Automated Cell Counter Research Articles

Whole gut imaging allows quantification of all enteric neurons in the adult zebrafish intestine.

A fundamental understanding of the enteric nervous system in normal and diseased states is limited by the lack of standard measures of total enteric neuron number. The adult zebrafish is a useful model in this context as it is amenable to in toto imaging of the intestine. We leveraged this to develop a technique to image and quantify all enteric neurons within the adult zebrafish intestine and applied this method to assess the relationship between intestinal length and total enteric neuron number. Dissected adult zebrafish intestines were immunostained in wholemount, optically cleared with refractive index-matched solution, and then imaged in tiles using light-sheet microscopy. Imaging software was used to stitch the tiles, and the full image underwent automated cell counting. Total enteric neuron number was assessed in relation to intestinal length using linear regression modeling. Whole gut imaging of the adult zebrafish intestine permits the visualization of endogenous and immunohistochemistry-derived fluorescence throughout the intestine. While enteric neuron distribution is heterogeneous between intestinal segments, total enteric neuron number positively correlates with intestinal length. Imaging of all enteric neurons within the adult vertebrate intestine is possible in models such as the zebrafish. In this study, we apply this to demonstrate a positive correlation between enteric neuron number and intestinal length. Quantifying total enteric numbers will facilitate future studies of enteric neuropathies and ENS structure in animal models and potentially in biopsied tissue samples.

What Is the Impact of Automated Synovial Cell Counting on Different Aseptic Causes and Periprosthetic Conditions Associated With Revision THA?

Studies have suggested that automated synovial cell counting may overestimate the white blood cell (WBC) count, resulting in false positive tests when evaluating patients for the possibility of periprosthetic joint infection (PJI) after THA. However, associations between WBC counts high enough to mimic PJI in patients whose arthroplasties are not infected but rather are experiencing a variety of aseptic problems-including but not limited to metallosis, polyethylene wear, and recurrent dislocation-have not, to our knowledge, been adequately addressed. In addition, there is a lack of analyses about the polymorphonuclear percentage (PMN%) when assessed by automated analyzers in this context. In the context of different indications for aseptic revision and different periprosthetic hip pathologic findings, we asked: (1) What were the synovial WBC count levels, and what proportion of values were above the 2018 International Consensus Meeting (ICM) cutoff (3000 cells/μL)? (2) What were the synovial PMN% levels, and what proportion of values were above the 2018 ICM (70%)? We retrospectively studied the preoperative cell count analyses of synovial fluid in patients who underwent revision THA for aseptic reasons at our tertiary referral arthroplasty center between January 2015 and December 2017. We considered all revisions performed on patients during that time potentially eligible, and after prespecified exclusions were applied (exclusions mainly included 15% [197 of 1306] sporadic missing data and 12% [155 of 1306] insufficient synovial fluid obtained in the aspirate), a total of 702 patients undergoing revision THA for aseptic reasons remained for the final analysis. As far as we know, no patients underwent re-revision for PJI at a mean follow-up of 46 ± 11 months, which tends to confirm our impression that indeed these hips did not have PJI. Cell count analyses were conducted using an automated analyzer. Clinical findings, preoperative radiographs, and surgical reports--confirmed by available histologic results--were used to establish diagnoses. We evaluated these hips considering the recommendations of the 2018 ICM (WBC count of 3000 cells/μL and PMN% of 70%) to see what proportion of them would have been characterized as likely having PJI on basis of those cutoff values. The mean WBC count for the entire cohort was 2120 ± 2395 cells/μL. The mean PMN% for the entire cohort was 36% ± 22%. Compared with aseptic loosening and recurrent dislocation, polyethylene wear had the highest mean WBC count (3817 ± 3711 cells/μL; p < 0.001). Of the investigated periprosthetic conditions, wear-induced synovitis had the highest value (4464 ± 3620 cells/μL; p < 0.001). Considering the ICM threshold, polyethylene wear showed the highest proportion of WBC counts above 3000 cells/μL among the indications for aseptic revision (60% [25 of 42]; p < 0.001). Of the periprosthetic conditions, wear-induced synovitis showed the highest proportion beyond the ICM cutoff (60% [50 of 83]; p < 0.001). The mean PMN% for aseptic causes ranged between 28% and 44% without differences among them (p = 0.12). Patients with metallosis had the highest mean PMN% of the periprosthetic conditions investigated (45% ± 25%; p = 0.007). Regarding the ICM threshold, metallosis resulted in the largest proportion of patients with a PMN% above 70% (21% [10 of 47]; p = 0.003), and that for wear-induced synovitis was 6% (5 of 83; p = 0.42) and for osteolysis was 3% (1 of 33; p = 0.51). There were no differences among aseptic loosening, recurrent dislocation, and polyethylene wear in terms of the proportion above 70% among the aseptic revision causes. Using automated cell counting, we found that WBC counts differ widely across indications for aseptic revision THA, and a high proportion of patients who underwent revision THA for aseptic reasons had WBC counts above the commonly used threshold of 3000 cells/μL. However, the PMN% was much less affected in several common indications for aseptic revision THA, making this measure more reliable for interpreting aspiration results using an automated analyzer. Based on the data distributions we observed, manual counting techniques might be considered in particular among patients with polyethylene wear, metal-on-metal bearing surfaces, or suspected metallosis. However, an elevated WBC count alone, observed using an automated analyzer in the context of polyethylene wear, should not be considered to be strongly suggestive of PJI, since that finding occurred so commonly among patients without infection. Level III, diagnostic study.

