Hematopoietic Stem/Progenitor Cells and Engineering: HEMATOPOIETIC STEM/PROGENITOR CELLS (HPC-A) CRYOPRESERVATION PROCESS VALIDATION USING CRYOSTOR CS10 FREEZE MEDIA

Background & Aim: During the COVID-19 pandemic, we performed HPC-A cryopreservation process validation using the CryoStor CS10 freeze media to replace the current 10% DMSO cryoprotectant (Control), which encountered severe backorder. Methods, Results & Conclusion(s): This process validation included phase I, phase II, and follow-up studies. Ten HPC-A collection cell product samples were cryopreserved in the phase I study using CS10 and Control (1:1) post-plasma depletion. Post-thaw viability tests using the 7-AAD method were performed on the cryopreserved samples for parallel comparison. In phase II, each of three patient HPC-A cell products was split evenly into CS10 and Control cryopreservation. The CS10 cryopreserved HPC-A cell products only were used for infusion. The recipients' engraftment outcomes of white blood cells (WBC), granulocytes (ANC), and platelets (Plts) were monitored. Post-thaw viability test was performed on the quality control samples from both groups. In the follow-up study, engraftment outcomes of WBC, ANC, and Plts were evaluated from ten recipients who received the CS10 cryopreserved HPC-A. In the phase I study, the post-thaw viability of the CS10 group was significantly higher than the Control group (p=0.002). All post-thaw viability results were above 60%, the current lab release criteria. In the phase II study, all cryopreserved cell products met cell product release criteria (> 60%). All engraftment results were within our center-established ranges except for the Pt b's platelet engraftment. Three recipients had not had any cell product infusion-related adverse events post infusion. Both CD34 and CD45 post-thaw viability results in the CS10 group were remarkably higher than the Control group, except for the patient c's CD34 viability. In the follow-up study, the total infused cell product volume ranged from 60 ml to 118 ml, and the WBC concentration in the cryopreserved cell products ranged from 134 to 440 (x10

FEATURES OF POST-VACCINATION HUMORAL IMMUNE RESPONSE IN THE PERSONS WHO UNDERWENT COVID-19 OF VARIOUS SEVERITY.

The studies on humoral immune response in the individuals who have undergone COVID-19 and vaccinated with anti-COVID vaccines allows us to assess the development of "hybrid" immunity, which contributes to understanding the mechanisms of its formation from the effector phase to the step of immunological memory. We assessed the relative and absolute contents of B cell populations and subpopulations, development of humoral immunity in the patients who suffered with COVID-19 of varying severity being thereafter vaccinated with "KoviVak" and "Sputnik V". The study involved volunteers (age 47.3+/-14.5 years) who beared COVID-19 asymptomatically (n = 32), at moderate severity (n = 21), or had severe form of the disease (n = 12), then being vaccinated with "KoviVak" and "Sputnik V" 6-9 months after their recovery. The groups of vaccinated persons consisted of those who beared severe disease being vaccinated with "KoviVak" (n = 6) or "Sputnik V" (n = 6);moderate cases, vaccinated with "KoviVak" (n = 10) and "Sputnik V" (n = 11);asymptomatic cases vaccinated with "KoviVak" (n = 10) and "Sputnik V" (n = 22). We have determined relative and absolute numbers of B lymphocytes (CD45+CD19+), B1 lymphocytes (CD45+CD5+CD19-CD27-), B2 lymphocytes (CD45+CD19+CD5-CD27-), total population of memory B cells (CD45+CD19+CD5-CD27+), non-switched (CD45+CD19+IgD+CD27+), and switched (CD45+CD19+IgD-CD27+) memory B cells;mature naive B lymphocytes (CD45+CD19+CD27-IgD+), plasmoblasts (CD45+CD19+ CD38+++IgD-CD27+), as well as presence of IgG to S(RBD)-SARS-CoV-2 protein. We have found that the humoral immunity among survivors of COVID-19 of varying severity is expressed for up to nine months. The largest number of volunteers who raised antibodies to SARS-CoV-2 S-protein was registered in the group of seriously ill patients. As soon as 1 month after "Sputnik V" vaccination and until the end of the observation, all the examined subjects in this group became seropositive. 4-5 months after injection of this vaccine, specific immunoglobulins were present in all patients who had asymptomatic or average-severity infection. All volunteers who received "KoviVak" had antibodies to the COVID-19 viral S protein from the beginning to the end of the study. Vaccination, especially with "KoviVak", contributed to the highest increase, both in relative and absolute numbers of memory B lymphocytes in asymptomatic patients. Less pronounced changes in the content of B lymphocytes in COVID-19 patients who had severe and moderate clinical course may be associated with higher levels of these cells prior to injection of the vaccines. A positive correlation was found between the number of memory B cells and presence of immunoglobulins to the S protein SARS-CoV-2 in all examined patients.Copyright © 2023 Russian Association of Allergologists and Clinical Immunologists, St. Petersburg Regional Branch (SPb RAACI). All rights reserved.

SUBPOPULATION COMPOSITION OF PERIPHERAL BLOOD LYMPHOCYTES IN COVID-19.

