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The question on the duration and effectiveness of post-infection vs post-vaccination SARS-CoV-2 immunity remains in the focus of numerous studies. The aim of the work was to examine the duration of maintained post-infection and post-vaccination SARS-CoV-2 immunity as well as formation of hybrid (vaccination after infection) and breakthrough (repeated disease or disease after vaccination) immunity in the context of an ongoing COVID-19 pandemic. 107 adults with mild or moderate COVID-19 3-18 months after the disease and 30 subjects vaccinated twice with the Sputnik V vaccine were examined 1-6 times. Antibodies against SARS-CoV-2 virus were determined by ELISA on the "SARSCoV-2-IgG quantitative-ELISA-BEST" test systems. The antibody avidity was measured by additional incubation with and without denaturing solution. Mononuclear cells were isolated from blood by gradient centrifugation, incubated with and without coronavirus S-protein for 20 hours, stained with fluorescently labeled antibodies, and the percentage of CD8highCD107a+ was counted using FACSCanto II cytometer. It was shown that in the group of convalescent and vaccinated subjects, the level of virus-specific antibodies decreased more deeply in individuals with initially high humoral response, but 9 months later the decrease slowed down and reached a plateau. The antibody avidity rose up to 50% and persisted for 18 months. Cellular immunity in recovered patients did not change for 1.5 years, while in vaccinated patients it gradually decreased 6 months later, but remained at detectable level. After revaccination, a significant increase in the level of antibodies, avidity up to 67.6% and cellular immunity returned to the initial level were noted. Hybrid immunity turned out to be significantly higher than post-infection and post-vaccination immunity. The level of antibodies increased to 1218.2 BAU/ml, avidity - to 69.85%, and cellular immunity - to 9.94%. Breakthrough immunity was significantly higher than that after the first disease. The level of antibodies rose to 1601 BAU/ml, avidity - up to 81.6%, cellular immunity - up to 13.71%. Using dynamic observation of four COVID-19 convalescents, it has been shown that in the context of the ongoing pandemic and active coronavirus mutation, natural boosting occurs both asymptomatically and as a result of a mild re-infection, which prevents disappearance of SARS-CoV-2 humoral and cellular immunity.Copyright © 2023 Saint Petersburg Pasteur Institute. All rights reserved.
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Background: mRNA vaccines have proven useful in protecting vulnerable populations against SARS-CoV-2 infection. However, certain therapeutics, specifically those used in cancer treatment, reduce mRNA vaccine-induced humoral responses against SARS-CoV-2. The effects on T cell responses are not well characterized. Here, we evaluate SARS-CoV-2 spike-specific T cell responses over the course of one year in solid tumor patients in BC, Canada. Method(s): 18 female, solid-tumor patients from the BC Cancer Agency were enrolled in this prospective, cohort study, with 7 patients receiving cytotoxic chemotherapy and 11 patients receiving non-cytotoxic treatments. Whole blood was collected 1-month (T1) and one-year +/- 1-month (T2) post series completion (2 mRNA doses). Antigen-induced marker assays (AIM assays) were used to quantify CD4+ and CD8+ T cell responses, where whole blood was stimulated with ancestral or omicron SARS-CoV2 Spike peptide pools or unstimulated for 48 hours at 37degree C, fluorescently stained for activation markers CD25 and OX40 (CD4+ T cells) or CD69 and CD137 (CD8+ T cells), and analyzed using a 5-laser flow cytometer. Phenotyping of antigen-specific CD4+ T cells was done in parallel to assess the frequency of spike-specific Tregs, Th1, Th2, Th9, Th17, and Th17.1 cells. Result(s): All individuals had detectable levels of spike-specific CD4+ T cells at T2, while only 72.2% of individuals had detectable levels of spike-specific CD8+ T cells. Treatment type did not significantly impact the magnitude or phenotype of T cell responses, including those to Omicron. However, increased age was associated with decreased ancestral CD8+ T cell responses at T2. Further, ancestral and omicron responses were significantly different at T2, with decreased magnitude and altered phenotype of omicron-specific CD4+ T cells. Conclusion(s): Here, we report that solid tumor patients, treated with either chemotherapy or biologics, mount robust T cell immunity to SARS-CoV-2 following vaccination. Additional data is needed to determine if these responses correlate with antibody levels and clinical illness.
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Neutrophilic granulocytes (NG) are the main drivers of pathological inflammation in COVID-19. Objective. To specify the mechanisms of immunopathogenesis of COVID-19 based on a comparative immunological study of the number and phenotype of CD16+SD62L+CD11b+CD63- and CD16+SD62L+CD11b+CD63+ subsets with an assessment of their effector functions against changing profile of NG-associated cytokines IL-8, IL-18, IL-17A, VEGF-A, IFNalpha, and IFNgamma. Patients and methods. In patients with moderate-to-severe and severe COVID-19, we determined IL-1beta, TNFalpha, IL-6, IL-8, IL-18, IL-17A, VEGF-A, IFNalpha, and IFNgamma (ELISA), the phenotype of CD16+SD62L+CD11b+CD63- and CD16+SD62L+CD11b+CD63+ subsets, NF-kappaB-NG (CYTOMICS FC500), phagocytically active NG (%), neutrophil extracellular traps (NETs), NG in apoptosis, and the activity of NADPH oxidase. Results. In COVID-19 against the background of IFNalpha and IFNgamma production blockade and high levels of NG-associated IL-8, IL-18, IL-17A, VEGF-A, a reduction in the number of mature and functionally active CD16brightSD62LbrightCD11bbrightCD63-NG subsets was revealed, as well as an increase in the number of CD16dimSD62LdimSD11bbrightCD63-NG subsets with an immunosuppressive phenotype and CD16brightSD62LbrightSD11bbrightCD63bright-NG subsets with high cytotoxic activity and ability to form NETs, a decrease in the percentage of phagocytically active NG and an increase in the activity of NADPH oxidase, NETs, and NG in apoptosis. Conclusion. IFNalpha deficiency provokes a hyperergic response of NG-associated cytokines, which leads to the formation of uncontrolled immune inflammation involving NG subsets with an immunosuppressive and cytotoxic phenotype, exacerbating the course of COVID-19. The use of recombinant IFNalpha-2b with antioxidants (Viferon) in the early stages of the disease can help to restore immune homeostasis, normalize the level of NG-associated cytokines, reduce NERTs, and achieve good clinical efficacy.Copyright © 2022, Dynasty Publishing House. All rights reserved.
