Issue highlights—November 2023

This new issue of Cytometry B (Clinical Cytometry) consists of five main manuscripts which contain original research in the field of clinical cytometry. A manuscript describing a simple (new) method for preservation of urinary cells for subsequent flow cytometric analyses (Freund et al., 2023) opens this issue of the journal. It is followed by three papers related to the application of flow cytometry in the field of acute leukemias. In the first two manuscripts distinct assays for measurable residual disease (MRD) monitoring in acute myeloblastic leukemia (AML) (Tettero et al., 2023) and B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) (Arunachalam et al., 2023) are (technically and clinically) validated, whereas the third one consists of a comparison and validation of four immunophenotypic scoring systems for the diagnosis of early-T precursor (ETP) acute lymphoblastic leukemia (ALL) (Basavaraju et al., 2023). The fifth article in this issue of Cytometry B revisits the application of flow cytometry for HLA-B27 typing through the comparison of 3 CE-IVD certified methods (Waeckel et al., 2023). Three letters to the editor complete the contents of the November issue of Cytometry B, in which different aspects of three clinically relevant flow cytometric assays for sepsis (Haem-Rahimi et al., 2023), drug-induced hypersensitivity (Ebo et al., 2023) and diagnostic screening of acute leukemias (Axler et al., 2023), are briefly addressed. In this section, I will summarize the contents and highlight the most relevant contributions of the above papers in four separate blocks related to the fields of (i) the flow cytometric analysis of samples with low cell viability, (ii) the flow cytometric diagnosis and monitoring of acute leukemias, (iii) HLA-B27 typing and (iv) the feasibility to measure HLADR expression levels on stabilized blood monocytes and blood circulating drug-specific T cells in the diagnostic work-up of sepsis and drug-hypersensitivity, respectively.

Flow cytometry assays used in diagnostic laboratories have mostly focused on blood samples and to a less extent also, in bone marrow and other tissue and body fluid specimens. Despite the high frequency of kidney and urinary tract diseases in the general population, and the frequent need for invasive diagnostic procedures (e.g., kidney biopsy), urine has been one of the less explored and used specimens among the distinct types of body fluids evaluated in (clinical) flow cytometry. Of note, urine samples are frequently obtained for conventional biochemistry assays, including analysis of proteinuria, and for the evaluation of the urine sediment by conventional, for example, cytomorphology. In contrast, flow cytometric analysis of urinary cells (e.g., immune cells, podocytes or epithelial cells) is rarely used in routine diagnostics, despite it has proven to provide valuable information in multiple kidney diseases (Abdulahad et al., 2009; Goerlich et al., 2020; Kopetschke et al., 2015; Mella et al., 2020; Zimmerman et al., 2019). The limited use of urine specimens for flow cytometry is due, at least in part, to the cellular alterations induced by the urine environment and the limited viability of urinary cells (Stachowski et al., 1998), which has contributed to make flow cytometric analyses of urine fully dependent on fresh samples. Thus, preservation of urine samples for delayed analysis of urine cells, currently remains an unmet need. In this issue of Cytometry B, Freund et al. (2023) developed a simple, two-step, preservation procedure for urine samples, based on gentle fixation of urinary cells in imidazolidinyl urea as fixative and MOPS to prevent precipitate formation. The method proved suitable for further flow cytometric analyses of urinary cells for up to 6 days, in the absence of significant numerical and phenotypic changes (Freund et al., 2023). Thereby, the proposed preservation method paves the way for further investigations that would explore more in-depth the potential utility of flow cytometry in both kidney and urinary tract diseases, not only in individual laboratories where a flow cytometer is available, but also in multicentric studies, as it might also facilitate shipment of samples from smaller centers to reference flow cytometry laboratories.

Flow cytometry immunophenotyping for the diagnosis of hematological neoplasms in general, and of acute leukemia in particular, is one of the most widely extended and world-wide established clinical applications of flow cytometry. Among acute leukemias, immunophenotypic criteria are essential for fast discrimination among BCP-ALL, T-ALL, AML, and ambiguous lineage acute leukemias with important clinical consequences (Arber et al., 2022; Khoury et al., 2022). Such distinction between acute leukemia cells from individual patients with maturation traits to the different major hematopoietic myeloid and lymphoid lineages needs to be fast and accurately achieved in routine diagnostics. At the same time, the definition of some specific subgroups of acute leukemias such as early-T precursor (ETP) ALL remains a challenge, due to its great heterogeneity and overlap with other acute leukemia subtypes such as pro-T ALL, near-ETP ALL, mixed lineage acute leukemia or undifferentiated acute leukemia (Genesca & la Starza, 2022). This later challenge has led to the proposal of flow cytometric scoring systems for better definition of ETP-ALL versus other acute leukemias with overlapping immunophenotypic features (Chandra et al., 2021; Inukai et al., 2012). In the November issue of Cytometry B, an ultra-rapid stain-no lyse flow cytometric method based on the Hoechst 34580 DNA dye and a set of eight additional markers (i.e., CD34, CD33, CD117, CD19, CD10, CD7, CD36 and CD45) was validated against the local routine flow cytometric stain-lyse-and-wash assay, based on a pilot single center study on 59 myelodysplastic syndrome (MDS) and acute leukemia samples (Axler et al., 2023). The method provides shorter time (30 min) to results with a promising high accuracy and deserves future extended validation in larger and more heterogeneous patient cohorts due to its inability to include in the screening highly specific cytoplasmic (Cy) markers such as CyCD3, CyMPO. Of note, in this November issue of the journal, Basavaraju et al. (2023) also report on a retrospective study in a cohort of 77 T-ALL patients enriched in ETP-ALL aimed at validating four different immunophenotypic scoring systems for ETP-ALL (vs. mixed phenotype acute leukemia and other non-ETP TALL, including those proposed by Chandra et al. (2021) and Inukai et al. (2012) (in addition to two extended variants of these two scoring systems with up to 11 markers). Interestingly, the scoring criteria based on the lowest number of markers (N = 5) proposed by Chandra et al. (2021), was the one which was associated with the highest predictive value, showing a sensitivity of between 91% and 94% and a specificity of between 100% and 96% for score cut-off values of 2.5 and 1.5, respectively (Basavaraju et al., 2023).

