Background: Daratumumab is an anti-CD38 immunotherapeutic drug that has increasingly been used to treat patients with heavily pre-treated and relapsed/refractory multiple myeloma. In so doing, the detection of CD38 antigen on plasma cells by flow cytometry is impeded. We hypothesized that alternative markers can be used in place or in addition to CD38 when detecting plasma cells post-treated with daratumumab.Methods: A total of 16 alternative markers were tested using 22 bone marrow aspirates from patients with plasma cell neoplasm. The ability of selected markers to discern plasma cells from other hematopoietic cells were evaluated. The stability of tested markers when stored at 4 or 25 C after T = 0, 24, 48, and 72 h was also established. Finally, selected markers were incorporated into a panel used for monitoring multiple myeloma measurable residual disease to test their utility to identify plasma cells in the presence of daratumumab and/or elotuzumab (anti-CD319) drugs.Results: Out of the 16 tested markers, CD319, CD54, CD229, CD317, and p63 were expressed by >90% of the plasma cells. Only CD319, CD54, and CD229 achieved 100% detection sensitivity. Further analysis showed that CD319 was better than CD229 and CD54 at resolving plasma cells from background hematopoietic cells, with CD54 being the worst (resolution metric, mean ± SD: CD319 [2.04 ± 0.86]; CD229 [1.47 ± 0.45]; and CD54 [1.22 ± 0.60]). CD229 was expressed by >90% of T lymphocytes, whereas CD319 was expressed preferentially by the CD8+ T cells and less frequently in CD4+ T cells. Additionally, CD229 was found on >60% of B and NK cells, as well as minor subsets of monocytes and granulocytes. CD319 was expressed on most NK cells and a minor subset of B cells, granulocytes, and monocytes. Even though CD229 and CD319 were expressed by different leukocyte subsets, their expression levels were highest on plasma cells. The expression of CD138 on plasma cells was significantly lower after storage at 4 C, while the expression levels of CD38, CD229, and CD319 remained stable at 4 or 25 C. Using limiting dilution experiments, the treatment of cells with daratumumab severely impeded the detection of CD38 antigen on plasma cells, whereas elotuzumab treatment did not block detection of CD319 on plasma cells.

Synopsis Plasma cell dyscrasia (PCD) is a heterogeneous disease which has seen a tremendous change in outcomes due to improved therapies. Over the last few decades, multiparametric flow cytometry has played an important role in the detection and monitoring of PCDs. Flow cytometry is a high sensitivity assay for early detection of minimal residual disease (MRD) that correlates well with progression-free survival and overall survival. Before flow cytometry can be effectively implemented in the clinical setting sample preparation, panel configuration, analysis, and gating strategies must be optimized to ensure accurate results. Current consensus methods and reporting guidelines for MRD testing are discussed.

Background Recent advances in therapeutic interventions have dramatically improved complete response rates in patients with multiple myeloma (MM). The ability to identify residual myeloma cells (e.g., measurable residual disease [MRD]) can provide valuable information pertaining to patient's depth of response to therapy and risk of relapse. Multiparametric flow cytometry is an excellent technique to monitor MRD and has been demonstrated to correlate with patient outcome post‐treatment. To achieve the high sensitivity (one abnormal cell in 105–106 cells) required for MRD evaluation, millions of cells have to be acquired and conventional immunophenotyping protocols are unable to attain these numbers, indicating the needs for alternative flow cytometric staining procedures. A bulk, “Pre‐lysis” method is the consensus approach for staining large number of cells, requires two red blood cell lysis steps, and can adversely affect epitope density. In this study, we tested the “Pooled‐tube” and “Dextran Sedimentation” staining procedures and correlated them with the “Pre‐lysis” method as potential alternative approaches. Methods A total of 22 bone marrow aspirates from patients with plasma cell (PC) dyscrasia were processed in parallel using the “Pre‐lysis,” “Pooled‐tube,” and “Dextran Sedimentation” techniques. Stain indices were calculated and compared to assess their impacts on staining performance for each antibody used in the consensus panel. The recovery of normal and abnormal PCs, mast cells, and B cell precursors was enumerated and compared after their counts were normalized using fluorescent beads. The limit of blank, limit of detection, and lower limit of quantification were established using serial dilution experiments. Results The staining performances of CD19 PECy7, CD27 BV510, CD81 APCH7, and CD138 BV421 were improved using the “Pooled‐tube” method when compared to “Pre‐lysis.” “Pre‐lysis” was better at resolving CD56 using clone C5.9 but our results demonstrated similar improvement can also be achieved by “Pooled‐tube” when alternative CD56 PE clones were used. “Dextran sedimentation” yielded similar staining results when compared to “Pre‐lysis” for all the markers analyzed. The “Pooled‐tube” method, when normalized to “Pre‐lysis,” recovered higher numbers of total PCs (1.2 ± 0.2 times higher; p = .049), normal PCs (1.4 ± 0.26; p = .007), mast cells (1.46 ± 0.27; p = .003), and B cell precursors (1.42 ± 0.3; p = .011), but not abnormal PCs (1.09 ± 0.2; p = .352). There was no evidence that the recovery of cells was different between “Pre‐lysis” versus “Dextran Sedimentation.” All three flow cytometric assays achieved a minimum sensitivity of 10−5 and approached that of 10−6 for detecting rare events. Conclusion Both “Pooled‐tube” and “Dextran Sedimentation” staining procedures were comparable to the “Pre‐lysis” method and are suitable high sensitivity flow cytometric approaches that can be used to process bone marrow samples for MM MRD testing.