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Background: Drug hypersensitivity reactions (DHRs) of the immediate type are diagnosed in approximately 1-2% per 100 thousand people. During the COVID-19 pandemic, the use of antibiotics increased, and cases of immediate reactions to these drugs became more frequent. However, due to the lack of medical centers which have the necessary conditions for carrying out provocation tests, the use of in vitro diagnostic methods for hypersensitivity reactions to antibiotics is becoming even more relevant during the pandemic. Flow CAST Basophil Activation Test (BAT) Flow Cytometry can be used for the in vitro detection of immediate type allergic reactions and hypersensitivities to suspected allergens in patients at risk for DHRs. The purpose is to study the possibility of diagnosing hypersensitivity reactions to antibiotics using BAT to antibiotics. Method(s): The Patient Questionnaire Card and the Patient Review Card were used to survey 32 (8.7%) individuals (f -56.3%, m -43.7%) who met the inclusion criteria (the presence of hypersensitivity reactions to beta-lactam antibiotics during the last 3 years). We used Flow CAST to identify the DHRs to beta-lactam antibiotics (Ceftriaxone (conc. 4 mg/ml);Cefuroxime (conc. 2.5 mg/ml);Amoxicillinum (conc. 2.5 mg/ml) from CAST Allergens for CASTFlow CAST) BUHLMANN LABORATORIES AG, Switzerland) in whole blood. Flow cytometric acquisition was performed on a flow cytometer BD FacsCalibur (USA), and 300 basophilic cells were analyzed. Result(s): The most common clinical manifestations included acute urticaria + angioedema (40.6%), generalized urticaria (28.1%), anaphylactic shock (21.9%), bronchospasm (9.4%). The percentage of patients diagnosed with an immediate reaction based on the time of its occurrence was 62.5%, whereas the percentage of patients diagnosed with an immediate reaction based on the clinical manifestations was 81.25%, which was confirmed by positive BAT results (p > 0.05). 68.75% of people with clinical manifestations of reactions to one antibiotic (ceftriaxone or amoxicillin) showed increased values on the BAT test to other beta antibiotics, which may indicate the presence of cross-reactivity between these groups of drugs. Conclusion(s): Diagnostics of immediate hypersensitivity reactions to antibiotics based on in vitro BAT is a highly accurate method. However, in cases of possible cross-reactivity between antibiotics and in cases of delayed reactions, in-depth studies are required.
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Background: Prevention of transfusion-associated infections remains a challenge in transfusion medicine. The Mirasol Pathogen Reduction Technology (PRT) System uses riboflavin plus UV light to inactivate residual white blood cells and nucleic acid-containing pathogens to reduce the risk of transmission of bacteria, viruses, parasites, novel pathogens e.g SARS-CoV-2. This demonstrates the cumulative quality data of Mirasol-treated PC produced under routine conditions in our institute in the last year Methods: One hundred sixteen whole blood derived PCs, resulting from the pooling of 5 buffy coats with 250ml PAS-E solution were treated with the Mirasol technology. PCs were mixed with 35 ml Riboflavin solution and illuminated with UV-light in accordance to manufacturer's instructions. For quality control (QC) assessment the following parameters were investigated post-production (PP) and at the end of shelf-life (EOS) at days 5, 6 or 7: pH (ABL80 FLEX Blood Gas Analyzer, at 37degreeC), platelet yield (Cell- Dyn Ruby, Abbott) and CD62P-positive cells with and without TRAP-6 (100muM) using the FACS methodology with FITC-labelled CD62P antibody (Cytomics FC 500 Flow Cytometer, Beckman Coulter). Result(s): Mirasol-treated PCs showed a mean pH of 7.3 at PP and ranged 7.1 to 7.0 at EOS1. Platelet yield PP and EOS were consistent with 3.0 to 3.1 x1011 platelets (PLT)/unit. Platelet activation measured with CD62P+ expression w/o TRAP-6 was 27.7% at PP and ranged from 46.9 to 55.8 at EOS1;CD62P+ expression induced by TRAP-6 was 79.3 at PP and ranged from 72.3 to 68.1 at EOS1. Conclusion(s): The QC data on Mirasol-treated PCs produced during the past year showed encouraging results: all pH values remained far above 6.4, platelet yields remained stable suggesting min cell loss, with EOS yields always above the threshold of 2.5x1011 PLT/unit. Rates of CD62P+cells increased with time, an upregulation of CD62P+ with TRAP-6 was still detectable at EOS up to Day 7. The presented results confirm the data of the initial Mirasol validation at our site, showing the robustness of the technology. (Table Presented).
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Background: Mucormycosis is a deadly fungal infection that emerges in patients affected with COVID-19. All fungal illnesses are caused by dysregulated adaptive immunity, but Myeloid-derived suppressor cells (MDSC) have added a new di-mension to the chronic inflammatory response. Objective(s): We attempted to enumerate the MDSC immune response in rhino-orbital mucormycosis patients before and after treatment and compared the data with healthy control. Method(s): A total of 3 ml of blood samples were taken in an EDTA vial from 20 patients with mucormycosis and 20 age-matched healthy control. A second blood sample was collected to examine the immune system post three months of treatment. Mycological identification was performed on nasal crust retrieved aftersurgery using KOH/culture.The expression of the MDSC marker was analyzed by immunostaining with the antibodies against CD14, HLA-DR, CD11b, CD33, CD66 (Biolegend). Flu-orescence profiles were recorded by Flow Cytometer (BD FACSAria TM III) and analyzed by Flow Jo s oftware (BD Biosciences). The percentage of positive cells is used to express the results.The GraphPad Prism (version 8, GraphPad s oftware, LaJolla, CA, USA) was used to analyze the data. All of the results were considered significant when P <.05. Result(s): All of the patients tested positive for Rhizopus arrhizus, which was confirmed by the culture. The percentages of Monocytic-MDSC (mMDSC: CD14 + HLA-DR-/low) cells were significantly high in patients compared to healthy control. In post-3-month treatment, the percentages of mMDSC were found significantly low and comparable with healthy control. Granulocytic MDSC (gMDSC: HLA-DR-/low CD33 + CD11b + CD66 +) cell population was higher in patients compared with healthy control and patients with post-3-month treatment. Conclusion(s): MDSC regulates T cells and other immune cells with a different mode of action. The findings in this study imminently indicatethe mechanism of immunedysregulation involvingMDSCpathways inmucormycosis andprovide evidence that restoration of immune balance causes reduction of MDSCcells may be considered a therapeutic option for long-term benefit.
