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This study aimed to perform histological, immunohistochemical, biomechanical, and wettability assessments of leukocyte- and platelet-rich fibrin (L-PRF) membranes obtained from the blood of healthy dogs. Ten client-owned Labrador Retriever dogs were enrolled. Blood samples were obtained from the external jugular vein using a vacuum tube without anticoagulant, which was immediately centrifuged at 400g for 12 min in a dedicated centrifuge. The L-PRF clot was removed from the tube, and the red clot was released from the buffy coat using a spatula. The membrane was produced using a PRF box. Histological examination identified the three portions of the L-PRF membranes. The first portion was composed mainly of red blood cells with the presence of a low number of leukocytes among them. The second portion was composed of white blood cells, mainly neutrophils. The third portion was composed of the fibrin network which was characterized by acidophilic staining. The immunohistochemical analysis showed that vascular endothelial growth factor and platelet-derived growth factor were expressed in all samples at different intensities, both in cellular components and fibrin mesh. The tensile test and wettability assessments were measured in membranes 30 min and 3 h after production. The 30 min L-PRF membranes supported twice the ultimate tensile strength compared to 3 h L-PRF membranes. The wettability of the 30 min sample membranes was statistically higher than the 3 h sample membranes. In conclusion, the centrifugation protocol allowed production of the L-PRF membrane using canine blood and this was confirmed by histological and immunohistochemical analysis. The mechanical resistance and wettability of the L-PRF membrane were significantly reduced over time.

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ABSTRACT Objective: The present study was conducted to investigate the effects of leukocyte-platelet-rich fibrin (L-PRF) on the rate of maxillary canine retraction for a period of 5 months. Methods: A split-mouth study was conducted on 16 subjects (9 males and 7 females; age range 17-25 years; mean age, 21.85±2.45 years) who required therapeutic extraction of bilateral maxillary first premolars. After the initial leveling and alignment, L-PRF plugs were placed in a randomly selected extraction socket (Experimental Group), and the other side served as a control (Control Group). Canine retraction was carried out by the activation of nickel-titanium (NiTi) closed-coil springs delivering 150 g of force. The rates of canine movement, canine rotation, tipping, root resorption, and molar movement were assessed at monthly intervals for five months (T0-T5). Pain, swelling and discomfort accompanying the procedure were assessed using a Likert scale. Results: The study revealed a significant increase in the rate of canine movement on the experimental side in the first two months, and significant molar anchorage loss was observed only in the first month for control side. There were no statistically significant differences between the groups regarding canine rotation, tipping, probing depth, root resorption, and pain perception. Conclusions: The use of L-PRF plugs in extraction sockets considerably enhanced the rate of canine movement only in the first two months, and long-term efficacy was not observed in this study.

RESUMO Objetivo: O presente estudo foi realizado para investigar os efeitos da fibrina rica em leucócitos e plaquetas (L-PRF) na taxa de retração do canino superior, durante um período de cinco meses. Métodos: Um estudo de boca dividida foi realizado em 16 indivíduos (9 homens e 7 mulheres; faixa etária de 17 a 25 anos; idade média de 21,85 ± 2,45 anos) que precisavam de extração terapêutica dos primeiros pré-molares superiores de ambos os lados. Após o nivelamento e o alinhamento iniciais, os plugs de L-PRF foram colocados em um alvéolo pós-extração, selecionado aleatoriamente (Grupo Experimental), e o outro lado serviu como controle (Grupo Controle). A retração do canino foi realizada pela ativação de molas fechadas de níquel-titânio (NiTi) com 150 g de força. As taxas de movimentação do canino, rotação, inclinação e reabsorção radicular do canino e movimentação do molar foram avaliadas em intervalos mensais durante cinco meses (T0-T5). A dor, o inchaço e o desconforto após o procedimento foram avaliados por meio de uma escala de Likert. Resultados: O estudo revelou um aumento significativo na taxa de movimentação do canino no lado experimental nos dois primeiros meses, e uma perda significativa de ancoragem do molar foi observada apenas no primeiro mês no lado controle. Não houve diferenças estatisticamente significativas entre os grupos, com relação à percepção da dor e rotação, inclinação, profundidade de sondagem e reabsorção radicular do canino. Conclusões: O uso de plugs de L-PRF em alvéolos pós-extração aumentou consideravelmente a taxa de movimentação do canino apenas nos dois primeiros meses, não sendo observada uma eficácia em longo prazo.

