Basic Hematology Course

2015
Flow Cytometric Immunophenotyping
Dr Prashant Tembhare
Tata Memorial Center, Mumbai
Email: docprt@gmail.com
 What is Flow Cytometry?

Cyto = cells

Metry = measurement

Flow = in a flow or a stream

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 Principles of flow cytometry
1. Measurement of physical properties i.e. size and complexity
(granularity).
Right Angle
Light Detector

Forward
c Light
Detector
LASER
BEAM

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 Principles of flow cytometry
2. Measurement of ANTIGENIC properties of cell surface and inside the cell
with the help of antibodies labeled with different fluorochromes.

LASER
BEAM

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 Instrument Components

1. Fluidics: Specimen, Sheath fluid, flow chamber.

2. Optics: Light source(s), mirrors, filters, detectors,

spectral separation

3. Electronics: Controls pulse collection, pulse

analysis, triggering, time delay, data display, gating,
sort control, light and detector control

4. Data Analysis: SOFTWARE - Data display & analysis,

multivariate/simultaneous solutions, identification of
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sort populations, quantitation
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 Fluidics
 Crosland-Taylor - Hydrodynamic focusing = coaxial flow
→ a narrow stream of cells flowing in a core within a wider sheath stream

• Provides a highly controlled fluid stream.

• Provides exact location of a cell in three dimensions
• Maintains sample handling compartment (Flow Cell)
• Forced under pressure through a conical nozzle assembly geometrically
designed to produce a laminar flow

• This fluid is SHEATH FLUID - Isotonic fluid

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 Fluidics

↓D by 10-40 = ↑V by 100-1600 fold

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 2. OPTICS

(a) LASER (argon)

(b) Dichroic Filters and Mirrors

(b) Photodiode

(d) PMT (photo multiplier tubes )

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 What is Fluorescence ?

HO O
= 488 nm = 520 nm
C
Incident CO2H
Light Energy Fluorescein Emitted Fluorescent
Molecule Antibody Light Energy

The fluorochrome absorbs energy from the laser.

• The fluorochrome releases the absorbed energy by:
vibration and heat dissipation.
Page  13 emission of photons of a longer wavelength.
 Mechanism of fluorochrome

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 Excitation & Emission

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 Fluorescence
Emitted fluorescence intensity is proportional to
binding sites

FITC FITC
FITC

FITC
FITC
Number of Events

FITC FITC
FITC

FITC
FITC

0 Log scale of Fluorescent Intensity

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 Emission Spectra

100%
FITC PE APC PerCP
Normalized Intensity

0%
400 500 600 700 800
Wavelength (nm)

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 Basic opticsc

A system of prisms and lenses

directs the laser light to the
interrogation point in the cuvette

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 Electronics

Compute pulse height

Perform calculations for pulse area and pulse width
Calculate ratios
Convert analog signals to proportional digital signals
Interface with the computer for data transfer

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 Optical to Digital

PMT Voltage Log amplification of signals

Signal
Out Analog to
2 Options for SSC and fluorescence channels Digital
Photon Converter
In
Linear amplification of signals

Voltage In

PMT compensation
Power Supply circuit

Levels 0–1000V Gain levels from 0–9.99

Amplifier output voltage
adjusted by slider control adjusted by slider control
ranging between 10mV to 10V
on computer on computer
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 Data Analysis by Software

Display Create Display Analyze

Plots Gates Statistics Statistics

Plot Types: Gate Types: Statistics Types: Results:

Histogram Polygon # of Events % positive for
Dot Ellipse % of Gated particular markers:
Contour Histogram % of Total -viable cells
Density Quadrant -immunophenotype
mean mean fluorescence intensity
geometric mean DNA content
standard deviation absolute counts
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 Sample processing
 Single cell suspension: all specimens with cells in suspension
PB, BMA, CSF, PF, BAL
Solid tissue
» Fine needle aspirations
» Tissue suspensions - slicing, mincing and teasing = Filtering

 Sample stabilization: Anticoagulant - EDTA or Heparin

 Enrichment of cells: For leucocytes - RBC Lysis - NH4CL or

- Density gradient centrifugation – Ficoll medium
 Antibody staining: Separate cells-wash-incubate with Ab-F in dark
 Acquisition: Acquire the stained cells at earliest or
Fixed and store in refrigerator
 Data Analysis: VIMP – Needs experience and knowledge
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 Common Clinical Applications
 Enumeration of lymphocyte subsets (CD4/CD8)
 Immunophenotyping of hematologic malignancies
 Minimal Residual Disease (MRD)
 Myelodysplatic Syndrome (MDS)
 HLA B27 typing
 PNH diagnosis (CD55-/CD59-)
 DNA/RNA analysis & Cell cycle studies
 Reticulocyte analysis
 Hemotopoietic stem cell (CD34+)analysis
 Platelet analysis
 Antigen quantitation e.g. CD20, CD22, CD33 etc

Other uncommon
 Microbiology
 Determination of drug resistance to chemotherapy
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 Analysis Approach

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 Management of Leukemia

 Accurate diagnosis and classification

 Knowledge of prognostic factors

 Monitoring response

 Diagnosis of early relapse at other sites

like CNS

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 ALL
naïve

B-lymphocytes

Plasma
Lymphoid cells
AUL progenitor T-lymphocytes

Mixed Lineage Leukemia

AML
Hematopoietic Myeloid Neutrophils
stem cell progenitor

Eosinophils

Basophils

Monocytes

Platelets

Red cells
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 FCM in diagnosis and classification
 Identification of blasts