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A resposta imune a infeccao por SARS-CoV-2 durante o periodo gravidico puerperal e as alteracoes que podem aumentar o risco de complicacoes maternas, fetais e neonatais ainda nao sao bem caracterizadas. Para isso, foram determinados, atraves de citometria de fluxo, niveis perifericos de linfocitos T-totais (CD3+), T-auxiliar (CD3+/CD4+), T-citotoxico (CD3+/CD8+), linfocitos B (CD19+), celulas NK (CD16+/56+) e NKT (CD3+/CD16-56+) em gestantes e puerperas com suspeita de COVID-19 com o objetivo de identificar potenciais alteracoes imunologicas induzidas pelo coronavirus. Foram utilizadas amostras de sangue periferico de mulheres que realizaram RT-PCR para COVID-19 entre maio de 2021 e marco de 2022. As amostras foram coletadas em tres maternidades publicas do Rio Grande do Norte na admissao para parto e no puerperio imediato nos casos suspeitos. O sangue foi coletado em tubos contendo EDTA para a realizacao da citometria de fluxo, utilizando o analisador de fluorescencia celular ativado (FACScan) e o software Cell Quest. Os linfocitos foram identificados por alta expressao de CD45 e baixa dispersao lateral, utilizando as seguintes combinacoes de 3 cores de anticorpo monoclonal: isotiocianato de uoresceina (FITC), ficoeritrina (PE) e Proteina Clorofila Piridina (PerCP). Cinquenta mulheres precisaram realizar o RT-PCR e 32 (64%) testaram positivo para COVID-19. Gestantes e puerperas infectadas pelo SARS-CoV-2 apresentaram niveis elevados de celulas T citotoxicas, mediana (ME) = 495,0;intervalo interquartil (IIQ) = 391,5, quando comparadas com pacientes nao infectadas, ME=356,2;IIQ=297,8;p=0.032. Com relacao a quantidade das celulas NK, ME=159,1;IIQ=220,4, e dos linfocitos B, ME=126,7;IIQ=186.4, as contagens foram significativamente mais baixas no grupo com menos de 30 dias de infeccao, em comparacao com o grupo em que o RT-PCR foi negativo, ME=280,8;IIQ=214,9;p=0,021 e ME=323,9;IIQ=365,5;p=0,045, respectivamente. Gestantes e puerperas com COVID-19 apresentam maior numero de linfocitos T citotoxicos no sangue periferico e menor numero de celulas NK e linfocitos B. Considerando que gravidez e pos-parto alteram fisiologicamente o sistema imunologico e que esse estudo transversal nao permite analisar causalidade entre infeccao e alteracoes celulares, mais estudos sao necessarios para elucidar as alteracoes causadas pela COVID-19 no sistema imunologico durante o periodo gravidico puerperal, para confirmar se a infeccao viral compromete a imunidade, aumentando o risco de complicacoes para o binomio mae-feto. Declaramos que nao houve apoio financeiro e (ou) material recebido para o desenvolvimento deste trabalho. Copyright © 2022
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Introduction: In Argentina, multiple sclerosis patients (MSp) are vaccinated against SARS-CoV-2 using different formulations upon availability, including viral vector/inactivated virus/mRNA vaccines, at distinct times between doses. The real-world effectiveness of these unique vaccination schedules is scarce, so asthe efficacy to mount an appropriate immune response even more in MSp under treatment (DMTs) Aims: To analyze the presence of reactive CD4+ and CD8+ T cells for SARS-CoV-2, IgG and IgM anti-Spike and anti-RBD, in MSp after receiving a 3rd vaccine dose Methods: 27 MSp and 9 healthy controls (HC) were included in this study. SARS-CoV-2-reactive T cells were analysed with a T Cell Analysis Kit from Miltenyi as described by the manufacturer. In brief, peripheral blood mononuclear cells (PBMCs) were cultured with a pool of lyophilized peptides, consisting of 15-mer sequences with 11 amino acids overlap, covering the complete protein coding sequence (aa 5-1273) of the surface or Spike glycoprotein (S) of SAR-CoV-2 and controls. After stimulation, the cells were stained with the live/dead marker, washed, fixed, permeabilized and stained for lineage and activation markers as well as cytokines. Cells were analysed using a flow cytometer. Doublets, debris, and dead cells as well as CD14+ and CD20+ cells were excluded. Cells were pregated on CD3 as well as CD4 and CD8. For reactive CD4 T cells CD154 and TNF-alpha were assessed on CD4+ T cells while TNF-alpha and IFN-gamma in CD8+ T cells Results: IgG antibodies (Ab) against S and RBD were found in all analysed HC, while in 22 and 20 out of a total of 27 MSp. Levels of IgG against S were lower in MSp vs HC. IgM levels against RBD were found in all HC and MSp, but 8 MSp had low levels of those Ab.There were no differences between HC and MSp in the % of reactive CD4+ T cells to S (p= 0.151). However, we found a lower % of reactive CD8+ T cells in MSp than HC (p= 0.026). Actually, CD8+ T cells were not detected in 4 out of 5 MSp treated with Fingolimod (FTY) but were present in all patients treated with monoclonal Ab, IFN or DMF. Furthermore, MSp treated with FTY had lower values of reactive CD4+ T cells and IgG anti-RBD than patients receiving other DMTs Conclusion(s): Most MSp vaccinated against SARS-CoV-2 present some humoral and cellular response to SARS-CoV-2. This humoral and cellular response would be lower in MSp treated with FTY.