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Our research group, through the analysis of miRNomes in platelet concentrates (PCs) stored in blood banks, identified and validated the miR-127 and miR-320a miRNAs as biomarkers of platelet storage lesions (PSLs) in PCs. In order to validate the miRNAs 127 and 320a methodologically, as PSL biomarkers in a large number of PC bags, we also evaluated important immunological markers involved in the platelet activation/aggregation process-the CD62P receptor (P-selectin), the surface glycoproteins (GP) IIb/IIIa, and the purinergic P2Y12 receptor-via flow cytometry. The miRNAs miR-127 and miR-320a were quantified by real-time quantitative PCR (RT-qPCR). To carry out this study, 500 collection tubes were used at the upper edge of the PC bags containing platelets. Each tube was divided into seven equal parts (totaling 3500 samples) for platelet analysis from 7 different storage days, where the 1st day represents the high-quality control, and the 7th day corresponds to the low-quality control of the platelets. After analyzing all parameters during storage days, it was concluded that the relative quantification of miR-320a below 0.50 and the CD62P receptor below 27.92% are reliable indicators of the absence of storage lesions in blood banks. We believe that the values found in the expression of the CD62P receptor legitimize the use of the miR-320a and miR-127 miRNAs to build a kit capable of accurately measuring whether the stored platelets are suitable for transfusion.

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Platelet-rich plasma (PRP) has been proposed as an agent to accelerate the healing process and stimulate the regenerative capacity of tissues due to its abundance of growth factors. A large variety of kits and protocols are available to obtain PRP by different cell-separation systems. However, the lack of standardization may lead to inconsistent results. The aim of this study was to characterize cellular composition, platelet parameters using the ADVIA 120 flow cytometer, and TGF-ß1 concentration from the PRP product obtained through a closed system, using simple centrifugation. Six clinically healthy horses were used in this study. The protocol in the closed system resulted in approximately 1.6-fold higher platelet and approximately 2.0-fold lower white blood cell concentrations in comparison with whole blood values. The evaluated system was efficient in concentrating platelets and in retrieving a small number of leukocytes, using a protocol of single centrifugation at low speed.

O plasma rico em plaquetas (PRP) tem sido proposto como um agente para acelerar o processo de cicatrização e estimular a capacidade regenerativa dos tecidos, devido à sua abundância de fatores de crescimento. Uma grande variedade de kits e de protocolos está disponível para obtenção de PRP, utilizando-se diferentes sistemas de separação de células. No entanto, a falta de padronização pode levar a resultados inconsistentes. O objetivo deste estudo foi caracterizar a composição celular, os parâmetros plaquetários pelo citômetro de fluxo ADVIA 120 e a concentração de TGF-ß1 do PRP obtido em sistema fechado, por centrifugação simples. Seis cavalos clinicamente saudáveis ​​foram usados ​​neste estudo. O protocolo no sistema fechado resultou em concentrações de plaquetas aproximadamente 1,6 vez maior e concentrações de leucócitos aproximadamente 2,0 vezes menores em comparação com os valores do sangue total. O sistema avaliado foi eficiente na concentração de plaquetas e na recuperação de um pequeno número de leucócitos, utilizando um protocolo de centrifugação única em baixa velocidade.

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PURPOSE: Platelet rich plasma (PRP) has been used in association with anterior cruciate ligament resconstruction (ACLR) to improve rehabilitation. The purpose was to systematically review the literature to compare the effects of PRP on ACLR in its objective and subjective outcomes. METHODS: A systematic review of the MEDLINE, Web of Science, Embase, Scopus, and Cochrane databases was performed. Two independent reviewers included all the English language literature of patients undergoing primary ACLR with autograft combined with PRP. The outcomes analyzed were graft ligamentization (MRI), tibial and femoral tunnel widening (MRI), knee laxity, IKDC, Lysholm, Tegner activity scale and visual analog scale. RESULTS: Nine studies were included with a total of 525 patients. PRP did not improve ligamentization of graft (standardized mean difference (SMD): 0.01 [95% CI: - 0.37; 0.39]), did not lead to lesser tunnel widening (SMD: 0.71 [95% CI: - 0.12; 1.54]), or lead to lesser knee laxity (raw mean difference: 0.33 [95% CI: - 0.84; 0.19]). Although there was statistical significance for PRP effects on Lysholm score and VAS (p < 0.01), their magnitude was limited. CONCLUSION: PRP showed no improvement in objective outcomes like ligamentization and less tunnel widening, while it showed just small improvements in terms of Lysholm, VAS and knee laxity. Therefore, there is not enough evidence to support a recommendation in favor of PRP and more research is needed. LEVEL OF EVIDENCE: I.