The pathogenesis of severe coronavirus infection COVID-19 is associated with activation of immune system, cytokine storm, impaired blood clotting, microvascular thrombosis, organ ischemia and multiple organ dysfunction syndrome. The role of various lymphocyte subpopulations in COVID-19 is still debated. The aim of our study was to analyze the subpopulational profile of peripheral blood lymphocytes in COVID-19 patients as compared with healthy donors. The study included 20 COVID-19 patients (11 males and 9 females,) and 26 healthy donors. Average age of the patients was 52 and 56 years, respectively. Clinical examinations were performed by standard laboratory methods. Peripheral blood lymphocytes were isolated in the Ficoll gradient. The cells were stained with antibodies to specific antigens of main lymphocyte populations, endothelial cells, and apoptotic cell markers. The analysis was performed by flow cytometry. The results showed that all patients had elevated C-reactive protein (14- to 35-fold), ferritin (1.2- to 13-fold), D-dimers (1.2- to 90-fold). 55% of men had a decrease in the absolute number of lymphocytes, in women this index was at the low normal limit. Cytometric analysis showed that, among peripheral blood lymphocytes, the proportion of functional cells expressing the CD45 marker ranged from 2 to 12% in 70% of patients, as compared with 80-99% among the donors. The proportion of CD45+ lymphocytes significantly correlated with the level of hemoglobin, but not with the levels of inflammatory biochemical markers. Among the functional lymphocytes of patients, there was a decrease in the proportion of CD3+, CD4+, CD8+T cells, increased proportion of natural killer CD56+ and the apoptotic (AnnexinV+) cell contents, but the proportion of CD19 and HLA-DR+B cells was not changed. Analysis of the lymphocyte (LC) subpopulations that did not express CD45 marker showed that this fraction contained different lymphocyte subsets with reduced expression of CD4, CD8, CD19, CD56 etc. in the blood of patients and donors. Higher percentage of endothelial cells expressing CD62P marker made the difference between patients and donors. Laboratory determination of lymphocyte subsets in blood samples of COVID-19 patients does not reflect the real severity pattern of the disease, thus requiring studies of the CD45-expressing functional cell populations.Copyright © Svirshchevskaya E.V. et al., 2023 The article can be used under the Creative Commons Attribution 4.0 License.

Characterization of Mesenchymal Cell from BAL of post COVID-19 patient whit fibrosis

Increasing number of severe COVID 19 patients develop pulmonary Fibrosis, but the management of this complication is still unclear due to a lack of clinical trials. Aim of this study was to characterize mesenchymal cells (MC) isolated from 10 broncho-alveolar lavage (BAL, at 2 months after discharge) from patients with COVID19 fibrosis (COVID19-f) and to compare them with those isolated from 8 patients with collagen tissue diseaseassociated interstitial fibrosis(CTD-ILD). BAL fluid (BALf) levels of TGFbeta, VEGF, TIMP2, RANTES, IL6, IL8, and PAI1 were assessed by ELISA. Primary MC foci were cultured and expanded in D-MEM +10% FBS, characterized by flow cytometry and osteogenic and adipogenic differentiation. Collagen 1 production (+/-TGF-beta) was tested by WB and mRNA expression. BALf cytokine and GF levels were comparable in the two groups. Efficiency of MC isolation from BAL was 100% in COVID-f compared to 65% in CTD-ILD. MC antigen surface expression of CD105, CD73, CD90 (>90%, respectively), CD45, CD34, CD19 and HLA-DR (<5%, respectively) was comparable. None of MC samples differentiated in adipocytes, while COVID19-f were positive for calcium deposition. COVID19-f MC showed at WB, higher Collagen 1 production with respect to CTD-ILD with TGF-beta stimulation. Our preliminary data suggest MC from COVID19-f share several features with CTD-ILD but might have a higher response to fibrogenic and differentiation signals.

Increased CD14+ monocytes and OPN+ positive airway macrophages in the BAL are associated with Post-COVID19 induced altered respiratory function

The long term consequences of severe COVID-19 in the lungs remain speculative, however interstitial abnormalities in these patients may arise. In this study previously identified fibrotic markers from the BAL were evaluated in PostCOVID-19 patients. 26 patients were referred for evaluation of respiratory symptoms and/or abnormalities on HRCT, on average 5.3 months from the acute disease phase, to the Post COVID-19 clinic. 20 patients showed persistent radiological findings with fibrotic changes and/or altered respiratory function. Bronchoalveolar lavage cellular composition was determined by MGG staining and CD45, CD14, CD11c, CD163 and Osteopontin staining. Airway monocytes were identified by SSClo/CD45+ parameters and surface expression of CD11c, CD14 and CD16 by flow cytometry. FVC% and DLCO% were used as measures of disease severity. Collectively, monocyte percentages in the BAL were associated with lower FVC% (Rs=-0.53,p=0.02). Importantly, patients with DLCO% below 60 showed higher monocyte infiltration (p=0.015). CD14 positivity on monocytes was more pronounced in patients with DLCO% below 60, while CD16 and CD11c were not associated with DLCO. Increased Osteopontin expression in airway macrophages was also linked with lower DLCO% levels (Rs=-0.661, p=0.019), in contrast to CD163 macrophage expression which tended to be higher in patients with higher DLCO%. Neutrophils were negatively associated with DLCO% in Post-COVID-19 patients (Rs=-0.62,p=0.01). Airway immune cell populations from BAL were associated with Post-COVID-19 induced altered respiratory function.

