Query: Flow cytometry

title: Quality and efficiency assessment of six extracellular vesicle isolation methods by nano flow cytometry
abstract: Extracellular vesicles (EVs) have sparked tremendous interest owing to their prominent potential in diagnostics and therapeutics. Isolation of EVs from complex biological fluids with high purity is essential to the accurate analysis of EV cargo. Unfortunately, generally used isolation techniques do not offer good separation of EVs from non EV contaminants. Hence, it is important to have a standardized method to characterise the properties of EV preparations, including size distribution, particle concentration, purity and phenotype. Employing a laboratory built nano flow cytometer (nFCM) that enables multiparameter analysis of single EVs as small as 40 nm, here we report a new benchmark to the quality and efficiency assessment of EVs isolated from plasma, one of the most difficult body fluids to work with. The performance of five widely used commercial isolation kits was examined and compared with the traditional differential ultracentrifugation (UC) Two to four orders of magnitude higher particle concentrations were observed for EV preparations from platelet free plasma (PFP) by kits when compared with the EV preparation by UC, yet the purity was much lower. Meanwhile, the particle size distribution profiles of EV preparations by kits closely resembled those of PFP whereas the EV preparation by UC showed a broader size distribution at relatively large particle size. When these kits were used to isolate EVs from vesicle depleted PFP (VD PFP) comparable particle counts were obtained with their corresponding EV preparations from PFP, which confirmed again the isolation of a large quantity of non vesicular contaminants. As CD9, CD63 and CD81 also exist in the plasma matrix, single particle phenotyping of EVs offers distinct advantage in the validation of EVs compared with ensemble averaged approaches, such as Western blot analysis. nFCM allows us to compare different isolation techniques without prejudice.

title: MIFlowCyt EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments
abstract: Extracellular vesicles (EVs) are small, heterogeneous and difficult to measure. Flow cytometry (FC) is a key technology for the measurement of individual particles, but its application to the analysis of EVs and other submicron particles has presented many challenges and has produced a number of controversial results, in part due to limitations of instrument detection, lack of robust methods and ambiguities in how data should be interpreted. These complications are exacerbated by the field's lack of a robust reporting framework, and many EV FC manuscripts include incomplete descriptions of methods and results, contain artefacts stemming from an insufficient instrument sensitivity and inappropriate experimental design and lack appropriate calibration and standardization. To address these issues, a working group (WG) of EV FC researchers from ISEV, ISAC and ISTH, worked together as an EV FC WG and developed a consensus framework for the minimum information that should be provided regarding EV FC. This framework incorporates the existing Minimum Information for Studies of EVs (MISEV) guidelines and Minimum Information about a FC experiment (MIFlowCyt) standard in an EV FC specific reporting framework (MIFlowCyt EV) that supports reporting of critical information related to sample staining, EV detection and measurement and experimental design in manuscripts that report EV FC data. MIFlowCyt EV provides a structure for sharing EV FC results, but it does not prescribe specific protocols, as there will continue to be rapid evolution of instruments and methods for the foreseeable future. MIFlowCyt EV accommodates this evolution, while providing information needed to evaluate and compare different approaches. Because MIFlowCyt EV will ensure consistency in the manner of reporting of EV FC studies, over time we expect that adoption of MIFlowCyt EV as a standard for reporting EV FC studies will improve the ability to quantitatively compare results from different laboratories and to support the development of new instruments and assays for improved measurement of EVs.

title: Impact of minimal residual negativity using next generation flow cytometry on outcomes in light chain amyloidosis
abstract: We evaluated bone marrow minimal residual disease (MRD) negativity in 44 patients with light chain (AL) amyloidosis using next generation flow cytometry (sensitivity =1 x 10 5; median events analyzed: 8.7 million, range: 4.8 to 9.7 million) All patients underwent MRD testing in 2 years from start of therapy (median: 7 months) The overall MRD negative rate was 64% (n 28) The MRD negative rate after one line of therapy was 71% (20/28) And, MRD negative rates were higher with stem cell transplant as first line therapy (86% 18/21) vs chemotherapy alone as first line treatment (29% 2/7) P .005. The MRD negative rate amongst patients in complete response was 75% (15/20) and in very good partial response, 50% (11/22) There were two patients in partial response/rising light chains (with renal dysfunction) who were MRD negative. There were no differences in baseline characteristics of MRD negative vs MRD positive patients, except younger age amongst MRD negative patients. Patients with MRD negativity were more likely to have achieved cardiac response at the time of MRD assessment, 67% (8/12) vs 22% (2/7) P .04. Renal response rates were similar in both groups. Progression free survival was assessed in the 42 patients achieving CR or VGPR. After median follow up of 14 months, the estimated 1 year progression free survival in MRD negative vs MRD positive patients was 100% (26 patients, 0 events) vs 64% (16 patients, five events) P .006, respectively. MRD assessment should be explored as a surrogate endpoint in clinical trials and MRD risk adapted trials may help optimize treatment in AL amyloidosis.

