Automated Cell Counter Research Articles

The prolyl-hydroxylase inhibitior Roxadustat upregulates HIF-1alpha and is associated with improved cardiac repair after myocardial infarction in mice

Abstract Introduction Activation of the CXCL12/CXCR4-axis is known to aid myocardial repair by attraction of reparative CXCR4+ cells through ischemia triggered hypoxia-inducible factor-1α (HIF-1α) activation of CXCL12 (1). To prolongate upregulation of CXCL12 we aimed to stabilize HIF-1α by administration of Roxadustat, the only prolyl hydroxylase inhibitor (PHI) clinically approved by the EMA with the purpose to augment myocardial remodelling and improve LV function. Methods To analyse the effects of HIF-1α mediated CXCL12 upregulation in vivo (BM & heart), optimal doses (50mg/kg i.p., every 72h) of the PHI Roxadusat (FG-4592) were administered to Bl6/J mice. CXCL12 and CXCR4 expression was analysed in-vitro in human umbilical vascular endothelial cells (HUVEC) after administration of FG-4592 (20µM to 1000µM) at different time points (1 to 24 hrs). Western Blots were performed and effects on HIF-1α -signalling were quantified by densitometry. We induced MI in BL6/J mice at the age of 8-10 weeks by LAD-ligation. Two groups (n=10) were treated with Roxadustat or saline for 28 days before assessment of cardiac function by echocardiography and evaluation by histology. Two other groups of n=5 mice were treated with Roxadustat or saline for one week and analysed regarding apoptotic-index by automated cell count in DAPI & Tunel stained sections. Results We were able to show a significant upregulation of HIF-1α after exposure of 50-500 μM Roxadustat for 4-24 hours in-vitro (HUVEC) and in-vivo (murine BL6/J hearts). Moreover, administration of 500 µM Roxadustat induced marked activation of CXCR4 and CXCL12, respectively. Infarct size (19.07%±7.86 vs. 24.14%±12,05; n= 10/8; p = 0.297), as well as LV-wall thickness in the infarcted myocardium (788.27µm±240.10 vs. 651.40µm±231.47; n=10/8; p = 0.240) revealed no significant differences 28 days after MI. Automated cell counting in the borderzone revealed a lowered apoptotic-index of Roxadustat treated animals (3.7%±1.1 vs. 6.3%±2.3; p = 0.071). Echocardiographic analyses showed a significantly preserved ejection fraction (EF) (46.1%±6.0 vs. 30.5%±9.3; n=10/9; p<0.001), reduced diastolic (4.18±0.32 vs. 4.77±0.40; p=0.002) and systolic (3.23±0.31 vs. 3.95±0.43; p<0.001) LV diameters in Roxadustat treated mice 28 days after MI compared to saline treated controls. Conclusion The clinically approved PHI Roxadustat revealed increased expression of HIF-1α associated with upregulation of CXCR4 and CXCL12. Administration of Roxadustat after MI revealed attenuated infarct remodelling and functional improvement showing its potential in ischemic heart disease.

Enhanced harvest performance predictability through advanced multivariate data analysis of mammalian cell culture particle size distribution.

The industry's pursuit for higher antibody production has led to increased cell density cultures that impact the performance of subsequent product recovery steps. This increase in cell concentration has highlighted the critical role of solids concentration in centrifugation yield, while recent product degradation cases have shed light on the impact of cell lysis on product quality. Current methods for measuring solids concentration and cell lysis are not suited for early-stage high-throughput experimentation, which means that these cell culture outputs are not well characterized in early process development. This article describes a novel approach that leveraged the data from a widely-used automated cell counter (Vi-CELL™ XR) to accurately predict solids concentration and a common cell lysis indicator represented as lactate dehydrogenase (LDH) release. For this purpose, partial least squares (PLS) models were derived with k-fold cross-validation from the particle size distribution data generated by the cell counter. The PLS models showed good predictive potential for both LDH release and solids concentration. This novel approach reduced the time required for evaluating the solids concentration and LDH for a typical high-throughput cell culture system (with 48 bioreactors in parallel) from around 7 h down to a few minutes.