Assessment of Cytokine (α-TNF) with Erythropoietin and their Correlation in Pulmonary Tuberculosis with Anaemia

Globally tuberculosis is the 9th leading cause of death worldwide. As pulmonary tuberculosis (PTB) is a chronic disease, anaemia of inflammation due to bacterial burden play a vital role in pathophysiology of anaemia. Inflammation interferes with erythropoietin (EPO) function.&#x0D; Methods: The present study was an analytical type of case control study. The study included 100 newly diagnosed anaemic PTB cases and 50 newly diagnosed non anaemic PTB controls. The PTB was confirmed by microscopic examination of sputum specimen for the detection of Acid-Fast Bacilli (AFB). Both cases and controls were subjected to hematological analysis by automated cell counter and serum α-TNF and EPO by ELISA method.&#x0D; Results: Statistically significant difference was observed in levels of both α-TNF and EPO in anemic and non-anemic PTB groups (p&lt;0.001). α-TNF (214.56±82.30) levels were found to be significantly higher in anaemic PTB group while EPO level (58.44 ±14.97) were found to be significantly higher in non anaemic PTB group. Significant inverse correlation (r1=cases, r2=controls) was observed between α-TNF and EPO (r=-0.257, p&lt;0.05) and α-TNF and Hb (r=-0.202, p&lt;0.05) in both the groups.&#x0D; Conclusion: Increased α-TNF with decreased EPO and hemoglobin infers that inflammation interferes with normal functioning of EPO and probably contributes in induction of anemia in tuberculosis patients.

Object detection for automatic cancer cell counting in zebrafish xenografts.

Cell counting is a frequent task in medical research studies. However, it is often performed manually; thus, it is time-consuming and prone to human error. Even so, cell counting automation can be challenging to achieve, especially when dealing with crowded scenes and overlapping cells, assuming different shapes and sizes. In this paper, we introduce a deep learning-based cell detection and quantification methodology to automate the cell counting process in the zebrafish xenograft cancer model, an innovative technique for studying tumor biology and for personalizing medicine. First, we implemented a fine-tuned architecture based on the Faster R-CNN using the Inception ResNet V2 feature extractor. Second, we performed several adjustments to optimize the process, paying attention to constraints such as the presence of overlapped cells, the high number of objects to detect, the heterogeneity of the cells’ size and shape, and the small size of the data set. This method resulted in a median error of approximately 1% of the total number of cell units. These results demonstrate the potential of our novel approach for quantifying cells in poorly labeled images. Compared to traditional Faster R-CNN, our method improved the average precision from 71% to 85% on the studied data set.

