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\nIntroduction: Serologic characterization of antihuman platelet antigen (HPA) alloantibodies is crucial in fetal neonatal alloimmune thrombocytopenia. The gold standard MAIPA assay requires fresh platelets from HPA-genotyped donors, which is challenging for some laboratories. Megakaryocytes express HPA epitopes and offer an alternative source for detecting anti-HPA antibodies. The objective of this study was to assess the efficacy of a novel assay called monoclonal antibody immobilization of megakaryocyte antigens (MAIMA) for detecting anti-HPA antibodies. Methods: CD34+ cells from buffy coats were differentiated into megakaryocytes in vitro. The performance of the MAIMA assay was evaluated using WHO reference reagents for HPA-1a, HPA-3a, and HPA-5b, along with sera samples from patients who had well-characterized anti-HPA antibodies. Results: The WHO anti-HPA-1a reference reagent showed similar binding to megakaryocytes and platelets in MAIMA and MAIPA, respectively. On the other hand, optical density (OD) values for the WHO anti-HPA-3a reference reagent were lower in MAIMA than in MAIPA. Anti-HPA-5b antibodies were not detectable in MAIMA. Patients’ sera containing anti-HPA-1a antibodies were successfully detected in MAIMA in all clinical samples. Moreover, OD values in MAIPA and MAIMA showed high correlation (r = 0.96, p < 0.001). MAIMA was reactive for samples with anti-HPA-3a as well as anti-HPA-3b; however, OD values were lower compared to MAIPA. Interestingly, all patient samples with anti-HPA-5b antibodies were tested negative in MAIMA. Conclusion: In vitro generated megakaryocytes can be used to detect anti-HPA-1a alloantibodies. However, despite this potential, they may be less suitable for the detection of alloantibodies against other HPAs such as HPA-5b. The identification of antibodies to human platelet antigens (HPA) is crucial. The standard test (MAIPA assay) is difficult because it requires fresh platelets from specific donors. This study investigated a new method, monoclonal antibody immobilization of megakaryocyte antigens (MAIMA), using in vitro generated megakaryocytes from CD34+ cells. The assay was tested against WHO reference reagents and patient samples with known anti-HPA antibodies. Results showed that MAIMA successfully detected anti-HPA-1a antibodies and correlated well with MAIPA. However, MAIMA had lower sensitivity for anti-HPA-3a and anti-HPA-5b antibodies. Notably, anti-HPA-5b antibodies were not detectable by MAIMA. In conclusion, in vitro generated megakaryocytes can effectively detect anti-HPA-1a antibodies. However, MAIMA may be less suitable for antibodies against other HPAs, such as HPA-5b. This study highlights the potential of MAIMA as an alternative assay and provides insight into its strengths and limitations in detecting specific anti-HPA antibodies. [ABSTRACT FROM AUTHOR]

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.

Background: Granulocyte concentrates (GCs) are usually prepared by single-donor apheresis after G-CSF pretreatment and have to be transfused within 24 h after cell collection because of the rapid decrease in pH and cell survival due to high lactate production by red blood cell contamination. GCs pooled from buffy coats of whole blood donations could improve the availability of these products. Methods to reduce red blood cell and platelet contamination may improve storability. We developed a manufacturing process for pooled GCs and investigated cell viability and functionality over time. Methods: Six ABO blood group-identical buffy coats were pooled. Subsequently, the red blood cells spontaneously sedimented after the addition of hydroxyethyl starch. The resulting leukocyte-enriched supernatant was washed twice with saline to reduce platelets and was resuspended in ABO-identical donor plasma. The leukocyte concentrate was transferred to a platelet storage bag and stored up to 72 h at 20–24°C w/o agitation. Cell count and viability, pH, blood gases, phagocytosis, and oxidative burst activity were monitored. Results: The number of red blood cells and platelets was reduced to 0.4% and 6.1% of the baseline levels. About 50% of the original present leukocytes could be extracted (n = 76). In the course of 72 h of storage, there were no significant changes in white blood cell counts (p = 0.12). The viability exceeded 98% during the entire period. The rate of granulocytes performing phagocytosis and oxidative burst remained above 95% anytime. Conclusion: GCs prepared from pooled buffy coats provide a precious alternative to granulocytes obtained from apheresis. Reduction of red blood cells and platelets by more than 90% extends the maximum shelf life of GCs from 24 h to 72 h. For a therapeutic dose of at least 1 × 1010 granulocytes, 15–20 buffy coats are required. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