Red Cells Distribution Width and Prognosis in Myelofibrosis Patients Treated with Ruxolitinib

Introduction Myelofibrosis (MF) is a BCR::ABL1-negative myeloproliferative neoplasm characterized primarily by genetic abnormalities and an aberrant production of pro-inflammatory cytokines (including IL-2, IL-6, TNF-α, and TGF-β among others) released from pathological hematopoietic stem cells leading to the deposition of reactive fibrosis in the bone marrow (BM), at the same time the development of extramedullary hematopoiesis, mainly in the spleen and liver, and finally the production of red blood cells (RBC) of often bizarre shape, i.e. dacryocytes. Consequently, increased variability in RBC volume, so-called anisocytosis, is a hallmark of MF. A small number of studies have already demonstrated that quantitative measurement of anisocytosis in terms of red cells distribution width (RDW) may have a negative prognostic role in newly diagnosed MF patients. Aim In this study we further evaluate the prognostic impact of RDW in a series of patients with primary (PMF) and secondary myelofibrosis (SMF) at diagnosis and during treatment with ruxolitinib (RUX). Methods Between October 2014 and May 2023, the RDW of 49 consecutive patients with PMF or SMF (32 males and 17 females; median age at RUX initiation, 70.5 years) were retrospectively assessed at MF diagnosis, at RUX start and after 3, 6, 12, 24 and 36 months from therapy initiation. All patients were treated with RUX according to current indications, requiring an IPSS risk of at least intermediate-1. Red cells distribution width was routinely reported by automated cell counters as part of RBC count, with an upper limit of normal of 14.5% used as a cut-off in our laboratory. At the time of RDW evaluation, complete blood cells count and spleen size [expressed as the distance from the left costal margin (BCM) in cm] were available for all patients. Results Among 49 patients, 24 had PMF (10 prefibrotic and 14 overt fibrotic) and 25 SMF [15 had post-polycythemia vera (PPV)- and 10 post-essential thrombocythemia (PET)-MF]. The JAK2V617F mutation was detected in 38 (77.6%) cases, CALR mutations in 8 (16.3%), and MPL in 1 (2%), while 2 patients (4.1%) were “triple-negative”. Elevated RDW values were present in all but 3 patients at MF diagnosis with a median RDW of 18.7% (range, 13.3% - 25.7%). RDW was higher in patients with splenomegaly ( p=0.04) and with JAK2V617F mutation ( p=0.09). Furthermore, looking specifically at JAK2V617F-mutated cases, the greater the RDW values, the higher the JAK2V617F allele burden. Interestingly, a higher RDW value at diagnosis also correlated with a shorter time from MF diagnosis to RUX start (-3.5 months per one RDW unit, 95%CI: -7.5 to +0.4; p=0.08). We observed a modest (<1%) increase in RDW during the first 6 months of RUX treatment. In a multivariable random-intercept model that considers all time points and contains the covariates time, RUX dose, and hemoglobin (Hb), we also observed a clear decrease in RDW with increasing Hb (slope: -0.35% per g/dL of Hb, 95%CI: -0.56 to -0.14; p=0.001). Median overall survival (OS) from MF diagnosis was 6.5 years with a median duration of RUX treatment of 33.3 months. The median RDW at diagnosis of 18.7% was used as a cut-off to identify two subgroups of patients [Group 1 (N=22): RDW 18.7% to 25.7%; Group 2 (N=23): RDW 13.3% to 18.6%] showing a difference in OS [Group 1 vs. 2: Hazard Ratio (HR) 2.74, 95%CI: 1.13 to 6.65; p=0.03] ( Figure 1); the median OS was 8.5 (14 deaths) and 11.8 (9 deaths) years for the Group 1 and 2, respectively. Using continuous RDW at diagnosis, the crude HR was 1.21 per one RDW unit (95%CI: 1.06 to 1.37; p=0.004). In a Cox model adjusted for gender, age, and Hb, the HR was 1.13 (95%CI: 0.99 to 1.29; p=0.06). Notably, RDW at diagnosis was associated with a 35% increase in infectious disease mortality in a crude analysis (HR 1.35, 95%CI: 1.07 to 1.71, 8 deaths; p=0.01) and a 27% increase (HR 1.27, 95%CI: 0.99 to 1.63; p=0.07) in the adjusted analysis. Conclusions Red cells distribution width may represent a good integrative measure of several disease processes and also has prognostic significance at the time of MF diagnosis and during RUX treatment. Even though our preliminary observations are limited by the small number of patients and need to be further confirmed by larger studies, we can speculate that RDW may help in the rapid detection of MF patients with a poor prognosis, which can only partially be improved from currently available targeted therapies with JAK inhibitors.

Hemoglobin profile and molecular characteristics of the complex interaction of hemoglobin Doi-Saket [α9(A7) asn > lys, HBA2:c.30C > a], a novel α2α1 hybrid globin variant, with hemoglobin E [β26(B8) Glu > lys, HBB:c.79G > A] and deletional α+-thalassemia in a Thai family