Hematological changes in hypothyroidism and hyperthyroidism in adults

Thyroid function disorders are among the most common endocrine diseases. Thyroid disorders can cause certain forms of anemia, more commonly in hypothyroidism. Slightly decreased TLC, relative neutropenia, relative lymphocytosis or relative eosinophilia may be found in thyroid disorders. These observations confirmed the association between thyroid gland dysfunction and hemopoiesis.1) To study the pattern of hematological changes in thyroid dysfunction 2) To correlate thyroid function tests with complete blood count &amp; red cell indices findings 3) To correlate complete blood count investigations and peripheral blood smear findings with serum TSH levels.It was an Observational study conducted during 18 months of period from March 2018 to October 2019 with 100 subjects of hypothyroidism, 80 subjects of hyperthyroidism and 100 euthyroid subjects as control group. CBC was done on automated cell counter. PBS was stained with Leishman stain and observed under microscope. TFTwas done by immunoassay method.It was done with SPSS Software.: Predominance of female was seen in hypothyroidism and hyperthyroidism. Significant decrease was seen in RBC, Hb, HCT, MCV and MCH in hypothyroid group, while in hyperthyroid group RBC, Hb and HCT were significantly decreased as compared with euthyroid control group. Both groups showed significant increase in RDW as compared with control group.It is important to carefully evaluate the thyroid hormones in cases of unexplained anemias. So, periodic evaluation for probable hematological changes should be done in all the patients with hypothyroidism and hyperthyroidism.

A Simple Approach for Counting CD4+ T Cells Based on a Combination of Magnetic Activated Cell Sorting and Automated Cell Counting Methods

Frequent tests for CD4+ T cell counting are important for the treatment of patients with immune deficiency; however, the routinely used fluorescence-activated cell-sorting (FACS) gold standard is costly and the equipment is only available in central hospitals. In this study, we developed an alternative simple approach (shortly named as the MACS-Countess system) for CD4+ T cell counting by coupling magnetic activated cell sorting (MACS) to separate CD4+ T cells from blood, followed by counting the separated cells using CountessTM, an automated cell-counting system. Using the cell counting protocol, 25 µL anti-CD4 conjugated magnetic nanoparticles (NP-CD4, BD Bioscience) were optimized for separating CD4+ T cells from 50 µL of blood in PBS using a DynamagTM-2 magnet, followed by the introduction of 10 µL separated cells into a CountessTM chamber slide for automated counting of CD4+ T cells. To evaluate the reliability of the developed method, 48 blood samples with CD4+ T cell concentrations ranging from 105 to 980 cells/µL were analyzed using both MACS-Countess and FACS. Compared with FACS, MACS-Countess had a mean bias of 3.5% with a limit of agreement (LoA) ranging from −36.4% to 43.3%, which is close to the reliability of the commercial product, PIMA analyzer (Alere), reported previously (mean bias 0.2%; LoA ranging from −42% to 42%, FACS as reference). Further, the MACS-Countess system requires very simple instruments, including only a magnet and an automated cell counter, which are affordable for almost every lab located in a limited resource region.

High-throughput lensless whole slide imaging via continuous height-varying modulation of a tilted sensor.

