Differential Blood Cell Research Articles

Advanced Bio-sensing Technologies for Sickle Cell Disease Diagnosis.

Sickle cell diseases are widespread in regions encompassing the Mediterranean, Middle East, sub-Saharan Africa, and specific parts of Asia, primarily due to the abnormal production of hemoglobin S. This genetic blood disorder stems from a mutation in the beta-globin gene, a crucial component of hemoglobin and the heme-containing protein found in red blood cells. Point mutations in the hemoglobin gene can be inherited as a heterozygous or homozygous pattern. These mutations disrupt the normal configuration of the protein, impeding its physiological function and altering the cell's shape, giving it a sickle-like appearance. The resulting sickle cells can lead to organ damage, intense physical discomfort, and anemia; in severe cases, the condition can be fatal. Early detection and effective treatment methods have the potential to progressively reduce the associated mortality rate over time. To diagnose sickle cell disease and its carrier states with unparalleled specificity, a variety of approaches have been developed. The most common method includes differential blood cell counts and their assessment, high-performance liquid chromatography (HPLC) and hemoglobin electrophoresis. Furthermore, innovative sensing technologies are currently under development, encompassing user-friendly, cost-effective and portable point-of-care devices that are capable of timely diagnosis at the genetic and molecular levels of these disorders. The review delves into a range of established and innovative strategies utilized in the detection of sickle cell disease, also underscoring the essential role played by diverse bio-sensing techniques in propelling the advancement of early diagnosis of SCD.

Chronische myelomonocytaire leukemie als toevallige vondst op de dienst geriatrie

Chronic myelomonocytic leukemia as an incidental finding at the geriatric department Persistent monocytosis may indicate a hematologic malignancy and merits a hematologic work-up. Chronic myelomonocytic leukemia (CMML) is a rare, but malignant disorder of the hematopoietic stem cell with an annual incidence of 1-2 persons per 100,000. The real incidence is probably higher because of underdiagnosis. The median age at the diagnosis is 65 to 75 years. The diagnosis is made based on the clinical symptoms, a peripheral blood analysis, a bone marrow aspirate and biopsy, as well as cytogenetic and molecular studies. The symptomatology is non-specific, but can be classified into a group with myelodysplastic (fatigue, anemia, infections) and a group with myeloproliferative (B-symptoms, hepatosplenomegaly) features. In the peripheral blood, by definition, one measures ≥ 0,5 x 109/l of monocytes, which account for more than 10% of the white blood cell differentiation. The bone marrow is often hypercellular with the presence of dysplasia. In 20% to 30% of the cases, cytogenetic abnormalities can be found, i.e. abnormalities in the karyotype. In the vast majority (90%), somatic mutations are present. The prognosis of patients with CMML is reserved with a median survival of about 3 years. One in 3 progresses to acute myeloid leukemia. The therapeutic options for CMML are limited. The only possible curative treatment is an allogeneic stem cell transplantation. The other options are primarily supportive and intended as symptom control.

Subclinical left ventricular dysfunction and laboratory predictor of activity in children with ulcerative colitis: A single-centre study.

We aimed to detect subclinical cardiac impairment in children with ulcerative colitis (UC) and test the association between absolute monocytic count (AMC) and lymphocyte-to-monocyte ratio (LMR) with disease activity. A group of children with UC and a comparable group as healthy controls were included. All children underwent history-taking, clinical examination and blood testing for complete blood counts with white blood cell differentials, LMR and erythrocyte sedimentation rate (ESR). Disease severity was assessed using the Paediatric UC Activity Index score. We used echocardiography for tissue Doppler, M-Mode, two-dimensional and three-dimensional (3D) speckle tracking echocardiography (STE) for left ventricular function assessment. Forty children were included, 20 with UC as cases, and 20 healthy controls. Disease activity was mild in 75% cases and moderate in 25% cases. Cases had significantly higher ESR than the control group (P < 0.001). Among cases, positive correlations were observed between monocytic, and platelet counts with left ventricular end-diastolic diameter (r = 0.5, P = 0.02; r = 0.5, P = 0.03). Children with UC had significantly lower ejection fraction and impaired left ventricular systolic function compared to the control group (P < 0.001) assessed by 3D STE, yet this observation was not reached by the conventional method (P = 0.3). In children with UC, 3D STE could detect subclinical left ventricular systolic dysfunction that conventional echocardiography could not. AMC and LMR showed no significant difference between children with UC and controls.