Cell-free extrachromosomal circular DNA (cf-eccDNA) has been proposed as a promising early biomarker for disease diagnosis, progression and drug response. Its established biomarker features are changes in the number and length distribution of cf-eccDNA. Another novel promising biomarker is a set of eccDNA excised from a panel of genes specific to a condition compared to a control. Deficiencies in two endonucleases that specifically target DNA, Dnase1 and Dnase1l3, are associated with systemic lupus erythematosus (SLE). To study the genic eccDNA profiles in the case of their deficiencies, we mapped sequenced eccDNA data from plasma, liver and buffy coat from Dnase1 and Dnase1l3 knockouts (KOs), and wild type controls in mouse. Next, we performed an eccDNA differential analysis between KO and control groups using our DifCir algorithm. We found a specific genic cf-eccDNA fingerprint of the Dnase1l3 group compared to the wild type controls involving 131 genes; 26% of them were associated with human chromosomal fragile sites (CFSs) and with a statistically significant enrichment of CFS-associated genes. We found six genes in common with the genic cf-eccDNA profile of SLE patients with DNASE1L3 deficiency, namely Rorb, Mvb12b, Osbpl10, Fto, Tnik and Arhgap10; all of them were specific and present in all human plasma samples, and none of them were associated with CFSs. A not so distinctive genic cf-eccDNA difference involving only seven genes was observed in the case of the Dnase1 group compared to the wild type. In tissue—liver and buffy coat—we did not detect the same genic eccDNA difference observed in the plasma samples. These results point to a specific role of a set of genic eccDNA in plasma from DNase KOs, as well as a relation with CFS genes, confirming the promise of the genic cf-eccDNA in studying diseases and the need for further research on the relationship between eccDNA and CFSs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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

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 < 0.05 (4049 hypomethylated but only 50 hypermethylated CpGs in discovery, and 63 hypomethylated but only 6 hypermethylated CpGs in validation). The results were the same even after controlling for age, storage duration as buffy coat prior to DNA extraction, and estimated cell type composition. Furthermore, we find that in both cohorts, CpGs have a greater likelihood to be hypomethylated the closer they are to a CpG island; except for CpGs at the CpG islands themselves which are less likely to be hypomethylated. Conclusion: Cryopreservation of DNA after a few years results in a detectable bias toward hypomethylation at the level of individual CpG methylation, though when analyzed in aggregate there is no detectable change in mean global methylation. Studies profiling methylation in cryopreserved DNA should be mindful of this hypomethylation bias, and more attention should be directed at developing more stable methods of DNA cryopreservation for biomedical research or clinical use. [ABSTRACT FROM AUTHOR]

BACKGROUND: Leukocytes are responsible for producing both immune and nonimmune adverse reactions, and therefore, various methods have been developed to remove them from the blood components before transfusion. AIM: The aim of this study was to analyze the quality parameters in leukoreduced red cell concentrates (RCCs) and investigate the efficiency of leukocyte removal and red cell recovery in the leukoreduction methods followed in our center. MATERIALS AND METHODS: The study evaluated the quality parameters in 112 RCCs prepared using buffy-coat reduction by the Terumo automatic component extractor II+ system, manual saline washing, and leukofiltration using the Leucolab filter system. RESULTS: With analysis, leukofiltration was found to be the most efficient in reducing leukocyte content in RCCs, achieving a mean leukoreduction of 99.99%. Buffy-coat reduction and saline washing achieved a leukoreduction of 78.54% and 82.67%, respectively. While filtration showed the least red cell recovery of 81.93% compared to 90.57% in buffy-coat reduction and 91.87% in saline washing methods. An analysis of hemoglobin content showed that none of the buffy-coat removed RCCs processed from 350-ml collections and underwent poststorage leukofiltration could meet the European Standards for minimum hemoglobin content. CONCLUSION: Filtration is found to be the better method for leukoreduction of RCCs. It is suggested to perform a single method of leukoreduction preferably leukofiltration for maximum red cell recovery. [ABSTRACT FROM AUTHOR]

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

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. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

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

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

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. [ABSTRACT FROM AUTHOR]

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

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.

