FLOW

CYTOMETRY
MODERATOR: DR. R.M. JAISWAL

By: Dr. Megha Gupta & Dr. Tashi Agarwal

FLOW CYTOMETRY

Definition:

Measuring properties of cell as they flow in a

fluid suspension across an illuminated light
path.
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
 Flow Cytometry
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.

The properties measured include a particles

related size, relative granularity or internal
complexity, and relative fluorescence intensity

Most commonly analyzed materials are:

blood,
bone marrow aspirate and
lymph node suspensions.
Principle of Flow Cytometry
Flow cytometer is composed of three main
components:

The Flow system (fluidics)

Cells in suspension are brought in single file past

The Optical system (light sensing)

a focused laser which scatter light and emit
fluorescence that is filtered and collected

The Electronic system (signal processing)

emitted light is converted to digitized values that
are stored in a file for analysis
The Flow System
One of the fundamentals of flow cytometry is the ability
to measure the properties of individual particles, which
is managed by the fluidics system.
When a sample is injected into a flow cytometer, it is
ordered into a stream of single particles.
The fluidic system consists of a FLOW CELL (Quartz
Chamber):
Central channel/ core - through which the sample is
injected.
Outer sheath - contains faster flowing fluid k/a
Sheath fluid (0.9% Saline / PBS) , enclosing the
Hydrodynamic Focusing

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.'
 The sample pressure is always higher than the
sheath fluid pressure, ensuring a high flow rate
allowing more cells to enter the stream at a given
moment.
High Flow Rate - Immunophenotyping analysis of
cells
Low Flow Rate - DNA Analysis
Sample
Tube

Sample
Sheath Pressure
Pressure (Variable)
Waste Sheath (Constant)
Tank Tank

Vacuum Line Pressure

OPTICS
After the cell delivery system, the need is to excite the
cells using a light source.
The light source used in a flow cytometer:
Laser (more commonly)
Arc lamp

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.

ARGON Lasers - 488nm wavelength (blue to blue

green)
When a light intersects a laser beam at the so called
'interogation 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.
OPTICS
a) LIGHT SCATTER
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.

OPTICS - 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).
OPTICS - 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 (eg. granules, cell
inclusions, etc.) Side scatter detector
Measuring cell granularity
 FSC
Detector
Laser Beam

Collection
Lens

SSC
Detector
Why FSC & SSC?

Granulocytes
Lymphocytes
SSC

Monocytes
RBCs, Debris,
Dead Cells
FSC

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.

The cells are labelled with fluorochrome-linked

antibodies or stained with fluorescent membrane,
cytoplasmic or nuclear dye.
Commonly used Fluorochromes

FLUOROCHROMES EMISSION
MAXIMUM
Fluorescein Isothiocynate (FITC) 530nm
Phycoerythrin (PE) 576nm
Peridin-chlorophyll alpha complex 680nm
(PerCP)
Allophycocyanin (APC) 660nm
Texas red 620nm
ECD( PE - Texas Red Tandem) 615nm
PC5 (PE - cyanin 5 dye tandem) 667nm
Optics
B) EMISSION OF FLUORESCENT LIGHT
(FLUORESCENCE)
As the fluorescent molecule present in or on the particle
is interrogated by the laser light, it will absorb energy
from the laser light and release the absorbed energy at
longer wave length.
Emitted photons pass through the collection lens and are
split and steered down specific channels with the use of
filters.
Emitted fluorescence intensity is proportional to the
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.
The system of filters ensures that each photodetector
receives light bands of various wavelengths.
Optical filters are designed such that they absorb or
reflect some wavelengths of light, while transmitting
others.
Types of filters
1. Long Pass 2. Short Pass
3. Band Pass 4. Dichroic
 Optics- Long Pass Filters
Transmit all wavelengths greater than specified
wavelength
Example: 500LP will transmit all wavelengths greater
than 500nm
Transmittance

400nm 500nm 600nm 700nm

Original from Cytomation Training Manual

 Optics- Short Pass Filter
Transmits all wavelengths less than specified
wavelength
Example: 600SP will transmit all wavelengths less
than 600nm.
Transmittance

400nm 500nm 600nm 700nm

Original from Cytomation Training Manual

 Optics- Band Pass Filter
Transmits a specific band of wavelengths
Example: 550/20BP Filter will transmit wavelengths
of light between 540nm and 560nm (550/20 = 550+/-
10, not 550+/-20)
Transmittance

400nm 500nm 600nm 700nm

Original from Cytomation Training Manual

Optics- Dichroic Filters
Long pass or short pass filters
Placed at a 45 angle of incidence
Part of the light is reflected at 90 , and part of the light is
transmitted and continues.

Detector 1
Detector 2

Dichroic Filter
OPTICS - DETECTORS
The photodetectors convert the photons to electrical
impulses.
Two common types of detectors used in flow cytometry:
Photodiode
used for strong signals, when saturation is a potential
problem (eg, forward scatter detector).
Photomultiplier tube (PMT)
more sensitive than photodiode but can be destroyed
by exposure to too much light.
used for side scatter and fluorescent signols.
ELECTRONICS

The electronic subsystem converts photons to

photoelectrons.

Measures amplitude, area and width of photoelectron

pulse.

