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• W4387472708 abstract "[Color figure can be viewed at wileyonlinelibrary.com] Despite the reality that my entire career has been spent devoted to flow cytometry, it is undeniable to me that the gold standard remains an image of the cell, and when visualized within tissue, the architectural relationships between cells. Thus my keen interest in bringing the Amnis technology into my research laboratory years ago and my current enthusiasm for the cell-imaging capacities of more recent technologies such as those from Becton-Dickinson (Cell-View), ThermoFisher (Attune CytPix) and other imaging flow cytometers currently or soon available. In this issue I am therefore extremely interested in Cyclic Analysis of Single-Cell Subsets and Tissue Territories (CASSATT) which identifies scanned slide images through multiple staining rounds, segments nuclei and assesses marker expression on each detected cell (Brockman et al., 2023). Cyclic immunochemistry (cycIHC) exploits multiple rounds of immunostaining and imaging for mapping and locating cells of interest with the speed and simplicity of brightfield microscopy, making the collection of entire tissue sections and slides possible. CASSATT employs registered scanned slide images across all rounds of staining, segments individual nuclei, and measures marker expression on each detected cell. It further explores the spatial relationships between cell populations and the odds of interaction frequencies between cell populations within tissue regions, helping to identify cells that interact or do not interact. In this report, a test dataset of six glioblastoma tissue sections were cyclically stained for eight biomarkers for a total of 48 scanned slide images. The authors describe an efficient workflow that provided answers to questions commonly encountered in discovery research in tissue specimens, such as the overall abundance of cells of interest within a tissue section as well as whether groups of cells were in close proximity. CASSATT gathered together and streamlined all steps necessary to produce single cell expression information from cycIHC datasets and did so utilizing an open source environment. In addition, CASSATT systematically analyzed the spatial relationships between cell populations and via unsupervised algorithms, identified clusters of cell niches. The persistence of measurable residual disease (MRD) is a strong indicator for adverse outcomes in acute myeloid leukemia (AML) and has been shown to be a valid surrogate marker for disease-free survival and overall survival, irrespective of patients' age, AML subtype, sample type, time of MRD assessment and MRD detection method (Short et al., 2020). Other hematopoietic malignancies such as B- and plasma cell lineages also benefit from MRD diagnostic assays (Chen et al., 2023; Gao et al., 2023; McMillan et al., 2023; Zhou et al., 2023). In a study by Wang et al. (2023) their experience working under CLSI HL62 guidelines and FDA IDE (investigational device exemption) approval in the validation of a 12-color AML MRD flow cytometry MRD assay is shared, including the details of panel design, analysis and interpretation. Prior to their recent cytometer upgrade to 12-marker capacity permitting more expansive testing, these investigators utilized an eight-marker AML MRD test designed to discriminate between normal and abnormal myelomonocytic precursors using analytical approaches combining the detection of leukemia-associated immunophenotypes (LAIP) and/or the identification of deviation from normal (DfN) methods. The eight-color assay was cross correlated with molecular testing, clinical outcomes, and resulted in multiple publications (Ouyang et al., 2015, 2016; Xu et al., 2017). In the current study, the assay accuracy was assessed by testing known positive and negative samples and correlating with the results of molecular genetic testing and follow-up bone marrow examination. The limit of detection (LOD) and limit of quantification (LOQ) were validated to a level between 0.01% and 0.1%, dependent on the numbers of cells evaluated and the degree of deviation from normal phenotypes. Assay linearity, precision and carry over studies were found to be acceptable. The clinical validity of the assay was tested in 61 patients in order that “trueness” could be determined by correlating with concurrent molecular genetic testing and or follow-up bone marrow examination results; the clinical test concordance was 93%, specificity 98% and sensitivity 83%. Ultimately, the most challenging aspects of the assay involved discerning differences between pre-leukemic cells (persistent clonal hematopoiesis) or underlying myelodysplastic clones from AML MRD with immunophenotypic switch or subclone selection, highlighting the need for further characterization of abnormal blasts bearing the potential for relapse. Processing as many monoclonal antibodies as possible in one staining tube in the clinical flow cytometry laboratory has been an on-going aspiration over my entire professional career. It is not as much of the “flow-geek” in me that wants such capacity but rather my contention that such practice clearly provides better medical care to our patients. This is especially true in the diagnosis of hematologic malignancies, where I contend that the tumors “…do not read the same books and journals” that we do related to linage markers, but rather do whatever they can to obfuscate their lineages and confuse us