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Background: There is an association between Coronavirus Disease 2019 (COVID-19) and coagulation abnormalities. Platelet monitoring is important for COVID-19 because abnormalities can occur in terms of quantity and quality. Impaired function of platelets can occur at the activation or aggregation stages. An increase in CD62P is associated with a 1.7-fold increased risk of venous thrombosis. In the event of thrombosis, platelet activation causes interactions between fibrinogen and GP IIb/IIIa molecules which will form intracellular bonds between platelets, causing platelet aggregation. This suggests the role of CD62P as a major marker of platelet activation and may mediate cancer cell adhesion, inflammation, and thrombosis Aims: This study aims to determine the description of platelet function as reflected in CD62P platelet expression in COVID-19 patients Methods: This study is a prospective study that take place from November 2020 to September 2021 at RSUP Dr. Sardjito, Yogyakarta, Indonesia. The subjects involved were adult patients aged over 18 years, men and women with confirmed COVID-19 through PCR swab results. Patients with leukemia, history of coagulation disease, and immunodeficiency were excluded from this study. Flowcytometry analysis using FACS Canto was used to measure CD62P expression on platelets. Antibody used was anti human monoclonal CD41 PE and CD62P FITC antibody. The CD62P examination was carried out on the first day of treatment. Patients were grouped according to the severity of COVID-19 as severe and non-severe. Mann Whitney test was used to compare CD62P platelet expression percentage between groups. Result(s): The CD62P platelet expression on day 1 of the deceased subjects were higher compared to the survived subjects (46.77% vs 43.38%;p = 0.04). On day 1, the severe subjects have a higher mean CD62P platelet expression compared to non-severe subjects (47.88 % vs 39.75%). Conclusion(s): CD62P platelet expression in deceased COVID-19 subjects is higher compared to survived subjects. (Figure Presented).
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Background: Infectious complications are a major cause of morbidity and mortality in Chronic Lymphocytic Leukaemia (CLL). Therapeutic approaches that deplete CLL cells also affect normal B-cells. Optimal treatment would result in eradication of CLL cells and recovery of normal immune function. FLAIR (ISRCTN01844152) is a phase III trial for previously untreated CLL comparing ibrutinib plus rituximab (IR) with fludarabine, cyclophosphamide and rituximab (FCR) and subsequently amended to also compare ibrutinib plus venetoclax (I+V) and ibrutinib alone (I) with FCR. Measurable residual disease (MRD) and normal B-cell levels were assessed at multiple timepoints. Aims: To assess the depletion of normal B-cells during treatment and recovery after end of treatment. Methods: Participants aged under 75 years with <20% TP53-deleted cells were initially randomised to FCR or IR and subsequently to FCR, IR, I+V or I with the IR arm closed after randomisation of 771 participants to FCR/IR. FCR was given for 6 cycles, while treatment in the IR, I and I+V arms continued for up to 6 years except in participants attaining <0.01% MRD who continued treatment for the time taken to achieved MRD <0.01% and then stopped if MRD remained <0.01%. Month (M) 24 was earliest permitted stopping point. MRD flow cytometry was performed according to ERIC guidelines (panel: CD19/5/20/43/79/81+ROR1, acquisition of 0.5-2.2 million cells, BD Biosciences Lyric). Additional analysis of normal B-cell subsets was performed in a cohort of >500 patients (panel: CD19 to identify B-cells, CD20/5/79b+ROR1 and CD3 to exclude CLL & contaminating cells, with CD27/ 38/IgD/IgM to characterise normal B-cell subsets using a Coulter Cytoflex LX). Results: Normal B-cells were undetectable during FCR treatment and only rarely detectable until 12 months after last FCR cycle. Circulating normal B-cells were reduced in number or undetectable in participants receiving ibrutinibcontaining regimens with greater depletion in the I+V and IR arms relative to I monotherapy. B-progenitors persist through FCR treatment but were depleted during I, I+R or I+V treatment. Normal B-cell levels at 24 and 36 months after randomisation, with time off-treatment if applicable, are shown in Figure 1. In the ibrutinib-containing arms (IR, I, and I+V), there was a trend towards fewer COVID-associated SAE at any time point for participants with detectable B-cells at 24M (4/181, 2.2%) compared to those with no detectable B-cells (14/344, 4.1%) and COVID-associated SAEs were not observed in FCR-treated participants who had recovered any level of normal B-cells by 24M (0/215). However, the data on COVID infections are limited and there was no apparent association between normal B-cell levels at 24M with the proportion of participants experiencing an infectious SAE overall. Assessment of normal B-cell subsets during ibrutinib-based treatment demonstrated a mix of naïve and memory B-cells. Serological response to COVID infection/vaccination in this cohort is currently being performed. Participants stopping I+V treatment at 24-30 months post-randomisation due to MRD eradication showed rapid recovery of normal naive B-cells within 6-12 months after end of treatment in the vast majority (>95%) of evaluable cases. Summary/Conclusion: Normal circulating B-cells are depleted during treatment with rituximab but can persist at a low level during I, IR or I+V treatment. Most patients in remission after treatment with FCR or I+V show recovery of normal B-cells at 12 months of stopping treatment.
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Background: The use of simulations has been steadily rising in popularity in the biosciences, not only due to the COVID-19 pandemic restricting access to physical labs and equipment but also in the face of rising student numbers. In this study, we describe the development and implementation of a novel, open-access interactive simulation used to not only supplement a laboratory class but to enhance the student learning experience. The simulation provides students with the opportunity to interact with a virtual flow cytometer, design a simple experiment and then critically analyse and interpret raw experimental data. Results: Results showed that this highly authentic assessment used a much broader range of the mark scheme acting as an excellent discriminatory for student ability compared to simple recall as assessed by multiple-choice questions. Overall, the student response to the new assessment was positive, highlighting the novelty of the assessment, however, some students did experience technical issues when the simulation was used for the first time. Conclusion: Simulations can play a crucial role in the student learning cycle by providing a rich, engaging learning environment, however, they need to be used to supplement other hands-on experiences to ensure that students acquire the necessary kinematic skills expected of a successful science graduate. Copyright © 2022 Francis, Ruckley and Wilkinson.
