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Filariasis is a very important public health problem endemic in tropical and subtropical countries. It is a debilitating disease with significant socioeconomic burden. In 2016, lymphatic filariasis was successfully eliminated from the Maldives, which was certified by WHO. We report a case of filariasis detected by a rapid antigen test while undergoing a screening program conducted by Health Protection Agency, Maldives in December 2023. The patient was asymptomatic and showed no peripheral blood eosinophilia. Conventional blood smears were negative. Nevertheless, buffy coat smear served as a promising test in detection of parasite, thus confirming the re-emergence of the disease in the country. Therefore, our study highlight the significance of buffy coat smears in detection of hemoparasites. We also focus on the importance of surveillance system in order to sustain the disease elimination goal that was already achieved.

Outcomes of haploidentical hematopoietic stem cell transplantation (haplo-SCT) have improved over time. Graft failure and graft-versus-host disease (GVHD), which were important complications in major human leukocyte antigen (HLA)-disparity stem cell transplantation, have significantly decreased. These improvements have led to an exponential increase in the use of haploidentical donors for transplantation, as well as in the number of publications evaluating haplo-SCT outcomes. Many studies focused on factors important in donor selection, novel conditioning regimens or GVHD prophylaxis, the impact of donor-specific anti-HLA antibodies (DSA), as well as strategies to prevent disease relapse post-transplant. DSA represents an important limitation and multimodality desensitization protocols, including plasma exchange, rituximab, intravenous immunoglobulin and donor buffy coat infusion, can contribute to the successful engraftment in patients with high DSA levels and is currently the standard therapy for highly allosensitized individuals. With regards to donor selection, younger donors are preferred due to lower risk of complications and better transplant outcomes. Moreover, recent studies also showed that younger haploidentical donors may be a better choice than older-matched unrelated donors. Improvement of disease relapse remains a top priority, and several studies have demonstrated that higher natural killer (NK) cell numbers early post-transplant are associated with improved outcomes. Prospective studies have started to assess the role of NK cell administration in decreasing post-transplant relapse. These studies suggest that the incorporation of other cell products post-transplant, including the administration of chimeric antigen receptor T-cells, should be explored in the future.

Abstract Background Blood-based DNA methylation has shown great promise as a biomarker in a wide variety of diseases. Studies of DNA methylation in blood often utilize samples which have been cryopreserved for years or even decades. Therefore, changes in DNA methylation associated with long-term cryopreservation can introduce biases or otherwise mislead methylation analyses of cryopreserved DNA. However, previous studies have presented conflicting results with studies reporting hypomethylation, no effect, or even hypermethylation of DNA following long-term cryopreservation. These studies may have been limited by insufficient sample sizes, or by their profiling of methylation only on an aggregate global scale, or profiling of only a few CpGs. Results We analyzed two large prospective cohorts: a discovery (n = 126) and a validation (n = 136) cohort, where DNA was cryopreserved for up to four years. In both cohorts there was no detectable change in mean global methylation across increasing storage durations as DNA. However, when analysis was performed on the level of individual CpG methylation both cohorts exhibited a greater number of hypomethylated than hypermethylated CpGs at q-value

The objective of this study was to compare, by qPCR, the circulating blood parasite load of Trypanosoma cruzi in the buffy coat, and in whole blood mixed with boiled and unboiled guanidine hydrochloride-EDTA buffer, of individuals with chronic ChD. The concentration and purity of DNA were evaluated in a Nanodrop Denovix DS-11FX Series Spectrophotometer (DeNovix Inc., Wilmington, NC, USA). The parasite load was determined with the Taqman® qPCR system using a Stratagene Mx3000P thermocycler (Agilent Technologies, Santa Clara, CA, USA) with Cruzi 1 and Cruzi 2 satellite primers. Student’s t-test with Bonferroni correction, Chi-squared (χ2) tests and Spearman’s correlation coefficient were applied. The concentration and purity of DNA were higher in the buffy coat. Parasite DNA was detected and quantifiable in the three types of samples in seven patients, without statistically significant differences in the parasite load obtained. Higher correlations were found between the total DNA concentrations and the parasite loads obtained in the samples of the buffy coat.

Objective To evaluate the quality of platelet concentrates (PCs) prepared by traditional and modified buffy coat centrifugal canning methods, and to explore the effect of modified buffy coat centrifugal canning method on improving the quality of PCs. Methods The buffy coat centrifugal canning methods was divided into traditional group and modified group. In the traditional group, the buffy coat component bag and empty bag were directly combined layer by layer and vertically canned for light centrifugation. In the modified group, a foam spacer was added between the buffy coat component bag and the empty bag for light centrifugation layer by layer. The effects of the two groups of centrifugal canning methods on the preparation quality of PCs were observed. Results The platelet content in PCs prepared by the modified buffy coat centrifugal canning method was significantly higher than that in the traditional group, and the mixed amount of red blood cells was lower than that in the traditional group (P0.05), the qualified rates of platelet content and mixed red blood cell in the modified group were higher than those in the traditional group (P

The buff-colored layer separating the plasma from red blood cells (RBCs) in centrifuged blood was named the “buffy coat” in the late 19th century. The division of platelets (PLTs) and leukocytes (WBCs) between the buffy coat, plasma, and RBC layers in centrifuged blood has not been described before. In this study, we centrifuged 8.5 mL anticoagulated blood samples at 1000× g for 1, 2, 3, 5, 10, and 20 min. We then divided each sample into ten layers and analyzed each layer for cellular composition and mean platelet volume (MPV). Our results show that even after 20 min of centrifugation, about 15% of platelets remain in the plasma layers and 65% in the RBC layers. We found that the platelet count achieved from aspiration of 1 mL volume was optimal, with aspiration beginning 1/2 mL below the buffy coat and extending 1/2 mL above the buffy coat rather than beginning at the buffy coat itself and aspirating only plasma. Using this method of aspiration, we found that the total platelet count means reached a maximum in the 1 mL around the buffy coat after only 5 min of centrifugation.

