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1.
Int J Mol Sci ; 22(21)2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34768764

RESUMO

Chronic inflammation is a pathological process where cells of the mesenchymal lineage become a major source of inflammatory mediators. Platelet-rich fibrin (PRF) has been shown to possess potent anti-inflammatory activity in macrophages, but its impact on mesenchymal cells has not been investigated. The aim of this study was, therefore, to expose mesenchymal cells to inflammatory cytokines together with lysates generated from liquid platelet-poor plasma (PPP), the cell-rich buffy coat layer (BC; concentrated-PRF or C-PRF), and the remaining red clot layer (RC), following centrifugation of blood. Heating PPP generates an albumin gel (Alb-gel) that when mixed back with C-PRF produces Alb-PRF. Membranes prepared from solid PRF were also subjected to lysis. We report here that lysates of PPP, BC, and PRF decreased the cytokine-induced expression of interleukin 6 (IL6) and nitric oxide synthase (iNOS) in the bone marrow-derived ST2 cells. Consistently, PPP, BC, and PRF greatly decreased the phosphorylation and nuclear translocation of p65 in ST2 cells. The inflammatory response caused by Pam3CSK4 was reduced accordingly. Moreover, PPP, BC, and PRF reduced the enhanced expression of inflammatory mediators IL6 and iNOS in 3T3-L1 pre-adipocyte mesenchymal cells, and iNOS and CCL5 in murine calvarial cells. Surprisingly, PRF lysates were not effective in reducing the inflammatory response of human gingival fibroblasts and HSC2 epithelial cells. The data from the present study suggest that both liquid PRF and solid PRF exert potent anti-inflammatory activity in murine mesenchymal cells.


Assuntos
Anti-Inflamatórios/metabolismo , Inflamação/tratamento farmacológico , Células-Tronco Mesenquimais/metabolismo , Fibrina Rica em Plaquetas/metabolismo , Animais , Buffy Coat/metabolismo , Plaquetas/metabolismo , Linhagem Celular , Citocinas/toxicidade , Fibroblastos/metabolismo , Gengiva/metabolismo , Humanos , Inflamação/induzido quimicamente , Camundongos , NF-kappa B/antagonistas & inibidores , Plasma/metabolismo , Cultura Primária de Células , Receptor 2 Toll-Like/agonistas , Fator de Transcrição RelA/metabolismo
2.
Front Immunol ; 12: 655122, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34408743

RESUMO

FOXP3+ regulatory T cells (Tregs) are central for maintaining peripheral tolerance and immune homeostasis. Because of their immunosuppressive characteristics, Tregs are a potential therapeutic target in various diseases such as autoimmunity, transplantation and infectious diseases like COVID-19. Numerous studies are currently exploring the potential of adoptive Treg therapy in different disease settings and novel genome editing techniques like CRISPR/Cas will likely widen possibilities to strengthen its efficacy. However, robust and expeditious protocols for genome editing of human Tregs are limited. Here, we describe a rapid and effective protocol for reaching high genome editing efficiencies in human Tregs without compromising cell integrity, suitable for potential therapeutic applications. By deletion of IL2RA encoding for IL-2 receptor α-chain (CD25) in Tregs, we demonstrated the applicability of the method for downstream functional assays and highlighted the importance for CD25 for in vitro suppressive function of human Tregs. Moreover, deletion of IL6RA (CD126) in human Tregs elicits cytokine unresponsiveness and thus may prevent IL-6-mediated instability of Tregs, making it an attractive target to potentially boost functionality in settings of adoptive Treg therapies to contain overreaching inflammation or autoimmunity. Thus, our rapid and efficient protocol for genome editing in human Tregs may advance possibilities for Treg-based cellular therapies.


Assuntos
Edição de Genes/métodos , Subunidade alfa de Receptor de Interleucina-2/genética , Receptores de Interleucina-6/genética , Linfócitos T Reguladores/metabolismo , Buffy Coat/citologia , Sistemas CRISPR-Cas/genética , Fatores de Transcrição Forkhead/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Voluntários Saudáveis , Humanos , Imunoterapia Adotiva/métodos , Cultura Primária de Células , RNA Guia/genética , Fatores de Tempo
3.
Transfusion ; 61(9): 2746-2755, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34331776

