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Keywords:

  • plasma cells;
  • multicolor flow cytometry;
  • light chain restriction;
  • multiple myeloma

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. LITERATURE CITED

Background:

Multicolor flow cytometry (MFC) is increasingly important for the diagnosis and minimal residual disease (MRD) assessment of patients with plasma cells (PC) dyscrasias, like multiple myeloma. Recently published information shows that immunophenotype of myeloma PC can change over time and normal PC are heterogeneous in the expression of CD19 and CD56. This implies that for a sensitive, reliable detection of MRD clonality assessment by the detection of cytoplasmic kappa and lambda light chains is advisable.

Methods:

Eight-color MFC was used to detect normal and myeloma PC by the expression of CD38 and CD138. Analysis of additional surface antigens like CD45, CD19, CD56, CD27, and the intracellular immunoglobulin light chain distribution were used to differentiate polyclonal from clonal PC.

Results:

Absence of cytoplasmic light chains expression in a PC subpopulation with an abnormal phenotype suggested the presence of non-secretory plasma cells in the bone marrow (BM) of this patient. This observation however, was contradicted by the presence of free lambda light chains in the patient's serum. After repeating the analysis with polyclonal antibodies against intracellular immunoglobulin light chains instead of monoclonal antibodies, the abnormal PC subpopulation appeared to express lambda light chains.

Conclusion:

These data illustrate that if clonality assessment of PC is included in disease monitoring, the use of polyclonal over monoclonal antibodies is preferred for the detection of intracellular immunoglobulin light chains. © 2012 International Clinical Cytometry Society


MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. LITERATURE CITED

The flow cytometric analysis as described in this case report was performed on a heparinized follow-up bone marrow aspirate from a multiple myeloma patient which was transferred to the University Medical Center Utrecht. The patient had prior been treated with an autologous and allogeneic bone marrow transplant and was receiving treatment with lenalidomide and dexamethasone at the time of referral. At the time of bone marrow sampling the serum M-protein concentration was 10 g/L IgG-lambda with an excess of serum-free lambda light chains (275 mg/L, Freelight (The Binding Site, Birmingham, UK) measured on an Image Nephelometer (Beckman Coulter, Miami, FL USA). For the flowcytometric analysis, whole bone marrow cells (1 × 106) were incubated with the following titrated monoclonal antibodies in a total volume of 180 μL for 15 min at room temperature in the dark: CD38 allophycocyanin (APC), CD138 PercP-Cy5.5, CD27 Horizon V500, CD56 Pe-Cy7 (all Becton Dickinson, Mountain View, CA), CD45 Pacific blue (DakoCytomation, Heverlee, Belgium) and CD19 APC-A750 (Beckman Coulter, Immunotech, Marseille, France). Subsequently, cells were washed and treated with IntraStain, according to manufacturer's instructions (DakoCytomation). Intrastain allows simultaneous detection of surface- and intracellular antigens. In this analysis, monoclonal antibodies kappa fluorescein isothiocyanate (FITC) and lambda phycoerythrin (PE) (Becton Dickinson, clones TB28-2 and 1-155-2, respectively) were used to detect cytoplasmic light chains. Using the same conditions, an additional staining on whole bone marrow cells (1 × 106) was performed using CD27 Horizon V500, CD138 PercP-Cy5.5, CD38 Pe-Cy7 (all Becton Dickinson), CD45 Pacific blue (DakoCytomation), CD19 APC-A750 (Beckman Coulter, Immunotech) in combination with staining for cytoplasmic plasma cell marker VS38c-FITC and cytoplasmic polyclonal rabbit IgG F(ab′)2 kappa APC and polyclonal rabbit IgG F(ab′)2 lambda PE (MultiMix triple-colour TC670, DakoCytomation).

Cell analysis was performed on a 3-laser Canto II flow cytometer (Becton Dickinson). Compensation beads were used to determine spectral overlap and compensation was automatically calculated using Diva software. Fluorescent labeled beads (CS&T beads, Becton Dickinson) were used to standardize the flow cytometer and verify optical path and stream flow. This procedure enables controlled standardized results and allows the determination of long-term drifts and incidental changes within the flow cytometer. CS&T beads were used before each analysis to verify optimal performance of the flow cytometer. No changes were observed which could affect the results. PC in the BM sample were identified as described (1) and shown in Figure 1. In short, CD38, CD138, and CD45 expression and light scatter characteristics were used to identify PC, including possible CD45 positive PC (Figs. 1A and 1B).