We report a new, to the best of our knowledge, lensless microscopy configuration by integrating the concepts of transverse translational ptychography and defocus multi-height phase retrieval. In this approach, we place a tilted image sensor under the specimen for introducing linearly increasing phase modulation along one lateral direction. Similar to the operation of ptychography, we laterally translate the specimen and acquire the diffraction images for reconstruction. Since the axial distance between the specimen and the sensor varies at different lateral positions, laterally translating the specimen effectively introduces defocus multi-height measurements while eliminating axial scanning. Lateral translation further introduces sub-pixel shift for pixel super-resolution imaging and naturally expands the field of view for rapid whole slide imaging. We show that the equivalent height variation can be precisely estimated from the lateral shift of the specimen, thereby addressing the challenge of precise axial positioning in conventional multi-height phase retrieval. Using a sensor with 1.67 µm pixel size, our low-cost and field-portable prototype can resolve the 690 nm linewidth on the resolution target. We show that a whole slide image of a blood smear with a 120mm2 field of view can be acquired in 18 s. We also demonstrate accurate automatic white blood cell counting from the recovered image. The reported approach may provide a turnkey solution for addressing point-of-care and telemedicine-related challenges.

Mean Platelet Volume and Platelet Volume Distribution Width in Canine Parvoviral Enteritis.

Bacterial translocation from the damaged intestinal tract, reported in canine parvoviral (CPV) enteritis, is thought to be responsible for the systemic inflammatory response resulting from coliform septicemia, which could ultimately progress to septic shock and death. Alterations in platelet indices, specifically mean platelet volume (MPV), is a consistent finding in critically ill people and dogs with and without sepsis. Increased MPV has been reported to be an indirect indicator of platelet activation and of bone marrow response in people and dogs with sepsis. The study aim was to compare admission MPV and platelet volume distribution width (PVDW) in dogs with CPV enteritis to that of healthy aged-matched control dogs. Forty-eight dogs with CPV enteritis and 18 healthy age matched control dogs were included. CPV infection was confirmed with electron microscopy and concurrent blood-borne infections were excluded using PCR. EDTA whole blood samples were analyzed on an automated cell counter, ADVIA 2120, within 30-60 min from collection. There was no significant difference for platelet count between the groups. The MPV for CPV infected dogs (median: 14.0; IQR: 12.2–15.1) was significantly higher compared to controls (11.3; IQR: 10.3–13.1, P = 0.002). The PVDW for CPV infected dogs (66.9; IQR: 64.2–68.8) was significantly higher compared to controls (63.3; IQR: 60.2–65.1, P < 0.001). These findings suggest that significant platelet activation is present in dogs with CPV enteritis which may play a role in the disease outcome, similar to people with sepsis. Further studies are required to investigate the prognosticating ability of MPV in dogs with CPV enteritis.

Considerations for Whole-Slide Analysis of Murine Xenografts Experiments.

Histochemistry of tumor sections is a widely employed technique utilized to examine cell death in preclinical xenograft animal models of cancer. However, this is under the assumption that tumors are homogeneous, leading to practices such as automatic cell counting across the entire section. We have noted that in our experiments the core of the tumor is largely or partially necrotic, and lacks evidence of vascularization (in contrast to the outer areas of the tumor). We note that this can bias and confound immunohistochemical analyses that do not take care to sample areas of interest in a way to take this into account. Design-based stereology with image analysis techniques is an alternative process that could be used to measure the volume of the necrotic region compared to the volume of the whole tumor.

Performance evaluation of automated cell counts compared with reference methods for body fluid analysis.

Cellular analysis of body fluids (BFs) can assist clinicians for the diagnosis of many medical conditions. The aim of this work is the evaluation of the analytical performance of the UF-5000 body fluid mode (UF-BF) analyzer compared to the gold standard method (optical microscopy, OM) and to XN-1000 (XN-BF), another analyzer produced by the same manufacturer (Sysmex) and with a similar technology for BF analysis. One hundred BF samples collected in K3EDTA tubes were analyzed by UF-BF, XN-BF and OM. The agreement was evaluated using Passing and Bablok regression and Bland-Altman plot analysis. The receiver operating characteristic (ROC) curves were selected for evaluating the diagnostic agreement between OM classification and UF-BF parameters. Comparison between UF-BF and OM, in all BF types, showed Passing and Bablok's slope comprised between 0.99 (polymorphonuclear cells count, PMN-BF) and 1.39 (mononuclear cells count, MN-BF), the intercepts ranged between 26.47 (PMN-BF parameter) and 226.80 (white blood cell count). Bland-Altman bias was comprised between 7.3% (total cell count, TC-BF) and 52.9% (MN-BF). Comparison between UF-BF and XN-BF in all BF showed slopes ranged between 1.07 (TC-BF and PMN-BF) and 1.16 (MN-BF), intercepts ranged between 8.30 (PMN) and 64.78 (WBC-BF). Bland-Altman bias ranged between 5.8 (TC-BF) and 21.1% (MN-BF). The ROC curve analysis showed an area under the curve ranged between 0.9664 and 1.000. UF-BF shows very good performance for the differential counts of cells in ascitic, pleural and synovial fluids and therefore it is useful to screen and count cells in this type of BF.