Tumor Neuroectodermico Primitivo (Pnet) Com Invasao Macica Em Medula Ossea Simulando Leucemia Aguda: Relato De Caso

Objetivos: Sabe-se que os tumores neuroectodermicos primitivos (PNETs) sao tumores raros envolvendo o sistema nervoso central, ossos ou tecidos moles, com pico de incidencia na adolescencia. O PNET e um tumor extremamente agressivo cuja sobrevida livre de doenca em 2 a 3 anos varia de 25 a 60%. Cerca de 30% dos pacientes apresentam metastase no momento do diagnostico. O prognostico varia de acordo com sitio acometido e a extensao da doenca ao diagnostico. O objetivo e relatar um caso raro de PNET, avaliado pela hematologia, com hipotese diagnostica inicial de leucemia aguda. Materiais e metodos: Coleta dos dados clinicos da paciente nas Unidades de Clinica Medica e Hematologia do HUCAM, bem como o levantamento em prontuario dos resultados de provas laboratoriais e exames especializados. Resultado: Paciente do sexo feminino, 19 anos, admitida no hospital com plaquetopenia grave. A paciente relatava mialgia, cefaleia e calafrios que iniciaram apos a terceira dose da vacinacao para Covid, evoluindo 2 dias apos com o surgimento de equimoses em membros e metrorragia. Ao exame fisico apresentava-se levemente palida, afebril, com sangramento cutaneo, sem linfonodomegalias perifericas ou visceromegalia. Os exames iniciais revelaram uma anemia normocitica (Hb-10.5g/dl), leucocitos-8200/mm3, plaquetopenia (12000/mm3), aumento de desidrogenase latica (DHL-4550), beta HCG negativo. A morfologia inicial do sangue periferico nao revelou alteracoes leucocitarias, porem o mielograma mostrou uma infiltracao intensa da medula ossea por celulas com caracteristicas imaturas, compativel com celulas blasticas. A imunofenotipagem estas celulas eram negativas para CD45, e para os marcadores de linhagens mieloide, linfoides B e T, e celulas dendriticas, e positivas para CD56. O diagnostico final foi de um tumor neuroectodermico primitivo com base na histopatologia da biopsia de medula. Discussao: Apresentamos o caso de uma paciente jovem com o diagnostico de PNET avancado, com manifestacoes hemorragicas cutaneo-mucosas de evolucao aguda, associado a plaquetopenia grave e infiltracao da medula ossea por celulas imaturas com caracteristicas blasticas, que faziam suspeitar fortemente de uma leucemia aguda, portanto, uma apresentacao atipica para um tumor solido raro e de comportamento agressivo, cujo diagnostico so foi possivel atraves de exames especializados. Conclusao: O diagnostico de certas neoplasias pode ser desafiador devido a sua rara incidencia e, por vezes, apresentacao clinica atipica, que pode simular outras doencas. O envolvimento primario do sangue periferico e da medula ossea suscita a avaliacao inicial do hematologista que, com base nos conhecimentos clinico e laboratorial, e capaz de estabelecer um raciocinio amplo e diferencial, cuja confirmacao requer o conhecimento de um especialista experiente para diagnostico assertivo e precoce. Copyright © 2022

Clinical, morphological and molecular biological examination of the myocardium in COVID-19 patients

The presence of coronavirus-associated myocarditis remains controversial despite elevations in cardiac troponin and natriuretic peptide in many patients. Aim. To assess the morphological changes in the myocardium of patients who died due to coronavirus disease 2019 (COVID-19) and compare them with the intravital level of cardiac biomarkers. Material and methods. A total of 420 hospital charts and 77 autopsies of those who died from COVID-19 were analyzed. In 15 of 77 cases (19%) with histologically suspected myocarditis, an immunohistochemical examination of the myocardium with antibodies to CD3, CD45, CD8, CD68, CD34, Ang1, VWF, VEGF, HLA-DR, MHC1, C1q, VP1 of enteroviruses was performed, and in 8 patients with immunohistochemically confirmed myocarditis (10%) — polymerase chain reaction for SARS-CoV-2. Results. Hemorrhage, intramural thrombosis, necrosis of non-coronary origin, myocardial infarction and lymphocytic myocarditis were detected in 43%, 10%, 17%, 19% and 10% of cases, respectively, without coronavirus N and E gene sequences in the myocardium. Dysplasia, hyperplasia and hypertrophy of the vascular endothelium, expression of Ang1, VWF, VEGF, MHC1, C1q, VP1 of enteroviruses were determined in 100, 100, 87, 100, 75 and 62% of cases of myocarditis, respectively. There were no significant correlations between inflammatory biomarkers and myo-carditis. Conclusion. The main morphological manifestation of COVID-19 in the myo-cardium is the so-called endotheliitis with dysplasia and endothelial activation, leading to hemorrhages, intramural thrombosis and necrosis. There is no con-vincing evidence of a direct involvement of coronavirus in myocarditis induction.

