Enumeration Of Cells Research Articles

Efficient diagnosis of hematologic malignancies using bone marrow microscopic images: A method based on MultiPathGAN and MobileViTv2

Background and objectivesHematologic malignancies, including the associated multiple subtypes, are critically threatening to human health. The timely detection of malignancies is crucial for their effective treatment. In this regard, the examination of bone marrow smears constitutes a crucial step. Nonetheless, the conventional approach to cell identification and enumeration is laborious and time-intensive. Therefore, the present study aimed to develop a method for the efficient diagnosis of these malignancies directly from bone marrow microscopic images. MethodsA deep learning-based framework was developed to facilitate the diagnosis of common hematologic malignancies. First, a total of 2033 microscopic images of bone marrow analysis, including the images for 6 disease types and 1 healthy control, were collected from two Chinese medical websites. Next, the collected images were classified into the training, validation, and test datasets in the ratio of 7:1:2. Subsequently, a method of stain normalization to multi-domains (stain domain augmentation) based on the MultiPathGAN model was developed to equalize the stain styles and expand the image datasets. Afterward, a lightweight hybrid model named MobileViTv2, which integrates the strengths of both CNNs and ViTs, was developed for disease classification. The resulting model was trained and utilized to diagnose patients based on multiple microscopic images of their bone marrow smears, obtained from a cohort of 61 individuals. ResultsMobileViTv2 exhibited an average accuracy of 94.28% when applied to the test set, with multiple myeloma, acute lymphocytic leukemia, and lymphoma revealed as the three diseases diagnosed with the highest accuracy values of 98%, 96%, and 96%, respectively. Regarding patient-level prediction, the average accuracy of MobileViTv2 was 96.72%. This model outperformed both CNN and ViT models in terms of accuracy, despite utilizing only 9.8 million parameters. When applied to two public datasets, MobileViTv2 exhibited accuracy values of 99.75% and 99.72%, respectively, and outperformed previous methods. ConclusionsThe proposed framework could be applied directly to bone marrow microscopic images with different stain styles to efficiently establish the diagnosis of common hematologic malignancies.

A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing.

CRISPR/Cas systems are some of the most promising tools for therapeutic genome editing. The use of these systems is contingent on the optimal designs of guides and homology-directed repair (HDR) templates. While this design can be achieved in silico, validation and further optimization are usually performed with the help of reporter systems. Here, we describe a novel reporter system, termed BETLE, that allows for the fast, sensitive, and cell-specific detection of genome editing and template-specific HDR by encoding multiple reporter proteins in different open-reading frames. Out-of-frame non-homologous end joining (NHEJ) leads to the expression of either secretable NanoLuc luciferase, enabling a highly sensitive and low-cost analysis of editing, or fluorescent mTagBFP2, allowing for the enumeration and tissue-specific localization of genome-edited cells. BETLE includes a site to validate CRISPR/Cas systems for a sequence-of-interest, making it broadly adaptable. We evaluated BETLE using a defective moxGFP with a 39-base-pair deletion and showed spCas9, saCas9, and asCas12a editing as well as sequence-specific HDR and the repair of moxGFP in cell lines with single and multiple reporter integrants. Taken together, these data show that BETLE allows for the rapid detection and optimization of CRISPR/Cas genome editing and HDR in vitro and represents a state-of the art tool for future applications in vivo.

Interstitial cystitis in common variable immunodeficiency: Could it be another autoimmune complication? A descriptive report

IntroductionCommon variable immunodeficiency (CVID) is considered the most common Human Inborn Error of Immunity (HIEI) and involves low IgG and susceptibility to infections. ESID 2014 criteria has expanded diagnostic criteria to include autoimmunity and lymphoproliferation. One less common and not well understood noninfectious complication of CVID is interstitial cystitis (IC), which may be caused by immune dysregulation. We hypothesized that patients with both CVID and IC may have reduced switched memory B cells. We conducted this study using data from United States Immunodeficiency Network (USIDNET) and compared results to patients at our institution to see if there were any variables of interest. Results and DiscussionSeventeen of 1446 patient (1.2%) with CVID in the USIDNET database had a diagnosis of possible interstitial cystitis and all were females. Patients in this group had normal average range for IgG (798 mg/dL) with IVIG replacement, along with normal WBC, platelet, IgM and basic T cell enumeration. B cell phenotyping data was available for only one patient, showing normal memory and switched memory B cells. Data from our institution showed similar normal ranges for all the above labs except one female patient who had low memory and switched memory B cell population and had the mildest IC-related disease course. One male patient had normal memory and switched memory B cell population with mild Tcell lymphopenia, however had the worst IC-related clinical outcome. ConclusionWe failed to observe a strong correlation between low switched memory B cells and IC in patients with CVID. IVIG, despite its immunomodulating effects, also appeared to lack any association. While the severity of IC and male gender for one of our patients may be coincidental, it does raise the suspicion that the phenotype of IC in male CVID patients may be different from their female counterparts. IC may be underreported in USIDNET, leading to limited data and prohibiting any clear causative conclusions we may draw this study. Nevertheless, we believe it is important to raise the awareness of this rare disease, so that we can be cognizant of its heterogeneous manifestations and impacts on morbidity and mortality.

