Flow Cytometric Assessment of Plasma Cell Myeloma, gating, key antigens and MRD

Overview

• Flow cytometry is an indispensable tool in the diagnosis, characterisation, and monitoring of plasma cell myeloma (PCM).
• It provides rapid, multiparametric, single-cell analysis of antigen expression, enabling identification of clonal plasma cells, quantification of disease burden, phenotypic characterisation of aberrant populations, and highly sensitive detection of minimal residual disease (MRD).
• While morphological assessment of the bone marrow aspirate remains the reference standard for plasma cell enumeration at diagnosis, flow cytometry complements and extends this assessment across the disease continuum, from initial workup through treatment monitoring and post-therapy surveillance.

Principles of Flow Cytometric Plasma Cell Identification

Instrument and Panel Design

• Modern PCM flow cytometry uses polychromatic platforms capable of simultaneously detecting 8-10 or more fluorochrome-conjugated antibodies per tube.
• Adequate sensitivity for MRD applications requires acquisition of a minimum of $1 \times 10^6$ total events per tube, with EuroFlow-based protocols recommending $\geq 2 \times 10^6$ events to achieve a sensitivity threshold of approximately $2 \times 10^{-5}$ (two neoplastic plasma cells per 100,000 total nucleated cells).

Core Gating Strategy

Plasma cells are initially identified by co-expression of CD38 (bright) and CD138, forming the backbone gate from which all further phenotypic interrogation proceeds:

$$\text{Plasma cell gate} = \text{CD38}^{\text{bright}} \cap \text{CD138}^{+}$$

Events within this gate are then assessed for markers distinguishing normal from neoplastic plasma cells.

Key Antigens and Their Significance

Specimen Processing and Pre-Analytical Considerations

Sample Type and Processing

Bone marrow aspirate is the primary specimen. Critical pre-analytical factors include:

• Time to processing: Ideally within 24 hours; delayed processing degrades CD138 expression
• Anticoagulant: EDTA is standard; heparin may interfere with some staining protocols
• Lipid-phase loss: Plasma cells associated with bone marrow fat are preferentially lost during density gradient centrifugation, the principal reason flow cytometry consistently underestimates total plasma cell percentage relative to morphology. Red cell lysis techniques that avoid density gradient processing are preferred for MRD applications
• Bone marrow trephine biopsy: Not processed directly by flow cytometry; assessed with CD138 and MUM-1 (IRF4) immunohistochemistry when aspirate quality is suboptimal

Peripheral Blood Immunophenotyping

• Flow cytometry applied to peripheral blood detects circulating plasma cells (CPCs).
• Detection of CPCs at any level is associated with a more aggressive clinical course.

Diagnostic thresholds for plasma cell leukaemia (PCL) differ between classification systems:

The ICC 2022 threshold is lower and captures a broader at-risk population; candidates should be familiar with both.

Diagnostic Application

Confirming Clonality and Cell Lineage

Flow cytometry confirms two fundamental diagnostic requirements:

1. Lineage: Population is of plasma cell origin based on CD38-bright/CD138-positive phenotype with cytoplasmic immunoglobulin expression
2. Clonality: Demonstrated by light chain restriction (κ-only or λ-only cytoplasmic staining)

Light chain restriction must be assessed using cytoplasmic (intracellular) staining, as surface light chains on plasma cells are unreliable due to passive adsorption from serum immunoglobulin.

Distinguishing Neoplastic from Normal/Reactive Plasma Cells

• The combination of aberrant marker expression alongside light chain restriction constitutes the immunophenotypic signature of neoplastic plasma cells.
• In reactive plasmacytosis (e.g. infection, connective tissue disease), plasma cells retain the normal phenotype.

Quantification of Plasma Cells

• Flow cytometric quantification underestimates plasma cell percentage due to lipid-phase loss.
• Morphological assessment of the aspirate and CD138 immunohistochemistry on the trephine biopsy remain the reference standards for the diagnostic ≥10% plasma cell threshold required for multiple myeloma diagnosis.
• Flow cytometry complements rather than replaces these methods at diagnosis.

MRD Assessment by Flow Cytometry

Rationale and Clinical Significance

MRD-negative status following treatment of newly diagnosed multiple myeloma is established as a surrogate for improved progression-free survival (PFS) and overall survival (OS). MRD negativity is incorporated into IMWG response criteria as a distinct category beyond complete response (CR):

Next-Generation Flow (NGF)

The EuroFlow consortium standardised NGF for myeloma MRD using two 8-colour tubes:

• Tube 1 (bulk identification): CD138, CD38, CD45, CD19, CD56, CD27, CD81, CD117
• Tube 2 (clonality/additional markers): CD138, CD38, CD45, CD19, cytoplasmic κ, cytoplasmic λ, CD200, CD28
• This approach achieves sensitivity of $2 \times 10^{-5}$ to $2 \times 10^{-6}$ when $\geq 2 \times 10^6$ events are acquired.
• NGF is considered equivalent in sensitivity to next-generation sequencing (NGS)-based MRD when $\geq 10^6$ events are collected, with the advantages of real-time results and no requirement for a patient-specific clonotypic baseline sequence.

MRD Threshold and Reporting

$$\text{MRD positive} = \text{detection of} \geq 1 \text{ aberrant PC per } 10^5 \text{ total nucleated cells}$$

• MRD negativity is defined as no detectable aberrant plasma cells at a sensitivity of at least $10^{-5}$.
• Results must explicitly state the sensitivity level achieved (i.e. events acquired), as inadequate cellularity invalidates a negative result.

