CD20 Flow Cytometry Marker Helps Separate MGUS From Multiple Myeloma

A routine audit of bone marrow samples in a large Australian diagnostic flow cytometry laboratory has uncovered a potentially useful clue for telling apart two related plasma cell disorders: monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). The retrospective study, published in Discover Immunity, reviewed every bone marrow specimen sent for plasma cell flow cytometry over a 12-month period and found that CD20, a surface molecule usually absent from mature plasma cells, was more frequently retained on MGUS plasma cells than on those of multiple myeloma. The authors suggest that this simple staining difference could help clinicians distinguish a generally benign precursor condition from an overt malignancy, particularly in cases where the clinical picture is ambiguous.

Flow cytometry is a cornerstone of modern haematological diagnostics. For plasma cell disorders, monotypic intracellular light chain staining is the hallmark feature that reveals clonality, meaning that a population of plasma cells is producing only one type of immunoglobulin light chain rather than a normal mixture. Although no international consensus panel exists for standard flow cytometric markers in plasma cell disorders, markers such as CD38, CD138 and CD45 are strongly encouraged, and plasma cells are generally identified on the basis of bright CD38 and/or CD138 expression.

The clinical stakes of distinguishing MGUS from myeloma are considerable. Multiple myeloma is diagnosed when at least 10 percent of bone marrow cells are clonal plasma cells by morphology, together with at least one so-called SLiM-CRAB myeloma-defining event. MGUS, by contrast, is characterised by fewer than 10 percent clonal bone marrow plasma cells and the absence of these features. MGUS is considered a benign precursor to more aggressive plasma cell disorders including smouldering myeloma and MM, and conversion to MM occurs at approximately 1 percent of cases per year. Because flow cytometry can significantly underestimate the proportion of plasma cells due to factors including sample processing, it has remained predominantly useful for the sensitive identification of clonal plasma cells rather than for quantification.

The study was conducted at the Flow Cytometry Laboratory of the Institute of Clinical Pathology and Medical Research, based at Westmead Hospital, which receives samples from western Sydney and greater western New South Wales, Australia, predominantly from adult hospitals. Over the 12-month audit period from 2024 to 2025, all patients who had a bone marrow specimen with a plasma cell flow cytometry panel requested were included, with duplicate patients excluded. Bone marrow samples were collected in heparinised tubes, transported at ambient temperature, and stained with a cocktail of antibodies targeting CD45, CD38, CD138, CD19, CD56, CD117, intracellular kappa and lambda light chains, and, where a concomitant standard B cell panel was run, CD20. Plasma cells were defined as CD38-high, CD138-high cells expressing intracellular light chains, and monoclonal plasma cells were defined as those exceeding the kappa to lambda ratio range of 0.4 to 4.0.

In total, 267 individual patient bone marrow samples had a plasma cell panel performed and sufficient detectable plasma cells for immunophenotyping. Of these, 189 cases, or 71 percent, had monoclonal plasma cell populations, including 127 cases of multiple myeloma and 23 cases of MGUS. Two smouldering myeloma cases were excluded from the MGUS-versus-MM comparison because of their low frequency in the cohort. Patients with monoclonal plasma cells tended to be slightly older, with a mean age of approximately 69 years, and the researchers used ordinal logistic regression to confirm that age did not confound the differential expression of the tested markers between monoclonal and polyclonal cases.

The broad phenotypic findings were consistent with established knowledge. Monoclonal plasma cells, whether from myeloma or other conditions, had dimmer CD45 expression than polyclonal plasma cells, were dim or positive for CD56 and CD117, and were negative for CD19. CD19 loss on myeloma plasma cells is a recognised feature and a very sensitive marker for identifying monoclonal plasma cells by flow cytometry; the authors note it may also indicate that malignant plasma cells originate from the long-lived CD19-negative plasma cell compartment of the bone marrow, with loss of the transcription factor Pax-5 potentially accounting for the down-regulation. Prior studies cited in the paper found myeloma plasma cells positive for CD19 and CD20 in only 5 percent and 25 percent of cases respectively.

