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Flow Cytometry

Introductory lecture

What Is Flow Cytometry?
• • • • Flow ~ particles (such as cells) in motion Cyto ~ cell Metry ~ measurement Measuring properties of cells in a fluid stream

•Flow cytometry uses the principles of light scattering , fluorescence to generate specific multi-parameter data from particles and cells in the size range of 0.5 – 40 m diameter. This process is performed at rates of thousands of cells per second.

MICROSCOPY

FLOW CYTOMETRY

FLUORESCENCE DETECTO R

FLUORESCENCE DET ECT OR

LASER

Parameter Means of Ana lysis Means of De tection Number of Cells Analyzed Rate of Analysis Sensitivity Data Type

Microscopy Cells immobilized on microscope slide, counted manually Eye Hundreds 100 cells per minute Low-only strong p rotein expression can be detected Qualitative-Cells scored as +/-

Flow Cytometry Cells suspended in liquid stream, pass single file in front of laser Electronic Thousands-M illions 2000-5000 cells/second High-Weak protein exp ression can be c learly identified. Quantitative-Fluorescenc e of each cell individu ally scored

Cytometry vs. Flow Cytometry
Cytometry • Localization of antigen is possible • Poor enumeration of cell subtypes • Limiting number of simultaneous measurements Flow Cytometry. • Cannot tell you where antigen is. • Can analyze many cells in a short time frame. • Can look at numerous parameters at once.

In flow cytometry, a sheath surrounds the sample to hydrodynamically focus the cells to the center, where they are illuminated by a laser. Forward scatter measures cell size. Site scatter measures cell complexity.

Fluorescence Activation Process (or Immunofluorescence)
Antibodies recognize specific molecules in the surface of some cells
FITC FITC Antibodies

Antibodies are artificially conjugated to fluorochromes

FITC FITC

When the cells are analyzed by flow cytometry, the cells expressing the marker for which the antibody is specific will manifest fluorescence. Cells who lack the marker will not manifest fluorescence

But not others

Fluorescence Compensation

1 Parameter Histogram
Positive

Negative
Count
6

Dimmer

Brighter

4

1

1 2 3 4 6 7

150 160 170 .. 190

Channel Number Fluorescence picked up from the FITC PMT

www.hmds.org.uk/pnh_review.html

2 Parameter Histogram
Single Positive PI Population Double Positive Population

PE FL

Negative Population

FITC FL

Single Positive FITC Population

Specimen Handling and Sample Preparation
• Single cell suspension: PB, BM, LN, etc. Blood and bone marrow may be anticoagulated with EDTA or heparin. Tissue specimens: transported and stored in tissue culture media. • Specimens of all types are commonly stored at room temperature prior to preparation. •RBC lysis: Analysis of white blood cells in PB or BM requires erythrocyte removal for efficient evaluation: ammonium chloride or commercial preparations • Once prepared, samples should be analyzed as soon as possible to avoid specimen and fluorochrome degradation, although fixation and refrigerated storage will delay degradation. • Number of cells: >=50,000 cells. For MRD evaluation, acquisition of a larger number (>250,000) events is often desirable.

Common Clinical Indications
• Diagnosis and Classification • Prognostic Factors • Therapeutic Targets

• Residual Disease Monitoring in Leukemia

Diagnosis and Classification
• Key in FCM interpretation is pattern recognition
– Mature:
• • • • B-cell T-cell NK cell Plasma cell

– Immature:
• Myeloid • B or T • Bilineages or no differentiation

Blood or bone marrow

Side Scatter

Granulocytes ? (Erythroids, Plasma Cells, Other)

? Blasts

Monocytes ? Lymphocytes ? CD45 Hematogones

Cells are gated based on side scatter and CD45 intensity

1st step: Recognize the abnormal cell population
Acute leukemia CLL/ mature Lymphoid leukemia

These are two examples of easy cases. For the cases with small tumor load or for MRD detection, a big panel of antibodies usually need to identify the abnormal cells. You need to know the normal cell phenotypes very well to design the panel and to interpret the results.

Side Scatter

Side Scatter

B-ALL

Surface marker expression during normal myeloid maturation

Immature hematopoietic malignancy
• CD45 • CD34, CD117, HLA-DR, TdT, CD1a

Blast Markers
• CD34 (often expressed on myeloid and lymphoid blasts; usually absent from promyelocytes) • CD117 (usually myeloid) • TdT (often lymphoid) • CD1a (early T-cells) • HLA-DR (early myeloblasts, monoblasts, Blymphoblasts, but not T-lymphoblasts or promyelocytes) • CD123

• Abnormal Blast ImmunoPhenotypes: Leukemia
Associated Phenotypes or Leukemia Associated Immunohenotypes (LAP or LAIP)
- Increased or decreased normal antigens - Asynchronous maturational expression - Aberrant antigen expression - Homogeneous

expression

Acute leukemias with a multilineage antigenic profile mixed phenotype acute leukemia , MPAL

Mature B-cell neoplasms

Prognostic Factors

In aggressive disease, the CLL cells usually express an unmutated immunoglobulin heavy-chain variable-region gene (IgVH) and the 70-kD zeta-associated protein (ZAP-70), whereas in indolent disease, the CLL cells usually express mutated IgVH but lack expression of ZAP-70. The presence of an unmutated IgVH gene is strongly associated with the expression of ZAP-70, ZAP-70 is a stronger predictor of the need for treatment in B-cell CLL.

N Engl J Med. 2004 Aug 26;351(9):893-901

Therapeutic Targets

Rituxan® (rituximab): anti-CD20 monoclonal antibody, is indicated for the treatment of patients with CD20-positive B-cell NHL /leukemia, some autoimmune disease. Alemtuzumab: anti-CD52 monoclonal antibody, is approved for use in the treatment of patients with CLL relapsed or refractory to fludarabine, or T-cell prolymphocytic leukemia. CD52 is expressed on a variety of hematopoietic cells, including normal and malignant T- and B-lymphocytes; CD52 is not expressed on hematopoietic stem cells. Others: Anti-CD38 antibody, anti-CD22, anti-CD30, anti-CD33, …

Residual Disease Monitoring

Minimal Residual Disease
Minimal residual disease (MDR) in acute leukemia: Presence of small number of abnormal blasts during or after treatment, at a level below the sensitivity of morphology
(<5%). – Detectable by flow cytometry, cytogenetics, or molecular

studies
The presence of MRD has been established as an important independent prognostic factor: Highly correlates with relapse and EFS.

EFS of 1971 patients and MRD at end of induction

Methods for Detecting MRD

(5%)

(5%) (1%) (0.01%) 10-4 (0.001%) 10-5 (0.0001%) 10-6

Population Identification
• Leukemia-Associated immunophenotype (LAIP)
- Advantages
• Conceptually simple and objective • Reduced reagent expense for follow up

- Disadvantages
• Requires pre-treatment sample to define LAIP • Requires immunophenotypic stability • Any event in pre-defined gate regarded as MRD

• Deviation from normal maturation
- Advantages
• Does not require pretreatment sample • Uniform reagent combinations utilized • Improved specificity through population identification • Less sensitive to immunophenotypic instability

- Disadvantages
• Requires detailed immunophenotypic knowledge (expert) • Subjective • More time consuming