A Novel Intravaginal Contraceptive Plug for Cats: A Preliminary Biocompatibility Assessment on Haematology and Vaginal Swab.

This preliminary study evaluated the biocompatibility of a novel degradable intravaginal plug contraceptive composed of PEG 4000 and chitosan in cats using haematological profiling and vaginal cytology. Five healthy, non-pregnant female cats were fully anaesthetised and fitted with an intravaginal plug (10 × 0.3 mm) using an applicator, following oestrogen administration 3 h prior. Blood samples were collected from the cephalic vein on days 0 (pre-insertion) and 3 and 7 (post-insertion). Vaginal cytology examinations were conducted on day 0 (pre- and post-oestrogen injection) and days 1, 3 and 7 post-insertion. Haematological parameters, including red blood cell count, haemoglobin levels, haematocrit values, total white blood cell count and differentiation, showed no significant changes after contraceptive insertion (p > 0.05). Vaginal cytology indicated an acute inflammatory response in one out of five subjects on day three post-insertion. The distribution of vaginal epithelial cells (parabasal, intermediate and superficial) remained unaffected by contraception. Oestrogen injection resulted in the dominance of superficial cells up to day 7 of observation (p < 0.05). Overall, PEG 4000 and chitosan-based intravaginal plug contraceptives demonstrated sufficient biocompatibility, indicating their potential as viable contraceptive options for feline use.

Essential role of Dhx16-mediated ribosome assembly in maintenance of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are vital for the differentiation of all mature blood cells, with their homeostasis being tightly regulated by intrinsic and extrinsic factors. Alternative splicing, mediated by the spliceosome complex, plays a crucial role in regulating HSC homeostasis by increasing protein diversity. This study focuses on the ATP-dependent RNA helicase DHX16, a key spliceosome component, and its role in HSC regulation. Using conditional knockout mice, we demonstrate that loss of Dhx16 in the hematopoietic system results in significant depletion of hematopoietic stem and progenitor cells, bone marrow failure, and rapid mortality. Dhx16-deficient HSCs exhibit impaired quiescence, G2-M phase cell cycle arrest, reduced protein synthesis, abnormal ribosome assembly, increased apoptosis, and decreased self-renewal capacity. Multi-omics analysis identified intron 4 retention in Emg1 mRNA in Dhx16 knockout HSCs, leading to reduced EMG1 protein expression, disrupted ribosome assembly, and nucleolar stress, activating the p53 pathway. Overexpression of Emg1 in Dhx16-deficient HSCs partially restored ribosome assembly and HSC function, suggesting Emg1 as a potential therapeutic target for ribosomopathies. Our findings reveal the critical role of Dhx16 in HSC homeostasis through the regulation of alternative splicing and ribosome assembly, providing insights into the molecular mechanisms underlying hematopoietic diseases and potential therapeutic strategies.

Protein phosphatase 2A regulates blood cell proliferation and differentiation in Drosophila larval lymph glands.

Protein phosphatase 2A (PP2A), one of the most abundant protein phosphatases, has divergent functions in multiple types of cells. Its inactivation has been closely associated with leukemia diseases. However, the physiological function of PP2A for hematopoiesis has been poorly understood in organisms. Drosophila hematopoiesis parallels the vertebrate counterpart in developmental and functional features but involves a much simpler hematopoietic system. Here, utilizing the Drosophila major larval hematopoietic organ lymph gland, we studied the function of PP2A for hematopoiesis in vivo. By knocking down the expression of Pp2A-29B that encodes the scaffold subunit of the PP2A holoenzyme complex, we found that PP2A silencing in the differentiating hemocytes resulted in their excessive proliferation. Furthermore, this PP2A inhibition downregulated the expression of Smoothened (Smo), a crucial component in the Hedgehog pathway, and smo overexpression was able to rescue the phenotypes of PP2A depletion, indicating that Smo functions as a downstream effector of PP2A to restrict the hemocyte proliferation. PDGF/VEGF-receptor (Pvr) overexpression also restored the Smo expression and lymph gland morphology of PP2A silencing, suggesting a PP2A-Pvr-Smo axis to regulate lymph gland growth and hemocyte proliferation. Moreover, inhibiting PP2A activity in the blood progenitor cells promoted their differentiation, but which was independent with Smo. Together, our data suggested that PP2A plays a dual role in the Drosophila lymph gland by preserving the progenitor population and restraining the hemocyte proliferation, to properly regulate the hematopoietic process.