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BACKGROUND: Anterior cruciate ligament reconstruction (ACLR) has a high incidence among sports players, and one important side effect of the surgery is graft donor site morbidity. Although some evidence suggests that application of platelet-rich plasma (PRP) during ACLR reduces pain and improves knee function, it is not a universal finding. PURPOSE: To perform a meta-analysis of previous studies testing the effects of PRP on donor site morbidity after ACLR. STUDY DESIGN: Systematic review and meta-analysis. METHODS: We reviewed PubMed (Medline), Web of Science, Embase, Scopus, and Cochrane databases to find studies testing the effects of PRP on the donor site of ACLR autograft. After identifying 4 studies, we conducted 2 meta-analyses, 1 for the effects of PRP on pain, assessed by visual analog scale (VAS), and the other for the functional knee scores. We also tested the ability of time after ACLR to predict the PRP-related reduction of pain. RESULTS: In the 4 studies identified, 157 patients were analyzed. Although the VAS score was lower with PRP at 6 months (raw mean difference [RMD], -0.97 [95% CI, -1.59 to -0.36]; P = .001) and 12 months (RMD, -0.61 [95% CI,-1.02 to -0.21]; P = .003), the effects of PRP disappeared at 24 months (RMD, -0.08 [95% CI,-0.38 to 0.22]; P = .586). A univariate regression analysis reinforced the ability of time after ACLR to predict the PRP-related reduction of VAS pain score (r2 = 0.98). However, knee function after ACLR was not improved by the use of PRP (standardized mean difference, 0.71 [95% CI,-0.17 to 1.60]; P = .114). CONCLUSION: PRP applied to a bone-patellar tendon-bone donor site could reduce knee pain within a year, and this reduction had a correlation with time, meaning that the effect of PRP decreased with time after surgery. However, pain reduction did not reach clinical relevance and did not lead to better functional knee scores.

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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

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The morphological characteristics of the autologous platelet concentrate (APC) of 31 dogs were evaluated after cooling and freezing in 6% DMSO. Blood from the jugular vein of each patient was collected and centrifuged at 191g for six minutes to obtain APC. In the fresh sample, the platelet count, MPV, PDW and cell morphology were evaluated. Four samples of each animal were sent for storage, one refrigerated at 4°C for seven days, another for 30 days and two more stored in a freezer at -80°C in the same time interval, using 6% DMSO as cryoprotectant. The conserved samples were submitted to the same laboratory analysis as the fresh sample. There was a difference between fresh and preserved samples for platelet count, cell concentration, MPV and PDW (P<0.05), except in the 30-day refrigerated group, which showed severe morphological changes. In the frozen group for seven days, no difference was observed in the percentage of activation (P>0.05). The results obtained lead to the conclusion that cryopreservation with 6% DMSO at -80°C for seven days is a favorable option for the maintenance of platelet concentrations and the morphological characteristics of APC in dogs.(AU)

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Platelet concentrate (PC) transfusions are widely used to save the lives of patients who experience acute blood loss. MicroRNAs (miRNAs) comprise a class of molecules with a biological role which is relevant to the understanding of storage lesions in blood banks. We used a new approach to identify miRNAs in normal human platelet sRNA-Seq data from the GSE61856 repository. We identified a comprehensive miRNA expression profile, where we detected 20 of these transcripts potentially expressed in PCs stored for seven days, which had their expression levels analyzed with simulations of computational biology. Our results identified a new collection of miRNAs (miR-486-5p, miR-92a-3p, miR-103a-3p, miR-151a-3p, miR-181a-5p, and miR-221-3p) that showed a sensitivity expression pattern due to biological platelet changes during storage, confirmed by additional quantitative real-time polymerase chain reaction (qPCR) validation on 100 PC units from 500 healthy donors. We also identified that these miRNAs could transfer regulatory information on platelets, such as members of the let-7 family, by regulating the YOD1 gene, which is a deubiquitinating enzyme highly expressed in platelet hyperactivity. Our results also showed that the target genes of these miRNAs play important roles in signaling pathways, cell cycle, stress response, platelet activation and cancer. In summary, the miRNAs described in this study, have a promising application in transfusion medicine as potential biomarkers to also measure the quality and viability of the PC during storage in blood banks.