Debela Abdeta Efa School of Veterinary Medicine, Wollega University, Nekemte, EthiopiaCorrespondence: Debela Abdeta EfaSchool of Veterinary Medicine, Wollega University, Nekemte, EthiopiaTel +251913125925Email debela.abdeta@gmail.comBackground: Bovine trypanosomosis remains a vital livestock disease and constraint which is intimidating livestock health and production, regardless of ongoing tsetse and trypanosomosis control struggles in Jimma Arjo district, East Wollega zone, Ethiopia.Methods: A cross-sectional study was carried out with the objective of determining prevalence of cattle trypanosomiasis and apparent tsetse fly density in six randomly selected peasant associations of Jimma Arjo District from April 2018 to January 2019.Results: From overall 819 arbitrarily selected cattle (n= 36; 4.39%), infection rate was recorded. Selected animals were invariably infested with different trypanosome species among which Tryapanosoma congolense (80.55%) was the most common, followed by T. vivax (11.11%), T. brucei (5.55%) respectively. Co-infection of T. vivax and T. congolense accounted for 2.77% of total infection rate. This finding indicates a statistically significant difference (p< 0.05) among good, medium, and poor body condition animals with respect to Tryapanosomosis infection rate. Poor body condition animals were highly infected with trypanosome parasite as compared to medium and good body condition score animals. This study shows statistically significant association was obtained between mean packed cell volume (PCV) and trypanosomiasis infection rate (P< 0.05). The lower mean PCV value (21.14%) were highly affected as compared with high mean PCV value animals (25.26%). The result of entomological survey, by using mono pyramidal traps deployed near animal grazing field and rivers of selected peasant association (PA), showed presence of four Glossina species namely Glossina morsitans, G. pallidipes, G. tachinoides, and G. fuscipes with high fly density per trap in Meta PA. Higher catches of G. fuscipes were registered as compared to other vectors.Conclusion: Generally, this study indicated the disease is still a main problem for livestock health and production in the study area and it necessitates disease and tsetse fly control.Keywords: bovine trypanosomiasis, buffy coat, Jimma Arjo, prevalence, tsetse flies

Abstract Background Both capillary and venous blood samples have been interchangeably used for the diagnosis of malaria in Ethiopia. However, Plasmodium parasites are thought to be more concentrated in capillary than in venous blood. Hence, selecting a sample source where parasites are more concentrated is indispensable approach in order to maximize the accuracy of blood film microscopy. Therefore, the present study aimed to compare the detection rate and the parasitemia level of Plasmodium species from conventional capillary and venous blood films, and buffy coat preparations. Methods A facility based cross-sectional study was conducted from Feburary to March 2020 among 210 febrile patients attending Hamusite health center, northwest Ethiopia. Capillary and venous blood samples were collected and buffy coat was prepared from each sample. Thin and thick blood films were prepared, stained, and examined microscopically following standard protocol. Data were analysed using Statistical Package for Social Sciences Software version 20 and Med-Calc software version 19.3. Results Capillary blood buffy coat (61/210, 29.0%) had significantly higher detection rate as compared to capillary (48/210, 22.9%) and venous (42/210, 20.0%) blood films (p

Buffy Coats, generated from a blood donor’s whole blood bag unit, are commonly used in biomedical research as a source of leukocytes due to the high number of cells that can be recovered from each Buffy Coat. Buffy Coats are leukocyte-enriched residual units obtained by centrifugation of whole blood. At the blood bank, blood can be processed using two different protocols according to the time interval between blood collection and processing. When blood collection and processing occur on the same day, it gives rise to Fresh Blood Buffy Coats. Alternatively, if blood processing only happens on the day after blood collection, Overnight Blood Buffy Coats are created. In this study, we aimed to address whether these two different Buffy Coat-processing protocols could differently impact monocyte function as antigen-presenting cells. For this purpose, we analyzed in the same experiment monocytes isolated from Fresh Blood and from Overnight Blood Buffy Coats. We assessed lipid antigen presentation by CD1d to invariant Natural Killer T (iNKT) cells. CD1d is a non-polymorphic MHC class I-like protein, which facilitates the study of antigen presentation among allogeneic samples. The results show that monocytes from Fresh Blood Buffy Coats have a better capacity to present antigens by CD1d, and consequently to activate iNKT cells, when compared to monocytes from Overnight Blood Buffy Coats. The differences observed were not explained by disparities in monocyte viability, CD1d expression, or basal activation state (monocyte expression of CD40 and CD80). Buffy Coats are a valid source of blood cells available daily. Hence, the type of protocol for Buffy Coat processing should be carefully considered in day-to-day research, since it may lead to different outcomes.

Granulocytes may be procured either as apheresis granulocyte concentrate (AGC) or as a pooled buffy coat granulocytes (PBCG) derived from whole blood donations. The chronic myelomonocytic leukemia patient with active pneumocystis pneumonia infection, received two transfusions of AGC and PBCG each, for prolonged severe neutropenia. AGCs had a higher mean white blood cells count per bag (1.63 × 1010 vs. 1.53 × 1010), neutrophil count per bag (1.05 × 1010 vs. 0.76 × 1010), and mean neutrophils percentage (62.45% vs. 49.25%), whereas PBCGs had a higher mean red blood cells volume per bag (196.1 vs. 62.5 ml) and platelet counts (5.03 × 1011 vs. 3.6 × 1011). The rise in patients' absolute neutrophil count was comparable with both AGC and PBCG transfusions. Granulocyte transfusions helped in the management of an acute neutropenic crisis. Whole blood-derived PBCGs may be a viable alternative in to AGCs. PBCGs have a lot of logistic issues and each blood center should have its standard operating procedure.

