Blood Fractions Research Articles

SNORD3A: a new possible circulating biomarker of human heart failure

Abstract Background Heart failure (HF) is a complex clinical syndrome and the leading cause of mortality, morbidity and hospitalization in industrialized countries. Human cardiac biopsies are invaluable resources for identifying cardiac signaling pathways, however blood fractions and peripheral blood mononuclear cells (PBMCs) could be used as a source of surrogate biomarkers of signaling pathway activation in human heart failure. Purpose In the present study we aimed to identify novel circulating biomarkers mirroring human myocardial signaling in HF and to study the role played by SNORD3A in cardiomyocyte survival and death. Methods Cardiac left ventricle samples and PBLs from 8 HF patients undergoing heart transplantation and 2 control patients (CTRL) were analyzed by RNA sequencing (RNASeq) to identify transcripts that were regulated in both hearts and PBLs. Five identified transcripts, KCNQOT1, MIAT, SCRN1, MALAT1 and SNORD3A, were then validated by quantitative real-time PCR in PBMCs and in LVs. Next, we analyzed the expression of the Small Nucleolar RNA (snoRNA) SNORD3A in LVs and PBMCs from 8-week-old wild type C57BL/6 mice with pressure overload-induced HF by transverse aortic constriction (TAC). Sham-operated mice (sham) were used as controls. To further investigate the role of SNORD3A in cardiomyocyte function and survival, SNORD3A antisense oligonucleotides (SNOaso) and scramble control sequences (GFPaso) were synthesized and tested in cardiomyoblasts H9C2 cells under normoxic or hypoxic conditions to evaluate SNORD3A transcription levels, cell death and protein synthesis. Results SNORD3A expression was significantly increased in both LVs and PBMCs from HF patients compared to control subjects. Consistent with these results, SNORD3A levels were increased both in PBMCs and LVs from TAC mice compared to sham. In vitro hypoxia significantly increased SNORD3A expression levels in H9C2 cells, compared to normoxia. After SNOaso transfection, SNORD3A transcripts were downregulated, and they were further reduced following 3-hour-hypoxia. Depletion of SNORD3A levels increased cardiomyocyte death and reduced protein synthesis in H9C2 cells. Conclusions Our data suggest that SNORD3A might be involved in the regulation of cardiomyocyte survival in HF and could be useful for diagnostic and prognostic applications in HF.

Proteomic analysis of post-COVID condition: Insights from plasma and pellet blood fractions

BackgroundPersistent symptoms extending beyond the acute phase of SARS-CoV-2 infection, known as Post-COVID condition (PCC), continue to impact many individuals years after the COVID-19 pandemic began. This highlights an urgent need for a deeper understanding and effective treatments. While significant progress has been made in understanding the acute phase of COVID-19 through omics-based approaches, the proteomic alterations linked to the long-term effects of the infection remain underexplored. This study aims to investigate these proteomic changes and develop a method for stratifying disease severity. MethodsUsing Sequential Window Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH-MS) technology, we performed comprehensive proteomic profiling of blood samples from 65 PCC patients. Both plasma and pellet (cellular components) fractions were analyzed to capture a wide array of proteomic changes associated with PCC. ResultsProteomic profiling revealed distinct differences between symptomatic and asymptomatic PCC patients. In the plasma fraction, symptomatic patients exhibited significant upregulation of proteins involved in coagulation, immune response, oxidative stress, and various metabolic processes, while certain immunoglobulins and proteins involved in cellular stress responses were downregulated. In the pellet fraction, symptomatic patients showed upregulation of proteins related to immune response, coagulation, oxidative stress, and metabolic enzymes, with downregulation observed in components of the complement system, glycolysis enzymes, and cytoskeletal proteins. A key outcome was the development of a novel severity scale based on the concentration of identified proteins, which correlated strongly with the clinical symptoms of PCC. This scale, derived from unsupervised clustering analysis, provides precise quantification of PCC severity, enabling effective patient stratification. ConclusionsThe identified proteomic alterations offer valuable insights into the molecular mechanisms underlying PCC, highlighting potential biomarkers and therapeutic targets. This research supports the development of tailored clinical interventions to alleviate persistent symptoms, ultimately enhancing patient outcomes and quality of life. The quantifiable measure of disease severity aids clinicians in understanding the condition in individual patients, facilitating personalized treatment plans and accurate monitoring of disease progression and response to therapy.