FASN inhibition sensitizes metastatic OSCC cells to cisplatin and paclitaxel by downregulating cyclin B1.

To investigate the potential effect of fatty acid synthase (FASN) inhibitor orlistat to enhance the effectiveness of chemotherapy drugs widely used to treat oral squamous cell carcinomas (OSCC), such as 5-fluorouracil, cisplatin, and paclitaxel. The OSCC SCC-9 LN-1metastatic cell line, which expresses high levels of FASN, was used for drug combination experiments. Cell viability was analyzed by crystal violet staining and automatic cell counting. Apoptosis and cell cycle were analyzed by flow cytometry with Annexin-V/7-AAD and propidium iodide staining, respectively. Cyclin B1, Cdc25C, Cdk1, FASN, and ERBB2levels were assessed by Western blotting. Finally, cell scratch and transwell assays were performed to assess cell migration and invasion. Inhibition of FASN with orlistat sensitized SCC-9 LN-1 cells to the cytotoxic effects of paclitaxel and cisplatin, but not 5-fluorouracil, which was accompanied by a significant reduction in cyclin B1. The suppression of proliferation, migration, and invasion of SCC-9 LN-1 cells induced by orlistat plus cisplatin or paclitaxel was not superior to the effects of chemotherapy drugs alone. Our results suggest that orlistat enhances the chemosensitivity of SCC-9 LN-1 cells to cisplatin and paclitaxel by downregulating cyclin B1.

Performance of Abbott Alinity hq hematology analyzer for automated cell counting of body fluids.

Body fluid cell counting and differentiation provide essential information for diagnosis and monitoring of diverse pathologies. We evaluated the performance of the newly launched Abbott Alinity hq hematology analyzer for automated cell counting in body fluids and compared red blood cell (RBC) and total nucleated cell (TNC) counts with the Cell-Dyn Sapphire automated hematology analyzer. Differential counts were compared with microscopic differentiation on cytocentrifuged preparations. Background concentration limits, limit of detection (LOD), linearity, imprecision, functional sensitivity and carryover were evaluated. For method comparison, we collected 172 body fluids (17 continuous ambulatory peritoneal dialysis fluids, 56 cerebrospinal fluids and 99 serous fluids). Background concentration limits were ≤1000 cells/μL for RBC counts and ≤3 cells/μL for TNC counts. The LOD was 1000 RBC/µL and 5 TNC/µL. Results from linear regression analysis revealed excellent linearity. Functional sensitivity was 3000 cells/µL for RBC counts and 50 cells/µL for TNC counts. Carryover was 0.6% and 0.1% for TNC and RBC, respectively. The Alinity hq shows good clinical performance. We demonstrated comparable performance for body fluid cell counting between the Alinity hq analyzer and the Cell-Dyn Sapphire. The Alinity hq can be very useful as a screening tool for body fluid cell counting.

Detection and classification of neurons and glial cells in the MADM mouse brain using RetinaNet.