Background An increasing number of α-hemoglobin (Hb) variants is causing various clinical symptoms; therefore, accurate identification of these Hb variants is important. Objective This study aimed to describe the molecular and hematological characteristics of novel Hb Doi-Saket that gives rise to a typical α+-thalassemia phenotype in carriers with and without other hemoglobinopathies. Materials and Methods Biological samples from a proband and his family members were analyzed. Hematological profiles were analyzed using a standard automated cell counter. Hb was analyzed by capillary electrophoresis and high-performance liquid chromatography. Mutations and globin haplotype were identified by DNA analysis. Novel diagnostic tools based on allele-specific polymerase chain reaction (PCR) and PCR–restriction fragment length polymorphism were developed. Results Hb analysis showed a major abnormal Hb fraction, moving slower than HbA, and a minor Hb fraction alongside HbA2 in the proband, his father, and son. DNA analysis of the α-globin gene identified the -α3.7 deletion and in cis the C > A mutation on codon 9 of the α2α1 gene, corresponding to Hb Doi-Saket [α9(A7) Asn > Lys]. This mutation could be identified using newly developed allele-specific PCR-based assays. The Hb Doi-Saket al.lele was significantly associated with haplotype [- + M + + 0 -]. Interaction of αDoi-Saket with βE globin chains led to a new Hb variant (HbE Doi-Saket). Phenotypic expression was clinically silent in heterozygotes and might present slight microcytosis. Conclusions Hb Doi-Saket emphasizes a great diversity present in α-globin gene. The mutation in this family from Thailand was linked to -α3.7 and caused mild microcytosis in the carriers. The combination of this variant with deletions in α genes might cause a severe clinical phenotype. Different methods of separation can provide useful information in diagnosis, and a complete molecular approach is needed for confirmation before considering patient management.

Assessment of Cartilage Resected from Osteochondral Lesions of the Talus for Intraoperative Reimplantation

Category: Basic Sciences/Biologics; Arthroscopy Introduction/Purpose: Osteochondral lesions of the talus (OLT) are frequently treated with microfracture and may be augmented with decellularized extracellular matrix or particulated juvenile cartilage. The damaged OLT cartilage is often discarded. Infrequently it is used as a source for autologous chondrocyte expansion and implantation in a subsequent procedure. This resected cartilage has demonstrated variable chondrocyte viability. The influence of cartilage type on chondrocyte viability, whether frayed fibrocartilage from in vivo repair or delaminated hyaline cartilage, remains unknown. Also, little has been reported on the use of the OLT cartilage as a source for immediate cartilage autograft. The purpose of this study is to evaluate chondrocyte quantity and viability from the excised cartilage of OLTs at the time of arthroscopic debridement and microfracture. Methods: Patients aged 18 to 65 undergoing arthroscopic debridement and microfracture of talar OLTs were included. IRB approval of the study protocol was obtained. At the time of ankle arthroscopy, the cartilage from the OLT was collected either by GraftNet™ Autologous tissue collector (GraftNet™ Autologous Tissue Collector, Arthrex; Naples, FL) or manual collection and mincing of the specimen with a scalpel. Chondrocytes were isolated by overnight digestion with 2% collagenase in tissue culture medium at 37°C. The isolated cells were stained with Trypan Blue and counted for total cell number and viability (%), using an automated cell counter. Data collection included patient age, size and location of the OLT, intraoperative surgeon assessment of cartilage type (hyaline cartilage, fibrocartilage, or mixed fibro-hyaline cartilage), histologic determination of cartilage type, weight of the specimen, and chondrocyte cell count and viability. Results: Fourteen OLT specimens were collected. The average size was 0.831 (SD ± 1.10) cm2, and average weight 0.19 (SD ± 0.17) grams. Overall, the average number of chondrocytes was 1.12 x106 (SD ± 1.13 x106) cells/mm2 and 3.99 x106 (SD ± 3.28 x106) cells/gram. The average viability was 72.36% (SD ± 12.25%). Using histologic determination of cartilage type, in hyaline specimens (n = 8, 57.14%) we found 1.08 x106 (SD ± 0.85 x106) chondrocytes/cm2, 70.73% (SD ± 11.73 %) viability. In fibrocartilage specimens (n = 2, 14.29%) we found 0.37 x106 (SD ± 0.10 x106) chondrocytes/cm2, 78.08% (SD ± 22.03%) viability. In mixed cartilage specimens (n = 4, 28.57%) we found 1.58 x106 (SD ± 1.80 x106) cells/cm2, 72.78% (SD ± 11.79%) viability. (Table 1) Conclusion: We found OLT chondrocyte viability of 72.36%, with a trend toward greater chondrocyte concentration and similar viability comparing hyaline and fibro cartilage, respectively. Cartilage type was consistent between intraoperative and histologic assessment in all but four specimens. OLT cartilage has the potential to serve as an immediate intraoperative chondrocyte autograft source for enhancement of talus cartilage repair procedures, eliminating the risk of donor site morbidity and additional surgeries as in culture-based methods. Further study with more patients may shed light on the accuracy of intraoperative cartilage classification, and differences in chondrocyte quantity and viability between cartilage types.

