FLOW CYTOMETRY, PRINCIPLE, APPLICATION, USE IN HAEMATOLOGY, COMPONENT OF FLOW CYTOMETRY, DATA INTERPRETATION, DATA ANALYSIS, CELL SHORTING ADVANTAGES AND DISADVANTAGES, IMMUNOLOGICAL CLASSIFICATION OF ACUTE LEUKEMIA

1. Prepared by Sanjeev Kumar
B.Sc MLT,PGIMER,CHD
FLOW CYTOMETRY
Definition:
Measuring properties of cell as they flow in a fluid suspension across an illuminated
light path.
Flow - fluid
Cyto - cells
Metry - measure
• Flow cytometry is a laser based, biophysical technology employed in cell counting,
sorting and biomarker detection by suspending cells in a stream of fluid and passing
them by an electronic detection apparatus.
• Measurement of cellular properties as they are moving in a fluids stream.
• Flow cytometry: Flow: cell move in single file & Cytometer: measurement of
numerous cell properties
• Unique ability to demonstrate protein expressed by cell at the rate of 5,000-10,000 cells
per second.
• This method allows the quantitative and qualitative analysis of several properties of cell
populations from virtually any type of fresh unfixed tissue or body fluid.
Most commonly analyzed materials are:
 blood,
 bone marrow aspirate and
 lymph node suspensions.
INTRODUCTION
 The concept of flow cytometry has been in existence for more than five decades.
 Flow cytometric immunophenotyping (FCI) first appeared in clinical laboratories in the
1980s, in the wake of the AIDS epidemic.
 Initially utilized to assess CD4 T-cells, the technique was soon applied to lymphoid and
eventually myeloid neoplasms.
 Current flow cytometers have the capability of simultaneously measuring multiple
parameters of individual cells in a cell suspension.
 Thus, a large number of cell specimens can be processed with a quick turnaround time.
 In addition, flow cytometry is also highly sensitive and can detect immunophenotype of
cells in a specimen with thousands of cells.
The parameters analyzed by flow cytometry include
 physical properties of cells; the size, cytoplasmic granularity, and amount of DNA
contents; and
 cell antigens/markers (surface, cytoplasmic, and nuclear) that can be recognized by
specific antibodies.
By using appropriate antibody panels, flow cytometry can reveal
 the cell type (hematopoietic, lymphoid, or nonhematopoietic),
 cell lineage (B- and T cells, natural killer cells, myeloid/ monocytic cells,
neuro/neuroendocrine cells, and epithelial cells),
 cell maturation stage (precursors vs. matured cells)

2. Prepared by Sanjeev Kumar
B.Sc MLT,PGIMER,CHD
FLOW CYTOMETRY
Basic mechanism
Biological sample
Label it with a fluorescent marker
Cells move in a linear stream through a focused light source (laser beam)
Fluorescent molecule gets activated and emits light that is filtered and detected by sensitive
light detectors (usually a photomultiplier tube)
Conversion of analog fluorescent signals to digital signals
Basic principle of FC
• Cell or particle incubated with fluorescent dye labeled antibodies in suspension is hydro
dynamically focused in a single file using sheath fluid under optimized pressure and when
a laser beam strikes a cell provide information about properties of cell.
It includes
• Relative size of cell
• Relative granularity or internal complexity, an
• Relative fluorescence intensity
• Based in these properties FC identifies cell population of interest

3. Prepared by Sanjeev Kumar
B.Sc MLT,PGIMER,CHD
FLOW CYTOMETRY
Component of FC
1. Fluidics-Flow chamber and sheath Fluid
2. Optics-laser, Light Sensing (FSC and SSC), Photomultiplier diode(PMT) and Filters-
Mirrors
3. Electronics-Software, hardware and Digital
Light
source
Flow
Chamber
Optical
system
Light
Detector
ElectronicComputer