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BACKGROUND: Platelets undergo structural, biochemical and functional alterations when stored, and platelet storage lesions reduce platelet function and half-life after transfusion. The objective of this study was to evaluate stored canine platelet concentrates with platelet aggregation, flow cytometry and biochemistry assays. Twenty-two bags of canine platelet concentrates were obtained by the platelet-rich plasma method and were assessed on days 1, 3 and 5 after collection. Parameters such as platelet counts, residual leukocytes, platelet swirling, glucose, lactate, pH, CD62P expression (platelet activation), JC-1 (mitochondrial function) and annexin V (apoptosis and cell death) were assessed. RESULTS: Over the five days of storage there was a significant decrease in glucose, HCO3, pCO2, ATP, pH, swirling and mitochondrial function, associated with a significant increase in lactate levels and pO2. At the end of storage pH was 5.9 ± 0.6 and lactate levels were 2.8 ± 1.2 mmol/L. Results of the quality parameters evaluated were similar to those reported in human platelets studies. The deleterious effects of storage were more pronounced in bags with higher platelet counts (> 7.49 × 1010/unit), suggesting that canine platelet concentrates should not contain an excessive number of platelets. CONCLUSIONS: Quality parameters of canine platelets under standard storage conditions were similar to those observed in human platelets. Our results have potential to be used for the routine evaluation and quality control in veterinary blood banks.

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The aims of the present study were (1) to describe the microscopic and ultrastructural appearance of equine platelet-rich fibrin (PRF) clots and (2) to determine the release and degradation of transforming growth factor beta 1 (TGF-ß1) and insulin-like growth factor type I (IGF-I) from PRF clots incubated over 14 days. Whole blood from six horses was collected into plain tubes and centrifuged at 240 g for 8 minutes. Clots were evaluated by histology and by both transmission and scanning electronic microscopy (TEM and SEM). Growth factor concentrations were measured by ELISA at 48-hour intervals over 14 days and analyzed by one-way repeated-measures ANOVA. Histology showed a clot composed by a fibrin layer and a cellular layer with platelets and leukocytes. Scanning electron microscopy showed the cells trapped by an incipient fibrin network at 1 hour. At day 8, these cells were embedded by an incipient fibrin network. At day 14, the leukocytes and platelet aggregates from the clot were imbibed in an organized web of fibrin fibrils. TEM exhibited platelets with preserved cytoplasm and alpha granules randomly scattered at day 8, and damaged platelets with interrupted cytoplasm and organelle emigration to the periphery at day 14. TGF-ß1 and IGF-I concentrations showed a progressive increase until day 14. TGF-ß1 was released from PRF clots in a gradual and controlled manner, and increasing its concentration for two weeks, which supports TEM findings indicating that platelets began disintegrating by day 14. Furthermore, IGF-I production and release from PRF clots is sustained over time.

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BACKGROUND: Platelet concentrate (PC) is one of the main products used in a therapeutic transfusion. This blood component requires special storage at blood banks, however, even under good storage conditions, modifications or degradations may occur and are known as platelet storage lesions. METHODS: This research was performed on scientific citation databases PubMed/Medline, ScienceDirect, and Web of Science, for publications containing platelet storage lesions. The results obtained mainly reveal the clinical applicability of miRNAs as biomarkers of storage injury and as useful tools for a problem affecting public and private health, the lack of PC bags in countries with few blood donors. The major studies listed in this review identified miRNAs associated with important platelet functions that are relevant in clinical practice as quality biomarkers of PC, such as miR-223, miR-126, miR-10a, miR-150, miR-16, miR-21, miR-326, miR-495, let-7b, let-7c, let-7e, miR-107, miR-10b, miR-145, miR-155, miR-17, miR-191, miR-197, miR-200b, miR-24, miR-331, miR-376. These miRNAs can be used in blood banks to identify platelet injury in PC bags. CONCLUSION: The studies described in this review relate the functions of miRNAs with molecular mechanisms that result in functional platelet differences, such as apoptosis. Thus, miRNA profiles can be used to measure the quality of storage PC for more than 5 days, identify bags with platelet injury, and distinguish those with functional platelets.

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Resumo Esta revisão tem por objetivo elencar as condições oftalmológicas em que tem sido utilizado o concentrado de plaquetas (CP), assim como as suas propriedades bioquímicas e fisiológicas. O CP possui tanto o potencial anticatabólico, presente no soro autólogo, quanto substâncias com propriedades anabólicas, que em conjunto são responsáveis pelos seus benefícios no tratamento de doenças da superfície ocular. Atualmente há um lapso de ensaios clínicos neste tema, tanto na oftalmologia como em outras áreas médicas, existindo mais estudos e relatos sobre o uso de soro autólogo. Em oftalmologia, o CP tem sido usado no tratamento do olho seco sintomático, úlceras corneanas, queimaduras oculares dentre outras aplicações, sendo uma alternativa eficaz em diversas patologias oculares; portanto, é evidente a importância de mais estudos nesse tema, para comprovar a efetividade do produto.

