Enumeration Of Cells Research Articles

South Asian adults with ASCVD exhibit a reduction in circulating vascular regenerative cell content compared to White European adults: a sub-analysis of the ORIGINS-RCE CardioLink-13 study

Abstract Background Compared to people of other races and ethnicities, South Asian (SA) adults are disproportionately affected by a higher burden of type 2 diabetes (T2D) and atherosclerotic cardiovascular disease (ASCVD). We previously found that compared to individuals of White European (WE) descent with T2D, SA individuals with T2D exhibited profoundly less vascular regenerative (VR) cell content. Given that VR cells critically regulate vessel homeostasis, we postulated that the "vascular regenerative cell exhaustion" phenomenon we had observed may also contribute to the prevalence of ASCVD in SA populations. Purpose The ORIGINS-RCE CardioLink-13 study compared the frequencies of VR cells and cell types in individuals of SA and WE ethnicities with either 1) T2D and cardiovascular-risk factors or 2) documented ASCVD. This sub-analysis was conducted to determine if there are differences in the frequency of circulating VR cells and cell types in SA and WE individuals with ASCVD. Methods Among the 120 self-identified SA and WE adult participants (≥40 years of age), 23 SA and 41 WE individuals had ASCVD. Peripheral blood samples were processed for pro-inflammatory vs. pro-vascular progenitor cell enumeration as well as measurement of reactive oxygen species (ROS) production using a unique high aldehyde dehydrogenase (ALDHhi) activity-based flow cytometry assay in combination with side scatter properties (SSC) and lineage specific cell surface markers to identify progenitor cell populations. Results Compared to samples from the WE participants, the SA adults had a lower frequency of ALDHhiSSClowCD133+ (59% vs. 48%, P<0.001) cells and ALDHhiSSClowCD34+CD133+ (56% vs. 45%, P<0.001) progenitor cells with previously established pro-angiogenic properties. We also observed that SA participants had a decreased frequency of ALDHhiSSClowCD34+CD133+CD45- (0.0019% vs. 0.0005%, P<0.05) cells which may represent a rare population of circulating endothelial progenitor cells with vasculogenic functions. SA participants also exhibited a shift in monocyte polarization from a reparative phenotype towards a pro-inflammatory state, as demonstrated by a decrease in ALDHhiSSCmidCD14+CD163+ (86% vs. 78%, P<0.01) monocytes and an increase in ALDHhiSSCmidCD86+CD163- (8.2% vs. 13%, P<0.01) cells. There were no significant differences in the frequency of circulating granulocytes (ALDHhiSSChi cells) suggesting comparable inflammatory granulocyte burden between SA and WE adults, with no meaningful difference in intracellular ROS production in ALDHhi progenitor cell subsets, as previously observed in participants with T2D. Conclusions South Asian adults with ASCVD demonstrate a loss of vascular regenerative cells suggesting a compromised ability to repair damaged vessels which may contribute to their elevated cardiovascular risk.

PSIV-28 Evaluation of Possible Interactions Between Platelets and Neutrophil in Cow Blood

Abstract Neutrophils have a central role in the inflammatory response and fight infection. Platelets are essential for hemostasis and thrombosis and fighting vascular damage. Studies in man show that the interaction between these cells can modulate their function. We aimed to assess the effect of possible relationships between the platelet count in whole blood on neutrophil activation. Blood samples (50 mL) from five clinically healthy Holstein Friesian cows at the NC A&T Dairy research unit were collected. Differential white blood cell counts were conducted using a VetScan (Zoetis). Neutrophils were isolated through differential centrifugation and hypotonic lysis of red blood cells. Duplicate samples of neutrophils were maintained in phosphate-buffered saline for 30 minutes at 37 °C. The total and viable neutrophil count was evaluated using TC20 (Bio-Rad). Samples were centrifuged. The supernatant was used to determine the concentration of total secreted protein using the Bicinchoninic acid assay (Pierce). Enumeration of bovine blood platelets and white blood cells was accomplished using the Vetscan study found that there was no significant difference observed in the total cell count or percent viability in neutrophils between the five cows (P > 0.05). However, protein concentration showed a significant change between animals. Correlations between total platelet count, neutrophil count, viability, and protein secretion were evaluated. No correlation was found between neutrophil count, protein secretion in isolated neutrophils and concentration of platelets in whole cow blood. The number of circulating platelets was not related to neutrophil viability or protein secretion. This approach can be used in further investigation using imaging and is needed to evaluate the effect and functional consequences of interactions between platelets and neutrophils in cow blood.

