Whole Blood Analysis Research Articles

Hematology and Biochemistry Panel Reference Intervals for Captive Saddleback Clownfish Amphiprion polymnus and Tomato Clownfish A.frenatus.

Reference intervals of blood analytes are important diagnostic tools that clinicians use as a basis for making clinical decisions and that have been documented for many fish species. Although the effects of dietary supplementation on hematology and blood biochemistry variables in clownfish have been shown, there are no published reference intervals to date for routine hematology and blood biochemistry panels for these popular cultured marine fish (including Amphiprion spp.). The objective of this study was to establish de novo reference intervals for selected hemogram data and, using whole-blood analysis, commonly utilized chemistry analytes in two species of aquacultured clownfish: the Tomato Clownfish Amphiprion frenatus and Saddleback Clownfish A.polymnus. This study is based on blood samples from 25 clinically normal Tomato Clownfish and 38 clinically normal Saddleback Clownfish from two clownfish production facilities in Florida. Guidelines by the American Society for Veterinary Clinical Pathology were followed to generate robust reference intervals from the data collected. We report descriptive statistics and reference intervals for several hematology (n=12) and biochemistry (n=8) analytes using a small sample size and a small amount of blood per sample. The blood analyte data from this study are comparable with hematology and biochemistry data that have been reported in other teleost species. These results provide baseline health data for clownfish species that can be used in various clinical settings while also serving as a springboard for further research studies.

Evaluation of Fumaric Acid and Maleic Acid as Internal Standards for NMR Analysis of Protein Precipitated Plasma, Serum, and Whole Blood.

Significant advances have been made in unknown metabolite identification and expansion of the number of quantifiable metabolites in human plasma, serum, and whole blood using NMR spectroscopy. However, reliable quantitation of metabolites is still a challenge. A major bottleneck is the lack of a suitable internal standard that does not interact with the complex blood sample matrix and also does not overlap with metabolite peaks apart from exhibiting other favorable characteristics. With the goal of addressing this challenge, a comprehensive investigation of fumaric and maleic acids as potential internal standards was made along with a comparison with the conventional standards, TSP (trimethylsilylpropionic acid) and DSS (trimethylsilylpropanesulfonic acid). Both fumaric acid and maleic acid exhibited a surprisingly high performance with a quantitation error <1%, while the TSP and DSS caused an average error of up to 35% in plasma, serum, and whole blood. Further, the results indicate that while fumaric acid is a robust standard for all three biospecimens, maleic acid is suitable for only plasma and serum. Maleic acid is not suited for the analysis of whole blood due to its overlap with coenzyme peaks. These findings provide new opportunities for improved and accurate quantitation of metabolites in human plasma, serum, and whole blood using NMR spectroscopy. Moreover, the use of protein precipitation prior to NMR analysis mirrors the sample preparation commonly used for mass spectrometry based metabolomics, such that these findings further strengthen efforts to combine and compare NMR and MS based metabolite data of human plasma, serum, and whole blood for metabolomics based research.

Initial In Vivo Evaluation of a Novel Amikacin-Deoxycholate Hydrophobic Salt Delivers New Insights on Amikacin Partition in Blood and Tissues.

In this study, an initial in vivo evaluation of a new amikacin-deoxycholate hydrophobic salt aimed at potentiating amikacin action against hard-to-treat lung infections was undertaken by quantifying, for the first time, amikacin in whole blood. Pharmacokinetic evaluation after intranasal administration in a murine model showed higher drug retention in the lungs compared to blood, with no significant differences between the salt and the free drug. Upon repeated administrations, the two treatments resulted in nonsignificant tissue damage and mild higher inflammation for the hydrophobic salt. Whole-blood analysis highlighted an unreported high partition of amikacin in blood components up to 48 h, while significant lung levels were measured up to 72 h. Such a new observation was considered responsible for the nearly overlapping pharmacokinetic profiles of the two treatments. To overcome such an issue, a dry powder in an inhalable form may be best suited. Moreover, if confirmed in humans, and considering the current once-a-day regimen for amikacin aerosols, important yet-to-be-explored clinical implications may be postulated for such amikacin persistence in the organism.