RESUMO

OBJECTIVES: Characterization of the proteasome and its stability in buffy-coat derived platelet concentrates (PCs) during storage. BACKGROUND: The proteasome plays a key role in cell homeostasis by processing misfolded or abnormal proteins and regulating the levels and activities of a high number of proteins contributing to cell cycle, survival, and proliferation. Controversial data exist, whether inhibition of the proteasome affects platelet function. Little is known about function, expression, and stability of the proteasome in PCs during storage, and the potential role of the platelet proteasome in storage lesions. STUDY DESIGN AND METHODS: PCs were produced by the buffy-coat method in additive solution and stored at room temperature under agitation. Platelet aggregation was monitored by light transmission aggregometry. Proteasome complexes were assessed by immunoprecipitation and immunoblotting, and proteasome activity was measured using fluorogenic substrates specific for the three different proteolytic activities over 7 days of storage. RESULTS: Proteasome inhibition led to a decreased platelet aggregation response after activation with collagen, ADP, TRAP-6, and thrombin. There were no changes in the expression of the catalytic active subunits as well as the proteasome activity during storage of PCs, comparing baseline and day 7. DISCUSSION: Platelet proteasome function is relevant for platelet aggregation in response to various agonists. The constitutive and stable expression of the active standard- and immunoproteasome in platelets makes it unlikely that loss of proteasome function is a relevant cause of storage lesions.


Assuntos
Plaquetas/citologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Buffy Coat/citologia , Plaquetas/metabolismo , Preservação de Sangue , Humanos , Ativação Plaquetária , Agregação Plaquetária , Testes de Função Plaquetária
4.
PLoS One ; 16(7): e0254615, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34297742

RESUMO

Leukocytes have an essential role in patient clinical trajectories and progression. Traditional methods of leukocyte enrichment have many significant limitations for current applications. It is demonstrated a novel 3D printing leukocyte sorting accumulator that combines with centrifugation to ensure label-free initial leukocyte enrichment based on cell density and size. The internal structure of leukocyte sorting accumulator (revealed here in a new design, leukocyte sorting accumulator-3, upgraded from earlier models), optimizes localization of the buffy coat fraction and the length of the period allocated for a second centrifugation step to deliver a higher recovery of buffy coats than earlier models. Established methodological parameters were evaluated for reliability by calculating leukocyte recovery rates and erythrocyte depletion rates by both pushing and pulling methods of cell displacement. Results indicate that leukocyte sorting accumulator-3 achieves a mean leukocytes recovery fraction of 96.2 ± 2.38% by the pushing method of layer displacement. By the pulling method, the leukocyte sorting accumulator-3 yield a mean leukocytes recovery fraction of 94.4 ± 0.8%. New procedures for preliminary enrichment of leukocytes from peripheral blood that avoid cellular damage, as well as avert metabolic and phase cycle intervention, are required as the first step in many modern clinical and basic research assays.


Assuntos
Procedimentos de Redução de Leucócitos/métodos , Leucócitos/citologia , Impressão Tridimensional/instrumentação , Buffy Coat/classificação , Buffy Coat/citologia , Centrifugação/instrumentação , Centrifugação/métodos , Humanos , Procedimentos de Redução de Leucócitos/instrumentação , Leucócitos/classificação
5.
Mol Biol Rep ; 48(5): 4247-4252, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34097204

RESUMO

Among the host restriction factors against HIV, SERINC5 has been described in vitro, but the mRNA level of SERINC5 in vivo has been little studied. We compare SERINC5 expression in subjects with HIV-1 (highly active antiretroviral treatment (HAART) and HAART-naïve) with and without suppression of viral load. A cross-sectional study was performed with 107 individuals distributed as follows: 24 with HAART-naïve and detectable viral load (> 50 copies/mL), 13 with HAART and detectable viral load (> 50 copies/mL), 50 with HAART and undetectable viral load (≤ 50 copies/mL), and 20 without HIV-1. SERINC5 expression in buffy coats was determined using RT-qPCR. The viral load was determined using real-time PCR and the amount of CD4 + and CD8 + T-lymphocytes was measured using flow cytometry. The data were normalized with the Shapiro-Wilk test and the Kruskal-Wallis test was subsequently performed. The relative expression was compared with a T-test and the remaining data with the Mann-Whitney U-test. ANCOVA multiple linear regression analysis was performed between characteristics of patients with SERINC5 expression. The mean and SD of the SERINC5 expression in the three groups with HIV-1 was 0.9 ± 0.2 and without HIV-1 was 1.7 ± 0.14 (P < 0.001). Multiple linear regression did not show the participation of CD4 +, CD8 + , viral load, infection time, or treatment time. No differences in the SERINC5 expression were found among the studied groups of patients with HIV-1. When comparing the groups with and without HIV-1 infection, SERINC5 was downregulation in the HIV-1 groups.