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The SARS-CoV-2 virus caused the COVID-19 pandemic is related to the SARS-CoV-1 and MERS coronaviruses, which were resulted in 2003 and 2012 epidemics. Antibodies in patients with COVID-19 emerge 7–14 days after the onset of symptoms and gradually increase. Because the COVID-19 pandemic is still in progress, it is hard to say how long the immunological memory to the SARS-CoV-2 virus may be retained. The aim of this study was to study a ratio between humoral and cellular immunity against the SARS-CoV-2 S protein in COVID-19 convalescents. There were enrolled 60 adults with mild to moderate COVID-19 2 to 12 months prior to the examination. The control group consisted of 15 adults without COVID-19 or unvaccinated. Specific antibodies to the SARS-CoV-2 virus were determined by ELISA with the SARS-CoV-2-IgG-ELISA-BEST kit. To determine the specific IgG and IgA subclasses, the anti-IgG conjugate from the kit was replaced with a conjugate against the IgG subclasses and IgA. Additional incubation with or without denaturing urea solution was used to determine the avidity of antibodies. Peripheral blood mononuclear cells were isolated by gradient centrifugation, incubated with or without coronavirus S antigen for 20 hours, stained by fluorescently labeled antibodies, and the percentage of CD8highCD107a cells was assessed on flow cytometer BD FACSCanto II. In the control group, neither humoral nor cellular immunity against the SARS-CoV-2 S protein was found. In the group of convalescents, the level of IgG antibodies against the SARS-CoV-2 S protein varies greatly not being strictly associated with the disease duration, with 57% and 43% of COVID-19 patients having high vs. low level of humoral response, respectively. A correlation between level of specific IgG and IgA was r = 0.43. The avidity of antibodies increased over time in convalescents comprising 49.9% at 6–12 months afterwards. No virus-specific IgG2 and IgG4 subclasses were detected, and the percentage of IgG1 increased over time comprising 100% 6–12 months after recovery. 50% of the subjects examined had high cellular immunity, no correlations with the level of humoral immunity were found. We identified 4 combinations of humoral and cellular immunity against the SARS-CoV-2 S protein: high humoral and cellular, low humoral and cellular, high humoral and low cellular, and vice versa, low humoral and high cellular immunity.
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Background: Recent studies reported that individuals with ABO blood type O are underrepresented among patients infected with severe acute respiratory syndrome coronavirus SARS-CoV-2 compared with controls. Our preceding study results indicated a lower proportion of individuals with blood type O accompanied by a higher incidence of blood type AB in patients hospitalized with COVID-19 than in healthy blood donors. Thus, we hypothesized that the variable susceptibility to infection with SARS-CoV-2 might be related to interference caused by circulating ABO antibodies and further may be influenced by the antibody titers which vary widely between individuals. Aims: Therefore, we aimed to investigate ABO antibody levels, including IgM, IgG and IgA subclasses, in the serum and saliva of Caucasians (n = 187), who recovered from mild COVID-19 and to compare them with those of individuals who had never been infected with the virus. Further, a possible association between ABO antibodies and virusspecific total antibody concentrations in the COVID-19 convalescents as well as a potential relationship between the total IgA secreted in saliva and anti-A/anti-B specific IgA in saliva specimens were addressed. Methods: The convalescent study participants were recruited between June 2020 and February 2021. Individuals who had been hospitalized with COVID-19 or who were pregnant were excluded from the study. Two samples were collected within 2 months after the diagnosis (median days: 44) and approximately 2 months later (median days: 66 days). Isotype specific anti-A and anti-B were determined by flow cytometry on a FACS Canto II. The results were compared with the levels in samples from blood and saliva donors. The antibodies specific to SARS-CoV-2 as well as total IgA in saliva were tested by ELISA. Results: COVID-19 convalescents had significantly lower levels of anti-A and anti-B IgM, IgG and IgA in their serum than control subjects (p < 0.001). ABO antibody levels tested in saliva of participants who previously suffered from COVID-19 did not differ significantly from antibody levels tested in saliva controls (p ≥ 0.338). ABO antibody levels remained stable over the period considered. No significant association between the level of ABO antibodies and SARS-CoV-2-specific antibodies was observed (-0.44 < rho < 0.42, p > 0.053). Total IgA in saliva was lower in convalescents than in controls (p = 0.038). Summary/Conclusions: We observed consistently lower serum concentrations of anti-A and anti-B in COVID-19 convalescents than in healthy controls, suggesting ABO antibodies to conferring a degree of protection against SARS-CoV-2 infection. There may be an increased susceptibility to SARS-CoV-2 infection due to individual preexisting low ABO antibody levels. However, the mechanism underlying our observation of significantly reduced ABO antibodies in the serum, but not in the saliva of affected individuals remains unresolved. Further studies to better understand the molecular mechanism underlying our observation are needed.
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Backgroung: COVID-19 pandemic (SARS-CoV-2) has affected an increasing number of people worldwide, with death rates higher than previous viral epidemics. It is possible that NK cells, known to have great cytokine secreting potential are competent at the onset of the condition and that in some individuals, the viral load is able to exhaust them. Balance between tolerant (CD27- CD11b-), secretory (CD27+ CD11b-/ CD27+ CD11b+) and cytotoxic (CD27- CD11b+) NK cells involved in the inflammatory response and their anti-SARS-CoV-2 activity are still not well established. Strategies that can restore function of NK cells against the virus are worth investigating. Here, we aimed to characterize NK cells frequency, functional subtypes and maturation in early phase of COVID-19 patients, by Multiparametric Flow Cytometry (MFC). Methods: Peripheral blood from 15 COVID-19 patients in early stage of infection (day 1-14, confirmed by RT-PCR), categorized according comorbidities in: G1 (not oncologic;n = 6), G2 (oncologic;n = 3), G3 (hematologic neoplasms;n = 3) and G4 (without comorbidities;n = 3), and 10 healthy samples enrolled the study. Clinical and laboratorial data were collected from electronic medical records. Samples were stained with CD45, CD19, CD3, CD56, CD11b, CD27, acquired on a FACS Canto II (BD Biosciences) and data analyzed with FlowJo V10 software. Results: A lower frequency of lymphocytes was observed in the disease when compared to controls (P < 0.0001) and frequency of NK cells were similar in both groups (P = 0.6605). Although frequency of CD27- CD11b- NK cells was lower in the disease (P = 0.0109), there was a significantly higher frequency of CD27+ CD11b- NK cells in COVID-19 samples when compared to controls (P < 0.0001), featuring a mostly immature profile in the disease. On the other hand, no statistical significance was observed regarding the frequencies of CD27+ CD11b+ (P = 0.1370) and CD27- CD11b+ NK cells with a more mature profile (P = 0.3094). Amongst disease groups, no statistical significance was found regarding frequency of NK cells and G1 showed lower frequency of CD27- CD11b- NK cells (P = 0.0226), while G3 group had an increased frequency of CD27+ CD11b- NK cells (P = 0.0238) when compared to the other groups and controls. Finally, no statistical significance was found in the frequency of CD27+ CD11b+ (P = 0.6691) and CD27- CD11b+ (P = 0.6270) NK cells between disease groups and controls. Conclusion: Although the frequency of NK cells did not show a significant difference between COVID-19 patients and healthy controls, our findings showed a possible change in their maturation profile, which seems to be inversely proportional to normal, with the frequency of CD27+ CD11b- NK cells considerably higher in the disease. This phenotype is directly associated with secretory function of a more immature NK cell and is responsible for triggering inflammatory responses that could lead to severe respiratory failure, what seems to be consistent with COVID-19 profile. A high frequency of cytotoxic cells was observed, which seemed to be similar to what we found in normal heathy samples. Even though unregulated maturation might be associated to a dysfunctional mature NK cell, additional studies of cytotoxicity and activation of NK cells in COVID-19 are required to affirm whether there is functional exhaustion or hyperactivation of the cytotoxic subtypes of these cells.
