Rapid Flow Cytometry Research Articles

Performance of Flow Cytometry-Based Rapid Assay in Detection of Carbapenemase-Producing Enterobacterales.

Carbapenemase-producing Enterobacterales are increasingly being recognized in nosocomial infections. The performance of a flow cytometry-based rapid assay for their detection and differentiation was evaluated. This is a disruptive phenotypic technology, phenotypic and growth-independent, that searches for the lesions produced by drugs acting on cells after a short incubation time. Overall, 180 Gram-negative bacteria were studied, and results were compared with those obtained molecularly by PCR and phenotypically by 'KPC, MBL and OXA-48 Confirm Kit'. This phenotypic method was used as reference for comparison purposes. Susceptibility to carbapenems (imipenem, meropenem, and ertapenem) was determined by standard broth microdilution. Overall, 112 isolates (62.2%) were carbapenemase producers, 41 KPCs, 36 MβLs, and 31 OXA-48, and 4 strains were KPC + MβL co-producers. Sixty-eight isolates were carbapenemase-negative. The percentage of agreement, sensitivity, and specificity were calculated according to ISO 20776-2:2021. The FASTinov assay showed 97.7% agreement with the reference method for carbapenemase detection. Discrepant flow cytometry results were obtained in four isolates compared with both reference and PCR results. The sensitivity and specificity of this new technology were 95.3% and 98.5%, respectively, for KPCs, 97.6% and 99.3% for MβLs, and 96.9% and 98% for OXA-48 detection. In conclusion, we describe a rapid flow cytometry assay with high accuracy for carbapenemase detection and the differentiation of various carbapenemases, which should impact clinical microbiology laboratories and patient management.

Simple and rapid flow cytometry assay for the detection of malignant epithelial cells in body fluids.

Morphology is routinely used for detecting malignant cells in body fluids, but it has limitations. Recently, flow cytometry (FCM) is used as an effective tool for studying non-haematological malignancies. The main objective of this study is to standardize a simple and rapid FCM test for the detection of malignant epithelial cells in body fluids. Body fluids that had been processed for cytology/cytology and FCM were enrolled in this prospective study. We developed a fluorescent-labelled, monoclonal antibody panel composed of cell surface markers for this FCM assay. We compared the results of cytology/cell block and FCM. A total of 121 fluid samples were studied. Comparing the diagnostic performance of cytology/cell block and FCM, 52 (43%) cases were positive and 60 (49.5%) cases were negative for carcinoma cells by both techniques. Nine cases showed discordant results between the two techniques. Six cases were cytology+ but FCM- and three cases were FCM+ cytology-. Clustered Epithelial Cell Adhesion Molecule (EpCAM)-positive events with high scatter properties were definitive for positive diagnosis by FCM. We studied PD-L1 expression in 13 cases by FCM. Six cases were reported as false negative by this FCM assay due to hypocellularity and lack of EpCAM expression in malignant cells. This FCM assay is simple, easier and cost-effective yielding sensitive results with no inter-observer variability. FCM would become a valuable tool to complement routine diagnostic cytology and reduces misdiagnosis.

Detection of antimicrobial resistance in

Antimicrobial resistance in Neisseria gonorrhoeae compromises gonorrhoea treatment and rapid antimicrobial susceptibility testing (AST) would be valuable. We have developed a rapid and accurate flow cytometry method (FCM) for AST of gonococci. The 2016 WHO gonococcal reference strains, and WHO Q, R and S (n = 17) were tested against seven clinically relevant antibiotics (ceftriaxone, cefixime, azithromycin, spectinomycin, ciprofloxacin, tetracycline and gentamicin). After 4.5 h incubation of inoculated broth, the fluorescent dye Syto™ 9 was added, followed by FCM analysis. After gating, the relative remaining population of gonococci, compared with unexposed growth control samples, was plotted against antimicrobial concentration, followed by non-linear curve regression analysis. Furthermore, the response at one single concentration/tested antibiotic was evaluated with the intention to use as a screening test for detection of resistant gonococci. A dose-dependent response was seen in susceptible isolates for all tested antimicrobials. There was a clear separation between susceptible/WT and resistant/non-WT isolates for ceftriaxone, cefixime, spectinomycin, ciprofloxacin and tetracycline. In contrast, for azithromycin, only high-level-resistant isolates were distinguished, while resistant isolates with MICs of 4 mg/L were indistinguishable from WT (MIC ≤ 1 mg/L) isolates. For gentamicin, all tested 17 isolates were WT and FCM analysis resulted in uniform dose-response curves. Using a single antibiotic concentration and a 50% remaining cell population cut-off, the overall sensitivity and specificity for resistance detection were 93% and 99%, respectively. By providing results in <5 h for gonococcal isolates, FCM-based AST can become a rapid screening method for antimicrobial resistance or antimicrobial susceptibility in gonococci.

