Human venous blood derivatives as fetal bovine serum substitute for fibroblast culture cells in a fibrin construct

Aim: Venous blood derivatives (VBDs) have been suggested as substitutes for Fetal Bovine Serum (FBS) to improve the clinical transition of cell-based therapies. The literature is not clear about which is the best VBDs substitute. The present study aimed to evaluate the influence of VBDs on cell viability and describe a new method to seed these cells in a 3D Platelet-Rich Fibrin (PRF). Methods: Blood was processed to obtain Platelet-Poor Plasma from PRF (P-PRF), Human Serum (HS), Platelet-Poor Plasma from PRP (P-PRP), activated-PRP (a-PRP), and Platelet lysate (PL). Cells were supplemented with each VBD at 10% and FBS at 10% was the control. Cell viability (fibroblast 3T3/NIH) test was evaluated with MTT assay in two ways: i) cell-seeded and expanded with VBD; ii) cell-seed with FBS and expanded with VBD. To seed the Fibrin construct, cells were suspended in PBS and dropped into the blood sample before performing Choukroun's protocol for PRF. Constructs were cultured for 7 days in VBD supplements and FBS. Histological and Immunohistochemical analysis with vimentin was performed. Cell viability was analyzed by one-way ANOVA. Results: VBD's production time was very heterogeneous. Cells expanded in HS and a-PRP has grown faster. VBD-supplemented culture media provided cell culture highly sensible to trypsin/EDTA 0.25%. Cells seeded and expanded with VBD presented viability comparable to FBS in HS, a-PRP, and P-PRP (p>0.05) and lower in P-PRF and PL groups (p<0.05). The viability of cell seed with FBS and expanded with VBD was similar between P-PRF, a-PRP, PL, and FBS (p>0.05) and lower in HS and P-PRP (p<0.005). PRF-seeded cells showed a positive expression of vimentin and were able to maintain all cells supplemented with VBD. Conclusion: VBD supplements were able to maintain fibroblast cells in 2D and 3D cultures. The new method of the fibrin-cell construct was efficient to insert the cells into the fibrin network

Effect of double dose plateletpheresis on target yield and donor platelet recovery

Abstract Introduction The demand for apheresis platelets has increased in the recent past and the shrinking donor pool has shifted the trend to collection of double-dose or higher yield of platelets. Objective The present study aimed to determine the effect of double-dose plateletpheresis on the target yield and donor platelet recovery. Methods The study was conducted on 100 healthy plateletpheresis donors, 50 of whom were in the study group, which underwent double-dose plateletpheresis (DDP), and 50 of whom were in the control group for single-donor plateletpheresis. Pre- and post-procedure samples of donors were subjected to a complete blood count. The DDP product was sampled for platelet yield and then split into two parts. Platelet yield, collection efficiency, collection rate, recruitment factor and donor platelet loss were calculated. Results The mean platelet yield in the SDP was 4.09 ± 1.15 × 1011 and in the DDP, 5.93 ± 1.04 × 1011. There was a significant correlation between the pre-donation platelet count and platelet yield. The total of platelets processed for the SDP were 5.42 ± 1.08 × 1011 and for the DDP, 7.94 ± 0.77 × 1011. The collection efficiency was 71.93 ± 25.14% in the SDP and 72.94 ± 16.28% in the DDP, while the collection rates were 0.78 × 1011 and 0.94 × 1011 per minute, respectively. The average recruitment factor observed was 0.98 in the SDP, while it was 0.99 in the DDP. The mean platelet loss observed in the SDP was 35.55 ± 8.53% and in the DDP, 37.76 ± 8.65%. Conclusion The double-dose plateletpheresis supplements the platelet inventory in developing countries where the apheresis donor pool is limited. It is prudent to ensure stringent donor selection criteria for donors donating high-yield platelet products, thus enhancing donor safety and retention.

Polimorfismos genéticos implicados en el desarrollo de la purpura trombocitopénica inmune / Genetic polymorphisms involved in the development of Immune Thrombocytopenic Purpura

Introducción: los cambios en el ácido desoxirribonucleico se conocen como mutaciones, estas dan lugar a los polimorfismos, los cuales generan variación alélica entre individuos y diversidad de la misma especie. Se ha sugerido que los polimorfismos genéticos en los mediadores inmunitarios desempeñan un papel fundamental en la patogénesis de muchos trastornos autoinmunes, como en la púrpura trombocitopénica inmune, siendo esta el tipo más común de púrpura trombocitopénica y, a menudo, se diagnostica como un tipo de trastorno autoinmune, debido a la destrucción de las plaquetas mediadas por el sistema inmunitario. Objetivo: realizar una revisión bibliográfica sobre el papel de los polimorfismos genéticos y su influencia en el desarrollo de la púrpura trombocitopénica inmune. Métodos: se realizó revisión literaria en inglés y español en PubMed y Elsevier, desde marzo hasta mayo del 2021, con el uso de combinación de palabras clave y términos MeSH, como púrpura trombocitopénica y polimorfismos genéticos. Se realizó análisis y resumen de la literatura encontrada. Conclusión: la púrpura trombocitopénica inmune es considerada como una patología multifactorial, causada por factores ambientales y genéticos, dentro de los cuales se encuentran los polimorfismos para los mediadores inmunitarios que pueden llevar a una exacerbación de la enfermedad o no intervenir en la misma.