HES1 is required for mouse fetal hematopoiesis

BackgroundHematopoiesis in mammal is a complex and highly regulated process in which hematopoietic stem cells (HSCs) give rise to all types of differentiated blood cells. Previous studies have shown that hairy and enhancer of split (HES) repressors are essential regulators of adult HSC development downstream of Notch signaling.MethodsIn this study, we investigated the role of HES1, a member of HES family, in fetal hematopoiesis using an embryonic hematopoietic specific Hes1 conditional knockout mouse model by using phenotypic flow cytometry, histopathology analysis, and functional in vitro colony forming unit (CFU) assay and in vivo bone marrow transplant (BMT) assay.ResultsWe found that loss of Hes1 in early embryonic stage leads to smaller embryos and fetal livers, decreases hematopoietic stem progenitor cell (HSPC) pool, results in defective multi-lineage differentiation. Functionally, fetal hematopoietic cells deficient for Hes1 exhibit reduced in vitro progenitor activity and compromised in vivo repopulation capacity in the transplanted recipients. Further analysis shows that fetal hematopoiesis defects in Hes1fl/flFlt3Cre embryos are resulted from decreased proliferation and elevated apoptosis, associated with de-repressed HES1 targets, p27 and PTEN in Hes1-KO fetal HSPCs. Finally, pharmacological inhibition of p27 or PTEN improves fetal HSPCs function both in vitro and in vivo.ConclusionTogether, our findings reveal a previously unappreciated role for HES1 in regulating fetal hematopoiesis, and provide new insight into the differences between fetal and adult HSC maintenance.

Performance evaluation of the digital morphology analyser Sysmex DI-60 for white blood cell differentials in abnormal samples

Sysmex DI-60 enumerates and classifies leukocytes. Limited research has evaluated the performance of Sysmex DI-60 in abnormal samples, and most focused on leukopenic samples. We evaluate the efficacy of DI-60 in determining white blood cell (WBC) differentials in normal and abnormal samples in different WBC count. Peripheral blood smears (n = 166) were categorised into normal control and disease groups, and further divided into moderate and severe leucocytosis, mild leucocytosis, normal, mild leukopenia, and moderate and severe leukopenia groups based on WBC count. DI-60 preclassification and verification and manual counting results were assessed using Bland–Altman and Passing–Bablok regression analyses. The Kappa test compared the concordance in the abnormal cell detection between DI-60 and manual counting. DI-60 exhibited notable overall sensitivity and specificity for all cells, except basophils. The correlation between the DI-60 preclassification and manual counting was high for segmented neutrophils, band neutrophils, lymphocytes, and blasts, and improved for all cell classes after verification. The mean difference between DI-60 and manual counting for all cell classes was significantly high in moderate and severe leucocytosis (WBC > 30.0 × 109/L) and moderate and severe leukopenia (WBC < 1.5 × 109/L) groups. For blast cells, immature granulocytes, and atypical lymphocytes, the DI-60 verification results were similar to the manual counting results. Plasma cells showed poor agreement. In conclusion, DI-60 demonstrates consistent and reliable analysis of WBC differentials within the range of 1.5–30.0 × 109. Manual counting was indispensable in examining moderate and severe leucocytosis samples, moderate and severe leukopenia samples, and in enumerating of monocytes and plasma cells.

Toxoplasma gondii suppress human cord blood cell differentiation to the NK cell population.