Geographical differences in sex-specific foraging behaviour and diet during the breeding season in the opportunistic Kelp Gull (Larus dominicanus)

Sexual segregation in feeding strategies has been widely reported in seabirds. Most seabirds occupy wide breeding distributional ranges, and dissimilar ecological settings may result in distinct environmental pressures on males and females leading to geographical differences in sexual segregation. Using GPS loggers and stable isotope analysis of whole blood (δ15N, δ13C), we assessed the occurrence of sexual differences in Kelp Gull (Larus dominicanus) foraging trip parameters, habitat use, isotopic niche and diet during the incubation period at three breeding locations in Argentina characterized by different foraging contexts. At Islote Arroyo Jabali Este, sexual differences were found in trip parameters and habitat use mainly associated with a significantly higher use by males of a refuse dump as foraging site. However, their isotopic niches were similar with both sexes consuming mostly recreational fish waste, suggesting it was mainly obtained by males at the dump and by females at shoreline areas used by fishers. At Punta Tombo, there were no sex differences in trip parameters nor in the main prey consumed consisting of fishery discards, although females used more shoreline areas and males presented a significantly larger isotopic niche width. In contrast, at Isla Vernaci Este, males and females showed similar trip parameters, habitat use, and isotopic niche width and diet composition. Results show geographical differences in the degree of sexual segregation, which may result from differences in the availability of local food resources, particularly anthropogenic food subsidies.

CRISPR/Cas9-mediated β-globin gene knockout in rabbits recapitulates human β-thalassemia

β-thalassemia, an autosomal recessive blood disorder that reduces the production of hemoglobin, is majorly caused by the point mutation of the HBB gene resulting in reduced or absent β-globin chains of the hemoglobin tetramer. Animal models recapitulating both the phenotype and genotype of human disease are valuable in the exploration of pathophysiology and for in vivo evaluation of novel therapeutic treatments. The docile temperament, short vital cycles, and low cost of rabbits make them an attractive animal model. However, β-thalassemia rabbit models are currently unavailable. Here, using CRISPR/Cas9-mediated genome editing, we point mutated the rabbit β-globin gene HBB2 with high efficiency and generated a β-thalassemia rabbit model. Hematological and histological analyses demonstrated that the genotypic mosaic F0 displayed a mild phenotype of anemia, and the heterozygous F1 exhibited typical characteristics of β-thalassemia. Whole-blood transcriptome analysis revealed that the gene expression was altered in HBB2-targeted when compared with WT rabbits. And the highly expressed genes in HBB2-targeted rabbits were enriched in lipid and iron metabolism, innate immunity, and hematopoietic processes. In conclusion, using CRISPR-mediated HBB2 knockout, we have created a β-thalassemia rabbit model that accurately recapitulates the human disease phenotype. We believe this tool will be valuable in advancing the investigation of pathogenesis and novel therapeutic targets of β-thalassemia and associated complications.

Pathomechanisms of HIV-Associated Cerebral Small Vessel Disease: A Comprehensive Clinical and Neuroimaging Protocol and Analysis Pipeline.

Rationale: We provide an in-depth description of a comprehensive clinical, immunological, and neuroimaging study that includes a full image processing pipeline. This approach, although implemented in HIV infected individuals, can be used in the general population to assess cerebrovascular health.Aims: In this longitudinal study, we seek to determine the effects of neuroinflammation due to HIV-1 infection on the pathomechanisms of cerebral small vessel disease (CSVD). The study focuses on the interaction of activated platelets, pro-inflammatory monocytes and endothelial cells and their impact on the neurovascular unit. The effects on the neurovascular unit are evaluated by a novel combination of imaging biomarkers.Sample Size: We will enroll 110 HIV-infected individuals on stable combination anti-retroviral therapy for at least three months and an equal number of age-matched controls. We anticipate a drop-out rate of 20%.Methods and Design: Subjects are followed for three years and evaluated by flow cytometric analysis of whole blood (to measure platelet activation, platelet monocyte complexes, and markers of monocyte activation), neuropsychological testing, and brain MRI at the baseline, 18- and 36-month time points. MRI imaging follows the recommended clinical small vessel imaging standards and adds several advanced sequences to obtain quantitative assessments of brain tissues including white matter microstructure, tissue susceptibility, and blood perfusion.Discussion: The study provides further understanding of the underlying mechanisms of CSVD in chronic inflammatory disorders such as HIV infection. The longitudinal study design and comprehensive approach allows the investigation of quantitative changes in imaging metrics and their impact on cognitive performance.