Nucleic acid content can be quantified by flow cytometry through the use of intercalating compounds; however, measuring the presence of specific sequences has hitherto been difficult to achieve by this methodology. The primary obstacle to detecting discrete nucleic acid sequences by flow cytometry is their low quantity and the presence of high background signals, rendering the detection of hybridized fluorescent probes challenging. Amplification of nucleic acid sequences by molecular techniques such as in situ PCR have been applied to single‐cell suspensions, but these approaches have not been easily adapted to conventional flow cytometry. An alternative strategy implements a Branched DNA technique, comprising target‐specific probes and sequentially hybridized amplification reagents, resulting in a theoretical 8,000‐ to 16,000‐fold increase in fluorescence signal amplification. The Branched DNA technique allows for the quantification of native and unmanipulated mRNA content with increased signal detection and reduced background. This procedure utilizes gentle fixation steps with low hybridization temperatures, leaving the assayed cells intact to permit their concomitant immunophenotyping. This technology has the potential to advance scientific discovery by correlating potentially small quantities of mRNA with many biological measurements at the single‐cell level. © 2016 by John Wiley & Sons, Inc.

The systematic modulation of mRNA and proteins governs the complicated and intermingled biological functions of our cells. Traditionally, transcriptomic technologies such as DNA microarray and RNA-Seq have been used to identify, characterize, and profile gene expression data. These are, however, considered bulk methods as they are unable to measure gene expression at the single cell level, unless the cells are pre-sorted. Branched DNA is a flow cytometry-based detection platform that has been developed recently to measure mRNA at the single cell level. Originally adapted from microscopy, the current system has been modified to achieve compatibility with the detection of surface and intracellular antigens using monoclonal antibodies conjugated to fluorochromes, thus permitting simultaneous detection of mRNAs and proteins. The Branched DNA method offers a variety of advantages when compared to traditional or standard methods used for the quantification of mRNA, such as (a) the detection of specific mRNA on a per cell basis, (b) an alternate detection tool when the measurement of a protein is technically infeasible (i.e. no quality antibody exists) or the epitope is not assessable, and (c) correlate the analysis of mRNA with protein. Compared to earlier attempts at measuring nucleic acid by flow cytometry, the hybridization temperature applied in the Branched DNA assay is much lower, thus preserving the integrity of cellular structures for further characterization. It also has greatly increased specificity and sensitivity. Here we provide detailed instruction for performing the Branched DNA method using it in a simple model system to correlate the expression of CD8 mRNA and CD8 protein by flow cytometry.

Recent interest in high‐sensitivity multiple myeloma (MM) measurable residual disease (MRD) testing is a direct consequence of the high‐quality responses achieved using novel therapeutic agents and better treatment strategies. Traditional diagnostic measures such as immunohistochemistry and morphology have detection sensitivities of only 10−2 to 10−3, which do not reliably predict progression‐free survival (PFS) or overall survival (OS) after these treatments. Contemporary monitoring of MM MRD has switched to more sensitive platforms such as allele‐specific oligonucleotide quantitative polymerase chain reaction (ASO‐qPCR), next‐generation sequencing (NGS), and multiparametric flow cytometry (MFC). Though both ASO‐qPCR and NGS have excellent detection sensitivities (10−5 to 10−6), both technologies have lower applicability when compared to MFC. Conventional MFC can easily reach a detection sensitivity of 10−4 and when optimized can achieve a sensitivity of 10−5 to 10−6. Current consensus guidelines require a minimum of 2 million and recommend 5 million events be acquired to reach a minimum sensitivity of 10−5. As conventional immunophenotyping protocols are unable to attain these numbers, alternative MFC staining procedures are required. This article describes two high‐sensitivity MFC approaches that can be used for MM MRD testing. © 2019 by John Wiley & Sons, Inc.

Background Multiple myeloma (MM) measurable residual disease (MRD) evaluated by flow cytometry is a surrogate for progression‐free and overall survival in clinical trials. However, analysis and reporting between centers lack uniformity. We designed and evaluated a consensus protocol for MM MRD analysis to reduce inter‐laboratory variation in MM MRD reporting. Methods Seventeen participants from 13 countries performed blinded analysis of the same eight de‐identified flow cytometry files from patients with/without MRD using their own method (Stage 1). A consensus gating protocol was then designed following survey and discussions, and the data re‐analyzed for MRD and other bone marrow cells (Stage 2). Inter‐laboratory variation using the consensus strategy was reassessed for another 10 cases and compared with earlier results (Stage 3). Results In Stage 1, participants agreed on MRD+/MRD− status 89% and 68% of the time respectively. Inter‐observer variation was high for total numbers of analyzed cells, total and normal plasma cells (PCs), limit of detection, lower limit of quantification, and enumeration of cell populations that determine sample adequacy. The identification of abnormal PCs remained relatively consistent. By consensus method, average agreement on MRD− status improved to 74%. Better consistency enumerating all parameters among operators resulted in near‐unanimous agreement on sample adequacy. Conclusion Uniform flow cytometry data analysis substantially reduced inter‐laboratory variation in reporting multiple components of the MM MRD assay. Adoption of a harmonized approach would meet an important need for conformity in reporting MM MRD for clinical trials, and wider acceptance of MM MRD as a surrogate clinical endpoint.

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.