A single-step, rapid, and versatile method for simultaneous detection of cell surface glycan profiles using fluorochrome-conjugated lectins.

Cell surface glycans play essential roles in diverse physiological and pathological processes and their assessment has important implications in biomedicine and biotechnology. Here we present a rapid, versatile, and single-step multicolor flow cytometry method for evaluation of cell surface glycan signatures using a panel of selected fluorochrome-conjugated lectins. This procedure allows simultaneous detection of cell surface glycans with a 10-fold reduction in the number of cells required compared with traditional multistep lectin staining methods. Interestingly, we used this one-step lectin array coupled with dimension reduction algorithms in a proof-of-concept application for discrimination among different tumor and immune cell populations. Moreover, this procedure was also able to unveil T-, B-, and myeloid cell subclusters exhibiting differential glycophenotypes. Thus, we report a rapid and versatile lectin cytometry method to simultaneously detect a particular repertoire of surface glycans on living cells that can be easily implemented in different laboratories and core facilities.

Mammary adipocyte flow cytometry as a tool to study mammary gland biology.

The mammary gland is a vital exocrine organ that has evolved in mammals to secrete milk and provide nutrition to ensure the growth and survival of the neonate The mouse mammary gland displays extraordinary plasticity each time the female undergoes pregnancy and lactation, including a sophisticated process of tertiary branching and alveologenesis to form a branched epithelial tree and subsequently milk-producing alveoli. Upon the cessation of lactation, the gland remodels back to a simple ductal architecture via highly regulated involution processes. At the cellular level, the plasticity is characterised by proliferation of mammary cell populations, differentiation and apoptosis, accompanied by major changes in cell function and morphology. The mammary epithelium requires a specific stromal environment to grow, known as the mammary fat pad. Mammary adipocytes are one of the most prominent cell types in the fat pad, but despite their vast proportion in the tissue and their crucial interaction with epithelial cells, their physiology remains largely unknown. Over the past decade, the need to understand the properties and contribution of mammary adipocytes has become more recognised. However, the development of adequate methods and protocols to study this cellular niche is still lagging, partially due to their fragile nature, the difficulty of isolating them, the lack of reliable cell surface markers and the heterogenous environment in this tissue, which differs from other adipocyte depots. Here, we describe a new rapid and simple flow cytometry protocol specifically designed for the analysis and isolation of mouse mammary adipocytes across mammary gland developmental stages.

The Association of Low CD4 Expression on Monocytes and Low CD8+ T-Cell Count at Hospital Admission Predicts the Need for Mechanical Ventilation in Patients With COVID-19 Pneumonia: A Prospective Monocentric Cohort Study.

Prospective observational monocentric cohort study. A university-affiliated hospital in Marseille, France. Thirty patients presenting with laboratory-confirmed COVID-19 pneumonia were enrolled within the first 48 hours of hospital admission and compared with 18 healthy controls. None. Whole-blood leukocytes were immunophenotyped with a rapid and simplified one-step flow cytometry method. Thirty-eight immune and five laboratory parameters were compared first between COVID-19 patients and controls and then between the COVID-19 patients who received or not MV during their stays. The variables that significantly discriminated MV from non-MV patients in univariate analysis were entered into a multiple stepwise logistic regression analysis. The COVID-19 patients were predominantly male (87%), aged 61 years (50-71 yr), and 93% received early corticosteroid therapy. Sixteen patients (53%) were managed with noninvasive respiratory support, and 14 (47%) required MV. Compared with controls, COVID-19 patients were characterized by an immune signature featuring: 1) decreased HLA-DR expression on monocytes; 2) reduced basophils, eosinophils, T-cells, NK cells, and nonclassical monocyte count; and 3) up regulation of CD169 on monocytes, CD64 on neutrophils, the adhesion/migration markers (CD62L and CD11b), and the checkpoint inhibitor CD274 on myeloid cells. Among the COVID-19 patients, those who received MV had lower level of CD4 and HLA-DR on monocytes, lower CD8+ T-cell count, and higher lactate dehydrogenase at hospital admission. In multivariate analysis, only CD4 on monocytes (p = 0.032) and CD8+ T-cell count (p = 0.026) were associated with MV requirement. The model combining these two variables provided an area under curve of 0.97 (95% CI, 0.83-0.99). The association of low CD4 on monocytes and low CD8+ T-cell count at hospital admission was highly predictive of the need for MV in hospitalized patients with COVID-19 pneumonia.