Elimination of the non-spectral matrix interferences in the analysis of bio-liquids by mass spectrometry with inductively coupled plasma

Systematic investigations of the impact of nitric acid, inorganic salts and organic compounds on the decrease in the analytical signals in the analysis of whole blood, serum and urine by mass spectrometry with inductively coupled plasma are performed. Two strategies for the sample pretreatment – dilution by different organic mixtures and acidic mineralization – are investigated. The maximum effect of the nitric acid and salts on the results of the determination of the diagnostically important elements P, Mn, Co, Cu, Zn, As, Se, Cd and Pb were found using synthetic model solutions. Analytes with ionization potentials (IP) above 9.0 eV are found to be affected most by these non-spectral interferences. The possibility of avoiding such interferences through simply specific tuning of the instrumental parameters is investigated. It is found that the greatest reduction of the interferences is seen for an increase in the RF power and a decrease in the velocity of the nebulized argon flow. Various strategies for selection of an internal standard (IS) for different tunings of the instrument are found. Two approaches for elimination of the non-spectral interferences are established: i) combination of a standard set of instrumental parameters and the use of the IS with IPs matched to the IPs of the analytes; ii) combination of “robust” instrumental parameters and the use of a single IS with any mass and IP. The first approach provides higher sensitivity but requires the use of different IS for different analytes. The second approach provides a much more flexible analytical performance for the routine determination of the various sets of analytes but at the expense of a modest decrease in the analytical sensitivity.The efficiency of the developed strategy is demonstrated by the analysis of standard reference materials of whole blood and urine and applied to the analysis of bio-liquids of patients suffering from different forms of a systematic inflammatory response syndrome (SIRS). The concentrations of Cu and Zn were found to be diagnostically important features.

RNA sequencing analysis reveals increased expression of interferon signaling genes and dysregulation of bone metabolism affecting pathways in the whole blood of patients with osteogenesis imperfecta

BackgroundOsteogenesis imperfecta (OI) is a rare genetic disorder in which the patients suffer from numerous fractures, skeletal deformities and bluish sclera. The disorder ranges from a mild form to severe and lethal cases. The main objective of this pilot study was to compare the blood transcriptional landscape of OI patients with COL1A1 pathogenic variants and their healthy relatives, in order to find out different gene expression and dysregulated molecular pathways in OI.MethodsWe performed RNA sequencing analysis of whole blood in seven individuals affected with different OI severity and their five unaffected relatives from the three families. The data was analyzed using edgeR package of R Bioconductor. Functional profiling and pathway analysis of the identified differently expressed genes was performed with g:GOSt and MinePath web-based tools.ResultsWe identified 114 differently expressed genes. The expression of 79 genes was up-regulated, while 35 genes were down-regulated. The functional analysis identified a presence of dysregulated interferon signaling pathways (IFI27, IFITM3, RSAD12, GBP7). Additionally, the expressions of the genes related to extracellular matrix organization, Wnt signaling, vitamin D metabolism and MAPK-ERK 1/2 pathways were also altered.ConclusionsThe current pilot study successfully captured the differential expression of inflammation and bone metabolism pathways in OI patients. This work can contribute to future research of transcriptional bloodomics in OI. Transcriptional bloodomics has a strong potential to become a major contributor to the understanding of OI pathological mechanisms, the discovery of phenotype modifying factors, and the identification of new therapeutic targets. However, further studies in bigger cohorts of OI patients are needed to confirm the findings of the current work.

Distinct whole-blood transcriptome profile of children with metabolic healthy overweight/obesity compared to metabolic unhealthy overweight/obesity.

Youth populations with overweight/obesity (OW/OB) exhibit heterogeneity in cardiometabolic health phenotypes. The underlying mechanisms for those differences are still unclear. This study aimed to analyze the whole-blood transcriptome profile (RNA-seq) of children with metabolic healthy overweight/obesity (MHO) and metabolic unhealthy overweight/obesity (MUO) phenotypes. Twenty-seven children with OW/OB (10.1 ± 1.3 years, 59% boys) from the ActiveBrains project were included. MHO was defined as having none of the following criteria for metabolic syndrome: elevated fasting glucose, high serum triglycerides, low high-density lipoprotein-cholesterol, and high systolic or diastolic blood pressure, while MUO was defined as presenting one or more of these criteria. Inflammatory markers were additionally determined. Total blood RNA was analyzed by 5'-end RNA-sequencing. Whole-blood transcriptome analysis revealed a distinct pattern of gene expression in children with MHO compared to MUO children. Thirty-two genes differentially expressed were linked to metabolism, mitochondrial, and immune functions. The identified gene expression patterns related to metabolism, mitochondrial, and immune functions contribute to a better understanding of why a subset of the population remains metabolically healthy despite having overweight/obesity. A distinct pattern of whole-blood transcriptome profile (RNA-seq) was identified in children with metabolic healthy overweight/obesity (MHO) compared to metabolic unhealthy overweight/obesity (MUO) phenotype. The most relevant genes in understanding the molecular basis underlying the MHO/MUO phenotypes in children could be: RREB1, FAM83E, SLC44A1, NRG1, TMC5, CYP3A5, TRIM11, and ADAMTSL2. The identified whole-blood transcriptome profile related to metabolism, mitochondrial, and immune functions contribute to a better understanding of why a subset of the population remains metabolically healthy despite having overweight/obesity.