A-135 Improving Cell Count Turnaround Times With the GloCyte® Automated Cell Counter for CSF: A Retrospective Study at MercyOne Des Moines Medical Center

Abstract Background Accurate, STAT cerebrospinal fluid (CSF) cell counts are critical in the diagnosis of meningitis, malignancy, demyelinating disease, and hemorrhage. Due to historical limitations with the linearity of automated analyzers, CSF cell counts have traditionally been performed manually with a hemocytometer and the aid of a brightfield optical microscope. Manual cell counting is time-consuming, subjective due to reliance on human interpretation, and requires a high degree of skill. This poses significant challenges for clinical laboratories of all sizes facing unprecedented med tech shortages. The purpose of this study was to evaluate the impact of implementing the GloCyte Automated Cell Counter for CSF, an FDA-cleared device with linearity down to 0 cells/µL, on result turnaround times. Methods Result turnaround time data for CSF specimens processed in the hematology lab at MercyOne Des Moines Medical Center, a 600-bed hospital in Des Moines, Iowa, was analyzed in a retrospective study of two cell counting methods- (1) manual cell counting using a Neubauer-Improved hemocytometer and (2) GloCyte, a benchtop cell counting device that uses fluorescence technology and highly specific reagents to automate RBC and TNC cell counts in less than 5 min. Turnaround times for 227 CSF specimens counted manually during an approximately six-month period prior to the implementation of GloCyte (January 2, 2017 to June 29, 2017) were compared to those for 133 specimens analyzed on GloCyte over a similar duration (January 4, 2021 to June 30, 2021). In this study, turnaround times were defined as the time between specimen arrival in the lab and result verification. Correlation between the methods was previously established in the MercyOne Des Moines Medical Center laboratory. Results The data demonstrated a significant, 16–30-minute improvement in turnaround times with GloCyte compared to the manual hemocytometer method. Median turnaround times, analyzed to adjust for add-ons that resulted in longer turnaround times pre- and post-GloCyte implementation, were 1:38h for specimens counted manually vs 1:08 h for specimens analyzed on GloCyte. The average turnaround time for the 227 CSF specimens counted manually was 2:03 h compared to 1:46 h for the 133 samples analyzed on GloCyte. The MercyOne Des Moines Medical Center laboratory processed 456 CSF cell counts in 2022, equating to a reduction in turnaround time of 122–228 h annually with the implementation of GloCyte. Conclusion This study demonstrates that GloCyte can be implemented to significantly improve turnaround times for CSF cell counts. The MercyOne Des Moines Medical Center laboratory realized several benefits from the reduction in turnaround times- GloCyte has (1) freed up significant tech time for other tasks, (2) standardized CSF cell counting to mitigate tech to tech variability, (3) increased lab staff satisfaction and decreased stress levels, and (3) increased physician satisfaction. For MercyOne Des Moines Medical Center’s sister site, MercyOne West Des Moines Medical Center, a 146-bed hospital that infrequently performs CSF cell counts, GloCyte has provided peace of mind in performing cell counts accurately with techs of all skill levels.

Targeted DNA Methylation Analysis Facilitates Leukocyte Counts in Dried Blood Samples.

Cell-type specific DNA methylation (DNAm) can be employed to determine the numbers of leukocyte subsets in blood. In contrast to conventional methods for leukocyte counts, which are based on cellular morphology or surface marker protein expression, the cellular deconvolution based on DNAm levels is applicable for frozen or dried blood. Here, we further enhanced targeted DNAm assays for leukocyte counts in clinical application. DNAm profiles of 40 different studies were compiled to identify CG dinucleotides (CpGs) with cell-type specific DNAm using a computational framework, CimpleG. DNAm levels at these CpGs were then measured with digital droplet PCR in venous blood from 160 healthy donors and 150 patients with various hematological disorders. Deconvolution was further validated with venous blood (n = 75) and capillary blood (n = 31) that was dried on Whatman paper or on Mitra microsampling devices. In venous blood, automated cell counting or flow cytometry correlated well with epigenetic estimates of relative leukocyte counts for granulocytes (r = 0.95), lymphocytes (r = 0.97), monocytes (r = 0.82), CD4 T cells (r = 0.84), CD8 T cells (r = 0.94), B cells (r = 0.96), and NK cells (r = 0.72). Similar correlations and precisions were achieved for dried blood samples. Spike-in with a reference plasmid enabled accurate epigenetic estimation of absolute leukocyte counts from dried blood samples, correlating with conventional venous (r = 0.86) and capillary (r = 0.80) blood measurements. The advanced selection of cell-type specific CpGs and utilization of digital droplet PCR analysis provided accurate epigenetic blood counts. Analysis of dried blood facilitates self-sampling with a finger prick, thereby enabling easier accessibility to testing.