Shedding light on the effects of blood on meniscus tissue: the role of mononuclear leukocytes in mediating meniscus catabolism

Traumatic meniscal injuries can cause acute pain, hemarthrosis (bleeding into the joint), joint immobility, and post-traumatic osteoarthritis (PTOA). However, the exact mechanism(s) by which PTOA develops following meniscal injuries is unknown. Since meniscus tears commonly coincide with hemarthrosis, investigating the direct effects of blood and its constituents on meniscus tissue is warranted. The goal of this study was to determine the direct effects of blood and blood components on meniscus tissue catabolism. Porcine meniscus explants or primary meniscus cells were exposed to whole blood or various fractions of blood for 3days to simulate blood exposure following injury. Explants were then washed and cultured for an additional 3days prior to collection for biochemical analyses. Whole blood increased matrix metalloproteinase (MMP) activity. Fractionation experiments revealed blood-derived red blood cells did not affect meniscus catabolism. Conversely, viable mononuclear leukocytes induced MMP activity, nitric oxide (NO) production, and loss of tissue sulfated glycosaminoglycan (sGAG) content, suggesting that these cells are mediating meniscus catabolism. These findings highlight the potential challenges of meniscus healing in the presence of hemarthrosis and the need for further research to elucidate the in vivo effects of blood and blood-derived mononuclear leukocytes due to both hemarthrosis and blood-derived therapeutics.

Amino acid distribution in blood following high-intensity interval exercise: a preliminary study.

This study investigated the effect of high-intensity interval exercise on total and individual amino acid concentrations in red blood cells (RBCs) and plasma. Seven males (31 ± 13yr) provided venous blood samples at rest, immediately and 15min and 30min following an 8-min high-intensity exercise bout. The exercise bout was 16 × 15s cycle efforts at 0.4N/kg of body mass and 90rpm, interspersed with 15s passive recovery. Total and individual amino acid concentrations of RBC and plasma and blood cell parameters were analysed. No significant differences for total amino acid concentrations between RBC and plasma were found. Individual amino acid analyses showed significant interaction effects for alanine and α-aminoadipic acid (P < 0.05), with plasma alanine significantly increased from baseline across the recovery period (P < 0.001). Blood fraction (group) effects showed greater concentrations of glycine, serine, asparagine, aspartic acid, glutamic acid, α-aminoadipic acid and ornithine in RBC, while greater concentrations of alanine, α-aminobutyric acid, valine, leucine, isoleucine, threonine, proline, phenylalanine, glutamine, tryptophan and cystine were found in plasma (P < 0.05). Comparable levels of histidine, lysine and tyrosine were observed between blood fractions. Significant differences in the variation of total amino acids in RBC were reported with higher variance at rest compared to following exercise (P = 0.01). Haemoglobin, pack cell volume and white blood cell count significantly increased immediately following exercise (P < 0.05) but returned to baseline after 15min recovery. These results support the notion of individualised amino acid transportation roles for RBC and plasma during exercise.

Identification and quantification of very small embryonic-like (VSEL) stem cells in fresh umbilical cord blood

The present study aimed to identify, quantify and compare the quantities of very small embryonic-like (VSEL) stem cells in the two main fractions of fresh umbilical cord blood (UCB). To do this, UCB obtained during caesarean section underwent gradient centrifugation. Nucleated cells isolated from the two main fractions – the mononuclear cell layer above the gradient, and the red blood cell pellet below the gradient – were immunofluorescently labelled to identify the VSELs (CD45−/CD133+/SSEA4+). These two cell fractions were then analysed via multiparameter sorting on a flow cytometer and the quantities of positively stained cells were compared.

КОЛОСТРАЛЬНЫЙ ИММУНИТЕТ ТЕЛЯТ НА ФОНЕ ИММУНОКОРРЕКЦИИ ОРГАНИЗМА КОРОВ-МАТЕРЕЙ

The aim of this work was to evaluate the effect of immunostimulating drugs Salus-P-E and Bovistim-K on the quality of colostrum and the immune status of calves after drinking colostrum. Work was carried out on first-calf heifers of the Holstein breed. Three groups of deep-calving cows were selected according to the principle of analogue groups of 10 animals each. Cows of the 1st experimental group were injected intramuscularly in the middle third of the neck with Salus-P-E at a dose of 10 ml three times 60, 30 and 15 days before the expected date of calving, the 2nd experimental group – Bovistim-K at the same time and dose, in the control group, biological preparations were not used. Colostrum sampling was carried out twice: within 60 minutes after calving and 24 hours after calving. In the second series of experiments, we studied the clinical and physiological state and blood parameters of newborn calves after drinking colostrum. The colostrum of cows of the 1st and 2nd experimental groups contained more immunoglobulins than control samples by 23,8 and 27,67 g/l; total protein - by 3,08 and 3,32%; the level of caseins – by 0,34 and 0,22%, respectively. Against the background of immunocorrection of the organism of deep-calving mother cows, there is an increase in the amount of hemoglobin, total protein, reserve alkalinity and certain changes in the ratio of protein fractions of blood of newborn calves. In the first day of life, an increase in the blood serum of calves of the experimental groups was found compared to the control: albumin by 7,9 and 8,4%; α-globulins – by 18,6 and 15,7%, β-globulins – by 22,1 and 16,9%, γ-globulins by 24,4 and 21,4%, respectively. Drinking high-quality colostrum (Brix over 24) contributed to an increase in average daily gains in calves of the 1st and 2nd experimental groups by 18,2 and 20,2%. The use of immunostimulating drugs Salus-P-E and Bovistim-K increases the immunocompetent properties of colostrum, which contributes to the formation of a high level of colostral immunity in the body of newborn calves, reducing morbidity and improving growth and survival rates.