 Enumeration of blasts

 Assignment of blast lineage

 Identification of abnormal blasts

 Subclassification
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 Identification of blasts 4
10 0.00%
262144 84.98%
 Low side light scatter
3
10
196608
 Weak CD45 expression

cyTdT-FITC

SSC-A
2
 Markers of immaturity 131072
10

such as CD34 and TdT 65536

10
1

 Lack markers of maturation 00

0.00%
10 0 10 1 10 2 3
5 4
15.02%
210 10
3 4
10 10 CD4510PerCP10 10
Myeloblasts - CD11b, CD15, CD16. CD45-ECD

B lymphoblasts – surface light chains 10

4

kappa/lambda 3

CD34 PerCP
10

T lymphoblasts – Surface CD3 10

2

1
10

0
10 0 1 2 3 4
10 10 10 10 10
CD45 FITC

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 Enumeration of Blasts
Flow cytometric count lower than manual count
 Dilution with peripheral blood
 Some blasts lack expression of CD34 and CD117
 CD45 expression may very

Flow cytometric count higher than manual count

 Loss of NRBCS during red cell lysis.
 Ficoll Hypaque separation
 Blast identifications may be difficult due to poor
preservation or may be disrupted during smear
preparation

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 Immunophenotypic markers
Markers of Immaturity – TdT, CD34

Lineage Specific markers

Myeloid - cMPO
B cell - cCD22/cCD79a
T cell - cCD3

Lineage Associated markers

Myeloid - Common CD13, CD33, CD117, CD11b, CD15
Monocytic - CD13, CD33, CD64, CD36, CD11c, CD11b, CD14, CD4, cLysozyme
Erythroid - CD36, CD71, CD105, CD235a (Glycophorin A), Hb
Megakaryocytic - CD36, CD41, CD42, CD61 andCD62
B cell - CD19, CD22, CD20, cCD79a, CD10, cIgM, sIg
T cell - Common CD1a, CD2, CD5, CD7, CD10
- Other CD4, CD8, CD3,
NK cell - CD16, CD56, CD57, CD94, KIR
PDC - CD123, CD4, CD56, CD68, CD33, CD43, BDCA,
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- Other on PB subset CD2, CD5, CD7
 Lineage Infidelity markers

(Leukemia associated immunophenotype; LAIP)

Lymphoid markers in AML - CD7, CD56, CD2, CD5 and CD19.
Myeloid markers in ALL – CD66c, CD13, CD33, CD117, CD15

Other Markers useful for MRD detection

Associated with AML – CD38, CD45, CD68, HLADR, CD123

Associated with ALL – CD9, CD24, CD25, CD58, CD72, CD73, CD81, CD8
CD123, CD200

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Case 1

36 yrs Male
Presented with pancytopenia

BM Aspirate
Hypercellular particles
Diff:
Blasts – 32%
Grans – 46%
Monocytes – 6%
Lymph – 2%
NRBCs – 12%

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 Flow cytometric Immunophenotyping

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Case 3 - 9 months child presented with high count

BM

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Case 4

11 yr male

- Fever - 2 month
- Hepatosplenomegaly

 HB- 9.7 gm/dl

 Platelets – 250 *109/L
 WBC – 2.3 * 109/L

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B Tube
 Myeloid Tube

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T tube
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Blasts
 Express dim to negative CD45, dim CD117, dim CD13, dim
CD15, moderate CD38
 Express moderate CD7, moderate CD2
 Express Cytoplasmic CD3
 Negative for CD34, CD4, CD8, CD5, CD1a, TdT, CD56, sTCR
ab and gamma delta
 Negative for B lymphoid, and other myeloid markers
 Negative for cytoplasmic MPO and CD79a

Early precursor T cell ALL

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 Approach to immunophenotyping CLPD

 Identification of lineage: expression of lineage specific

markers.

 B cell lineage- CD 19 or CD20 (CD20 may be lost after

treatment with rituximab).

 Immunoglobulin Light chain restriction

 T cell lineage- CD7, CD3, CD2, CD5 (many markers may

be lost in null cell phenotype)

 TCR V beta repertoire restricted usage

 NK cell – CD7, cytoCD3, CD2, CD16, CD56, CD57

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 Case 4

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 Immunophenotypic approach to B CLPD diagnosis

CD200

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 Plasma cell dyscrasia
Wide spectrum- from MGUS to Multiple
myeloma
Evolving roles of flow cytometry
1. Diagnosis
2. Prognostication
3. Minimal residual disease
4. Risk of progression from MGUS to MM
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 Abnormal Plasma Cells in Myeloma

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 Reporting

 Calculate percentages of tumor cells of viable cells

 A descriptive report with levels of antigen expression
 If flow results are diagnostic of a distinct disease entity
– CLL & HCL
 If not, enlist a list of possible entities with suggestion of
additional studies that might be of diagnostic value such as
– immunohistochemistry,
– Conventional cytogenetic,
– fluorescence in situ hybridization (FISH), and
– Molecular diagnostic studies
 Additional prognostic information
 Identification of targets for potential directed therapy
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 THANK YOU!

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