High Dimensional Profiling of Patient-Derived Multiple Myeloma Bone Marrow Specimens Treated with an FcRH5-Targeted Bispecific Ex Vivo

Background: Cevostamab is an FcRH5 (Fc receptor-homolog 5) targeted T cell-dependent bispecific (TDB) currently being evaluated in the clinic, with promising activity and favorable safety profile as a monotherapy in patients (pts) with heavily pre-treated Relapsed/Refractory multiple myeloma (MM) (NCT03275103, Trudel et al. ASH 2021). Cevostamab selectively engages and activates T cells in the proximity of MM cells in the bone marrow (BM) to drive tumor cell killing. To dissect the transcriptional and functional impact of cevostamab treatment on the MM BM tumor microenvironment (TME) and identify factors linked to enhanced killing, we established an ex vivo TDB killing assay with MM pt BMMC (BM mononuclear cells) samples followed by detailed characterization of immune and tumor cells. Methods: We tested commercially procured frozen BMMC samples from 7 pts with Stage I or Stage III Newly Diagnosed Multiple Myeloma (NDMM) with no known prior treatments (except a paired, longitudinally collected sample) and median age of 67.5 years. BMMC samples containing endogenous T and MM cells were treated ex vivo with 10 ug/mL of either a non-targeted TDB (considered as baseline) or a research grade cevostamab surrogate (FcRH5 TDB) for 16 to 48 hours (hr). T cell enumeration and MM killing (CD138 + CD38 +) was evaluated at 48 hours by flow cytometry. Ex vivo response was normalized by T cell quantity and defined as % tumor cell killing per 10 3 T cells present in the assay, which was used to classify samples as Responders (R) (>= 10%) or Suboptimal Responders (SoR) (< 10%). Single cell RNA-seq (scRNA-seq) was performed at 16, 24 and 48 hr post treatment. Tumor-specific genes enriched in SoRs at baseline were investigated for prognostic value by examining their bulk gene expression profile in baseline tumor samples (CD138-enriched) from pts (N=1150) treated with immunomodulatory drug, proteasome inhibitors, or combinations thereof (NCT01454297, MMRF CoMMpass IA22). Results: We first examined the T cell compartment by scRNA-seq profiling. Upon FcRH5 TDB treatment, clustering analysis suggested that CD4 and CD8 T cells undergo extensive transcriptional changes compared to control TDB (Fig 1A). A TNF and LTA-expressing CD4 memory cluster was enriched upon treatment in both SoRs and Rs, but to a greater extent in Rs. A similar trend was observed with an IFNG and CCL4-enriched CD8 effector-like proliferating T cell cluster. Transcriptional profiles of MM cells suggested inter- and intra-pt tumor heterogeneity at baseline. MM cells in SoRs at baseline had higher expression of genes like CD44, SRGN, CCND2 and SLAMF1. Of these genes, higher expression of CD44 or SRGN was found to be significantly associated with worse outcome to 1L treatments. Exposure to FcRH5 TDB altered tumor cells from all pts (Figure 1B), resulting in significant upregulation of genes such as HLA-E, SOCS1, and TNFSF10. Increase in CXCL9 expression upon treatment was found to be higher in Rs than SoRs. Discussion: Our work provides a novel ex vivo approach for unbiased, high dimensional pharmacodynamic profiling of functional and transcriptomic changes in pt samples by combining TDB treatment with scRNA-seq. Consistent with the expected mechanism of action and preliminary biomarker analysis reported for cevostamab (Nakamura et al. ASH 2020), FcRH5 TDB led to increased expression of genes in T cells associated with infiltration, activation and effector function. It also induced transcriptional changes in MM cells regardless of the ex vivo response status, including upregulation of genes associated with T and NK cell infiltration and antigen presentation. For certain genes, a greater magnitude of these changes was observed in Rs. These identified genes, and enriched clusters will be examined in clinical RNAseq data from cevostamab-treated pts. It remains to be determined whether our results reflect treatment effects that are applicable to other targeted bispecifics in MM. Overall, our assay setup can serve as a platform to generate novel hypotheses, corroborate clinical observations, identify pt-specific features driving improved or dampened response, and test novel combinations for improved killing. Figure 1 : UMAP (Uniform Manifold Approximation and Projection) plot of all (A) T cells and (B) tumor cells shows distinct clustering of cells by treatment type for all pts (red: FcRH5 TDB; blue: non-targeted TDB)

CD34+ Megakaryocytes Associate with Myelodysplastic Syndromes and Related Cytogenetic Abnormalities, but Not Other Hematological Disorders