Validation of a rapid potency assay for cord blood stem cells using phospho flow cytometry: The IL-3-pSTAT5 assay.

Public cord blood banks (CBBs) are required to measure cord blood units (CBUs) potency before their release, allowing for the identification of units that may be unsuitable for haematopoietic transplantation. We have developed a rapid flow cytometry assay based on the measurement of STAT-5 phosphorylation of CD34+ stem cells in response to IL-3 stimulation. To adapt the assay from a research setting to its implementation within our CBB regulated operations, we proceded with a full method validation and a correlation comparison of the IL-3-pSTAT5 assay results with the colony-forming unit assay (CFU) results. A total of 60 CBUs cryopreserved in vials were analysed by flow cytometry to determine the sensitivity, specificity, intra-assay precision, robustness, reproducibility, and inter-laboratory agreement of the assay. The CFU assay was also done on the same samples for comparison purposes. The assay threshold was established at 50% CD34+CD45+pSTAT5+, which provides a 100% sensitivity and a 98.3% specificity. An average intra-assay CV of 7.3% was determined. All results met our qualitative results acceptance criteria regarding the inter-user and inter-laboratory agreements, IL-3 stimulation time, post-thaw incubation delay and staining time. The IL-3-pSTAT5 assay results correlated well with the total CFU determined using the CFU assay (r2 = 0.82, n= 56). This study shows that our rapid flow cytometry assay can be successfully validated and that the potency data obtained display good sensitivity, specificity and robustness. These results demonstrate the feasibility of implementing this assay within CBB operations, as a validated potency assay.

A novel, rapid and sensitive flow cytometry method reveals degradation of promoter proximal paused RNAPII in the presence and absence of UV.

RNA polymerase II (RNAPII) is emerging as an important factor in DNA damage responses, but how it responds to genotoxic stress is not fully understood. We have developed a rapid and sensitive flow cytometry method to study chromatin binding of RNAPII in individual human cells through the cell cycle. Indicating enhanced transcription initiation at early timepoints, levels of RNAPII were increased at 15–30min after UV-induced DNA damage. This was particularly evident for the S5 phosphorylated form of RNAPII (pRNAPII S5), which is typically associated with promoter proximal pausing. Furthermore, degradation of pRNAPII S5 frequently occurs, as its levels on chromatin were strongly enhanced by the proteasome inhibitor MG132 with and without UV. Remarkably, inhibiting pause release with 5,6-dichloro-1-beta-ribo-furanosyl benzimidazole (DRB) further promoted UV-induced degradation of pRNAPII S5, suggesting enhanced initiation may lead to a phenomenon of ‘promoter proximal crowding’ resulting in premature termination via degradation of RNAPII. Moreover, pRNAPII S2 levels on chromatin were more stable in S phase of the cell cycle 2h after UV, indicating cell cycle specific effects. Altogether our results demonstrate a useful new method and suggest that degradation of promoter proximal RNAPII plays an unanticipated large role both during normal transcription and after UV.

