FLOW CYTOMETRY
AND LABORATORY
AUTOMATION
Reporters:
Doreen Tactacon Angie Lee Lungay
Learning Outcomes
1. List the major components of a flow cytometer and
describe their functions
2. Differentiate between intrinsic parameters (FSC and
SSC) and extrinsic parameters (fluorescence)
3. Identify key clinical applications of flow cytometry
including HIV monitoring and leukemia phenotyping
4. Distinguish T-cell, B-cell, and NK-cell populations
based on CD marker expression
5. Compare batch analyzers versus random-access
analyzers and define key validation terms
What is Flow Cytometry?
· Technology that analyzes individual cells as they pass through a laser beam
· Measures two types of properties:
· Intrinsic: Physical characteristics (size, granularity)
· Extrinsic: Presence of proteins using fluorescent-labeled antibodies
· Key advantage: Analyzes thousands of cells per second, detecting rare
populations
The Core Components of a Flow Cytometer
· Fluidics System:
· Uses hydrodynamic focusing to align cells single file through the laser
· Laser Light Source:
· Provides light to excite cells and fluorescent labels
· Optics and Photodetectors:
· Collect scattered and emitted light
· Convert light signals into electrical pulses for analysis
Intrinsic Parameters: Light Scatter Extrinsic Parameters: Fluorescence
· Forward Scatter (FSC): · Fluorochromes: Molecules that absorb light and
· Measures light scattered in forward direction emit longer wavelengths
· Indicates relative cell SIZE · Each fluorochrome has unique absorption and
· Side Scatter (SSC): emission patterns
· Measures light scattered at 90-degree angle · The Process:
· Indicates internal GRANULARITY or complexity · Fluorescent-labeled antibodies bind to specific cell
markers
These two parameters alone can differentiate · Laser excites the fluorochrome
lymphocytes, monocytes, and neutrophils in lysed blood · Detectors measure emitted fluorescence
· Multiple fluorochromes allow several markers to be
analyzed on one cell simultaneously
Histograms vs. Dot Plots
· Single-Parameter Histogram:
· One parameter (x-axis) versus number of events (y-axis)
· Determines percentage of positive cells for a single marker
· Dual-Parameter Dot Plot:
· Two parameters plotted against each other
· Each dot represents one cell
· Identifies populations based on co-expression of markers
Gating and Quadrant Analysis
· Gating:
· Isolates a specific cell population for further analysis
· Filters out debris and isolates subpopulations of interest
· Quadrant Analysis:
· Divides dot plot into four quadrants
· Separates positive from negative events on each axis
· Quadrant 2: Cells positive for both markers (e.g., CD3+CD4+ T-helper cells)
Clinical applipications
Malignant Immunophenotyping
Nonmalignant Leukemia and Lymphoma:
Immunophenotyping · Classifies cancers by lineage (B-cell,
· HIV/AIDS Monitoring: T-cell, myeloid)
· Enumeration of CD4+ T-cells to stage disease · Essential for diagnosis and post-
and guide treatment therapeutic monitoring
· Monitors response to antiretroviral therapy · Minimal Residual Disease Detection:
· Inherited Immunodeficiencies:
· Identifies small numbers of abnormal
· Bruton's agammaglobulinemia: Lack of mature
B cells (CD19+)
cells
· Chronic Granulomatous Disease: Defective · Rare cell events can be detected
neutrophil oxidative burst statistically
· Paroxysmal Nocturnal Hemoglobinuria: · Example:
· Cells lack GPI anchors for surface proteins · Chronic Lymphocytic Leukemia: Cells
· Diagnosed by detecting missing anchor proteins positive for both CD5 and CD20
Important CD Antigens
CD3: All mature T-cells
· CD4: T-helper cells, monocytes; HIV receptor
· CD8: Cytotoxic T-cells
· CD19/CD20: B-cells
· CD16/CD56: Natural Killer cells
· CD34: Hematopoietic stem cells, immature leukemias
· CD45: All leukocytes (pan-leukocyte marker); expression varies with
maturity
Types of Automated Analyzers
· Batch Analyzers:
· Perform one type of test at a time
· Cannot process STAT samples
immediately
· Ideal for high-volume, single-test labs
· Random-Access Analyzers:
· Perform multiple different tests in one Ensuring Quality Results
run
· Can process STAT samples immediately · Accuracy: Measures what it claims to
· Offer greater flexibility for varied test measure
Immunoassay Analyzers: menus · Precision: Reproducibility on repeated
testing
· Goals of Automation: · Analytic Sensitivity: Lowest detectable
· Reduce manual tasks and errors amount of analyte
· Handle large sample volumes · Analytic Specificity: Negative result when
· Decrease turnaround time analyte is absent
· Reportable Range: Range of values that
· Key Benefits:
generate positive results
· Greater accuracy and precision · Reference Interval: Normal values found
· Reduced human error in healthy individuals
· Faster results
· More testing with less staff
Conclusion
· Flow cytometry measures size, granularity, and fluorescence of individual
cells
· Forward scatter indicates size; side scatter indicates complexity
· Fluorochrome-labeled antibodies detect specific cell markers
· Immunophenotyping diagnoses HIV, leukemias, lymphomas, and
immunodeficiencies
· Automation improves accuracy, precision, and turnaround time
· Batch analyzers run one test; random-access analyzers run multiple tests
· New methods require validation of accuracy, precision, sensitivity,
specificity, reportable range, and reference interval
THANK
YOU