Background: CD34 is a transmembrane phosphoglycoprotein that is a marker of hematopoietic stem cells and is used clinically to aid in enumeration of blast cells for identification of advanced myeloid neoplasms. Megakaryocytes are differentiated cells from the myeloid lineage that reside in the bone marrow and generally stain negative for CD34 (CD34-). However, CD34 positive (CD34+) megakaryocytes have been observed in hematological disorders such as myelodysplastic syndrome, myeloproliferative neoplasms, and acute leukemias. CD34+ megakaryocytes are proposed to be relatively specific for malignant hematologic conditions and have been reported to associate with a worse prognosis. However, the general clinical, biochemical, and cytogenic characteristics for bone marrow cases of CD34+ megakaryocytes versus CD34- megakaryocytes remains unclear. Objective: We sought to determine the clinical, biochemical, and cytogenetic characteristics associated with finding CD34+ megakaryocytes on bone marrow biopsies. Methods: We identified 281 independent, index bone marrow biopsy cases between 2011-01-01 and 2017-12-31 from Vancouver General Hospital, Canada that underwent CD34 immunohistochemistry at clinical request. The clinical, biochemical, and cytogenetic characteristics of patients were compared based on megakaryocytes being CD34+ vs CD34- on immunohistochemistry. Cases of CD34+ megakaryocytes were defined as having >5% of megakaryocytes staining CD34+, but sensitivity analyses were performed using different percentage cut-offs of 10, 30, and 50%. Results: Out of 281 cases, 134 had CD34+ megakaryocytes (58.2% male sex; mean age = 64.1 years [SD = 15.2 years]) and 147 had CD34- megakaryocytes (57.1% male sex; mean age = 60.0 years [SD = 16.6 years]). There was a significant difference in the distribution of diagnostic categories observed between cases of CD34+ versus CD34- megakaryocytes: myelodysplastic syndromes 35.1% (n=47) versus 21.8% (n=32), acute myeloid leukemias 20.9% (n=28) versus 21.1% (n=31), non-diagnostic etiology 17.9% (n=24) versus 14.3% (n=21), and myeloproliferative neoplasms 10.4% (n=14) versus 19.7% (n=29) (Figure A; Chi-square p-value=0.0004). Sensitivity analyses using different percentage cut-offs to define cases of CD34+ from CD34- megakaryocytes yielded similar results. The differences in biochemical characteristics between cases of CD34+ versus CD34- megakaryocytes were generally unremarkable. The median (IQR) for biochemical parameters between cases of CD34+ and CD34- megakaryocytes were hemoglobin 99 (28) versus 100 (34) g/L, mean corpuscular volume 94 (14) versus 93 (10) fL, neutrophil count 2.20 (3.68) versus 2.46 (3.63) x10 9/L, platelet count 98 (158) versus 112 (194) x10 9/L, and bone marrow blasts 7 (4) versus 3 (14)%. Lastly, there were notable trends in cytogenetic results associated with 90 cases with CD34+ versus 84 cases with CD34- megakaryocytes including: complex karyotypes for 15.6% (n=14) versus 6.0% (n=5), isolated deletion 5q for 7.8% (n=7) versus 1.2% (n=1), and/or isolated trisomy 8 for 5.6% (n=5) versus 2.4% (n=2). Similar cytogenetic results were observed when cases were restricted to only cases of CD34+ versus CD34- megakaryocytes with a diagnosis of myelodysplastic syndrome (Figure B). Conclusion: The presence of CD34+ megakaryocytes is more common in cases of myelodysplastic syndrome relative to other neoplastic hematologic disorders, but does not appear to help with discriminating between neoplastic and non-neoplastic hematological disorders ( e.g. non-diagnostic etiology). Cases with CD34+ megakaryocytes tend to associate with complex karyotype and deletion 5q abnormalities relative to cases with CD34- megakaryocytes.

Metataxonomic analysis of milk microbiota in the bovine subclinical mastitis.

Subclinical mastitis is one of the most widespread diseases affecting dairy herds with detrimental effects on animal health, milk productivity, and quality. Despite its multifactorial nature, the presence of pathogenic bacteria is regarded one of the main drivers of subclinical mastitis, causing a disruption of the homeostasis of the bovine milk microbial community. However, bovine milk microbiota alterations associated with subclinical mastitis still represents a largely unexplored research area. Therefore, the species-level milk microbiota of a total of 75 milk samples, collected from both healthy and subclinical mastitis-affected cows from two different stables, was deeply profiled through an ITS, rather than a traditional, and less informative, 16S rRNA gene microbial profiling. Surprisingly, the present pilot study not only revealed that subclinical mastitis is characterized by a reduced biodiversity of the bovine milk microbiota, but also that this disease does not induce standard alterations of the milk microbial community across stables. In addition, a flow cytometry-based total bacterial cell enumeration highlighted that subclinical mastitis is accompanied by a significant increment in the number of milk microbial cells. Furthermore, the combination of the metagenomic and flow cytometry approaches allowed to identify different potential microbial marker strictly correlated with subclinical mastitis across stables.

Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells.