4. Prepared by Sanjeev Kumar
B.Sc MLT,PGIMER,CHD
FLOW CYTOMETRY
Fluidics-Flow chamber
 One of the fundamentals of flow cytometry is
the ability to measure the properties of
individual particles, which is managed by the
fluidics system.
 Heart of instrument.
 It is designed to deliver cells in single file at
point of measurement.
 Sample is injected into the center of sheath fluid.
 Flow chamber wall made up of quartz cuvette.
 Once the sample is injected into a stream of
sheath fluid within the flow chamber, they are
forced into the center of the stream forming a
single file by the PRINCIPLE OF
HYDRODYNAMIC FOCUSING.
 'Only one cell or particle can pass through the laser beam at a given moment.'
 As the sheath fluid moves, it creates a massive drag effect on the narrowing central
chamber. This alters the velocity of the central fluid whose flow front becomes parabolic
with greatest velocity at its centre and zero velocity at the wall. The effect creates a single
file of particles and is called hydrodynamic focusing.
 Closed camber -used for analysis.
 Open chamber -ample stream emerges into open chamber- used for analyzing and sorting
cell
Optical- Light Sensing
• Laser or an arc lamp or LED
• Commonly used laser is air cooled argon ion laser producing blue light at 448 nm
• Laser produce a high intensity beam of monochromatic light
• Occur at the point of interception of stationary light and cell.
• Occur light scattering and emission of fluorescence light.
• Factors that affect light scattering are the cell's membrane, nucleus, and any granular
material inside the cell.
• Light that is scattered in the forward direction (along the same axis the laser is traveling)
is detected in the Forward Scatter Channel.
• Laser light that is scattered at 90° to the axis of the laser path is detected in the Side
Scatter Channel.
• Why Lasers are more common?
• They are highly coherent and uniform. They can be easily focused on a very small
area (like a sample stream). They are monochromatic, emitting single
wavelengths of light.

5. Prepared by Sanjeev Kumar
B.Sc MLT,PGIMER,CHD
FLOW CYTOMETRY
When a light intersects a laser beam at the so called 'interrogation point' two events occur:
a) light scattering
b) emission of light (fluorescence)
Fluorescence is light emitted during decay of excited electron to its basal state.
 When light from a laser interrogates a cell, that cell scatters light in all directions.
 The scattered light can travel from the interrogation point down a path to a detector.
FORWARD SCATTER (FSC)
• Light that is scattered in the forward direction (along the same axis the laser is traveling)
is detected in the Forward Scatter Channel.
• The intensity of this signal has been attributed to cell size, refractive index (membrane
permeability)
SIDE SCATTER (SSC)
 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 (e.g. granules, cell inclusions, etc.)
Side scatter detector
Measuring cell
granularity
FSC
Detector
Collection Lens
SSC Detector
Laser
Beam

6. Prepared by Sanjeev Kumar
B.Sc MLT,PGIMER,CHD
FLOW CYTOMETRY
Why FSC & SSC?
Study of FSC and SSC allows us to know the differentiation of different types of cells.
 The light scattered in the forward direction is proportional to the square of the radius of a
sphere, and so to the size of the cell or particle.
Filters and Mirrors
• Emission optics : A series of filters and mirrors are used to separate and direct the light
of different wavelengths to the corresponding detectors.
• Optical filters are designed such that they absorb or reflect some wavelengths of light,
while transmitting other
• 3 types of filters
 Long Pass filter
 Short Pass filter
 Band Pass filter
LONG PASS FILTER
Transmit all wavelengths greater than specified wavelength.
SHORT PASS FILTER
Transmits all wavelengths less than specified wavelength.
BAND PASS FILTER
Transmits a specific band of wavelengths.

7. Prepared by Sanjeev Kumar
B.Sc MLT,PGIMER,CHD
FLOW CYTOMETRY
Dichroic Filters
 Can be a long pass or short pass filter
 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.
Detectors
There are two main types of photo detectors used in flow cytometer
 Photodiodes
 Used for strong signals, when saturation is a potential problem (e.g. FSC detector)
 Photo Detectors usually have a band pass filter in front of them to only allow a
specific band width of light to reach it.
 These are sensitive instrument and ideal for scatter and fluorescence detection.
 The amount of light or the number of photons are proportional to the amount of
fluorochrome present in the cell.
 Photo multiplier tubes (PMT)
 More sensitive than a Photodiode, but can be destroyed by exposure to too much
light.
 PMT is used for detecting small amounts of fluorescence emitted from
fluorochromes (e.g. SSC & fluorescent signals)
 PMT Convert the incident light into electronic pulses.
 Once PMT generates a pulse, the signal needs to be amplified & ultimately digitized
for computer analysis by use of amplifiers & analogue-to- digital converters.
(ADCs). Silicon photodiodes are usually enough to measure forward scatter.
 Electronic of FC
• When light hits a photo detector a small
current (a few microamperes) is
generated. Its associated voltage has an
amplitude proportional to the total
number of light photons received by the
detector.
• This voltage is then amplified by a
series of linear or logarithmic
amplifiers, and by analogue to digital
convertors (ADCs), into electrical
signals large enough (5–10 volts) to be
plotted graphically.
• Computer system is used for analysis,
presentation & storage of cytometric
data.This raw data in computer permits
repeated analysis, often with different
gating or compensation.

