Introduction: The Zika Virus (ZIKV) is a single-stranded RNA genome virus, belonging to the family Flaviviridae, genus Flavivirus. Outbreaks around the world have demonstrated that the presence of asymptomatic viremic blood donors provides an increase in the risk of transfusion transmission (TT) and nucleic acid test (NAT) screening has been proposed to ensure the blood safety. This study implemented an "in-house" method to detect ZIKV RNA in blood sample donations. Methods: Primary plasma tubes are submitted to nucleic acid extraction on an automated platform. After extraction, the NAT set-up is performed in the robotic pipettor, in which an amplification mixture containing primers and probes for ZIKV and Polio vaccine virus (PV) are added in duplex as an internal control. The real-time polymerase chain reaction is then performed in a thermocycler, using the protocol established by the supplier. Results: From May 2016 to May 2018, 3,369 samples were collected from 3,221 blood donors (confidence coefficient 95%), of which 31 were considered false positive (0.92%), as they did not confirm initial reactivity when repeated in duplicates and 14 (0.42%) had their results invalid due to repeat failure in the internal control, 4 (0.12%), due to insufficient sample volume and 2 (0.05%), due to automatic pipettor failures. No Zika RNA reactive sample was identified. Conclusion: The test showed feasible to be incorporated into the blood screening routine. Our data do not indicate the need to screen for ZIKV RNA in São Paulo during the evaluated period. However, a generic NAT system covering a group of flaviviruses which are circulating in the region, such as DENV and YFV, among others, could be a useful tool.

The relevance of a bank with genotyped platelets donors

ABSTRACT Objective: Describe the clinical and laboratory characteristics and the transfusion strategy of patients at Hospital Israelita Albert Einstein with platelet refractoriness and identify their etiological characteristics. Standardize the platelet immunofluorescence technique by flow cytometry as a test for platelet compatibility in immune platelet refractoriness in transfusion support. Methods: Review of medical records of refractory platelet patients followed at HIAE from January 2011 to May 2017. Clinical-demographic data, laboratory data and identification of the use of compatible genotyped platelets for patients in need of transfusion therapy were collected. The analyzed patients were classified according to the etiology of their platelet refractoriness. To standardize the FC-PIFT technique, blood group O platelets were incubated with serum from blood group AB donors and anti-IgG monoclonal antibody to determine the negative control. In order to verify the influence of the ABO system, monoclonal anti-IgG antibodies were incubated with blood group A or B platelets and with blood group O donor serum with isohemagglutinins below and above 1/64. Results: A total of 47 patients were evaluated, a 51% (24/47) preponderance of associated immune and non-immune factors (NIPR + IPR). The most common causes of NIPR + IPR were splenomegaly (54%) and the development of HLA antibodies (88%), consistent with the literature. For patients who required therapeutic transfusion, only a small portion received compatible genotyped platelets. Conclusion: Although 60% of patients could benefit from the therapeutic transfusion of genotyped platelets, only 10% were actually transfused with this type of blood component. This reaffirms the need for investments in a bank of genotyped platelet donors.

Platelet antibodies identification: comparison between two laboratory tests

ABSTRACT Introduction: Platelet antibody identification is indispensable for diagnosing the human platelet antigen (HPA) or human leukocyte antigen (HLA) immunization, mostly because it can restrict the compatibility and results of transfusions. Correct detection of these antibodies is of utmost importance for the diagnosis and treatment. Method: We present 16 platelet alloimmunization results, comparing two tests with different technologies: the MAIPA (monoclonal antibody immobilization of platelet antigens), as a reference technique, and a bead-based assay, the Pak-Lx. Results: Eleven samples (68.75%) showed agreement in both techniques. Two tests were false negatives in the Pak-Lx: a pan-reactivity in GPIIbIIIa and an anti-HPA-9b. On the other hand, the Pak-Lx was more sensitive to detect a decreasing anti-HPA-5b. The Pak-Lx found an anti-HPA-2b positive, but with a low median fluorescent intensity (MFI), suggesting a false-positive result. Moreover, in one case, the MAIPA was negative for a positive Pak-Lx HLA. Conclusion: Antibody platelet diagnosis can sometimes be challenging. The methods seemed similar, the Pak-Lx being faster and simpler than the MAIPA, and they can be complementary to solve clinical issues.

Preclinical Evaluation of the Tumorigenic and Immunomodulatory Properties of Human Bone Marrow Mesenchymal Stromal Cell Populations with Clonal Trisomy 5.