Lead (Pb) exposure assessment in dried blood spots using Total Reflection X-Ray Fluorescence (TXRF)

Lead (Pb) exposure is often determined through the analysis of whole blood though venipuncture poses ethical, economic, and logistical barriers. Dried Blood Spots (DBS) may help overcome such barriers though past studies measuring Pb in DBS have been challenged with quality control, small sample volumes, and other issues. Total Reflection X-Ray Fluorescence (TXRF) may help address some of these challenges but has yet to be used to measure Pb in DBS. As such, the aim of the current study was to develop, validate, and apply a method to analyze Pb in DBS samples using TXRF for use in human biomonitoring studies. First, we developed a novel method (tested a range of parameters), and then used blood reference materials to validate the method against performance criteria listed in ICH Q2A and Q2B and the European Bioanalysis Forum. Finally, we applied the method to two populations who exemplify divergent conditions (41 university members with relatively low Pb exposures sampled in a clinical environment; 40 electronic waste workers with relatively high Pb exposures sampled in a contaminated field setting). The limits of detection and quantification of the method were 0.28 and 0.69 μg/dL, respectively. The overall precision and accuracy of the method were 15% and 111%, respectively. The mean (±SD) DBS Pb levels by TXRF in the university members and e-waste workers were 0.78 (±0.46) and 3.78 (±3.01) μg/dL, respectively, and these were not different from Pb measures in venous whole blood using ICP-MS. Bland-Altman plot analyses indicated good agreement between DBS Pb measures by TXRF versus whole blood Pb measures by ICP-MS in both groups. By combining data from the two population groups, there was no significant constant bias (intercept of 0.02 μg/dL) or proportional bias (slope was −0.02) between the two measures, and the lower and upper LoA were −0.86 and 0.91 μg/dL, respectively, with a LoA range of 1.77 μg/dL. These results demonstrate that TXRF-based analysis of Pb content in DBS is a good alternative to the gold standard (i.e., ICP-MS analysis of whole blood), and helps overcome some of the challenges associated with current methods.

Inaccuracy of Sodium Measurement in Patients with Severe Hypernatremia.

We observed discordant sodium results from a patient with severe hypernatremia such that whole-blood analysis produced results up to 9.6 mmol/L higher than plasma sodium obtained on the same collection. We investigated this bias by comparing other patients' sodium results and performing comparisons of 3 blood gas and 2 chemistry analyzers. First, the laboratory information system was queried for whole-blood sodium results >160 mmol/L, which were used for comparison against plasma results from the same collection. Second, whole blood was collected from a healthy donor, a portion of which was spiked with sodium chloride to generate 8 samples with target concentrations of 140 to 185 mmol/L. Whole-blood sodium was measured in duplicate on the ABL90, RAPIDPoint 500, and GEM 4000. Plasma sodium was then measured in duplicate on the Architect c8000 and Cobas c702. Finally, plasma was injected on the blood gas analyzers to measure sodium in singleton. Overall, 53 paired results from patients showed a significant positive bias on the ABL90 relative to Vitros when sodium was >160 mmol/L. The magnitude of difference was insignificant within the reference range but increased proportionately with concentration. The magnitude and pattern of positive bias in ABL90 sodium results were consistent with the observation in patient results. In severe hypernatremia, sodium results produced by blood gas and plasma analyzers can differ significantly.