Assuntos
Buffy Coat/metabolismo , Regulação para Baixo/genética , Infecções por HIV/sangue , Infecções por HIV/genética , HIV-1/genética , Proteínas de Membrana/genética , Carga Viral/métodos , Adolescente , Adulto , Idoso , Terapia Antirretroviral de Alta Atividade/métodos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Estudos Transversais , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Masculino , Proteínas de Membrana/sangue , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Resultado do Tratamento , Adulto Jovem
6.
Sci Immunol ; 6(60)2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172588

RESUMO

CD1c presents lipid-based antigens to CD1c-restricted T cells, which are thought to be a major component of the human T cell pool. However, the study of CD1c-restricted T cells is hampered by the presence of an abundantly expressed, non-T cell receptor (TCR) ligand for CD1c on blood cells, confounding analysis of TCR-mediated CD1c tetramer staining. Here, we identified the CD36 family (CD36, SR-B1, and LIMP-2) as ligands for CD1c, CD1b, and CD1d proteins and showed that CD36 is the receptor responsible for non-TCR-mediated CD1c tetramer staining of blood cells. Moreover, CD36 blockade clarified tetramer-based identification of CD1c-restricted T cells and improved identification of CD1b- and CD1d-restricted T cells. We used this technique to characterize CD1c-restricted T cells ex vivo and showed diverse phenotypic features, TCR repertoire, and antigen-specific subsets. Accordingly, this work will enable further studies into the biology of CD1 and human CD1-restricted T cells.


Assuntos
Apresentação do Antígeno , Antígenos CD1/metabolismo , Antígenos CD36/metabolismo , Glicoproteínas/metabolismo , Subpopulações de Linfócitos T/imunologia , Buffy Coat , Antígenos CD36/antagonistas & inibidores , Voluntários Saudáveis , Humanos , Células Jurkat , Ligantes , Lipídeos/imunologia , Cultura Primária de Células , Multimerização Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/metabolismo
7.
BMC Infect Dis ; 21(1): 576, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34130649

RESUMO

BACKGROUND: Both capillary and venous blood samples have been interchangeably used for the diagnosis of malaria in Ethiopia. However, Plasmodium parasites are thought to be more concentrated in capillary than in venous blood. Hence, selecting a sample source where parasites are more concentrated is indispensable approach in order to maximize the accuracy of blood film microscopy. Therefore, the present study aimed to compare the detection rate and the parasitemia level of Plasmodium species from conventional capillary and venous blood films, and buffy coat preparations. METHODS: A facility based cross-sectional study was conducted from Feburary to March 2020 among 210 febrile patients attending Hamusite health center, northwest Ethiopia. Capillary and venous blood samples were collected and buffy coat was prepared from each sample. Thin and thick blood films were prepared, stained, and examined microscopically following standard protocol. Data were analysed using Statistical Package for Social Sciences Software version 20 and Med-Calc software version 19.3. RESULTS: Capillary blood buffy coat (61/210, 29.0%) had significantly higher detection rate as compared to capillary (48/210, 22.9%) and venous (42/210, 20.0%) blood films (p < 0.001). However, no significant difference was observed between capillary and venous blood films (p = 0.070) in detecting Plasmodium species. The highest and the lowest mean asexual stage parasite counts were found in capillary blood buffy coat (4692.88) and venous blood (631.43) films, respectively showing significant variations (p < 0.001). Mean gametocyte count was also highest in capillary blood buffy coat (3958.44). As compared to capillary blood buffy coat, the sensitivity of venous blood buffy coat, capillary blood film and venous blood film were 73.8, 78.7, 68.9%, respectively. CONCLUSION: Capillary blood buffy coat samples showed the highest sensitivity in detecting and quantitating malaria parasites that its use should be promoted in clinical settings. However, conventional capillary and venous blood films could be used interchangeably.


Assuntos
Testes Hematológicos/métodos , Malária/diagnóstico , Plasmodium/isolamento & purificação , Adolescente , Adulto , Animais , Buffy Coat/parasitologia , Coleta de Amostras Sanguíneas/métodos , Capilares/parasitologia , Criança , Estudos Transversais , Etiópia , Feminino , Humanos , Malária/sangue , Masculino , Microscopia/métodos , Parasitemia/parasitologia , Parasitos , Veias/parasitologia , Adulto Jovem
8.
BMJ Open ; 11(4): e042519, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33931406

RESUMO

INTRODUCTION: HIV coinfection presents a challenge for diagnosis of visceral leishmaniasis (VL). Invasive splenic or bone marrow aspiration with microscopic visualisation of Leishmania parasites remains the gold standard for diagnosis of VL in HIV-coinfected patients. Furthermore, a test of cure by splenic or bone marrow aspiration is required as patients with VL-HIV infection are at a high risk of treatment failure. However, there remain financial, implementation and safety costs to these invasive techniques which severely limit their use under field conditions. METHODS AND ANALYSIS: We aim to evaluate blood and skin qPCR, peripheral blood buffy coat smear microscopy and urine antigen ELISA as non-invasive or minimally invasive alternatives for diagnosis and post-treatment test of cure for VL in HIV-coinfected patients in India, using a sample of 91 patients with parasitologically confirmed symptomatic VL-HIV infection. ETHICS AND DISSEMINATION: Ethical approval for this study has been granted by The Liverpool School of Tropical Medicine, The Institute of Tropical Medicine in Antwerp, the University of Antwerp and the Rajendra Memorial Research Institute of Medical Science in Patna. Any future publications will be published in open access journals. TRIAL REGISTRATION NUMBER: CTRI/2019/03/017908.