Abstract The aim of this review is to list the ophthalmological conditions in which platelet concentrate (CP) has been used, as well as its biochemical and physiological properties. The CP has both anticatabolic potential, present in autologous serum, and substances with anabolic properties, which together are responsible for its benefits in the treatment of ocular surface diseases. There is currently a shortage of clinical trials in this area, both in ophthalmology and other medical areas, with more studies and reports on the use of autologous serum. In ophthalmology, CP has been used in the treatment of symptomatic dry eye, corneal ulcers and ocular burns, among other applications, being an effective alternative in several ocular pathologies; therefore, it's evident the importance of more studies in this topic to prove the efficiency of this product.

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PURPOSE: Low-level laser therapy (LLLT) can increase bone metabolism, cell proliferation, and maturation, and reduce inflammation, while platelet concentrate (PC) assists bone healing process by releasing proteins and growth factors. Here, we evaluated the efficacy of combined LLLT and PC therapy in the healing of critical-size bone defects. MATERIALS AND METHODS: Calvarial critical-size defects 5 mm in diameter were made in 48 Wistar rats. Bones were removed, milled, and used as autogenous bone grafts. Animals were randomized into four groups: LP (LLLT + PC), PC, L (LLLT), and C (control, autogenous graft only). Animals were sacrificed at day 30 and 60 post-surgery. Specimens were submitted to radiographic (digital and conventional), histological, histomorphometric, and immunohistochemical analyses. RESULTS: Digital radiography was shown to be a better image analysis method compared with conventional radiography. Histological analysis demonstrated a significant difference in bone formation between animals in group L (p = 0.049) at day 60 than in other groups. Higher rates of inflammatory infiltrates and fibrosis were observed in the LP and PC groups at days 30 and 60, whereas the groups not receiving PC showed a higher rate of bone maturity. The inflammatory processes were reduced in the animals in the L group, together with new bone formation and maturation. Groups L and C had higher scores of positive osteocalcin immunostaining in bone and extracellular matrix. CONCLUSIONS: LLLT reduces inflammation and contributes to increased bone formation. PC treatment was shown to maintain connective tissue and to induce fibrosis during bone repair. Combined LLLT and PC treatment did not improve bone repair.

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OBJECTIVE: Human blood is the only source of red blood cells, platelets and plasma, and includes the clotting factors. Transfusion of concentrated erythrocyte and blood products is a simple form of organ transplant, the benefits of blood transfusion are real, and the life of the patients depends on how is used. to know the transfusion adherence to the recommendations in the Hospital of High Specialty of Veracruz. METHOD: For a period of 12 months an audit took place in the Transfusion Service of the Hospital of High Specialty of Veracruz, México, on a basis of 3 168 requests for transfusion from which 2314 corresponded to erythrocyte concentrate, 220 to platelet concentrate, 493 to fresh frozen plasma and 41 to cryoprecipitate. RESULTS AND CONCLUSIONS: An analysis of concordance was made with the different established regulations for a right indication and the results showed that 2171 (67.26%) were appropriate and 1037 were inadequate, which means that the lack of academic training in medicine transfusional affects the risk for patients and cost for Health Institutions.

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Fifteen male New Zealand rabbits were used in this study, with the aim of storing their platelet-rich plasma (PRP) for 30 days at 4-6 C to investigate its conservation and viability during this period. Thirty samples of PRP were prepared and sorted into three equal groups (G1, G2, and G3), and every three days a sample was taken out for evaluationof the number of platelets, mean platelet volume (MPV), pH of the plasma, aggregation post addition of calcium thromboplastin, and for the presence of bacterial and fungal contamination. Results suggested that, for the number of platelets, there was no linear relationship over time. However, when comparing the number of platelets pre-storage to that post-storage, a statistical difference was observed. The hemogram MPV variables, pre and post-storage, also did not relate with time however, there was a statistical difference between the MPV of hemogram and MPV pre-storage, and between MPV pre-storage and MPV post-storage. From the pH evaluation, no influence of time on the variables was found, but statistical differences were found in the samples after storage between 30 and 6 days, 30 and 24 days, and 30 and 27 days. Platelet aggregation occurred within twenty seconds in all samples, independent of storage time. There was no growth of bacteria or yeast in any sample; however, mold growth occurred in the samples stored for 21 days from

