Cytometry Part B: Clinical Cytometry最新文献

The publication of Clinical and Laboratory Standards Institute's guideline H62 has provided the flow cytometry community with much-needed guidance on development and validation of flow cytometric assays (CLSI, 2021). It has also paved the way for additional exploration of certain topics requiring additional guidance. Flow cytometric analysis of rare matrices, or unique and/or less frequently encountered specimen types, is one such topic and is the focus of this manuscript. This document is the result of a collaboration subject matter experts from a diverse range of backgrounds and seeks to provide best practice consensus guidance regarding these types of specimens. Herein, we define rare matrix samples in the setting of flow cytometric analysis, address validation implications and challenges with these samples, and describe important considerations of using these samples in both clinical and research settings.

The advent of high-dimensional imaging offers new opportunities to molecularly characterize diagnostic cells in disorders that have previously relied on histopathological definitions. One example case is found in tuberous sclerosis complex (TSC), a developmental disorder characterized by systemic growth of benign tumors. Within resected brain tissues from patients with TSC, detection of abnormally enlarged balloon cells (BCs) is pathognomonic for this disorder. Though BCs can be identified by an expert neuropathologist, little is known about the specificity and broad applicability of protein markers for these cells, complicating classification of proposed BCs identified in experimental models of this disorder. Here, we report the development of a customized machine learning pipeline (BAlloon IDENtifier; BAIDEN) that was trained to prospectively identify BCs in tissue sections using a histological stain compatible with high-dimensional cytometry. This approach was coupled to a custom 36-antibody panel and imaging mass cytometry (IMC) to explore the expression of multiple previously proposed BC marker proteins and develop a descriptor of BC features conserved across multiple tissue samples from patients with TSC. Here, we present a modular workflow encompassing BAIDEN, a custom antibody panel, a control sample microarray, and analysis pipelines-both open-source and in-house-and apply this workflow to understand the abundance, structure, and signaling activity of BCs as an example case of how high-dimensional imaging can be applied within human tissues.

Hematologic neoplasms with germline predisposition have been increasingly recognized as a distinct category of tumors over the last few years. As such, this category was added to the World Health Organization (WHO) 4th edition as well as maintained in the WHO 5th edition and International Consensus Classification (ICC) 2022 classification systems. In practice, these tumors require a high index of suspicion and confirmation by molecular testing. Flow cytometry is a cost-effective diagnostic tool that is routinely performed on peripheral blood and bone marrow samples. In this review, we sought to summarize the current body of research correlating flow cytometric immunophenotype to assess its utility in diagnosis of and clinical decision making in germline hematologic neoplasms. We also illustrate these findings using cases mostly from our own institution. We review some of the more commonly mutated genes, including CEBPA, DDX41, RUNX1, ANKRD26, GATA2, Fanconi anemia, Noonan syndrome, and Down syndrome. We highlight that flow cytometry may have a role in the diagnosis (GATA2, Down syndrome) and screening (CEBPA) of some germline predisposition syndromes, although appears to show nonspecific findings in others (DDX41, RUNX1). In many of the others, such as ANKRD26, Fanconi anemia, and Noonan syndrome, further studies are needed to better understand whether specific flow cytometric patterns are observed. Ultimately, we conclude that further studies such as large case series and organized data pipelines are needed in most germline settings to better understand the flow cytometric immunophenotype of these neoplasms.

Over the last 15 years activity of diagnostic flow cytometry services have evolved from monitoring of CD4 T cell subsets in HIV-1 infection to screening for primary and secondary immune deficiencies syndromes and assessment of immune constitution following B cell depleting therapy and transplantation. Changes in laboratory activity in high income countries have been driven by initiation of anti-retroviral therapy (ART) in HIV-1 regardless of CD4 T cell counts, increasing recognition of primary immune deficiency syndromes and the wider application of B cell depleting therapy and transplantation in clinical practice. Laboratories should use their experience in standardization and quality assurance of CD4 T cell counting in HIV-1 infection to provide immune monitoring services to patients with primary and secondary immune deficiencies. Assessment of immune reconstitution post B cell depleting agents and transplantation can also draw on the expertise acquired by flow cytometry laboratories for detection of CD34 stem cell and assessment of MRD in hematological malignancies. This guideline provides recommendations for clinical laboratories on providing flow cytometry services in screening for immune deficiencies and its emerging role immune reconstitution after B cell targeting therapies and transplantation.

Multicolor flow cytometry (MFC) is crucial in detecting occult or minimal bone marrow (BM) involvement by non-Hodgkin lymphomas (NHL), which may not be detected using trephine biopsy or imaging studies. Detection of low-level BM involvement can be challenging without definite immunophenotypic aberrancies. We studied the utility of CD305 in MFC detection of minimal BM involvement by B-NHL, especially in the absence of aberrancies by commonly used markers. The study included 1084 consecutive BM samples submitted for the staging of B-NHLs (excluding CLL) over two years. Samples were studied for morphological, immunophenotypic, and histopathological assessment. MFC studies were performed using 10–13 color MFC, including CD305-antibody (clone, DX26). Minimal BM involvement was defined with a cutoff of ≤10% lymphoma cells in viable cells on MFC assessment. Of 1084, 148 samples revealed overt morphological involvement by B-NHL and were excluded from analysis. BM samples of 172/936 patients were morphologically negative but revealed involvement using MFC independently. Corresponding trephine biopsy involvement was detected in only 79/172 (45.9%) patients. On MFC, 23/172 samples showed BM involvement with >10% lymphoma cells, and 149/172 (86.6%) samples revealed minimal involvement. In 54/149 (36.24%) samples, lymphoma cells were detected only with aberrant loss of CD305 expression. In 78 of the remaining 95 samples (82.1%), it provided an immunophenotypic aberrancy addition to other markers and supported the results. CD305 is a highly useful marker in the flow cytometric assessment of minimal BM involvement by B-NHL. MFC is a superior modality to trephine biopsy in detecting low-level BM involvement.