title: Assessment of minimal residual disease using multiparametric flow cytometry in patients with AL amyloidosis.
abstract: Despite achieving a hematologic complete response after treatment, many patients with AL amyloidosis do not attain recovery of organ function and/or experience hematologic relapse. A persistent plasma cell clone producing amyloidogenic light chains at levels below the detection threshold of traditional serologic methods is hypothesized to impede organ response in some patients. Assessment of minimal residual disease (MRD) may therefore have clinical importance as a more stringent treatment response tool for patients in a hematologic complete response. We used 2 tube, 10 color combination multiparametric flow cytometry to assess for MRD at a minimum sensitivity of 1 in 105 nucleated cells. Of 65 patients in hematologic complete response, 36 (55% were found to have a residual clonal plasma cell population in the bone marrow. Comparing the MRD negative and MRD positive groups, renal response was observed in 88% vs 64% (P .06) cardiac response in 75% vs 59% (P .45) and any organ response in 90% vs 75% (P .20) of patients. Depth of organ response as measured by the percent decrease in 24 hour proteinuria and brain natriuretic peptide was 96% vs 91% (P .16) and 55% vs 46% (P .66) respectively. These data suggest a possible correlation between MRD negativity and higher probability of organ response after treatment in AL amyloidosis. Future prospective studies with a larger cohort are needed to determine the clinical relevance of these improvements.

title: Virtual freezing fluorescence imaging flow cytometry
abstract: By virtue of the combined merits of flow cytometry and fluorescence microscopy, imaging flow cytometry (IFC) has become an established tool for cell analysis in diverse biomedical fields such as cancer biology, microbiology, immunology, hematology, and stem cell biology. However, the performance and utility of IFC are severely limited by the fundamental trade off between throughput, sensitivity, and spatial resolution. Here we present an optomechanical imaging method that overcomes the trade off by virtually freezing the motion of flowing cells on the image sensor to effectively achieve 1000 times longer exposure time for microscopy grade fluorescence image acquisition. Consequently, it enables high throughput IFC of single cells at >10,000 cells s 1 without sacrificing sensitivity and spatial resolution. The availability of numerous information rich fluorescence cell images allows high dimensional statistical analysis and accurate classification with deep learning, as evidenced by our demonstration of unique applications in hematology and microbiology. High throughput imaging flow cytometry suffers from trade offs between throughput, sensitivity and spatial resolution. Here the authors introduce a method to virtually freeze cells in the image acquisition window to enable 1000 times longer signal integration time and improve signal to noise ratio.

title: Flow Cytometry Identifies Risk Factors and Dynamic Changes in Patients with COVID 19
abstract: SARS CoV 2 infection, first documented in Wuhan (China) at the end of 2019, rapidly spread to become a pandemic disease (COVID 19) The infection is highly contagious, and is characterized by a highly heterogeneous clinical phenotype, ranging from fully asymptomatic to severe disease and a high fatality rate  While the reasons for such phenotypic heterogeneity remain ill defined, some patterns have emerged. In particular, in individuals with severe clinical course, the disease often manifests in two waves  In the first phase, patients present typical clinical symptoms of a respiratory tract infections, whereas in the second phase (approximately 8 12 days after onset of symptoms) sudden worsening of respiratory status occurs. At this stage, diffuse ground glass opacities are observed on chest computed tomography. Lymphopenia, elevated D dimer levels, and prolonged prothrombin time are common laboratory abnormalities in these critically ill patients [2, 3] Although the infection has been documented in subjects of all ages, the mortality rate is particularly high among elderly 70 years) individuals. Pre existing co morbidities (such as diabetes, hypertension, and chronic lung disease) are associated with worse outcome. The mechanisms underlying worsening of the disease are object of intense investigation. Increased levels of interleukin 6 (IL 6) and tumor necrosis factor a (TNF a) have been documented in severely infected individuals [2 4] Along with lymphopenia, these observations have led to hypothesize that dysregulation of immune responses may play an important role in the pathology of the disease. However, limited data are available on the immune status of COVID 19 patients. In particular, it remains unknown whether the study of immune parameters at the time of admission to the hospital may help distinguish those who will progress to respiratory failure and will require admission to intensive care units (ICU) and possibly die, from those with moderate symptoms, who will not require invasive ventilation and will eventually recover.