Background: The dermal aging process and the formation of deep wrinkles are a biological involution that also involves the regeneration system of cells immersed in the extracellular matrix and the papillary dermis. The progressive loss of niches of adult stem cells (MSCs) is more evident after the first third of life; it increases the phenotypic expression and the characteristics of the tissue senescence process. The purpose of this study was to clinically demonstrate that in viable micrograft there may be an improvement of deep wrinkles and surrounding tissues. Methods: This study involved 11 female patients who underwent the correction of deep dermal wrinkles through a suspension containing 0.8 mL of viable micrografts in a 5 mL plasma gel scaffold, obtained from the centrifugation of a 20 cc venous sample peripheral blood, gelled by heat in a dry steriliser and the buffy coat coming from the same venous sample, in order verify overtime the improvement of the interested anatomical area. Individual signs of wrinkles and the degree of correction obtained for each treatment and each area were objectively evaluated by using a 10-0 visual analog scale (VAS), Modified Vancouver scale and Berardesca's scale. Results: With this technique excellent results were obtained. In fact, wrinkles were improved, as well as surrounding tissues, even after 60 days, as shown by the Berardesca's, VAS and Modified Vancouver scales. Conclusion: This retrospective clinical evaluation allowed us to consider the excellent clinical results obtained with this method for the treatment of deep wrinkles and surrounding tissues, through a suspension of progenitors with MSCs derived from adipose tissue (ADSCa) in a not inflammatory plasma gel scaffold combined with buffy coat.

BACKGROUND AND OBJECTIVES: Platelet concentrates (PCs) can be prepared in several different ways, and they can be stored over few days before the use. Regulated on activation, normal T cells expressed and secreted (RANTES) levels in these concentrates may vary depending on the type of preparation and duration of storage of this component. We measured RANTES levels in platelet supernatants in different preparations and with different storage duration.MATERIALS AND METHODS: Fifteen PCs were prepared by platelet-rich plasma (PRP) and buffy coat (BC) method each. Forty-two single donor platelets (SDPs) were prepared using cell separators Cobe Spectra, Trima Accel, and Amicus. Filtered PCs were prepared using labside and bedside filters. The supernatants were collected after 1, 18, 65, and 112 h of preparation. SDP samples were taken on the 0 day, 3rd day, and 5th day. In filtered PC, pre- and post-filtration samples were taken, and aliquots were frozen at − 56°C for the measurement of RANTES.RESULTS: RANTES at 1 h was 1210 ± 560 pg/ml in PRP-PC, 1384 ± 463 pg/ml in BC-PC. At 112 h, 1617 ± 451 pg/ml and 1949 ± 134 pg/ml, respectively. In SDP, 0-day level was 1850 ± 278 pg/ml and >2000 pg/ml on 5th day. In prestorage, filtered PC RANTES was 1035 ± 496 pg/ml, and in the poststorage sample, it was 310 ± 508 pg/ml. With bedside filters, presample showed 1243 ± 832 pg/ml and postsample showed 556 ± 748 pg/ml.CONCLUSION: The concentration of RANTES increased continuously from 1 h to 5 days of storage in all PCs. After 65 h, BC-PC showed higher levels of RANTES compared to PRP-PC. Filtered PRP-PCs appear to be the best in terms of low RANTES to prevent allergic reactions and cultures negative.

Objective: investigation of the extra-low-frequency (ELF) stimulation effect on blood-cell proteins, that causes variation in its electrostatic-state. A hypothesis that this results in the conformational change in the blood-cell proteins which could enhance immune activity is explored. Since HIV-1 and host-cell engage through charge-charge interactions, an electrical-pulse may cause charge redistribution, hypothetically resulting in host-cell proteins to be isolated from viral access. Methods: Buffy coat samples were exposed to ELF square waveform pulses of 5Hz, 10Hz and 1MHz, for 2-hours, and were then examined using immunofluorescence technique. The expression of glycoprotein CD4, and co-receptor protein CCR5, were investigated. Also, the binding activity of the N-terminal domain of CCR5 and the distribution of the nuclear-pore-complex (NPC) transport factor, FGNup153 were investigated. Comparison with control samples were carried out. Results: Increased CD4 count, which could enhance the immune system. In addition, the inability of N-terminus-specific antibody 3A9 to bind to CCR5 N-terminal, could be due to the interactions with the ELF electric-field, which may also hypothetically inhibit HIV-1 attachment. Furthermore, the electrostatic interactions between the ELF pulse and the FGNup153 induces redistribution in its disorder sequence and possibly causes conformational change. This could possibly prevent large virus particle transport through the NPC. Conclusion: Novel concept of ELF stimulation of blood cellular proteins has been developed leading to transformation of immune activity. Clinical-Impact: The translational aspect is the use of ELF as an avenue of electro-medicine and the results are a possible foundation for the clinical application of ELF stimulation in immune response.

Background: We aimed to assess the extent to which the buffy coat DNA methylome is representative of methylation patterns in constitutive white blood cell (WBC) types in normal pregnancy.Methods: A comparison of differential methylation of buffy coat DNA vs DNA isolated from polymorphonuclear (PMN) and lymphocytic fractions was performed for each blood sample obtained within 24 h prior to delivery from 29 normotensive pregnant women. Methylation profiles were obtained using an Illumina Human Methylation 450 BeadChip and CHaMP bioinformatics pipeline. A subset of differentially methylated probes (DMPs) showing discordant methylation were further investigated using statistical modeling and enrichment analysis.Results: The smallest number of DMPs was found between the buffy coat and the PMN fraction (2.96%). Pathway enrichment analysis of the DMPs identified biological pathways involved in the particular leukocyte lineage, consistent with perturbations during isolation. The comparisons between the buffy coat and the isolated fractions as a group using linear modeling yielded a small number of probes (∼29,000) with discordant methylation. Demethylation of probes in the buffy coat compared to derived cell lines was more common and was prevalent in shelf and open sea regions.Conclusion: Buffy coat is representative of methylation patterns in WBC types in normal pregnancy. The differential methylations are consistent with perturbations during isolation of constituent cells and likely originate in vitro due to the physical stress during cell separation and are of no physiological relevance. These findings help the interpretation of DNA methylation profiling in pregnancy and numerous other conditions.

