Flow cytometry is a technology that combines fluidics, optics, electronics, and computer analysis, allowing for the simultaneous analysis of multiple cellular characteristics at the single-cell level. Its development began in the late 19th century and has evolved significantly, enabling applications in clinical laboratories such as immunophenotyping, leukemia detection, and organ transplantation. Key advancements include the introduction of lasers for detecting fluorescence and the use of monoclonal antibodies for specific cell identification.

1. Flowcytometry
Dr.Dnyaneshwar Patale
SR PDCC Apheresis
ILBS

2. Outline
 History
 Basics of Flow
 Def
 Fluidics
 Optics
 Electronics
 Data analysis
 Application

3. Historical Highlights
 Flow cytometry was initially conceived as a practical methodology to count
blood cells.
 In 1879 Lord Rayleigh observed that fluid emerging from an orifice breaks
into a series of droplets. Cell sorting is based on the physics of droplet
formation.
 In 1934, A. Moldavan reported the development of the first device that could
count red blood cells automatically while in flow. (Science. 1934 Aug
24;80(2069):188-9 PHOTO-ELECTRIC TECHNIQUE FOR THE COUNTING
OF MICROSCOPICAL CELLS)
 In 1949, Wallace Coulter filed a patent entitled, “Means for Counting
Particles Suspended in a Fluid”. The patent was issued in 1953. This lead to
the development of the “Model A” Coulter Counter. Today, clinical
hematology laboratory instruments used to count blood cells employ the
principles developed by Coulter.

4.  In 1965, Mack J. Fulwyler reported the first flow cytometry instrument
capable of sorting cells. He sorted cells based on their Coulter volume by
using a Coulter cell sizing instrument and modifying the electrostatic ink
jet droplet deflection technique developed by Richard G. Sweet. (Rev.
Sci. Instrum. 36, 131 (1965); http://dx.doi.org/10.1063/1.1719502 High
Frequency Recording with Electrostatically Deflected Ink Jets)
 In 1968 Wolfgang Göhde designed a fluorescence based flow cytometer
(ICPII) in 1968 which was commercialized by Partec in 1969. In 2000 he
turned his efforts toward developing a program to provide low cost CD4
tests for people with AIDS in Africa.
 Lou Kamentsky and Myron Melamed worked on distinguishing cancer
cells from normal cells using differences in the absorption and scattering
of light. Kamentsky designed the Rapid Cell Spectrophotometer (RCS),
which measured nucleic acid content and cell size.
 Leonard Herzenberg, an immunologist at Stanford, used the RCS
prototype realizing how useful this technology would be in cell biology,
He coined the term ‘FACS’ – Fluorescence Activated Cell Sorter. Becton
Dickinson (BD) owns the FACS trade name and launched the first
commercial instrument, FACS-1 in the early 1970’s.
 Today, flow cytometers with 5 lasers capable of analyzing or sorting cells
labeled with 18 fluorochromes is possible. Soon instruments capable of
50 parameters may be possible.

5. What Is Flow Cytometry?
 Flow ~ motion
 Cyto ~ cell
 Metry ~ measure

6. Flow Cytometry Background
 A system that integrates electronics, fluidics, optics, laser technology and computer
analysis in a single platform.
 Allows for simultaneous multiparametric analysis of physical/chemical/biological
characteristics of cells or particles at the single-cell level by detecting fluorescence
intensity as they travel in suspension one by one past a sensing point
Flow chamber and
fluidics
Emission light
Optical components
Detectors/amplifiers (PMT)
Digital (computer/software)
Excitation light
Lasers
Mixed cells with fluorescence label

7. Optics
Fluidics
Electronics
Mechanics of a Flow
Cytometer
• Cells in suspension are brought
in single file past
• a focused laser where they
scatter light and emit
fluorescence that is filtered and
collected
• then converted to digitized
values that are stored in a file
for analysis

