Evaluation of measurable residual disease in multiple myeloma by multiparametric flow cytometry: Current paradigm, guidelines, and future applications.

Multiple myeloma (MM) is a heterogeneous group of mature B-cell diseases that are typically characterized by the presence and accumulation of abnormal plasma cells (PCs), which results in the excess production of monoclonal immunoglobulin and/or light chain found in the serum and/or urine. Multiparametric flow cytometry (MFC) is an indispensable tool to supplement the diagnosis, classification and monitoring of the disease due to its high patient applicability, excellent sensitivity and encouraging results from various clinical trials. In this regard, minimal or, more appropriately, measurable residual disease (MRD) negativity by MFC has been recognized as a powerful predictor of favourable long-term outcomes. Before flow cytometry can be effectively implemented in the clinical setting for MM MRD testing, sample preparation, panel configuration, analysis and gating strategies must be optimized to ensure accurate results. This manuscript will discuss the current consensus guidelines for flow cytometric processing of samples and reporting of results for MM MRD testing. We also discuss alternative approaches to detect plasma cells in the presence of daratumumab treatment. Finally, there is a lack of information describing the subclonal distribution of myeloma cells based on their protein expression. The advent of high-dimensional analysis may assist in following the evolution of antigen expression patterns on abnormal plasma cells in patients with relapsed/refractory disease. This in turn can help identify clonal subtypes that are more aggressive for potential informed decision. An analysis using t-SNE to identify the emergence of PCs subclones by MFC, along with the analysis of their immunophenotypic profiles are presented as a future perspective.

Calibration and characterization of intracellular Asante Potassium Green probes, APG-2 and APG-4.

The response of fluorescent ion probes to ions is affected by intracellular environment. To properly calibrate them, intracellular and extracellular concentrations of the measured ion must be made equal. In the first, computational, part of this work, we show, using the example of potassium, that the two requirements for ion equilibration are complete dissipation of membrane potential and high membrane permeability for both potassium and sodium. In the second part, we tested the ability of various ionophores to achieve potassium equilibration in Jurkat and U937 cells and found a combination of valinomycin, nigericin, gramicidin and ouabain to be the most effective. In the third part, we applied this protocol to two potassium probes, APG-4 and APG-2. APG-4 shows good sensitivity to potassium but its fluorescence is sensitive to cell volume. Because ionophores cause cell swelling, calibration buffers had to be supplemented with 50 mM sucrose to keep cell volume constant. With these precautions taken, the average potassium concentrations in U937 and Jurkat cells were measured at 132 mM and 118 mM, respectively. The other tested probe, APG-2, is nonselective for cations; this is, however, a potentially useful property because the sum [K+] + [Na+] determines the amount of intracellular water.

Standardization, Evaluation, and Area-Under-Curve Analysis of Human and Murine Treg Suppressive Function.

FOXP3+ T-regulatory (Treg) cells have important roles in immune homeostasis, and alterations in their number and function can predispose to diseases ranging from autoimmunity to allograft rejection and tumor growth. Reliable identification of human Tregs remains a persistent problem due to a lack of specific markers. The most definitive Treg characterization currently involves combined assessment of phenotypic, epigenetic and functional parameters, with the latter typically involving in vitro Treg suppression assays. Unfortunately, suppression assays are frequently performed using differing methods and readouts, limiting comparisons between studies. We provide a perspective on our experience with human and murine Treg suppression assay conditions, including Treg data obtained in clinical transplant studies, Tregs isolated from healthy donors and treated with epigenetically active compounds, and Tregs from standard murine strains (C57BL/6 and BALB/c). We provide detailed descriptions and illustrations of typical problems, shortcomings and troubleshooting; describe new modifications and approaches; and present a new method for calculation of suppressive assay data using a modified area-under-curve (AUC) method. This method allows us to directly compare Treg suppressive function between multiple patients (such as in clinical transplant studies), to reliably track changes in Treg function from the same person over time, or compare effects of Treg-modulating compounds tested with different healthy donors Tregs in separate or combined experimental settings.

Flow cytometry in preclinical drug development.

Flow cytometry has many applications in clinical medicine allowing rapid and highly specific characterization of cells in solution (e.g., peripheral blood) or from dissociated tissues. The data generated from these analyses may be used to diagnose and monitor progression of disease as well as aid in prognostication of selected pathologic processes. In recent years, flow cytometric techniques have established a foothold in preclinical drug development providing an ability to identify and characterize both cell morphology and function, as well as to more clearly assign observed alterations in one or more cell attributes as intended or unexpected effects of new biopharmaceutical entities. The inclusion of flow cytometric evaluation assays (some described in this chapter) during preclinical drug development has increased and enhanced the detail of data generated to support the safety and efficacy of new biopharmaceuticals. Flow cytometry analyses used in preclinical drug development that are described in this chapter include immunophenotyping, peripheral blood cross-reactivity, binding activity and stability and cell receptor dynamics.