Assuntos
Coinfecção , Infecções por HIV , Leishmaniose Visceral , Buffy Coat , Ensaio de Imunoadsorção Enzimática , Infecções por HIV/complicações , Humanos , Índia , Leishmaniose Visceral/complicações , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/tratamento farmacológico , Estudos Prospectivos , Sensibilidade e Especificidade
9.
Mol Biol Rep ; 48(4): 3059-3068, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33929647

RESUMO

The expression of human and microbial genes serves as biomarkers for disease and health. Blood RNA is an important biological resource for precision medicine and translational medicine. However, few studies have assessed the human transcriptome profiles and microbial communities composition and diversity of peripheral blood from different cell isolation methods, which could affect the reproducibility of researches. We collected peripheral blood from three healthy donors and processed it immediately. We used RNA sequencing to investigate the effect of three leukocyte isolation methods including buffy coat (BC) extraction, red blood cell (RBC) lysis and peripheral blood mononuclear cell (PBMC) isolation with the comparison with whole blood (WB), through analyzing the sensitivity of gene detection, the whole transcriptome profiling and microbial composition and diversity. Our data showed that BC extraction with high globin mRNA mapping rate had similar transcriptome profiles with WB, while RBC lysis and PBMC isolation depleted RBCs effectively. With the efficient depletion of RBC and distinct compositions of leukocyte subsets, RNA-seq of RBC lysis and PBMC isolation uniquely detected genes from specific cell types, like granulocytes and NK cells. In addition, we observed that the microbial composition and diversity were more affected by individuals than isolation methods. Our results showed that blood cell isolations could largely influence the sensitivity of detection of human genes and transcriptome profile.


Assuntos
Células Sanguíneas , Separação Celular/métodos , RNA-Seq , Buffy Coat , Eritrócitos , Humanos , Leucócitos Mononucleares , Microbiota/genética , Análise de Sequência de RNA , Transcriptoma
10.
Transfus Apher Sci ; 60(3): 103110, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33736955

RESUMO

BACKGROUND: Platelets (PLTs) stored at 20-24 °C have a short shelf life of only 5 days, which can result in their restricted availability. PLT cryopreservation extends the shelf life to 2 years. METHODS: We implemented a method of PLT freezing at -80 °C in 5-6% dimethyl sulfoxide. Buffy-coat-derived leucodepleted fresh PLTs blood group O (FP) were used for cryopreservation. Cryopreserved pooled leucodepleted PLTs (CPP) were thawed at 37 °C, reconstituted in PLT additive solution SSP + and compared to FP regarding PLT content, PLT concentration, pH, volume, PLT loss, anti-A/B antibody titre, total protein, plasma content, and PLT swirling. Clot properties were evaluated via rotational thromboelastometry. PLT microparticle number and surface receptor phenotype were assessed via flow cytometry. RESULTS: CPP met the required quality parameters. The mean freeze-thaw PLT loss was 22.24 %. Anti-A/B antibody titre and plasma content were significantly lower in CPP. CPP were characterised by faster clot initiation and form stable PLT clots. The number of PLT microparticles increased 25 times in CPP and there were more particles positive for the activation marker CD62 P compared to FP. CONCLUSION: Thawing and reconstitution are easy and fast processes if platelet additive solution is used. Low anti-A/B antibody titre and plasma content make possible the use of CPP of blood group O reconstituted in SSP + as universal ABO products, including clinical situations where washed PLTs are required. Clot properties evaluated via rotational thromboelastometry demonstrated that CPP retain a significant part of their activity compare to FP and are haemostatically effective.


Assuntos
Buffy Coat/metabolismo , Plaquetas/metabolismo , Criopreservação/métodos , Hemostasia , Humanos
11.
J Vis Exp ; (168)2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33616094

RESUMO

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high mortality, despite new therapeutic options such as transcatheter aortic valve replacement (TAVR). The complete pathways that start with valve calcification and lead to severe aortic stenosis remain only partly understood. By providing a close representation of the aortic valve cells in vivo, the assaying of T lymphocytes from stenotic valve tissue could be an efficient way to clarify their role in the development of calcification. After surgical excision, the fresh aortic valve sample is dissected in small pieces and the T lymphocytes are cultured, cloned then analyzed using fluorescence activated cell sorting (FACS). The staining procedure is simple and the stained tubes can also be fixed using 0.5% of paraformaldehyde and analyzed up to 15 days later. The results generated from the staining panel can be used to track changes in T cell concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific T cell subtypes of interest. In this study, we show the isolation of T cells, performed on fresh calcified aortic valve samples and the steps of analyzing T cell clones using flow cytometry to further understand the role of adaptive immunity in CAVD pathophysiology.


