1. Basics of Flow Cytometry
Prashant Tembhare 1

2. Flow = in a flow or a stream
What is Flow Cytometry?
Cyto = cells
Metry = measurement
Flow = in a flow or a stream
Flow Cytometry is the automated measurement of Physical, Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system. 2

3. Flow cytometry Flow cytometer is an instrument that
- illuminates cells as they flow in front of a light source &
- detects and correlates the signals from the illumination.
Unique Ability – rapid analysis of thousands of cells
cells flow at a velocity of 5–50 m/s
Analyze cells/second
- simultaneous illustration of multiple antigens
Two major principles 1. Measurement of physical properties
2. Measurement of antigenic properties

4. 4

5. Principles of flow cytometry Forward Light Detector
1. Measurement of physical properties i.e. size and complexity (granularity).
Right Angle
Light Detector
Forward Light Detector c
Forward Scatter (FSC): Diffracted light
FSC is related to cell surface area.
FSC is detected along axis of incident light in the forward direction.
Side Scatter (SSC): Reflected and Refracted light
SSC is related to cell granularity and complexity.
SSC is detected at 90° to the laser beam.
Factors that affect light scatter: Shape, surface, size, granularity, internal complexity, refractive index.
Notice that the forward scatter signal is the light that is diffracted (bends around the cell) and the
side scatter is the light that is refracted at greater angles, typically off internal components of the cell.
LASER BEAM 5

6. 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. c
LASER BEAM 6

7. Instrument Components
Fluidics: Specimen, Sheath fluid, flow chamber.
Optics: Light source(s), mirrors, filters, detectors, spectral separation
Electronics: Controls pulse collection, pulse analysis, triggering, time delay, data display, gating, sort control, light and detector control
Data Analysis: SOFTWARE - Data display & analysis, multivariate/simultaneous solutions, identification of sort populations, quantitation

9. Fluidics Crosland-Taylor - Hydrodynamic focussing = 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 9

10. Fluidics
↓D by = ↑V by fold

11. HYDRODYNAMIC FOCUSING

12. OPTICS LASER (argon) Dichroic Filters and Mirrors (b) Photodiode
(d) PMT (photo multiplier tubes ) 12

13. Emitted Fluorescent Light Energy
What is Fluorescence ? HO O
 = 488 nm
 = 520 nm C
Incident Light Energy
CO2H
Emitted Fluorescent Light Energy
Fluorescein Molecule
Antibody
A fluorochrome absorbs laser light (energy) and gets excited. The excited fluorochrome releases that energy in three ways,
vibration, heat dissipation, and photon emission. These photons are of a longer wavelength (different color) than the incident laser light.
The photon emission is referred to as fluorescence.
The fluorochrome absorbs energy from the laser.
The fluorochrome releases the absorbed energy by:
vibration and heat dissipation.
emission of photons of a longer wavelength. 13

14. Mechanism of fluorochrome

15. Fluorescence
Emitted fluorescence intensity is proportional to binding sites
FITC
FITC
FITC
FITC
FITC
FITC
FITC
FITC
Number of Events
Fluorescence intensity emitted from a cell which binds fluorochrome-labeled antibody is proportional to the quantity of binding sites.
FITC
FITC
Log scale of Fluorescent Intensity 15

16. Emission Spectra 100% Normalized Intensity 0% 400 500 600 700 800
FITC PE
APC
PerCP
Normalized Intensity
Fluoroscence Chart of Fluorochromes used in immunofluorescence measurements on the FACSCalibur.
This graphic shows the emission spectra of fluorochromes for which the FACSCalibur has been optimized to detect.
With the use of optical filters, the majority of the signal from a fluorochrome can be routed to its respective detector.
But notice how each fluorochrome emits a spectrum of wavelengths that overlap each other. Because of this overlap
we must electronically compensate the detected signals. 0%
400
500
600
700
800
Wavelength (nm) 16

17. Emission Spectra 100% Normalized Intensity 0% 400 500 600 700 800
Cascade Blue
FITC
Alexa 430 PE PI
APC
PerCP
PerCP-Cy5.5
PE-Cy7
Normalized Intensity
Fluoroscence Chart of Fluorochromes used in immunofluorescence measurements on the BD LSR II. 0%
400
500
600
700
800
Wavelength (nm) 17