P10.30.B COMBINATION TREATMENT WITH ERIBULIN AND TTFIELDS INDUCES APOPTOSIS AND G2/M CELL CYCLE ARREST IN HUMAN GLIOBLASTOMA CELLS

Abstract BACKGROUND TTFields has emerged to a first line treatment in newly diagnosed glioblastoma (GBM) patients. Several preclinical studies showed that the alternating electric fields inhibit proliferation and survival of human glioblastoma cells by disruption of the spindle apparatus and impairment of mitotic segregation. Eribulin is an antimitotic drug, that is currently used in the third line treatment of breast cancer. As it is also able to inhibit the pro-mitotic function of TERT, which is mutated in 80% of human glioblastoma, the drug was also shown to have a radiosensitizing and antiproliferative effect on human glioblastoma and ovarian cancer cell lines. This study aims to evaluate the effect of the combination of Eribulin and TTFields on the survival and cell cycle of glioblastoma cells. MATERIAL AND METHODS U87, A172 and four patient derived glioblastoma cell lines were cultured and plated to 80% confluence. Cells were then treated with TTFields at a frequency of 200kHz for 24 hours and/or received the ED50 and 0.5-fold ED50 of Eribulin. Controls remained untreated respectively. After 24 hours cells were harvested and counted by automatic cell count. Survival and cell cycle status were evaluated by FACS analysis. RESULTS The rate of early apoptotic cells was 82.28% in the group treated with TTFields and Eribulin. With TTFields treatment only 50.99% and with Eribulin alone 14.38% of cells were in an early apoptotic state. Untreated control cells had an apoptosis rate of 4.25%. The combination of TTFields and Eribulin was significantly more effective than the single treatment modalities (p>0.05). Treatment with TTFields increased the rate of cells an G2/M status. This effect was even stronger in the group with combination treatment. CONCLUSION Combination treatment of the antimitotic drug Eribulin with TTFields is able to improve the inhibitory effect of the drugs on cell survival. Furthermore the combination therapy effects the cell cycle of glioblastoma cells inducing G2/M arrest. Future preclinical studies should focus on the antimitotic effect and the influence of the treatment on genome integrity for a more detailed insight into the mechanisms of action.

A Comparative and Critical Analysis for In Vitro Cytotoxic Evaluation of Magneto-Crystalline Zinc Ferrite Nanoparticles Using MTT, Crystal Violet, LDH, and Apoptosis Assay.

Zinc ferrite nanoparticles (ZFO NPs) are a promising magneto-crystalline platform for nanomedicine-based cancer theranostics. ZFO NPs synthesized using co-precipitation method are characterized using different techniques. UV-visible spectroscopy exhibits absorption peaks specific for ZFO. Raman spectroscopy identifies Raman active, infrared active, and silent vibrational modes while Fourier transforms infrared spectroscopic (FTIR) spectra display IR active modes that confirm the presence of ZFO. X-ray diffraction pattern (XRD) exhibits the crystalline planes of single-phase ZFO with a face-centered cubic structure that coincides with the selected area electron diffraction pattern (SAED). The average particle size according to high-resolution transmission electron microscopy (HR-TEM) is 5.6 nm. X-ray photoelectron spectroscopy (XPS) signals confirm the chemical states of Fe, Zn, and O. A superconducting quantum interference device (SQUID) displays the magnetic response of ZFO NPs, showing a magnetic moment of 45.5 emu/gm at 70 kOe. These ZFO NPs were then employed for comparative cytotoxicity evaluation using MTT, crystal violet, and LDH assays on breast adenocarcinoma epithelial cell (MCF-7), triple-negative breast cancer lines (MDA-MB 231), and human embryonic kidney cell lines (HEK-293). Flow cytometric analysis of all the three cell lines were performed in various concentrations of ZFO NPs for automated cell counting and sorting based on live cells, cells entering in early or late apoptotic phase, as well as in the necrotic phase. This analysis confirmed that ZFO NPs are more cytotoxic towards triple-negative breast cancer cells (MDA-MB-231) as compared to breast adenocarcinoma cells (MCF-7) and normal cell lines (HEK-293), thus corroborating that ZFO can be exploited for cancer therapeutics.

Hematological Normality, Serum Biochemistry, and Acute Phase Proteins in Healthy Beef Calves in the Brazilian Savannah.