8. The Fluidics System
“Cells in suspension flow single file”
 Cells must flow one-by-one into the
cytometer to do single cell analysis
 Accomplished through a pressurized
laminar flow system.
 The sample is injected into a sheath fluid
as it passes through a small orifice
(50um-300um)

9. Hydrodynamic Systems
Sample in
Sheath
Sheath in
Laser beam
Piezoelectric
crystal oscillator
Fluorescence
Sensors
Scatter Sensor
Core
Sheath
Signal
direction
Flow Chamber

10. 10 psi
10.2 psi
10 psi
10.4 psi
10 psi
10.8 psi
• Difference in pressure between sample and sheath
• This will control sample volume flow rate
• The greater the differential, the wider the sample core.
• If differential is too large, cells will no longer line up single
file
• Results in wider CV’s and increase in multiple cells passin
through the laser at once » » » Faster cell analysis BUT lo
Fluidics- Sample Differential

11. Hydrodynamic
Focusing
V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3
Notice how the ink is focused
into a tight stream as it is
drawn into the tube under
laminar flow conditions.
PBS/Sheath
Sample/cells/core
Laminar flow
Hydrodynamic
Focusing
Laminar flow occurs when a fluid flows in parallel layers, with no
disruption between the layers

12. Incoming
Laser
Sample
Sheath Sheath
Sample
Core
Stream
Low Differential High Differential or “turbulent flow
Laser
Focal Point

13. Sample Preparation
Samples must be in single cell suspension
Solid tissue requires mechanical dissociation and often
enzymatic digestion
Adherent cell lines require detachment from the culture dish and
dissociation
Cell aggregates must be filtered out
Red cells should be lysed
Well prepared single cell suspensions yield good data

14. Common specimens suitable for flow
cytometry analysis include
Peripheral blood,
Bone marrow,
Body fluids,
Cerebrospinal fluid,
Urine,
Lymph node (cells or freshtissues),
Any fine-needleaspirates,
Fresh tissues suspicious for hematopoietic and lymphoiddisorders.

15. Interrogation
 Light source needs to be focused on the
same point where cells are focused.
 Light source
99%=Lasers

16. Lasers
Light amplification by stimulated emission of
radiation
 Lasers provide a single wavelength of light
(monochromatic)
 They can provide milliwatts to watts of power
 Low divergence
 Provide coherent light
 Gas, dye, or solid state
Coherent: all emiting photons have same wavelength,
phase and direction as stimulation photons

17. Light collection
 Collected photons are
the product of laser light
scattering or bouncing
off cells
 488nm
 Information associated
with physical attributes
of cells (size, granularity,
refractive index)
Scatter Fluorescence
VS
 Collected photons are
product of excitation with
subsequent emission
determined by fluorophore
 350nm-800nm
 Readout of intrinsic
(autofluorescence) or
extrinsic fluorescence
(intentional cell labeling)

18. Colors
 488 nm wavelength is the most commonly used type of laser in Flow Cytometers
 Many 5 laser instruments have these additional lasers
◦ 355 nm UV
◦ 405 nm Violet
◦ 640 nm Red
◦ 561 nm Yellow-Green
http://antonine-education.co.uk/

19. Forward Scatter
FSC
Detector
Laser Beam
Original from Purdue University Cytometry Laboratories

20. Different size cells
Particle or cell size (log scale) FSC
Number
of
events
small large
0.1 1 10 100 1000
0.9
20
90
200
700
While forward light scatter is not always related to cell size, in
The majority of cases between 1-20 microns, it is a reasonable estimate

21. Side Scatter
FSC
Detector
Collection
Lens
SSC
Detector
Laser Beam
Original from Purdue University Cytometry Laboratories

22. Side Scatter
Laser light that is scattered at 90
degrees to the axis of the laser path is
detected in the Side Scatter Channel
The intensity of this signal is
proportional to the amount of cytosolic
structure in the cell (eg. granules, cell
inclusions, drug delivery nanoparticles.)
Side Scatter=SSC=RALS=90 degree
Scatter