Since the 1990s flow cytometry has emerged, together with PCR-based conventional and next-generation molecular techniques, as one of the most well-suited and widely used approaches for MRD monitoring of patients with acute leukemia after therapy has been initiated, due to its short time to results, high accuracy and sensitivity (Schuurhuis et al., 2018; van Dongen et al., 2015). From the clinical point of view, MRD has proven informative for the evaluation of the quality of CR (complete response) and as an independent prognostic factor that is currently used for both patient risk re-stratification after (initial) therapy, and for immediate treatment decisions (Berry et al., 2017; van Dongen et al., 2015). Thus, MRD monitoring by flow cytometry is currently a well-established method in both childhood and adulthood ALL, particularly in BCP-ALL, where several assays, including high-sensitive and standardized next-generation flow (NGF) procedures, are currently available and broadly used (Berry et al., 2017; van Dongen et al., 2015). However, still (e.g., technical, access) restrictions exist in many centers around the world which promote the design of local strategies that require clinical validation against patient outcome in the real-world settings. An example of such clinical validation of flow-MRD is provided in this new issue of Cytometry B by Arunachalam et al. (2023) based on a large series of 450 children and adults diagnosed with BCP-ALL and treated at a single institution in Vellore (India) in which the independent prognostic value of flow-MRD is confirmed based on real-world data.

In contrast to BCP-ALL, at present, a consensus high-sensitive MRD approach for AML is still missing (Schuurhuis et al., 2018; Short et al., 2020). In fact, due to important advances achieved in recent years in the treatment of AML, with the availability of new drug candidates (DiNardo et al., 2023), there is an urgent need for a reliable, robust and reproducible flow cytometric MRD assay for use in AML both in clinical trials and routine diagnostics (Short et al., 2020). In this issue of Cytometry B, Tettero et al. (2023) evaluated an MRD assay in terms of adherence to the In Vitro Diagnostic Regulations (IVDR) in Europe using a consensus panel of four 8-color tubes that contained a backbone of five common markers (CD13, CD34, CD45, CD117 and HLADR) combined in each of the four tubes with three additional and distinct markers per tube. Their interest focused on the definition of specific features of the assay in multicentric settings, including its accuracy, analytical specificity, and sensitivity. Overall, their results showed that the assay is reproducible and provides accurate MRD detection and quantitation at the 0.1% (10⁻³) threshold, similarly to other 12-color flow cytometry validated assays which obtained FDA IDE (investigational device exemption) approval (Wang et al., 2023).

Extensive data has accumulated over decades which demonstrates an association between the Human Leukocyte Antigen B27 (HLAB-27) and several chronic inflammatory diseases, such as ankylosing spondylitis, juvenile rheumatoid arthritis psoriatic arthritis, inflammatory bowel disease-associated spondyloarthritis and acute anterior uveitis (Sibley, 2016). This association has pushed for a simple, fast, and cost-effective assay for HLA-B27 typing in these subjects, flow cytometry playing an important role in this regard due to its broader availability, high speed, relative simplicity, and lower cost (Zeng et al., 2018). Thus, screening for HLA-B27 remains as one of the most long-standing diagnostic applications of flow cytometry (Barath et al., 2022; Zeng et al., 2018) for which different CE-IVD labeled methods have been developed and are available. In this issue of Cytometry B, Waeckel et al. (2023) compared the performance of three CE-IVD labeled flow cytometry assays against the molecular biology gold standard. Their results show that despite the high sensitivity and specificity of the methods and their satisfactory performance, there is still room for improvement with some of the evaluated assays, genotyping remaining critical for cases in whom the flow cytometry assay provides inconclusive results.

Two letters to the editor complete the contents of this November issue of Cytometry B. These two letters provide interesting contributions related with two distinct clinical applications of flow cytometry: (i) the diagnostic work-up and monitoring of patients at risk of sepsis (Haem-Rahimi et al., 2023); and (ii) the diagnosis of drug-induced hypersensitivity (Ebo et al., 2023).

Reduced expression of HLADR on blood monocytes has been recognized as a sign of monocyte deactivation in sepsis-induced immunosuppression. Because of this, HLADR expression on blood monocytes has become a robust marker in the settings of sepsis (e.g., to enroll patients in clinical trials and to support and guide immunotherapy in patients admitted at intensive care units). However, the earliest tests developed required fresh EDTA-anticoagulated samples collected and processed under strict conditions, in order to obtain reliable and reproducible results as regards HLADR expression levels on blood monocytes and the pre-established robust cut-offs for sepsis. Here, Haem-Rahimi et al. (2023) confirm previous data reported in this journal (Hamada et al., 2022; Quadrini et al., 2021) about the feasibility to use stabilized blood samples and translate into the stabilized blood settings, the pre-established cut-off values for the definition of HLADR expression levels on fresh blood monocytes, in the diagnostic work-up and monitoring of monocyte deactivation during sepsis. In turn, Ebo et al. (2023) report here on the potential utility of flow-based T-lymphocyte activation tests in the diagnostic work-up of patients with drug-induced hypersensitivity based on their interesting findings in a patient who carried bleomycin-specific T cells.