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient disease since CTCs are rare and heterogeneous. CTCs are detached from the primary tumor, enter the blood circulation system, and potentially grow at distant sites, thus metastasizing the tumor. Since CTCs carry similar information to the primary tumor, CTC isolation and subsequent characterization can be critical in monitoring and diagnosing cancer. The enumeration, affinity modification, and clinical immunofluorescence staining of rare CTCs are powerful methods for CTC isolation because they provide the necessary elements with high sensitivity. Microfluidic chips offer a liquid biopsy method that is free of any pain for the patients. In this work, we present a list of protocols for clinical microfluidic chips, a versatile CTC isolating platform, that incorporate a set of functionalities and services required for CTC separation, analysis, and early diagnosis, thus facilitating biomolecular analysis and cancer treatment. The program includes rare tumor cell counting, clinical patient blood preprocessing, which includes red blood cell lysis, and the isolation and recognition of CTCs in situ on microfluidic chips. The program allows the precise enumeration of tumor cells or CTCs. Additionally, the program includes a tool that incorporates CTC isolation with versatile microfluidic chips and immunofluorescence identification in situ on the chips, followed by biomolecular analysis.

The Therapeutic Potential of Oxyhydrogen Gas in Oncology: A Study on Epstein–Barr Virus-Immortalised B-Lymphoblastoid (TK6) Cells

Cancer is a leading cause of mortality worldwide. B-cells are a keystone of the adaptive immune response and are essential for the presentation of tumor-associated antigens to various types of T-cells. Approximately 1.5% of global cancer cases, including breast and gastric carcinomas and both Hodgkin’s and non-Hodgkin’s lymphomas, are linked with prior Epstein–Barr Virus (EBV) infection. Such properties make EBV-infected lymphocytes ideal models for understanding the effect of oxyhydrogen gas on dysfunctional cell cycling. The aim of this study is to assess the effects of the direct infusion of oxyhydrogen gas on the replicative capacity of EBV-immortalised B-lymphocytes. Oxyhydrogen gas was directly infused into cell culture media. Cells were incubated in 95% air and 5% CO2 for up to 72 h. Cell enumeration was assessed with and without the addition of mitogenic growth stimuli, and subsequent cell-cycle analysis was performed. Cell enumeration: An initial trend of replicative inhibition of TK6 cells is noted with a single oxyhydrogen treatment at the 24 and 48 h time points. The daily addition of oxyhydrogen-infused media showed statistically relevant data at 24 and 48 h but not at 72 h. In mitogen-stimulated cells, a non-statistical trend of inhibition was observed at 24, 48 and 72 h. Analysis details a significant increase in DNA in the Sub G1 phase, indicating increased apoptosis.

Qualification of the differential leukocyte count and immunophenotyping in cryopreserved ex vivo whole blood assay.

We developed a flow cytometry-based assay, termed Differential Leukocyte Counting and Immunophenotyping in Cryopreserved Ex vivo whole blood (DLC-ICE), that allows quantification of absolute counts and frequencies of leukocyte subsets and measures expression of activation, phenotypic and functional markers. We evaluated the performance of the DLC-ICE assay by determining inter-operator variability for processing fresh whole blood (WB) from healthy donors collected at multiple clinical sites. In addition, we assessed inter-operator variability for staining of fixed cells and robustness across different anticoagulants. Accuracy was evaluated by comparing DLC-ICE measurements to real-time cell enumeration using an accredited hematology analyzer. Finally, we developed and tested the performance of a 27-colour immunophenotyping panel on cryopreserved fixed WB and compared results to matched fresh WB. Overall, we observed <20% variability in absolute counts and frequencies of granulocytes, monocytes and lymphocytes (T, B and NK cells) when fresh WB was collected in different anti-coagulant tubes, processed or stained by independent operators. Absolute cell counts measured across operators and anti-coagulants using the DLC-ICE method exhibited excellent correlation with the reference method, complete blood count (CBC) with differential, measured using a hematology analyzer (r2 > 0.9 for majority of measurements). A comparison of leukocyte immunophenotyping on fresh WB versus DLC-ICE processed blood yielded equivalent and linear results over a wide dynamic range (r2 = 0.94 over 10-104 cells/μL). These results demonstrate low variability across trained operators, high robustness, linearity and accuracy, supporting utility of the DLC-ICE assay for large cohort studies involving multiple clinical research sites.

B-142 Development and Clinical Validation of Artificial Intelligence Assisted Flow Cytometry Diagnosis for Acuteleukemia