23. Why Look at FSC v. SSC
 Since FSC ~ size and SSC ~ internal
structure, a correlated measurement between
them can allow for differentiation of cell types
in a heterogenous cell population
FSC
SSC
Lymphocytes
Monocytes
Granulocytes
RBCs, Debris,
Dead Cells
LIVE
Dead

27. Optics- Filters
 Different wavelengths of light are scattered
simultaneously from a cell
Need to split the light into its specific wavelengths in
order to measure and quantify them independently.
This is done with filters
 Optical filters are designed such that they
absorb or reflect some wavelengths of light,
while transmitting others
 3 types of filters
Long Pass
Short Pass
Band Pass
Dichroic

28. Optical Filters

29. Optics- Dichroic Filters
 Long pass or short pass filters
 Placed at a 45º angle of incidence
 Part of the light reflected at 90º , and part
of the light is transmitted and continues
on.
Dichroic Filter
Detector 1
Detector 2

30. IMMUNOPHENOTYPING
ANALYSIS
Requires
Antibodies.
Fluorochromes.

31.  Highly specific monoclonal antibodies are usedthat are
produced by cloned antibody secreting cells.
 Antibodies are based on cluster of differentiation (CD)- a
protocol used for identification anddistinction of cell surface
antigens.
 Using CD system wecan identify cells by the presence or
absence of particular surface markers for e.g. CD3+ or CD20-
etc.
ANTIBODY

32. FLUOROCHROMES
Fluorochromes are substances that can be excited by
certain light source (such as laser) and emita
fluorescent signal at a single wavelength.
Fluorescent dyes can directly bind to certain cellular
content, such as DNA and RNA, and allow us to
perform quantitative analysis on individualcells.
However, in mostcases fluorochromes are conjugated
with monoclonal antibodies, which specifically target
cellular antigens/markers.

33. Understanding Fluoroscence
The fluorescent
molecule is excited
by the excitation
source (laser). This
imparts energy to
electrons in the
molecule which in
then released as
the molecule
relaxes. The
energy is released
as light.
e-
Excited
Resting
e-
e-
e-
e-
state
Mechanism of
Fluorscence

34. Fluorescence Detection
 Fluorochromes on the cell surface or inside the cell are
excited by the laser beam as the cell passes the
interrogation point.
 These fluorochromes then release energy as they leave
their excited state.
 The energy release is in the form of a photon with a
specific wavelength, longer than the excitation
wavelength.
 These photons of light are steered and collected by
optical lens and filters at specific wavelengths.

35. Characteristics of fluorochromes commonly
used in flow cytometry.
FLUOROCHROMES CONJUGATED TO ANTIBODIES EXCITATION WAVELENGTH(NM) EMISSIONWAVELENGTH(NM)
Fluorescein isothiocyanate(FITC) 488 530
Phycoerythrin (PE) 488 580
PE-Texas Red 488 615
PE-Cy5 488 670
Peridinin chlorophylprotein(PerCP) 488 670
Allophycocyanin (APC) 633 670
APC-Cy7 633 767
Interestingly, although some of them can be excited by the same light source, the
different fluorochromes may emit fluorescent signals with different
wavelengths/colors. Thus, multiple fluorochromes can be simultaneouslyexcited bya
light source and detected by their emission fluorescent signals with different
wavelengths, respectively.

36. How do I choose my
Fluorochromes ?
• Antibody availability
• Fluorochrome brightness
• Excitation source
• Emission filters
• Other fluorochromes/ Signals present in my
sample (spectral overlap)

37. Electronics
 Detectors basically collect photons of
light and convert them to an electrical
current
 The “electronics” process light signal
and convert the current to a digitized
value that computer can graph

38. Detectors
 There are two main types of photo
detectors used in flow cytometry
Photodiodes
 Used for strong signals, when saturation is a
potential problem (eg. FSC detector)
Photomultiplier tubes (PMT)
 Used for detecting small amounts of fluorescence
emitted from fluorochromes.
 Incredible Gain (amplification-up to 10million times)
 Low noise