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Introdução: O novo coronavírus, SARS-CoV-2, causador da doença COVID-19, foi detectado em 31 de dezembro de 2019 em Wuhan, na China. A COVID-19 apresenta alta morbidade e mortalidade, e aproximadamente 15% dos casos confirmados progridem para fase severa da doença. A síndrome respiratória aguda grave (SRAG) e a falência múltipla de órgãos são as principais causas de morte pela COVID-19, provocadas pela resposta inflamatória sistêmica descontrolada. Estudos recentes sugerem que pacientes com COVID-19 internados em UTI apresentaram contagens reduzidas de linfócitos TCD4 e TCD8, mas ainda não está clara a relação das demais células como preditoras de gravidade da doença. Objetivo: Avaliar possíveis diferenças no perfil linfocitário de pacientes positivos para COVID-19 com quadro severo e não severo da doença. Material e métodos: Pacientes admitidos no Hospital de Clínicas de Porto Alegre (HCPA) entre junho/2020 e maio/2021 foram divididos em dois grupos quanto à severidade da COVID-19;grupo severo (GS): saturação de SpO2 < 93% em ar ambiente e/ou aumento da frequência respiratória > 30 batimentos/min e/ou relação PaO2/FiO2 ≤ 300 mmHg e grupo não severo (GNS): pacientes que não se enquadraram nesses critérios. A análise imunofenotípica da população linfocitária foi realizada na amostra de sangue periférico do hemograma no momento da admissão dos pacientes com resultado de RT-PCR positivo para o vírus. Foram adquiridos 100.000 eventos na região dos linfócitos no citômetro de fluxo FACSCanto II®. A aquisição foi realizada no software FACSDiva e os dados analisados no software Infinicyt TM 2.0. Foi utilizada plataforma dupla para a obtenção de valores absolutos das células, a partir da contagem de leucócitos. Resultados: Foram incluídos no estudo 72 indivíduos, com média de idade de 60,2 anos (25-94), desses, 39 foram do sexo masculino (54,2%). Foram incluídos 25 (18%) pacientes no GNS e 47 (82%) no GS. Em relação ao GNS, o GS apresentou um aumento na contagem de neutrófilos/μL (P = 0,002) e na relação neutrófilos/linfócitos (P < 0,001);além disso, apresentou diminuição na contagem de linfócitos/μL totais (P = 0,043). Quanto às subpopulações linfocitárias, as contagens de células CD3/μL (P = 0,020), CD8/μL (P = 0,020) e NK/μL (P = 0,029) encontraram-se diminuídas, assim como das células TCD4/μL de memória central (P = 0,047), memória efetora (P = 0,020), e TCD8/μL de memória central (P = 0,002), memória efetora (P = 0,013) e efetora tardia (P = 0,032). Não foram encontradas diferenças significativas na contagem de CD4/μL, nem de linfócitos B entre os dois grupos. Discussão: Sabe-se que os linfócitos TCD8 desempenham função importante no clearance viral, sendo capazes de secretar moléculas como perforina, granzimas e IFN-γ. A redução dos linfócitos tem sido explicada tanto pela ação direta do vírus, como pela redistribuição das células para os órgãos alvo. Nossos resultados confirmaram a redução do número de linfócitos TCD8 e do número de células NK no grupo de indivíduos com a forma severa da COVID-19, o que sugere que a resposta imune contra as infecções virais depende da ativação de linfócitos TCD8 e que isto é crítico para a recuperação dos pacientes. Entretanto, outros estudos avaliando marcadores de ativação celular e em demais amostras, como lavado broncoalveolar, são necessários para confirmar esses achados.