Abstract Background Flow cytometry is one of the most commonly used diagnostic tools in hematopathology. Manual flow cytometry data analysis is vulnerable to substantial inter-operator variation and typically takes a technologist 20 min to analyze a case. In the last decade, artificial intelligence (AI) technology has advanced dramatically and become widely used in image processing. Previously, we created an AI-assisted workflow for classifying white blood cell subsets in peripheral blood. The goal of this study is to evaluate the automated flow cytometry analysis system for diagnosing acute leukemia. Methods A total of 117 cases were retrieved from the archived diagnostic cases, consisting of three diagnostic categories: 20 normal cases, 37 acute lymphoblastic leukemia and 60 acute myeloid leukemia. Flow cytometry data manually analyzed by a pathologist is the gold standard. The same flow cytometry data was processed by the flow cytometry analysis software DeepFlow® using proprietary AI algorithm. The final diagnosis and blast cell count of AI and manual analysis are compared by Pearson correlation coefficient. Results The AI-assisted blast cell enumeration showed moderate correlation as compared to manual evaluation (r = 0.8, Fig. 1). Manual adjustment for enumeration of CD34 negative blast population with uncommon immunophenotype is required for 7 cases, including 4 CD34−/CD117+ acute myeloid leukemia and 3 CD34−/TDT+ lymphoblastic leukemia. The AI analysis effectively recognized cell populations and provided correct diagnoses on 94% of cases (110/117). With pathologists’ review, all cases were classified into the correct diagnostic categories. Conclusion Our study demonstrated that AI can accurately recognize the flow cytometry pattern of leukemia and make the proper diagnosis. Additional training for CD34 negative blast gating and other uncommon blast immunophenotypes is being conducted. Ultimately, AI will decrease the demand on manual analysis by technologists and pathologists, standardize the analysis process, and provide accurate diagnosis.

A-075 Development and Validation of Image-based Cell Counter Microscanner C3 for CD34-positive Hematopoietic Stem Cell Enumeration

Abstract Background Accurate measurement of CD34-positive hematopoietic stem cells (HSCs) is critical for successful peripheral blood stem cell transplantation (PBSCT). Flow cytometry is by far the gold standard for measuring CD34-positive stem cells. However, its complicated procedure requires time, cost and highly-trained operators, thus the necessity of simpler and cheaper alternative method is emerging. We have developed a new image-based cell counter, Microscanner C3 (MSC3, Biozentech, South Korea) as an alternative method for enumeration of CD34-positive HSCs and evaluated its performance. Methods 100 µl of apheresis product was stained with CD45-BB515 and CD34-PE antibodies (Becton Dickinson, USA) at room temperature in the dark. Red blood cell lysis with 500 µl of OptiLyze C solution (Beckman Coulter, USA) was followed by centrifugation. After the removal of supernatants, the sample was resuspended with 500 µL PBS, which then infused to filter paper imbedded BZ-1 microchip (Biozentech, South Korea). Thirty-fields of the loaded microchip was auto-scanned by MSC3 and its dedicated software analyzed the images and yielded CD34-positive cell fraction out of CD45-positive cells as percentage. Results of thirty-six samples were compared to that of FacsLyric (Beckton Dickinson, USA) flow cytometry and accuracy and imprecision (pentaplicate measurements for three samples), repeatability (two experimenters performed triplicate measurements for three samples each), linearity (triplicate measurements for five levels), limit of detection (hexaplicate measurements for five levels) and specificity (using whole blood specimen from three donors with monoclonal gammopathy, thrombocytosis or monocytosis) was evaluated. Results CV% values of inaccuracy, imprecision and repeatability did not exceed 15%. The method was found to be linear across the measuring range. LOD was evaluated to be 7 cells/µL and no background interference was detected. The difference between the measured fraction of MSC3 and FacsLyric was within allowable limit, with an R-value greater than 0.98. Conclusion We developed a reliable method for quantifying CD34-positive HSCs based on image analysis. The limitation involved in MSC3 applications in CD34-positive HSCs is that, like dual-platform flow cytometry, the results should be combined with the results of conventional hematology analyzer in order to define absolute HSC numbers. However, with its simpler and faster procedures, MSC3 exhibited a great potential to be used as an alternative to conventional flow cytometry in CD34-positive HSCs enumeration and other applications as well.

High-Throughput Viability Testing of Microbial Communities in a Probiotic Product Using Flow Cytometry

There is growing scientific and commercial interest in multi-species probiotic products due to their potential benefits in maintaining gut health. Determining the viability of probiotic microorganisms in these products is essential to ensure that they confer maximal health benefits. The gold standard for enumerating probiotic viability is the plate count method. However, this may be inaccurate for enumerating mixed probiotic populations, with recognised limitations including difficulty measuring metabolically active yet unculturable, very slow growing microbes, microencapsulated, enteric coated microbes, or multi-strain formulations that require differing growth media. Here, we developed a flow-cytometry-based approach using SYTOXTM Green dye to assess the viability of probiotic microorganisms in a multi-species, fibre-containing probiotic product and compared this to the traditional plate count method. This method was suitable for enumerating both total bacterial cells and the viable cell fraction in the complete product mixture, and could also be used to assess how stressors, such as gastric digestion and exposure to bile acids, affect bacterial cell viability. Flow cytometry measurements routinely detected higher viable cell counts than plate counting. This work demonstrates that flow cytometry assays can be established as a suitable method for rapid enumeration of viable cells in complex, multi-species probiotics.