The ability to automatically detect and classify populations of cells in tissue sections is paramount in a wide variety of applications ranging from developmental biology to pathology. Although deep learning algorithms are widely applied to microscopy data, they typically focus on segmentation which requires extensive training and labor-intensive annotation. Here, we utilized object detection networks (neural networks) to detect and classify targets in complex microscopy images, while simplifying data annotation. To this end, we used a RetinaNet model to classify genetically labeled neurons and glia in the brains of Mosaic Analysis with Double Markers (MADM) mice. Our initial RetinaNet-based model achieved an average precision of 0.90 across six classes of cells differentiated by MADM reporter expression and their phenotype (neuron or glia). However, we found that a single RetinaNet model often failed when encountering dense and saturated glial clusters, which show high variability in their shape and fluorophore densities compared to neurons. To overcome this, we introduced a second RetinaNet model dedicated to the detection of glia clusters. Merging the predictions of the two computational models significantly improved the automated cell counting of glial clusters. The proposed cell detection workflow will be instrumental in quantitative analysis of the spatial organization of cellular populations, which is applicable not only to preparations in neuroscience studies, but also to any tissue preparation containing labeled populations of cells.

Correlation between Increased Serum Vascular Endothelial Growth Factor Levels and 30-Day-Outcome After Acute Myocardial Infarction

Vascular Endothelial Growth Factor (VEGF) plays an important role in the process of regeneration and vascular repair. Atherogenesis and angiogenesis contribute to the formation of coronary collateral circulation as an alternative source of blood supply during the repairing process of Acute Myocardial Infarction (AMI). This study aimed to investigate the elevation of VEGF and its correlation to Peripheral Blood Mononuclear Cells (PBMC) and Major Adverse Cardiovascular st nd th Events (MACE). Serum VEGF measurements were carried out on 1 , 2 , and the 5 -day onset of angina on 20 patients using st th Chemwell Analyzer based on ELISA method. PBMC was counted on days 1 and 5 according to CBC results from Automatic Cell Counter Sysmex XN-1000i. The major adverse cardiovascular event was recorded 30 days after AMI onset. It was found nd that serum VEGF level in this study was 169.3±34.5 pg/mL on the 1st day, 217.0±49.7 pg/mL on the 2 day, and th th 249.2±48.5 pg/mL on the 5 day. Serum VEGF levels increased gradually and the highest value was found on the 5 day st (p=0.000). There was no correlation between elevated serum VEGF levels with PBMC on the 1 day (p=0.429, r=-0.035) and th the 5 day of AMI (p=0.225, r=+0.081). There was no correlation between elevated serum VEGF levels with incidence of MACE on 30 days after onset of AMI (OR=0.959, 95% CI, p=0.302). Serum VEGF concentrations are increased in Acute Myocardial Infarction and can be used as a marker of myocardial injury. However, this study was unable to prove its role in the outcome of AMI

Evaluation of thrombocytopenia: A single-center experience

To evaluate the common etiologies and bleeding manifestations in patients of thrombocytopenia and its clinical presentation, methods of investigation, and impact of various modes of management. Total 104 patients with platelet count less than l00,000 per cu.mm. with age, more than 12 years admitted to hospital between January 2010 to October 2011 were included in this study. Platelet count on automated cell counter less than 100,000 per cu.mm. and confirmed in peripheral smear were included in the study. All EDTA samples were processed in Abacus junior 5 hematology Analyser. Peripheral blood smear review was done for all cases. Clinical history and physical examination were collected from patients and medical record files.The present study included 56 male patients and 48 female patients. The most common bleeding manifestation was petechial rash 8.6%, hemoptysis and traumatic bleeding account for 1.9 % each, whereas 74% of patients did not present with any bleeding issues. Total 66 patients presented with symptoms of fever, 73 had signs of pallor, 22 patients had splenomegaly, 16 patients had hepatomegaly. Total 37.5% of patients were diagnosed with malaria and 1 patient (0.96%) was diagnosed with plasma cell leukemia. Chronic liver disease, megaloblastic anaemia, ITP (Idiopathic thrombocytopenia) and chronic renal failure account for 9.6%, 13.4%, 5.7% and 3.8% respectively. Total 16 patients had platelet counts less than 20000/cu.mm. and 37 patients had platelet count between 60000-80000/cu.mm. Malaria (39 patients) was the major cause of thrombocytopenia. Out of 39 patients with malaria 10 patients had P. Falciparum, 27 had P. Vivax and 2 patients had both. Patients with a platelet count less than 100,000/cu.mm have very high chances of bleeding manifestation. Cutaneous bleed is the most common manifestation. Malaria can be present with signs of thrombocytopenia. Timely and accurate diagnosis is the key to the management of thrombocytopenia.

