Why Flow Cytometry is Performed
Flow cytometry is performed to rapidly identify, characterize, and quantify specific cell populations through multiparametric analysis of surface and intracellular markers, enabling precise diagnosis, prognostication, and treatment monitoring in hematologic malignancies. 1
Primary Clinical Applications
Flow cytometry serves three essential diagnostic functions in clinical hematology:
Diagnosis and Classification of Hematologic Malignancies
Flow cytometry enables rapid diagnosis and classification of acute leukemias and chronic lymphoproliferative disorders through simultaneous assessment of multiple cell surface antigens, providing definitive immunophenotyping within hours rather than days. 1
The technique allows differential diagnosis between neoplastic and reactive conditions by identifying aberrant immunophenotypes and establishing clonality through light chain restriction analysis. 2, 1
In multiple myeloma and plasma cell disorders, flow cytometry identifies phenotypically abnormal plasma cells using markers like CD138, CD38, and CD45, distinguishing malignant from reactive plasmacytosis with greater specificity than immunohistochemistry alone. 2, 1
Prognostic Risk Stratification
Flow cytometry identifies independent prognostic markers that predict disease progression, particularly in MGUS and asymptomatic myeloma, where the ratio of aberrant to normal plasma cells determines risk. 2, 1
Patients with ≥95% phenotypically aberrant plasma cells face significantly higher risk of progression to active myeloma, enabling risk-adapted treatment strategies. 1
Specific antigen expression patterns (CD56, CD45, CD117, CD28) provide additional prognostic information that influences treatment decisions. 2
Minimal Residual Disease (MRD) Detection and Treatment Monitoring
Quantitative MRD assessment by flow cytometry predicts treatment outcomes and determines stringent complete remission as defined by the International Myeloma Working Group. 2, 1
The technique achieves sensitivity of 1 in 10⁴ to 10⁵ cells when adequate cell numbers are analyzed, though this requires at least 10-20 positive events to call a sample positive. 2
Flow cytometry provides objective, reproducible quantification of residual disease burden, superior to morphological assessment alone for predicting relapse risk. 2
Technical Advantages Over Conventional Methods
Speed and Efficiency
Flow cytometry analyzes thousands of cells per second, providing results within hours compared to days required for conventional cytogenetic or molecular techniques. 2, 3
Fast sample throughput enables timely clinical decision-making, particularly important in acute leukemias requiring rapid diagnosis. 2
Multiparametric Analysis
The ability to simultaneously measure 10-30+ parameters on individual cells provides comprehensive phenotypic characterization impossible with microscopy or immunohistochemistry. 3, 4, 5
Combined assessment of cell size, granularity, and multiple fluorescent markers enables precise identification of rare cell populations within heterogeneous samples. 2, 6
Objectivity and Reproducibility
Unlike morphological assessment, flow cytometry provides quantitative, objective data that reduces inter-observer variability, though gating strategies still require experienced interpretation. 2
Automated counting and recording of fluorescent events improves reproducibility compared to manual microscopy. 2
Critical Limitations and Caveats
Sensitivity Constraints
Flow cytometry sensitivity is fundamentally limited by the requirement for 10-20 positive events, meaning sensitivity cannot exceed 1 in 10⁴ cells when only 1-2 × 10⁵ cells are available. 2
Post-chemotherapy samples with low cellularity may not provide sufficient cells for optimal sensitivity, potentially missing low-level disease. 2
Loss of Morphological Context
Flow cytometry analyzes suspended cells without cytomorphological features beyond size and granularity, potentially missing diagnostic information visible by microscopy. 2
The technique cannot document antigen expression and genetic abnormalities on two-dimensional images as comprehensively as fluorescence microscopy. 2
Sample Quality Dependence
Discrepancies between flow cytometry and morphological plasma cell counts often result from using secondary aspirates of poorer quality than primary aspirates. 2
Expression of adhesion molecules and counting errors can impact accuracy of cell enumeration. 2
Essential Technical Requirements
Optimal Marker Panels
CD38, CD138, and CD45 should all be included in at least one tube for plasma cell identification and enumeration, with primary gating based on CD38 vs. CD138 expression. 1
A minimal panel for detecting abnormal plasma cells must include CD19 and CD56, while preferred panels add CD20, CD117, CD28, and CD27. 1
Cytoplasmic kappa and lambda light chains are essential for clonality assessment. 1