Toxoplasma gondii is an obligate intracellular protozoan parasite that can invade all mammalian cells. It is well established that natural killer (NK) cells have critical protective roles in innate immunity during infections by intracellular pathogens. In the current study, we conducted an in vitro experiment to evaluate NK cell differentiation and activation from human umbilical cord blood mononuclear cells (UCB-MNCs) after infection with T. gondii tachyzoites. UCB-MNCs were infected by fresh tachyzoites of type I (RH) or type II (PTG) strains of T. gondii pre-expanded in mesenchymal stem cells for 2 weeks in a medium enriched with stem cell factor,Flt3, IL-2, and IL-15. Flow cytometry analysis and western blot analysis were performed to measure the CD57+, CD56+, and Granzyme A (GZMA). Data revealed that incubation of UCB-MNCs with NK cell differentiation medium increased the CD57+, CD56+, and GZMA. UCB-MNCs cocultured with PTG tachyzoites showed a significant reduction of CD56+ and GZMA, but nonsignificant changes, in the levels of CD56+ compared to the control UCB-MNCs (p > .05). Noteworthy, 2-week culture of UCB-MNCs with type I (RH) tachyzoites significantly suppressed CD57+, CD56+, and GZMA, showing reduction of NK cell differentiation from cord blood cells. Our findings suggest that virulent T. gondii tachyzoites with cytopathic effects inhibit NK cell activation and eliminate innate immune responses during infection, and consequently enable the parasite to continue its survival in the host body.

A comparative study of blood cell count in four automated hematology analyzers: An evaluation of the impact of preanalytical factors.

Differential white blood cell counts are frequently used in diagnosis, patient stratification, and treatment selection to optimize therapy responses. Referral laboratories are often used but challenged with use of different hematology platforms, variable blood shipping times and storage conditions, and the different sensitivities of specific cell types. To extend the scientific literature and knowledge on the temporal commutability of blood samples between hematology analyzers, we performed a comparative ex-vivo study using four of the most utilized commercial platforms, focusing on the assessment of eosinophils given its importance in asthma management. Whole blood from healthy volunteers with and without atopy (n = 6+6) and participants with eosinophilic asthma (n = 6) were stored under different conditions (at 4, 20, 30, and 37°C, with or without agitation) and analyzed at different time points (3, 6, 24, 48 and 72h post-sampling) in parallel on the Abbott CELL-DYN Sapphire, Beckman Coulter DxH900, Siemens ADVIA 2120i and Sysmex XN-1000V. In the same blood samples, eosinophil-derived neurotoxin (EDN), eosinophil activation and death markers were analyzed. All platforms gave comparable measurements of cell differentials on fresh blood within the same day of sampling. However, by 24 hours, significant temporal and temperature-dependent differences were observed, most markedly for eosinophils. None of the platforms performed perfectly across all temperatures tested during the 72 hours, showing that handling conditions should be optimized depending on the cell type of interest and the hematology analyzer. Neither disease status (healthy vs. asthma) nor agitation of the sample affected the cell quantification result or EDN release. The eosinophil activation markers measured by flow cytometry increased with time, were influenced by temperature, and were higher in those with asthma versus healthy participants. In conclusion, hematology analyzer, time window from sampling until analysis, and temperature conditions must be considered when analyzing blood cell differentials, particularly for eosinophils, via central labs to obtain counts comparable to the values obtained in freshly sampled blood.

Semen and haematological characteristics of cocks drenched varying levels of Turmeric Powder (TP)

Emerging evidence from in vivo as well as in vitro studies indicates that botanicals play noteworthy roles in the treatment, prevention and management of livestock diseases. Use of natural compounds in botanicals has been proposed as potential alternative to conventional therapeutic options. Therefore, this study aimed to evaluate the effect of varying levels of turmeric powder (Curcuma longa) on semen characteristics and haematological indices of cocks. Turmeric (C. longa) rhizomes were obtained from a local market in Saki in Oyo State, Nigeria, in March 2023. They were washed, skin scraped and air-dried at room temperature for 10 hours, and then oven-dried at 40°C for 12 hours. Afterwards, the dried turmeric rhizomes were ground into powder using a blender. The product was kept in an air-tight container until use. The experimental material was drenched at 0.00 g (T1), 0.50 g (T2), 1.00 g (T3) and 1.50 g (T4) after 2 weeks of acclimatization. Semen volume, sperm cell progressive motility, sperm cell liveability, acrosome integrity, sperm cell concentration and normal sperm cell were evaluated for semen characteristics. Haematological parameters measured were: PCV, RBC, WBC Hb, MCV, MCH, MCHC and white blood cell differentials. Data obtained were subjected to one-way analysis of variance. Semen volume (0.34 – 0.37 ml), sperm cell progressive motility (68.33 – 80%), sperm cell liveability (46.66 – 85.00%), acrosome integrity (50.00 – 85%) and normal sperm cell (66.66 – 90%) showed significant difference (p&lt;0.05) in favour of cocks on highest level (1.50 g) of turmeric powder, while sperm cell concentration (28.33 - 40.00 X109/ml) showed no significant difference (p&gt;0.05). Also PCV (36.00 – 40.33%), RBC (3.55 – 3.74 X106/ml), WBC (19.01 – 19.71 X109/ml), Hb (11.66 – 13.00 dl), MCV (100.53 – 109.53 ⴄ), MCH (32.57 – 35.31pg) and MCHC (32.00 – 32.37%) showed no significant differences (p&gt;0.05) between treatments. In conclusion turmeric powder up to 1.50 g showed an improvement in all the semen characteristics measured without negative effect on haematological characteristics of cocks.