Transcriptional Interaction Between RelA and cJUN

Rationale: The SARS-CoV-2 pandemic has underscored the need for novel anti-infectious strategies, including host-directed therapeutics, against existing and emerging respiratory pathogens. We have reported that an aerosolized therapeutic comprised of a Toll-like receptor (TLR)-2/6 agonist, Pam2CSK4, and a TLR-9 agonist, ODN M362, stimulate pathogen-agnostic innate immune responses in lung epithelial cells. This therapeutic (“Pam2-ODN”) promotes synergistic microbicidal activity and host survival benefit against pneumonia caused by a wide range of pathogens. Here, we study the immunomodulatory signaling mechanisms required to effect this inducible epithelial resistance. Methods: Bioinformatic analysis of transcriptional responses from human and mouse lung epithelium al cells to influenza A H1N1 or SARS-CoV-2 (GSE147507) or Pam2-ODN (GSE289984, GSE26864) were analyzed using R and IPA software to identify essential transcription factors (TFs). Lung cell population dynamics were studied for TFs related to Pam2-ODN immunomodulatory signaling using high-throughput imaging flow cytometry (IFC). Human or mouse lung epithelial cells were stimulated with PBS or Pam2-ODN and single or dual inhibitors of TFs before challeng with influenza A H3N2 (IAV) or coronavirus OC43 (CoV) to compare the epithelium-specific transcriptional control of relevant TFs using in-cell western blotting, IFC and hemagglutination for viral burdens. Results: Functional enrichment analysis revealed RelA and cJUN to be major immunomodulatory TFs of Pam2-ODN and activators of leukocyte- and epithelial-derived antiviral immune mechanisms targeting replication of influenza A and SARS-CoV-2. Cell population dynamics studied from mouse lungs confirmed activation of RelA and cJUN in CD45+, EpCAM- leukocytes and in CD45-, EpCAM+ epithelial cells, with predominant activation of the lung epithelium and none or minimal activation of structural cell populations such as fibroblasts or endothelial cells. Studies of epithelium-specific signaling in vitro revealed co-activation of RelA-(pS536) and cJun- (pS73) TFs with Pam2-ODN, and earlier onset of cJUN phosphorylation and nuclear translocation with Pam2-ODN after IAV or CoV infection. Individual or dual inhibition of RelA and/or cJUN activity in vitro disrupted the antiviral activity of Pam2-ODN of IAV infected cells. Conclusion: Pam2-ODN induces unique, pathogen-agnostic protective signaling in lung epithelial cells that involves cooperative activation of RelA and cJUN. This combined TF signaling mechanism is not observed in other structural lung cell populations after Pam2-ODN exposure. Further, the phospho-regulation dynamics of RelA and cJUN are not replicated by IAV or CoV infection alone, suggesting a novel therapeutic process that can be leveraged to protect individuals against pneumonia. (Figure Presented).

ANTIVIRAL RESPONSE OF NATURAL KILLER CELLS IN EARLY STAGE OF COVID-19 PATIENTS

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.

MACROGLOBULINEMIA DE WALDENSTRÖM: RELATO DE CASO

Introdução: A macroglobulinemia de Waldenstrom (MW) é uma patologia linfoproliferativa neoplásica maligna das células plasmáticas e linfócitos B, normalmente responsável pela síntese das cadeias pesadas de imunoglobulinas. Diferente da leucemia linfoblástica aguda, os linfócitos mantêm a capacidade de se diferenciar e amadurecer em células plasmáticas. A MW possui características que variam de células linfoides maduras a plasmócitos. Imunofenotipagem de células obtidas a partir da medula óssea, dos linfonodos ou do sangue periférico de pacientes com essa doença mostram IgM citoplasmática detectável em células plasmáticas e imunoglobulina superficial na maioria dos linfócitos. Objetivo: Apresentar um caso de MW. Material e métodos: Revisão de prontuáriros médicos e literatura. Descrição do caso: Paciente do sexo masculino, 61 anos, apresentou epistaxe de grande volume com repetição, fadiga e astenia, além de perda de 15 quilos em 6 meses. Na admissão hospitalar apresentava os seguintes exames: anemia com hemoglobina 5,4 g/dL, hemácias de 1,45 milhão/mm3, plaquetopenia (87.000/mm3), presença de esplenomegalia importante, roleaux eritrocitário, esplenomegalia e linfonodomegalia abdominal. Solicitada avaliação com hematologista por paciente referir repetidas anemias ao longo da vida e histórico de transfusões de repetição. Seguem os resultados da investigação com especialisgta: Mielograma apresentou células linfoides de tamanho pequeno e aspecto maduro, algumas apresentando aspecto plasmocitoide. Imunofenotipagem de sangue periférico com CD20/CD38, CD38, CD200, CD19/CD200 positivo fraco;CD19, CD45, CD45/CD22, KAPPA, CD20: positivo de alta intensidade;KAPPA cito- plasmático e CD43 positivo. Dosagem de Imunoglobulinas: IgM 10.100 mg/dL, IgG 242 mg/dL e IgA 91,5 mg/dL. Pico monoclonal IgM/Lambda na imunofixação sérica. Exame de Coombs direto negativo. Como terapêutica, iniciou-se Ciclofosfamida associado a Rituximabe. O paciente evoluiu ao óbito devido ao Covid-19 durante a internação. Discussão: A MW é uma neoplasia rara, sendo que o paciente deste relato enquadra-se na idade média do diagnóstico de MW, que ocorre em torno dos 60 anos, maioria do sexo masculino. Grande parte dos pacientes apresenta sintomas como fadiga, fraqueza e sangramento (principalmente epistaxe). Outras manifestações como perda de peso, distúrbios visuais e fenômeno de Raynaud são menos comuns. Ao exame físico, encontra-se rotineiramente hepatoesplenomegalia e linfadenopatia. Proteinúria de Bence Jones está presente em um quarto de todos os pacientes com MW, devido a uma lesão predominantemente glomerular com depósitos de IgM e material amiloide. A função plaquetária está prejudicada pelo revestimento de plaquetas com IgM, e alguns pacientes podem apresentar defeitos da cascata de coagulação. Os níveis de IgM são marcadamente aumentados, e a crioglobulina pode ser detectada em alguns pacientes. O tratamento deve ser individualizado de acordo com as manifestações clínicas de cada paciente e seu curso clínico é variável. Causas de morte relacionam-se geralmente a hiperviscosidade, anemia, hemorragia, trombose e infecções, sendo que neste caso a infecção por Covid-19 atuou como fator definidor. Conclusão: É oportuno o relato de um caso de MW devido a raridade de seu diagnóstico e importância de estabelecer diagnóstico diferencial com doenças de maior prevalência.