Study of Release of Biologically Active Compounds from Cord Blood Under Different Conditions of Low-Temperature Impact

Here, we have studied the impact of cord blood destruction method on composition of the cord blood-derived low molecular fractions, and compared the cryodestruction with other methods of cell destruction before extracting. Human cord blood was destroyed by rapid or slow freezing / warming, hypotonic lysis and thermal destruction. The obtained substance was used to produce the cord blood fraction (CBF) by multi-stage ultrafiltration and lyophilization. Dry weight, CBF composition and total protein content in them were evaluated by chromatographic profiles (gel permeation and reverse-phase high-performance liquid chromatography). The CBFs, obtained by diff erent techniques for cord blood destruction were established to diff er in the content and molecular weights of the components. These fi ndings suggest the possibility to vary the amount and range of low molecular weight compounds in lyophilized cord blood fractions by using low temperatures and combining diff erent regimens of freezing / warming. Key words: cryodestruction, cord blood, biologically active substances, low-molecular fraction, low temperatures

An Integrated Multimodal Framework for Noninvasive TCL Disease Detection and Monitoring

Background: Early noninvasive identification of therapeutic responses could help accelerate the development of more effective therapies for T-cell lymphoma (TCL), and to improve patient outcomes. However, standardized noninvasive disease-monitoring strategies remain elusive for the most common mature T-cell tumors. Therefore, the development of highly sensitive and accurate disease monitoring methods for TCL represents a strong unmet clinical need. We tackled this challenge using an integrated liquid biopsy strategy leveraging both cell-free DNA (cfDNA) and cell-free RNA (cfRNA), by simultaneously interrogating tumor-specific aberrations, including somatic mutations, clonotypic TCR rearrangements, and TCL gene expression signatures. Methods: We studied 83 serial blood samples from 36 patients diagnosed with TCL and treated in the US or Japan, including 63 longitudinal samples from 30 cases from Stanford University receiving standard TCL therapies (NCT00398177), and 20 blood samples from 6 subjects with relapsed/refractory TCL treated in Kyushu University hospital. Cell-free DNA samples for all cases were profiled to simultaneously detect somatic mutations by CAPP-Seq (Newman et al 2016 Nature Biotech) as well as clonotypic VDJ rearrangements by SABER (Shukla et al 2020 ASH, Sworder et al 2022 Cancer Cell). Therefore, an integrated CAPP-Seq targeted panel was designed for hybrid capture to target recurrently mutated genes in diverse T-cell neoplasms along with TCR rearrangements at 4 loci. Captured libraries were sequenced to a median deduplicated depth of ~3000x. For CAPP-Seq, single nucleotide variants (SNVs) were identified by the analysis of tumor tissue or pre-treatment cfDNA; constitutional germline variants and clonal hematopoiesis (CHIP) variants were censored using matched DNA from either CD4+ T-cell depleted PBMCs or buccal swabs. For SABER, tumor clonotypic VDJ sequences were defined as most prevalent clone in tumor tissue or pretreatment plasma cfDNA. Plasma cfRNA was profiled for a subset of patients (20 serial samples from 13 patients) using RARE-Seq (Nesselbush et al, in preparation). We then measured correlations between radiographic disease status and circulating disease levels, as measured using mutant ctDNA and clonotypic TCR molecules, and cfRNA gene expression signatures. Results: Among the 36 TCL subjects, all cases (100%) could be noninvasively detected in plasma cfDNA using either somatic mutations (mutant ctDNA only: 86%) or using clonotypic cell-free TCR rearrangements (cfTCR only: 88%). When considering the concentrations of these distinct tumor reporters, mutant ctDNA levels (hGE/mL plasma) and cfTCR levels (# of clones/mL plasma) were significantly correlated (Pearson R = 0.65, p&amp;lt;0.01, Fig. 1A). When considering longitudinal measurements during and after therapy, circulating levels of both ctDNA and clonotypic cfTCR were significantly lower in patients experiencing objective responses (CR/PR) than in patients experiencing SD/PD (p&amp;lt;0.01). Moreover, when comparing acellular plasma versus cellular (PBMC) blood fractions, circulating disease levels were significantly higher in plasma and often became undetectable in PBMCs (Fig. 1B). When considering TCR repertoires, CDR3 diversity was significantly lower at timepoints of measurable disease (pre-treatment, SD, PD). Furthermore, clonotypic tumor cell-free TCR (tumor cfTCR) fragments were significantly shorter than non-tumor cfTCR molecules (p&amp;lt;0.01). When considering cfRNA, a gene expression signature of activated CD4 memory T-cells was strongly correlated with both mutant ctDNA and tumor cfTCR levels (R=0.85, 0.64), and was higher at timepoints of active TCL disease. Conclusions: Noninvasive TCL detection and monitoring appears feasible through liquid biopsies interrogating cfDNA and cfRNA, with advantages over PBMCs. The complementarity for disease detection using tumor-specific somatic mutations, clonotypic TCR rearrangements, and gene expression levels, strongly suggests synergies to enable an integrated multimodal liquid biopsy framework for TCL. This novel platform should allow more accurate assessment of disease status and MRD, and to inform risk-adapted treatment strategies in TCL.