Monomethyl auristatin E (MMAE), a payload for multiple antibody drug conjugates (ADCs), demonstrates differential red blood cell partitioning across human and animal species

Background: Monomethyl auristatin E (MMAE) has been used as a payload for several Food and Drug Administration (FDA) approved antibody-drug conjugates (ADCs). It is known that MMAE is released from the ADC following binding, internalisation and proteolytic degradation in target tissues. A striking discrepancy in systemic MMAE levels has been observed across species with 50-fold higher MMAE levels in human than that in rodents when normalised by ADC dose with unknown mechanism. Hypothesis and purpose: Multiple factors could affect systemic MMAE levels such as production and elimination of unconjugated MMAE following ADC dosing. In this study, we have explored whether MMAE displays differential red blood cell (RBC) partitioning across species that may contribute to the different MMAE levels seen between human and animals. Experiments: To determine MMAE RBC partitioning, tritium labelled MMAE ([3H]-MMAE) was incubated in whole blood from mice, rats, monkeys and humans in vitro, then RBC partitioning was determined and compared across species. To test whether MMAE released from the ADC would show any difference in RBC partitioning, pinatuzumab vedotin or polatuzumab vedotin was administered to mice, rats, and monkeys. MMAE levels were measured in both blood and plasma, and the ratios of MMAE levels were calculated as blood-to-plasma ratio (in vivo RBC partitioning). Results: Our in vitro data showed that unconjugated MMAE has a species-dependent RBC partitioning with strong RBC partitioning in mouse, rat, followed by monkey blood, whereas minimal RBC partitioning was seen in human blood. Incubation of 2 nM of MMAE in mouse blood resulted in a blood-to-plasma ratio of 11.8 ± 0.291, followed by rat, monkey, and human at 2.36 ± 0.0825, 1.57 ± 0.0250, and 0.976 ± 0.0620, respectively. MMAE RBC partitioning is also concentration-dependent, with an inverse relationship between RBC partitioning and MMAE concentration (higher RBC partitioning at lower concentration). In vivo dosing of pinatuzumab vedotin in mouse displayed systemic MMAE at about a 5-fold higher blood concentration compared to plasma concentration once MMAE reached a pseudo-equilibrium, while systemic MMAE from blood and plasma concentration showed a 1.65-fold difference in rat. Implication and conclusion: These data demonstrated that MMAE has a distinct RBC partitioning across different species, which may contribute to, at least in part, to the differential in the systemic MMAE levels observed in vivo between preclinical and clinical studies. These findings highlight the importance of fully characterising the ADME properties of both the ADC and its payload, to enable better translation from animals to human for ADC development.