LABORATORY MARKERS FOR PREDICTING AGGRAVATION AND POOR PROGNOSIS IN PATIENTS WITH COVID-19

Introduction: Covid-19 is an infectious disease with systemic involvement, which causes intense changes in the blood system, such as neutrophilia and lymphopenia, as well as changes in coagulation function and the concentration of acute phase proteins. Infected patients require laboratory follow-up to assist in clinical and therapeutic management. It is important to define efficient parameters to predict the clinical course of the disease, especially when the overall symptoms are becoming worse, in an attempt to anticipate therapeutic measures and to ensure the most appropriate assistance. Purpose: To correlate neutrophil and lymphocyte counts and their subtypes with the severity and outcome of patients with Covid-19. Materials and methods: Patients hospitalized for severe Covid-19, of both genders and without evidence of bacterial pneumonia, seen at the CHC-UFPR between April and June 2020, were included. Lymphocyte subpopulation analysis was performed by multiparametric flow cytometry (MFC) on whole blood sample using antibodies against CD45, CD3, CD4, CD8 and CD19. A BD FACSCanto™ II cytometer and Infinicyt™ 2.0 analysis software were used. ROC curve and other statistical relationships were performed with IBM SPSS™ v. 25 software. Results: Patients were divided as moderate (not intubated, n = 41) and severe (intubated, n = 35). From the median total leukocyte, neutrophil and lymphocyte counts and their subsets, we define the cutoff values with the highest correlation with hospital discharge. Patients with lymphocyte counts higher than 489/μL, CD4 counts higher than 326 and CD8 counts higher than 121 had a greater chance of evolving with a better prognosis (p < 0.001). Patients who had neutrophil-to-lymphocyte ratio (NLR) higher than 15.2 showed greater correlation with worse prognosis. Patients with lymphopenia below cutoff values are 40 to 55% more likely to be intubated and 50 to 63% to progress to death. Patients with NLR higher than 15.2 have 53.1% more chances of being intubated and 78.1% of evolving to death. Discussion: Laboratory evaluation is essential in the follow-up of patients with Covid-19. In addition to routinely used biochemical markers, cellular analysis can provide valuable information about the clinic and its progression. Lymphopenia and neutrophilia are common parameters in patients with severe disease, so NLR analysis presents itself as an objective scale for stratification of infected patients with a high correlation with possible outcomes. Associated with this, assessment of the immune profile with low levels of T-cells and especially low levels of positive CD4 cells has been associated with worse prognosis in patients with severe Covid-19. Conclusion: We conclude that analysis of the neutrophil/lymphocyte ratio routinely obtained from the complete blood count may provide relevant prognostic information for patients with Covid-19. In addition, flow cytometry analysis of CD4 and CD8 T-lymphocytes can complement the screening of patients with Covid-19 by providing information on the immune profile of the disease.

Immunological profile and diagnostic indices of inflammation as prognostic markers of clinical outcome in patients with SARS-CoV2 infection

Background The clinical course of pneumonia caused by SARS-CoV-2 is quite peculiar and is characterized by a rapid deterioration of the clinical condition of patients. The aim of this study is to characterize immunological dysfunctions in COVID-19 patients and correlate them with markers of hyperactivation of the inflammatory response, in an attempt to find threshold values indicative of patient outcome. Methods A total of 100 patients were recruited. All patients had a positive PCR test for SARS-COV-2 from nasopharyngeal sample. Patients were grouped into those who did not require mechanical ventilation (n=72) and those who required mechanical ventilation (n=28). Blood samples were collected and analyzed at the point of admission in all patients. Patient immune phenotyping was performed in whole blood samples of patients by flow cytometric analysis. The flow cytometric analysis was performed in an Aquios cytometer (Aquios -Beckman Coulter CA, USA). Antibodies used for cell staining are TETRA-1 Panel (CD45, CD4, CD8, CD3). Data were analyzed using flow cytometric analysis software. Data from the routine biochemical assessment performed in the first 24 hrs after admission to infectious diseases unit will be collected. Results Both the percentage and the absolute number of neutrophils were higher in patients needing ICU care than non-ICU patients, whereas absolute lymphocyte count, and especially the percentage of lymphocytes, presented a deep decline in critical patients. There was no difference between the two groups of patients for CD4 Tlymphocytes, neither in percentage of lymphocyte nor in absolute number, however for CD8 T-cells the differences were significant for both parameters which were in decline in ICU patients. There was a firm correlation between the highest values of inflammation indicators with the decrease in percentage of CD8 T-lymphocytes. This effect was not seen with CD4 cells. Conclusion Our results describe the immune response of severe COVID-19 patients and highlight the value of a novel ratio of CD4/CD8 as a putative marker of poor prognosis. Nevertheless, further research is warranted in order to fully comprehend the transition of the different stages of COVID-19 progression in the context of successful combat of this novel disease.

