Can flow cytometry for detecting plasma cell dyscrasias (plasma cell disorders) produce false positives?

Flow Cytometry Can Produce False Positives in Detecting Plasma Cell Dyscrasias

Yes, flow cytometry for detecting plasma cell dyscrasias can produce false positives due to technical limitations and interpretation challenges. 1

Common Causes of False Positives

• Gating strategy limitations: Using CD38 vs. CD138 for primary gating can lead to high contamination rates with non-plasma cells, inhibiting the ability to accurately demonstrate abnormal phenotypes 1
• Inadequate antibody panels: Insufficient marker combinations may fail to properly distinguish between normal and neoplastic plasma cells, leading to misinterpretation 2
• Sample preparation issues: Density gradient centrifugation can cause variable increases or decreases in plasma cell percentages compared to whole blood/marrow approaches, potentially leading to inaccurate enumeration 1
• Technical limitations: Two-color immunophenotypical analyses are not feasible for accurate plasma cell identification, as at least two antigens are required to properly gate plasma cells 1

Optimal Technical Approaches to Minimize False Positives

• Use four or more detectors: The European Myeloma Network recommends using at least four-color flow cytometry to more easily identify and reproduce abnormal populations 1

• Proper gating strategy: The combined use of CD38, CD138, and CD45 together with light scatter characteristics provides the optimal detection rate and concordance between different operators 1

  • The primary gate should be set using CD38 vs. CD138 expression rather than CD38 vs. CD45 to ensure CD45+ plasma cells are not excluded 1

• Adequate event acquisition: A minimum of 1,000 total events should be analyzed to achieve sufficient sensitivity for detecting abnormal plasma cells 2

• Proper sample preparation: Any whole blood/marrow approach is suitable for plasma cell enumeration and phenotyping, but density gradient centrifugation should be avoided 1

Distinguishing Normal from Abnormal Plasma Cells

• Normal plasma cells typically express:

  • CD19+/CD56- phenotype 2
  • Balanced kappa:lambda light chain ratios (between 1:2 and 4:1) 2

• Abnormal plasma cells typically show:

  • CD19-/CD56+ phenotype (in approximately 95% and 75% of myeloma cases, respectively) 1
  • Monoclonal restriction with kappa:lambda ratios of >4:1 or <1:2 2
  • Abnormal CD81 expression (95% sensitivity, 100% specificity for abnormal plasma cells) 3

Strategies to Improve Accuracy

• Use standardized approaches: The European Myeloma Network recommends standardized approaches with defined specificity and sensitivity, along with suitable quality control schemes 4
• Include multiple markers: Essential markers include CD19 and CD56, with recommended additional markers such as CD117, CD20, CD28, CD27, CD81, and CD200 1
• Proper controls: Controls for staining should be in accordance with standard flow cytometry procedures; isotype controls are not required for gating reagents as they are controlled internally 1

Clinical Implications of False Positives

• Misdiagnosis risk: False positives could lead to unnecessary treatment for a plasma cell dyscrasia that isn't present 5
• Prognostic implications: The ratio between phenotypically normal and aberrant plasma cells can predict the risk of disease progression in MGUS and asymptomatic myeloma, so false positives could lead to inaccurate risk stratification 1
• Treatment response assessment: Inaccurate flow cytometry results could affect minimal residual disease (MRD) assessment, which is increasingly used as a surrogate endpoint for survival 6

By understanding these limitations and following recommended technical approaches, laboratories can minimize the risk of false positives in flow cytometry for plasma cell dyscrasias, improving diagnostic accuracy and patient outcomes.