What is Flow Cytometry
Flow cytometry is a laser-based analytical technique that measures physical and biochemical properties of individual cells as they pass single-file through a laser beam, enabling rapid, multiparametric analysis of cell populations through detection of scattered light and fluorescence signals. 1
Core Technical Principles
Flow cytometry operates by suspending cells in a fluid stream and passing them one by one across laser interrogation points. 2 The technology measures:
- Light scattering properties that reflect cell size and internal granularity 1
- Fluorescence emission from fluorochrome-conjugated antibodies or fluorescent probes bound to specific cellular markers 3
- Multiple parameters simultaneously on individual cells, allowing analysis of up to 50 different markers in a single tube with modern instruments 4
The fundamental mechanism involves particles passing through a laser beam, scattering light and emitting fluorescence signals to multiple measurement channels that are then collected and analyzed. 1
Primary Clinical Applications
Hematologic Malignancies
Flow cytometry is mandatory for diagnosis and monitoring of acute leukemias and chronic lymphoproliferative disorders, as established by the International Myeloma Working Group. 5 Specific applications include:
- Differential diagnosis between neoplastic plasma cell disorders and reactive plasmacytosis 5
- Immunophenotyping of hematologic cells through cell surface antigen determinations 6
- Minimal residual disease detection with sensitivity reaching 1 in 10⁴ to 10⁵ cells when adequate cell numbers are analyzed 1
Multiple Myeloma and Plasma Cell Disorders
For plasma cell analysis, the technique enables:
- Enumeration of plasma cells in bone marrow with demonstration of phenotypically abnormal, monoclonal populations 1
- Clonality assessment through kappa/lambda light chain ratio analysis 5
- Prognostic stratification based on relative proportions of abnormal versus normal plasma cells 1, 5
The European Myeloma Network recommends CD38, CD138, and CD45 should all be included in at least one tube for plasma cell identification, with primary gating based on CD38 versus CD138 expression. 5
Key Advantages Over Traditional Methods
Flow cytometry provides several distinct benefits compared to conventional microscopy and immunohistochemistry:
- Speed: Results available within hours, with fast sample throughput 1
- Objectivity: A positive result is defined as a cluster of at least 10-20 flow cytometric events and does not depend solely on cytomorphological skills of the observer 1
- Quantitative precision: Allows relatively quantitative, multiparametric analysis at the single cell level 3
- Cell sorting capability: Physical separation of cell subpopulations based on different parameters at exceptionally high speeds approaching 100% purity 2
- Multiparametric analysis: Simultaneous assessment of multiple markers provides more specific information than can be obtained by immunohistochemistry alone 1
Critical Limitations and Technical Considerations
Sensitivity Constraints
The requirement for at least 10-20 events to call a sample positive limits sensitivity. 1 To reach sensitivity of 1 in 10⁵ cells, 1-2 × 10⁶ cells are required for analysis, which is not always feasible especially in post-chemotherapy samples. 1
Morphological Limitations
Flow cytometry analyzes suspended cells for size, granularity, and antigen expression, but no other cytomorphological features can be recorded. 1 This contrasts with immunocytology where detailed cellular morphology can be assessed. 1
Observer-Dependent Elements
Despite being more objective than microscopy, flow cytometry is not completely objective—correct distinction between positive events and high background depends on the observer's personal experience with the instrument and applied antibodies. 1
Sample Quality Issues
For extracellular vesicles and small particles, instrument settings used for cell analysis are inappropriate and must be optimized specifically, including trigger channel, threshold, detector voltages, and flow rate. 1 The fluorescence and scatter sensitivity of different flow cytometry instrument designs can vary by more than an order of magnitude. 1
Common Pitfalls to Avoid
- Insufficient cell numbers: Inadequate sample volume or insufficient flow cytometry events can lead to false negatives or reduced diagnostic accuracy 7
- Inadequate antibody panels: Using minimal markers when comprehensive phenotyping is needed 7
- Delayed processing: Samples must be processed promptly to prevent cellular degradation 7
- Ignoring specimen adequacy: Particularly important to confirm marrow elements are present in follow-up samples 5
- Misinterpreting negative results: After treatment, clonality assessment is only informative when combined with immunophenotype to detect abnormal cells 5