An Optimized Method of Collecting Murine Peripheral Blood and Dilution Correction for Accurate Blood Cell Enumeration.

Accurate measurement of whole blood counts from mice is an essential quantitative tool across the fields of vascular cell biology. In particular, the measurement of platelet counts can be challenging as the process relies upon good phlebotomy technique, the inclusion of a sufficient amount of the appropriate anticoagulant, and very often dilution of the sample to meet the sample volume requirements of an automated analyzer. To minimize sample dilution, blood collection tubes pre-coated with the anticoagulant can be used; however, these are expensive and prone to blood clotting issues. Here, we describe a simple dilution correction method that accurately calculates blood-to-anticoagulant dilutions to generate appropriate volumes for automated blood cell analysis while minimizing blood clotting. We also discuss some simple steps that can be incorporated into blood collection methods to avoid artefacts during blood collection. Blood count data analysis involving volume correction and clot exclusion can significantly reduce variable blood cell count values among healthy untreated littermates. It also detects subtle changes in blood cell counts, mainly of platelets and RBCs in experimental settings, which can be masked in the absence of careful and precise volume correction. Blood count analysis with volume correction precisely determines mouse whole blood cell counts for investigators. The decreased variability in cell count values reduces the number of experimental animals required for meaningful analysis. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: An optimized method of collecting murine peripheral blood and dilution correction for accurate blood cell enumeration.

T-cell chronic active EBV diagnosed in a 65-year-old male presenting as idiopathic hypereosinophilic syndrome

A 65-year-old male from Nigeria, admitted with dehydration and painful oral ulcers, is seen by allergy/immunology for hypereosinophilia.The patient had idiopathic eosinophilia (mean 5,000 cells/uL) ongoing for nine years. There were no evident drug or infectious causes. The eosinophilia peaked at 28,000 cells/µL, accompanied by parotid swelling, pustular skin rash, and elevated serum-IgG4 level (400 mg/dL). Rheumatology evaluated for IgG4-related disease and hypereosinophilic syndrome. This work-up demonstrated elevated IgE (39,000 IU/mL) and tryptase (40 ng/mL) levels. Bone marrow biopsy showed a hypercellular marrow with erythroid hyperplasia and eosinophilia. Submandibular gland, skin, and excisional lymph node biopsies demonstrated IgGsecreting plasma cells, lymphocytic/eosinophilic infiltrates, and lymphoid hyperplasia, respectively. All biopsies stained negative for IgG4. Treatment for idiopathic hypereosinophilia was initiated with corticosteroids, rituximab, and mycophenolate mofetil (MMF). Symptomatic improvement was attained on MMF (1000 mg twice daily) with the addition of mepolizumab (300 mg every 4 weeks).He unfortunately progressed to high fevers with elevated ferritin (7,286 ug/L) and soluble IL-2Ra (31,276.7 pg/mL) levels. He did not meet full criteria for hemophagocytic lymphohistiocytosis and improved after corticosteroids. EBV PCR from blood was >220,000 IU/mL.Dedicated immune work-up showed a non-specific population of lymphocyte-like cells without clear lineage markers (CD2dim/- surface CD3-CD4-CD8-CD7-CD56-CD49d-CD294dim) on peripheral blood flow cytometry, with otherwise normal T, B, and NK cell enumeration. There was reduced cytotoxic killing function of CD8+ T cells and NK cells by chromium-51 release assay. Sorted peripheral blood mononuclear cells demonstrated positive EBV viral load in B cells (1,018,239 IU/mL), T cells (857,418 IU/mL),NK cells (237,459), and myeloid cells (532,641 IU/mL). EBV-encoded RNA was positive within lymph node T cells, most consistent with a diagnosis of T-cell CA-EBV. The Invitae 407 gene immunodeficiency panel was sent; however, a pathogenic variant was not identified. The patient was seen by hematology/oncology with plans for hematopoietic stem cell transplant. Unfortunately, he succumbed to complications from CA-EBV and multiorgan failure.We describe a male with lymphocytic-variant hypereosinophilic syndrome and a longstanding constellation of inflammatory and lymphoproliferative symptoms due to CA-EBV. His course initially eluded his care team and only after careful multidisciplinary discussion was a diagnosis reached.

