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Transfusion ; 61(4): 1247-1257, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33481275


BACKGROUND: Irradiation of red blood cells (RBCs) inactivates residual donor T lymphocytes to prevent transfusion-associated graft-vs-host disease (TA-GVHD) but can have adverse effects on recipients and inventory management. Reported incidence of TA-GVHD is lower when leukoreduced RBCs and older blood products are transfused; therefore, the impact of leukoreduction and storage was evaluated as an alternative prevention strategy. STUDY DESIGN AND METHODS: Effectiveness of leukoreduction filters on white blood cell (WBC) proliferation was evaluated by filtering buffy coat (BC) products and isolating residual WBCs. Additionally, leukoreduced RBCs were spiked with 5 × 106 WBCs on Day 21 of hypothermic storage, then stored and processed on Days 7, 14, and 21 to obtain residual WBCs to investigate the impact of hypothermic storage on their viability and proliferative ability. Viability of residual WBCs was assessed by staining with annexin V and an antibody cocktail for flow cytometry analysis. Proliferative ability was assessed by placing carboxyfluorescein diacetate succinimidyl ester-labeled residual WBCs into culture for 6 days with phytohemagglutinin before flow cytometry assessment. RESULTS: Filtration of BC units depleted WBCs, particularly T lymphocytes, to 0.001% ± 0.003% cells/unit, although proliferative activity remained consistent with prefiltration levels of WBCs. WBCs in stored RBCs remained viable even on Day 21 of storage; however, the proliferative activity decreased to 0.24% ± 0.41%. CONCLUSIONS: Hypothermic storage of RBCs for 21 days or more is sufficient to inactivate T lymphocytes, which may help prevent TA-GVHD when irradiated RBCs are not available.

Criobiologia/métodos , Eritrócitos/fisiologia , Procedimentos de Redução de Leucócitos/métodos , Reação Transfusional/prevenção & controle , Preservação de Sangue/métodos , Proliferação de Células/fisiologia , Proliferação de Células/efeitos da radiação , Transfusão de Eritrócitos/efeitos adversos , Eritrócitos/efeitos da radiação , Filtração , Citometria de Fluxo/métodos , Humanos , Incidência , Procedimentos de Redução de Leucócitos/estatística & dados numéricos , Leucócitos/imunologia , Linfócitos T/imunologia , Linfócitos T/efeitos da radiação , Fatores de Tempo , Reação Transfusional/epidemiologia , Reação Transfusional/imunologia
Cell Commun Signal ; 18(1): 155, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948210


BACKGROUND: Thrombospondin-1 (TSP-1), a Ca2+-binding trimeric glycoprotein secreted by multiple cell types, has been implicated in the pathophysiology of several clinical conditions. Signaling involving TSP-1, through its cognate receptor CD47, orchestrates a wide array of cellular functions including cytoskeletal organization, migration, cell-cell interaction, cell proliferation, autophagy, and apoptosis. In the present study, we investigated the impact of TSP-1/CD47 signaling on Ca2+ dynamics, survival, and deformability of human red blood cells (RBCs). METHODS: Whole-cell patch-clamp was employed to examine transmembrane cation conductance. RBC intracellular Ca2+ levels and multiple indices of RBC cell death were determined using cytofluorometry analysis. RBC morphology and microvesiculation were examined using imaging flow cytometry. RBC deformability was measured using laser-assisted optical rotational cell analyzer. RESULTS: Exposure of RBCs to recombinant human TSP-1 significantly increased RBC intracellular Ca2+ levels. As judged by electrophysiology experiments, TSP-1 treatment elicited an amiloride-sensitive inward current alluding to a possible Ca2+ influx via non-selective cation channels. Exogenous TSP-1 promoted microparticle shedding as well as enhancing Ca2+- and nitric oxide-mediated RBC cell death. Monoclonal (mouse IgG1) antibody-mediated CD47 ligation using 1F7 recapitulated the cell death-inducing effects of TSP-1. Furthermore, TSP-1 treatment altered RBC cell shape and stiffness (maximum elongation index). CONCLUSIONS: Taken together, our data unravel a new role for TSP-1/CD47 signaling in mediating Ca2+ influx into RBCs, a mechanism potentially contributing to their dysfunction in a variety of systemic diseases. Video abstract.

