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1.
Hematology ; 29(1): 2293492, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38193467

RESUMO

OBJECTIVE: To explore the feasibility of using a disposable platelet storage bag containing a leukocyte filter to prepare leukocyte-depleted pooled platelet concentrates with the buffy coat method. METHODS: 150 bags of whole blood samples (400 mL/bag) were stored overnight at 22 ± 2°C, and buffy coats were separated on Day 2, then 5 units of ABO homotypic buffy coat and 1 unit of plasma were pooled into a disposable platelet storage bag containing a leukocyte filter to prepare leukocyte-depleted pooled platelet concentrates and stored in a Platelet Agitator. On Day 2, 4, 5 and 7 after the collection of whole blood, platelet content, pH value, pO2, pCO2, glucose (GLU), ATP, and other quality indicators were measured. RESULTS: The quality indicators of leukocyte-depleted pooled platelet concentrates met the requirements for leukocyte-depleted aphaeresis platelets in the Chinese national standard Quality Requirements for Whole Blood and Blood Components (GB18469-2012). With the prolongation of storage time, MPV and PDW of platelets gradually increased, pH value, bicarbonate, and GLU gradually decreased, LA, LDH, and ATP gradually increased, pO2 slightly increased, pCO2 decreased, and HSR had no significant change. ESC decreased significantly on Day 7, CD62p decreased first and then increased, sP-selectin and GP V increased first and then decreased, but the results on Day 7 were higher than those on Day 2. CONCLUSION: The quality of leukocyte-depleted pooled platelet concentrates prepared by the buffy coat method using disposable platelet storage bags containing a leukocyte filter was comparable to that of leukocyte-depleted apheresis platelets, and could be used clinically.


Assuntos
Plaquetas , Leucócitos , Humanos , Trifosfato de Adenosina , Glucose , Buffy Coat
2.
Blood Transfus ; 21(2): 157-167, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-35302481

RESUMO

BACKGROUND: Platelet transfusions can be associated with adverse reactions, such as febrile non-haemolytic transfusion reaction (FNHTR). It has been suggested that damage-associated molecular patterns (DAMP) and complement play a role in FNHTR. This study investigated the nature of DAMPs and complement activation products contained in platelet concentrates during storage, with a specific focus on different platelet storage solutions. MATERIALS AND METHODS: Buffy coats (BC) from healthy donors were pooled (15 BC per pool) and divided into three groups of the same volume. After addition of different storage solutions (plasma, platelet additive solutions [PAS]-C or PAS-E; n=6 for each group), BC pools were processed to platelet concentrates (PC). Leukoreduced PCs were stored on a shaking bed at 20-24°C and sampled on days 1, 2, 6 and 8 after collection for selected quality parameters: platelet activation, DAMPs (High Mobility Group Box 1 [HMGB1], nucleosomes), and complement activation products. RESULTS: During storage, equal levels of free nucleosomes and increasing concentrations of HMGB1 were present in all groups. Complement activation was observed in all PC. However, by day 8, the use of PAS had reduced C3b/c levels by approximately 90% and C4b/c levels by approximately 65%. DISCUSSION: Nucleosomes and HMGB1 were present in PCs prepared in plasma and PAS. Complement was activated during storage of platelets in plasma and in PAS. The use of PAS is associated with a lower amount of complement activation products due to the dilution of plasma by PAS . Therefore, PC in PAS have less complement activation products than platelets stored in plasma. These proinflammatory mediators in PC might induce FNHTR.


Assuntos
Preservação de Sangue , Ativação do Complemento , Plasma , Transfusão de Plaquetas , Soluções , Reação Transfusional , Humanos , Fatores de Coagulação Sanguínea/análise , Plaquetas , Preservação de Sangue/efeitos adversos , Preservação de Sangue/métodos , Ativação do Complemento/imunologia , Proteína HMGB1/análise , Nucleossomos/imunologia , Ativação Plaquetária/imunologia , Transfusão de Plaquetas/efeitos adversos , Transfusão de Plaquetas/métodos , Soluções/efeitos adversos , Soluções/farmacologia , Soluções/uso terapêutico , Reação Transfusional/etiologia , Reação Transfusional/prevenção & controle , Plasma/química , Plasma/imunologia , Buffy Coat/química , Buffy Coat/citologia
3.
Prep Biochem Biotechnol ; 53(6): 591-598, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36121058

RESUMO

During the last few years, an important element in the improvement of the molecular biology techniques has been the necessity for availability of high quality and functionality DNA. Several DNA extraction procedures with different results in both performance and quality, have been proposed. In this study our objective was to determine the most reliable extraction method that balances DNA quantity, and to assess the sample quantification of the fluorometric DNA quantification methods. For this, blood extracted by venopunction from 20 healthy volunteers was used to obtain DNA from buffy coat, and 4 commercial DNA extraction kits were assessed as well as two fluorometric DNA quantification methods with protocols of different complexity. Results suggest that manual methods achieve higher quality and larger yields of DNA. DNA purity obtained with the 4 extraction kits evaluated through the 260/280 and 260/230 ratio showed that the Qiacube kit fulfilled the criteria established in this work, followed very close by the Flexigene kit. On the other hand, the fluorometric DNA methods used in the samples quantification showed a higher variability when using QuantiFlour method, obtaining better results probably due to the simplicity of this protocol.