Investigation of Clonal Hematopoiesis in New Japanese Cohort

Introduction: Clonal hematopoiesis (CH) is an age-related change in which blood cells with somatic mutations are clonally expanded. CH is known to be a predisposing factor for various age-related diseases. However, the characteristics of blood cells with somatic mutations derived from CH at the single-cell level is not fully understood. Objective: We performed this study to explore the comprehensive properties of mutant cells derived from CH. Methods: We enrolled 51 healthy elderly individuals (male, 13; female, 38) from the Kashiwanoha cohort for this study. To investigate somatic mutations frequently mutated in CH, we designed a custom panel targeting 49 genes. Targeted deep sequencing (TDS) was performed on mononuclear cells, and T, B, and monocyte fractions of peripheral blood (PB). Error-correction process was performed on TDS data. The error-correction process to the TDS data was as follows: Firstly, read families which have more than 5 identical unique molecular identifier (UMI) amplicon were included. Second, at each position, nucleotides were compared and a consensus nucleotide was called if at least 90% the nucleotides were identical. If the agreement was below 90%, the nucleotides were changed to “N ”at that position. Third, if the “N” constituted less than 10% of a read family, it was recognized as a consensus read. Single-cell multiome analysis (sc Multiome) was performed on PB samples of 43 individuals by 10xGenomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression. Sc Multiome analysis was conducted using Seurat and Signac for quality control, integration, and clustering. Additionally, we employed long-read sequencing with the PromethION platform to identify CH mutations in the sc Multiome libraries. Results: The median age of this cohort was 72 years old (range, 50 - 85). After error correction was applied to the TDS data, a total of 56 mutations were detected in 34 individuals ( DNMT3A R882, 1; DNMT3A nonR882, 16; TET2, 10; GNAS, 4; STAT3, 3; KRAS, 1; MYD88, 1; others, 20). The median VAF was 0.0091 (range, 0.003 to 0.164). The number of mutated genes in each individual increased with age, and individuals of under the age of 69 had significantly fewer mutations compared to those over 70 years old. Regarding the TDS data of each fraction, DNMT3A mutations were detected in both monocyte and B-cell fractions in 5 cases, while they were restricted to T-cell fraction in one case. In 3 cases, mutations were detected in all fractions. TET2 mutations were present in both monocyte and B-cell fractions in all 5 cases. On the other hand, KRAS, STAT3 and MYD88 mutations were restricted to T-, T-, and B-cell fractions, respectively. In sc Multiome data, the median number of cells included was 7931 (range, 2998 to 13413). By the sc Multiome long read sequencing, 39 mutations were detected in 22 individuals ( DNMT3A R882, 1; DNMT3A nonR882, 5; TET2, 6; GNAS, 1; STAT3, 3; KRAS, 1; others, 22). The median rate of DNMT3A mutated cells was 0.007(range, 0.003 to 0.014). Similarly, the median TET2 mutated cell population rate was 0.007(range, 0.004 to 0.044). The average cell number of B cells, T cells, monocytes, and NK cells in sc Muliome data were 955 (range, 305 to 2275), 3875 (range, 955 to 7726), 1812 (range, 352 to 4750), and 868 (range, 289 to 6022) respectively. Among B cells, 16 mutations were detected in 13 individuals ( DNMT3A nonR882, 2; TET2, 2; STAT3, 3; others, 9). For T cells, 27 mutations were detected in 20 individuals ( DNMT3A R882, 1; DNMT3A nonR882, 4; TET2, 4; GNAS, 1; STAT3, 3;others, 12). In Monocytes cells 24, mutations were detected in 16 individuals ( DNMT3A R882, 1; DNMT3A nonR882, 4; TET2, 5; GNAS, 1; STAT3, 3;others, 10). Lastly, among NK cells, 23 mutations were detected in 17 individuals ( DNMT3A nonR882, 5; TET2, 4; GNAS, 1; STAT3, 3;others, 10). The mutated cell populations in each cell fraction were 0.037(range, 0.014 to 0.208), 0.013 (range, 0.002 to 0.180), 0.013(range, 0.003 to 0.178), and 0.043(range, 0.009 to 0.284). Conclusion: This study clarified the detailed distribution of CH-derived mutant cells in PB of elderly individuals. We identified 17 mutations that expanded in myeloid fraction and 12 mutations expanded in lymphoid fraction. Sc Multiome analysis combined with long-read sequencing allowed us to gain a deeper understanding of CH mutated cell populations. These findings provide the valuable insights into properties of CH mutant cells.

Microfluidic Blood Separation: Key Technologies and Critical Figures of Merit.