Cryopreservation of Allogeneic Hematopoietic Cell Products During COVID-19 Pandemic: Graft Characterization and Engraftment Outcomes

The COVID-19 pandemic triggered the deployment of unfamiliar measures to safeguard successful allogeneic hematopoietic cell transplantation (allo-HCT). Among these measures, cryopreservation offered logistical benefits that could outlast the pandemic, including graft availability and timely clinical service. The purpose of this study was to evaluate graft quality and hematopoietic reconstitution in patients transplanted with cryopreserved allogeneic stem cell products during the COVID-19 pandemic. We evaluated 44 patients who underwent allo-HCT using cryopreserved grafts consisting of hematopoietic progenitor cells (HPC) apheresis (A) and bone marrow (BM) products at Mount Sinai Hospital. Comparative analyses of 37 grafts infused fresh during the one-year period preceding the pandemic were performed. Assessment of cellular therapy products included total nucleated cell and CD34+ cell enumeration, viability, and post-thaw recovery. The primary clinical endpoint was the evaluation of engraftment (absolute neutrophil count [ANC] and platelet count) and donor chimerism (presence of CD33+ and CD3+ donor cells) at day +30 and +100 post-transplant. Adverse events related to cell infusion were also analyzed. Patient characteristics were comparable between the fresh and cryopreserved groups with 2 exceptions in the HPC-A cohort: the number of patients in the cryopreserved group that received haploidentical grafts was 6 times that in the fresh group, and the number of patients in the fresh group with a Karnofsky performance score >90 was double that in the cryopreserved group. The quality of HPC-A and HPC-BM products was not affected by cryopreservation, and all grafts met the release criteria for infusion. The pandemic did not affect the time between collection and cryopreservation (median, 24 hours) and time in storage (median, 15 days). Median time to ANC recovery was significantly delayed in recipients of cryopreserved HPC-A (15 vs 11 days, P=.0121), and there was a trend toward delayed platelet engraftment (24 vs 19 days, P=.0712). The delay in ANC and platelet recovery was not observed when only matched graft recipients were compared. Cryopreservation did not affect the ability of HPC-BM grafts to engraft and reconstitute hematopoiesis, and there was no difference in the rates of ANC and platelet recovery. Achievement of donor CD3/CD33 chimerism was not affected by cryopreservation of either HPC-A or HPC-BM products. Graft failure was observed in only 1 case, a recipient of cryopreserved HPC-BM. Three recipients of cryopreserved HPC-A grafts died before ANC engraftment from infectious complications. Remarkably, 22% of our studied population had myelofibrosis, and almost half received cryopreserved HPC-A grafts with no graft failure observed. Finally, patients receiving cryopreserved grafts were at a higher risk of infusion-related adverse events than those receiving fresh grafts. Cryopreservation of allogeneic grafts results in adequate product quality with minimal impact on short-term clinical outcomes, except for an increased risk of infusion-related adverse events. Cryopreservation is a safe option in terms of graft quality and hematopoietic reconstitution with logistical benefits, but additional data are needed to determine long-term outcomes and assess whether this is a suitable strategy for at-risk patients.

Albumin-based solution is the ideal post-thawing suspension medium for cord blood hematopoietic stem cells: A stability and proliferative evaluation.

Cryopreservation and thawing protocols represent key factors for the efficacy of cellular therapy products, such as hematopoietic stem cells (HSCs). While the HSC cryopreservation has already been standardized, the thawing procedures have been poorly studied. This study aimed to evaluate the thawing and washing protocol of cord blood (CB) derived HSCs or the HPC(CB), by selecting the optimal thawing solution and determining CD34+ cells' stability over time. Seven cryopreserved CB products were thawed, washed, and resuspended in three different solutions (10% Dextran40 in NaCl equally prepared with 5% human albumin; 5% human albumin in PBS/EDTA; and normal saline) and stored at 4°C (±2°C). Mononuclear cell (MNC) count, CD45+/CD34+ cell enumeration, and cell viability were tested at 0, 1, 2, 4, 6, 8, 12, 24, 36, and 48 h. The protocol with the selected solution was further validated on additional 10 CB samples. The above parameters and the colony-forming unit (CFU) assay were analyzed at time points 0, 2, 4, 6, and 8 h. The results showed that the 5% human albumin was the most suitable thawing solution. MNCs were stable up to 4 h (p = 0.009), viable CD45+ cells were unstable even at 2 h (p = 0.013), and viable CD34+ cells were stable until 6 h (p = 0.019). The CFU assay proved the proliferative potential up to 8 h, although significantly decreased after 4 h (p = 0.013), and correlated with the viable CD34+ cell counts. We demonstrated that the post-thawed and washed HPC(CB) using 5% human albumin is stable for up to 4 h.