DNA Content Analysis and Proliferation Studies

Ploidy Assessment

Flow cytometry measures plasma cell DNA content using intercalating dyes (e.g. propidium iodide, DAPI) combined with plasma cell identification markers, classifying tumours into:

• Ploidy status correlates with FISH findings and is an independent prognostic variable.
• The proportion of normal residual plasma cells in the same specimen also provides important prognostic information.

Plasma Cell Labelling Index (PCLI)

• The PCLI measures the S-phase fraction of plasma cells using bromodeoxyuridine (BrdU) incorporation followed by immunofluorescent detection.
• A PCLI ≥3% identifies a high-proliferation phenotype associated with aggressive behaviour.
• PCLI is not widely accessible and is not required for routine diagnostic workup.

Phenotypic Considerations in Special Situations

Post-Daratumumab Therapy

Daratumumab targets CD38 and causes significant downregulation of CD38 expression on neoplastic and normal plasma cells, haematopoietic progenitors, and natural killer cells, creating challenges for:

• MRD assessment: CD38-dim or CD38-negative residual plasma cell populations may be missed by standard CD38-based gating; panels must incorporate CD138 as an independent identifier, and alternative backbone markers (e.g. VS38c, CD319/SLAMF7) may be required
• Interpretation window: CD38 re-expression typically recovers 3-6 months after daratumumab cessation; MRD assessments during active therapy require adjusted gating strategies
• NK cell interference: Daratumumab-associated NK cell depletion (CD38+ NK cells) may complicate gating in peripheral blood panels

Nonsecretory Myeloma

• Nonsecretory myeloma (approximately 3% of cases) produces no detectable M-protein in serum or urine.
• Flow cytometry confirming clonal plasma cell immunophenotype is essential for diagnosis and monitoring.
• The serum free light chain (FLC) assay is abnormal in >60% of nonsecretory cases and may serve as a biochemical monitoring tool, but bone marrow flow cytometry-based MRD assessment remains more sensitive for tracking disease.

Plasma Cell Leukaemia

• In primary PCL, peripheral blood flow cytometry characterises the circulating clone.
• The immunophenotype frequently differs from typical PCM: higher rates of CD20 expression, more frequent CD56 negativity, and CD45 positivity, partially overlapping with lymphoplasmacytic lymphoma or late-stage B-cell lymphomas.
• Clonal cytoplasmic light chain restriction and CD38-bright/CD138-positive phenotype confirm plasma cell lineage.

POEMS Syndrome and AL Amyloidosis

• Plasma cell burdens are often low (<10% by morphology) in both conditions.
• Flow cytometry, with its ability to detect small clonal populations against a background of normal plasma cells, may establish clonality when morphological assessment is equivocal.
• In AL amyloidosis, the clone is frequently λ-restricted, often CD56-negative, and may be CD20-positive, a phenotypic pattern associated with t(11;14) and cyclin D1 overexpression, with therapeutic implications (venetoclax activity in the t(11;14) subgroup, though not recommended in other cytogenetic subtypes due to adverse overall survival effects).

Differential Diagnosis Aided by Flow Cytometry

Quality Assurance and Standardisation

Robust plasma cell flow cytometry requires:

• Standardised panels: EuroFlow-validated 8-colour NGF protocols ensure inter-laboratory reproducibility
• Adequate events: Minimum $1 \times 10^6$ for diagnosis; $\geq 2 \times 10^6$ for MRD at $10^{-5}$ sensitivity
• Normal plasma cell reference: Residual normal plasma cells (CD19+/CD45+/CD56−/polytypic) in the same specimen serve as an internal quality control population
• Reporting standards: Results should state (a) total events acquired, (b) number of plasma cell events detected, (c) sensitivity level achieved, (d) MRD-positive or MRD-negative conclusion, and (e) technical limitations (e.g. inadequate plasma cell events, prior daratumumab exposure)

Relationship to Other Diagnostic Modalities

Summary of Key Points for Examination

• The backbone plasma cell gate uses CD38-bright and CD138 co-expression; all phenotypic analysis proceeds from this gate
• Neoplastic plasma cells are characteristically CD19-negative, often CD56-positive, CD45-negative/dim, and light chain-restricted on cytoplasmic staining
• Flow cytometry underestimates plasma cell percentage compared to morphology due to preferential loss of lipid-phase-associated plasma cells during specimen processing
• NGF achieves MRD sensitivity of $2 \times 10^{-5}$ (or better with higher event counts) and is incorporated into IMWG response criteria
• MRD-negative status after treatment for newly diagnosed MM is associated with improved long-term survival
• Daratumumab therapy causes CD38 downregulation, requiring panel adaptation (CD138-based gating, alternative markers such as VS38c or CD319/SLAMF7) for valid MRD assessment
• DNA ploidy analysis identifies hyperdiploid vs. non-hyperdiploid groups with independent prognostic significance; proportion of normal residual PCs also provides prognostic information
• PCLI using BrdU incorporation measures plasma cell proliferation; not widely available and not required in most patients
• Peripheral blood flow cytometry for CPCs has prognostic relevance; ICC 2022 lowers the PCL threshold to $\geq 0.5 \times 10^9$/L or ≥5% CPCs (compared with WHO 2022 threshold of ≥20%)
• Conventional karyotype analysis is technically difficult in PCM due to the slow-growing nature of plasma cells; FISH is the preferred cytogenetic modality