Assuntos
Estenose da Valva Aórtica/patologia , Valva Aórtica/citologia , Valva Aórtica/patologia , Buffy Coat/efeitos da radiação , Calcinose/patologia , Separação Celular/métodos , Células Alimentadoras/citologia , Citometria de Fluxo/métodos , Linfócitos T/citologia , Valva Aórtica/metabolismo , Células Cultivadas , Células Alimentadoras/metabolismo , Humanos , Linfócitos T/metabolismo
12.
Transfusion ; 61(2): 627-633, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33174258

RESUMO

BACKGROUND: Buffy coat (BC) platelets (PLTs) have been used globally for many years. In 2004 Canadian Blood Services (CBS) made the decision to transition from PLT-rich plasma (PRP) to BC PLTs. We reviewed the benefits and manufacture process of BC and the implementation challenges involved. STUDY DESIGN AND METHODS: A literature review was performed in the following areas: BC efficacy, donor population shifts, production and good stewardship of PLTs, logistic considerations with overnight holds, advantages of the overnight hold, the CBS experience, licensure and standards, and changes needed to produce BC PLTs in the United States. The aim was to analyze current practice and identify possible actions for blood centers and hospitals. RESULTS: Implementation of BC would offer an additional source of PLTs to address the growing elderly population and the declining apheresis donor base. Substantial logistic, operational, and financial benefits were seen when CBS transitioned to BC with overnight hold. CONCLUSIONS: Buffy coat blood products are widely used throughout the world. Recent conversion from PRP to BC by CBS showed that conversion can be accomplished with planning, communication, and partnership from all stakeholders. In conclusion, BC PLTs are worth serious consideration in the United States, but regulatory barriers in the United States will need to be addressed.


Assuntos
Bancos de Sangue/organização & administração , Buffy Coat/citologia , Plaquetas , Transfusão de Plaquetas , Doadores de Sangue , Preservação de Sangue , Canadá , Humanos , Licenciamento , Transfusão de Plaquetas/legislação & jurisprudência , Transfusão de Plaquetas/normas , Fatores de Tempo , Estados Unidos
13.
Transfus Apher Sci ; 60(1): 103014, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33262053

RESUMO

INTRODUCTION: The overnight storage of the buffy coat (BC) at room temperature has logistic and operational advantages for the blood centre. The present study aimed to evaluate the impact of an overnight hold (stored) of BC at room temperature in comparison with the 2-hour hold (fresh) of buffy coats on the platelet concentrate (PC) characteristics. METHODS: A total of 60 BCs were included in the study, 30 PCs (fresh) were prepared after two hours holding time of the BCs and the other 30 PCs (stored) were prepared after the overnight BC storage at room temperature. The primary endpoint of PCs evaluation was the platelet yield, volume, pH, WBC count, RBC count, and platelet swirling in the PC and the secondary endpoints were glucose concentration, lactate, LDH, and sterility of the PCs. All the tests were performed on the day+1 of the blood collection. RESULTS: There was no difference concerning the volume, RBC count, and swirling between the two groups (P>0.05). The PCs from the fresh BC had higher pH and glucose concentration (P<0.05). On the other hand, the overnight hold of BC produced higher platelet counts, WBC counts, lactate, and LDH levels (P<0.05). All the 60 PCs did not record any bacterial growth on the culture media for the sterility results. CONCLUSION: The overnight hold of BC produces a higher platelet yield with higher storage lesions. This may also allow better supervision, ensuring better quality control.


Assuntos
Buffy Coat/metabolismo , Plaquetas/metabolismo , Preservação de Sangue/métodos , Humanos , Temperatura , Fatores de Tempo
14.
Transfusion ; 61(2): 568-578, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33202065

RESUMO

BACKGROUND: Manufacture of platelet concentrates (PCs) and plasma may fail to remove all residual red blood cells (rRBCs). Measuring rRBCs for compliance to guidelines has proven challenging, leading to an absence of a consensus methodology. Sysmex hematology analyzers with the Blood Bank mode (BB mode) analysis option offer the potential for automated rRBC counting. We therefore performed a two-site appraisal of the system. STUDY DESIGN AND METHODS: Performance characteristics were determined using platelet and plasma samples spiked with RBCs. Sample stability (n = 47) and the impact of sample type were also assessed. Components (platelets, n = 1474; plasma, n = 77) prepared using different routine manufacturing methods were tested to assess variation in rRBC concentration. RESULTS: Linearity studies up to 19 000 RBCs/µL demonstrated good correlation between expected and observed results (R2 ≥ 0.9731), and flow cytometric results also correlated well with BB mode (R2 = 0.9400). Precision analysis gave a limit of quantitation of 6 to 7 RBCs/µL, and carryover was 0.03%. Ethylenediaminetetraacetic acid and plain tube results were not significantly different (P ≥ 0.10), and samples were stable up to 24 hours. Apheresis PCs produced at two sites had lower rRBC concentrations (medians, 17 and 13 RBCs/µL) than those produced with the buffy coat method either manually (median, 681 RBCs/µL) or with the automated Terumo Automated Centrifuge and Separator Integration process (median, 81 RBCs/µL). All PCs failing visual inspection as having RBCs ≥4000 RBCs/µL were also detected by the BB mode. CONCLUSION: The BB mode had acceptable performance characteristics and has the potential for integration into a fully automated process control system for rRBC enumeration in plasma and PCs.