Blood is a complex sample comprised mostly of plasma, red blood cells (RBCs), and other cells whose concentrations correlate to physiological or pathological health conditions. There are also many blood-circulating biomarkers, such as circulating tumor cells (CTCs) and various pathogens, that can be used as measurands to diagnose certain diseases. Microfluidic devices are attractive analytical tools for separating blood components in point-of-care (POC) applications. These platforms have the potential advantage of, among other features, being compact and portable. These features can eventually be exploited in clinics and rapid tests performed in households and low-income scenarios. Microfluidic systems have the added benefit of only needing small volumes of blood drawn from patients (from nanoliters to milliliters) while integrating (within the devices) the steps required before detecting analytes. Hence, these systems will reduce the associated costs of purifying blood components of interest (e.g., specific groups of cells or blood biomarkers) for studying and quantifying collected blood fractions. The microfluidic blood separation field has grown since the 2000s, and important advances have been reported in the last few years. Nonetheless, real POC microfluidic blood separation platforms are still elusive. A widespread consensus on what key figures of merit should be reported to assess the quality and yield of these platforms has not been achieved. Knowing what parameters should be reported for microfluidic blood separations will help achieve that consensus and establish a clear road map to promote further commercialization of these devices and attain real POC applications. This review provides an overview of the separation techniques currently used to separate blood components for higher throughput separations (number of cells or particles per minute). We present a summary of the critical parameters that should be considered when designing such devices and the figures of merit that should be explicitly reported when presenting a device's separation capabilities. Ultimately, reporting the relevant figures of merit will benefit this growing community and help pave the road toward commercialization of these microfluidic systems.

Integrative blood-based characterization of oxidative mitochondrial DNA damage variants implicates Mexican American’s metabolic risk for developing Alzheimer’s disease

Alzheimer’s Disease (AD) continues to be a leading cause of death in the US. As the US aging population (ages 65 +) expands, the impact will disproportionately affect vulnerable populations, e.g., Hispanic/Latino population, due to their AD-related health disparities. Age-related regression in mitochondrial activity and ethnic-specific differences in metabolic burden could potentially explain in part the racial/ethnic distinctions in etiology that exist for AD. Oxidation of guanine (G) to 8-oxo-guanine (8oxoG) is a prevalent lesion and an indicator of oxidative stress and mitochondrial dysfunction. Damaged mtDNA (8oxoG) can serve as an important marker of age-related systemic metabolic dysfunction and upon release into peripheral circulation may exacerbate pathophysiology contributing to AD development and/or progression. Analyzing blood samples from Mexican American (MA) and non-Hispanic White (NHW) participants enrolled in the Texas Alzheimer’s Research & Care Consortium, we used blood-based measurements of 8oxoG from both buffy coat PBMCs and plasma to determine associations with population, sex, type-2 diabetes, and AD risk. Our results show that 8oxoG levels in both buffy coat and plasma were significantly associated with population, sex, years of education, and reveal a potential association with AD. Furthermore, MAs are significantly burdened by mtDNA oxidative damage in both blood fractions, which may contribute to their metabolic vulnerability to developing AD.

Rejuvenating effects of young extracellular vesicles in aged rats and in cellular models of human senescence

Rejuvenation of an old organism was achieved in heterochronic parabiosis experiments, implicating different soluble factors in this effect. Extracellular vesicles (EVs) are the secretory effectors of many cells, including cardiosphere-derived cells (CDCs) with demonstrated anti-senescent effect. 1. To determine the role of EVs (versus other blood fractions) on the rejuvenating effect of the young blood. 2. To evaluate the anti-aging properties of therapeutically administered EVs secreted by young-CDCs in an old organism. Neonatal blood fractioned in 4 components (whole blood, serum, EV-depleted serum and purified EVs) was used to treat old human cardiac stromal cells (CSPCs). CDCs were generated from neonatal rat hearts and the secreted CDC-EVs were purified. CDC-EVs were then tested in naturally-aged rats, using monthly injections over 4-months period. For validation in human samples, pediatric CDC-EVs were tested in aged human CSPCs and progeric fibroblasts. While the purified EVs reproduced the rejuvenating effects of the whole blood, CSPCs treated with EV-depleted serum exhibited the highest degree of senescence. Treatment with young CDC-EVs induce structural and functional improvements in the heart, lungs, skeletal muscle, and kidneys of old rats, while favorably modulating glucose metabolism and anti-senescence pathways. Lifespan was prolonged. EVs secreted by young CDCs exert broad-ranging anti-aging effects in aged rodents and in cellular models of human senescence. Our work not only identifies CDC-EVs as possible therapeutic candidates for a wide range of age-related pathologies, but also raises the question of whether EVs function as endogenous modulators of senescence.

Leukocyte differentiation in bronchoalveolar lavage fluids using higher harmonic generation microscopy and deep learning.