The Brazilian savannah region, characterized by high average temperatures, well-defined rainy and dry seasons, soil with low productive potential, and high pressure for parasitic diseases, is home to the highest percentage of the beef herd, which is the world's largest commercial beef producer. Therefore, breeds that present rusticity combined with productivity are the focus of research in cattle breeding in the region. Considering their geographic particularities and their effects on the animals' blood parameters, the objective was to study the behavior of hematological variables, serum biochemistry, and acute phase proteins in beef calves at weaning age. Jugular blood samples were collected in a single day from 30 weaning calves (about eight months old and 200 kg of body weight) from the following breeds: Nellore, Senepol, Nellore × Aberdeen Angus cross, Nellore × Senepol cross, and Nellore × Aberdeen Angus × Senepol cross. Hematological data were obtained using an automatic cell counter, serum biochemical measurements were obtained using commercial kits, and the electrophoretogram was obtained using the SDS-page technique. In general, the results were consistent with data already published in similar situations regarding health status, age, and level of metabolic activity. However, differences observed between groups can be explained by differences observed in other concurrent variables like temperament. The pure zebu breed was more reactive than pure taurine. While crossbreds showed intermediate values, and parasitic infestation, the pure taurine breed with higher parasite infestation, while the zebu breed had lower values, which produced effects in some laboratory tests, and generated differences between breeds. In conclusion, the reference intervals available for healthy animals can be routinely used without interference from the geographic region for animals produced without nutritional failures as long as changes are recorded in pathological, infectious, metabolic, or nutritional deficiency situations. However, it is suggested that a study covering a larger number of herds may demonstrate a greater geographic effect on the studied variables.

ROI-BMC-DNNet: An efficient automatic analysis model of whole-slide scanned bone marrow aspirate images for the diagnosis of hematological disorders

Cell differential counting in the scanned whole-slide image (WSI) is essential but still challenging in the intelligent diagnosis of hematological disorders based on bone marrow aspirate smear images. The huge size and the complex content of WSI pose multiple obstacles to training an efficient and accurate analysis model. This paper proposes a novel analysis framework termed ROI-BMC-DNNet. To capture patch images in the cell well-spread areas, we provide a pyramid segmentation network based on prior pathological knowledge to automatically locate regions of interest (ROI) that pathologists usually manually focus on to count nucleated cells. Specifically, we describe a simple but effective alignment method for multiscale ROI, aiming to maintain the accuracy of the semantic and the spatial information of the pyramid network. Furthermore, we design a patch sampling algorithm and a patch quality evaluating network to ensure the effectiveness of the sampled patches. Additionally, we provide a cell detection model to realize the automatic differential counting of the nucleated cells in the sampled patches. Experiments are conducted on the datasets of scanned WSIs from 120 patients. Our method achieves 92.27% retrieval precision for the high-quality patches (patches with nucleated cells with better morphology and without particles). When the missed nucleated cells are evaluated, the detection Recall and Precision of the bone marrow nucleated cells from the WSIs are 87.13% and 87.90% respectively. Notably, the recognition Recall and Precision of the blast cell are 91.37% and 90.47%, respectively. The results confirm that the proposed method is promising to work as an auxiliary diagnostic tool for hematological disorders.

Platelet count and platelet indices in patients of pregnancy-induced hypertension at a rural tertiary care hospital in western India

Background: Pregnancy-induced hypertension is a serious complication during pregnancy causing maternal and fetal morbidity and mortality. Pregnancy-induced hypertension associated with thrombocytopenia can help in predicting the severity of the disease. The study aimed to study the platelet count, and platelet indices in Pregnancy induced hypertension patients and compare them with normotensive pregnant women. Materials and methods: This was a case control study, conducted on patients visiting antenatal care (ANC) at a Tertiary care rural hospital in western India. Study population included a total of 82 patients, which were divided into 2 groups with 41 patients in each control (healthy pregnant women) and study groups (pregnant women with PIH). Patients with chronic hypertension and known case of thrombocytopenia were excluded from the study. The blood samples collected were processed on fully automated cell counter for estimation of platelet count and indices. Results: The platelet count in the study group ranged from 186434.1±81086.9 whereas in the control group it was 268659±72206.9, showing that the platelet count was significantly lower in women with Pregnancy induced hypertension than in the control group. The MPV, PDW, systolic BP, diastolic BP was found to be significantly elevated in the study group as compared to the control group. Conclusions: The present study concludes that blood parameter such as platelets count and platelet indices can be considered as an early, economical and easily available parameter for assessment of severity of PIH cases.

Safety and feasibility of platelet transfusion through long catheters in the neonatal intensive care unit: an in vitro study

ObjectiveTo assess the safety and feasibility of platelet transfusion through small-bore long lines used in the neonatal intensive care unit (NICU), including double-lumen umbilical venous catheters (UVCs) and 24 G...