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Figure 1. Characterization of PC. A: PC are positive for CD38 and CD138. B: Based on CD45 expression two PC subpopulations (1 and 2) are discerned. C: PC subpopulation 1 is positive for CD19. D: PC subpopulation 2 is negative for CD19. E: PC in subpopulation 1 stain for cytoplasmic kappa or lambda using monoclonal antibodies. F: PC in subpopulation 2 do not stain for cytoplasmic kappa or lambda using monoclonal antibodies. G: PC in subpopulation 1 stain for cytoplasmic kappa or lambda using polyclonal antibodies. H: PC in subpopulation 2 stain for cytoplasmic lambda using polyclonal antibodies. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. LITERATURE CITED

Recent advances in multicolor flow cytometry allow a detailed analysis of PC in BM of normal individuals and patients with multiple myeloma. Markers that are shared by PC, like CD38 and CD138, are used to identify PC in both normal and patient samples (2, 3). Technical and data analysis guidelines have been published to identify normal and abnormal PC using flow cytometry (1). There is heterogeneity in cell surface antigen expression on normal PC (2), nevertheless differential expression of selected surface makers form the basis for discrimination between normal and abnormal PC. Markers expressed on most normal PC, in addition to CD38 and CD138, include CD19, CD27, and CD45 (4). Myeloma PC may differ from normal PC in the expression of CD19 (5), adhesion molecules like CD56 (6), Lymphocyte Function Antigen 1 (LFA-1)-1 (7) and CD44 splice variants (8), co-stimulatory molecules like CD27 (9) and CD28 (10), and myeloid cell associated antigens like CD33 and CD117 (11). In a recently described statistical model, the absence of CD19 and CD27 on myeloma PC permitted a reproducible detection of minimal residual disease (MRD) (12). These data suggest that immunophenotype alone can provide a reliable sensitive detection of residual myeloma PC. Although in general this may be true, caution is advised since the immunophenotype of myeloma PC can change over time (13) and normal PC are heterogeneous in the expression of CD19 and CD56 (14). This raises the question whether immunophenotype alone, in the absence of a clonality assessment by the detection of cytoplasmic kappa and lambda light chains, can provide a reliable sensitive detection of MRD.

In this report, a follow-up bone marrow sample of myeloma patient was analyzed for the presence of residual abnormal PC by multicolor flow cytometry (MFC). Based on CD38 and CD138 expression, 2.6% plasma cells are detected in the bone marrow of the patient. These PC (Fig. 1A) can be separated in two subpopulations (PC subpopulation 1 (0.4%) and 2 (2.2%) respectively) based on the expression of CD45 (Fig. 1B). Both populations also differ in CD19 expression (Figs. 1C and 1D), are negative for CD56 (Figs. 1C and 1D), and positive for CD27 (data not shown). Analysis of intracellular light chain expression using monoclonal antibodies revealed polyclonality in PC subpopulation 1 and absence of kappa or lambda expression in PC subpopulation 2 (Figs. 1E and 1F). The presence of kappa or lambda positive PC in subpopulation 1 implies these to be normal PC and excludes the possibility of a staining artifact. Absence of cytoplasmic light chains in PC subpopulation 2 might indicate the presence of non-secretory plasma cells in the BM of this patient. However, in the serum of this patient an access of free lambda light chains (275 mg/L) was found. This prompted us to repeat the flow cytometry analysis with a different set of antibodies, including polyclonal antibodies specific for immunoglobulin light chains. The results as depicted in Figures 1G and 1H clearly show that, in contrast to the monoclonal immunoglobulin light chain antibodies (Figs. 1E and 1F), the polyclonal antibodies stained both PC subpopulations (Figs. 1G and 1H) identifying only lambda positive PC in subpopulation 2. This finding suggests that in this patient the epitope recognized by the monoclonal anti-lambda light chain antibody is not expressed on the pathological immunoglobulins. These data furthermore illustrate that if clonality assessment of PC is included in disease monitoring, the use of polyclonal over monoclonal antibodies is preferred for the detection of intracellular immunoglobulin light chains.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. LITERATURE CITED
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