Cytogenetic aberrations may emerge in human mesenchymal stromal cells (MSC) during ex vivo expansion for cell therapy. We have detected clonal trisomy 5 in two distinct autologous MSC products expanded from bone marrow which, based on the current quality control criteria, could not be released for clinical use. Although a safety concern, it is still unclear to what extent recurrent aneuploidies detected in MSC products may affect the threshold for neoplastic transformation or the medicinal properties of these cells. We have carried out an exploratory preclinical study to evaluate these MSC products with clonal trisomy 5, regarding their oncogenic and immunomodulatory potential. Cell population growth in vitro was reduced in MSC cultures with clonal trisomy 5 compared with the population growth of their euploid MSC counterparts, based on a lower cumulative population doubling level, reduced cell proliferation index, and increased senescence-associated beta-galactosidase activity. Subcutaneous injection of clinically relevant amount of MSC population, either with or without clonal trisomy 5, did not generate tumors in immunodeficient mice within a follow-up period of six months. Most importantly, MSC population with clonal trisomy 5 kept immunomodulatory properties upon interferon gamma (IFNγ) licensing, displaying overexpression of IDO, CXCL9, CXCL10, and CXCL11, in a similar fashion than that of IFNγ-licensed euploid MSC. Our findings suggest that bone marrow MSC products with clonal trisomy 5 may retain their therapeutic potential, based on poor tumor initiating capability and preserved immunomodulatory potency. This preclinical evidence may further support the definition of release criteria of autologous MSC products for cell therapy under critical clinical scenarios. This trial is registered with Clinical Study registration number: RBR-29x2pr.

HLA-DRB1 and cytokine polymorphisms in Brazilian patients with myelodysplastic syndromes and its association with red blood cell alloimmunization.

OBJECTIVE(S): This study aimed investigate association of HLA-DRB1 and cytokine polymorphisms with red blood cell(RBC) alloimmunization in Brazilian Myelodysplastic syndrome(MDS) patients with prior exposure to RBC transfusion. BACKGROUND: MDS patients are at risk RBC alloimmunization due to chronic RBC transfusion. However, differences in immune response of MDS transfused patients are not completely known. METHODS/MATERIALS: A retrospective cohort of 87 polytransfused patients with MDS including 28 alloimmunized (PA) and 59 non-alloimmunized (PNA) was evaluated in three Brazilian reference hospitals. HLA-DRB1genotype was performed by polymerase chain reaction (PCR)-SSOP (Luminex platform) and cytokine polymorphisms analysed by PCR and TaqMan assays. RESULTS: While HLA-DRB1 allele frequencies did not differ between groups, IL17A 197G > A SNP and IL4 polymorphisms showed significant correlation with RBC alloimmunization. IL17A 197A allele A and AA genotype were significantly more frequent in PA than PNA(A, 46.4% versus 27.1%, p = 0.012; OR = 2.3; 95%CI = 1.1-4.9; AA, 25% versus 6.8%, p = 0.041; OR = 6.2; 95%CI 1.3-30.8). Moreover, significant association of alloimmunization to Rh antigens with IL17A 197A allele and AA genotype was also identified in PA group(A, 45% versus 27.1%, p = 0.036; OR = 2.5; 95% CI 1.1-5.7; AA, 30% versus 6.8%, p = 0.042; OR = 7.9; 95%CI 1.5-42.3). Genotype A1A2 of IL4 intron 3 was overrepresented in PA(50% versus 16.9%, p = 0.009; OR = 4.97; 95%CI 1.6-15.5). Similarly, IL4-590 CT genotype was overrepresented in PA(53.6% versus 28.8%, p = 0.049; OR = 3.3; 95%CI 1.2-9.3). CONCLUSIONS: This study showed no association regarding HLA-DRB1 alleles for RBC alloimmunization risk or protection, however the IL17A 197G>A, IL4 intron 3 and IL4 590C>T SNP was significantly associated to RBC alloimmunization risk in this cohort of Brazilian MDS patients.

Hospital red blood cell and platelet supply and utilization from March to December of the first year of the COVID-19 pandemic: The BEST collaborative study.

BACKGROUND: At the start of the coronavirus disease 2019 (COVID-19) pandemic, widespread blood shortages were anticipated. We sought to determine how hospital blood supply and blood utilization were affected by the first wave of COVID-19. STUDY DESIGN AND METHODS: Weekly red blood cell (RBC) and platelet (PLT) inventory, transfusion, and outdate data were collected from 13 institutions in the United States, Brazil, Canada, and Denmark from March 1st to December 31st of 2020 and 2019. Data from the sites were aligned based on each site's local first peak of COVID-19 cases, and data from 2020 (pandemic year) were compared with data from the corresponding period in 2019 (pre-pandemic baseline). RESULTS: RBC inventories were 3% lower in 2020 than in 2019 (680 vs. 704, p < .001) and 5% fewer RBCs were transfused per week compared to 2019 (477 vs. 501, p < .001). However, during the first COVID-19 peak, RBC and PLT inventories were higher than normal, as reflected by deviation from par, days on hand, and percent outdated. At this time, 16% fewer inpatient beds were occupied, and 43% fewer surgeries were performed compared to 2019 (p < .001). In contrast to 2019 when there was no correlation, there was, in 2020, significant negative correlations between RBC and PLT days on hand and both percentage occupancy of inpatient beds and percentage of surgeries performed. CONCLUSION: During the COVID-19 pandemic in 2020, RBC and PLT inventories remained adequate. During the first wave of cases, significant decreases in patient care activities were associated with excess RBC and PLT supplies and increased product outdating.