Quinze coelhos machos da raça Nova Zelândia foram utilizados neste experimento, com a finalidade de armazenar o plasma rico em plaquetas (PRP) durante 30 dias a 4-6C para investigar a sua conservação e viabilidade durante este período. Trinta amostras de PRP foram preparadas e divididos em três grupos iguais (G1, G2 e G3), e de três em três dias foi retirada uma amostra para avaliação do número de plaquetas, volume plaquetário médio (VPM), pH do plasma, agregação após adição de tromboplastina cálcica, e para a presença de contaminação bacteriana e fúngica. Os resultados sugerem que, para o número de plaquetas, não houve uma relação linear com o tempo. No entanto, quando se compara o número de plaquetas pré-armazenamento com o pós-armazenamento, foi observada diferença estatística. As variáveis VPM no hemograma, pré e pós-armazenamento, também não se relacionou com o tempo, porém, houve diferença estatística entre o VPM do hemograma e pré-armazenamento, e entre VPM pré-armazenamento e pós-armazenamento. A partir da avaliação do pH, não houve influência do tempo sobre as variáveis, porém foram encontradas diferenças estatísticas nas amostras após o armazenamento entre 30 e 6, 30 e 24, e 30 e 27 dias. A agregação de plaquetas ocorreu no prazo de vinte segundos, em todas as amostras, independente do tempo de armazenamento. Não houve crescimento de bactérias ou fungos em qualquer a

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Fifteen male New Zealand rabbits were used in this study, with the aim of storing their platelet-rich plasma (PRP) for 30 days at 4-6 C to investigate its conservation and viability during this period. Thirty samples of PRP were prepared and sorted into three equal groups (G1, G2, and G3), and every three days a sample was taken out for evaluationof the number of platelets, mean platelet volume (MPV), pH of the plasma, aggregation post addition of calcium thromboplastin, and for the presence of bacterial and fungal contamination. Results suggested that, for the number of platelets, there was no linear relationship over time. However, when comparing the number of platelets pre-storage to that post-storage, a statistical difference was observed. The hemogram MPV variables, pre and post-storage, also did not relate with time however, there was a statistical difference between the MPV of hemogram and MPV pre-storage, and between MPV pre-storage and MPV post-storage. From the pH evaluation, no influence of time on the variables was found, but statistical differences were found in the samples after storage between 30 and 6 days, 30 and 24 days, and 30 and 27 days. Platelet aggregation occurred within twenty seconds in all samples, independent of storage time. There was no growth of bacteria or yeast in any sample; however, mold growth occurred in the samples stored for 21 days from

Quinze coelhos machos da raça Nova Zelândia foram utilizados neste experimento, com a finalidade de armazenar o plasma rico em plaquetas (PRP) durante 30 dias a 4-6C para investigar a sua conservação e viabilidade durante este período. Trinta amostras de PRP foram preparadas e divididos em três grupos iguais (G1, G2 e G3), e de três em três dias foi retirada uma amostra para avaliação do número de plaquetas, volume plaquetário médio (VPM), pH do plasma, agregação após adição de tromboplastina cálcica, e para a presença de contaminação bacteriana e fúngica. Os resultados sugerem que, para o número de plaquetas, não houve uma relação linear com o tempo. No entanto, quando se compara o número de plaquetas pré-armazenamento com o pós-armazenamento, foi observada diferença estatística. As variáveis VPM no hemograma, pré e pós-armazenamento, também não se relacionou com o tempo, porém, houve diferença estatística entre o VPM do hemograma e pré-armazenamento, e entre VPM pré-armazenamento e pós-armazenamento. A partir da avaliação do pH, não houve influência do tempo sobre as variáveis, porém foram encontradas diferenças estatísticas nas amostras após o armazenamento entre 30 e 6, 30 e 24, e 30 e 27 dias. A agregação de plaquetas ocorreu no prazo de vinte segundos, em todas as amostras, independente do tempo de armazenamento. Não houve crescimento de bactérias ou fungos em qualquer a

RESUMO

Para padronização de uma técnica manual para a obtenção de plasma rico em plaquetas (PRP) autólogo em bovinos com custo reduzido (método manual) e de boa qualidade (capacidade de concentrar plaquetas, alta concentração de fatores de crescimento e contaminação reduzida com leucócitos e eritrócitos), que poderá ser utilizado como um agente modulador da resposta imune de vacas com diferentes enfermidades, 450 ml de sangue total de nove vacas clinicamente saudáveis e com perfil hematológico normal foi coletado em bolsas de sangue CPDA-1 e processado dentro de quatro horas após a coleta. O sangue foi separado em alíquotas para avaliar 8 protocolos (P) de centrifugação dupla que variaram quanto a velocidade e o tempo de centrifugação. A contagem de plaquetas, eritrócitos e leucócitos na suspensão obtida (PRP) foi realizada pelo método manual em câmara de Neubauer: P5 (400g e 800g ambos durante 10 min) foi o protocolo com maior número de plaquetas, seguido por P3 (120g e 473g ambos durante 10 min), P4 (300g e 640g durante 10 min cada), P6 (640g durante 10 min e 640g durante 5 min), P8 (640g durante 5 min e 120g durante 10 min) e P7 (720g e 720g durante 5 min) e diferentes (p<0,05) dos menores valores encontrados em P1 (120g e 240g, ambos por 5 minutos) e P2 (120g e 473g ambos por 5 min). Em relação aos eritrócitos, P8, P7, P6, P5 e P4 apresentaram menores concentrações e maiores valores (p<0,05) foram observados em P3 e P2. Menores quantidades de leucócitos foram observadas em P5, P6, P8 e P7 com o maior valor obtido em P2 (p<0,05). Todos os protocolos (P1 a P8) foram eficientes em concentrar plaquetas sendo o valor mais baixo (3,65±0,79) observado em P1. Em relação aos fatores de crescimento ao se mensurar TGF- 1, os protocolos P1 e P8 evidenciaram valores mais elevados. De acordo com os resultados obtidos os protocolos P5 e P8 apresentaram os melhores resultados para confecção de PRP bovino.(AU)