Introduction: Changes in deoxyribonucleic acid are known as mutations, these give place to polymorphisms, which generate allelic variation between individuals and provide diversity among same species. Genetic polymorphisms in immune mediators have been suggested to play a key role in the pathogenesis of many autoimmune disorders, such as immune thrombocytopenic purpura, this being the most common type of thrombocytopenic purpura and is often diagnosed as a type of autoimmune disorder, due to the destruction of platelets mediated by the immune system. Objective: To execute a bibliographic review on the role of genetic polymorphisms and their influence on the development of immune thrombocytopenic purpura. Methods: A literary review in English and Spanish was performed in PubMed and Elsevier from March to May 2021, with the use of a combination of keywords and MeSH terms such as Thrombocytopenic Purpura and genetic polymorphisms. Analysis and summary of the literature found was executed. Conclusion: Immune thrombocytopenic purpura is considered a multifactorial pathology, caused by environmental and genetic factors, among which are polymorphisms for immune mediators that can lead to an exacerbation of the disease or not intervene in the same.

Platelet-rich fibrin improves the osteoneogenesis in non-critical defects in calvaria: a histological and histometric study

Purpose: The aim of this study was to evaluate the effect of platelet-rich fibrin (PRF) and autograft on non-critical bone repair. Methods: Four bone defects (8.3 × 2 mm) were produced on the calvarium of 15 rabbits. The surgical defects were treated with either autograft, autograft associated to PRF, PRF alone, and sham. Animals were euthanized on the second, fourth or sixth posteoperative week. Histological analyses for presence of bone development on deffect was evaluated comparing the groups treated with autograft and without the autograft separately within the same period. Mann-Whitney's tests were used to compare the percentage of bone repair in each post-operative period for autograft × autograft + PRF groups and also for control × PRF groups (α = 5%). Results: No differences were observed between the groups that received autograft and autograft associated to PRF on the second and fourth postoperative week, but areas treated with PRF demonstrated significant osteogenesis when compared to sham group on the fourth and sixth weeks. The groups that received PRF (with autograft or alone) demonstrated an enlarged bone deposition when compared to their control group. Conclusions: The use of PRF may influence bone repair and improve the bone deposition in late period of repair demonstrating osteoconductive and osteogenic properties.

Prediction of Rupture by Complete Blood Count in Tubal Ectopic Pregnancies Treated with a Single-Dose Methotrexate Protocol / Predição de ruptura por hemograma completo em gestações ectópicas tubárias tratadas com protocolo de dose única de metotrexato

Abstract Objective The availability of reliable and inexpensive markers that can be used to determine the risk of rupture during methotrexate (MTX) treatment in ectopic pregnancies (EPs) is considerable. The aim of the present study is to investigate the role of systemic inflammatory markers such as leukocytes (or white blood cells, WBCs), the neutrophil-to-lymphocyte ratio (NLR), and platelet distribution width (PDW), which are among the parameters of the complete blood count (CBC), in the prediction of rupture of EPs under MTX treatment. Materials and Methods A total of 161 patients with tubal EP who underwent a single-dose methotrexate (MTX) protocol were retrospectively analyzed, and the control group (n = 83) included patients cured by MTX, while the ruptured group (n = 78) included patients who were operated on for tubal rupture during the MTX treatment. The features of EP, beta-human chorionic gonadotropin (β-hCG) levels, sonographic findings, and CBC-derived markers such as WBC, NLR, and PDW, were investigated by comparing both groups. Results The NLR was found to be higher in the ruptured group, of 2.92 ± 0.86%, and significantly lower in the control group, of 2.09 ± 0.6%. Similarly, the PDW was higher (51 ± 9%) in the ruptured group, and it was significantly lower a (47 ± 13%) in the control group (p < 0.05). Other CBC parameters were similar in both groups (p > 0.05). Conclusion Systemic inflammation markers derived from CBC can be easily applied to predict the risk of tubal rupture in Eps, since the CBC is an inexpensive and easy-to-apply test, which is first requested from each patient during hospitalization.

Resumo Objetivo A disponibilidade de marcadores confiáveis e baratos que podem ser usados para determinar o risco de ruptura durante o tratamento com metotrexato (MTX) em gestações ectópicas (GEs) é considerável. O objetivo do presente estudo é investigar o papel de marcadores inflamatórios sistêmicos, como leucócitos (ou glóbulos brancos, glóbulos brancos), a relação neutrófilo-linfócito (NLR) e largura de distribuição de plaquetas (PDW), que estão entre os parâmetros do hemograma completo (hemograma), na predição de ruptura de PEs sob tratamento com MTX. Materiais e Métodos Foram analisados retrospectivamente 161 pacientes com EP tubária submetidas a protocolo de dose única de metotrexato (MTX), sendo que o grupo controle (n = 83) incluiu pacientes curadas com MTX, enquanto o grupo roto (n = 78) incluíram pacientes operadas por ruptura tubária durante o tratamento com MTX. As características de EP, beta-gonadotrofina coriônica humana (β-hCG), achados ultrassonográficos e marcadores derivados de CBC, como WBC, NLR e PDW, foram investigados comparando os dois grupos. Resultados A RNL foi maior no grupo roto, de 2,92 ± 0,86%, e significativamente menor no grupo controle, de 2,09 ± 0,6%. Da mesma forma, o PDW foi maior (51 ± 9%) no grupo roto, e foi significativamente menor a (47 ± 13%) no grupo controle (p < 0,05). Outros parâmetros do hemograma foram semelhantes em ambos os grupos (p > 0,05). Conclusão Marcadores inflamatórios sistêmicos derivados do hemograma podem ser facilmente aplicados para predizer o risco de ruptura tubária na Eps, uma vez que o hemograma é um exame de baixo custo e fácil aplicação, solicitado primeiramente a cada paciente durante a internação.