Improved diagnostics of “concealed” metastases in patients with сТ1–2N0М0 oral squamous cell carcinoma

Introduction. Oral squamous cell carcinoma is associated with a high risk of regional metastasis even in early stages (T1–2N0M0). Morphological examination reveals concealed metastases in 20 % of removed clinically unaffected lymph nodes.Objective – to evaluate the effectiveness of flow cytometry for detection of concealed metastases in sentinel lymph nodes as an indication for cervical lymph node dissection in patients with stage сT1–2N0M0 oral squamous cell carcinoma.Materials and methods. This study included 27 patients, including 13 patients (48.1 %) with stage T1N0M0 cancer and 14 patients with stage T2N0M0 cancer (51.9 %). At the first stage, all participants underwent primary tumor removal and cervical lymph node dissection with sentinel lymph node examination. After mobilization, half of these lymph nodes was used for routine morphological examination followed by immunohistochemical examination, while the second half of them was analyzed using flow cytometry. Then we compared the results obtained.Results. We examined 46 removed sentinel lymph nodes from 27 patients. Conventional histological examination revealed 4 concealed metastases (8.7 %). Polymerase chain reaction allowed us to detect another 16 metastases (37.8 %). Levels I, II, and III lymph nodes were most frequently affected. Flow cytometry demonstrated that 20 % of patients with a &lt;4 mm invasion had concealed metastases, whereas patients with a 4 to 8 mm invasion had concealed metastases in 60 % of cases. Median follow-up time was 20.4 ± 11.7 months (range: 2.2 to 42.5 months; median 19.5 months). All participants were alive during this time. Seventeen out of 27 patients with stage T1–2N0M0 disease (63 %) were found to have concealed metastases, while the remaining ten patients (37 %) had no metastasis. One patient with stage T1N0M0 oral floor squamous cell carcinoma underwent the removal of the primary tumor and sentinel lymph node. Morphological examination and flow cytometry showed no metastatic lesions. After 14.8 months, the patient developed ipsilateral regional CN3 metastasis. The three-year progression-free survival rate was 94.7 ± 5.1 %.Conclusion. Highly sensible and rapid flow cytometry can become the method of choice in the diagnosis of metastases and deciding on cervical lymph node dissection in patients with cN0 disease. The study is still ongoing.

Rapid potency assessment of autologous peripheral blood stem cells by intracellular flow cytometry: the PBSC-IL-3-pSTAT5 assay

Background aimsThe current gold standard for stem cell product potency assessment, the colony-forming unit (CFU) assay, delivers results that are difficult to standardize and requires a substantial amount of time (up to 14 days) for cellular growth. Recently, the authors developed a rapid (<24 h) flow cytometry assay based on the measurement of intracellular phosphorylated STAT5 (pSTAT5) in CD34+ cord blood stem and progenitor cells in response to IL-3 stimulation. The present work presents a novel adaptation of the protocol for use with autologous peripheral blood stem cells (PBSCs) and a performance comparison with the CFU assay. MethodsThe flow cytometry intracellular staining assay was optimized for PBSCs, and patient samples were analyzed using the PBSC-IL-3-pSTAT5 and CFU assays. Warming events were also simulated to emulate impaired potency products. ResultsOptimization led to minor protocol adjustments, such as removal of the red blood cell lysis step, the addition of a formaldehyde fixation step and an increase in anticoagulant concentration. The PBSC-IL-3-pSTAT5 assay discriminated between normal and impaired samples and identified 100% (18 of 18) of the impaired samples, thus showing better specificity than the CFU assay. ConclusionsThe updated IL-3-pSTAT5 potency assay has several important advantages, such as accelerating the release of autologous stem cell products and enabling the detection of potentially impaired products. The assay could also be used to rapidly assess the potency of any cryopreserved allogeneic stem cell product, such as those processed during the coronavirus disease 2019 pandemic.

Quantitative Evaluation of Proliferative Potential Using Flow Cytometry Reveals Intratumoral Heterogeneity and Its Relevance to Tumor Characteristics in Vestibular Schwannomas.

This study sought to explore the existence and clinical significance of intratumoral heterogeneity of proliferative potential in vestibular schwannoma (VS). Rapid intraoperative flow cytometry was utilized with raw samples to measure the proliferative ability of VS. The proliferation index (PI) was defined as the ratio of the number of cells with greater than normal DNA content to the total number of cells. A total of 66 specimens (26 from the intrameatal portion and 40 from the cisternal portion) were obtained from 34 patients with VS. There was a moderate correlation between the PI and MIB-1 labelling index values (R = 0.57, p < 0.0001). In contrast, the patterns of heterogeneity, represented by the proportion of intrameatal PI to cisternal PI, were associated with tumor size (p = 0.03). In addition, preoperative hearing tended to be poor in cases where the intrameatal PI was higher than the cisternal PI (p = 0.06). Our data demonstrated the presence of intratumoral heterogeneity of proliferative potential in VS and its relationship with tumor characteristics. The results of this study may advocate the resection of the intrameatal portion of large VSs treated with planned subtotal resection, especially in cases of poor preoperative hearing function.