ABSTRACT
Background: Since the first case of COVID19 infection in 2019, this RNA virus has led an unprecedented pandemic that infected more than 232 million people. Although the disease is studied extensively, much remains poorly understood. Here, we performed the first correlation study on the peripheral blood morphology and immunophenotype of the white blood cells (WBCs) from COVID19 patients. Design: A total of 52 samples from COVID19 patients and 15 blood samples as control group were analyzed. COVID19 patients were divided into two groups based on clinical severity, severe (respiratory failure) or non-severe (hospitalized but stable). The controls were the patients with negative COVID19 results by PCR and antibody tests. The WBC morphology was examined either by blood smear review or via CellaVision DM analyzer captured images. Navios flow cytometer and Beckman Kaluza C software were used for immunophenotype analysis. Two-tailed T-test was performed on the COVID19 groups and the control group Results: Almost all COVID19 patients showed marked neutrophilia and lymphopenia on the CBC tests. Morphologically, the neutrophils showed irregularities like hypogranulation, toxic granules and pseudo Pelger-Huet anomaly (Fig 1A). In severe COIVD19 group, there was an increase in neutrophils with immatures phenotypes, showing CD33 positivity while CD10, CD13 and CD16 negative (Fig 1B). Conversely, the CD10(+) mature neutrophils aberrantly expressed CD56 (Fig 1B). The percentage of CD56(+) neutrophils was significantly higher in both COVID19 groups, suggesting a stronger cellular adhesion and interaction. The monocytes from the COVID19 patients had increased cytoplasm with cytoplasmic protrusion and vacuolization (Fig 2A). Phenotypically they were positive for CD13, CD33, CD38 and HLA-DR. The lymphocytes were also atypical, including increased cytoplasm with large granules and vacuoles. Phenotypically, they are activated, expressing CD38, HLA-DR, and mainly α/β subtype. Giant platelets with cytoplasmic vacuoles and projections were easily seen. Platelet aggregations were observed (Fig 2B). These platelets were CD45(-) and expressed CD61 at lower-than-normal intensity, while expressing increased CD42b intensity when compared to the control group on a log scale. Conclusions: Despite being a small study, we were able to correlate the morphologic and phenotypic alterations of the WBCs in COVID19 patients. As such, this helped to explain some of the clinical hematologic manifestation of the disease. (Figure Presented).
ABSTRACT
BACKGROUND AND AIM: The source of COVID-19, which was declared a pandemic in March 2020, is coronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Our immune system to eliminate infectious agents such as SARS-COV-2;It uses cellular elements such as lymphocytes, monocytes/macrophages, neutrophils, dendritic cells, NK cells and platelets, dissolved molecules such as cytokines, chemokines, acute phase proteins and complements that work in harmony with each other. Cytokines are regulatory proteins. They undertake tasks such as proliferation, differentiation and activation of cellular elements such as leukocytes. In this study, it was aimed to investigate relationship between cytokines and immunophenotypic characters of leukocyte subgroups in COVID-19 patients. METHODS: In this study, 51 COVID-19 patients (18-71 years) were included who outpatient applied to Mersin University Hospital and were positive for COVID-19 according to PCR (Bio-Speedy RT-qPCR Detection Kit, Bioeksen, Turkey) analysis. Samples taken on the application day were included in the study. Immunophenotype analyzes were measured by flow cytometry using the FACSCalibur (Becton Dickinson, USA) instrument. Cytokine levels (IL-1β and IL-6) were studied with the ELISA method (DSX-4-Plate Automated, Dynex, USA). RESULTS: Correlations of cytokines and leukocyte subgroups were examined, respectively, in CD3+ T-lymphocytes (r=-0,034-r=-0,096), CD4+ T-Helper (r=-0,035-r=-0,074), CD8+ T-cytotoxic (r=0,097-r=0,135), CD19+ B-lymphocytes (r=0,020-r=0,09), HLA-DR+ lymphocytes (r=0,064-r=0,006), CD56+ NK cells (r=-0,023-r=0,57). No correlation was observed between IL-1β and IL-6 and leukocyte subgroups (P>0.05). A strong positive correlation was found between IL-1β and IL-6 (r=0.894, p<0.05). CONCLUSIONS: New approaches are needed in the treatment of COVID-19 patients, especially in patients with cytokine storm. Due to do there was no significant correlation between IL-1β and IL-6 and the immunophenotypic characters of leukocyte subgroups, and a strong positive correlation was observed between IL-1β and IL-6, it was thought that monitoring proinflammatory cytokines in patients would be of clinical benefit.
ABSTRACT
Red Blood Cells from COVID-19 Patients Show Evidence of Increased Oxidative Stress and Increased Lactate Influx Corona Disease 19 (COVID-19) is caused by SARS-CoV-2, a novel, highly infectious, single stranded RNA virus. In severe cases, excess oxidative stress produced by a ‘cytokine storm’ may generate excess reactive oxygen species (ROS) and lead to tissue damage in the lungs and elsewhere. As the potential role of RBCs in the pathophysiology of COVID-19 remains controversial (1), we investigated for evidence of increased oxidative stress and increased thrombotic tendency in RBCs from patients with COVID-19 infection. Following ethical approval and written informed consent, we used flow cytometry (BD FACSCanto II) to measure baseline RBC ROS following incubation with 2‘-7‘-dichlorofluorescein diacetate (DCF). RBC ROS were also measured following pre-incubation with hydrogen peroxide (H2O2) (2mM) +/- antioxidant N-acetyl cysteine (NAC) (0.6mM). We also measured RBC surface expression of adhesion molecules CD44, CD47 and CD242, as well as CD147. Results were expressed as mean +/- standard deviation (SD). RBC ROS were measured in 22 COVID-19 positive patients and in 10 age matched healthy controls. One patient died from respiratory failure, whilst only 3 others required ITU admission for continuous positive airway pressure (CPAP) or intubation. There was no statistical difference in mean basal RBC DCF mean fluorescence intensity (MFI) levels between COVID-19 positive patients and controls. However, mean increase in RBC DCF MFI following H2O2 incubation was significantly higher in the COVID-19 positive group (1105.7+/-336.3) compared to the control group (843.4+/-256.7)( p= 0.042). The increase in RBC DCF MFI in the COVID-19 positive group correlated with CRP (p=0.014) but not with D-dimer, serum ferritin or any complete blood count (CBC) parameters. Incubation of RBC with 0.6 mM NAC for 30 minutes prior to H2O2 exposure caused a mean reduction in DCF MFI of 26.7% in the COVID-19 positive group. RBC expression of CD44, CD47, CD242 and CD147 were measured In a separate cohort of COVID-19 positive patients (n=32), and in 22 age matched controls. There were no statistically significant differences in mean expression levels of CD44, CD47 and CD242 between the 2 groups. However, mean RBC CD147 MFI expression was higher in the COVID-19 group (1319.64+/-374.76) compared to controls (1061.59+/-253.33) (p=0.018). There was no significant correlation between RBC CD147 MFI and D-dimer, CRP, serum ferritin or any CBC parameters in the COVID-19 positive group. However, 21 of the 32 COVID-19 positive patients had blood lactate levels measured and there was a positive correlation between CD147 MFI expression and blood lactate (R=0.56, p=0.0077). Induction of oxidative stress by H2O2 resulted in a greater increase in ROS in RBCs from COVID-19 patients compared to controls and with correlation to CRP, despite the fact that there were very few patients with severe disease in the study. This suggests a role for oxidative stress in disease pathogenesis. Pre-incubation with NAC attenuated this increase in ROS, suggesting a possible role for antioxidants in therapy. Increased RBC cell surface expression of adhesion molecules CD44, CD47 and CD242 can facilitate RBC interaction with platelets and/or endothelial cells, potentially contributing to thrombosis. We found no increase in their expression in COVID-19 patients compared to controls although RBCs may contribute to thrombosis in COVID-19 infection by other means (1). CD147 is tightly associated with and enables proper expression of monocarboxylate transporter 1, the lactate transporter for RBCs. We found increased surface expression of CD147 on RBCs of COVID-19 patients, whilst CD147 expression showed a moderate correlation with serum lactate levels, suggesting that RBCs in COVID-19 infection may be acting as a lactate sink to protect against lactic acidosis. In summary, our study suggests that COVID-19 infection causes increased oxidative stress and increased lactate influx i RBCs. Further studies are warranted into the role of RBCs in COVID-19 infection. Reference: (1) Murphy P, Glavey S, Quinn J. Anemia and red blood cell abnormalities in COVID-19. Leuk Lymphoma 2021;62:1539 Disclosures: Quinn: Takeda: Honoraria. Glavey: Abbvie: Research Funding;Celgene and BMS company: Research Funding;Janssen: Honoraria, Research Funding;Amgen: Honoraria, Research Funding.