with their “unfaithful” antigenic co-expressions. The more markers we can apply to such malignancies, along with built in internal positive and negative controls (provided by comingling normal cells), the better diagnosticians we become. These are not the only advantages of minimizing the number of staining tubes, in that when samples are pauci-cellular, more diagnostic information also becomes further available utilizing the least number of tubes or wells cells are processed in. The more analytic power the clinical flow cytometry laboratory can harness, the more flexibility we can offer and better questions we can ask such as those described just above related to MRD as well as so many other areas (Estevam et al., 2021; Quirós-Caso et al., 2022; Shameli & Roshan, 2022; Sanjabi & Lear, 2021). Such hardware, reagents and software already exists in our research flow cytometry laboratories; we are all left waiting for these to move fully into the clinical realm, and to see advances in this area brings us all closer to far better laboratory practice. In the submission of Hammerich et al. (2023), we learn of the application of a three-laser spectral flow cytometer demonstrating the analysis of a 31-marker clinically oriented testing panel. The standard 3-laser Aurora utilized by this group resolved 31 fluorochromes excited off a 405, 488, and 640 nm lasers. All fluorochromes and titrated antibodies used in the study were commercially available simplifying modification of the panel and facilitating replacement of markers of interest with others that might be more appropriate. In fact since not all detectors were utilized in this study, it is possible to hypothesize that in the future a few more conjugated antibodies might possibly expand the breadth of this platform's analytic capacity. In conclusion, I find this to be a great moment in clinical flow diagnostics… except of course that I want at least one if not two- to -three more lasers in the instrument than those discussed in this article . It is my privilege and honor to usher into our Journal a new type of peer-reviewed submission we refer to as the “Best Practice” category. While presently new to our Journal pages, in past years such informative pieces in the form of numbered Modules have been available to the clinical flow laboratory community, originating from our own ICCS Quality and Standards Committee. The current submission from Devitt et al. (2023) underscores the importance of flow cytometric assay validation which provides confidence that such assays yield reliable results that can be trusted by clinical caregivers in their determining critical medical decisions. For example, the authors provide our readership much needed descriptions, explanations and distinguishing characteristics of both IVD (in vitro diagnostic) or LDT (laboratory developed) tests. IVD tests are developed by manufacturers who optimize, validate, and submit such assays to a regulatory body, such as the FDA. The regulatory body approves the validation and clears it for clinical use permitting the manufacturer to sell the assay to laboratories for testing patient samples. Laboratories must follow the manufacturer's described standard operating procedures and verify that they can reproduce the manufacturer's performance specifications in their lab. In contrast to IVD tests, Devitt et al. (2023) focusses on LDT assays which are developed, optimized and validated in individual laboratories using that specific laboratories equipment, reagents and staff. There is currently no requirement for an outside regulatory body to approve LDTs in the United States, although federal legislation to change this paradigm (VALID act) has been suggested and such potential changes must be closely monitored by many impacted medical societies. The laboratory must validate performance specifications unique to their in-house-developed assay. Once validated, the laboratory can perform the assay on patient samples; however, tests may not be sold to another facility. Any change to an IVD assay that deviates from the manufacturer instructions may render the test an LDT and require validation. The authors continue with other aspects of LDTs such as describing their reporting structures (quantitative, semi- quantitative, and qualitative or mixed) and providing examples of each. The concepts of testing accuracy, precision, limits of detectability, stability along with helpful examples of each concept round out this incredibly helpful reference document. I trust this is a great start to our Journal publishing's future contributions in this new category! Four “Letters to the Editor” generally related to hematologic malignancy round off this issue of Clinical Cytometry (Li et al., 2023; Martin-Moro & Garcia-Vela, 2023; Panda et al., 2023a, 2023b). With that I would like to thank all who submit articles to our Journal, as well as our splendid Associate Editors and talented Editorial Board who are involved in the important task of reviewing submitted articles. Additionally, hats off to our friends at Wiley, the ICCS and ESCCA. And a special thanks to Doris Regal with whom I have the pleasure of working with in making this Journal a success!" @default.
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• W4387472708 title "Issue highlights—September 2023" @default.
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• W4387472708 doi "https://doi.org/10.1002/cyto.b.22145" @default.
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