title: OMIP 069: Forty Color Full Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Major Cell Subsets in Human Peripheral Blood
abstract: This 40 color flow cytometry based panel was developed for in depth immunophenotyping of the major cell subsets present in human peripheral blood. Sample availability can often be limited, especially in cases of clinical trial material, when multiple types of testing are required from a single sample or timepoint. Maximizing the amount of information that can be obtained from a single sample not only provides more in depth characterization of the immune system but also serves to address the issue of limited sample availability. The panel presented here identifies CD4 T cells, CD8 T cells, regulatory T cells, gd T cells, NKT like cells, B cells, NK cells, monocytes and dendritic cells. For each specific cell type, the panel includes markers for further characterization by including a selection of activation and differentiation markers, as well as chemokine receptors. Moreover, the combination of multiple markers in one tube might lead to the discovery of new immune phenotypes and their relevance in certain diseases. Of note, this panel was designed to include only surface markers to avoid the need for fixation and permeabilization steps. The panel can be used for studies aimed at characterizing the immune response in the context of infectious or autoimmune diseases, monitoring cancer patients on immuno or chemotherapy, and discovery of unique and targetable biomarkers. Different from all previously published OMIPs, this panel was developed using a full spectrum flow cytometer, a technology that has allowed the effective use of 40 fluorescent markers in a single panel. The panel was developed using cryopreserved human peripheral blood mononuclear cells (PBMC) from healthy adults (Table 1) Although we have not tested the panel on fresh PBMCs or whole blood, it is anticipated that the panel could be used in those sample preparations without further optimization.

title: Panel Design and Optimization for High Dimensional Immunophenotyping Assays Using Spectral Flow Cytometry
abstract: Technological advances in fluorescence flow cytometry and an ever expanding understanding of the complexity of the immune system have led to the development of large (20+ parameters) flow cytometry panels. However, as panel complexity and size increase, so does the difficulty involved in designing a high quality panel, accessing the instrumentation capable of accommodating large numbers of parameters, and analyzing such high dimensional data. A recent advancement is spectral flow cytometry, which in contrast to conventional flow cytometry distinguishes the full emission spectrum of each fluorophore across all lasers, rather than identifying only the peak of emission. Fluorophores with a similar emission maximum but distinct off peak signatures can therefore be accommodated within the same flow cytometry panel, allowing greater flexibility in terms of panel design and fluorophore detection. Here, we highlight the specific characteristics of spectral flow cytometry and aim to guide users through the process of building, designing, and optimizing high dimensional spectral flow cytometry panels using a comprehensive step by step protocol. Special considerations are also given for using highly overlapping dyes, and a logical selection process for optimal marker fluorophore assignment is provided.

title: Monocyte HLA DR Measurement by Flow Cytometry in COVID 19 Patients: An Interim Review
abstract: Several months after the sudden emergence of SARS CoV 2 and COVID 19, the understanding of the appropriate host immune response to a virus totally unknown of human immune surveillance is still of major importance. By international definition, COVID 19 falls in the scope of septic syndromes (organ dysfunction due to dysregulated host response to an infection) in which immunosuppression is a significant driver of mortality. Sepsis induced immunosuppression is mostly defined and monitored by the measurement of decreased expression of HLA DR molecules on circulating monocytes (mHLA DR) In this interim review, we summarize the first mHLA DR results in COVID 19 patients. In critically ill patients, results homogenously indicate a decreased mHLA DR expression, which, along with profound lymphopenia and other functional alterations, is indicative of a status of immunosuppression.

title: Comparison of MALDI TOF mass spectrometry analysis of peripheral blood and bone marrow based flow cytometry for tracking measurable residual disease in patients with multiple myeloma
abstract: Matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI TOF MS) may soon replace routine electrophoretic methods for monitoring monoclonal proteins in patients with multiple myeloma. To further evaluate the clinical utility of this assay, we compared the performance of MALDI TOF MS head to head with an established bone marrow based measurable residual disease assay by flow cytometry (Flow BM MRD) using Memorial Sloan Kettering Cancer Center's 10 color, single tube method. Our results suggest that MALDI TOF MS adds value to bone marrow based MRD testing and may be most useful for early detection of relapse in peripheral blood compared to current electrophoretic methods.