The contribution of platelets to peripheral BDNF elevation in children with autism spectrum disorder

Brain-derived neurotrophic factor (BDNF), a key peptide in neurocognitive development, has been reported to be elevated in the serum of children with autism spectrum disorder (ASD). In a few studies, however, no differences or the converse have been documented. As a secondary analysis of a natural history study, we examined differences in ELISA serum BDNF between a group of children aged 1 to 9 years (69% white) with ASD (n = 94) and those with typical development (n = 52) or non-ASD developmental delay (n = 21), while accounting for the potential confounding effects of platelet quantity. Platelet counts were measured within 4 h of blood draw using an automated cell counter. Taqman single nucleotide polymorphism (SNP) assays were used to genotype 11 SNPs within the BDNF locus. Unadjusted mean BDNF concentration was higher in children with ASD than in children with typical development (standardized mean difference = 0.23; 95% CI 0.07, 0.38), but not children with non-ASD developmental delay. The magnitude of this difference was reduced after adjusting for platelet count (standardized mean difference = 0.18; 95% CI 0.02, 0.33). Although some BDNF SNPs were related to BDNF concentration, the distributions of these genotypes did not differ across diagnostic groups. This study replicates previous work suggesting that average serum BDNF concentration is higher in ASD compared to typical development, and extends that work by highlighting the potentially confounding role of platelet counts. The etiology of platelet count differences warrants further elucidation. Nonetheless, our results suggest that elevation in BDNF may be partially explained by higher platelet counts in children with ASD, an association that should be considered in future analysis and interpretation.Registration: NCT00298246

P10.01 Effect of mesenchymal cells on infiltration and phenotype of T cells in a 3D glioblastoma spheroid model

Abstract BACKGROUND Glioblastoma (GBM), the most malignant primary brain tumor with a median survival less than two years, is characterized by aggressive invasive growth, presence of hypoxic and necrotic regions, and T-cell dysfunction. Mesenchymal cells with characteristics of cancer associated fibroblasts are present in GBMs, but it remains to be established whether they contribute to T cell sequestration in the tumor stroma and functional inactivation similarly to extracranial tumors. Murine models do not reflect several aspects of human immune system and 2D cell culture systems often fail to recapitulate complex interactions in the tumor microenvironment. 3D spheroids prepared from patient-derived cell cultures offer higher degree of complexity and biological relevance. The aim of this study was to establish a 3D GBM spheroid model to determine the effect of patient derived mesenchymal cells on human T cell infiltration and phenotype. MATERIAL AND METHODS Glioma stem-like cell and mesenchymal cell cultures were derived from human GBMs. Naïve T lymphocytes were isolated from buffy coats of healthy donors by Ficoll paque gradient separation, followed by negative magnetic isolation techniques using Naïve Pan T Cell Isolation Kit (Miltenyi Biotec). 3D spheroids were prepared by embedding the cells in 20 µl of Geltrex (Thermo Fisher Scientific). T cells activated by magnetic beads with anti-CD2, anti-CD3 and anti-CD28 antibodies (Miltenyi Biotec) and IL-2 were subsequently added to the spheroids. After 5 days of co-culture, spheroids were dissociated, and viable cells were counted using automated cell counter (Biorad). Expression of a T-cell activation marker CD69 and immune checkpoint molecules LAG3, and PD-1 in CD3+ cells was analyzed by flow cytometry. Mesenchymal, stem cell and lymphocyte markers TE-7, Nestin and CD45 respectively, were analyzed by immunohistochemistry (IHC) on frozen spheroid sections. RESULTS We prepared spheroids comprising glioma stem-like cells with and without mesenchymal cells. Glioblastoma-derived cells were evenly dispersed throughout the spheroids as demonstrated by IHC. T cells infiltrated into both types of spheroids, but their number in spheroids containing mesenchymal cells was about 40% (p=0.002) lower compared to the ones without mesenchymal cells. In both types of spheroids, the presence of activated allogeneic T cells led to more than a 50% reduction of the glioblastoma-derived cells. Ongoing studies evaluate the effect of mesenchymal cells on the phenotype of spheroid infiltrating T cells. CONCLUSION We have successfully established a 3D spheroid model to study the role of mesenchymal cells in GBM microenvironment. Our results indicate that mesenchymal cells may influence the infiltration of T cells and modulate their phenotype. SUPPORT Project was financed by the Center for Tumor Ecology (CZ.02.1.01/0.0/0.0/16_019/0000785).