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.

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.

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: Platelet-rich plasma is an autologous blood preparation which is used in various medical specialties because of its regenerative properties. There is a wide variation in platelet-rich plasma preparation protocols and attaining the ideal platelet yield (>1 million platelets/μL) in a clinic setting can be challenging. We aimed at analyzing the centrifuge spin rates at which to attain an ideal platelet-rich plasma yield and also to study the effect of inclusion of the buffy coat after the first spin on the final platelet concentration in platelet-rich plasma.Methods: Seventy-five whole blood samples were obtained and divided into two groups - (1) leukocyte-rich platelet-rich plasma group and (2) leukocyte-poor platelet-rich plasma group. Samples in both groups were centrifuged using the dual spin method, at one of three centrifugation speed combinations (initial "soft" spin and second "hard" spin speeds, respectively): (1) 100 g/400 g, (2) 350 g/1350 g and (3) 900 g/1800 g. Platelet, red blood cell (RBC) and white blood cell (WBC) counts in both groups were compared.Results: The 100 g/400 g spin gave a high platelet yield (increase of 395.4 ± 111.1%) in the leukocyte-poor-platelet-rich plasma group, while in the leukocyte-rich platelet-rich plasma group both 100 g/400 g and 350 g/1350 g spins resulted in significantly higher yields with an increase of 691.5 ± 316.3% and 738.6 ± 193.3%, respectively.Limitations: The study was limited by a smaller sample size in the pure platelet-rich plasma (leukocyte-poor platelet-rich plasma) group.Conclusion: Ideal platelet yields can be achieved with both the 100 g/400 g as well as the 350 g/1350 g spins using the buffy coat inclusion method while the 100 g/400 g spin for "pure" platelet-rich plasma accomplishes a near-ideal platelet count with significantly reduced contamination with other cells. [ABSTRACT FROM AUTHOR]

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 (BC-PC), and Single Donor Platelets units (SDP). 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, BC-PC and apheresis methods Materials and Methods: Laboratory based observational study done at rural, tertiary care, post graduate, 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, 52units BC-PC method and the remaining were 52 units of SDP prepared by apheresis method. Quality parameters studied include volume, pH, swirling, White Blood Cell count (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, pCO2, 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. A score of 5 was obtained for 58 out of 156 cases for SDP, 56 out of 156 cases for BC-PC, 42 out of 156 cases for PRP-PC units with respective percentage of 37.1%, 33.3% and 26.9%, 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. [ABSTRACT FROM AUTHOR]

Bovine trypanosomiasis is a major threat to the livestock sector of Cameroon. The main objective of this study was to determine the prevalence of bovine trypanosomes and vectors in the SODEPA Ranch of Ndokayo. A cross sectional parasitological survey was carried out in the late dry season, where blood was collected through the jugular vein of 390 cattle. The trypanosome detection in cattle blood was carried out using the buffy coat technique (BCT). An entomological prospection was carried out in the late dry season and early rainy season using two unbaited trap types notably Nzi (n = 4) and Vavoua (n = 4). The overall bovine trypanosomiasis prevalence was 2.82% and species specific prevalence included: Trypanosoma congolense (63.6%), Trypanosoma brucei (18.2%) and mixed species infections (T. congolense + T. brucei and T. brucei + T. vivax) (18.2%). Sixteen (16) species of flies were identified and grouped under six genera: Stomoxys, Ancala, Tabanus, Chrysops, Hematopota and Glossina. The overall trap apparent density (ADT) was 3.92 flies/trap/day, with a higher ADT in early rainy season than in the late dry season. The highest species richness was noticed with the genus Tabanus, while the highest ADT was found in Stomoxyini. The Nzi trap recorded a higher mean ADT for most fly vectors than Vavoua, except for Stomoxyini that rather recorded a higher ADT with the Vavoua trap. The present findings will instruct fly control authorities of Cameroon to better manage trypanosomiasis and vectors in Ndokayo.

