Issue highlights—February 2024

It is a pleasure to usher in the first issue of Cytometry Part B: Clinical Cytometry for the New Year. I would like to take this opportunity to wish the International Society for Clinical Cytometry, the European Society for Clinical Cell Analyses, and Cytometry Part B, continued success in 2024. Also, I would like to thank all the people who make each issue of our journal possible, the submitting authors, the reviewers, the Editorial Board, the Associate Editors, Deputy Editor, Janos Kappelmayer, and our Editor-in-Chief, Fred Preffer. And last, but certainly not least, special thanks to our Managing Editor, Doris Regal who somehow makes it all come together, each and every issue.

In this issue, the importance of multiparametric flow cytometry in clinical diagnosis and drug development is highlighted with many of the papers echoing my passion for standardization, validation, and quality control.

The paper from the laboratories of Wang et al. (2024), “Standardization of Flow Cytometric Detection of Antigen Expression,” is the result of a collaboration between the National Institute of Standards and Technology (NIST) and the National Cancer Institute (NCI) and promises to be one of the most important papers of the year (Tian et al., 2024). This point is highlighted by the Commentary on the paper by Bruce Davis, “Editorial on IVD cellular assay validation” (Davis, 2024). Both documents are ones that everyone conducting cytometry, in any setting, needs to read and re-read. They bring us one step closer to understanding what is required in order to achieve reproducible and quantitative flow cytometry data across platforms and across laboratories.

These manuscripts highlight the increased importance of accurately measuring antigen expression levels when treating patients with novel immunotherapies. Antigen density measurements not only impact patient selection, but are also instrumental in determining treatment efficacy and patient outcomes. The Tian et al. paper ultimately concludes that assay standardization is a critical requirement to enable broad clinical utility and impact of this novel class of therapies. A good part of the paper focuses on the inherent variability and subjectivity in qualitative estimates of antigen density (e.g., dim, moderate, bright) and the resulting need for quantitative measurements of cell surface antigen expression. Common methods for determining antigen density such as geometric mean fluorescence intensity (GeoMFI) and antibodies bound per cell (ABC) appear to be straightforward; however, result comparability across different instrument platforms, reagent lots, operators, and laboratories has not yet been demonstrated. Using a systematic, well-thought-out approach, this team evaluated assay variability of flow cytometric quantitation and then describe procedures and quality control practices whereby highly reproduceable antigen expression measurements can be achieved even across different platforms.

The paper from James Jacobberger's team addresses one of my favorite topics, the development and validation of a flow cytometric biomarker assay (Woost et al., 2024). Their assay detects DNA methyltransferase 1 (DNMT1) protein levels in blood and bone marrow cell subpopulations defined by immunophenotype and cell cycle state. The context of use for this assay would be as a biomarker for hypomethylating therapies in order evaluate the targeted molecular pharmacodynamic effects and better understand treatment-resistance/failure.

Miatell et al. reported on the modification of an existing lyse/no wash for the quantitation of HLA-DR on peripheral blood monocytes (Miatell et al., 2024). Their modifications include adding additional antibodies to the assay and decreasing the blood volumes so that the assay would be suitable for pediatric patients.

Several papers address the role of immunophenotyping by flow cytometry which is an integral part of the diagnosis and classification of leukemias/lymphomas.

Owing to their role in in acute myeloid leukemia (AML) measurable residual disease (MRD) detection and prognostic value, Mizuta et al., evaluated CD34+, CD38− cells in patients without abnormal blasts, with idiopathic thrombocytopenic purpura, malignant lymphoma, and myeloid malignancies (AML, AML with myelodysplasia-related changes, MDS with excess blasts, therapy-related myeloid neoplasms) (Mizuta et al., 2024). They also investigated the co-expression of CD45RA, CD22, CD33, CD123 in the CD34+, CD38− population. Their conclusions suggest monitoring CD45RA expression in the CD34+, CD38− population could be beneficial in myeloid malignancy diagnosis.

The association of the expression of orphan receptor tyrosine kinase type 1 (ROR1) with chronic B lymphocytic leukemia (CLL) has been used for diagnosis and in the follow-up of minimal residual disease (MRD) research. Using a large data set of 767 samples, Flávia Arandas de Sousa et al., evaluate the expression pattern of ROR1 in other subtypes of mature B lymphoid neoplasms (NLBM) (De Sousa et al., 2024). They observed predominant ROR1 expression in CD5+/CD10− NLBM. They conclude that it is important to evaluate ROR1 expression in the diagnosis of NLBM.

Castillo et al. explore the value using T cell Receptor Beta Constant Region 1 (TRBC1) for determining T cell clonality and the diagnostic potential for identify T-cell non-Hodgkin lymphomas (T-NHL) (Castillo et al., 2024). The manuscript presents results from a study in which samples from 59 patients were screened with the standard EuroFlow lymphoid screening tube (LST) and with a custom-designed T-cell clonality assessment tube which includes CD45/TRBC1/CD2/CD7/CD4/TCRγδ/CD3. The conclusions were that integrating TRBC1 into routine lymphoma screening strategies via flow cytometry could provide a robust method for T-cell clonality assessment.

The manuscript by Boris et al. aims to reach a better understanding of seven leukemia-associated phenotype (LAP) markers in B ALL: CD9, CD21, CD66c, CD58, CD81, CD123, and NG2 (Boris et al., 2024). They evaluated peripheral blood leukocytes from apparently healthy donors, normal precursor B regenerative cells in the bone marrow, and lymphoblasts in the peripheral blood and bone marrow of B acute lymphoblastic leukemia (B-ALL) patients at diagnosis. The expression profiles of these markers were also evaluated in normal B cell differentiation and compared with B lymphoblasts in order to establish a synopsis of their expression in normal hematogones. They concluded that CD21, CD66c, CD123, and NG2, markers not expressed by normal regenerative B populations, aid in the identification of residual blasts from the hematogones during and after treatment of these patients.

In the future issues of Cytometry Part B—Clinical Cytometry, we look forward to seeing more high-quality manuscripts which describe advances in clinical and quantitative flow cytometry with the aim of advancing patient care and treatment and enabling the evaluation of novel therapies.