Reccurence of atypical hemolytic uremic syndrome after COVID-19 vaccination

Background: Atypical Hemolytic Uremic Syndrome (aHUS) is a complement-mediated thrombotic microangiopathy. Pathophysiological mechanism involves uncontrolled complement activation due to a genetic or acquired anomaly coupled with a triggering event. We report a case of aHUS recurrence following COVID-19 vaccination. Material and methods: Whole blood (EDTA) was collected and processed with CD46-PE, CD45-PerCP, isotype control-PE markers. Staining was measured through median fluorescence intensity and expressed as CD46/isotype ratio. Sanger sequencing was used for identification of variants in CD46 gene. All the participants provided informed written consent. Results: Proband (P) is a 39-year-old woman admitted for nausea, vomiting, epigastric pain and haematuria, three days after first dose of ChAdOx1 nCov-19 vaccine. Laboratory testing showed MAHA (Hb:8.8g/dL, Ht:26%), thrombocytopenia (80x109/mm3) and acute kidney injury (Cr:2.15mg/dL, Ur:92mg/dL). P and three of her siblings have experienced recurrent TMA episodes since childhood. In 2019, genetic study from P's sister (S) identified two heterozygous variants in CD46, one pathogenic (Glu179Gln) and one of uncertain significance (Cys94Tyr). We demonstrated that P carries the same variants and observed a 50% decrease of CD46 expression in both P and S (fig.1). Platelet transfusion, corticosteroids and 9 sessions of plasmapheresis contributed to rapid recovery of P. Discussion: Glu179Gln was reported to increase CD46 expression on granulocytes in aHUS patient and to reduce C4b cofactor activity1. We observed that combination of Glu179Gln and Cys94Tyr was associated with low levels of CD46 on cell surface. Conclusion: This case report supports the evidence of COVID-19 vaccine as a precipitating event for aHUS recurrence.

Novel Calr Mutation in Acute Lymphoblastic Leukemia

Introduction: Acute lymphoblastic leukemia in adults represent amajor therapeutic challenge even in modern era. Constantly evolvinggenomics has led to a better risk stratification and prognostication.Aims &Objectives: Here we present a novel mutation in calreticulingene in a patient with Precursor B lineage Acute lymphoblasticleukemia.Materials &Methods: A 19 year old boy presented with fever,jaundice and pancytopenia. Initial investigation revealed a hemoglobin of 5.8 g/dl with a total leukocyte count of 700 cells/mm3 andplatelet count of 11,000/mm3. Differential count showed 95% lymphocytes and 5% neutrophils and no blasts with mildanisopoikilocytosis on PBF. Bone marrow biopsy demonstartedreduction granulocytic and erythroid lineage with adequatemegakaryocytes and occasional collections of immature appearingcells, whose charcter was not able to be definitely ascertained. PETCT showed FDG avid lymph nodes on both sides of diaphragm, withPET guided biopsy was suggestive of non specific lymphocyticinflammatory infiltrate. A diagnosis of hemophagocytic lymphohistiocytosis (HLH) was made according to HLH 2004 criteria (Fever,cytopenias, hypertriglyceridemia, splenomegaly and elevated serumferritin levels). However workup for primary HLH and primaryimmune deficiencies were negative.Clinical exome sequencing forprim was postive for mvk transcript (c.808G >A). He was treatedwith IVIG and short course steroids. Repeat bone marrow was normocellular with mild erythroid prominence and adequaterepresentation of granulocytic and megakaryocytic lineage elements.He was under regular follow up thereafter.Result: He developed mild Covid illness a month after discharge. Amonth after recovery from illness, he presented with easy fatiguabilityand pancytopenia (Hb: 7.1 g/dl, TLC: 900 cells/mm3 and Plateletcount: 1.46 lakhs) with presence of occasional blast in PBF. Repeatbone marrow was markedly hypercellular with 76% blast.Megakaryocytes were relatively preserved. On flow cytometry, blastswere positive for CD 10, CD 19, CD 20, cyto CD 79a, CytoCD22, CD34, CD 45, HLA DR and TDT and negative for MPO CD 2 CD 3 CD13 aand CD 33 consistent with Precursor B Lineage acute lymphoblastic leukemia. Multiplex RT-PCR for recurrent geneticabnormalities (ALL) were negative. A never reported CALR (TYPE1) mutation was found in this patient. CALR plays an important rolein cell proliferation, apoptosis and immune responses. Patient wastreated with modified BFM regimen and Rituximab, attained CR postinduction and currently in consolidation phase of therapy.Conclusions: CALR mutation has never been reported before in acase of acute lymphoblastic leukemia. Long term follow up of patient is required to conclude whether the novel mutation has prognostic andtherapeutic implications.