In diseases such as interstitial lung diseases (ILDs), patient diagnosis relies on diagnostic analysis of bronchoalveolar lavage fluid (BALF) and biopsies. Immunological BALF analysis includes differentiation of leukocytes by standard cytological techniques that are labor-intensive and time-consuming. Studies have shown promising leukocyte identification performance on blood fractions, using third harmonic generation (THG) and multiphoton excited autofluorescence (MPEF) microscopy. To extend leukocyte differentiation to BALF samples using THG/MPEF microscopy, and to show the potential of a trained deep learning algorithm for automated leukocyte identification and quantification. Leukocytes from blood obtained from three healthy individuals and one asthma patient, and BALF samples from six ILD patients were isolated and imaged using label-free microscopy. The cytological characteristics of leukocytes, including neutrophils, eosinophils, lymphocytes, and macrophages, in terms of cellular and nuclear morphology, and THG and MPEF signal intensity, were determined. A deep learning model was trained on 2D images and used to estimate the leukocyte ratios at the image-level using the differential cell counts obtained using standard cytological techniques as reference. Different leukocyte populations were identified in BALF samples using label-free microscopy, showing distinctive cytological characteristics. Based on the THG/MPEF images, the deep learning network has learned to identify individual cells and was able to provide a reasonable estimate of the leukocyte percentage, reaching >90% accuracy on BALF samples in the hold-out testing set. Label-free THG/MPEF microscopy in combination with deep learning is a promising technique for instant differentiation and quantification of leukocytes. Immediate feedback on leukocyte ratios has potential to speed-up the diagnostic process and to reduce costs, workload and inter-observer variations.

Исследование клеточного состава богатой тромбоцитами плазмы для оптимизации ее использования в хирургии макулярных разрывов. Часть 1

Currently, the use of platelet-rich plasma (PRP) is one of the successful areas of tissue engineering and cell therapy. In medicine PRP is widely used in ophthalmosurgery, in particular for macular hole (MH) closure. Purpose. To evaluate cellular composition of PRP in order to optimize its use in macular hole surgery. Material and methods. This study included platelet-rich plasma analysis of 20 patients (6 male and 14 female) who underwent surgical treatment of macular hole in Eye Microsurgery Center in the period from April till July 2021. Patients' age ranged from 57 to 82 (69,0±1.2) years. Ycellbio-Kit was used to obtain PRP. As a result of centrifugation 2 main blood fractions were obtained: plasma and erythrocyte layer. The study of the contents of the test tube was performed on 4 levels. The main attention was paid to the quantitative composition of platelets and leukocytes, which are the most important and significant in MH surgery. Results. The following platelet and leukocyte count in the general blood test and PRP were obtained as a result of the study. General blood test – platelet concentration was 140 to 285•10³/µL (215.85±9.23), P-PRP (pure platelet-rich plasma) – 699 to 5681•10³/µL (2600.4±329.9), L-PRP (plasma containing platelets and leukocytes) – 1995 to 9989•10³/µL (6555.8±506.7) (p&lt;0.001). General blood test – white blood cell concentration was 3.83 to 7.73•10³/µL (5.58±0.29), P-PRP was 0.02 to 7.08•10³/µL (1.49±0.58), L-PRP was 6.12–494.69•10³/µL (99.3±38.1) (p&lt;0.01). Conclusion. Platelet-rich plasma has regenerative activity and contractile properties in both fractions – in P-PRP and L-PRP, which simplifies the process of PRP extraction from the test tube and expands the possibilities for its use in MH surgery and other retinal pathologies associated with retinal tears. Given the lowest leukocyte concentration, the P-PRP fraction may be optimal for MH surgery. Key words: macular rupture, platelet-enriched plasma, platelets, leukocytes

Validation of a whole exome/whole transcriptome liquid biopsy assay with correction for clonal hematopoiesis.

e15049 Background: Liquid biopsies have become integral to the management of patients with advanced malignancies, however significant deficiencies exist in currently used platforms. Liquid biopsy data often show unacceptably high discordance compared to gold standard tissue-based data; this is often due to false positives (FP) resulting from clonal hematopoiesis (CH) or increased false negatives due to inherently poor sensitivity as well as the confounding intra-patient or tumor stage dependent ‘non-shedder’ problem. Caris Assure is a novel whole exome (WES) and whole transcriptome (WTS) NGS assay performed on plasma and buffy coat (BC) fractions of whole blood that maximizes the number of unique nucleic acids, thereby increasing sensitivity as well as identifying incidental germline variants and correcting for CH. Methods: Matched tissue and whole blood specimens were obtained from 184 patients with solid tumors. Whole blood was collected in PAXgene ccfDNA tubes. DNA &amp; RNA were co-extracted from matched plasma, BC, and FFPE tumor tissue specimens and prepared for NGS using hybrid bait capture methodology. Plasma and BC samples were tested using the Caris Assure WES/WTS assay as well as orthogonal assays. Tissue specimens were tested using Caris’ validated WES/WTS tissue assay. Results were compared between the plasma assays and matched tissues as well as using a majority-rules calling model. A positive plasma assay variant was counted a true positive if it was also identified in the matched tissue or the orthogonal plasma result. Similarly, a negative result was considered a true negative if either of the other two test results were also negative. Results: When data were analyzed from patients with metastatic disease and were focused on only those genes included in the limited orthogonal liquid panel, and tissue was treated as the gold standard, Caris Assure achieved a PPA for SNVs and INDELS of 81.4% and PPV of 85.1%, while the orthogonal assay achieved a PPA and PPV of 79.1% and 53.1%. When the majority rules approach was used, Assure PPA and PPV were 93.3% and 93.3%, respectively, compared to 91.1% and 64.1%. Germline variants detected by Caris Assure had 100% PPA &amp; PPV when compared to the external germline assay. There were 444 pathogenic/likely pathogenic mutations detected across all assays. Of those, 77 (17.3%) were derived from CH and identified as such in the Caris Assure results, though they are inaccurately reported as tumor-derived variants using the comparator assay. Using Caris Assure corrected for CH-derived false positives, the correlation between plasma and tissue TMB was very strong (r = 0.9) when tissue and blood specimens were collected within 4 weeks of each other. Conclusions: The novel Caris Assure liquid biopsy platform demonstrates high sensitivity and specificity for somatic variants, while mitigating against FPs from CH, and identifying incidental germline pathogenic alterations.