Effect of oil exposure stages on the heat resistance of Salmonella enterica serovar Enteritidis phage type 30 in peanut flour

The oil in low-moisture foods (LMFs) shows protective effects on bacteria during thermal processing. However, the circumstances under which this protective effect strengthens remain unclear. This study aimed to understand which step of the oil exposure to bacterial cells (inoculation, isothermal inactivation, or recovery and enumeration step) in LMFs can enhance their heat resistance. Peanut flour (PF) and defatted PF (DPF) were selected as the oil-rich and oil-free LMF models. Salmonella enterica Enteritidis Phage Type 30 (S. Enteritidis) was inoculated into four designated PF groups representing different oil exposure stages. It was isothermally treated to obtain heat resistance parameters. At a constant moisture content (aw,25°C = 0.32 ± 0.02) and controlled aw,85°C (0.32 ± 0.02), S. Enteritidis exhibited significantly high (p < 0.05) D values in oil-rich sample groups. For instance, the heat resistance values of S. Enteritidis in the PF-DPF and DPF-PF groups were D80°C of 138.22 ± 7.45 min and 101.89 ± 7.82 min; however, the D80°C in the DPF-DPF group was 34.54 ± 2.07 min. The oil addition after the thermal treatment also helped injured bacterial recovery in the enumeration. For instance, the D80°C, D85°C, and D90°C values in the DFF-DPF oil groups were 36.86 ± 2.30, 20.65 ± 1.23, and 7.91 ± 0.52 min, respectively, which were higher than those in the DPF-DPF group at 34.54 ± 2.07, 17.87 ± 0.78, and 7.10 ± 0.52 min. We confirmed that the oil protected S. Enteritidis in PF in all three stages: desiccation process, heat treatment, and recovery of bacterial cells in plates.

Abstract SY30-02: Oncosomes in the early stages of breast cancer

Abstract Comprehensive analysis of cellular, acellular and plasma components of the Liquid Biopsy provides the opportunity to investigate cancer evolution and the interactions with the immune system at the systemic level in the individual patient. This presentation will discuss early findings supporting the relevance of oncosomes, large extracellular vesicles, in early-stage breast cancer and its potential utility in early detection and cell-level staging of the disease when combined with the cellular profiles. Breast cancer (BC) affects 1 in every 8 women in the United States and is currently the most prevalent cancer worldwide. Precise staging at diagnosis and prognosis are essential components for the clinical management of BC patients. We set out to evaluate the feasibility of a next generation liquid biopsy platform to stratify late-stage BC, early-stage BC, and age- and gender matched controls using peripheral blood samples. Utilizing 5 biomarkers, we can identify rare circulating events with epithelial, mesenchymal, endothelial and hematological origin. We detected a higher level of CTCs in late-stage patients, compared to the early-stage and normal donors. We observed more tumor-associated oncosomes in the early-stage, compared to late-stage and the normal donor groups. Overall, we were able to detect reproducible patterns in the enumeration of rare cells and oncosomes of cancer vs. normal donors with an AUC of 99%. An initial concept for cell-level staging with the liquid biopsy was applied to early-stage vs. late-stage BC samples and reached an AUC of 91% allowing for robust stratification. Citation Format: Peter Kuhn. Oncosomes in the early stages of breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr SY30-02.

Microbial communities in freshwater used for hydraulic fracturing are unable to withstand the high temperatures and pressures characteristic of fractured shales.

Natural gas is recovered from shale formations by hydraulic fracturing, a process known to create microbial ecosystems in the deep subsurface. Microbial communities that emerge in fractured shales include organisms known to degrade fracturing fluid additives and contribute to corrosion of well infrastructure. In order to limit these negative microbial processes, it is essential to constrain the source of the responsible micro-organisms. Previous studies have identified a number of potential sources, including fracturing fluids and drilling muds, yet these sources remain largely untested. Here, we apply high-pressure experimental approaches to assess whether the microbial community in synthetic fracturing fluid made from freshwater reservoir water can withstand the temperature and pressure conditions of hydraulic fracturing and the fractured shale environment. Using cell enumerations, DNA extraction and culturing, we show that the community can withstand high pressure or high temperature alone, but the combination of both is fatal. These results suggest that initial freshwater-based fracturing fluids are an unlikely source of micro-organisms in fractured shales. These findings indicate that potentially problematic lineages, such as sulfidogenic strains of Halanaerobium that have been found to dominate fractured shale microbial communities, likely derive from other input sources into the downwell environment, such as drilling muds.

Volumetric imaging of human mesenchymal stem cells (hMSCs) for non-destructive quantification of 3D cell culture growth.