Introduction: One of the most important responsibility of Blood Bank is to ensure uninterrupted supply of quality blood products, namely Platelet Concentrate (PC), which are in high demand particularly during pandemic. In India, PCs are of three types prepared by different methods like Platelet Rich Plasma-Platelet Concentrate (PRP-PC), Buffy Coat Platelet Concentrates (BCPC), and Single Donor Platelets (SDP) units. Hence, establishing optimum quality standards across all the three types of PC remains a challenge in order to maximum benefit to recipients. Aim: To assess and compare the quality parameters of platelet concentrate prepared by PRP-PC, BC-PC and apheresis methods (SDP). Materials and Methods: This laboratory based observational study was conducted at rural, tertiary care, postgraduate, teaching hospital located at South India from December 2018 to November 2019. A total of 156 PCs were studied with 52 units belonging to PRP method, 52 units BC-PC method and the remaining were 52 units of SDP prepared by apheresis method. Quality parameters include volume, pH, swirling, White Blood Cell (WBC) count/bag, platelet count/bag and platelet indices. Platelet indices include Mean Platelet Volume (MPV), Platelet Distribution Width (PDW), and Platelet Large Cell Ratio (PLCR). Arterial Blood Gas (ABG) analysis was also done to assess (pH, partial pressure of Carbon Dioxide (pCO2 ), plasma bicarbonate (pHCO3-)). The p-value less than 0.05 were considered statistically significant. Statistical Package for the Social Sciences (SPSS) software version 23.0 used for statistical analysis. Results: All the 156 PC units were analysed as per the Director General of Health Services (DGHS) India, criteria. Among the 156 platelet concentrates scored as per DGHS criteria, a score of 5 was obtained for 34.61% [18/52], 30.76 [16/52] and 11.5% [6/52] SDP, BC-PC and PRP-PC units, respectively. All the PC units were sterile and Red Blood Cell (RBC) contamination was within acceptable limits. Conclusion: In this study, quality parameters of SDP was better than BC-PC and PRP-PC but with improvement and more standardisation of procedures and storage, BC-PC units can give a yield similar to that of SDP units.

Transcriptome studies have reported the dysregulation of cell cycle-related genes and the global inhibition of host mRNA translation in COVID-19 cases. However, the key genes and cellular mechanisms that are most affected by the severe outcome of this disease remain unclear. For this work, the RNA-seq approach was used to study the differential expression in buffy coat cells of two groups of people infected with SARS-CoV-2: (a) Mild, with mild symptoms; and (b) SARS (Severe Acute Respiratory Syndrome), who were admitted to the intensive care unit with the severe COVID-19 outcome. Transcriptomic analysis revealed 1009 up-regulated and 501 down-regulated genes in the SARS group, with 10% of both being composed of long non-coding RNA. Ribosome and cell cycle pathways were enriched among down-regulated genes. The most connected proteins among the differentially expressed genes involved transport dysregulation, proteasome degradation, interferon response, cytokinesis failure, and host translation inhibition. Furthermore, interactome analysis showed Fibrillarin to be one of the key genes affected by SARS-CoV-2. This protein interacts directly with the N protein and long non-coding RNAs affecting transcription, translation, and ribosomal processes. This work reveals a group of dysregulated processes, including translation and cell cycle, as key pathways altered in severe COVID-19 outcomes.

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

A study was conducted from August 3 to September 5, 2014 and from February 6 to March 7, 2015 at the Izeli area of the Mushie Ranch in the Democratic Republic of Congo. The objective was to study the influence of certain determinants that may influence trypanosome infection status in N’dama cattle. The sample of 324 cattle selected using the systematic sampling technique was obtained according to the Thrusfield (2005) formula because of 162 in the dry season and as much for the rainy season. The study focused on 4 herds including Selection, Nganzaka, Dwe and Wulu-Wulu. The parasitological diagnosis made by the buffy coat technique and confirmed by molecular biology analyzes (PCR) made it possible to determine the rate of trypanosome infection (Prevalence). This rate has been studied in relation to certain determinants, including the age, season, hematocrit, rearing environment, herd health status, zootechnical category and management behavior. The conclusion of the study reveals that season, hematocrit, breeding environment (site), herd health status, zootechnical category, and herd management have a significant effect on infection status trypanosome (p˂0.05) in contrast to age (p>0.05).

A study was conducted in 2014 on N’dama cattle in the Izeli sector of the Mushie ranch in DR Congo. The objectives were: (1) to identify the different causes of mortality of pathological and non-pathological origin, (2) to determine the number of cases, the frequency and the mortality rate for each cause according to whether calves, adult cattle (including cows) and cows apart. The methodology consisted of taking into account all verified mortality cases recorded during the year. The causes of death were identified through a few techniques including buffy coat, PCR, clinical examinations, autopsy reports, post mortem examinations and the use of documentary technique (annual report of the ranch). The Excel software was used for data entry while the SPSS 21 for their processing. The% allowed to determine the frequency and the mortality rate for each cause. The results revealed 23 causes of death that resulted in the death of 283 cattle, of which 45.99% (n = 129) were of pathological origin and 54.41% (n = 154) non-pathological.

A study was conducted at the Izeli sector of the Mushie ranch (Mai-Ndombe province) in the Democratic Republic of Congo from August 3 to September 5, 2014 and from February 6 to March 7, 2015. The objective was to study the relationship between (1) hematocrit and anemia status, (2) hematocrit and trypanosome infection status, and (3) anemia and trypanosome infection status on N’Dama cattle raised on the ranch. The 324 cattle sample, determined by the systematic sampling technique, was obtained according to the formula of Thrusfield (2005). Four herds were selected on the basis of criteria set in advance: Selection, Nganzaka, Dwe and Wulu-Wulu. Parasitological diagnosis using the buffy coat technique determined the hematocrit for each sampled cattle and then identified positive cattle. Parasitological results were supplemented by molecular biology (PCR) analyzes that confirmed trypanosome infection in each flock. The results of the study reveal a significant correlation between hematocrit and anemia status (p> 0.05), a non-significant correlation between hematocrit and trypanosome infection status (p˂0.05) and finally a significant correlation between anemia and trypanosome infection (p> 0.05).