8. Prepared by Sanjeev Kumar
B.Sc MLT,PGIMER,CHD
FLOW CYTOMETRY
Data Analysis
In a Flow- cytometer data is analyses as-
 Single parameter
 Multiple parameter
 In a single parameter, we analyses only one
parameter on each cell & prepared a scatter gram
FSC-SSC
 In multiple parameter, we analyses more than
one parameter on each cell & prepared a scatter gram
FL1-FL2
Data Interpretation
The digitized data, stored in a computer software & interpret as-
 Histogram
 Dot-plot analysis
 Histogram relies on the
measurement of pulses of a
given value & their
assignment to channels
which represent diff.
Voltage levels. The
process of counting each
pulse in the appropriate
channel is known as ADC.
 Software present in the flow
cytometer, interpret the signals in
dot plot analysis as-
FSC
SSC
Single Parameter Graph
Multiple parameter
FSC
SSC

9. Prepared by Sanjeev Kumar
B.Sc MLT,PGIMER,CHD
FLOW CYTOMETRY
Cell sorting by FC
separating cells according to subtype or
epitope expression for further biological
studies.
After the sample is hydrodynamically
focused, each particle is probed with a
beam of light. The scatter and fluorescence
signal is compared to the sort criteria set
on the instrument. If the particle matches
the selection criteria, the fluid stream is
charged as it exits the nozzle of the fluidics
system.
Electrostatic charging actually occurs at a
precise moment called the ‘break-off
point’, which describes the instant the
droplet containing the particle of interest
separates from the stream. To prevent the
break-off point happening at random distances from the nozzle and to maintain
consistent droplet sizes, the nozzle is vibrated at high frequency. The droplets eventually
pass through a strong electrostatic field, and are deflected left or right based on their
charge.
The speed of flow sorting depends on several factors including particle size and the rate
of droplet formation. A typical nozzle is between 50–70 μM in diameter and, depending
on the jet velocity from it, can produce 30,000–100,000 droplets per second, which is
ideal for accurate sorting. Higher jet velocities risk the nozzle becoming blocked and
will also decrease the purity of the preparation

10. Prepared by Sanjeev Kumar
B.Sc MLT,PGIMER,CHD
FLOW CYTOMETRY
Clinical Applications in Hematology
Today, flow cytometer has become a principal tool for the diagnosis of various disorders
in clinical practice. Some applications of flow cytometer are as follow-
 For the diagnosis of Acute Leukemia.
 For the diagnosis of CLPD.
 For the diagnosis of PNH.
 For the detection of MRD.
 For the diagnosis of Platelet abnormality.
 Detection of fetal-cells in mother-circulation.
 For reticulocyte count.
IMMUNOLOGICAL CLASSIFICATION OF ACUTE
LEUKEMIA
Acute Lymphoblastic Leukemia (ALL) Tdt+ve-
B-cell lineage (CD19+ve, CD79a+ve &CD22+ve)-
Pro B-ALL( CD10-, CD15+)
Common ALL (CD10+, cIgM- )
Pre B ALL ( cIg M+ )
Mature B ALL (sIg +)
T-Cell lineage ( cCD3+, CD7+)-
Pro T-ALL( CD3+, CD7+)
Pre T-ALL(CD2+, CD5+)
Corticol T-ALL( CD1a+)
Mature T-ALL (CD3+)
Acute Myeloid Leukemia (AML)-
AML(M0-M5) (anti MPO+ ,CD13+, CD33+, CD117+ )
Pure erythroid leukaemia (antiglycophorin A+, antiblood group antigen+, CD36+ )
Megakaryoblastic leukaemia ( CD41+, CD42+, CD61+)