18. Fluorescent Light absorption
Control
Absorption
No blue/green light
red filter

19. Dichroic Filters Can be a long pass or short pass filter or band pass
Filter is placed at a 45º angle to the incident light
Part of the light is reflected at 90º to the incident light, and part of the light is transmitted and continues on.
Dichroic
Filter
Detector 1
Detector 2

20. Coulter optical system - Elite
PMT4
PMT3
PMT2
PMT1
575 BP
525 BP
488 BP
PMT5 L L L D
675 BP
632 BP D D 5 2 5
488 BK 5 9 6 4
APC
The Elite optical system uses 5 side window PMTs and a number of filter slots into which any filter can be inserted
PMT6 TM
PMT7

22. Optical Design Laser Sample Dichroic Filters Flow cell Bandpass
PMT 5
PMT 4
Sample
PMT 3
Dichroic
Flow cell
Filters
PMT 2
Scatter
PMT 1
Laser
Sensor
Bandpass
Filters

23. 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
FACSCalibur Electronics.
We’ll discuss how the optical signal becomes an electronic signal, how the electronic pulse is evaluated,
and how the pulse values are converted to digital values so that the computer can work with the data. 23

24. Electronics: Triggering on a voltage pulse
Laser
Voltage
Time
Laser
Voltage
The creation of a pulse applies to all channels, scatter or fluorescence. When the cell first encounters the laser beam
it starts to scatter light or in the case of fluorescence, starts to fluoresce. When the cell is in the center of the beam,
the maximum amount of scatter or fluorescence, is achieved. As the cell leaves the laser the signal drops back off.
Time
Voltage
Laser
Time 24

25. Optical to Digital PMT Voltage Signal Out Log amplification of signals
Analog to Digital Converter
2 Options for SSC and fluorescence channels
Photon In
Linear amplification of signals
Voltage In
Photons strike PMT (Photo Multiplier Tube). PMT output current is converted to a voltage pulse by a preamplifier (not shown).
The resultant voltage pulse is proportional to the number of photons impinging upon the detector.
PMT gain can be adjusted from 0 volts to 1000 volts.
The operator of a flow cytometer adjusts PMT voltage until the population is clearly visible on the display.
Before the signals are passed to the ADC, they are routed through compenstion circuits, log or linear amplifiers (operator determined),
Q: If you increased the FL2 PMT voltage, in which direction would you expect the FL2 population to move, right or left?
A: The population would move to the right.
Once the voltage pulse is generated, it is digitized and sent to memory.
PMT Power Supply
compensation circuit
Levels 0–1000V
adjusted by slider control on computer
Gain levels from 0–9.99
adjusted by slider control on computer
Amplifier output voltage ranging between 10mV to 10V 25

26. Data Analysis by Software
Display Plots
Create Gates
Display Statistics
Analyze 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
Data analysis consists of displaying data from a list-mode file in a plot, and then measuring the distribution of the events within the plots. Gates and statistics can be used to obtain information about subset populations.
Plot Types: Gate Types: Statistics Types: Results:
Histogram Polygon # of Events % positive for
Dot Ellipse % of Gated particular markers
Contour Histogram % of Parent -viable cells
Density Quadrant % of Total -immunotphenotye
mean mean fluorescence intensity
geometric mean DNA content
standard deviation absolute counts 26

27. 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 – Transport at RT
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

28. Clinical Applications of Flow Cytometry
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
Cell Function analysis

29. Analysis Approach 29

30. FCM in management of Acute Leukemia
Accurate diagnosis and classification
Knowledge of prognostic factors
Monitoring response
Diagnosis of early relapse at other sites like CNS

31. Mixed Lineage Leukemia
ALL
Hematopoietic
stem cell
Neutrophils
Eosinophils
Basophils
Monocytes
Platelets
Red cells
Myeloid
progenitor
Lymphoid
B-lymphocytes
T-lymphocytes
Plasma
cells
germinal center
naïve
AUL
Mixed Lineage Leukemia
AML

32. FCM in diagnosis and classification
Identification of blasts
Enumeration of blasts
Assignment of blast lineage
Identification of abnormal blasts
Subclassification

33. Identification of blasts
Low side light scatter
Weak CD45 expression
Markers of immaturity
such as CD34 and TdT
Lack markers of maturation
Myeloblasts - CD11b, CD15, CD16.
B lymphoblasts – surface light chains
kappa/lambda
T lymphoblasts – Surface CD3