Liquid platelet-rich fibrin (PRF) is produced by fractionation of blood without additives that initiate coagulation. Even though liquid PRF is frequently utilized as a natural source of fibrinogen to prepare sticky bone, the concentration of fibrinogen and the overall amount of “clottable PRF” components have not been evaluated. To this aim, we prepared liquid PRF at 300, 700, and 2000 relative centrifugal force (RCF), for 8 min and quantified the fibrinogen levels by immunoassay. We report here that, independent of the RCF, the fibrinogen concentration is higher in the platelet-poor plasma (PPP) compared to the buffy coat (BC) fraction of liquid PRF and further decreases in the remaining red fraction. We then determined the weight of the clotted PRF fractions before and after removing the serum. The PPP and BC fractions consist of 10.2% and 25.3% clottable matrix suggesting that more than half of the weight of clottable BC is caused by cellular components. Our data provide insights into the distribution of fibrinogen in the different fractions of liquid PRF. These findings suggest that PPP is the main source of clottable fibrinogen, while the BC is more a cell source when it comes to the preparation of sticky bone.

Diagnosis of anaplasmosis is challenging considering the great variation in clinical signs and the limitations of the available diagnostic assays, while the detection of carrier animals that play a significant role in disease epidemiology as reservoirs is of great significance. In this study, we evaluated the diagnostic accuracy of a newly developed indirect immunofluorescent assay (Ag-IFAT) for the detection of A. phagocytophilum antigens in buffy coat specimens, alone and in combination with cytology, using PCR as a reference. Blood samples were collected from 138 sheep of the Chios breed from six farms in Greece. A buffy coat was extruded from the centrifuged blood. Buffy coat smears were used for cytological examination and the Ag-IFAT assay. The Ag-IFAT assay presented excellent specificity (100%) and high sensitivity (85.4%) for the detection of A. phagocytophilum antigens in buffy coats, and it has an almost perfect agreement with PCR and cytology (κ value = 0.88 and 0.85, respectively). A. phagocytophilum antigens are likely to be detected using Ag-IFAT in a PCR-positive animal, as indicated by the good performance of the assay. Overall, this assay presents high diagnostic accuracy, and it could be used for the detection of animals during the early stage of infection.

In Brazil, Trypanosoma vivax is present in several states. The disease is endemic in Pantanal and Minas Gerais. In Paraná there is still no report of the parasite, but due to the state borders with Mato Grosso do Sul, São Paulo, Paraguay and Argentina, it is believed that the protozoan circulates in the region without diagnosis. The objective of this study was to investigate the prevalence of T. vivax in dairy cattle in the western region of Paraná. For this purpose, 600 blood samples and 400 serum samples were collected from dairy cattle, distributed in 60 and 40 properties, respectively. While buffy coat smears were performed on blood samples, serum samples were used in Indirect Immunofluorescence Reaction. All samples, in both techniques, presented negative results for T. vivax. These results indicate that the studied hemoprotozoan is not circulating among the cattle in the western region of Paraná. However, future work evolving beef cattle must be carried out and preventive measures should be adopted in order to avoid the entry of the parasite in the State of Paraná.

Cord blood DNA methylation is associated with numerous health outcomes and environmental exposures. Whole cord blood DNA reflects all nucleated blood cell types, while centrifuging whole blood separates red blood cells, generating a white blood cell buffy coat. Both sample types are used in DNA methylation studies. Cell types have unique methylation patterns and processing can impact cell distributions, which may influence comparability. We evaluated differences in cell composition and DNA methylation between cord blood buffy coat and whole cord blood samples. Cord blood DNA methylation was measured with the Infinium EPIC BeadChip (Illumina) in eight individuals, each contributing buffy coat and whole blood samples. We analyzed principal components (PC) of methylation, performed hierarchical clustering, and computed correlations of mean-centered methylation between pairs. We conducted moderated t-tests on single sites and estimated cell composition. DNA methylation PCs were associated with individual (PPC1 = 1.4 × 10−9; PPC2 = 2.9 × 10−5; PPC3 = 3.8 × 10-5; PPC4 = 4.2 × 10-6; PPC5 = 9.9 × 10-13, PPC6 = 1.3 × 10−11) and not with sample type (PPC1-6>0.7). Samples hierarchically clustered by individual. Pearson correlations of mean-centered methylation between paired samples ranged from r = 0.66 to r = 0.87. No individual site significantly differed between buffy coat and whole cord blood when adjusting for multiple comparisons (five sites had unadjusted P

Solid platelet-rich fibrin (PRF), consisting of coagulated plasma from fractionated blood, has been proposed to be a suitable carrier for recombinant bone morphogenetic protein 2 (BMP2) to target mesenchymal cells during bone regeneration. However, whether solid PRF can increase the expression of BMPs in mesenchymal cells remains unknown. Proteomics analysis confirmed the presence of TGF-β1 but not BMP2 in PRF lysates. According to the existing knowledge of recombinant TGF-β1, we hypothesized that PRF can increase BMP2 expression in mesenchymal cells. To test this hypothesis, we blocked TGF-β receptor 1 kinase with SB431542 in gingival fibroblasts exposed to PRF lysates. RT-PCR and immunoassays confirmed that solid PRF lysates caused a robust SB431542-dependent increase in BMP2 expression in gingival fibroblasts. Additionally, fractions of liquid PRF, namely platelet-poor plasma (PPP) and the buffy coat (BC) layer, but not heat-denatured PPP (Alb-gel), greatly induced the expression of BMP2 in gingival fibroblasts. Even though PRF has no detectable BMPs, PRF lysates similar to recombinant TGF-β1 had the capacity to provoke canonical BMP signaling, as indicated by the nuclear translocation of Smad1/5 and the increase in its phosphorylation. Taken together, our data suggest that PRF can activate TGF-β receptor 1 kinase and consequently induce the production of BMP2 in cells of the mesenchymal lineage.

The pandemic caused by the SARS-CoV-2 infection affects many aspects of public health knowledge, science, and practice around the world. Several studies have shown that SARS-CoV-2 RNA in plasma seems to be associated with a worse prognosis of COVID-19. In the present study, we investigated plasma and buffy RNA in patients with COVID-19 to determine its prognostic value. A prospective study was carried out in patients hospitalized for COVID-19, in which RNA was analyzed in plasma and the buffy coat. Morphological and immunohistochemical studies were used to detect the presence of SARS-CoV-2 in the buffy coat. In COVID-19 patients, the obtained RNA concentration in plasma was 448.3 ± 31.30 ng/mL. Of all the patients with positive plasma tests for SARS-CoV-2, 46.15% died from COVID-19. In four cases, tests revealed that SARS-CoV-2 was present in the buffy coat. Abnormal morphology of monocytes, lymphocytes and neutrophils was found. An immunohistochemical study showed positivity in mononuclear cells and platelets. Our results suggest that SARS-CoV-2 is present in the plasma. This facilitates viral dissemination and migration to specific organs, where SARS-CoV-2 infects target cells by binding to their receptors. In our study, the presence of plasma SARS-CoV-2 RNA was correlated with worse prognoses.