Evaluación de la eficacia en la colecta de plaquetas por un separador celular / Evaluation of the efficacy in the collection of platelets by a cell separator / Avaliação da eficiência da coleta de plaquetas por um separador de células separador de células

La aféresis es el procedimiento más utilizado para la obtención de concentrados plaquetarios de alto rendimiento, calidad y para mejorar las terapias transfusionales en pacientes trombocitopénicos, oncohematológicos,cirugias e incluso, en pacientes con factores clínicos adversos a la refractariedad. Objetivo. Determinar la eficacia de un separador celular en la colecta de plaquetas en un Instituto Nacional de Salud de Lima. Material y métodos. Estudio descriptivo; la muestra fue de 80 concentrados plaquetarios, obtenidos por plaquetoaferesis y utilizando el equipo de separador celular americano. La colecta de plaquetas se realizó en un servicio de Hemoterapia y Banco de Sangre de una institución de salud de Lima, durante los meses de febrero a julio de 2018. La eficacia se realizó evaluando el rendimiento, la eficiencia y el cumplimiento de estándares de calidad aprobados. Uno de los parámetros utilizados fue el recuento de plaquetas y leucocitos residuales, procesados en el analizador hematológico. Resultados. Las evaluaciones fueron: concentración promedio de plaquetas por concentrado plaquetario (rendimiento)= 3,4 x 1011 plaquetas /ml, recuento de leucocitos residuales = 0,07 x 10 6 leucocitos/ml, volumen promedio de sangre procesado = 2480 ml, volumen final promedio = 217,5 ml, eficiencia en la colecta = 56,9 a 63,9 %, el tiempo medio por procedimiento de colecta = 72 minutos. Conclusiones. Los concentrados plaquetarios obtenidos con el procedimiento de plaquetoaferesis cumplen con los estándares de calidad nacional e internacionales, por lo que, se concluye que este procedimiento es eficaz en la colecta de productos de alta calidad que logran la eficacia en la transfusión.

Apheresis is the most widely used procedure to obtain high yield and quality platelet concentrates and to improve transfusion therapies in thrombocytopenic patients, oncohematological patients, surgical patients and even patients with adverse clinical factors to refractoriness. Objective. To determine the efficacy of a cell separator in the collection of platelets in a National Health Institute in Lima. Material and methods. Descriptive study; the sample consisted of 80 platelet concentrates, obtained by plateletpheresis and using American cell separator equipment. The platelet collection was performed in a Hemotherapy and Blood Bank service of a health institution in Lima, during the months of February to July 2018. Effectiveness was performed by evaluating performance, efficiency and compliance with approved quality standards. One of the parameters used was the residual platelet and leukocyte count, processed in the hematological analyzer. Results. The evaluations were: average platelet concentration per platelet concentrate (yield)= 3.4 x 1011 platelets/ml, residual leukocyte count = 0.07 x 10 6 leukocytes/ml, average volume of blood processed = 2480 ml, average final volume = 217.5 ml, collection efficiency = 56.9 to 63.9 %, average time per collection procedure = 72 minutes. Conclusions. The platelet concentrates obtained with the plateletpheresis procedure comply with national and international quality standards, therefore, it is concluded that this procedure is effective in the collection of high quality products that achieve transfusion efficiency.

A aférese é o procedimento mais utilizado para obter concentrados plaquetários de alto rendimento e alta qualidade e para melhorar as terapias transfusionais em pacientes trombocitopênicos, oncohematológicos, cirúrgicos e até mesmo pacientes com fatores clínicos adversos à refratariedade. Objetivo. Para determinar a eficácia de um separador de células na coleta de plaquetas em um Instituto Nacional de Saúde em Lima. Material e métodos. Estudo descritivo; a amostra consistiu de 80 concentrados de plaquetas, obtidos por plaquetaferese e utilizando equipamento separador de células americano. A coleta de plaquetas foi realizada em um serviço de Hemoterapia e Banco de Sangue de uma instituição de saúde em Lima, durante os meses de fevereiro a julho de 2018. A eficácia foi avaliada através da avaliação do desempenho, eficiência e conformidade com os padrões de qualidade aprovados. Um dos parâmetros utilizados foi a contagem residual de plaquetas e leucócitos, processada no analisador hematológico. Resultados. As avaliações foram: concentração média de plaquetas por concentrado de plaquetas (rendimento) = 3,4 x 1011 plaquetas/ml, contagem de leucócitos residuais = 0,07 x 10 6 leucócitos/ml, volume médio de sangue processado = 2480 ml, volume final médio = 217,5 ml, eficiência da coleta = 56,9 a 63,9%, tempo médio por procedimento de coleta = 72 minutos. Conclusões. Os concentrados de plaquetas obtidos com o procedimento de plaquetférese atendem aos padrões de qualidade nacionais e internacionais, portanto, conclui-se que este procedimento é eficaz na coleta de produtos de alta qualidade que alcançam eficiência transfusional.