The adoption of cell-based therapies into the clinic will require tremendous large-scale expansion to satisfy future demand, and bioreactor-microcarrier cultures are best suited to meet this challenge. The use of spherical microcarriers, however, precludes in-process visualization and monitoring of cell number, morphology, and culture health. The development of novel expansion methods also motivates the advancement of analytical methods used to characterize these microcarrier cultures. A robust optical imaging and image-analysis assay to non-destructively quantify cell number and cell volume was developed. This method preserves 3D cell morphology and does not require membrane lysing, cellular detachment, or exogenous labeling. Complex cellular networks formed in microcarrier aggregates were imaged and analyzed in toto. Direct cell enumeration of large aggregates was performed in toto for the first time. This assay was successfully applied to monitor cellular growth of mesenchymal stem cells attached to spherical hydrogel microcarriers over time. Elastic scattering and fluorescence lightsheet microscopy were used to quantify cell volume and cell number at varying spatial scales. The presented study motivates the development of on-line optical imaging and image analysis systems for robust, automated, and non-destructive monitoring of bioreactor-microcarrier cell cultures.

Abstract P5-06-01: A Multi-center Clinical Study to Harvest and Characterize Circulating Tumor Cells from Patients with Metastatic Breast Cancer Using the Parsortix® PC1 System in support of FDA clearance

Abstract Background: Circulating tumor cells (CTCs) captured from the blood of cancer patients may serve as a non-invasive surrogate source of tumor material to investigate tumor characteristics in real-time. However, the only FDA-cleared CTC assay is limited to the enumeration of surface marker-defined epithelial cells and not designed for further characterization of the CTCs identified. The Parsortix® PC1 system is a semi-automated microfluidic device capable of capturing and harvesting CTCs from peripheral blood based on cell size and deformability, making it cell-surface marker agnostic. Here, we demonstrate that the Parsortix® PC1 system enables the enrichment and capture of CTCs from the blood of patients with metastatic breast cancer (MBC) and their interrogation using evaluation techniques commonly available in clinical laboratories. Methods: As part of a multicenter clinical trial (NCT03427450), peripheral blood samples from 216 patients with MBC and 205 healthy volunteers (HVs) were prospectively collected at four different clinical sites located throughout the United States. Each subject provided two separate blood samples collected into K2EDTA Vacutainer® tubes to be processed using the Parsortix® PC1 system on the same day. The cells harvested from one of the blood samples collected from each subject by the Parsortix® PC1 system were deposited onto cytology slides using a cytocentrifugation method and stained with Wright-Giemsa reagents using an automated stainer. The stained slides were subjected to cytopathological evaluation by a board-certified pathologist to enumerate CTCs. As proof of principle, cells harvested from the second blood sample were evaluated using one of three additional techniques: molecular profiling by qRT-PCR, RNA sequencing, or cytogenetic analysis of HER2 amplification by FISH. Results: Cytologic examination identified one or more cells as a CTC in 48.5% (95% CI of 41.5 – 55.4%) of the 194 patients with MBC and 9.9% (95% CI of 6.4 – 14.9%) of the 192 HVs. The results from the qRT-PCR evaluation (102 HVs and 74 MBC patients) showed differential expression of cancer-related genes (KRT19, EPCAM, and TWIST1) in the patients with MBC compared to the HVs. Results from the RNA sequencing (53 HVs and 16 MBC patients) showed differential expression of several genes involved in the Kegg Cancer Pathway in the patients with MBC compared to the HVs. The results from the HER2 FISH evaluation (38 HVs and 101 MBC patients) showed that while the majority of the CTC identified had normal HER2/CEP17 ratios, detection of HER2 amplification was possible. Conclusions: The Parsortix PC1 system is capable of capturing and harvesting CTCs from the peripheral blood of patients with MBC. Harvested cells can be evaluated using standard orthogonal methodologies such as gene expression and FISH to identify and characterize CTCs. Based in part on the above results, the FDA granted a De Novo classification request (DEN200062) for the Parsortix PC1 device in May of 2022. Citation Format: Evan Cohen, Gitanjali Jayachandran, Richard Moore, Massimo Cristofanilli, Julie E. Lang, Joseph Khoury, Michael F. Press, Heather McBride, Kyu Kwang Kim, Negar Khazan, Qiang Zhang, Youbin Zhang, Roberta Guzman, Michael C. Miller, James Reuben, Naoto T. Ueno. A Multi-center Clinical Study to Harvest and Characterize Circulating Tumor Cells from Patients with Metastatic Breast Cancer Using the Parsortix® PC1 System in support of FDA clearance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-06-01.

Abstract P4-07-24: Circulating tumor cells enumeration and Health Related Quality of Life of patients treated with first-line chemotherapy for HER2 negative metastatic breast cancer