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.

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.

Density gradient centrifugation is a conventional technique widely utilized to isolate bone marrow mononuclear cells (BM-MNC) from bone marrow (BM) aspirates obtained from pediatric B-cell acute lymphoblastic leukemia (B-ALL) patients. Nevertheless, this technique achieves incomplete recovery of mononuclear cells and is relatively time-consuming and expensive. Given that B-ALL is the most common childhood malignancy, alternative methods for processing B-ALL samples may be more cost-effective. In this pilot study, we use several readouts, including immune phenotype, cell viability, and leukemia-initiating capacity in immune-deficient mice, to directly compare the density gradient centrifugation and buffy coat processing methods. Our findings indicate that buffy coat isolation yields comparable BM-MNC product in terms of both immune and leukemia cell content and could provide a viable, lower cost alternative for biobanks processing pediatric leukemia samples.

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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. [ABSTRACT FROM AUTHOR]

Detection of donor-specific anti-HLA antibodies (DSA) has been associated with graft rejection in all forms of transplantation. The mechanism by which DSA increase the risk of graft failure remains unclear. We hypothesized that complement-binding DSA are associated with engraftment failure in hematopoietic stem cell transplantation (HSCT) and analyzed 122 haploidentical transplant recipients tested prospectively for DSA. Retrospective analysis to detect C1q binding DSA (C1q+DSA) was performed on 22 allosensitized recipients. Twenty-two of 122 patients (18%) had DSA, 19 of which were women (86%). Seven patients with DSA (32%) rejected the graft. Median DSA level at transplant for patients who failed to engraft was 10,055 mean fluorescence intensity (MFI) versus 2065 MFI for those who engrafted (P = .007). Nine patients with DSA were C1q positive in the initial samples with median DSA levels of 15,279 MFI (range, 1554 to 28,615), compared with 7 C1q-negative patients with median DSA levels of 2471 MFI (range, 665 to 12,254) (P = .016). Of 9 patients who were C1q positive in the initial samples, 5 patients remained C1q positive at time of transplant (all with high DSA levels [median, 15,279; range, 6487 to 22,944]) and experienced engraftment failure, whereas 4 patients became C1q negative pretransplant and all engrafted the donor cells (P = .008). In conclusion, patients with high DSA levels (>5000 MFI) and complement-binding DSA antibodies (C1q positive) appear to be at much higher risk of primary graft failure. The presence of C1q+DSA should be assessed in allosensitized patients before HSCT. Reduction of C1q+DSA levels might prevent engraftment failure in HSCT.

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

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.

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

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).

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. [ABSTRACT FROM AUTHOR]

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.

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

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

Human biobanks are collections of biological samples and health information that allow the organization of biomedical research for upgrading the knowledge of human disorders from different diseases (cancer, allergies, rare diseases, etc.), and reach real answers for diagnosis and treatment. A wide range of samples can be stored in these biorepositories such as hair, nails, urine, tissue, whole blood, red blood cells, buffy coat, plasma, serum, DNA, and RNA. Among these, buffy coat and whole blood are widely used by researchers because they can obtain DNA and RNA from these matrices. Some preliminary studies have been performed on animals to evaluate the quality and functionality of the nucleic acids obtained from some of these matrices, although more in-depth studies are needed in this area. In this study, blood samples extracted by venipuncture from 30 healthy volunteers were used to obtain DNA from buffy coat and whole blood. The purity and integrity of the nucleic acids obtained were assessed by spectrophotometry, fluorimetry, and agarose electrophoresis, and functionality was assessed by PCR and real-time PCR. Another aspect tested in this study was based on the comparison between short-term and long-term storage at −80°C and fresh samples from both matrices to evaluate the storage conditions at the biobank. Results showed differences in the yield obtained from both matrices as a function of the storage time, although the functionality of all the obtained DNA remained intact. [ABSTRACT FROM AUTHOR]

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.