Report of a clinical and laboratory management of cell therapy for knee cartilage in the face of mycoplasma contamination.

To describe a case of autologous chondrocyte implantation after cell culture contamination by Mycoplasma pneumoniae and the measures taken to successfully complete cell therapy in a patient with focal chondral lesion. A 45-year-old male patient, complaining of chronic pain on the knee and no history of trauma. He had a chondral lesion in the trochlear region of the femur and clinical tests compatible with pain in the anterior compartment of the knee. Conservative treatment failed to alleviate symptoms. Surgical treatment was indicated, but due to the size of the lesion, membrane-assisted autologous chondrocyte implantation was the technique of choice. Cartilage biopsies were collected from the intercondylar region of the distal femur. After isolation, chondrocytes were expanded ex vivo in a trained laboratory, for three weeks, and seeded onto a commercially available collagen membrane prior to implantation in the knee. Two days before surgery, a cell culture sample tested positive for Mycoplasma pneumoniae. The source of contamination was found to be autologous blood serum, extracted from the patient´s peripheral vein, and used to supplement the cell culture medium. After treating the patient with antibiotics, all procedures were repeated and the new final cell product, free from contaminants, was successfully implanted. We discuss the strategies available to deal with this situation, and describe the results of this particular case, which led to modifications in the autologous chondrocyte implant protocol.

Matrix-assisted autologous chondrocyte transplantation for treatment of focal chondral lesions in the knee: the Hospital Israelita Albert Einstein experience.

OBJECTIVE: Phase 1 clinical trial to determine feasibility, safety, and efficacy of a new advanced cell therapy product for treatment of knee articular cartilage injuries. METHODS: Three participants with knee focal chondral lesions were included, with no signs of osteoarthritis. Chondrocytes were obtained through knee arthroscopy, cultured in collagen membrane for 3 weeks at the laboratory, subjected to tests to release the cell therapy product, and implanted. All patients underwent a specific 3-month rehabilitation protocol, followed by assessments using functional and imaging scales. The main outcome was the incidence of severe adverse events. RESULTS: Three participants were included and completed the 2-year follow-up. There was one severe adverse event, venous thrombosis of distal leg veins, which was no associated with therapy, was treated and left no sequelae. The clinical and radiological scales showed improvement in the three cases. CONCLUSION: The preliminary results, obtained with the described methodology, allow concluding that this product of advanced cell therapy is safe and feasible. ReBEC platform registration number: RBR-6fgy76.

Senescence State in Mesenchymal Stem Cells at Low Passages: Implications in Clinical Use.

Mesenchymal stem cells (MSCs) are multipotent cells found in various tissues and are easily cultivated. For use in clinical protocols, MSCs must be expanded to obtain an adequate number of cells, but a senescence state may be instituted after some passages, reducing their replicative potential. In this study, we report a case where MSC derived from an elderly donor acquired a senescence state after three passages. The bone marrow was aspirated from a female patient submitted to a cell therapy for the incontinency urinary protocol; MSCs were cultivated with DMEM low glucose, supplemented with 10% autologous serum (AS) plus 1% L-glutamine and 1% antibiotic/antimycotic. Senescence analysis was performed by ß-galactosidase staining after 24 and 48 h. Controls were established using BM-MSC from healthy donors and used for senescence and gene expression assays. Gene expression was performed using RT-PCR for pluripotency genes, such as SOX2, POU5F1, NANOG, and KLF4. MSC telomere length was measured by the Southern blotting technique, and MSCs were also analyzed for their capacity to differentiate into adipocytes, chondrocytes, and osteocytes. The patient's MSC expansion using AS displayed an early senescence state. In order to understand the role of AS in senescence, MSCs were then submitted to two different culture conditions: 1) with AS or 2) with FBS supplementation. Senescence state was assessed after 24 h, and no statistical differences were observed between the two conditions. However, patients' cells cultured with AS displayed a higher number of senescence cells than FBS medium after 48 h (p = 0.0018). Gene expression was performed in both conditions; increased expression of KLF4 was observed in the patient's cells in comparison to healthy controls (p = 0.0016); reduced gene expression was observed for NANOG (p = 0.0016) and SOX2 (p = 0.0014) genes. Telomere length of the patient's cells was shorter than that of a healthy donor and that of a patient of similar age. Osteocyte differentiation seemed to be more diffuse than that of the healthy donor and that of the patient of similar age. MSCs could enter a senescence state during expansion in early passages and can impact MSC quality for clinical applications, reducing their efficacy when administered.