34. 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

35. 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
- Other - CD11b, CD15
Monocytic - CD13, CD33, CD64, CD68, CD117, 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,
- Other on PB subset CD2, CD5, CD7

36. Lineage Infidelity markers
(Leukemia associated immunophenotype; LAIP)
Lymphoid markers in AML - CD7, CD56, CD2, CD5 and CD19.
Myeloid markers in ALL – CD13, CD33, CD117, CD15
Other Markers useful for MRD detection
Associated with AML – CD38, CD45, CD68, HLADR
Associated with ALL – CD9, CD24, CD25, CD52, CD58, CD81, CD123

37. AML M0

38. AML M2
t(8;21)(q22;q22) RUNX1-RUNX1T1

39. AML M5a

40. AML Monocytic differentiation (M5b)

41. AML M6

42. AML M7

43. B - ALL

44. T - ALL

45. Biphenotypic or mixed lineage leukemia
Borowitz M, Bene M, Harris N and Matutes E, (2008) Acute leukaemias of ambiguous lineage., World Health Organization Classification of Tumours IARC Press, Lyon, pp. 150–155.

47. EG Weir and MJ Borowitz. Leukemia (2007) 21, 2264–2270.
Bi-lineal Leukemia

48. Hematogones Antigens Early (St-1) Intermediate (st 2 & 3) Mature
B cells
TdT + -
CD34
CD10
bright
dim
CD19
intermediate
CD22
CD20
(-/+) weak
CD38
variable
CD45
CD58
CD81
Cyt IgM
K/L
-/+

49. ALL in various cluster patterns

50. Role of flow cytometry in CLPD & MM
Diagnosis
Staging of lymphoma – Bone marrow involvement or body fluids
Prognostication eg Zap 70 in CLL
Minimal residual disease
Diagnosis of relapse

51. detection of abnormal immunophenotype
Analysis Approach
Isolation of cells using lineage specific markers like CD19 for B cells and CD3 for T cells
detection of abnormal immunophenotype
Clonality evaluation eg kappa or lambda
Note size of cells – FSC

52. Antibody panels- B CLPD
Mature B cells
CD19, CD20, CD22, cyto79a, CD79b
Mature T cells
CD2, CD3, CD4, CD5, CD7, CD8, TCR αβ/γδ
NK cells
CD2, cytoCD3, CD7, vCD8, CD16, CD56, vCD57, CD94, CD158 (KIRs)
Plasma cells
CD138, bCD38, CD19, cyto79a, cyto-Kappa, cyto-Lambda
Clonality markers
B cells - sKappa, sLambda,
PCs - cyto-Kappa, cyto-Lambda
T cells – TCR V beta repertoire
Other important Markers
CD45, CD38, HLADR, Granzyme, Perforin, TIA

53. Disease oriented B CLPD MM – CD19, CD20, CD27, CD45, CD56, CD81, CD117
CLL – CD19,CD5, CD23, d-n CD20, d-n CD22, d-n FMC7, CD43, CD81, CD200
HCL – CD11c, CD25, CD103, CD123
FCL/DLBCL – CD10
MCL – CD5 & CCD
MM – CD19, CD20, CD27, CD45, CD56, CD81, CD117
T CLPD
ATLL/CTCL – CD25, CD26, CD27
AILT – CD10
ALCL – CD30
EATCL – CD103

54. 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

55. CLL

56. MANTLE CELL LYMPHOMA

58. HAIRY CELL LEUKEMIA

59. ATLL
PERIPHERAL T CELL LYMPHOMA - NOS

60. Immunophenotype of plasma cells
Normal plasma cells
Specific markers- CD138, CD38 (strong)
B cell lineage – weak CD19, strong CD27
Moderate expression of CD45
Neoplastic plasma cells
Aberrant expression- CD20, bCD56, CD28, CD117, CD200
Loss of CD19, CD27, CD45, CD81
Surface/Cytoplasmic light chain restriction

61. Multiple Myeloma

63. Immunophenotyping in Myelodysplastic Syndrome
Normal Granulocytic Maturation
Granulocytic dysplasia in MDS

64. Immunophenotyping in Myelodysplastic Syndrome
Normal Monocytic Maturation
Monocytic dysplasia in MDS

65. Paraxysmal Nocturnal Hemoglobinuria (PNH)

66. THANK YOU!