39. • Fluorescent emissions are detected as a voltage pulse
from photomultiplier tube (PMT) detectors
• The area, voltage and height of the voltage pulse is
measured
Voltage
Laser
Laser
Laser
time
time
time
Voltage
Voltage
1.
2.
3.
Flow Cytometry: Signal Conversion
in PMT
Photon  Current  Voltage  Digital Signal

41. Forward light scatter (FSC) and side light scatter (SSC) . FSC collects
light at 180° from the point at which the laser beam intersects the cells .It
is correlated with cell size.and thus can distinguish normal lymphocytes
(small), monocytes (intermediate), and neoplastic cells (generally they
are large in size).
SSC collects right-angle light at 90° and is correlated with cytoplasmic
granularity and nuclearconfiguration.
 The combination of both FSC and SSC can distinguish normal
lymphocytes, granulocytes, and monocytes.
 The detection of lymphocytes and monocytes provides a reliable
internal control to evaluate the size of the cells of interest.
Basic parameters and Windows
of cell population

42. CD45 & SSC
• As the first step, it is most important todeterminewhetherthecells
of interest arehematopoietic.
• Generally speaking, all hematopoietic/lymphoid cells express
CD45 antigens (CD45+).
• Thus, a histogramof CD45 on a logarithmicscalevs. SSC on a linear
scale is indispensableasa starting pointof flow cytometryanalysis.
• Based onantigenexpression, cells aredivided into CD45+ and
CD45– groups.
• Among the CD45+ group, the cells can further separated into
subgroups based on expression levelsof CD45 and intensity of
cytoplasmicgranularity.

43. Compensation
 Fluorochromes typically fluoresce over a
large part of the spectrum (100nm or more)
 A detector may “see” fluorescence from
more than one fluorochrome. (referred to as
bleed over)
 You need to “compensate” for this bleed
over so that ONE detector reports signal
from only ONE fluorochrome

45. Histograms
Flow cytometry data can be plotted in several different ways:
• the axes of the graphs represent fluorescence intensity data, usually
plotted on a log scale
• for histograms, the y axis is cell number
Flow Cytometry: Data Analysis

46.  By cell distribution in the CD45vs. SSC. This is most
useful in a specimen containing mixed cell populations .
Thegrouped cells in individual windows represent different
celllineages.
 By cell size: In FSC vs. SSC histograms, neoplastic cells
(usually large in size) can be gated by using lymphocytes
(small) and monocytes (intermediate) as an internal size
control . Once thecells of interest aregated, furtheranalysis
of cell lineage can be performed.
 By cell lineage-specific antigens (immunophenotype) :
If cells are CD45+ but do not fit into particular windows in
the CD45 vs. SSC histogram, identification of lineage-
specific antigen expression is needed
Types of Gating

47. APPLICATION
Applications in Clinical Laboratories
 Immunophenotyping (HIV)
 CD4 absolute counts
 Leukemia and lymphoma immunophenotyping
 Cell cycle and ploidy analysis of tumors
 Reticulocyte enumeration
 Flow cross-matching (organ transplantation)
 Stem cell enumeration
 Residual white blood cell detection
 (QC platelet, red blood cells)

48. Applications in Blood Banking
 Fetomaternal Hemorrhage Testing
 Flow in AIHA Associated with a Negative DAT
 HLA Antibody Detection
 CD34+ cell enumeration and viability
 Platelet receptor defects (Bernard Soulier sndrome
and Glanzmann )
 Storage lesion studies
 Pretransfusion compatibility testing

49. Platelet analysis by flow…
 Identification of inherited disorders
 Monitoring of anti-platelet therapy
 Monitoring of clinical course of
disease
 Monitoring of platelet production in
thrombocytopenia
 Identification of patients at risk of
thrombosis
 Diagnosis of HIT