Assuntos
Contagem de Células Sanguíneas/instrumentação , Transfusão de Componentes Sanguíneos , Contagem de Eritrócitos/métodos , Eritrócitos , Anticoagulantes , Automação , Buffy Coat/citologia , Remoção de Componentes Sanguíneos/métodos , Ácido Edético , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Humanos
15.
Transfusion ; 61(2): 546-556, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33345368

RESUMO

BACKGROUND: Cryopreserved platelets show a reduced recovery and viability after freezing and thawing including several ultrastructural and phenotypic deteriorations compared with liquid-stored platelets. It is suggested that using Controlled-Rate Freezing (CRF) can reduce variability and optimize the functionality profile for cells. The objective of the study is to compare cellular, metabolic, phenotypic and functional effects on platelets after cryopreservation using different freezing rate protocols. STUDY DESIGN AND METHODS: To evaluate the possible effects of different freezing rate protocols a two-experimental study comparing diverse combinations was tested with a pool and split design. Uncontrolled freezing of platelets in materials with different thermal conductivity (metal vs cardboard) was evaluated in experiment 1. Experiment 2 evaluated uncontrolled vs a controlled-rate freezing protocol in metal boxes. All variables were assessed pre and post cryopreservation. RESULTS: Directly after thawing, no major differences in platelet recovery, LDH, ATP, Δψ, CD62P, CD42b, platelet endothelial cell adhesion molecule and sCD40L were seen between units frozen with different thermal conductivity for temperature. In contrast, we observed signs of increased activation after freezing using the CRF protocol, reflected by increased cell surface expression of CD62P, PAC-1 binding and increased concentration of LDH. Agonist induced expression of a conformational epitope on the GPIIb/IIIa complex and contribution to blood coagulation in an experimental rotational thromboelastometry setup were not statistically different between the groups. CONCLUSION: The use of a uncontrolled freezing rate protocol is feasible, creating a platelet product comparable to using a controlled rate freezing equipment during cryopreservation of platelets.


Assuntos
Buffy Coat/citologia , Plaquetas , Preservação de Sangue/métodos , Criopreservação/métodos , Difosfato de Adenosina/farmacologia , Coagulação Sanguínea , Plaquetas/química , Plaquetas/citologia , Plaquetas/fisiologia , Ligante de CD40/farmacologia , Separação Celular , Sobrevivência Celular , Centrifugação , Colágeno/farmacologia , Criopreservação/instrumentação , Dimetil Sulfóxido , Humanos , Fatores Imunológicos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Refrigeração/instrumentação , Condutividade Térmica , Tromboelastografia
16.
Rev. cienc. salud ; 19(3): 1-13, 2021. graf, tab
Artigo em Espanhol | LILACS, COLNAL | ID: biblio-1367531

RESUMO

Introducción: los concentrados plaquetarios (CPG) son hemocomponentes lábiles afectados por varios factores, desde el método de obtención hasta las condiciones de almacenamiento, que provocan una paulatina pérdida de funcionalidad, por lo que es necesario evaluar parámetros de calidad que garanticen la viabilidad de las plaquetas durante los días de almacenamiento, con el propósito de monitorear el mantenimiento de las características funcionales de las plaquetas. Materiales y métodos: estudio descriptivo transversal, con un tamaño muestral de 64 cpq, evaluados a los 3, 5 y 7 días de almacenamiento. Los parámetros monitoreados fueron físicos, de almacenamiento y porcentaje de la activación plaquetaria mediante la medición de P-selectina (cd62) por citometría de flujo. Se aplicó el estadístico de chi cuadrado, Anova de un factor, Kruskall-Wallis y correlación de Pearson. Resultados: existen diferencias significativas al séptimo día con relación al tercer y quinto día de almacenamiento, especialmente en el parámetro de formación de remolino plaquetario o swirling (p < 0.005) y agregados plaquetarios (p = 0.001). La activación plaquetaria aumentó significativamente (p = 0.001) desde el quinto día. Conclusiones: la viabilidad de los cpq difiere con los días de almacenamiento, por lo que es necesario evaluar pH, formación de remo-linos y agregados a todos los cpq antes de ser transfundidos como indicativos de activación plaquetaria y disminución de su funcionalidad