Abstract Background: In patients with metastatic breast cancer (mBC), Circulating Tumor Cells (CTC) counts have a strong prognostic impact on progression free survival (PFS) and overall survival (OS). Changes 4 weeks after the start of a new line of therapy, inform on treatment efficacy. Despite improvements in systemic treatment, metastatic BC remains mainly uncurable with alteration of health-related quality of life (HRQOL) during the course of the disease. The aim of this work was to assess impact of clinical factors and biological factors as CTC on HRQOL. Methods: The French cohort COMET is a prospective study including first line HER2 negative patients receiving weekly paclitaxel and bevacizumab according to EMA approved combination. The aim of this cohort was to evaluate clinical, biological and radiological parameters associated with patients’ outcome (CTC, CEC, serum markers, ctDNA, pharmacogenomic polymorphisms, metabolomic parameters, visceral fat assessed by initial CTscan, serum estradiol level, and quality of life). HRQOL was assessed at baseline, at every cycle until progression and then every 3 months up to death using the EORTC QLQ-C30 questionnaire and its breast cancer specific module, the EORTC QLQ-BR23. Five dimensions of HRQOL were analyzed for the primary analyses: Global health status (GHS), physical functioning (PF), Emotional functioning (EF), fatigue (FA) and pain (PA). Time until definitive deterioration (TUDD) in HRQOL was defined as the interval between inclusion and the first decrease in HRQOL score ≥ 5 compared to baseline HRQOL score with no further improvement or in case of death. CTC counts were determined using the standard CellSearch system [Menarini Silicon Biosystems]. Results: Out of 510 patients included in COMET study, 432 patients with available HRQOL data were analyzed in this study. At baseline, patients reported a mean score for GHS of 57.6 (SD=22.7), for PF of 75.8 (23.2), for EF of 62.2 (25.8), for FA of 42.2 (29.60) and for PA of 38.1 (31.5). The Median TUDDs for the 5 targeted dimensions was 10.1 months [7.5-16.9] for GHS, 6.1 months [4.1-8.9] for PF, 21.6 [18.7-31.2] for EF, 10.8 [6.2-16.6] for FA and 13.6[10.1-22.5] months for PA. CTC counts were available in 261 patients at base line and in 229 patients after 4 weeks of treatment, before second cycle of chemotherapy. CTC high count was independent of main clinical and biological characteristics except lobular subtype. We confirmed the poor outcome of patients with high CTC count at base line and after one cycle of treatment with the threshold of &amp;gt; 4CTC/7.5 ml of blood. Out of the 5 dimensions of HRQOL, TUDD of EF was significantly correlated with a high CTC level at base line (p=0.0262) and even more with still an elevated count of CTC after one cycle of chemotherapy(p=0.0137). There was no association of CTC with the other dimensions of HRQOL. Conclusion: This is the first study ever reporting an analysis of QoL and CTC. We observed an association of high CTC count with one component of HRQOL scale. This suggests that CTC could be complementary to clinical factors that could influence HRQOL in HER2 negative metastatic BC treated with first line chemotherapy. Citation Format: Jean-Yves Pierga, Oumar Billa, Sandrine Dabakuyo, Jérôme Lemonnier, Frédérique Berger, Olivier Trédan, William Jacot, Anthony Gonçalves, Marc Debled, Christelle Levy, Christelle Jouannaud, Marie-Ange Mouret-Reynier, Jean-Marc Ferrero, Florence Dalenc, Fatima-Zohra Toumi, Franck Bonnetain, Francois-Clement Bidard, Shufang Renault. Circulating tumor cells enumeration and Health Related Quality of Life of patients treated with first-line chemotherapy for HER2 negative metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-24.

Lattice Enumeration with Discrete Pruning: Improvements, Cost Estimation and Optimal Parameters

Lattice enumeration is a linear-space algorithm for solving the shortest lattice vector problem (SVP). Extreme pruning is a practical technique for accelerating lattice enumeration, which has a mature theoretical analysis and practical implementation. However, these works have yet to be applied to discrete pruning. In this paper, we improve the discrete pruned enumeration (DP enumeration) and provide a solution to the problem proposed by Léo Ducas and Damien Stehlé regarding the cost estimation of discrete pruning. We first rectify the randomness assumption to more precisely describe the lattice point distribution of DP enumeration. Then, we propose a series of improvements, including a new polynomial-time binary search algorithm for cell enumeration radius, a refined cell-decoding algorithm and a rerandomization and reprocessing strategy, all aiming to lift the efficiency and build a more precise cost-estimation model for DP enumeration. Based on these theoretical and practical improvements, we build a precise cost-estimation model for DP enumeration by simulation, which has good accuracy in experiments. This DP simulator enables us to propose an optimization method of calculating the optimal parameters of DP enumeration to minimize the running time. The experimental results and asymptotic analysis both show that the discrete pruning method could outperform extreme pruning, which means that our optimized DP enumeration might become the most efficient polynomial-space SVP solver to date. An open-source implementation of DP enumeration with its simulator is also provided.

Longitudinal analysis of mucosa-associated invariant T cells in sepsis reveals their early numerical decline with prognostic implications and a progressive loss of antimicrobial functions.