Non-alcoholic fatty liver disease and thrombocytopenia IV: its association with granulocytopenia

ABSTRACT Introduction: We have previously shown that some patients present thrombocytopenia (less than 100 × 109/L platelets) in non-alcoholic fatty liver disease (NAFLD). To further explore the nature of this association, we have now analyzed the association of thrombocytopenia with neutropenia (less than 0.5 × 109/L granulocytes) in NAFLD. Material and methods: Persons with NAFLD were prospectively accrued in the study after February 2018. The presence of NAFLD was defined by both serologic determinations (Fibromax ®) and liver transient elastography (TE/Fibroscan ®). Results: In 123 consecutive patients with NAFLD without cirrhosis, thrombocytopenia was identified in 20 (16%), whereas neutropenia was identified in 9 (7%). In the subset of 20 patients with NAFLD and thrombocytopenia, granulocytopenia was identified in 5 (25%), whereas in the subset of 9 patients with granulocytopenia, thrombocytopenia was identified in 5 (55%). We found a significant association between thrombocytopenia and both leukopenia and granulocytopenia (OR 8.25, 95% CI 1.9-34.2, p = 0.004). Conclusions: Both thrombocytopenia and neutropenia were identified in persons with NAFLD and, as there is a significant relationship between these two variables, we speculate that this finding may support the possibility of hypersplenism being involved in the cytopenias found in NAFLD without cirrhosis.

Influence of blood/anticoagulant ratio and storage temperature on platelet parameters using Cell Dyn Ruby® analyzer / Influência da relação sangue/anticoagulante e da temperatura de armazenamento em parâmetros plaquetários utilizando o analisador Cell Dyn Ruby®

Introduction: The pre-analytical phase directly influences the laboratory result, such as the method of collection, transport, and storage of biological samples. Therefore, the stability of the biological sample is a crucial and determinant aspect of the quality of results in a clinical laboratory. Studies show that some platelet parameters may suffer alterations in the presence of altered blood/anticoagulant ratio, with increased storage time and/or variations in storage temperature, possibly leading to unrepresentative results. This study aimed to investigate the reliability of platelet parameters regarding the effect of the anticoagulant/blood ratio and storage temperature in samples stored for up to 24 hours after collection using the Cell Dyn Ruby® equipment.Methodology: A total of 351 blood samples were evaluated under different analysis periods: 2, 5, 12, and 24 hours and storage methods: at room temperature (25 °C) and 4 °C, in addition to the analysis of anticoagulant/blood ratio. The Platelet parameters selected were: PLT (total platelet count), MPV (mean platelet volume), PDW (Platelet Distribution Width) and PCT (plateletcrit). The imprecision of the results was evaluated by the CVa (%) within the maximum allowed analytical variation, as well as by the mean difference of the results concerning the baseline sample (2 hours). Results: The total platelet count was the only parameter evaluated that showed reproducibility of results in all conditions analyzed. Regarding the other platelet parameters, it could be observed an imprecision of results emitted by Cell Dyn Ruby® after five hours of storage, both at room and refrigerated temperature. Conclusion: This study demonstrates that pre-analytical factors, such as storage temperature and storage time, can affect the variability of platelet parameters, which may produce erroneous results. Thus, the correct blood/anticoagulant ratio must be respected to avoid the late processing of the sample (AU)

Introdução: O resultado laboratorial é diretamente influenciado por etapas da fase pré-analítica, como método de coleta, transporte e armazenamento das amostras biológicas. Desta forma, a estabilidade da amostra biológica é um aspecto crucial e determinante para a qualidade dos resultados de um laboratório clínico. Estudos demonstram que alguns parâmetros plaquetários podem sofrer modificações na presença da relação sangue/anticoagulante alterada, com aumento do tempo de estocagem e/ou alterações na temperatura de armazenamento, podendo levar a resultados não representativos. Portanto, o objetivo desse estudo foi investigar a confiabilidade dos parâmetros plaquetários com relação ao efeito da relação anticoagulante/sangue e da temperatura de armazenamento, em amostras estocadas por até 24 horas após a coleta, utilizando o equipamento Cell Dyn Ruby®. Métodos: Foram avaliados 351 hemogramas, em diferentes tempos de análise: 2, 5, 12 e 24 horas e formas de estocagem: à temperatura ambiente (25°C) e à 4°C, além da relação anticoagulante/sangue. Foram selecionados os seguintes parâmetros plaquetários: PLT (contagem total de plaquetas), VPM (volume plaquetário médio), PDW (amplitude de variação do tamanho das plaquetas) e PCT (plaquetócrito). A confiabilidade dos resultados foi avaliada pelo CVa (%), dentro da variação analítica máxima permitida, assim como pela diferença de média dos resultados em relação à amostra de base (2 horas). Resultados: A contagem total de plaquetas foi o único parâmetro avaliado que apresentou reprodutibilidade de resultados em todas as condições analisadas. Em relação aos demais parâmetros plaquetários, foi observado imprecisão dos resultados emitidos pelo Cell Dyn Ruby®, a partir de 5 horas de estocagem, tanto em temperatura ambiente quanto refrigerada. Conclusão: Este estudo demonstra que fatores pré-analíticos, como a temperatura de armazenamento e o tempo de estocagem, podem afetar a variabilidade dos parâmetros plaquetários, podendo produzir resultados equivocados. Deste modo, deve-se respeitar a correta relação sangue/anticoagulante e evitar o processamento tardio da amostra (AU)