Abstract 21 Machine Learning Approach for Automatic Enumeration of Cord Blood Stem Cells by Flow Cytometry

Abstract Introduction Stem cell laboratories measure the number of viable CD34+ and CD45+ cells in their products by flow cytometry following a standard protocol (ISHAGE). Although the ISHAGE protocol is well documented, its correct application can be challenging. Automated gating algorithms have been created to address this issue, but they have limitations and can only be used in certain conditions. Recent developments in artificial intelligence (AI) and machine learning (ML) have made these approaches more accessible and practical to use. For flow cytometry data, the Cytobank platform now offers the Automatic Gating Algorithm (AGA), an AI-ML-based feature allowing users to easily implement their own gating strategies. Objectives The performance of the AGA based on the ISHAGE protocol gating was evaluated using cord blood post-thaw flow cytometry data. Methods In this study, the AGA was trained using 29 flow cytometry cord blood data files analyzed according to the ISHAGE gating strategy. Using the model, an inference analysis was conducted on two series of 12 data samples acquired from two independent operators. The enumeration of viable CD34+ cells was manually performed and compared to the AI analyses. Results The mean differences and standard deviation in vCD34+ counts between the manual and the automatic gating are 6.0% ± 2.7 for Operator #1 and 7.7% ± 2.1 for Operator #2. The correlation coefficients between vCD34+ results are 0.9981 for Operator #1 vs AI, and 0.9699 for Operator #2 vs AI. These results show that the AI inference provides results comparable to human manual gating, and are within the accepted difference of 10% between duplicates as defined by ISHAGE convention. Discussion This study provides a testing ground for using AI-ML-based automatic gating and supports its use in stem cell laboratories with acceptable performance and accuracy. The application of AI and ML for vCD34+ enumeration can potentially lead to the development of more widely applicable automatic gating algorithms. This could improve standardization by providing a common tool for analyzing flow cytometry data.

(Invited) hand-Held Florescence Microscope for Single Micro/Nano Particle Imaging

Fluorescence microscopes are commonly used to detect micro and nano biological entities (blood cells, microbes, functionalized particles etc.). Several experimental assays have been developed for biomarker measurements using this microscopy instrument. Common examples include cell viability analysis, surface receptor based specific blood cell enumeration, phagocytosis determination etc. However, the laboratory-based bench top fluorescence microscope suffers from high capital cost, maintenance, experienced staff for its operation and lack of portability. To address several of these challenges, here we have developed a hand-held, portable, 3D printed fluorescence microscope to enable single cell enumeration and nanoparticle imaging operated by smartphone.3D printed platform is composed of top and bottom portions. The bottom portion includes excitation optics including an LED and excitation filter. While the top portion houses an emission filter, and a lens, while a smartphone can be placed on top portion for imaging. The excitation and emission filters can be swapped out for different fluorophores used. We have used the platform for imaging leukocytes collected from the whole blood. Blood samples were procured from Robert Wood Johnson Medical Hospital. We ran the leukocytes on our photonics platform and laboratory florescence microscope concurrently for a comparative analysis and found a high correlation of R2 = 0.99 in between cell counting results. Further, swapping different lenses can achieve high resolution for the imaging system. The resolution was verified by US Airforce Resolution Chart. This allowed us to image fluorescent nanoparticles up to 800nm at various concentrations and excitation modalities.This platform can be used for single-cell analysis, specific leukocytes enumeration and nanoparticles imaging. The demonstrated results can enable significant applications to develop many biomedical and diagnostic assays.

Using convective mixing in mesocosms to study climate-driven shifts in phytoplankton community distributions

With climate change predicted to alter water column stability and mixing across the world’s oceans, a mesocosm experiment was designed to ascertain how a natural phytoplankton community would respond to these changes. As a departure from other mesocosm experiments, we used heating and cooling to produce four different climate-inspired mixing scenarios ranging from well-mixed water columns representative of typical open turbulence (ϵ = 3 x 10-8 m2/s3) through to a quiescent water column with stable stratification (ϵ = 5 x 10-10 m2/s3). This method of turbulence generation is an improvement on previous techniques (e.g., grid, shaker, and aeration) which tend to produce excessive dissipation rates inconsistent with oceanic turbulence observations. Profiles of classical physical parameters used to describe turbulence and mixing (turbulent dissipation rate, buoyancy frequency, turbulent eddy diffusivity, Ozmidov scale) were representative of the profiles found in natural waters under similar mixing conditions. Chlorophyll-a profiles and cell enumeration showed a clear biological response to the different turbulence scenarios. However, the responses of specific phytoplankton groups (diatoms and dinoflagellates) did not conform to the usual expectations: diatoms are generally expected to thrive under convective, turbulent regimes, while dinoflagellates are expected to thrive in converse conditions, i.e., in stable, stratified conditions. Our results suggest that responses to mixing regimes are taxon-specific, with no overwhelming physical effect of the turbulence regime. Rather, each taxon seemed to very quickly reach a given vertical distribution that it managed to hold, whether actively or passively, with a high degree of success. Future studies on the effects of climate change on phytoplankton vertical distribution should thus focus on the factors and mechanisms that combine to determine the specific distribution of species within taxa. Our convection-based mesocosm approach, because it uses a primary physical force that generates turbulence in open waters, should prove a valuable tool in this endeavor.

Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays

In a precarious world of rapidly growing pandemics, the field of vaccine production has witnessed considerable growth. Bacillus Calmette-Guérin (BCG) is a live-attenuated vaccine and a part of the immunization program in 157 countries. The quality control is based on a potency test through viable cell enumeration. The colony-forming unit (CFU) assay is the official method, however, it often yields fluctuating results, suffers from medium cracking, and requires lengthy analysis (~ 28 days). Flow cytometric analysis was proposed earlier, but it was coupled with a Coulter counter for measuring the entire bacterial population (live/dead). In the present study, thiazole orange/propidium iodide dyes supplemented with fluorogenic reference beads were employed for viable counting, eliminating the need for a Coulter counter. Both the flow cytometry and the colorimetric technique employing tetrazolium salt were validated and compared to the CFU assay. The colorimetric assay displayed high precision, accuracy, and a strong positive correlation with the CFU assay. The flow cytometry assay demonstrated high precision and a notable ability to distinguish different forms of BCG cells (live, injured, and dead). It also exhibited a perfect positive correlation with the CFU assay. Both methods reduced the analysis time by > 26 days and eliminated the need for human intervention by automating the test.

Methicillin Resistance of Food-Borne Biofilm-Forming Staphylococci

The aim of this study was to evaluate the biofilm-forming ability and the resistance of planktonic cells and biofilm to methicillin (MIC and MBC, and log CFU/cm2 reduction, respectively). Thirty-four isolates were used, including coagulase-positive Staphylococcus (CPS) and coagulase-negative Staphylococcus (CNS), recovered from ready-to-eat (RTE) foods. Biofilm-forming ability was based on enumeration of viable cells from biofilms formed on three types of surfaces, namely stainless steel, polystyrene, and glass. Thirteen of the thirty-four isolates (38.2%) were methicillin-resistant (MIC higher than 4 µg/mL). Staphylococcus aureus (CPS) and Staphylococcus saprophyticus (CNS) were the species that showed the highest percentage of resistance (50% and 71.4%, respectively). Staphylococcus warneri was the only species susceptible to methicillin. In 20 of the 34 isolates, the MBC value was twice the MIC value. The CPS (S. aureus) and the CNS (the other species) were not significantly different (p &gt; 0.05) on biofilm-forming ability on the three surfaces used. However, when comparing the averages obtained for the biofilm-forming ability of the isolates, the values obtained (log CFU/cm2) in polystyrene were significantly higher (p &lt; 0.05) than those obtained with the isolates on stainless steel. On the other hand, data from biofilm-forming ability of the isolates on glass and stainless steel are positively and significantly correlated with each other (r = 0.54; p = 0.02). It was not possible to determine the concentration of methicillin that promotes biofilm removal since log reductions were less than 3 log CFU/cm2. The results of this study indicate that foodborne CPS and CNS can form biofilms on different types of material. As these biofilms are resistant to high concentrations of methicillin, their occurrence in food environments and their spread to medical settings can result in staphylococcal food poisoning or, in the worst-case scenario, septicemia, respectively. Good hygiene and good manufacturing practices (GHP and GMP) are therefore mandatory to prevent contamination with Staphylococcus spp.

Thermal bubble-driven impedance-based high-throughput cell counting chip design

Cell identification and enumeration are important methods within clinical and research laboratories for rapid diagnosis of relevant diseases. However, there are still many shortcomings in the current cell counting methods. In order to improve the performance of cell counting, a novel impedance-based cell counting chip based on thermal bubble drive was designed in this study. The chip is fabricated by whole-wafer processing and provides the driving force for cells through a combination of thermal bubble nozzles and microfluidic channels, integrating 100 individual detection units on a 38 mm2 size chip. Experimental and theoretical analyses have demonstrated that the chip can achieve high throughput detection of 45 000 beads/s under extreme conditions. A fourfold difference in detection voltage was obtained for both 14 and 7 µm diameter polystyrene beads. The linear fit coefficient of determination between the cell number measured by the chip and the cell number observed in reality was above 0.999 for both polystyrene beads and 211H cells, and the counting accuracy exceeded previous studies. It turns out that the chip achieves portable, low-cost, high-throughput, and high-accuracy cell counting, which is conducive to the development of impedance cell counting.