When a patient’s platelet count is low, the sample is assessed for platelet clumps to distinguish between thrombocytopenia and pseudothrombocytopenia. Microscopic examination of blood smear is the standard method for identifying platelet clumps at Diakonhjemmet Hospital AS, but the method is time-consuming and may be challenging due to artefacts. The purpose of this project was to evaluate if two alternative methods, direct microscopic examination of EDTA whole blood and buffy coat, were suitable for identification of platelet clumps. In addition, the samples were analyzed by Sysmex XE-5000 before and after removal of 10 uL buffycoat, to investigate if platelet and leukocyte parameters were influenced to an extent that supplementary analysis after removal would not be possible. We included 57 samples with platelet counts lower than 90 x 109/L. Microscopic examination of buffy coat had a sensitivity of 97 % when compared to blood smear. In addition, the method was time-saving and easier to perform. Direct microscopic examination of whole blood was the easiest method to perform and most time-saving. However, the sensitivity was 64 % when compared to blood smear, and detecting possible platelets clumps was challenging due to the amount of erythrocytes present. Based on this, we do not recommended whole blood as an alternative method for identifying platelet clumps. Furthermore, the results show that there was no statistically significant difference in platelet and leukocyte parameters before and after removal of buffy coat. In addition, the observed difference in percent was less than the analytical variation, which indicates that the differences are of no clinical significance. We conclude that buffy coat may be used as an alternative method to blood smear to identify platelet clumps.

Currently, the only method for identifying infective hosts with Leishmania infantum to the vector Lutzomyia longipalpis is xenodiagnosis. More recently, quantitative polymerase chain reaction (qPCR) has been used to model human reservoir competence by assuming that detection of parasite DNA indicates the presence of viable parasites for infecting vectors. Since this assumption has not been proven, this study aimed to verify this hypothesis. The concentration of amastigotes in the peripheral blood of 30 patients with kala-azar was microscopically verified by leukoconcentration and was compared to qPCR estimates. Parasites were identified in 4.8 mL of peripheral blood from 67% of the patients, at a very low concentration (average 0.3 parasites/mL). However, qPCR showed 93% sensitivity and the estimated parasitaemia was over a thousand times greater, both in blood and plasma, with higher levels in plasma than in blood. Furthermore, the microscopic count of circulating parasites and the qPCR parasitaemia estimates were not mathematically compatible with the published proportions of infected sandflies in xenodiagnostic studies. These findings suggest that qPCR does not measure the concentration of circulating parasites, but rather measures DNA from other sites, and that blood might not be the main source of infection for vectors.

Many molecular methods have been used for diagnose is of canine leishmaniasis. These methods mainly rely on tissue specimens provided by invasive approaches for molecular detection of leishmaniasis. The aim of this study was to evaluate two noninvasive sampling methods detection of Leishmaniasis using PCR method. For this purpose, a total of 60 dogs were selected and equally divided into three groups: pet dogs with cutaneous lesions (Group 1); pet dogs without cutaneous lesions (Group 2) and stray dogs (Group 3). After physical examination of dogs, eye swab and buffy coat samples were collected from each dog, also, tissue smears were prepared from the lesions of dogs in group 1. DNA of eye swab and buffy coat samples were extracted and used to detection of Leishmania species using PCR and semi-nested PCR tests. Blood smears were stained by Giemsa method for microscopic evaluation. Out of 60 dog, the DNA of four swab samples related to the dogs of Group3 and one swab and DNA of buffy coat sample in a dog in group 1 was infected with Leishmania infantum. Leishman bodies was only observed in tissue smear of one dog in group 1. Three positive samples were sequenced for confirmation of results. Based on the results of this study, Leishmania infantum infection is common among dogs in Mashhad area and it seems that ocular swab specimens can be used as a noninvasive method for molecular detection of leishmania infection in dogs.

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

Immediately after dental implant insertion, blood will be in direct contact and interact with the implant surface and activates inflammatory responses and complement cascades within seconds. The aim of the present study was to determine the ability of fluoride-modified titanium surfaces to activate complement cascades using the human buffy coat as model. The buffy coats were exposed to hydrofluoric acid-modified surfaces for a short time and its responses were compared to controls. Identification and quantification of complement cascade biomarkers were conducted using ELISA kits and multianalyte profiling using Luminex. A lower level of C3 at 30 min and increased levels of C4, MIP-4, CRP, and pigment epithelium-derived factor at 360 min were found on modified surfaces as compared to controls. We found no significant differences in the levels of C3a, C5a, C Factor H, α2M, ApoA1, ApoC3, ApoE, Prealbumin, α1AT, and SAP in modified surfaces in the buffy coats. We conclude that titanium surfaces treated with hydrofluoric acid modify the levels of specific biomarkers related to the complement cascade and angiogenesis and, thus, tissue growth, remodeling and repair, as this may play a role in the enhanced clinical performance of fluoride-modified Ti dental implants.

In Brazil, Trypanosoma vivax is present in several states. The disease is endemic in Pantanal and Minas Gerais. In Paraná there is still no report of the parasite, but due to the state borders with Mato Grosso do Sul, São Paulo, Paraguay and Argentina, it is believed that the protozoan circulates in the region without diagnosis. The objective of this study was to investigate the prevalence of T. vivax in dairy cattle in the western region of Paraná. For this purpose, 600 blood samples and 400 serum samples were collected from dairy cattle, distributed in 60 and 40 properties, respectively. While buffy coat smears were performed on blood samples, serum samples were used in Indirect Immunofluorescence Reaction. All samples, in both techniques, presented negative results for T. vivax. These results indicate that the studied hemoprotozoan is not circulating among the cattle in the western region of Paraná. However, future work evolving beef cattle must be carried out and preventive measures should be adopted in order to avoid the entry of the parasite in the State of Paraná.