Antioxidant Effects of Vitamin C on Some Hematological Parameters of Male Wistar Rats Exposed to Lead Acetate

The study investigated the effects of vitamin C on platelet parameters, white blood cell count and white blood cells differentials in Wistar rats exposed to lead acetate. A total of twenty-four male Wistar rats weighing between 160g and 200g were utilized. The experimental animals were divided into four groups of six rats each (n=6). Group 1 served as the control group (received normal feed and water), group II received 10mg/kg body weight of lead acetate, group III received 100mg/kg body weight of Vitamin C, and group IV received 10mg/kg body weight of lead acetate followed by 100mg/kg body weight of Vitamin C. Lead and Vitamin C, along with normal feed, were administered for four weeks. Blood samples were collected via jugular puncture and stored in EDTA bottles for analysis to determine the blood profile of the rats. The results showed significant increase in Platelet Count (PLT) in group III and a significant decrease in Mean Platelet Volume (MPV) in group IV (Pb + vitamin C). The Mean Platelet Width (MPW) showed decrease in groups 2, 3, and 4 compared to the control group, although this decrease was not statistically significant. The study also noted an elevated level of white blood cells (WBC) in response to the antioxidant treatment, indicating a potential positive impact on immune function. In conclusion, this study demonstrates the therapeutic effect of Vitamin C against the toxic effects of lead on platelet parameters and white blood cell count and differentials.

RIO-kinase 2 is essential for hematopoiesis.

Regulation of protein synthesis is a key factor in hematopoietic stem cell maintenance and differentiation. Rio-kinase 2 (RIOK2) is a ribosome biogenesis factor that has recently been described an important regulator of human blood cell development. Additionally, we have previously identified RIOK2 as a regulator of protein synthesis and a potential target for the treatment of acute myeloid leukemia (AML). However, its functional relevance in several organ systems, including normal hematopoiesis, is not well understood. Here, we investigate the consequences of RIOK2 loss on normal hematopoiesis using two different conditional knockout mouse models. Using competitive and non-competitive bone marrow transplantations, we demonstrate that RIOK2 is essential for the differentiation of hematopoietic stem and progenitor cells (HSPCs) as well as for the maintenance of fully differentiated blood cells in vivo as well as in vitro. Loss of RIOK2 leads to rapid death in full-body knockout mice as well as mice with RIOK2 loss specific to the hematopoietic system. Taken together, our results indicate that regulation of protein synthesis and ribosome biogenesis by RIOK2 is essential for the function of the hematopoietic system.

The Evaluation of Artificial Intelligence Technology for the Differentiation of Fresh Human Blood Cells From Other Species' Blood in the Investigation of Crime Scenes.

The current study used the deep machine learning approach to differentiate human blood specimens from cow, goat, and chicken blood stains based on cellmorphology. A total of 1,955 known Giemsa-stained digitized images were acquired from the blood of humans, cows, goats, and chickens. To train the deep learning models, the well-known VGG16, Resnet18, and Resnet34 algorithms were used. Based on the image analysis, confusion matrices were generated. Findings showed that the F1 score for the chicken, cow, goat, and human classes were all equal to 1.0 for each of the three algorithms. The Matthews correlation coefficient (MCC) was 1 for chickens, cows, and humans in all three algorithms, while the MCC score was 0.989 for goats by ResNet18, and it was 0.994 for both ResNet34 and VGG16 algorithms. The three algorithms showed 100% sensitivity, specificity, and positive and negative predictive values for the human, cow, and chicken cells. For the goatcells, the data showed 100% sensitivity and negative predictive values with specificity and positive predictive values ranging from 98.5% to 99.6%. These data showed the importance of deep learning as a potential tool for the differentiation of the species of origin of fresh crime scene blood stains.

Sialic acid in the regulation of blood cell production, differentiation and turnover.

Sialic acid is a unique sugar moiety that resides in the distal and most accessible position of the glycans on mammalian cell surface and extracellular glycoproteins and glycolipids. The potential for sialic acid to obscure underlying structures has long been postulated, but the means by which such structural changes directly affect biological processes continues to be elucidated. Here, we appraise the growing body of literature detailing the importance of sialic acid for the generation, differentiation, function and death of haematopoietic cells. We conclude that sialylation is a critical post-translational modification utilized in haematopoiesis to meet the dynamic needs of the organism by enforcing rapid changes in availability of lineage-specific cell types. Though long thought to be generated only cell-autonomously within the intracellular ER-Golgi secretory apparatus, emerging data also demonstrate previously unexpected diversity in the mechanisms of sialylation. Emphasis is afforded to the mechanism of extrinsic sialylation, whereby extracellular enzymes remodel cell surface and extracellular glycans, supported by charged sugar donor molecules from activated platelets.