Platelet antibodies identification: comparison between two laboratory tests

ABSTRACT Introduction: Platelet antibody identification is indispensable for diagnosing the human platelet antigen (HPA) or human leukocyte antigen (HLA) immunization, mostly because it can restrict the compatibility and results of transfusions. Correct detection of these antibodies is of utmost importance for the diagnosis and treatment. Method: We present 16 platelet alloimmunization results, comparing two tests with different technologies: the MAIPA (monoclonal antibody immobilization of platelet antigens), as a reference technique, and a bead-based assay, the Pak-Lx. Results: Eleven samples (68.75%) showed agreement in both techniques. Two tests were false negatives in the Pak-Lx: a pan-reactivity in GPIIbIIIa and an anti-HPA-9b. On the other hand, the Pak-Lx was more sensitive to detect a decreasing anti-HPA-5b. The Pak-Lx found an anti-HPA-2b positive, but with a low median fluorescent intensity (MFI), suggesting a false-positive result. Moreover, in one case, the MAIPA was negative for a positive Pak-Lx HLA. Conclusion: Antibody platelet diagnosis can sometimes be challenging. The methods seemed similar, the Pak-Lx being faster and simpler than the MAIPA, and they can be complementary to solve clinical issues.

Immature Platelet Fraction and Thrombin Generation: Preeclampsia Biomarkers / Fração de plaquetas imaturas e geração de trombina: Biomarcadores da pré-eclâmpsia

Abstract Preeclampsia, a human pregnancy syndrome, is characterized by elevated blood pressure and proteinuria after the 20th week of gestation. Its etiology remains unknown, and its pathophysiological mechanisms are related to placental hypoperfusion, endothelial dysfunction, inflammation, and coagulation cascade activation. Recently, the role of the complement system has been considered. This syndrome is one of the main causes of maternal and fetal mortality and morbidity. This article discusses the hypothesis of preeclampsia being triggered by the occurrence of inadequate implantation of the syncytiotrophoblast, associated with bleeding during the first stage of pregnancy and with augmented thrombin generation. Thrombin activates platelets, increasing the release of antiangiogenic factors and activating the complement system, inducing the membrane attack complex (C5b9). Immature platelet fraction and thrombin generation may be possible blood biomarkers to help the early diagnosis of preeclampsia.

Resumo A pré-eclâmpsia, uma síndrome da gestação humana, é caracterizada por elevação da pressão arterial e proteinúria patológica após a 20ª semana de gestação. Sua etiologia permanece desconhecida, e seus mecanismos fisiopatológicos estão relacionados à hipoperfusão placentária, disfunção endotelial, inflamação, e ativação da cascata de coagulação. Recentemente, o papel do sistema do complemento foi considerado. Essa síndrome é uma das principais causas de morbidade e mortalidade materna e fetal. Este artigo discute a hipótese de a pré-eclâmpsia ser desencadeada pela ocorrência da implantação inadequada do sinciciotrofoblasto, associada ao sangramento durante o primeiro trimestre da gravidez com aumento da geração de trombina. A trombina ativa plaquetas, aumentando a liberação de fatores antiangiogênicos na circulação e ativando o sistema do complemento, especialmente o complexo de ataque de membrana (C5b9). Portanto, a fração de plaquetas imaturas e a geração de trombina podem ser possíveis biomarcadores sanguíneos para auxílio no diagnóstico precoce da pré-eclâmpsia.

Development of a Device to Obtain Platelet-Rich Plasma (PRP) / Desenvolvimento de um dispositivo para obter plasma rico em plaquetas (PRP)

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.

Comparación clínica y laboratorial de la fiebre amarilla severa versus dengue grave en Perú / Clinical and laboratory differences between severe yellow fever and severe dengue in emergency patients

En los centros de Emergencia con poco apoyo de laboratorio, es difícil diferenciar a los pacientes con dengue grave y fiebre amarilla severa. El objetivo fue comparar el perfil clínico y de laboratorio de los pacientes con dengue grave y fiebre amarilla severa en Urgencias. Se realizó un estudio observacional retrospectivo de pacientes con diagnóstico confirmado de dengue y fiebre amarilla en el período 2018 a 2020 atendidos en la Unidad de Emergencia del Hospital Carrión, Huancayo-Perú. Se evaluaron un total de 35 pacientes, 11 pacientes (31,4%) fueron diagnosticados con fiebre amarilla severa y 24 pacientes (68,5%) con dengue grave. La media de los resultados de laboratorio con fiebre amarilla severa fueron bilirrubina indirecta 4,7 ml/dL, aspartato transaminasa 4463 UI/L, transaminasa aminotransferasa 4329 UI/L, creatinina 4,9 mg/dl. En pacientes con dengue grave el hematocrito promedio fue 51,8, hemoglobina 17,6 g/dl, plaquetas 24 × 103/mm. En pacientes con síndrome ictérico-febril la presencia de bradicardia, bilirrubina indirecta elevada y transaminasas muy elevadas debe hacer sospechar de fiebre amarilla; mientras que los pacientes que acuden por ascitis, derrame pleural, aumento de hematocrito y deficiencia de plaquetas, se debe tratar como dengue grave sobre todo en Unidades de Emergencia con poco apoyo de laboratorio(AU)

In Emergency centers with little laboratory support, differentiating patients with dengue and yellow fever is difficult. The Aim was to compare the clinical and laboratory profile of patients with severe dengue and severe yellow fever in the Emergency unit. We conducted a retrospective observational study of patients with a confirmed diagnosis of dengue and yellow fever in the period 2018 to 2020 treated in the Emergency Unit of the Carrión hospital, Huancayo-Peru. A total of 35 patients were evaluated, 11 patients (31.4%) were diagnosed with severe yellow fever and 24 patients (68.5%) with severe dengue. The mean laboratory results in patients with severe yellow fever were indirect bilirubin 4.7 ml/dL, aspartate transaminase 4463 IU/L, transaminase aminotransferase 4329 IU/L, creatinine 4.9 mg / dl. In patients with severe dengue were hematocrit 51.8, hemoglobin 17.6 g / dl, platelets 24 × 103 / mm. In patients with syndrome jaundice and fever the presence of bradycardia, elevated indirect bilirubin, and very elevated transaminases should be suspicious for yellow fever; while in patients who come for ascites, pleural effusion, increased hematocrit and platelet deficiency, it should be treated as severe dengue especially in Emergency Units with little laboratory support(AU)