Machine learning assistive rapid, label-free molecular phenotyping of blood with two-dimensional NMR correlational spectroscopy

Translation of the findings in basic science and clinical research into routine practice is hampered by large variations in human phenotype. Developments in genotyping and phenotyping, such as proteomics and lipidomics, are beginning to address these limitations. In this work, we developed a new methodology for rapid, label-free molecular phenotyping of biological fluids (e.g., blood) by exploiting the recent advances in fast and highly efficient multidimensional inverse Laplace decomposition technique. We demonstrated that using two-dimensional T1-T2 correlational spectroscopy on a single drop of blood (<5 μL), a highly time- and patient-specific ‘molecular fingerprint’ can be obtained in minutes. Machine learning techniques were introduced to transform the NMR correlational map into user-friendly information for point-of-care disease diagnostic and monitoring. The clinical utilities of this technique were demonstrated through the direct analysis of human whole blood in various physiological (e.g., oxygenated/deoxygenated states) and pathological (e.g., blood oxidation, hemoglobinopathies) conditions.

Transcriptome analysis of peripheral whole blood identifies crucial lncRNAs implicated in childhood asthma

BackgroundAsthma is a chronic disorder of both adults and children affecting more than 300 million people heath worldwide. Diagnose and treatment for asthma, particularly in childhood asthma have always remained a great challenge because of its complex pathogenesis and multiple triggers, such as allergen, viral infection, tobacco smoke, dust, etc. It is thereby great significant to deeply investigate the transcriptome changes in asthmatic children before and after desensitization treatment, in order that we could identify potential and key mRNAs and lncRNAs which might be considered as useful RNA molecules for observing and supervising desensitization therapy for asthma, which might guide the diagnose and therapy in childhood asthma.MethodsIn the present study, we performed a systematic transcriptome analysis based on the deep RNA sequencing of ten asthmatic children before and after desensitization treatment, including identification of lncRNAs using a stringent filtering pipeline, differential expression analysis and network analysis, etc.ResultsFirst, a large number of lncRNAs were identified and characterized. Then differential expression analysis revealed 39 mRNAs and 15 lncRNAs significantly differentially expressed which involved in two biological processes and pathways. A co-expressed network analysis figured out a desensitization-treatment-related module which contains 27 mRNAs and 21 lncRNAs using WGCNA R package. Module analysis disclosed 17 genes associated to asthma at distinct level. Subsequent network analysis based on PCC figured out several key lncRNAs probably interacted to those key asthma-related genes, i.e., LINC02145, GUSBP2. Our functional investigation indicated that their functions might involve in immune, inflammatory response and apoptosis process.ConclusionsOur study successfully discovered many key noncoding RNA molecules related to pathogenesis of asthma and relevant treatment, which may provide some clues for asthmatic diagnose and therapy in future.

Are CMV-predictive scores in inflammatory bowel disease useful in clinical practice?

Concurrent cytomegalovirus (CMV) in inflammatory bowel disease (IBD)-related colitis is an important scenario associated with high rates of colectomy and other morbidity. Due to the low incidence of CMV, testing of all patients is associated with an unacceptably high consumption of resources and delay in treatment. Therefore, several predictive scores have been developed to identify patients at risk for a CMV infection. We performed a retrospective single center study in a German University hospital including all IBD patients with available data on CMV-PCR analysis in whole blood between 2010 and 2018 and evaluated 2 prognostic scores for CMV infection for their diagnostic accuracy. In the study, 907 patients with IBD and CMV-PCR were identified. Of them, 21 patients (2.3 %) had a positive CMV-PCR (≥ 1000 copies/mL), 14 of them in ulcerative colitis and 7 in Crohn's disease. The Berlin Score identified 667 patients (73.1 %) as potentially CMV-positive, resulting in a positive predictive value of 2.5 % and a negative predictive value of 98.3 %. In contrast, the Münster Score identified 60 patients as potentially CMV-positive, resulting in a PPV of 20 % and an NPV of 99.4 %. Scoring systems can help to identify patients at risk for a CMV infection and minimize resource consumption and delay in treatment. Due to low incidence, a 2-step-algorithm, consisting of the Münster Score followed by a CMV-PCR if the score indicates a CMV infection, is preferable.

Diagnosis of papillary thyroid carcinoma by 1H NMR spectroscopy-based metabolomic analysis of whole blood.