Mono-dysplasia Score Based on Automated Cell Counter (Sysmex) – A Novel Parameter for Differentiating Reactive Monocytosis from Hematological Malignancies

INTRODUCTION: In India, there is a high burden of infections such as tuberculosis, dengue, and malaria which are common causes of monocytosis. This increases the workload of smear examination by the pathologist. “Mono-dysplasia score” is obtained with a simple complete blood count on an automated cell counter and includes the parameters neutrophil (Ne) and monocyte (Mo) count and Ne-WX which is a Ne dispersion parameter. It is operator independent, objective, and does not require a high level of expertise. AIMS: The aims of the study were to assess the utility of Monoscore/mono-dysplasia score calculated using research parameters of Sysmex XN automated cell counter, as a screening tool for differentiating reactive monocytosis from hematological malignancies associated with monocytosis. MATERIALS AND METHODS: Samples sent in EDTA vacutainer for routine hemogram fulling the criteria for monocytosis (WHO criteria – absolute monocyte count ≥1 × 109/L and accounting for ≥10% of the total white blood cell count) were included in the study. Monoscore was calculated using the formula established by Schillinger et al. Flow cytometry, bone marrow examination, etc., were done as and when needed as standard-of-care tests to establish a final diagnosis. RESULTS: One thousand two hundred and fifty-seven samples were analyzed out of which 41 samples were chronic myelomonocytic leukemia and 126 were other hematological malignancies (HD) including acute leukemias, myelodysplastic syndrome, myeloproliferative neoplasm, etc. Using receiver operating characteristics curves, we established the cutoff 0.212 which showed a sensitivity of 97.6% and specificity of 96.4% to differentiated reactive monocytosis form HD. CONCLUSIONS: A sample showing monocytosis and Monoscore <0.212 and without any other flags can be safely auto-authorized without peripheral blood smears examination, reducing the burden of slides to be reviewed.

Clinical value of serum soluble receptor for advanced glycation end products in evaluating the condition of patients with bronchial asthma.

Objective: This study focused on the clinical value of serum soluble receptor for advanced glycation end products (sRAGE) levels in evaluating the severity of bronchial asthma (BA). Methods: Serum sRAGE expression was measured by using enzyme-linked immunosorbent assay, eosinophils (EOS) count was measured by using an automatic blood cell counter, and forced expiratory volume in 1 second (FEV1) was measured by pulmonary function analyzer in 120 patients with BA, 40 patients with non-BA pulmonary disease, and 40 healthy controls. Receiver operating characteristic curves were used to analyze the clinical value of sRAGE expression levels, EOS counts, and FEV1 level to assess the severity of illness in the patients with BA. Results: Compared with the healthy controls and the patients without BA, the patients with BA had the lowest serum sRAGE expression level (47.36 ± 6.3 ng/L versus 75.3 ± 6.3 ng/L versus 67.5 ± 5.06 ng/L; p < 0.05), the highest EOS count (231.2 ± 18.3 106/L versus 175.9 ± 15.6 106/L versus 197.8 ± 19.6 106/L; p < 0.05), and the lowest FEV1 level (1.19 ± 0.15 L versus 1.57 ± 0.2 L versus 1.3 ± 0.17 L; p < 0.05). Correlation analysis revealed that the serum sRAGE expression levels were notably negatively correlated with the EOS counts (r value of -0.471, p < 0.05) but significantly positively linked to FEV1 levels (r value of 0.362, p < 0.05). Serum sRAGE expression levels could help in accurately diagnosing patients with severe BA (area under the receiver operating characteristic curve (AUC) = 0.904), whereas prediction in the patients with mild BA was achieved by EOS counts (AUC = 0.857). Conclusion: The serum sRAGE level has potential value in diagnosing the severity of BA, which is conducive to identifying patients with severe BA and guiding in development of new therapeutic strategies.

Utility of automated cell counter histogram in diagnosis of thrombocytopenia and pseudothrombocytopenia

Background: Automated hematology analyzers have become mainstream of complete blood count (CBC) during the past two decades; they are potential, more accurate to produce results of CBCs. However, often automated analyzers can give false results too, specially, falsely low platelet count due to platelets aggregates, which have to be confirmed on peripheral smears. Aims and Objectives: The aim and objective of the study was to diagnose thrombocytopenia (TCP) cases and differentiate them from pseudothrombocytopenia (PTCP) cases by analyzing platelet histogram and to determine the sensitivity and specificity of platelet histograms for diagnosing TCP and PTCP. Materials and Methods: The present study was conducted in the Department of Pathology at Shyam Shah Medical College, Rewa, M.P., after obtaining ethical clearance from the Institutional Ethics Committee. It was prospective study conducted for a period of 15 months from January 2021 to May 2022 on 1000 samples. Results: The sensitivity and specificity of platelet histogram flags and presence of multiple peaks to diagnose TCP and PTCP cases were calculated as 73.60% and 93.60%, respectively. The present study also interpret that mean platelet volume and platelet distribution width cannot use as indicator to differentiate between TCP and PTCP. Conclusion: The present study concludes that analysis and interpretation of histogram more specifically platelet histogram flagging provide clue for early detection of PTCP cases prior peripheral smear examination and also helpful in differentiating them from true TCP cases. These platelet histogram flagging can be used as a screening parameter for the detection of PTCP and these are helpful in preventing unnecessary stress for clinician, patients, and their relatives. However, the peripheral smear examination will remain the gold standard to differentiate TCP from, PTCP.