Sars-Cov-2 T-Cell Response in Allogeneic Hematopoietic Stem Cell Recipients Following Two Doses of BNT162b2 Vaccine

Introduction: Virus-specific humoral and cellular immune responses act synergistically to protect from viral infection. In our recent observational monocentric study of 117 hematopoietic stem cell adult recipients, we found that 54% and 83 % patients achieved a humoral response after two doses of BNT162b2 anti-SARS-CoV-2 messenger RNA vaccine (Pfizer BioNTech), respectively. Here, we evaluated the T-cell response against the SARS-Cov-2 spike protein after two doses of BNT162b2 vaccine in some allografted patients from the same cohort and compared these results to those from healthy controls. Methods: To quantify SARS-CoV-2 specific T-cells, we used an INFg ELISpot assay that detects these cells after activation of peripheral blood mononuclear cells (PBMC) with 3 peptide pools covering the whole protein sequence of the spike glycoprotein (Prot _S1;_S+ and _S PepTivator peptide pools, Miltenyi Biotec, Bergisch Gladbach, Germany). EBV and CMV specific T-cells were also quantified as controls. The immunophenotype of PBMC was determined by flow cytometry, after dead cell exclusion, with monoclonal antibodies identifying the following surface antigens: CD45, CD3, CD14, CD19 and HLA-DR. The frequencies of spot-forming units (SFU) were reported as per 10 6 CD3+ T-cells. Results: Samples from 46 allografted patients (acute myeloblastic leukemia, N=27, myelodysplastic syndrome, N=19) and 16 healthy controls were available. Characteristics of the population are given in Table 1. All fully vaccinated healthy donors became seropositive and developed a positive T-cell response to spike peptide pools even though variable frequencies were observed. The median response was 195 SFU/10 6 T-cells. By comparison, the frequency of EBV-specific T-cells was 774 SFU/10 6 T-cells (Figure 1). In the group of patients, 78% (n=36/46) had achieved a humoral response after the second dose of vaccine. Among these humoral responders (HR), 89% (n=32/36) also had a positive anti-spike T-cell response with variable frequencies (median =119 SFU/10 6 T-cells. For 8 patients, this T cell response was higher than that of controls (>800 SFU/10 6 T-cells) (Figure 1), which is equivalent to more than 1 specific T-cell per microliter of blood (Figure 2). The humoral responders (HR) who did not develop a T-cell response (11%, n=4/36) had a median time from transplant to vaccination of 523 days compared to 1032 days for cellular responder patients. Among the 10 patients who were non humoral responders (NHR) (22%, n=10/46), 4 (40%) developed a cellular immunity, including one with a very high T cell response (1333 SFU/10 6 T-cells). As expected, the absence of humoral response was observed in patients who were within one year of the transplant. Of note, somehow unexpectedly, patients often presented a high frequency of EBV- and CMV-specific T cells (Figures 1 & 2). As expected, PBMC immunophenotypic analysis revealed that CD3+ frequencies were lower in patients compared to those of controls but were similar between HR and NHR. NHR had very low frequencies of B cells and interestingly, they had an elevated frequency of CD14+ monocytes with low/neg HLA-DR expression potentially corresponding to myeloid-derived suppressor cells (MDSCs) (Figure 3). Conclusion: In this series, 89% of allografted patients who developed an anti-spike humoral response also presented an anti-SARS-Cov-2 cellular immunity. Interestingly, anti-SARS-Cov-2 specific T-cells could be detected in 40% of NHR patients. Although a larger group of patients is required to confirm these results, it remains to be determined whether this T-cell response is protective against SARS-Cov-2 infection as previously demonstrated for CMV (Litjens et al, 2017). Finally, the role of potential immunosuppressive MDSCs must be explored in patients who develop no sign of T-cell response after vaccination. [Formula presented] Disclosures: Moreau: Oncopeptides: Honoraria;Celgene BMS: Honoraria;Sanofi: Honoraria;Abbvie: Honoraria;Janssen: Honoraria;Amgen: Honoraria.

Optimizing Viable CD34 + Enumeration in Cryopreserved HPC Samples for Implementation of an External QA Program - Relevance to the Current Pandemic