Introduction: Platelet concentrates (cpq) are labile blood components affected by several factors from the method of production to storage conditions that cause a gradual loss of functionality. For this reason, it is necessary to evaluate the platelet quality parameters that guarantee the viability during the storage days, with the purpose of monitoring the maintenance of the functional characteristics of the platelets. Materials and methods: This cross-sectional descriptive study had a sample size of 64 platelet concen-trates, evaluated at 3, 5, and 7 days of storage. The monitored parameters were the physical storage parameters and percentage of platelet activation by measuring P-selectin (cd62) via flow cytometry. The chi-square statistic, one-way Anova, Kruskal­Wallis test, and Pearson correlation were applied. Results: Significant differences were observed on the 7th day in relation to the 3rd and 5th day of storage, espe-cially in the swirling parameter (p < 0.005) and platelet aggregates (p = 0.001). The platelet activation increased significantly (p = 0.001) on the 5th day. Conclusions: Based on the findings of this study, the viability of the platelet concentrates differs with the days of storage. For this reason, it is necessary to evaluate the swirling, pH, and aggregates to all platelet concentrates before being transfused as an indication of platelet activation and decreased functionality


Introdução: os concentrados de plaquetas (cpq) são hemocomponentes lábeis afetados por diversos fato-res, desde o método de obtenção até as condições de armazenamento que ocasionam uma perda grada-tiva de funcionalidade, sendo necessário avaliar os parâmetros de qualidade que garantem a viabilidade das plaquetas ao longo dos dias de armazenamento, a fim de monitorar a manutenção das características funcionais das plaquetas. Materiais e métodos: estudo descritivo transversal, com tamanho de amostra de 64 cpq, avaliados aos três, cinco e sete dias de armazenamento. Os parâmetros monitorados foram físicos, de armazenamento e porcentagem de ativação plaquetária pela dosagem de P-selectin (cd62) por citometria de fluxo. Os testes estatísticos aplicados incluíram o teste qui-quadrado, anovade um fator, teste de Kruskall-Wallis e correlação de Pearson. Resultados: há diferenças significativas no sétimo dia em relação ao terceiro e quinto dia de armazenamento, principalmente no parâmetro formação de rede-moinhos ou swirling de plaquetas (p < 0.005) e agregados plaquetários (p = 0.001). A ativação plaquetária aumentou significativamente (p = 0.001) a partir do quinto dia. Conclusões: a viabilidade dos concentra-dos de plaquetas difere com os dias de armazenamento, por isso é necessário avaliar o pH, a formação de redemoinhos e agregados a todos os concentrados de plaquetas antes de serem transfundidos como indicativo de ativação plaquetária e diminuição de sua funcionalidade


Assuntos
Humanos , Buffy Coat , Plaquetas , Ativação Plaquetária , Análise de Variância , Parâmetros de Referência
17.
Indian J Pathol Microbiol ; 63(4): 642-644, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33154326

RESUMO

A leukemic phase of anaplastic lymphoma kinase positive anaplastic large cell lymphoma (ALK+ ALCL) is rare. The leukemic cells morphologically appear as small to intermediate-sized cells with cerebriform and cloverleaf-like nuclei and are misdiagnosed as other T-Cell lymphomas/leukemia with similar morphology. We describe a case where the diagnosis of leukemic ALK+ ALCL was aided by immunohistochemistry performed on the cell blocks prepared from the peripheral blood buffy coat specimen. The diagnosis of ALK+ ALCL was further confirmed on the biopsy of a cutaneous nodule of this patient. We found the method of immunohistochemistry on peripheral blood buffy coat cell block very useful and suggest that it may be used as an alternative method to flowcytometry in low resource settings.


Assuntos
Quinase do Linfoma Anaplásico/genética , Linfoma Anaplásico de Células Grandes/sangue , Linfoma Anaplásico de Células Grandes/diagnóstico , Adulto , Buffy Coat , Técnicas Histológicas , Humanos , Imuno-Histoquímica , Masculino , Manejo de Espécimes
18.
PLoS Genet ; 16(11): e1009090, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33147208