The incidence rate of thyroid carcinoma, especially papillary thyroid carcinoma (PTC), has increased significantly over time. As a primary pathway for metastasis, the lymphatic system is an important prognostic factor for PTC patients. Although the metabolic changes in PTC patients have been investigated in extensive studies, few studies focused on the whole blood metabolic profiling of PTC patients. In this study, we investigated the 1H NMR-based metabolic profiles of whole blood samples that were obtained from healthy individuals and PTC patients, with or without lymph node metastasis. The estimation of the predictive potential of metabolites was evaluated using multivariate statistical analyses, which revealed that the whole blood carries information that is sufficient for distinguishing between PTC patients and healthy individuals. However, PTC patients were not well classified as positive or negative according to the lymph nodes. We did not find a metabolite that could discriminate the presence of lymph node metastasis. Further studies with larger sample sizes are needed to elucidate significant metabolites to indicate the presence of lymph node metastasis in patients with PTC.

Abstract 2854: Multi-omic considerations: Exploring LBgard blood tubes for proteomic analysis

Abstract Expanding the number of -omic (genomic, proteomic, etc.) analyte classes, a single blood draw will serve to increase the scientific value of that sample by affording additional multi-variant analyses. The detection and monitoring of many chronic diseases and cancers requires multi-factorial/multi-analyte measurement. Specialized blood collection tubes (e.g., liquid biopsy tubes) are routinely used in the development of minimally invasive methods to monitor key cancer biomarkers in blood. The suitability of plasma isolated from liquid biopsy blood tubes for quantitative proteomics studies needs further exploration. Herein we performed proteomic analyses of whole blood collected and stored in LBgard Blood Tubes. In this study, matched blood samples from 3 healthy donors were collected in LBgard Blood Tubes and K3EDTA (EDTA) tubes, which is the standard blood collection tube used for plasma proteomic analyzes. Blood collected in LBgard Blood Tubes was stored for either 5 days or 7 days at ambient temperature. Following high-abundant plasma protein depletion, plasma proteins were analyzed in quantitative discovery proteomics by liquid chromatography tandem-mass spectrometry (TMT-LC-MS/MS). Over 800 unique proteins were identified by mass spectrometry in the plasma samples analyzed. Of those, &amp;gt;90% were found to be of similar abundance between EDTA Day 0 plasma samples and plasma isolated from whole blood collected in LBgard Blood Tubes. After storage of the LBgard Blood Tube samples for up to 7 days at ambient temperature, &amp;gt;85% of the proteins demonstrated similar abundance in both stored LBgard Blood samples and EDTA tube samples processed immediately after collection. Samples were also evaluated via ELISA to quantify the levels of proteins known to be cancer related. LBgard Blood Tubes prevented changes in the plasma concentrations of several protein biomarkers known to be sensitive to preanalytical variability in samples stored for 5 days at ambient temperature as compared to samples stored in EDTA tubes under the same conditions. Utilizing tandem-mass tag spectrometry and immunoassay methodologies, we have determined the compatibility of LBgard Blood Tube for determining proteomic profiles of blood samples. Additionally, blood collected in LBgard Blood Tubes was shown to have consistent concentrations of protein after storing the collected blood sample for several days, with enhanced protein detection as compared to EDTA tubes. Citation Format: Jacob M. Vasquez, Joel Desharnais. Multi-omic considerations: Exploring LBgard blood tubes for proteomic analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2854.

Quantification of circadian rhythm in mitochondrial DNA copy number in whole blood, and identification of factors that influence it

Mitochondrial DNA (mtDNA), the genetic material in mitochondria, encodes key genes related to the respiratory chain and ATP production. To accurate quantification mtDNA content in whole blood is important for various disease states. Absolute quantitative Real-time PCR and platelet contamination erase method were used for mtDNA copy number analysis in whole blood. In the quantitative study of mtDNA content, it was found that whole blood mtDNA copy number showed a fluctuating rhythm during a 24-h period due to dynamic changes in white blood cells combined with platelets. However, when isolated white blood cells were used, or absolute whole blood mtDNA was calculated, the circadian rhythm pattern of mtDNA disappeared. In this study, a feasible method that can accurately quantify mitochondrial DNA in small blood samples was established, and it was found that two factors which greatly influenced mtDNA copy number were sampling time and platelets in blood.

Prospective Evaluation of Feline Sourced Platelet-Rich Plasma Using Centrifuge-Based Systems.