A pediatric case of EDTA-related pseudothrombocytopenia and implementation of the histograms by the automated cell counters

Abstract Objectives Platelet aggregation in the presence of ethylenediaminetetraacetic acid (EDTA), is called EDTA-related pseudothrombocytopenia (EDTA-PTP), resulting in low platelet count by automatic cell counters. Case presentation Herein, we present a case of a 5-year-old female, who was referred to our laboratory due to persistent thrombocytopenia. Conclusion Our case report discusses the efficiency of the histograms and flag warnings of the cell counter, indicating the importance of these variables.

A rapid Focus-Forming Assay for quantification of infectious adenoviral vectors

Currently available methods to titrate adenoviral vectors (AdV) in the absence of a gene reporter such as GFP, are either time-consuming or not very reproducible. A Focus-Forming Assay (FFA) for quantification of infectious AdV particles followed by automated focus counting was developed using new monoclonal antibodies (mAbs) against the human adenovirus type 5. Briefly, in this method, 96-well plates of HEK293A cells were infected with 2-fold dilutions of AdV at seeding time. Forty eight hours post-infection, the cells were fixed with methanol. The cells were then incubated with each mAb followed by a FITC conjugated anti-mouse antibody. The plates were scanned and positive cells counted using an automated fluorescence microscopy system. The results of the FFA were compared with the plaque assay and the TCID50 assay. The titer of six different recombinant AdV were compared using the FFA along with a commercial kit. The results were similar, but in contrast to the commercial kit for which the stained cells are counted manually, the software automatically counts the positives cells in the FFA. The automatic counting of positive cells makes the FFA a more precise and reliable assay compared to the commercial kit for titration of AdV.

Eosinopenia as a Marker of Sepsis in Intensive Care Unit Patients

Sepsis is one of the most common cases of mortality and morbidity in the intensive care unit (ICU). This study was done to evaluate eonsinopenia as a marker of sepsis in intensive care unit patients. This cross sectional study was carried out in the Department of Clinical Pathology, Bangabandhu Sheikh Mujib Medical University, Dhaka from March 2012 to February 2013. Absolute Eosinophil Count (AEC) was done by automated cell counter and rechecked manually microscopically. In this study 74 suspected case of sepsis were enrolled from intensive care unit, BSMMU, Dhaka. Out of these patients 34 were included in the infection group and 40 in the non-infection group depending on blood culture report. The area under ROCcurve was 0.765 in current study. At the cutoff valve of AEC&lt; 40 cell/cumm, the sensitivity and specificity of AEC for diagnosis of sepsis was 72.5% and 61.8% respectively. This present data revealed that decreased absolute eosinophil count was significantly associated with sepsis. So eosinopenia may be a reliable marker for early diagnosis of sepsis.