MICy: a Novel Flow Cytometric Method for Rapid Determination of Minimal Inhibitory Concentration.

ABSTRACTEarly initiated adequate antibiotic treatment is essential in intensive care. Shortening the length of antibiotic susceptibility testing (AST) can accelerate clinical decision-making. Our objective was to develop a simple flow cytometry (FC)-based AST that produces reliable results within a few hours. We developed a FC-based AST protocol (MICy) and tested it on six different bacteria strains (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis) in Mueller-Hinton and Luria–Bertani broth. We monitored the bacterial growth by FC to define the optimal time of AST. All bacteria were tested against 12 antibiotics and the MIC values were compared to microdilution used as reference method. McNemar and Fleiss’ kappa inter-observer tests were performed to analyze the bias between the two methods. Susceptibility profiles of the two methods were also compared. We found that FC is able to detect the bacterial growth after 4-h incubation. The point-by-point comparison of MICy and microdilution resulted in exact match above 87% (2642/3024) of all measurements. The MIC values obtained by MICy and microdilution agreed over 80% (173/216) within ±1 dilution range that gives a substantial inter-observer agreement with weighted Fleiss’ kappa. By using the EUCAST clinical breakpoints, we defined susceptibility profiles of MICy that were identical to microdilution in more than 92% (197/213) of the decisions. MICy resulted 8.7% major and 3.2% very major discrepancies. MICy is a new, simple FC-based AST method that produces susceptibility profile with low failure rate a workday earlier than the microdilution method.IMPORTANCE MICy is a new, simple and rapid flow cytometry based antibiotic susceptibility testing (AST) method that produces susceptibility profile a workday earlier than the microdilution method or other classical phenotypic AST methods. Shortening the length of AST can accelerate clinical decision-making as targeted antibiotic treatment improves clinical outcomes and reduces mortality, duration of artificial ventilation, and length of stay in intensive care unit. It can also reduce nursing time and costs and the spreading of antibiotic resistance. In this study, we present the workflow and methodology of MICy and compare the results produced by MICy to microdilution step by step.

IOTG-03. Rapid intraoperative flow cytometry of brain tumor useful for surgical decision-making

Abstract Flow cytometry is a measuring device frequently used in basic research, we have developed a fully automatic flow cytometer device that performs cell isolation, staining of cell nuclei, and measurement for intraoperative diagnosis. Placing a small specimen in a reagent containing PI calculates, the total number of cells, the DNA histogram, and the Malignancy Index (MI), which means in the proliferation phase, the ratio of the number of cells (S, G2, M) / total number of cells. The major feature of this device is that calculating takes only 10 minutes by thoroughly reviewing the process. We here introduce our study of this device, rapid diagnosis, WHO grading, prognosis in glioma, and differentiation from malignant lymphoma (PCNSL), then how to use it in decision making in the operation. In the analysis of 323 gliomas, the threshold between the peripheral brain and the tumor was 7%, and the MI value was correlated with WHO grade (II 13%, III 35%, IV 47%) (J Neurosurg 2013). A correlation was also found between the MI value and the number of residual tumors (Brain Tumor Path 2018). Furthermore, it was also useful for differentiating from PCNSL with many S-phase cells (World Neurosurg 2018). In addition, the relationship between the presence of DNA aneuploidy and poor prognosis in 102 grade II patients (Clin Neuro Neurosurg 2018), and the high MI in 102 grade IV patients showed a good prognosis and an inverse correlation with expectations (Neurosurg 2018). Intraoperative flow cytometry has enabled various intraoperative decision-making support by converting intraoperative histology, which was an analog-like transmission, into digitized histological information. In the future, the development of this research makes expectations for more accurate diagnosis and prediction by artificial intelligence and the development of other departments.