Fractal dimension of pulmonary gas and blood distribution assessed by synchrotron K-edge subtraction imaging: effect of bronchoconstriction.

We analyzed the fractal dimension (Df) of lung gas and blood distribution imaged with synchrotron radiation K-edge subtraction (KES), in six anesthetized adult New Zealand White rabbits. KES imaging was performed in upright position during stable Xe gas (64% in O2) inhalation and iodine infusion (Iomeron, 350 mg/mL), respectively, at baseline and after induced bronchoconstriction by aerosolized methacholine (125 mg/mL, 90 s) and bronchodilator (salbutamol, 10 mg/mL, 90 s) inhalation, at two axial image levels. Lung Xe and iodine images were segmented, and maps of regional lung gas and blood fractions were computed. The Df of lung gas (DfXe) and blood (DfIodine) distribution was computed based on a log-log plot of variation coefficient as a function of region volume. DfXe decreased significantly during bronchoconstriction (P < 0.0001), and remained low after salbutamol. DfIodine depended on the axial image level (P < 0.0001), but did not change with bronchoconstriction. DfXe was significantly associated with arterial [Formula: see text] (R = 0.67, P = 0.002), and negatively associated with [Formula: see text] (R = -0.62, P = 0.006), respiratory resistance (R = -0.58, P = 0.011), and elastance (R = -0.55, P = 0.023). These data demonstrate the reduced Df of gas distribution during acute bronchoconstriction, and the association of this parameter with physiologically meaningful variables. This finding suggests a decreased complexity and space-filling properties of lung ventilation during bronchoconstriction, and could serve as a functional imaging biomarker in obstructive airway diseases.NEW & NOTEWORTHY Here, we used an energy-subtractive imaging technique to assess the fractal dimension (Df) of lung gas and blood distribution and the effect of acute bronchoconstriction. We found that Df of gas significantly decreases in bronchoconstriction. Conversely, Df of blood exhibits gravity-dependent changes only, and is not affected by acute bronchoconstriction. Our data show that the fractal dimension of lung gas detects the emergence of clustered rather than scattered loss of ventilatory units during bronchoconstriction.

Obstetric outcomes in Jehovah's Witnesses: case series over nine years in a London teaching hospital.

The aim of the study is to assess obstetric and fetal outcomes of Jehovah's Witnesses (JWs) mothers in an inner city teaching hospital, as well as to examine the acceptance rates of various blood fractions and blood transfusion alternatives. Case series to evaluate the maternal and fetal outcomes of JWs over a nine period between 2013 and 2021. There were 146 pregnancies extracted from our database, of which 10 were early pregnancy losses. Data from 136 deliveries > 24weeks' gestation were assessed, with a mean maternal age and gestational age of 30.26 (± 5.4) years and 38.7 (± 5.3) weeks, respectively. 57% had normal vaginal deliveries, 8% had instrumental births and 35% had caesarean births. Mean estimated blood loss at caesarean was 575 (± 305.6) mls, while the overall mean estimated loss was 427.8 (± 299.8) mls. Cell salvage was performed in all caesarean sections but autologous transfusion was only necessary for 26%. Consultant presence was documented in 62% of caesarean births. The mean birthweight and 5-min Apgar scores were 3.31 (± 0.05) kg and 9.1 (± 0.09), respectively. There were no maternal deaths or admissions to the adult intensive care unit and the most serious complication was a uterine rupture following a trial of scar, after which the baby required cooling for hypoxic ischaemic encephalopathy. Pregnant JWs received obstetric care led by senior clinicians, with optimisation of haematinics, minimizing of blood loss at delivery and access to technology such as cell salvage.

Evaluation of Platelet-Rich Plasma by means of PRGF®-Endoret® protocol in leukemia cats: PDGF-BB and TGF-ß1 valuation.