Assuntos
Buffy Coat , DNA , Humanos , DNA/isolamento & purificação
4.
Vox Sang ; 117(7): 913-919, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35405038

RESUMO

The buffy coat method as a source for platelet concentrates was developed in the 1970s and is still used in many blood centres around the world. Development of the method sparked various technological advances in blood collection, processing and storage. At the time, the need for platelet concentrates sharply increased because of better treatment regimens for (onco)haematological diseases, which forced blood centres to standardize and automate their production processes as much as the technology would allow. In this review, a historical overview of the Dutch experiences is provided in the context of the international developments.


Assuntos
Plaquetas , Preservação de Sangue , Buffy Coat , Humanos
5.
Rev. bras. ortop ; 57(2): 289-294, Mar.-Apr. 2022. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1387991

RESUMO

Abstract Objective To present an innovative device that applies the double centrifugation method to obtain platelet-rich plasma (PRP), assessing whether there was an effective increase in the concentration of platelets. Method Ten volunteers underwent blood collection. The samples were separated in 20 ml syringes, sealed and subjected to the double centrifugation protocol at 1,100 revolutions per minute (rpm) for 15 minutes, resulting in the separation of red blood cells, plasma with platelets, and leukocytes. Then, 10 ml syringes were added to remove 9 ml, respecting the "buffy coat" parameter, collecting 8 ml above and 1 ml below for the second centrifugation and transferring again to the 20 ml syringe. The plasma was again centrifuged at 1,550 rpm for 10 minutes; as a result, it was divided into two parts: at the top, consisting of low platelet plasma (LPP), and at the bottom, by the platelet button. Part of the LPP was discarded, leaving only 3 ml with the platelet button. The cells were then counted. Results This innovative device was able to increase the concentration of platelets by almost three times compared with the baseline. In addition, the preparation time for the PRP was adequate, lasting only 35 to 40 minutes. Conclusions Platelet-rich plasma was successfully obtained by the double centrifuge protocol, allowing its clinical use. In addition, obtaining through the presented device promotes greater applicability in the preparation of PRP in specific centers, furthermore, being a quick and economical way to obtain PRP.


Resumo Objetivo Apresentar um dispositivo inovador que aplique o método de centrifugação dupla para obter plasma rico em plaquetas (PRP), avaliando se houve um aumento efetivo na concentração de plaquetas. Método Dez voluntários foram submetidos a coleta de sangue. As amostras foram separadas em seringas de 20 mL, seladas e submetidas ao protocolo de centrifugação dupla a 1.100 revoluções por minuto (rpm) por 15 minutos, resultando na separação de hemácias, plasma com plaquetas e leucócitos. Em seguida, foram adicionadas seringas de 10 mL para remover 9 mL, tendo como parâmetro a "buffy coat", coletando 8 mL acima e 1 mL abaixo para a segunda centrifugação e transferindo novamente para a seringa de 20 mL. O plasma foi novamente centrifugado a 1.550 rpm por 10 minutos; como resultado, foi dividido em duas partes: na parte superior, consistindo em plasma pobre em plaquetas (PPP), e na parte inferior, pelo botão plaquetário. Parte do PPP foi descartada, restando apenas 3 mL com o botão de plaquetas. As células foram então contadas. Resultados Este dispositivo inovador foi capaz de aumentar a concentração de plaquetas em quase 3 vezes relação a linha de base. Além disso, o tempo de preparo do PRP foi adequado, com duração de apenas 35 a 40 minutos. Conclusões O PRP foi obtido com sucesso pelo protocolo de centrifugação dupla, permitindo seu uso clínico. Além disso, a obtenção através do dispositivo apresentado promove maior aplicabilidade no preparo do PRP em centros específicos, além de ser, uma forma rápida e econômica de obter PRP.


Assuntos
Humanos , Perfil de Saúde , Plaquetas , Plasma Rico em Plaquetas , Buffy Coat , Equipamentos e Provisões
6.
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
7.
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 de Cinetoplastídeos/genética , Fatores de Tempo
8.
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
9.
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
10.
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 de 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
11.
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
12.
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
13.
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
14.
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
15.
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
16.
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
17.
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
18.
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
19.
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
20.
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
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