For standardization of manual technique to obtain autologous platelet-rich plasma (PRP) in cattle with reduced cost (manual method) and good quality (ability to concentrate platelets, high level of growth factors and reduced contamination with leukocytes and erythrocytes), that may be used as a modulating agent of the immune response of cows chronically infected with various diseases, 450ml of whole blood from nine clinically and hematologically healthy cattle were collected in CPDA-1 bags and processed within four hours after collection. The blood was divided in aliquots to evaluate 8 protocols (P) of double centrifugation which varied as the speed and time of centrifugation. Platelet, erythrocytes and leukocytes counts in PRP were performed by manual method in a Neubauer chamber. The highest concentration of platelets was obtained in P5 (400g and 800g both for 10 min), followed by (p>0.05) P3 (120g e 473g ambos durante 10 min), P4 (300g e 640g durante 10 min cada), P6 (640g durante 10 min e 640g durante 5 min), P8 (640g durante 5 min e 120g durante 10 min) and P7 (720g and 720g both for 5 min) and different (p <0.05) than the protocols that had lower rates at P1 (120g to 240g, both for 5 minutes) and P2 (both 120g and 473g for 5 min). As for erythrocytes, P8, P7, P6, P5, P4 showed lower concentrations with higher values (p <0.05) observed in P3 and P2. Lesser values of leukocytes were found in P5, P6, P7 and P8 with the biggest value (p <0.05) obtained in P2. All protocols (P1 to P8) were efficient to concentrate platelets and the lowest value (3.65±0.79) was found in P1. Regarding TGF-ß1, the P1 and P8 protocols demonstrated the highest values. According to results, P5 and P8 protocols showed the best results for production of PRP in bovine.(AU)

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RESUMO

Abstract: For standardization of manual technique to obtain autologous platelet-rich plasma (PRP) in cattle with reduced cost (manual method) and good quality (ability to concentrate platelets, high level of growth factors and reduced contamination with leukocytes and erythrocytes), that may be used as a modulating agent of the immune response of cows chronically infected with various diseases, 450ml of whole blood from nine clinically and hematologically healthy cattle were collected in CPDA-1 bags and processed within four hours after collection. The blood was divided in aliquots to evaluate 8 protocols (P) of double centrifugation which varied as the speed and time of centrifugation. Platelet, erythrocytes and leukocytes counts in PRP were performed by manual method in a Neubauer chamber. The highest concentration of platelets was obtained in P5 (400g and 800g both for 10 min), followed by (p>0.05) P3 (120g e 473g ambos durante 10 min), P4 (300g e 640g durante 10 min cada), P6 (640g durante 10 min e 640g durante 5 min), P8 (640g durante 5 min e 120g durante 10 min) and P7 (720g and 720g both for 5 min) and different (p 0.05) than the protocols that had lower rates at P1 (120g to 240g, both for 5 minutes) and P2 (both 120g and 473g for 5 min). As for erythrocytes, P8, P7, P6, P5, P4 showed lower concentrations with higher values (p 0.05) observed in P3 and P2. Lesser values of leukocytes were found in P5, P6, P7 and P8 with the biggest value (p 0.05) obtained in P2. All protocols (P1 to P8) were efficient to concentrate platelets and the lowest value (3.65±0.79) was found in P1. Regarding TGF-1, the P1 and P8 protocols demonstrated the highest values. According to results, P5 and P8 protocols showed the best results for production of PRP in bovine.