Image-guided core needle biopsies (IG-CNB) represent a minimally invasive approach for obtaining tissue in patients with lymphadenopathy and suspected lymphoma. Despite their utility, diagnostic challenges persist, with lower efficacy compared with excisional biopsies. Our study aimed to evaluate the potential utility of incorporation of flow cytometry (FC) alongside immunohistochemistry (IHC) when performing IG-CNB for suspected lymphoproliferative diseases. Analyzing 170 consecutive cases, guided by ultrasound (n = 94) or computer tomography (n = 76), we employed a diagnostic algorithm, already established in our laboratory practice, utilizing three antibody cocktail-equipped tubes tailored for defining lymphomas, particularly those of B-cell origin. FC expedited the diagnostic process, yielding presumptive results in 87.6% of cases within 48 h, with a positive predictive value of 98%. Addition of FC to routine IHC enhanced the diagnostic rate from 91.2% to 95.3%, reducing IG-CNB failure rate by 45%, from 8.8% to 4.7%. This enhancement was particularly notable for deep-seated sites and in the setting of suspected disease recurrences. Consequently, FC emerges as a valuable adjunctive tool, allowing for the improvement of diagnostic performance, with a particular focus on the ability to quantify the expression of surface markers for targeted therapies, and holding the potential to diminish the necessity for repeat excisional biopsies subsequent to IG-CNB procedures.

The flow cytometry-based evaluation of TRBC1 expression has been demonstrated as a rapid and specific method for detecting T-cell clones in sCD3-positive TCRαβ+ mature T-cell lymphoma. The aim of the study was to validate the utility of surface (s) TRBC1 and cytoplastic (cy) TRBC1 assessment in detecting clonality of sCD3-negative peripheral T-cell lymphomas (PTCLs), as well as exploring the existence and characteristics of sCD3-negative clonal T-cell populations with uncertain significance (T-CUS). Evaluation of sTRBC1 and cyTRBC1 were assessed on 61 samples from 37 patients with sCD3-negative PTCLs, including 26 angioimmunoblastic T-cell lymphoma (AITL) patients and 11 non-AITL patients. The sCD3-negative T-CUS were screened from 1602 patients without T-cell malignancy and 100 healthy individuals. Additionally, the clonality of cells was further detected through T-cell gene rearrangement analysis. We demonstrated the monotypic expression patterns of cyTRBC1 in all sCD3-negative PTCLs. Utilizing the cyTRBC1 evaluation assay, we identified a novel and rare subtype of sCD3-negative T-CUS for the first time among 13 out of 1602 (0.8%) patients without T-cell malignancy. The clonality of these cells was further confirmed through T-cell gene rearrangement analysis. This subset exhibited characteristics such as sCD3-cyCD3 + CD4 + CD45RO+, closely resembling AITL rather than non-AITL. Further analysis revealed that sCD3-negative T-CUS exhibited a smaller clone size in the lymph node and mass specimens compared to AITL patients. However, the clone size of sCD3-negative T-CUS was significantly lower than that of non-AITL patients in both specimen groups. In conclusion, we validated the diagnostic utility of cyTRBC1 in detecting sCD3-negative T-cell clonality, provided a comprehensive analysis of sCD3-negative T-CUS, and established a framework and provided valuable insights for distinguishing sCD3-negative T-CUS from sCD3-negative PTCLs based on their phenotypic properties and clone size.

Clinical flow cytometry laboratories require quality control materials for assay development, validation, and performance monitoring, including new reagent lot qualification. However, finding suitable controls for populations with uncommonly expressed antigens or for rare populations, such as mast cells, can be difficult. To that end, we evaluated synthetic abnormal mast cell particles (SAMCP), developed together with, and manufactured by, Slingshot Biosciences. The SAMCP's were designed to phenotypically mimic abnormal neoplastic mast cells: they were customized to have the same light scatter and autofluorescence properties of mast cells, along with surface antigen levels of CD45, CD33, CD117, CD2, CD25, and CD30 consistent with that seen in mast cell disease. We evaluated several performance characteristics of these particles using ARUP's high sensitivity clinical mast cell assay, including limit of detection, off-target activity and FMO controls, precision, scatter properties of the particles utilizing several different cytometer platforms, and particle antigen stability. The phenotype of the SAMCP mimicked abnormal mast cells, and they could be distinguished from normal native mast cells. FMO controls demonstrated specificity of each of the markers, and no off-target binding was detected. The limit of detection of the particles spiked into normal bone marrow was found to be ≤0.003% in a limiting dilution assay. The mast cell particles were found to perform similarly on Becton Dickinson Lyric, Cytek Aurora, and Beckman Coulter Navios and CytoFLEX platforms. Within run and between run precision were less than 10% CV. SAMCP were stable up to 13 days with minimal loss of antigen fluorescence intensity. The SAMCP's were able to successfully mimic neoplastic mast cells based on the results of our high sensitivity mast cell flow cytometry panel. These synthetic cell particles represent an exciting and innovative technology, which can fulfill vital needs in clinical flow cytometry such as serving as standardized control materials for assay development and performance monitoring.