The novel and clinically interesting result emerged when the researchers compared MGUS and myeloma cases directly. MGUS plasma cells showed a broadly similar phenotype to myeloma plasma cells but were more likely to display dim to positive CD20 expression, with around a third of MGUS cases expressing some CD20. This association persisted when only untreated myeloma cases were considered, indicating that treatment did not significantly alter plasma cell CD20 expression. The statistical test for this comparison yielded a chi-squared value of 9.04 with two degrees of freedom and a p value of 0.011. However, the authors are candid about the marker’s limits: dim to positive CD20 had poor sensitivity of 15.0 percent for distinguishing myeloma from MGUS, meaning most myeloma cases would not be caught by CD20 alone, but it displayed moderate specificity of 71.4 percent.

Why might MGUS plasma cells hold on to CD20 when myeloma plasma cells typically do not? CD20 is a molecule commonly expressed on mature B cells, believed to be important in the organisation of the B cell receptor complex, and is also found on a small subset of T cells in blood and tissue. It is normally lost or reduced during plasma cell development, and plasmablasts, the short-lived precursors that in some cases migrate to the bone marrow to mature into plasma cells, also down-regulate CD20 as part of normal development. Experimental genetic deletion of CD20 reprograms a resting naive B cell into an antibody-secreting plasma cell, suggesting CD20 acts as a gatekeeper of B cell fate. The authors propose several explanations for CD20 retention in MGUS: the plasma cells may be somewhat immature, there may be delayed loss of CD20, or there may be aberrant expression.

Beyond diagnostics, the finding touches on therapy. CD20 is commonly targeted by therapeutic monoclonal antibodies such as rituximab in haematological malignancies and autoimmune disorders. In experimental models, rituximab effectively depletes CD20-positive plasmablasts, particularly those associated with autoantibodies, and in documented cases of MGUS associated with neuropathies, rituximab produced clinically favourable responses and decreases in total IgM levels. The authors note that although MGUS cases are not actively treated, CD20 expression on a subset of cases raises the possibility of using anti-CD20 therapy to prevent progression or to treat monoclonal gammopathies of clinical significance or related disorders such as smouldering myeloma. They argue their findings justify routine inclusion of CD20 in flow cytometric assessment of plasma cell disorders, a marker that is often not part of standard panels.

The study’s authors are careful to spell out its limitations. It was restricted to a single laboratory site, which may limit generalisability, since subtle variations in antibody cocktails and flow cytometer configurations can influence plasma cell staining patterns. The 12-month window also yielded only 23 MGUS patients, a limited number for drawing firm conclusions, and replication in other cohorts with more MGUS patients is required. The project was approved as a quality assurance project by the Western Sydney Local Health District Research Office. Even so, within a large real-world laboratory cohort of 267 patients, the work offers a concrete, testable addition to the diagnostic toolkit: when a monoclonal plasma cell population keeps its CD20, the balance of evidence may tilt away from myeloma and toward its more indolent precursor, with potential implications for how patients are classified and managed.

Subject of Research: Flow cytometric CD20 expression as a diagnostic discriminator between MGUS and multiple myeloma plasma cells

Article Title: CD20 by diagnostic flow cytometry helps discriminate monoclonal gammopathy of undetermined significance and multiple myeloma plasma cells

Article References: Lee, A. Y. S. (2026). CD20 by diagnostic flow cytometry helps discriminate monoclonal gammopathy of undetermined significance and multiple myeloma plasma cells. Discover Immunity, 3(1), Article 8. https://doi.org/10.1007/s44368-026-00026-x

Image Credits: AI Generated

DOI: 10.1007/s44368-026-00026-x

Keywords: flow cytometry, CD20, multiple myeloma, MGUS, plasma cells, bone marrow, immunophenotyping, diagnostic markers, haematology, retrospective study, CD19, CD45