Sepsis-elicited immunosuppression elevates the risk of secondary infections. We used a clinically relevant mouse model and serial peripheral blood samples from patients to assess the antimicrobial activities of mucosa-associated invariant T (MAIT) cells in sepsis. Hepatic and splenic MAIT cells from B6-MAITCAST mice displayed increased CD69 expression and a robust interferon-γ (IFNγ) production capacity shortly after sublethal cecal ligation and puncture, but not at a late timepoint. Peripheral blood MAIT cell frequencies were reduced in septic patients at the time of intensive care unit (ICU) admission, and more dramatically so among nonsurvivors, suggesting the predictive usefulness of early MAIT cell enumeration. In addition, at ICU admission, MAIT cells from sepsis survivors launched stronger IFNγ responses to several bacterial species compared with those from patients who subsequently died of sepsis. Of note, while low human leukocyte antigen (HLA)-DR+ monocyte frequencies, widely regarded as a surrogate indicator of sepsis-induced immunosuppression, were gradually corrected, the numerical insufficiency of MAIT cells was not resolved over time, and their CD69 expression continued to decline. MAIT cell responses to bacterial pathogens, a major histocompatibility complex-related protein 1 (MR1) ligand, and interleukin (IL)-12 and IL-18 were also progressively lost during sepsis and did not recover by the time of ICU/hospital discharge. We propose that MAIT cell dysfunctions contribute to post-sepsis immunosuppression.

Immunophenotypic Profile of Multiple Myeloma: A Tertiary Care Centre Experience.

Background Immunophenotyping and enumeration of plasma cells (PCs) by flow cytometry are deemed to be prognostically significant. However, PCs enumeration by flow cytometry is challenging owing to discrepancy with morphology and PCs loss during sample processing. Enumeration and differentiation of abnormal plasma cells (APCs) and normal plasma cells (NPCs) is difficult because abnormal antigen expression can be seen in subsets of NPCs. This is particularly true when a limited panel of antibodies are relied upon. Aims and purpose To study the immunophenotypic profile of newly diagnosed multiple myeloma (MM) cases by flow cytometry and evaluate the sensitivities and specificities of individual antigens and combinations. Methods We studied immunophenotype of PCs in newly diagnosed MM cases ( n = 48) and control cases ( n = 10) by a 6-color, 3-tube flow cytometry panel. The sensitivities and specificities of antigens in MM were evaluated and compared with control cases. Results Majority of MM cases ( n = 43) had < 3% NPCs. CD19 was the most sensitive (100%) and CD81 was the most specific marker (100%) for differentiating APCs from NPCs. CD38 MFI came out as a useful marker for APCs identification. In combination, CD19 and CD81 had a higher sensitivity and specificity to detect APCs. Conclusion NPCs may show aberrant antigen expression. A combination of multiple markers including CD81 and CD38 MFI should be used for accurate APC detection.

Circulating Tumor Cell Enumeration for Serial Monitoring of Treatment Outcomes for Locally Advanced Esophageal Squamous Cell Carcinoma.

We aim to reveal the clinical significance and potential usefulness of dynamic monitoring of CTCs to track therapeutic responses and improve survival for advanced ESCC patients. Peripheral blood (PB) (n = 389) and azygos vein blood (AVB) (n = 13) samplings were recruited prospectively from 88 ESCC patients undergoing curative surgery from 2017 to 2022. Longitudinal CTC enumeration was performed with epithelial (EpCAM/pan-cytokeratins/MUC1) and mesenchymal (vimentin) markers at 12 serial timepoints at any of the pre-treatment, all of the post-treatments/pre-surgery, post-surgery follow-ups for 3-year, and relapse. Longitudinal real-time CTC analysis in PB and AVB suggests more CTCs are released early at pre-surgery and 3-month post-surgery into the circulation from the CTRT group compared to the up-front surgery group. High CTC levels at pre-treatments, 1-/3-month post-surgery, unfavorable changes of CTC levels between all post-treatment/pre-surgery and 1-month or 3-month post-surgery (Hazard Ratio (HR) = 6.662, p < 0.001), were independent prognosticators for curative treatment. The unfavorable pre-surgery CTC status was independent prognostic and predictive for neoadjuvant treatment efficacy (HR = 3.652, p = 0.035). The aggressive CTC clusters were more frequently observed in AVB compared to PB. Its role as an independent prognosticator with relapse was first reported in ESCC (HR = 2.539, p = 0.068). CTC clusters and longitudinal CTC monitoring provide useful prognostic information and potential predictive biomarkers to help guide clinicians in improving disease management.