50. APPLICATION
Research Laboratories
 Immune function studies
 Hematopoietic stem cells
 Multi-drug resistance studies (cancer)
 Kinetics studies (cell function) Platelet analysis
(coronary disease)
 Environmental sample analysis
 Flow and FISH

51. APPLICATION
Immunophenotyping
 Is the term used in the identification of cells by
labeling with monoclonal antibodies identified as
cluster designations (CD)
 Used to determine
Cell lineage
Activation status
Adhesion migration and homing capacity of cells
Ability to respond to stimuli
Ability to respond to stimuli

52. Propidium iodide (PI) - Cell
Viability
How the assay works:
 PI cannot normally cross the cell membrane
 If the PI penetrates the cell membrane, it is assumed to be
damaged
 Cells that are brightly fluorescent with the PI are damaged or
dead
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
Viable Cell Damaged Cell

53. Minimum Residual Disease
 Patients that appear to be in complete
remission can be found to have residual
tumors cells that are too few in number to
be counted by standard techniques.
 Flow cytometry can detect these rare cells
before they proliferate and cause the
patient to relapse.

54. Flow Cytometric Crossmatch
(FCXM)
 Flow cytometry has become a valuable tool to
assess potential solid organ allograph
recipients.
 It is now recognized as the laboratory
procedure of choice. Circulating
alloantibodies at levels too low to be detected
by standard methods can be detected by a
flow cytometric crossmatch (FCXM).
 This means that transplants done based on a
negative FCXM are more successful.

55. Diagnosis of PNH
Conventional laboratory tests for the diagnosis of PNH
include the sugarwater testand the Ham’s acid hemolysis test .
Antibodies to CD55 and CD59 are specific for decay-
accelerating factor and membrane-inhibitor of reactive lysis,
respectively, and can be analyzed by flow cytometry to makea
definitive diagnosis of PNH.

56. DNA studies
• These assays involve measurements of fluoroscence
associated with DNA to determine
• Stage of cell cycle
• Apoptosis
• Gene Transfection
• Chromosomal Aberrations
• Endoreduplication
• Doubling Time

57. Leukemia and Lymphoma
 Lymphomas are tumors of the immune
system, primarily in the lymph nodes,
spleen, and bone marrow.
 Flow Cytometry has been used since
the late 1970's to diagnose and
classify human lymphomas.
 A standard panel of fluorescent
labeled monoclonal antibodies is used
for this purpose.

58. Fluorescence-Activated Cell Sorting (FACS)
v
laser PMT
Mixture of
cells to be
sorted
New
drop empty drop
+
+
+
+
+
+
+
+
+
_
_
_
_
_
_
_
_
+
+
+
_
_
_
FACS: a specialized type of flow cytometry to sort a
heterogeneous mixture of cell suspension
Features
• Sort up to 4 populations of interests
• 15 fluorescence color simultaneous on the same
cell
• Sort different types cells
o Primary BM, PBMC, mouse splenocytes
o Any types of cell lines
o Large fragile cells like activated neutrophiles, lung
DCs
o Sticky and hard to sort cells (e.g. solid tumor cells,
neuron cells)
• Multi-purpose sorting
o 7ml round bottom tube, 15ml conical tubes.
o Tissue culture plates, 96 well PCR plates
o Microscope slides including multiwell chamber slides
o Single cell sorting
o Different modes to maximize sort purity (99% for
qPCR) or recovery (for assays requiring large
number of cells
• sterile sorting, sample agitation, temperature
nozzl
e

59. CONCLUSION
Flow cytometry is a powerful technique forcorrelating
multiplecharacteristics on singlecells. This qualitative
and quantitative technique has made the transition
from a research tool to standard clinicaltesting.
Each instrument sub system –Fluidics, optics ,electronics
play critical role in accuracy of data that is collected
Smaller, less expensive instruments and anincreasing
number of clinically useful antibodies are creating
more opportunities for routine clinical laboratories to
use flow cytometry in thediagnosis and management
of disease