Resumo: Para padronização de uma técnica manual para a obtenção de plasma rico em plaquetas (PRP) autólogo em bovinos com custo reduzido (método manual) e de boa qualidade (capacidade de concentrar plaquetas, alta concentração de fatores de crescimento e contaminação reduzida com leucócitos e eritrócitos), que poderá ser utilizado como um agente modulador da resposta imune de vacas com diferentes enfermidades, 450 ml de sangue total de nove vacas clinicamente saudáveis e com perfil hematológico normal foi coletado em bolsas de sangue CPDA-1 e processado dentro de quatro horas após a coleta. O sangue foi separado em alíquotas para avaliar 8 protocolos (P) de centrifugação dupla que variaram quanto a velocidade e o tempo de centrifugação. A contagem de plaquetas, eritrócitos e leucócitos na suspensão obtida (PRP) foi realizada pelo método manual em câmara de Neubauer: P5 (400g e 800g ambos durante 10 min) foi o protocolo com maior número de plaquetas, seguido por P3 (120g e 473g ambos durante 10 min), P4 (300g e 640g durante 10 min cada), P6 (640g durante 10 min e 640g durante 5 min), P8 (640g durante 5 min e 120g durante 10 min) e P7 (720g e 720g durante 5 min) e diferentes (p 0,05) dos menores valores encontrados em P1 (120g e 240g, ambos por 5 minutos) e P2 (120g e 473g ambos por 5 min). Em relação aos eritrócitos, P8, P7, P6, P5 e P4 apresentaram menores concentrações e maiores valores (p 0,05) foram observados em P3 e P2. Menores quantidades de leucócitos foram observadas em P5, P6, P8 e P7 com o maior valor obtido em P2 (p 0,05). Todos os protocolos (P1 a P8) foram eficientes em concentrar plaquetas sendo o valor mais baixo (3,65±0,79) observado em P1. Em relação aos fatores de crescimento ao se mensurar TGF- 1, os protocolos P1 e P8 evidenciaram valores mais elevados. De acordo com os resultados obtidos os protocolos P5 e P8 apresentaram os melhores resultados para confecção de PRP bovino.

RESUMO

INTRODUÇÃO: Devido à sepse bacteriana associada à transfusão de concentrados plaquetários (CPs) ter sérias consequências clínicas para os pacientes, alguns procedimentos têm sido incorporados na preparação e no controle de qualidade dos componentes sanguíneos para reduzir o risco da contaminação bacteriana. Este artigo descreve a prevalência da contaminação bacteriana dos CPs que foram transfundidos, o espectro bacteriano detectado com seu perfil de sensibilidade aos antimicrobianos e as reações transfusionais nos receptores. MÉTODOS: Um total de 292 CPs (278 randômicos e 14 por aférese), proveniente do Hemocentro do Estado do Rio Grande do Sul (HEMORGS) de Santa Maria foi testado. As quantidades de 100μL e 200μL foram coletadas da porção tubular da bolsa de plaquetas e semeadas utilizando dois tipos de metodologias. RESULTADOS: Em cinco unidades(1,7 por cento; 5/292) foram isoladas bactérias pela metodologia qualitativa e apenas uma pela quantitativa. Staphylococcus epidermidis foi o microrganismo identificado em todas as amostras. Dois pacientes apresentaram sepse associada à transfusão com desfecho fatal. CONCLUSÕES: A contaminação bacteriana pelas transfusões de CPs constitui-se num importante problema de saúde pública devido a sua associação com altas taxas de morbidade e mortalidade. Neste estudo, somente microrganismos gram-positivos foram isolados sendo que nenhuma amostra obtida por aférese apresentou contaminação.

INTRODUCTION: Bacterial sepsis associated with the transfusion of platelet concentrates (PCs) results in serious clinical implications for patients. Given these implications, certain procedures have been integrated into the preparation and quality control of blood components to reduce the risk of bacterial contamination. This article describes the prevalence of bacterial contamination on transfused PCs, the bacterial spectrum detected and their antimicrobial susceptibility profile and transfusion reactions in receptors. METHODS: A total of 292 PCs (278 random and 14 per apheresis) from the Blood Center of the State of Rio Grande do Sul (HEMORGS), located in the city of Santa Maria, were tested. Quantities of 100μL and 200μL were collected from platelet bag tubing and seeded using two methodologies. RESULTS: Using the qualitative methodology, bacteria were isolated in five units (1.7 percent; 5/292), while only one was isolated using the quantitative methodology. Staphylococcus epidermidis was the microorganism identified in all samples. Two patients died of transfusion-related sepsis. CONCLUSIONS: Bacterial contamination due to PC transfusion is considered a major public health problem due to its association with high rates of morbidity and mortality. In this study only gram-positive microorganisms were isolated and none of the samples obtained by apheresis presented contamination.

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