Leaf extract of Coffea arabica L. reduces lipid peroxidation and has anti-platelet effect in a rat dyslipidemia model

Abstract This study aimed to evaluate the antioxidant potential of the Coffea arabica Lineu (L.) leaf extract and its effects on platelet aggregation of dyslipidemic rats. The extract was obtained by the percolation of C. arabica L. leaves in hydroethanolic solution 70% (v/v). The mass spectrometry FIA-ESI-MS² suggested the presence of chlorogenic acid, rutin acid, and quinic acid. The DPPH• radicals scavenging capacity was demonstrated (IC50 = 0.06 mg/mL). The extract was administered to rats by gavage (300 mg/kg/day) for 56 days. Dyslipidemia was induced by administering Triton WR-1339 (300 mg/kg body weight) on the 54th day. On day 56, blood was collected by puncturing the abdominal aorta artery and the aortic artery was removed. Lipid profile, markers of renal and hepatic injury, lipid peroxidation, and platelet aggregation tests were carried out. The ingestion of extract reduced the lipid peroxidation (aorta and plasma) and platelet aggregation in dyslipidemic rats. The extract did not affect markers of renal and hepatic function as analyzed in this study, suggesting neither impaired liver nor kidney function in these animals. Therefore, our results demonstrate that the extract of leaves of C. arabica L. show antioxidant potential in vitro and in vivo as well as anti-platelet aggregation in dyslipidemic animals

99m Technetium-HMPAO-labeled platelet scan in practice: Preparation, quality control, and biodistribution studies

Abstract There is no biodistribution or imaging data on 99mtechnetium (Tc)-hexamethyl propylamine oxime (HMPAO)-labeled platelets in the literature. The current study aimed to present updated information about the clinical procedures for preparation and use of labeled platelets. Following two-step centrifugation at 1500 and 2500 rpm, the platelets were extracted from whole blood into platelet-rich plasma (PRP) above the buffy coat and then from PRP into a platelet pellet at the bottom of the tube. The 99mTc-HMPAO-labeled platelets were inspected for purity, viability, release of 99mTc from platelets, and sterility. Also, microscopic examination and thin layer chromatography (TLC) were performed. Biodistribution was assessed following necropsy in BALB/c mice and through imaging of New Zealand rabbits. The separation ratio was estimated at 98%, and radiochemical purity was measured to be 80%. The labeling efficiency was above 30% in more than half of the assays (range: 17-43%). The release of 99mTc from platelets was 9% per hour at 37ºC. After 24 hours, stability was estimated at 54% in the human serum. The target organs of mice included the spleen and liver. In rabbits, the imaging results indicated liver as the target organ. Thyroid uptake was negligible up to 90 minutes. Based on the findings, extraction of platelets and labeling them with 99mTc-HMPAO is a feasible and safe approach in routine practice.

Alteraciones hematológicas como consecuencia de COVID-19 y sus vacunas / Hematological changes as a consequence of COVID-19 and its vaccines

El virus SARS-CoV-2 continúa infectando a millones de individuos en el mundo. Aunque los síntomas más frecuentes observados en los pacientes con COVID-19 son fiebre, fatiga y tos, en los casos severos la hipercoagulabilidad y la inflamación son dos condiciones que pueden producir complicaciones y causar daño en órganos, poniendo en riesgo la vida del paciente. Con el fin de clasificar a los pacientes durante el triaje, se han explorado diferentes marcadores hematológicos, incluidos el recuento de plaquetas, linfocitos y eosinófilos, y la relación neutrófilos/ linfocitos, entre otros. Por su parte, para la evaluación de las coagulopatías, se vienen determinando marcadores como el dímero D y el fibrinógeno. En esta revisión se abordan las coagulopatías y los parámetros hematológicos en pacientes con COVID-19, al igual que las anormalidades en la coagulación como la trombocitopenia trombótica inmune inducida por las vacunas contra el SARS-CoV-2

The SARS-CoV-2 virus continues to infect millions of individuals around the world. Although the most frequent symptoms observed in patients with COVID-19 are fever, fatigue and cough, in severe cases hypercoagulability and inflammation are two conditions that can cause complications and organ failure, putting the patient's life at risk. In order to classify patients during triage, different hematological markers have been explored, including platelet, lymphocyte, and eosinophil counts, and the neutrophil/lymphocyte ratio, among others. Furthermore, for the evaluation of coagulopathies, markers such as D-dimer and fibrinogen are being evaluated. This review addresses the coagulopathies and hematological parameters in patients with COVID-19, as well as coagulation abnormalities such as immune thrombotic thrombocytopenia induced by SARS-CoV-2 vaccines

Blood components use at two private hospitals in Belo Horizonte, Minas Gerais between July 2017 and June 2019