ABSTRACT
[Formula presented] Background and Objective The rapid spread of the COVID -19 pandemic and the high variability of the course of the disease make it essential to search for early predictors of outcome. The objective of our study is to predict severe SARS COV2 pneumonia using early cytometric profiles Material and Methods Prospective and observational study of adults with confirmed COVID-19 infection admitted on Emergency Department (ED). We collected epidemiological, clinical and laboratory data of every patient until they were discharged or died. Multiparametric flow cytometry (FC) analysis of T-lymphocytes (CD4, CD8, CD4 activated, CD8 activated, naïve (Tn), central-memory (Tcm), effector-memory (Tem), effector (Te) and Th17 subsets), B-lymphocytes (naïve, memory, transitional subsets, and assessment of clonality), NK cells, plasmablasts, p-DCs (plasmacytoid dendritic cells), m-DCs (myeloid dendritic cells), basophils, and monocytes (MO1, MO2, MO3, slan+ MO3) was performed on whole peripheral blood collected on EDTA, before immunosuppressive therapy was started. We designed a 7-tube 8-color experimental panel. Cell surface staining of 2 × 106 cells was performed and at least 500 000 total events were acquired for the assessment of plasmablasts, p-DCs, m-DCs, basophils, and the monocyte subsets;for the study of B, T and NK-lymphocyte populations we acquired at least 100 000 total events (FACS Canto II;BD Biosciences). Severity was assessed on the basis of World Health Organization´s (WHO) international 10 level ordinal scale (WHOs) and also according to 4 respiratory status, based on SpO2(peripheral blood oxygen saturation)/FIO2 (fraction of inspired oxygen) ratio (SpFi). SpFi group 1 >452, SpFi2 2: 315-452;SpFi 3 236-315, SpFi 4: <236 (respiratory distress) Results 53 patients were included: epidemiological and clinical data available on table 1. 23 patients (43.39%) arrived to SpFi4 status. WHOs >6 (WHO 6:oxygen by non invasive ventilation or high flow) was achieved by 20 patients (37.7%) Good prognosis (meaning SpFi1 as the worse respiratory status in the follow -up) was associated to cytometric profiles: there was a significant increase in CD3, p-DCs, m-DCs, basophils, monocytes, Tcm, % of lymphocytes and CD3/CD19 ratio whereas there was a significant reduction in CD19, % of neutrophils and % Neutrophils/% lymphocyte ratio. In the SpFi4 group, there was a significant reduction of CD3, p-DCs, m-DCs, plasmablasts and CD3/NK ratio. In patients starting in SpFi group 1-2 in the ED but progressing to SpFi 3-4 during the follow up (27 patients), there was a statistically significant relation with Tn, Te and Tn/Te ratio (Tn/Te ratio <0.717: OR 13.5 (p 0.002, [95% CI 2.552-71.403). Initial SpFi1 patients that evolved to SpFi 3-4 during follow up (10 patients), presented a Tn/Te ratio < 0.717 with an OR 11.556 (p =0.005, [95% CI 2.059-64.853]). Plasmablasts < 0.075 and CD3/NK <5.71, were identified as independent risk factors for SpFI4 during follow up. After multivariable analysis, both variables kept their significance: CD3/NK (OR 11,247, p=0.005) and plasmablasts (OR 12,524, p=0.004). About prediction of WHOs >6, multivariable analysis showed CD3/NK <5.71 (OR 22,240 [95%CI 2,340-211,342] p= 0.007) and plasmablasts<0.075 (OR 28.635 [95% CI 3,187-257,301] p=0.003). A score (0,1,2) comprising both risk factors, was significantly predictive of SpFI4, regardless of the initial respiratory status, age or days from symptoms onset. In our cohort, only 1/15 (6.7%) patients with 0 points (neither plasmablasts nor CD3/NK score), arrived to SpFi4. However, 10/11 (90.9%) patients with 2 points, reached to SpFi4 respiratory status (C-index = 0.837) Same score was applied to predict WHOs > 6: 90.9% with 2 points progressed to WHO>6 and 0/15 patients with 0 points reached the same goal (C-index = 0.872) An incidental finding of 4 indolent B-lymphoproliferative disorders (2CLL-like MBLs and 2non -CLL-like MBL), was found, and they were associated with older age and progression to death. Conclusions Flow cytometry on whole pe ipheral blood samples of SARS-COV2 pneumonia patients, collected before corticosteroid or immunosuppressive therapy, could identify cytometric patterns associated to prognosis. Plasmablasts, mDCs and pDCs levels as well as CD3/NK ratio, are associated to a worse respiratory status, while Tn/Te ratio could detect non-severe patients who will require high-flow oxygen devices during follow up. [Formula presented] Disclosures: No relevant conflicts of interest to declare.