Feline leukemia virus (FeLV) is a chronic disease that leads to the weakening of a cat's immune system. Platelet-rich plasma (PRP) offers therapeutic effects for multiple diseases, the use of PRP and growth factors (GFs) determination could be an alternative treatment to improve the quality of life in these patients. The objectives of this study were to determine and compare the concentration of platelets (PLTs), red blood cells (RBCs) and white blood cells (WBCs) between samples of whole blood (WB), PRP and platelet-poor plasma (PPP) fractions, and to evaluate the concentration of platelet-derived growth factor BB (PDGF-BB) and transforming growth factor β1 (TGF-β1) in both fractions in FeLV cats using a PRGF®-Endoret® protocol previously standardized in this species. WB was collected from 11 asymptomatic FeLV-positive cats. PRP and PPP was obtained following PRGF®-Endoret® technology according to centrifugation at 265 g for 10 min. Cellular components, RBCs, WBCs, PLTs, and the PDGF-BB and TGF-β1 concentrations in PRP and PPP fractions were determined. PLT in the PRP fraction was statistically higher than WB and PPP fraction, with no statistical differences between WB and PPP. PLT concentration increased 1.4 times in PRP fraction compared to WB. Mean platelet volume (MPV) did not differ significantly between the WB, PRP, and PPP fractions. Compared to WB, the absolute numbers of RBCs and WBCs were decreased by 99% and more than 95% in the PRP and PPP fractions, respectively. TGF-ß1 concentrations increased in PRP vs. PPP, with no changes in PDGF-BB. Based on the degree of PLT enrichment and the absence of RBCs and WBCs, this blood product could be classified as a Pure Platelet-Rich Plasma (P-PRP). The presence of GFs in PRP and PPP samples suggests that the PRGF®-Endoret® methodology is suitable for obtaining PRP in FeLV cats, despite future studies are necessary to optimize the technique, standardize the results and assess clinical efficacy.

DETERMINATION OF THE OPTIMUM PLATELET-RICH PLASMA METHOD FOR THE ESSENTIAL AMOUNT OF PLATELETS

Objective: Platelet-rich plasma (PRP) is the cellular plasma component containing a higher platelet concentration obtained by centrifugation of autologous peripheral blood. PRP has an auxiliary role in regenerative medicine treatment. Current treatment protocols for tissue repair strategies may be globally insufficient. PRP treatment via Platelet-released growth factors (GF) supports inflammation, proliferation, and wound healing. PRP studies have progressed and have proposed different formulations in recent years. Although various methods and devices are used, there are few and different standardized protocols to obtain PRP with the highest platelet concentration. Although anticoagulants containing citrate are primarily preferred in PRP production To provide effective platelet concentration in PRP, we noticed differences in the literature regarding PRP tube volumes, anticoagulant-citrate, and blood amounts. We aimed to obtain the most appropriate platelet concentration in PRP treatment by using different concentration ratios of Acid Citrate dextrose (ACD) solution A and different blood amounts and introducing a new treatment method to the literature. Methods: The blood of 30 individuals aged between 30 and 75 was taken into 10cc laboratory tubes, and 15cc and 18cc hourglass-shaped PRP tubes containing 8% ACD-solution A. Blood fractions were separated by centrifugation, buffy coat area was collected, and count platelet amount by Hematology Analyzer. Results: The platelet amount per µl was statistically significantly higher in 18cc blood compared to 15cc and 10cc. Platelet amounts obtained from 15 and 18cc peripheral blood with 8% ACD-solution A were above the effective treatment amount recommended in the literature. Conclusion: It was observed that the amount of used blood volume was correlated with the amount of obtained platelets.

Manipulating omega-3 and omega-6 fatty acids in sheep: depletion of long-chain omega-3 polyunsaturated fatty acids by feeding different grain types

Context Plasma fatty acid profiles provide evidence of short-term feeding, while red blood cells (RBC) indicate longer-term feeding effects. Information on the rate of change in fatty acid profiles of sheep blood over time after alterations in diet is lacking. This study monitored the short- and longer-term effects of feeding grain-based diets to lambs on changes to omega-3 polyunsaturated fatty acids (PUFA) in blood fractions over 21 days. The data provide a guide to managing omega-3 PUFA availability for adequate tissue accretion of these fatty acids. Aims To determine the rate of depletion over 21 days of omega-3 PUFA in the blood fractions of sheep fed on grain-based diets. Methods Blood samples were taken from ewes taken off pasture and fed grain-based diets for 21 days. Fatty acid profiles in the blood plasma and RBC were used to monitor the rate of depletion of omega-3 PUFA. Key results Immediate changes in plasma omega-3 PUFA concentrations occurred from the first day of grain feeding. The omega-3 PUFA content declined continuously on all five grains, but with substantial differences among them. RBC omega-3 PUFA contents displayed great variation across individual sheep but depletion was not significant. Conclusions Plasma omega-3 PUFA depletion immediately on grain feeding indicated a lower availability for tissue accretion in growing sheep following short-term changes in diet. The lack of change in RBC omega-3 PUFA concentrations over 21 days indicated a differential accumulation of these fatty acids in this lipid depot compared with plasma, and changes in other tissues should also be examined. Implications The effect of these short- and long-term changes on LCn-3PUFA accumulation in meat and reproductive tissues warrants further investigation.