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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.

Animal trypanosomosis is a significant livestock disease with economic and social repercussions, reducing the supply of animal products and restricting the utilization of animals for traction and transportation. In Ethiopia, it is prevalent and poses a major hindrance to the advancement of animal production. This repeated cross-sectional study was aimed at assessing seasonal variation in bovine trypanosomosis prevalence and tsetse fly density and identifying the potential risk factors in the Loka Abaya and Derara districts of the Sidama National Regional State. Blood samples were collected from 964 cattle, 484 samples during the dry season, and 480 during the wet season. The buffy coat method was employed to analyze these samples. Furthermore, 78 standard NGU traps were set up at various locations in the two districts during both seasons for entomological investigation. The overall apparent prevalence of trypanosomosis was 9% (95% CI 7.3–11.0), without a significant difference (p > 0.05) between the dry season (7.4%) and wet season (10.6%). The apparent prevalence was significantly higher in Loka Abaya (11.8%) than in Derara (6.3%) district (OR = 2.04; p = 0.003) and in cattle with black coat color (29%) than in mixed color (6.8%) (OR = 5.3; p < 0.001). The majority of infections were caused by Trypanosoma congolense (70%), followed by T. vivax (29%), and mixed infections (1%) with the two species. The average packed cell volume (PCV) was significantly (p < 0.0001) lower in infected animals (20.7 ± 4%) compared to uninfected ones (25.5 ± 5.4%), in cattle examined during the dry season (24.1 ± 6%) versus the wet season (26.1 ± 4.7%), in cattle sampled from the Loka Abaya district (24.2 ± 5.5%) versus Derara district (26 ± 5.3%), and in cattle with poor body condition (23.6 ± 5.7%) compared to those with good body condition (26.5 ± 5.3%). A total of 5282 flies were captured during the study, with 4437 (84%) being tsetse flies (Glossina pallidipes), 439 (8.3%) Tabanids, 190 (3.6%) Stomoxys spp., and 216 (4.1%) Musca spp. The apparent density (AD) of G. pallidipes was 28.4 flies/trap/day, showing no statistically significant difference between wet (32.1) and dry (24.6) seasons (p > 0.05). The AD of G. pallidipes was significantly (p < 0.001) higher in the Loka Abaya district (57.3) than in the Derara district (0.9). The study highlights a moderate trypanosomosis apparent prevalence and high AD of G. pallidipes, showing significant variation between the study districts but no seasonal difference. The observed apparent prevalence of trypanosomosis and tsetse fly density notably affects animal health and productivity. As a result, strategies for vector control like insecticide-treated targets, trypanocidal medications for infected animals, and community-based initiatives such as education and participation in control programs are recommended. [ABSTRACT FROM AUTHOR]

Background and Objectives: Regulatory requirement of fixed holding time (6 h) of whole blood (WB) at room temperature, that is, 22–24°C (RT) results in sub‐optimal component separation. The aim was to evaluate the platelet concentrates (PC) prepared by both platelet rich plasma (PRP) and buffy coat (BC) methods after overnight hold (18–24 h) at RT. Materials and Methods: A prospective experimental study was performed. A total of 48 WB units collected were divided into four groups (12 each) control‐1 (C1) and test‐1 (T1) for PRP and control‐2 (C2) and test‐2 (T2) for the BC method. Control groups were processed within 6 h, and in test groups, components were prepared after overnight hold, followed by evaluation of quality parameters. Results: Irrespective of the method used, all PCs had similar volume, platelet yield, swirling, no bacterial contamination, RBC contamination, PaO2 and PaCO2 levels. PCs in the T1 group had significant differences in glucose and MPV values on d1, which were resolved by d5 of storage. PCs in T2 has significant differences in pH, glucose, and MPV levels throughout storage. PRBC in test and control groups had similar quality parameters till d42 of storage. FFPs in all tests were noninferior to the concurrent control groups till 3 months of storage. Conclusion: Overnight holding of WB had no lasting deleterious changes. Though a few biochemical parameters in the test groups were significantly different, they can be accepted to improve the logistics of component separation. Overall PRP method seemed to have a better result than the BC method after an overnight hold. [ABSTRACT FROM AUTHOR]

\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]

Preparing maximum platelets from all daily blood donations is essential to maintain adequate stock of platelets that may be feasible by storing buffy coat overnight. The overnight stored buffy coat method is prevalent in Europe and was not studied much in India. Therefore, a comparative study was planned to compare RDPs prepared from fresh and overnight stored buffy coat methods. In this study total of eighty RDPs were prepared. Forty RDPs were prepared by each method by matching similar platelet counts in whole blood in pairs and avoiding other bias. Quality analysis was done in various hematological, biochemical, metabolic, and activation parameters and with National guideline. RDPs prepared by the overnight stored buffy coat method had statistically higher mean platelet counts, lesser WBC contamination, lesser glucose concentration, higher lactate concentration, lesser pH, and higher MPV & PDW than RDPs prepared by the fresh buffy coat method. P selectin levels were lesser in the overnight stored category. The platelet yield in RDPs (extraction from the whole blood) was more in the overnight stored category. Compared with the national guideline, 87.5% of units passed the quality criteria in the fresh category compared to 100% of units in the overnight category. RDPs prepared from overnight stored buffy coats not only had better quality parameters but also were in a less activated state. Because of better quality and feasibility in blood centers, the overnight stored buffy coat method may also be used along with the traditional preparation method. [ABSTRACT FROM AUTHOR]

Acetaminophen and ibuprofen are widely used over-the-counter medications to reduce fever, pain, and inflammation. Although both drugs are safe in therapeutic concentrations, self-medication is practiced by millions of aged patients with comorbidities that decrease drug metabolism and/or excretion, thus raising the risk of overdosage. Mitochondrial dysfunction has emerged as an important pathomechanism underlying the organ toxicity of both drugs. Assessment of mitochondrial oxygen consumption in peripheral blood cells is a novel research field Cu several applications, including characterization of drug toxicity. The present study, conducted in human platelets isolated from blood donor-derived buffy coat, was aimed at assessing the acute, concentration-dependent effects of each drug on mitochondrial respiration. Using the high-resolution respirometry technique, a concentration-dependent decrease of oxygen consumption in both intact and permeabilized platelets was found for either drug, mainly by inhibiting complex I-supported active respiration. Moreover, ibuprofen significantly decreased the maximal capacity of the electron transport system already from the lowest concentration. In conclusion, platelets from healthy donors represents a population of cells easily available, which can be routinely used in studies assessing mitochondrial drug toxicity. Whether these results can be recapitulated in patients treated with these medications is worth further investigation as potential peripheral biomarker of drug overdose. [ABSTRACT FROM AUTHOR]

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]

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 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.

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.

Measuring circulating tumor cells (CTCs) or circulating cancer stem cells (CCSCs) in blood, which shed from primary tumors, is a noninvasive method to screen and/or diagnose patients with a high risk of developing metastatic cancers or relapse. Here, we describe an optimized and standardized laboratory method for isolating CCSCs from human blood samples, using cancer-specific stem cell biomarkers (Kantara et al., Lab Invest 95:100-112, 2015). When performing this technique, 0-1 circulating epithelial tumor cells/mL blood should be expected in patients free of malignant adenocarcinomas compared to over 3 circulating cancer stem cells/mL blood in patients positive for malignant adenocarcinomas (Kantara et al., Lab Invest 95:100-112, 2015)., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)