The buffy coat is obtained routinely for disseminated histoplamosis (DH) diagnosis in Ceará, Brazil. The aim of this study is to describe the accuracy of staining smears for Histoplasma in the buffy coat of AIDS-patients with DH. From 2012−2013, all results of stained buffy coat smears and culture for fungi performed at São José Hospital were recorded. In total, 489 buffy coats of 361 patients were studied; 19/361 (5.3%; 95%CI = 2.9−7.6%) had positive direct examination stained smears for Histoplasma and 61/361 (16.9%; 95%CI = 13.0−20.8%) had growth in culture. For those with positive Histoplasma cultures, the CD4 count was significantly lower (139.3 vs. 191.7cells/µL; p = 0.014) than others, and death was 18%. The sensitivity and specificity of stained smears was 25.9% and 100%, respectively. A second test, performed up to 36 days from the first one, increased the sensitivity of stained smears to 32.2%. Stained smears of buffy coat have low accuracy; nonetheless, they are easy to perform and can give a quick diagnosis in low-resource endemic areas. Despite the decrease in mortality, it is not yet to the low levels observed in areas that have better and more efficient methods.

Context: Melioidosis and Brucellosis are important endemic infections among people in India, especially in rural settings. Conventional detection techniques have several limitations. Only a few studies exist on the prevalence of Melioidosis and Brucellosis in rural area especially in India. Aim: We sought to evaluate detection of Burkholderia pseudomallei and Brucella spp. among patients presenting febrile illness. Material and Methods: Previously described polymerase chain reaction ( PCR) assays for both pathogens were evaluated with Deoxyribonucleic acid extracts of buffy coat samples collected from 301 patients recruited prospectively. Data was not amenable to statistical analysis. Results: The PCR showed specific amplification and no non-specific amplification with heterologous Gram-negative bacilli. The lower limit of detection of the assay for B. pseudomallei was determined to be 1 colony-forming unit /mL and for Brucella it was 1.95 x 10 3 plasmids per microliter. Blood culture in automated blood culture system was negative for all the samples. This prospective study carried out in southern India for the first time. PCR for Brucella was positive in 1% of the patient samples whereas 0.3% was positive for B. pseudomallei. Conclusion: The finding of Brucella and Burkholderia infections in our populations leads us to suggest that tests for Brucella and B. pseudomallei should also form part of a diagnostic platform for patients with Pyrexia of unknown origin in tropical developing countries.

Random donor platelet (RDP) is not sufficient to improve the platelet count in most thrombocytopenic patients. Single donor platelet (SDP) or buffy coat pooled platelet (BCPP) are the two choices to provide a full therapeutic dose of platelets. However, there are constraints in the preparation of SDP due to stringent donor selection procedure, time required for procedure, and need of special expensive equipments and kits. BCPP is widely practiced, especially in the European countries, since 1995. In India, we decided to adopt the procedure of buffy coat pooling of platelets, especially for economically backward patients and for emergencies. This study was prospectively conducted from September 2009 to September 2010. A total of 129 units of BCPP [tested prior for viral markers by enzyme-linked immunosorbent assay (ELISA) and individual donor nucleic acid amplification test (ID-NAT)] were issued to 129 patients suffering from dengue and were included in this study. For comparison between efficacy of SDP and BCCP, patients were divided into two groups of 50 each. The post-transfusion platelet counts of the patients were noted after 2 hours of transfusion for each type of component. The platelet yield varied from 2.5 to 4.4 Χ 10ΉΉ in BCPP samples. The samples analyzed were sterile without any contamination. The different biochemical parameters were analyzed in detail. The observed post-transfusion platelet recovery and corrected count increment (CCI) at 1 hour and 24 hours after BCPP transfusion were similar to that after SDP transfusion. Hence, we concluded that BCPP can be a low cost alternative to SDP in the times of emergencies like dengue and non-affordability by the patient for SDP.

In this study, the use of fibrin rich in leukocytes and platelets (L-PRF) was explored to heal osteomyelitis ulcers in a diabetic foot. The goal was to standardize the utilization of L-PRF in patients with osteomyelitis to direct it for healing. L-PRF was obtained autologously from the peripheral blood of the diabetic patients (n = 3) having osteomyelitis and skin lesions for at least six months. The L-PRF and supernatant serum were inserted into the skin lesion to the bone after a surgical debridement. The evolution of lesions over time was analyzed. All three patients showed positivity to the Probe-to-Bone test and Nuclear Magnetic Resonance detected cortico-periosteal thickening and/or outbreaks of spongy cortical osteolysis in adjacency of the ulcer. The infections were caused by Cocci Gram-positive bacteria, such as S. Aureus, S. β-hemolytic, S. Viridans and Bacilli; and Gram-negative such as Pseudomonas, Proteus, Enterobacter; and yeast, Candida. The blood count did not show any significant alterations. To date, all three patients have healed skin lesions (in a patient for about two years) with no evidence of infection. These preliminary results showed that L-PRF membranes could be a new method of therapy in such problematic diseases. Overall, the L-PRF treatment in osteomyelitis of a diabetic foot seems to be easy and cost-effective by regenerative therapy of chronic skin lesions. In addition, it will improve our understanding of wound healing.