Performance Assessment of Sysmex DI-60: Is Digital Morphology Analyzer Reliable for White Blood Cell Differentials in Body Fluids?

Few studies have evaluated digital morphology (DM) analyzers on body fluids (BF). We evaluated the performance of a DM analyzer, Sysmex DI-60 (Sysmex, Kobe, Japan) for white blood cell (WBC) differentials in BF samples. In five BF samples (two pleural fluids and three ascites) containing a single, dominant cell type (>80%, neutrophils, lymphocytes, macrophages, abnormal lymphocytes, and malignant cells in each sample), we evaluated the precision of the DI-60 and compared the WBC differentials and turnaround times (TAT) between DI-60 and manual counting. The precision of the DI-60 pre-classification and verification was excellent (%CV, 0.01-3.16%). After verification, the DI-60 showed high sensitivity, specificity, and efficiency (ranges: 90.8-98.1%, 96.8-97.9%, and 92.5-98.0%, respectively) for the dominant cell types in neutrophil- and lymphocyte-dominant samples. For all samples, the DI-60 and manual counting showed high correlations for major cell types (neutrophils, lymphocytes, macrophages, and others, r = 0.72 to 0.94) after verification. The agreement between the pre-classification and verification of the DI-60 was strong in the neutrophil-dominant sample (κ = 0.81). The DI-60 showed a significantly longer TAT (min: s) than manual counting for all samples (median TAT/slide: 6:28 vs. 1:53, p < 0.0001), with remarkable differences in abnormal lymphocyte- and malignant cell-dominant samples (21:05 vs. 2:06; 12:34 vs. 2:25). The DI-60 may provide reliable data in neutrophil- and lymphocyte-dominant BF samples. However, it may require longer times and higher workloads for WBC differentials especially in BF samples containing atypical cells. Further improvement would be needed before applying DM analyzers for routine clinical practice in BF analysis.

Integration of transcription regulation and functional genomic data reveals lncRNA SNHG6’s role in hematopoietic differentiation and leukemia

BackgroundLong non-coding RNAs (lncRNAs) are pivotal players in cellular processes, and their unique cell-type specific expression patterns render them attractive biomarkers and therapeutic targets. Yet, the functional roles of most lncRNAs remain enigmatic. To address the need to identify new druggable lncRNAs, we developed a comprehensive approach integrating transcription factor binding data with other genetic features to generate a machine learning model, which we have called INFLAMeR (Identifying Novel Functional LncRNAs with Advanced Machine Learning Resources).MethodsINFLAMeR was trained on high-throughput CRISPR interference (CRISPRi) screens across seven cell lines, and the algorithm was based on 71 genetic features. To validate the predictions, we selected candidate lncRNAs in the human K562 leukemia cell line and determined the impact of their knockdown (KD) on cell proliferation and chemotherapeutic drug response. We further performed transcriptomic analysis for candidate genes. Based on these findings, we assessed the lncRNA small nucleolar RNA host gene 6 (SNHG6) for its role in myeloid differentiation. Finally, we established a mouse K562 leukemia xenograft model to determine whether SNHG6 KD attenuates tumor growth in vivo.ResultsThe INFLAMeR model successfully reconstituted CRISPRi screening data and predicted functional lncRNAs that were previously overlooked. Intensive cell-based and transcriptomic validation of nearly fifty genes in K562 revealed cell type-specific functionality for 85% of the predicted lncRNAs. In this respect, our cell-based and transcriptomic analyses predicted a role for SNHG6 in hematopoiesis and leukemia. Consistent with its predicted role in hematopoietic differentiation, SNHG6 transcription is regulated by hematopoiesis-associated transcription factors. SNHG6 KD reduced the proliferation of leukemia cells and sensitized them to differentiation. Treatment of K562 leukemic cells with hemin and PMA, respectively, demonstrated that SNHG6 inhibits red blood cell differentiation but strongly promotes megakaryocyte differentiation. Using a xenograft mouse model, we demonstrate that SNHG6 KD attenuated tumor growth in vivo.ConclusionsOur approach not only improved the identification and characterization of functional lncRNAs through genomic approaches in a cell type-specific manner, but also identified new lncRNAs with roles in hematopoiesis and leukemia. Such approaches can be readily applied to identify novel targets for precision medicine.