Objective: To evaluate the hematologic components of platelet-rich plasma (PRP) generated using feline blood with two commercially available centrifuge-based systems1,2.Materials and methods: Twenty healthy adult cats were enrolled in this prospective study from November 2018 to January 2019. Feline blood samples were obtained for analysis of whole blood (WB) cellular components and preparation of PRP product. PRP was prepared using two commercial systems and complete blood count (CBC) testing was performed on both WB and PRP samples. The cellular composition of the PRP product was compared to the WB sample for each patient.Results: Both systems showed significant decrease of median RBC concentration in PRP products compared to WB samples (P = 0.002 for both systems). System 1 significantly decreased median WBC concentration (P = 0.002). System 2 decreased WBC concentration, though statistical significance was not reached (P = 0.63). Median platelet concentration was decreased by 3% using System 1, and increased by 187% using System 2. Platelet aggregation presented a challenge with 8/20 (40%) of samples demonstrating platelet aggregation.Clinical relevance: Commercial systems available for generation of PRP may be useful for creating a feline sourced product and in this study showed promise in decreasing RBC and WBC concentration. Neither system tested achieved 2–5 times platelet concentration from baseline. Platelet aggregation presented a significant obstacle to reliable generation of PRP products using feline blood. This treatment modality may be particularly beneficial for feline patients with osteoarthritis and soft tissue injuries, though first characterizing the PRP product made using feline blood is critical to validate its use in further clinical studies.

Research Highlights

Research Highlights

P0934THE IMPACT OF HAEMODIALYSIS INITIATION ON CKD-ASSOCIATED MYELOID CELL DYSREGULATION

Abstract Background and Aims Abnormalities of the myeloid immune cell lineages are well described in people with both Chronic Kidney Disease (CKD) and End Stage Renal Disease (ESRD). Increased numbers and proportions of the intermediate (CD14++CD16+) and nonclassical (CD14+CD16+) monocyte subsets and increased neutrophil numbers have been reported in both settings. These changes are one aspect of the chronic inflammatory state which exists in people with CKD and ESRD and which contributes to complications including cardiovascular disease and protein-energy malnutrition. The effects of transition from advanced CKD to dialysis on myeloid cell populations has not been well studied. We investigated these changes by measuring major leucocyte population numbers and monocyte subset numbers and proportions in adults with Stage 5 CKD prior to and after haemodialysis initiation. Method Adults with stage 5 CKD and a non-emergent indication for dialysis initiation (in-centre haemodialysis) were recruited after informed consent. Blood samples were collected within the week prior to dialysis initiation and follow-up samples were collected prior to a dialysis session at one week and one month after haemodialysis initiation. Major leucocyte population numbers were determined by two-colour flow cytometric analysis of whole blood on an Accuri™ Flow Cytometer. Peripheral blood mononuclear cells were isolated and stained for multi-colour flow cytometric analysis on a Canto II™ flow cytometer. The absolute number of each monocyte subset/ml blood was calculated based on the above values. Results Ten individuals (six male, four female) with a mean age of 78 ± 6.4years were enrolled and completed all follow-ups. The mean eGFR was 9 ± 1.9ml/min at the time of haemodialysis initiation. Total monocyte numbers had not changed after one month of haemodialysis (Figure 1A) (3.6x105 ± 1.6x105cells/ml) compared to initiation (5.2x105 ± 3.2x105, p=0.11). However, the proportion of nonclassical monocytes was markedly increased after one week of haemodialysis (16.8% (IQR 12.8-21.0%) compared to 11.2% (IQR 9.3-12.3%) at initiation, p=0.007) (Figure 1B). At one month the proportion of nonclassical monocytes was maintained (17.1% (IQR 14.5-20.5%)) but had not increased further compared to the one week timepoint (p=0.97). This proportionate change was not reflected in the absolute nonclassical count. There were no significant changes in the proportion of classical or intermediate monocytes. Neutrophil numbers were reduced at one month (3.6x106 ± 1.3x106cells/ml) compared to initiation (4.8X106 ± 1.6x106cells/ml, p=0.04) (Figure 1A). Total lymphocyte numbers did not change significantly after dialysis initiation. Conclusion Haemodialysis initiation is associated with an increase in the proportion of nonclassical monocytes without significant increases in the absolute number of any monocyte subset. A reduction in total neutrophil number also occurs one month after dialysis initiation. It has been previously been shown that progression of CKD results in increasing abnormalities of the myeloid cell lineages. Here we have demonstrated that transition from advanced CKD to haemodialysis results in further modulation of myeloid cell numbers and myeloid cell subset proportions.