Evaluation of hemoglobin concentration, red blood cell indices and microscopic examination of red blood cell in Sudanese blood donors in Gezira state (Sudan) - 2023

Objective: To detect hemoglobin level and Red blood cell indices in apparently healthy Sudanese male, to establish safety for both donor and recipient. To perform hemoglobin estimation and Red blood cell indices for blood donors using automated machine (Blood cell counter). Material and Methods: Venous blood samples were taken from 200 apparently healthy males donors and Hemoglobin level was measured using an automated cell counter (Sysmex KN21), accompanied by peripheral blood films were assessed to detect any abnormalities. Results: The study revealed that the mean hemoglobin values were 14.5 g/dl ±1.2076, with minimum count (10.1 g/dl) and maximum count 17.8 g/dl. Hemoglobin less than 12.5 g/dl was obtained in 30 donors (6%) and they were reported as fit for blood donation using copper sulphate for hemoglobin estimation. Those 30 donors actually they are not fit for blood donation because their hemoglobin concentration must be more than 12.5 g/dl. Conclusion: The study revealed that a significant number of anemic donors were not detected by estimation of Hb by copper sulphate method.

A comparative study of red cell histogram along with CBC parameters and peripheral blood smear in various anemias

: The red blood cell (RBC) parameters generated by cell counters can aid in morphological categorization of anemia, the significance of microscopic analysis of peripheral blood smears (PBSs) cannot be overlooked when it comes to interpreting the underlying cause of anemia. Therefore, both RBC parameters and microscopic examination of PBSs are essential for a comprehensive assessment of anemia.This study aimed to evaluate and compare the results obtained through two different methods: examination of peripheral blood smears (PBS) and automated cell counter generated parameters. The objective was to identify any differences or similarities in the diagnostic outcomes produced by each method. Over the course of one year, from December 2020 to November 2021, a hospital-based, prospective study was conducted. The study aimed to evaluate and compare the PBS findings and RBC parameters and histograms generated by a cell counter in 1000 anemic patients.In a sample of 1000 cases, we compared the typing of anemia using an automated analyzer and peripheral smear examination. Out of these cases, 690 showed consistent typing results between the two methods, while 310 showed discordant typing. The automated analyzer diagnosed majority of cases 70.1% as microcytic anemia and Normocytic, dimorphic, and macrocytic cases were found in 13.5%, 7.8%, and 4.3% of cases, respectively. Additionally, a normal histogram was observed in 17.6% of cases.We have concluded that while the automated analyzer is useful in reducing the overall workload through its graphical representation of anemia typing, it should always be confirmed through microscopy. The automated analyzer is a helpful tool, it should not be relied upon solely for accurate anemia typing. Microscopy remains an essential method for confirming the results of automated analysis.

Comparative analysis of total protein, casein, lactose, and fat content in milk of cows suffering from subclinical and clinical mastitis caused by Streptococcus spp.

The aim of the study was to analyse the total protein (TP), casein (CAS), lactose (LAC), and fat content of milk from cows with subclinical (SCM) and clinical mastitis (CM) caused by Streptococcus spp. A total of 60 milk samples from diseased cows and 30 milk samples from healthy cows were included in the study. Milk samples were taken from Holstein-Friesian cows from four dairy farms in Lublin Province. The bacteriological examination of the milk was performed and the somatic cells count in 1 mL of milk was determined using a SomaCount FC automatic cell counter. Determination of TP, CAS, LAC, FAT and FA levels in milk was carried out using a DairySpec FT automated Fourier transform infrared spectrometer. Total protein in milk from HE was significantly higher than in milk from cows with mastitis (4.04% vs 3.57% in milk from SCM cows and 3.7% in milk from CM cows, P = 0.001). The CAS level was 2.73% in milk from CM cows and 2.92% in milk from SCM cows vs 3.30% in milk from HE cows, P = 0.001. The changes in CAS and TP in milk resulted in a significant difference in the CAS/TP ratio (81.7% in milk from HE cows vs 73.8% in milk from CM cows). A decrease in levels was also recorded for LAC (4.8% in milk from HE cows vs 4.51% in milk from SCM cows and 4.01% in milk from CM cows, P = 0.001). The fat level was significantly higher in milk from healthy cows than in milk from cows with mastitis (4.0% vs 2.3% in milk from SCM cows and 1.64% in milk from CM cows, P = 0.001). It should be emphasised that the decrease in the levels of TP, LAC and FAT was significant not only in milk from CM cows but also in milk from SCM cows. This is very unfavourable, because the reduction in the main milk components results in poor quality dairy products and impairs line processes.

WED-344 - Validation of automatic cell counting based on impendaciometry and flow cytometry in ascitic fluid

WED-344 - Validation of automatic cell counting based on impendaciometry and flow cytometry in ascitic fluid