Current trends in colistin susceptibility testing methods in enteric Gram-negative bacilli and other non-fermentative pathogens

Polymyxins, an age-old sort of cationic polypeptide antibiotic, are regarded as final-resort drugs in handling infections caused by multidrug-resistant Gram-negative bacteria. An exact assessment of colistin susceptibility is crucial due to increasing colistin resistance and absence of other antibiotics in curing multidrug resistance pathogens. Both Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing have investigated colistin broth microdilution and offer particular recommendations, but this method has a challenging perform in routine daily practice. It is time consuming and requires ≥48 h. In this issue, several alternative rapid tests are developed such as ResaPolymyxin Acinetobacter/Pseudomonas NP. The sensitivity and specificity of 100% for the Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test to Enterobacterales have been reported. Colistin broth disk elution test and colistin agar test are another novel potential methods that were validated by the Clinical laboratory standard institutes subcommittee for colistin screening in the Enterobacterales and Pseudomonas aeruginosa. In addition, recently, Rapid flow cytometry (FASTinov) method was introduced which requires only 2 h compared with conventional methods. This review will take a look at current trends on methods for colistin resistance measurements.

Results of a Multicenter Evaluation of a Rapid Flow Cytometry Potency Assay for Cryopreserved Cord Blood Units: A BEST Collaborative Study

Results of a Multicenter Evaluation of a Rapid Flow Cytometry Potency Assay for Cryopreserved Cord Blood Units: A BEST Collaborative Study

Cryptococcal antigen carriage among HIV infected children aged 6 months to 15 years at Laquintinie Hospital in Douala

Up to 15% of deaths of people living with HIV is attributable to meningeal cryptococcosis, with nearly 75% occuring in sub-Saharan Africa. Although rare in children, it is a major cause of morbidity and mortality in people living with HIV. A strong association between cryptococcal antigenemia and the development of meningeal cryptococcosis has been shown in adults. Thus, in 2018, the World Health Organization published an updated version of its guidelines for the diagnosis, prevention and management of cryptococcal infection in adults, adolescents and the HIV-infected child. To determine the prevalence of cryptococcal antigenemia and to identify its determinants in children infected with HIV. An analytical cross-sectional study was carried out at the approved treatment center of Laquintinie hospital in Douala over a period of 4 months. Children were recruited consecutively after informed parental consent. Cryptococcal antigenemia and CD4 assay were performed using a Cryptops® immunochromatographic rapid diagnostic test and flow cytometry, respectively. The data collected included the socio-demographic, clinical and paraclinical variables of the children, as well as their antecedents. Data analysis was performed using Epiinfo software version 3.1 and SPSS 21.0. The significance threshold was set at 5%. A total of 147 children were enrolled. The mean age was 9.8 ± 4.09 years. The majority were on antiretroviral therapy (142, 96.60%). Only 13 (8.80%) were in severe immunosuppression. No child showed signs of meningeal cryptococcosis. The prevalence of cryptococcal antigenemia was 6.12%. Severe immunosuppression [OR: 10.03 (1.52-65.91), p = 0.016] and contact with pigeons [OR: 9.76 (1.14-83.65), p = 0.037] were independent factors significantly associated with the carriage of the cryptococcal antigen. We recommend screening for cryptococcal antigenemia and routine treatment with fluconazole of all HIV positive children with cryptococcal antigen whether symptomatic or not.

Demonstration of a Single-Parameter, Rapid Flow Cytometry Assay in Assessing Natural Killer Cell Cytotoxic Function