Dentre os métodos para a obtenção de hemocomponentes destaca-se o método do plasma rico em plaquetas (PRP) e o método da extração da camada leucoplaquetária (ECLP). Este estudo tem por objetivo comparar os métodos do PRP e da ECLP na produção de hemocomponentes. Foram processadas 88 bolsas de sangue total (ST) pelo método do PRP, 130 bolsas triplas pelo método da ECLP (ECLPT) e 215 bolsas coletadas em bolsas quádruplas pelo método da ECLP (ECLPQ) com o uso de extrator automático. Encontramos diferença estatisticamente significante na quantidade de Hb total /unidade entre ECLPT e ECLPQ (p=0,005) e entre ECLPT e PRP (p=0,007) no ST. Houve diferença estatisticamente entre ECLPT e ECLPQ (pThe most commonly used methods for blood component processing are the "plasma rich in platelets method" (PRP), and the Buffy-coat extraction method (BC).The purpose of this study was to compare these two methods in the processing of blood components. Eighty-eight whole blood units (WB) were processed by the PRP method, 130 blood units were processed by the BC triple blood bag method (BCT) and 215 blood units were collected in quadruple blood bags by the BC method (BCQ) using an automatic extractor. A statistically significant difference was observed in the number in the total Hb per unit of WB between the BCT and BCQ methods (p=0.005) and between the BCT and PRP methods (p=0.007). There were also statistically significant differences between the BCT and BCQ methods (p

The morphological identification of Trypanosoma cruzi is currently considered to have a high specificity, but its sensitivity, which depends on the volume of the sample examined, is rather low. Trypanosome developmental stages suspended in blood, reduviid feces, and culture media are routinely searched for by means of fresh film examination (about 2 µL). High speed centrifugation of blood samples separates the buffy coat, where most trypomastigotes concentrate. As the parasites are transparent and colorless, their detection is mostly dependent on their motility. The fluorescent vital stain acridine orange has been used to enhance image contrast, as exemplified by the QBC (Quantitative Buffy Coat) technique. Staining blood, buffy coat, reduviid feces, and culture media samples with methylene blue (also a vital dye) is a means of producing sharp, well contrasted images of motile or non-motile T. cruzi developmental stages, only standard laboratory microscopes being required. Slides previously coated with a thin layer of methylene blue are used to stain fresh blood films. Photomicrographs exemplify the results of methylene blue staining applied to living and fixed parasites.A identificação morfológica de Trypanosoma cruzi tem alta especificidade, segundo é geralmente aceito; entretanto, sua sensibilidade, dependente do volume da amostra examinada, é baixa. Formas evolutivas de T. cruzi suspensas em sangue, fezes de reduviídeos e meios de cultura são rotineiramente pesquisadas em esfregaços a fresco (cerca de 2 µL). Centrifugação de amostras de sangue a altas velocidades produz a separação do creme leucocitário, onde se concentram as formas tripomastigotas. Em preparações a fresco, a motilidade das formas tripomastigotas e epimastigotas de T. cruzi, protozoário transparente e incolor, facilita sua detecção. Laranja de acridina, corante vital fluorescente, tem sido usada para acentuar o contraste das imagens de parasitas. Disto é exemplo a técnica QBC (Quantitative Buffy Coat). A coloração por meio de azul de metileno (também um corante vital), de amostras de sangue, de fezes de reduviídeos ou de meios de cultura permite obter imagens nítidas e contrastadas de formas evolutivas de T. cruzi com ou sem motilidade. Microscópios de uso geral em laboratórios permitem o exame dos parasitas corados. Uma camada bem delgada de azul de metileno colocada sobre a parte central da lâmina limpa (por meio da evaporação de solução diluída do corante) é usada para corar as preparações a fresco. O aspecto dos parasitas corados em materiais frescos ou previamente fixados pode ser observado em fotomicrografias.

Trypanosomosis is a parasitic disease that causes serious economic losses in livestock, especially in sub-Saharan countries. This study was conducted from October 2010 to March 2011 in the Diga and Sasiga districts of the East Wollega zone in western Ethiopia to determine the prevalence of bovine trypanosomosis and its vectors. A total of 386 blood samples were collected from randomly selected animals. Packed cell volume (PCV) was determined and samples were examined for the presence of trypanosomes using the buffy coat technique. Out of 386 blood samples, 8.55% tested positive for trypanosomes. The majority of the infections were caused by Trypanosoma congolense (72.73%), followed by Trypanosoma vivax (27.27%). There were no statistically significant differences (p > 0.05) between districts, altitudes, sexes and ages, but the prevalence was significantly higher (p < 0.05) in cattle which were in poor body condition. The mean PCV value of infected animals (21.45 ± 3.62 s.d.) was significantly lower (p < 0.05) than that of non-infected animals (26.60 ± 4.60 s.d.). A total of 1151 flies were caught by deploying 21 monoconical shaped traps. Of these flies, 822 (71.42%) were Glossina, whilst the remaining flies were either Stomoxys (17.20%) or Tabanus (11.38%). The overall apparent densities of tsetse and biting flies were 1.45 and 0.58 flies per trap per day, respectively. In conclusion, this study confirmed that trypanosomes and their vectors are prevalent and still pose a threat to cattle production in the area. Therefore, proper strategies have to be designed and implemented to minimise their effect on livestock production.

Fisheries and other human activities pose a global threat to the marine environment. Marine protected areas (MPAs) are an emerging tool to cope with such threats. In the Dutch Wadden Sea, large MPAs (covering 31% of all intertidal flats) have been created to protect shellfish-eating birds and allow recovery of important habitats. Even though shellfish fishing is prohibited in these areas, populations of shellfish-eating birds in the Wadden Sea have declined sharply. The role of shellfish fisheries in these declines is hotly debated, therefore, we investigated the effectiveness of MPAs for protecting oystercatcher (Haematopus ostralegus) populations. Shellfish stocks (cockles, Cerastoderma edule) were substantially higher in the MPAs, but surprisingly this has not resulted in a redistribution of wintering oystercatchers. Oystercatchers in unprotected areas had less shellfish in their diet and lower condition (a combined measure of mass and haematological parameters), and their estimated mortality was 43% higher. It is likely, therefore, that shellfish fishing explains at least part of the 40% decline in oystercatcher numbers in recent years. Condition and mortality effects were strongest in males, and the population sex ratio was female biased, in agreement with the fact that males rely more on shellfish. The unprotected areas apparently function as an "ecological trap," because oystercatchers did not respond as anticipated to the artificial spatial heterogeneity in food supply. Consequently, the MPAs are effective on a local scale, but not on a global scale. Similar problems are likely to exist in terrestrial ecosystems, and distribution strategies of target species need to be considered when designing terrestrial and marine protected areas if they are to be effective.