ABSTRACT Introduction: A retrospective ecological longitudinal study was carried out with data on blood components use from two private hospital units that belong to the same organization located in Belo Horizonte between July 2017 and June 2019. Objectives: To describe the monthly series of red blood cells, platelets and plasma use and the rate of blood components use for general hospitalizations in the health network, from the perspective of time series. Methods: A total of 15 time series were created with monthly data related to the use of blood components. The stationarity of the series was verified by the unit root test, the trend, by the Cox-Stuart test and seasonality, by the Fisher test (significance levels of 10% for the first test and 5% for the last two). Results: All series tested positive for the trend component and showed an increasing trend for the use of blood components. Ten series showed statistically significant seasonality and eight series were identified as non-stationary. The percentage of transfusions of blood components due to hospitalization at hospitals 1 and 2 was 29% (22% at hospital 1 and 38.9% at hospital 2). Conclusion: This study was able to describe the components of blood components use dynamics, from the perspective of time series at hospitals. Due to the growing trend in demand for blood components and their high cost, we propose the reduction of blood components use and the expanded use of alternative blood transfusion strategies.

Lisado plaquetario autólogo y su efectividad en pacientes con epicondilitis / Autologous platelet lysate and its effectiveness in patients with epicondylitis

Introducción: La epicondilitis constituye uno de los motivos de consulta más frecuentes tanto en la asistencia primaria como especializada y sin duda alguna, es uno de los problemas que tiene mayor repercusión en la persona que la padece. El tratamiento de las epicondilitis constituye un reto para la medicina debido a enormes implicaciones sanitarias, sociolaborales y el dolor e impotencia funcional que provoca. Objetivo: Evaluar la efectividad del lisado plaquetario autólogo como alternativa de tratamiento en pacientes enfermos con epicondilitis. Método: Se realizó un estudio cuasi experimental analítico longitudinal prospectivo en el que se evaluó el uso de lisado plaquetario autólogo como alternativa de tratamiento en pacientes con epicondilitis. El universo estuvo constituido por los pacientes que acudieron a consulta de Ortopedia y traumatología con el diagnóstico de epicondilitis, durante el periodo comprendido entre octubre de 2014 y julio de 2018. La muestra quedo constituida por 80 pacientes que cumplieron con los criterios de inclusión y exclusión. Resultados: El grupo de edad entre 36-56 años y del sexo femenino son los de mayor representación en padecer esta enfermedad. Las infiltraciones de lisado plaquetario autólogo aportan mejores resultados al convencional y se observa la mayor representación de pacientes que tuvieron una remisión total. Las complicaciones fueron mucho más evidentes en el tratamiento convencional. También es relevante el costo-beneficio del tratamiento con lisado plaquetario autólogo. Conclusiones: El tratamiento con lisado plaquetario autólogo puede ser una alternativa para mejorar la calidad de vida de los pacientes con epicondilitis(AU)

Introduction: Epicondylitis is one of the most frequent reasons for attending consultation in both primary and specialized care; while it is undoubtedly one of the problems with the greatest impact on the person who suffers from it. The managment epicondylitis is a challenge for medicine, due to the enormous health-related and social implications, as well as the pain and functional impotence that it causes. Objective: To assess the effectiveness of autologous platelet lysate as a treatment alternative in patients with epicondylitis. Method: A prospective, longitudinal, analytical and quasiexperimental study was carried out, in which the use of autologous platelet lysate as an alternative treatment in patients with epicondylitis was assessed. The universe consisted of patients who attended the orthopedics and traumatology consultation, during the period between October 2014 and July 2018, with a diagnosis of epicondylitis. The sample was made up of eighty patients who met the inclusion criteria; exclusion criteria were also considered. Results: The age group between 36 and 56 years, together with the female sex, are the most represented with respect to suffering from this disease. Infiltrations of autologous platelet lysate provide better outcomes than the conventional one, while greater representation of remitted patients is observed. Complications were much more evident in conventional treatment. The cost-benefit relationship of treatment with autologous platelet lysate is also relevant. Conclusions: Treatment with autologous platelet lysate can be an alternative to improve the quality of life of patients with epicondylitis(AU)

Profile of apheresis donors at Professor Alberto Antunes University Hospital of the Federal University of Alagoas

Hemotherapy services play a key role in attracting donors and providing safe blood to the population. The apher-esis platelet collection procedure is a relatively simple, safe and important procedure for increasing the stocks of these services. However, the recruitment and retention of these donors still represent a major challenge. Objec-tive: Evaluating the profile of donors of blood components by apheresis in the Transfusion Unit of Professor Alberto Antunes University Hospital - UFAL, as well as knowing the hematological parameters pre- and post-donation, the occurrence of the main adverse events related to the procedure and the difficulties faced by the donor. Method:This was a cross-sectional observational study. We analyzed a total of 160 forms of apheresis donors from March 2017 to June 2018. The data were tabulated using the Excel program, and then analyzed in order to determine the objectives. Results: Most donors were male (93.13%), aged between 25 and 40 years (48.75%) and brown (25.62%). There was a slight prevalence of singles (49.37%) and 73.75% were from Maceió. The most prevalent ABO and Rh phenotyping was O+ (39.3%). Most of the procedures were simple platelet collection (75.60%) and the occurrence of adverse events during donations was 30.63%. Conclusion: Evaluation of apheresis donor profile and the knowledge of the possible side effects related to the procedure provided a better understanding of this type of donation and may improve the capture and retention processes of these individuals, minimizing the effects of lack of blood for Alagoana population. (AU)