Manipulation of neonatal ruminal populations at birth results in sustained effects on microbial populations and measures of health and production in merino and suffolk lambs

The efficiency of the microbial-driven fermentation process in the rumen is closely related to the efficiency of the ruminant host. This experiment examined the effect of manipulating the neonatal microbiota on postnatal development, immune function and productivity (liveweight gain and wool growth parameters). The trial examined the differences between naturally inoculated lambs (maternal control, n = 21) and those given ruminal fluid, which had been collected from ruminally-cannulated ewes, fed either a roughage diet (roughage, n = 14), or a grain-based diet (high grain, n = 14). At lambing, newborn lambs were tagged and weighed. Inoculation lambs received 10 ml of rumen fluid per inoculation, daily, for a total of 7 inoculations. Live weight, body condition scores, wool growth (greasy and clean wool weights), wool length, mean fibre diameter, and differential blood cell counts, and IgG and IgA in both blood and saliva) were quantified from birth until week 18 (slaughter). Total feed intake was recorded from weaning. Ruminal fluid was collected by stomach tube from 12 lambs from each treatment group and their dams (n = 7–8 per treatment, with the lower number of ewes coming about through ewes that had twins) at time of weaning (week 10). The pH of the ruminal fluid, the density of ciliated protozoa in ruminal fluid, and the composition of the ruminal bacterial populations were analysed. At slaughter (week 18), ileal samples were taken from the same focal lambs as were used for microbial analysis. The artificial inoculations resulted in significant differences in ruminal bacterial genera to those of naturally inoculated lambs at time of weaning (some 3 months post-lambing). Significant differences were also apparent in ileal bacterial communities at slaughter, between lambs which had received the inoculum from roughage-fed ewes and naturally inoculated lambs. Both the efficiency (growth, condition, feed conversion efficiency, carcass weight) and indices of health (mortalities, red blood cell count, blood haemoglobin concentration, haematocrit, and eosinophil counts) of the inoculated animals, were affected negatively by the inoculations ( < 0.05). There was a significant interaction between inoculum source and breed in terms of effects on all parameters measured. The effects of the high grain inoculation were significantly affected by breed, with high grain merinos found to have reduced mean fibre diameter (P < 0.05) and increased wool growth relative to the maternal control (P = 0.035). This effect was not noted in the suffolks. In summary, we have demonstrated that long-term effects on ruminal and intestinal microbiota can be generated by intervention in the normal process of ruminal inoculation in neonatal lambs. Moreover, there appeared to be genetic differences in response to artificial inoculation. Whilst the effects of artificial inoculation in this trial were negative in terms of indices of health and production, further studies of maternal diet and its effects on the natural establishment of the neonatal microbiota are warranted given the persistence of the effects and the interaction with breed.

Baseline haematological parameters in three common Australian frog species.

Amphibians are experiencing declines globally, with emerging infectious diseases as one of the main causes. Haematological parameters present a useful method for determining the health status of animals and the effects of particular diseases, but the interpretation of differential cell counts relies on knowing the normal ranges for the species and factors that can affect these counts. However, there is very little data on either normal haematological parameters or guides for blood cell types for free-ranging frog species across the world. This study aims to 1) create a visual guide for three different Australian frog species: Litoria paraewingi, Limnodynastes dumerilii, and Crinia signifera, 2) determine the proportions of erythrocytes to leukocytes and 3) differential leukocytes within blood smears from these three species and 4) assess the association between parasites and differential counts. We collected blood samples from free-ranging frogs and analysed blood smears. We also looked for ectoparasites and tested for the fungal disease chytridiomycosis. Overall, we found that the differentials of erythrocytes to leukocytes were not affected by species, but the proportions of different leukocytes did vary across species. For example, while lymphocytes were the most common type of leukocyte across the three species, eosinophils were relatively common in Limnodynastes dumerilii but rarely present in the other two species. We noted chytridiomycosis infection as well as ectoparasites present in some individuals but found no effect of parasites on blood parameters. Our results add baseline haematological parameters for three Australian frog species and provide an example of how different frog species can vary in their differential blood cell counts. More information is needed on frog haematological data before these parameters can be used to determine the health status of wild or captive frogs.