RESUMO

Interferon ß (IFN-ß) is a cytokine that induces a global antiviral proteome, and regulates the adaptive immune response to infections and tumors. Its effects strongly depend on its level and timing of expression. Therefore, the transcription of its coding gene IFNB1 is strictly controlled. We have previously shown that in mice, the TRIM33 protein restrains Ifnb1 transcription in activated myeloid cells through an upstream inhibitory sequence called ICE. Here, we show that the deregulation of Ifnb1 expression observed in murine Trim33-/- macrophages correlates with abnormal looping of both ICE and the Ifnb1 gene to a 100 kb downstream region overlapping the Ptplad2/Hacd4 gene. This region is a predicted myeloid super-enhancer in which we could characterize 3 myeloid-specific active enhancers, one of which (E5) increases the response of the Ifnb1 promoter to activation. In humans, the orthologous region contains several single nucleotide polymorphisms (SNPs) known to be associated with decreased expression of IFNB1 in activated monocytes, and loops to the IFNB1 gene. The strongest association is found for the rs12553564 SNP, located in the E5 orthologous region. The minor allele of rs12553564 disrupts a conserved C/EBP-ß binding motif, prevents binding of C/EBP-ß, and abolishes the activation-induced enhancer activity of E5. Altogether, these results establish a link between a genetic variant preventing binding of a transcription factor and a higher order phenotype, and suggest that the frequent minor allele (around 30% worldwide) might be associated with phenotypes regulated by IFN-ß expression in myeloid cells.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica/imunologia , Interferon beta/genética , Células Mieloides/metabolismo , Alelos , Animais , Buffy Coat/citologia , Células Cultivadas , Humanos , Interferon beta/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Knockout , Células Mieloides/imunologia , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células , Regiões Promotoras Genéticas , Locos de Características Quantitativas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
Sci Rep ; 10(1): 20312, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33219265

RESUMO

Diagnostic leukapheresis (DLA) enables to sample larger blood volumes and increases the detection of circulating tumor cells (CTC) significantly. Nevertheless, the high excess of white blood cells (WBC) of DLA products remains a major challenge for further downstream CTC enrichment and detection. To address this problem, we tested the performance of two label-free CTC technologies for processing DLA products. For the testing purposes, we established ficollized buffy coats (BC) with a WBC composition similar to patient-derived DLA products. The mimicking-DLA samples (with up to 400 × 106 WBCs) were spiked with three different tumor cell lines and processed with two versions of a spiral microfluidic chip for label-free CTC enrichment: the commercially available ClearCell FR1 biochip and a customized DLA biochip based on a similar enrichment principle, but designed for higher throughput of cells. While the samples processed with FR1 chip displayed with increasing cell load significantly higher WBC backgrounds and decreasing cell recovery, the recovery rates of the customized DLA chip were stable, even if challenged with up to 400 × 106 WBCs (corresponding to around 120 mL peripheral blood or 10% of a DLA product). These results indicate that the further up-scalable DLA biochip has potential to process complete DLA products from 2.5 L of peripheral blood in an affordable way to enable high-volume CTC-based liquid biopsies.


Assuntos
Dispositivos Lab-On-A-Chip , Leucaférese/instrumentação , Neoplasias/diagnóstico , Células Neoplásicas Circulantes , Buffy Coat/citologia , Linhagem Celular Tumoral , Humanos , Biópsia Líquida/instrumentação , Biópsia Líquida/métodos , Neoplasias/sangue
20.
Int J Mol Sci ; 21(20)2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-33076376

RESUMO

Solid platelet-rich fibrin (PRF) is produced with centrifugation tubes designed to accelerate clotting. Thus, activated platelets may accumulate within the fibrin-rich extracellular matrix even before centrifugation is initiated. It can thus be assumed that platelets and their growth factors such as transforming growth factor-ß (TGF-ß) are trapped within PRF independent of their relative centrifugal force (RCF), the gravitation or g-force. To test this assumption, we prepared PRF membranes with tubes where clotting is activated by a silicone-coated interior. Tubes underwent 210 g, 650 g and 1500 g for 12 min in a horizontal centrifuge. The respective PRF membranes, either in total or separated into a platelet-poor plasma and buffy coat fraction, were subjected to repeated freeze-thawing to prepare lysates. Gingival fibroblasts were exposed to the PRF lysates to provoke the expression of TGF-ß target genes. We show here that the expression of interleukin 11 (IL11) and NADPH oxidase 4 (NOX4), and Smad2/3 signaling were similarly activated by all lysates when normalized to the size of the PRF membranes. Notably, platelet-poor plasma had significantly less TGF-ß activity than the buffy coat fraction at both high-speed protocols. In contrast to our original assumption, the TGF-ß activity in PRF lysates produced using horizontal centrifugation follows a gradient with increasing concentration from the platelet-poor plasma towards the buffy coat layer.


Assuntos
Buffy Coat/metabolismo , Fibroblastos/efeitos dos fármacos , Membranas Artificiais , Fibrina Rica em Plaquetas/química , Fator de Crescimento Transformador beta/farmacologia , Coagulação Sanguínea , Células Cultivadas , Centrifugação/métodos , Fibroblastos/metabolismo , Gengiva/citologia , Gravitação , Humanos , Interleucina-11/genética , Interleucina-11/metabolismo , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Silicones/química , Proteínas Smad/genética , Proteínas Smad/metabolismo
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