Assessing cytotoxic function of immune effector cells, such as natural killer (NK) cells, is necessary to determine their potency. Various cytotoxicity assays measuring target cell death have been reported, however these assays typically feature a number of limitations that curb their broad applicability. Quantitative assays of NK cell killing traditionally measure the release of chromium or a fluorescent dye from labelled target cells. These assays typically show low reproducibility and often underestimate cell death for many cell types. Recent strategies aimed at ensuring manufacture of high NK cell doses commonly include NK cell co-culture with antigen presenting cells as feeder cells for their expansion. Such expansion platforms result in a final NK cell product with a high content of dead or dying feeder cells. This end product also complicates the accurate measurement of target cell death in NK cell cytotoxicity assays. Here we report a rapid flow cytometry assay for quantifying target cell killing after single-color labeling of the target cells prior to co-incubation with NK cells. In this assay, target cells are first labeled with calcein-AM (CAM), a green fluorescent dye. CAM-labeled target cells are then co-incubated with feeder cell expanded-NK cells in 96-well plates using various NK cell-to-target ratios. After 4 hours of NK cell-target cell co-incubation, a fixed volume of the cell suspension was analyzed using a flow cytometer capable of reading 96-well plates. The live, CAM-labeled target cells were gated and quantitated for all the treatment conditions to determine the number of CAM-labeled target cells present per fixed volume.Using this assay, we observed highly consistent cytotoxicity results against both suspension and adherent cell targets. The accuracy of this assay was also demonstrated by NK cell dose-dependent responses. Importantly, the presence of dead feeder cells in the NK cell suspension does not interfere with the analysis of this assay. In addition, the gating strategy is simple, consistent, and the analysis between treatment groups does not require cumbersome calculations or compensation that could introduce variability. Assessment of target cell death was also shown to be more reliable and consistent when compared with the measurement of CAM release using a microplate reader. The latter assay has previously been shown to be highly variable with inconsistent results depending on target cell and CAM dilution. Our flow cytometry assay consistently identifies live cells, avoiding errors due to variability in dye intake and dye release detection. DisclosuresNo relevant conflicts of interest to declare.

An Optimized and Standardized Rapid Flow Cytometry Functional Method for Heparin-Induced Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a thrombocytopenia caused by heparin and mediated by an atypical immune mechanism leading to a paradoxical high thrombotic risk, associated with severe morbidity or death. The diagnosis of HIT combines a clinical scoring of pretest probability and laboratory testing. First-line routine tests are antigen binding assays detecting specific antibodies. The most sensitive of these tests have a high HIT-negative predictive value enabling HIT diagnosis to be ruled out when negative. However, HIT-positive predictive value is low, and a functional assay evaluating the pathogenicity of the antibodies should be performed to exclude false-positive results. In contrast to screening assays, functional assays are highly specific but technically challenging, and are thus performed in referral laboratories, where platelet activation is detected using radioactive serotonin (serotonin release assay, SRA) or visually (heparin-induced platelet activation, HIPA). Flow cytometry is a possible alternative. It is, however, currently not widely used, mostly because of the lack of standardization of the published assays. This article describes and discusses the standardization of a HIT flow cytometry assay (HIT-FCA) method, which subsequently led to the development and commercialization of a CE-marked assay (HIT Confirm®, Emosis, France) as a suitable rapid HIT functional test.

CD169 and CD64 could help differentiate bacterial from CoVID-19 or other viral infections in the Emergency Department.

The identification of a bacterial, viral, or even noninfectious cause is essential in the management of febrile syndrome in the emergency department (ED), especially in epidemic contexts such as flu or CoVID‐19. The aim was to assess discriminative performances of two biomarkers, CD64 on neutrophils (nCD64) and CD169 on monocytes (mCD169), using a new flow cytometry procedure, in patients presenting with fever to the ED during epidemics. Eighty five adult patients presenting with potential infection were included during the 2019 flu season in the ED of La Timone Hospital. They were divided into four diagnostic outcomes according to their clinical records: no‐infection, bacterial infection, viral infection and co‐infection. Seventy six patients with confirmed SARS‐CoV‐2 infection were also compared to 48 healthy volunteers. For the first cohort, 38 (45%) patients were diagnosed with bacterial infections, 11 (13%) with viral infections and 29 (34%) with co‐infections. mCD169 was elevated in patients with viral infections, with a majority of Flu A virus or Respiratory Syncytial Virus, while nCD64 was elevated in subjects with bacterial infections, with a majority of Streptococcus pneumoniae and Escherichia coli. nCD64 and mCD169 showed 90% and 80% sensitivity, and 78% and 91% specificity, respectively, for identifying patients with bacterial or viral infections. When studied in a second cohort, mCD169 was elevated in 95% of patients with SARS‐CoV‐2 infections and remained at normal level in 100% of healthy volunteers. nCD64 and mCD169 have potential for accurately distinguishing bacterial and acute viral infections. Combined in an easy and rapid flow cytometry procedure, they constitute a potential improvement for infection management in the ED, and could even help for triage of patients during emerging epidemics.

Rapid Isolation and Flow Cytometry Analysis of Murine Intestinal Immune Cells after Chemically Induced Colitis

Rapid Isolation and Flow Cytometry Analysis of Murine Intestinal Immune Cells after Chemically Induced Colitis