11. Prepared by Sanjeev Kumar
B.Sc MLT,PGIMER,CHD
FLOW CYTOMETRY
Panel of monoclonal Abs for the Diagnosis of Acute leukemia
ALL AML
B-lineage T-lineage
First Line CD19,CD22,
CD79a,
CD10
CD7,
CD2,
cyCD3
CD13, CD33,
CD117, anti-
MPO
TdT, HLA-DR, CD34
Second
Line
cymu, SmIg CD1a,
CD5,
CD8,
anti-TCR
CD41, CD42,
CD61,
anti-
glycophorin A
Chronic Lymphoproliferative Disorder (CLPD)
 Chronic Lymphocytic Leukemia(CLL).
 Prolymphocytic Leukemia(PLL).
 Hairy cell Leukemia(HCL).
 Splenic lymphoma with villous lymphocyte (SLVL).
 Adult T-cell Leukemia /Lymphoma.
Panel of monoclonal Abs for the diagnosis of lymphoid
disorders
B-Cell T-Cell
First Line SmIg (kappa /lambda)
CD19, CD23, FMC7
mCD79b, mCD22, CD5
CD2,
CD5,
Second
Line
CD11c, CD25, CD103
CD123, CD38, CD138, CyIg
CD3, CD4, CD7, CD8

12. Prepared by Sanjeev Kumar
B.Sc MLT,PGIMER,CHD
FLOW CYTOMETRY
PNH
 Acquired hematological disorder characterized by anemia, intravascular hemolysis
specially at night while patient is sleeping as well as tendency to thrombosis and
infection.
 Results from the mutation in the PGPA gene in the haemopoietic stem cells, which leads
to reduced / absent synthesis of GPI proteins.
 Increased sensitivity of erythrocytes to complement-mediated cell lysis due to
deficiency of membrane-bound GPI anchored proteins which normally function as the
inhibitors of reactive hemolysis. GPI-anchored proteins include: complement regulatory
proteins like DAF (decay accelerating factor, CD55), MIRL (membrane inhibitor of
reactive lysis, CD59),
 Used to detect the deficiency of GPI anchored proteins on RBC & WBC (neutrophils).
Reticulocyte enumeration
 This method provides excellent discrimination between reticulocytes and mature RBCs,
with greater precision, sensitivity, and reproducibility than the traditional method.
 The flow cytometric enumeration of reticulocytes uses fluorescent dyes that bind the
residual RNA, such as thiazole orange
 The fluorescence intensity is directly proportional to the amount of RNA and related to
the immaturity of the RBC, a reticulocyte maturity index can be given.
Fetomaternal Hemorrhage
 The use of flow cytometry for the detection of fetal cells is much more objective,
reproducible, and sensitive than the other traditional method.
 Fluorescent labeled antibodies to the rhesus (D) antigen can be used, or more recently,
antibodies directed against hemoglobin F.
 This method has the ability to distinguish fetal cells from Adult-cells (adult red cells
with small amounts of hemoglobin F).
 This intracellular approach, which uses permeabilization of the red cell membrane and
an antibody to the g chain of human hemoglobin, is precise and sensitive.
 These are the few applications of flow cytometer, but now days a flow cytometer is
broadly used in DNA analysis, cell cycle analysis, chromosome analysis, cell-culture
studies, detection of blood-parasites & bacteria in blood and body fluids etc. The main
principle is the same, only the change in the nature of sample & monoclonal Ab’s
tagged to cell surface.

13. Prepared by Sanjeev Kumar
B.Sc MLT,PGIMER,CHD
FLOW CYTOMETRY
Advantages of flow cytometry
• Rapid assessment of large no. of cells
• Multi parameter analysis
• High accuracy & reproducibility
• Objective analysis
• Ability to analyze many samples quickly
• Capable of data reduction
• Permanent data storage
• Ability to reanalyze data
• Requires relatively small sample
Disadvantage
• Expensive
• Need trained Flow cytometrist.
• Difficult to obtain the antibodies according to panel list.
• Chance of auto fluorescence and false negative result in case of deteriorate reagent