Os serviços de hemoterapia desempenham um papel fundamental na atração de doadores e no fornecimento de sangue seguro à população. No entanto, o recrutamento e a retenção desses doadores ainda representam um gran-de desafio. Objetivo: Avaliar o perfil dos doadores de hemocomponentes por aférese na Unidade de Transfusão do Hospital Universitário Professor Alberto Antunes - UFAL, bem como conhecer os parâmetros hematológicos pré e pós-doação, a ocorrência dos principais eventos adversos relacionados à procedimento e as dificuldades enfrenta-das pelo doador. Método: Estudo observacional transversal. Foram analisadas 160 fichas de doadores de aférese de março de 2017 a junho de 2018. Os dados foram tabulados no programa Excel e analisados para determinar os objetivos. Resultados: A maioria dos doadores era do sexo masculino (93,13%), com idade entre 25 e 40 anos (48,75%) e parda (25,62%). Houve uma leve prevalência de solteiros (49,37%) e 73,75% eram de Maceió. A feno-tipagem ABO e Rh mais prevalente foi O+ (39,3%). A maioria dos procedimentos foi de coleta simples de plaquetas (75,60%) e a ocorrência de eventos adversos durante as doações foi de 30,63%. Conclusão: A avaliação do perfil do doador de aférese e o conhecimento dos possíveis efeitos colaterais relacionados ao procedimento proporcio-naram uma melhor compreensão sobre esse tipo de doação e podem ajudar a melhorar os processos de captura e retenção desses indivíduos, minimizando os efeitos da falta de sangue para a população Alagoana.(AU)

Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates / Otimização de um método baseado em PCR em tempo real para rastreamento de bactérias em concentrados de plaquetas

Abstract Bacterial contamination of blood components remains a major challenge in transfusion medicine, particularly, platelet concentrates (PCs) due to the storage conditions that support bacterial proliferation. In this study, we develop a rapid, sensitive and specific real-time PCR protocol for bacterial screening of PCs. An internally controlled real-time PCR-based method was optimized and validated with our proprietary 16S Universal PCR Master Mix (IBMP/Fiocruz), which targets a conserved region of the bacterial 16S rRNA gene. Nonspecific background DNA was completely eliminated by treating the PCR Master Mix with ethidium monoazide (EMA). A lower limit of detection was observed for 10 genome equivalents with an observed Ct value of 34±1.07 in calibration curve generated with 10-fold serial dilutions of E. coli DNA. The turnaround time for processing, including microbial DNA purification, was approximately 4 hours. The developed method showed a high sensitivity with no non-specific amplification and a lower time-to-detection than traditional microbiological methods, demonstrating it to be an efficient means of screening pre-transfusion PCs.

Resumo A contaminação bacteriana dos componentes sanguíneos é um grande desafio na medicina transfusional, principalmente nos concentrados de plaquetas (PCs) devido às condições de armazenamento que favorecem a proliferação bacteriana. Neste estudo, desenvolvemos um protocolo de PCR em tempo real rápido, sensível e específico para a triagem bacteriana de PCs. Um método baseado em PCR em tempo real, controlado internamente, foi otimizado e validado com um Master Mix Universal PCR 16S (IBMP / Fiocruz), que detecta uma região conservada do gene 16S rRNA bacteriano. O background de DNA não específico foi completamente eliminado tratando a PCR Master Mix com monoazida de etídio (EMA). O limite de detecção inferior observado foi de 10 cópias equivalentes do genoma com um valor de Ct 34 ± 1,07, a curva de calibração foi gerada com diluições seriada de 10 vezes do DNA de E. coli. O tempo de processamento, incluindo a purificação microbiana do DNA, foi de aproximadamente 4 horas. O método desenvolvido mostrou alta sensibilidade sem amplificação inespecífica e menor tempo de detecção do que os métodos microbiológicos tradicionais, demonstrando ser um meio eficiente de triagem de PCs pré-transfusionais.

Transfusion of ABO non-identical platelets increases the severity of trauma patients at ICU admission

ABSTRACT Background: Transfusion of ABO-compatible non-identical platelets (PTLs), fresh plasma (FP) and red blood cells (RBCs) has been associated with increased morbidity and mortality of recipients. Trauma victims are frequently exposed to ABO non-identical products, given the need for emergency transfusions. Our goal was to evaluate the impact of the transfusion of ABO non-identical blood products on the severity and all-cause 30-day mortality of trauma patients. Methods: This was a retrospective single-center cohort, which included trauma patients who received emergency transfusions in the first 24 h of hospitalization. Patients were divided in two groups according to the use of <3 or ≥3 ABO non-identical blood products. The patient severity, measured by the Acute Physiology and Chronic Health Evaluation (APACHEII) score at ICU admission, and the 30-day mortality were compared between groups. Results: Two hundred and sixteen trauma patients were enrolled. Of these, 21.3% received ≥3 ABO non-identical blood products (RBCs, PLTs and FP or cryoprecipitate). The transfusion of ≥3 ABO non-identical blood products in the first 24 h of hospitalization was independently associated with a higher APACHEII score at ICU admission (OR = 3.28 and CI95% = 1.48-7.16). Transfusion of at least one unit of ABO non-identical PTLs was also associated with severity (OR = 10.89 and CI95% = 3.38-38.49). Transfusion of ABO non-identical blood products was not associated with a higher 30-day mortality in the studied cohort. Conclusion: The transfusion of ABO non-identical blood products and, especially, of ABO non-identical PLTs may be associated with the greater severity of trauma patients at ICU admission. The transfusion of ABO non-identical blood products in the trauma setting is not without risks.