Hybrid machining of P20 die steel masters for the development of polymer-based microfluidic devices to study the effect of surface roughness on bacterial activity

Compared to the traditional machining and finishing process, a hybrid technique consisting of mechanical micro-milling and electropolishing is advantageous in maintaining dimensional accuracy and fine surface quality without compromising the form. This is a cost-effective, less tedious technique that can fabricate high-quality masters for microfluidic devices. P20-die steel masters are fabricated to evaluate the method with this technique. The dimensional accuracy and surface quality of the masters are studied. It is found that the peak-to-valley surface roughness (Rt) of the masters is reduced by 50%. These die steel masters are then used to make polydimethylsiloxane (PDMS)-based microfluidic devices that could be used for particle separation. As biofouling and bacterial growth are undesirable in most of the microfluidic devices, the fabricated PDMS devices are tested for bacterial growth and adhesion. The growth of DH5α Escherichia coli bacteria in the devices fabricated using the electropolished masters is evaluated. Bacterial growth and adhesion are monitored for 0, 4 and 12 h, and it is found that the growth and adhesion in these devices are reduced by 15–20% and 40–50%, respectively, compared to the devices produced using non-electropolished masters. Enumeration of the bacterial cells in the samples flown is carried out by evaluating the optical density of the sample fluids by UV-visible spectroscopy and quantifying the bacterial cells using the McFarland 0.5 standard.

Outcomes and clinicopathologic characteristics associated with disseminated tumor cells in bone marrow after neoadjuvant chemotherapy in high-risk early stage breast cancer: the I-SPY SURMOUNT study.

Disseminated tumor cells (DTCs) expressing epithelial markers in the bone marrow are associated with recurrence and death, but little is known about risk factors predicting their occurrence. We detected EPCAM+/CD45- cells in bone marrow from early stage breast cancer patients after neoadjuvant chemotherapy (NAC) in the I-SPY 2 Trial and examined clinicopathologic factors and outcomes. Patients who signed consent for SURMOUNT, a sub-study of the I-SPY 2 Trial (NCT01042379), had bone marrow collected after NAC at the time of surgery. EPCAM+CD45- cells in 4mLs of bone marrow aspirate were enumerated using immunomagnetic enrichment/flow cytometry (IE/FC). Patients with > 4.16 EPCAM+CD45- cells per mL of bone marrow were classified as DTC-positive. Tumor response was assessed using the residual cancer burden (RCB), a standardized approach to quantitate the extent of residual invasive cancer present in the breast and the axillary lymph nodes after NAC. Association of DTC-positivity with clinicopathologic variables and survival was examined. A total of 73 patients were enrolled, 51 of whom had successful EPCAM+CD45- cell enumeration. Twenty-four of 51 (47.1%) were DTC-positive. The DTC-positivity rate was similar across receptor subtypes, but DTC-positive patients were significantly younger (p = 0.0239) and had larger pretreatment tumors compared to DTC-negative patients (p = 0.0319). Twenty of 51 (39.2%) achieved a pathologic complete response (pCR). While DTC-positivity was not associated with achieving pCR, it was significantly associated with higher RCB class (RCB-II/III, 62.5% vs. RCB-0/I; 33.3%; Chi-squared p = 0.0373). No significant correlation was observed between DTC-positivity and distant recurrence-free survival (p = 0.38, median follow-up = 3.2years). DTC-positivity at surgery after NAC was higher in younger patients, those with larger tumors, and those with residual disease at surgery.

The Impact of Surgery on Circulating Malignant Tumour Cells in Oral Squamous Cell Carcinoma.

The extent to which surgical management of oral squamous cell carcinoma (OSCC) disseminates cancer is currently unknown. To determine changes in numbers of malignant cells released into systemic circulation immediately following tumour removal and over the first seven post-operative days. An observational study from March 2019 to February 2021. This study was undertaken at Queen Mary University Hospital, Hong Kong. Patients with biopsy-proven oral SCC were considered for eligibility. Patients under 18 years of age, pregnant or lactating women and those unable to understand the study details or unable to sign the consent form were excluded. Twenty-two patients were enrolled (12 male and 10 female) with mean age of 65.5 years. Primary tumour management was performed in accord with multi-disciplinary team agreement. Anaesthesia and post-operative care were unaltered and provided in accord with accepted clinical practice. Three types of malignant cells detected in peripheral blood samples were enumerated and sub-typed based on the presence of chromosomal aneuploidy and immunohistochemical characteristics. To test the hypothesis that malignant cells are released by surgery, the numbers of single circulating tumour cells (CTCs), circulating tumour microemboli (CTM) and circulating endothelial cells (CTECs) were recorded pre-operatively, upon tumour removal and the second and seventh post-operative days. Of a potential 88 data collection points, specimens were not obtainable in 12 instances. Tumour removal resulted in a statistically significant increase in CTCs and a non-statistically significant rise in CTMs. CTCs, CTMs and CTECs were detected in the majority of patients up to the seventh post-operative day. Individual patients demonstrated striking increases in post-operative CTCs and CTECs numbers. Surgical management of OSCC has a significant impact on the systemic distribution of cancer cells. Malignant cells persisted post-operatively in a manner independent of recognised staging methods suggesting differences in tumour biology between individuals. Further investigation is warranted to determine whether circulating malignant cell enumeration can be used to refine risk stratification for patients with OSCC.