Antígeno CD47/metabolismo , Deformação Eritrocítica , Eritrócitos/citologia , Transdução de Sinais , Trombospondina 1/metabolismo , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Sobrevivência Celular , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Humanos
Transfus Apher Sci ; 59(5): 102942, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32943325


Extracellular vesicles (EVs) have been shown to be involved in various physiological and pathophysiological processes. With respect to Transfusion Medicine, the accumulation of EVs in blood products during hypothermic storage is an indicator of the storage lesion and reportedly correlates with adverse effects after transfusion, including but not limited to immunomodulation, activation of coagulation, endothelial activation, and others. To optimally reduce such an impact on blood product quality degradation and improve post-transfusion outcomes, better methods for detection, enumeration, characterisation by size and phenotype, and functional involvement of EVs in different pathophysiological and physiological processes are required. Currently, Imaging Flow Cytometry (IFC) technology provides the most comprehensive assessment of EV subsets in different body fluids. The unique ability of IFC to detect EVs of 20 nm size by registration of a single pixel of fluorescence signal makes this approach highly promising for comprehensive studies of EVs. In this review, we will focus on the recent breakthrough and advantages of using the ImageStreamX MKII IFC platform for the detection and characterisation of EVs and its future prospects for routine application of IFC in Transfusion Medicine.

Vesículas Extracelulares/metabolismo , Citometria de Fluxo/métodos , Medicina Transfusional/métodos , Humanos
Proc Natl Acad Sci U S A ; 117(35): 21381-21390, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32839303


Stored red blood cells (RBCs) are needed for life-saving blood transfusions, but they undergo continuous degradation. RBC storage lesions are often assessed by microscopic examination or biochemical and biophysical assays, which are complex, time-consuming, and destructive to fragile cells. Here we demonstrate the use of label-free imaging flow cytometry and deep learning to characterize RBC lesions. Using brightfield images, a trained neural network achieved 76.7% agreement with experts in classifying seven clinically relevant RBC morphologies associated with storage lesions, comparable to 82.5% agreement between different experts. Given that human observation and classification may not optimally discern RBC quality, we went further and eliminated subjective human annotation in the training step by training a weakly supervised neural network using only storage duration times. The feature space extracted by this network revealed a chronological progression of morphological changes that better predicted blood quality, as measured by physiological hemolytic assay readouts, than the conventional expert-assessed morphology classification system. With further training and clinical testing across multiple sites, protocols, and instruments, deep learning and label-free imaging flow cytometry might be used to routinely and objectively assess RBC storage lesions. This would automate a complex protocol, minimize laboratory sample handling and preparation, and reduce the impact of procedural errors and discrepancies between facilities and blood donors. The chronology-based machine-learning approach may also improve upon humans' assessment of morphological changes in other biomedically important progressions, such as differentiation and metastasis.

Bancos de Sangue , Aprendizado Profundo , Eritrócitos/citologia , Humanos
Transfusion ; 60(11): 2633-2646, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32812244


BACKGROUND: Characteristics of red blood cells (RBCs) are influenced by donor variability. This study assessed quality and metabolomic variables of RBC subpopulations of varied biologic age in red blood cell concentrates (RCCs) from male and female donors to evaluate their contribution to the storage lesion. STUDY DESIGN AND METHODS: Red blood cell concentrates from healthy male (n = 6) and female (n = 4) donors were Percoll separated into less dense ("young", Y-RCCs) and dense ("old", O-RCCs) subpopulations, which were assessed weekly for 28 days for changes in hemolysis, mean cell volume (MCV), hemoglobin concentration (MCHC), hemoglobin autofluorescence (HGB), morphology index (MI), oxygen affinity (p50), rigidity, intracellular reactive oxygen species (ROS), calcium ([Ca2+ ]), and mass spectrometry-based metabolomics. RESULTS: Young RCCs having disc-to-discoid morphology showed higher MCV and MI, but lower MCHC, HGB, and rigidity than O-RCCs, having discoid-to-spheroid shape. By Day 14, Y-RCCs retained lower hemolysis and rigidity and higher p50 compared to O-RCCs. Donor sex analyses indicated that females had higher MCV, HGB, ROS, and [Ca2+ ] and lower hemolysis than male RBCs, in addition to having a decreased rate of change in hemolysis by Day 28. Metabolic profiling indicated a significant sex-related signature across all groups with increased markers of high membrane lipid remodeling and antioxidant capacity in Y-RCCs, whereas O-RCCs had increased markers of oxidative stress and decreased coping capability. CONCLUSION: The structural, functional, and metabolic dissimilarities of Y-RCCs and O-RCCs from female and male donors demonstrate RCC heterogeneity, where RBCs from females contribute less to the storage lesion and age slower than males.

Doadores de Sangue , Preservação de Sangue , Senescência Celular , Eritrócitos , Estresse Oxidativo , Adulto , Eritrócitos/classificação , Eritrócitos/citologia , Eritrócitos/metabolismo , Feminino , Humanos , Masculino