Background The current External Quality Assurance (EQA) programs for haemopoietic progenitor cell (HPC) involve enumeration of total CD34 + cells in fixed samples, which does not align with clinical practice where analysis of viable CD34 + cells (vCD34 +) is required. The COVID-19 pandemic forced a fundamental change in the global procurement of allogeneic HPC for transplantation. To better meet the emergent challenges of transporting cryopreserved allogeneic HPC during pandemics, there is an urgent need for EQA programs to evaluate reproducibility and harmonization of vCD34 + HPC enumeration between collection and transplant centres. A successful vCD34 + EQA program will require cost-effective distribution of cryopreserved reference samples (CRS) with acceptable reproducibility and specificity. This study aims to evaluate the feasibility of distribution of CRS to participating facilities for vCD34 + enumeration using dry ice, instead of liquid nitrogen which is not suitable for CRS transport due to logistical and cost implications. Method: A 15 ml sample was cryopreserved from each of 10 HPC harvests from consented transplant donors (SVH HREC approval #10/07). Cryopreserved HPC samples were either stored on dry ice for 1-4 days, or on dry ice for one day followed by liquid nitrogen (LN 2) storage for 1-3 days to assess optimal conditions for vCD34 + EQA. For viable CD34 + measurement by flow cytometry, the single platform assay was performed using Trucount tubes containing CD45-FITC/CD34-PE and 7-AAD viability exclusion dye. The optimum transportation condition was validated in pilot and multi-center national studies which involved transport of CRS on dry ice to 12 recipient centers in 5 of the 6 Australian states. Results: Dry ice and LN 2 transport and storage conditions were simulated at the central laboratory and the vCD34 + enumerated. It was found that a combination of one day on dry ice followed by LN 2 storage stabilized the viability compared to continuous storage on dry ice. A successful pilot study confirmed the effect on vCD34 + count of shipping two CRS from central Lab to two interstate laboratories. For the national multicenter study, the transportation distances ranged from 0.5 - 4,000 km (median 513 km) with transit times ranging from 1- 26 hours (median 22.5 hours). Eight of 12 centers (67%) returned comparable results that were within ±10% of the median. There was no significant difference between samples tested immediately upon arrival or after subsequent LN 2 storage (p=0.41). There was no significant relationship between comparability of vCD34 + counts and the sample transit time (R=0.67, p=0.07) nor distance travelled (R = 0.19, p=0.55), showing that laboratory outcome was unrelated to sample transport. Conclusion: Dry ice distribution of cryopreserved HPC for up to 26 hours results in a stable CRS. The estimated cost of safer and more convenient dry ice delivery is >20-fold lower than LN 2. This feasibility study illustrates that an EQA utilizing this mode of transport and storage of CRS is suitable for inter-facility harmonization and standardization forming the basis of an EQA programs for vCD34 + HPC enumeration. Disclosures: No relevant conflicts of interest to declare.

Safety of pooled mesenchimal stem cells in patients with severe COVID-19 pneumonia

Background: COVID-19 is a disease caused by the SARS-CoV-2 virus that is often associated with pneumonia and acute respiratory distress syndrome (ARDS). Pathogenesis of COVID-19 is closely related to the host ' s response to the virus. Impaired regulation of immunity has been observed in patients with severe COVID-19 pneumonia. Mesenchymal stem cells (MSCs), having multipotency, ability to selfrenew, and ability to evade immune response may be useful in in the treatment of COVID-19 pneumonia. MSCs have an immunomodulatory effect on almost all types of immunocompetent cells: T-and B-lymphocytes, natural killer cells, monocytes and macrophages, dendritic cells, neutrophils. This study assesses the safety and tolerability of pooled allogeneic mesenchymal stem cells (poolMSC) in COVID 19 pneumonia. Method: The study included 5 patients with PCR confirmed COVID-19 pneumonia-4 men and 1 woman, average age 62.4 years. Pooled MSC (pMSC) was a mixture of 3 cultures of olfactory mucosa-derived MSCs (OM-MSCs) obtained from the mucous membrane of the middle nasal passage of healthy volunteers. MSCs were tested for viability (>95%), immunophenotype CD90 + CD105 + CD73 + CD31-CD45-HLA-DR-, and sterility. pMSC at a dosage of 1×10 6 cells per kg of body weight were suspended in 100 ml of saline and injected intravenously over 60 minutes. Results: All patients were carefully examined at baseline before pMSC infusion. Clinical examination was done on the day of infusion of pMSC with a skin test to pMSC was performed. In the absence of systemic and local allergic reactions pMSC were injected. Three patients received one pMSC infusion, and two patients received two pMSC infusions at an interval of 4 days. None of the patients had any adverse reactions to the pMSC skin test or infusion. Conclusion: Assessment of pMSC infusion demonstrated good tolerance and safety of intravenous use in patients with severe pneumonia caused by the SARS-CoV-2 and complicated by ARDS.

Clinical and morphological characteristics of COVID-19-associated myocarditis

Background: COVID-19 is accompanied by the development of a wide range of cardiovascular lesions. The goal: to study the clinical and morphological features of SARS-CoV-2-associated myocarditis (SCM), determining the presence of viral RNA and proteins in myocardial tissue. Methods: The study was based on 32 autopsies with a confirmed diagnosis of myocarditis. The average age of the patients was 72.7±15.5 years. Men predominated in the group (53%). The immunohistochemical determination of the surface markers of CD45, CD3, CD20, CD 68 inflammatory infiltrates and SARS-CoV-2 nucleocapsid and spike protein has been done. Detection of coronavirus RNA was performed. Results: The clinical manifestations SCM included heart failure and variety of rhythm disturbances. Increased level of anticardiac antibodies was detected. Lymphomacrophage infiltrates (more than 7 CD3+ T-lymphocytes, more than 14 CD45+ lymphocytes and more than 7 CD68+ macrophages per 1 mm2) were found in 100% of cases. RNA of the virus was detected in myocardial tissue. Virus proteins were identified in macrophages of the inflammatory infiltrate and cardiomyocytes. Conclusion: The results suggest persistence of the virus.