Analytical errors in nucleated red blood cell enumeration.

Enumeration of nucleated red blood cells (NRBCs) in peripheral blood of dogs and cats is performed by manual counting during blood film evaluation. Automated methods have increased precision and accuracy; however, most analyzers cannot distinguish leukocytes and NRBCs. The Sysmex XN-V Series may distinguish NRBCs and leukocytes; however, analytical errors occur. We aimed to investigate cases with discrepant automated and manual NRBC counts, and to evaluate reasons for analytical errors. Data from samples with increased NRBCs were collected retrospectively and compared with manual counts performed on blood films using Spearman's correlation, Passing-Bablok agreement analysis, and Bland-Altman comparisons. Precision of the automated method and interobserver agreement of manual counts were evaluated. Cases with discrepant results were investigated. Agreement between the methods was good at ≤1NRBC ×109 /L in dogs and cats, and inadequate at ≥1NRBC ×109 /L. The automated method demonstrated a negative proportional difference to the manual method. Precision was good for the automated method (overall CV 7.1%) and interobserver agreement for the manual method was poor overall (mean CV 27.3%, range 0%-106.1%). Inaccuracies in NRBC enumeration by the automated method occurred with high hematocrits, the mergence of the cell fragments and leukocyte clouds, and the presence of earlier erythroid precursors. NRBC enumeration by the WNR channel on the Sysmex XN-1000 V is precise and has good agreement with manual counts in canine and feline blood samples at ≤1NRBC ×109 /L. Manual film review is indicated for samples with ≥1NRBC ×109 /L, earlier erythroid precursors, samples from greyhounds and dehydrated patients, and if gating errors are noted.

Candida albicans Adhesins Als1 and Hwp1 Modulate Interactions with Streptococcus mutans.

Candida albicans and Streptococcus mutans are known to synergistically interact with each other in the oral cavity. For example, glucosyltransferase B (GtfB), secreted by S. mutans, can bind to the C. albicans cell surface, promoting dual-species biofilm formation. However, the fungal factors mediating interactions with S. mutans are unknown. The C. albicans adhesins Als1, Als3, and Hwp1 are key players in C. albicans single-species biofilm formation, but their roles, if any, in interacting with S. mutans have not been assessed. Here, we investigated the roles of the C. albicans cell wall adhesins Als1, Als3, and Hwp1 on forming dual-species biofilms with S. mutans. We assessed the abilities of the C. albicans wild-type als1Δ/Δ, als3Δ/Δ, als1Δ/Δ/als3Δ/Δ, and hwp1Δ/Δ strains to form dual-species biofilms with S. mutans by measuring optical density, metabolic activity, cell enumeration, biomass, thickness, and architecture of the biofilms. We observed that the C. albicans wild-type strain formed enhanced dual-species biofilms in the presence of S. mutans in these different biofilm assays, confirming that C. albicans and S. mutans synergistically interact in the context of biofilms. Our results reveal that C. albicans Als1 and Hwp1 are major players in interacting with S. mutans, since dual-species biofilm formation was not enhanced when the als1Δ/Δ or hwp1Δ/Δ strains were cultured with S. mutans in dual-species biofilms. Als3, however, does not seem to play a clear role in interacting with S. mutans in dual-species biofilm formation. Overall, our data suggest that the C. albicans adhesins Als1 and Hwp1 function to modulate interactions with S. mutans and could be potential targets for future therapeutics.

Evaluation of Semen Self-Sampling Yield Predictors and CTC Isolation by Multi-Color Flow Cytometry for Liquid Biopsy of Localized Prostate Cancer.

Liquid biopsy (LB) for prostate cancer (PCa) detection could represent an alternative to biopsy. Seminal fluid (SF) is a source of PCa-specific biomarkers, as 40% of ejaculate derives from the prostate. We tested the feasibility of an SF-based LB by evaluating the yield of semen self-sampling in a cohort of >750 patients with clinically localized PCa. The overall SF collection yield was 18.2% (39% when considering only compliant patients), with about a half of the patients (53.15%) not consenting to SF donation. Independent favorable predictors for SF collection were younger age and lower prostate volume. We implemented a protocol to enrich prostate-derived cells by multi-color flow cytometry and applied it on SF and urine samples from 100 patients. The number of prostate-enriched cells (SYTO-16+ PSMA+ CD45-) was variable, with higher numbers of cells isolated from SF than urine (p value < 0.001). Putative cancer cells (EpCAMhigh) were 2% of isolated cells in both specimens. The fraction of EpCAMhigh cells over prostate-enriched cells (PSMA+) significantly correlated with patient age in both semen and urine, but not with other clinical parameters, such as Gleason Score, ISUP, or TNM stage. Hence, enumeration of prostate-derived cells is not sufficient to guide PCa diagnosis; additional molecular analyses to detect patient-specific cancer lesions will be needed.