It amplifies pulse either linearly or logarithmically and

then digitalizing the amplified pulse.
Electronics- Creation of a Voltage Pulse

Time
 Data Analysis- Plot Types
There are several plot choices:
Single Color Histogram

Fluorescence intensity (FI) versus the number of cells

counted.
Two Color Dot Plot

FI of parameter 1 versus FI of Parameter 2

Two Color Contour Plot

Concentric rings form around populations. The more

dense the population, the closer the rings are to each
other
Two Color Density Plot

Areas of higher density will have a different color than

 Plot Types
Contour Plot Density Plot

Histogram

Greyscale Density Dot Plot

www.treestar.com
DATA ANALYSIS - GATING

Gating is in essence electronic window that sets

upper and lower limits on the type and amount
of material that passes through.
Selection of only a certain population of cells
for analysis on a plot.
Allows the ability to look at parameters specific
to only that subset.
Interpretation of Graphs

An important tool for evaluating data is the dot

plot.

The instrument detects each cell as a point on

an X-Y graph. This form of data presentation
looks at two parameters of the sample at the
same time.
Three common modes for dot plots
are:

Forward scatter (FSC) vs. side scatter (SSC)

To look at the distribution of cells based upon size &
granularity
Single color vs. side scatter
To visualize the expression of the fluorescence of the
cells
Two-color fluorescence plot.

To differentiate between those cells that express only one

of the particular fluorescent markers, those that express
neither, and those that express both.
used to discriminate dead cells from the live ones that
are expressing the desired fluorescence.
 When to say an antigen is positive
or negative?
A sample that has some
cells single positives for
CD8 along the x-axis
(green arrow)
some single positives for
CD4 along the y-axis (red
arrow).
Upper right quadrant of the
plot - cells positive for both
fluorescent markers
(purple arrow).
Lower left quadrant - cells
negative for both markers
(orange arrow).
 How to differentiate dim & bright
expression of an antigen?

Dim : cells are BRIGHT

present more towards
the origin(0) on x(red) DIM
- y axis (pink) Y-axis
CD4

Bright : cells are

present away from
the origin(0) on X-axis
x(green) & y(yellow) CD8

axis.
WHAT IS UNIQUE IN
FLOWCYTOMETRY

MULTIPARAMETRIC
RAPID ANALYSIS OF LARGE NUMBER OF
CELLS
INFORMATION AT A SINGLE CELL LEVEL
DETECTION OF RARE CELL POPULATIONS
ALLOWS PHYSICAL ISOLATION OF CELLS
OF INTEREST
USES OF
FLOWCYTOMETRY
APPLICATIONS

ANALYSIS
Immunophenotyping

Dyes that bind to nucleic acids (DNA, RNA)

Functional assays
CELL COUNTING
CELL SORTING
CLINICAL APPLICATIONS

Absolute CD4 counts

HIV/AIDS

HLA B27 assay

Joint Pain

Hematological Diagnosis and Classification

Detection of MRD
Malignancies
DNA Ploidy
Solid Tumours S Phase fraction

Primary TBNK
Immunodeficiency Phagocytic function defect
disorders
Cont..

Reticulocyte count
Hemolytic anaemia PNH
Osmotic fragility assay

Feto- maternal Hemorrhage

Fetal Hb detection treatment response in Sickle Cell Anemia

Platelet receptor assays (Platelet count, GT,

Bleeding Disorders BSS)
Platelet function assay (CD62P, PAC-1)

Transfusion and CD34 STEM CELL COUNTS

Residual WBC count in leukodepleted blood
Transplant packs
Flow cytometry Crossmatch
Host Immune Surface markers in PMN, Monocytes
response in Sepsis Cytokine response
CLPD ON
FLOWCYTOMETRY
Objectives

Diagnosis of lymphoma
Classification of lymphoma
Ploidy analysis
B Cell Lymphoma
Flow cytometric approach to the diagnosis
and classification of B- cell lymphoid
neoplasms.
B CELL DIFFERENTIATION
 How to differentiate between
Normal and Neoplastic B
cells

1) Imunoglobulin light 2) Aberrant antigen

chain class restriction. expression.

MONOCLONALITY CD 13, CD 33, CD 5 ON B

CELLS
Normal, polyclonal B-cells are a
mixture of kappa-B-cells and
lambda-B-cells.
A B-cell carries either kappa- or
lambda-light chain on its surface. And
normal polyclonal B-cells are a mixture
of kappa-B-cells and lambda B-cells as
can be seen in the left-hand figure.

Monoclonal mature B-cells are either

kappa or lambda.
If a malignant B-cell clone proliferates
this will result in a B-cell population
consisting of either only kappa- or only
lambda-B-cells. The latter case (i.e.
lambda-monoclonal B-cells) is
symbolized in the left-hand figure.
Expression of CD5
The arrow in the right panel
points to the abnormal,
strong expression of CD5 by
B-cells. CD5 expression as
strong as this can usually
only be found on T-cells.
Normal B-cells show no or
only a weak expression of
CD5 (left-hand panel)

Weak expression of CD20

The B-cells in the right panel
show only a weak
expression of CD20 (arrow).
For comparison: normal
CD20 expression in the left-
hand panel.
APPROACH TO B CELL
LYMPHOMA 1. FOLLICULAR: FMC7 +
CD5 2. DLBCL
3. BURKITT: CD23 - FMC7 +
4. B-ALL: CD23 - FMC7 -
POSITIVE NEGATIVE

CD23 - CD23 + FMC7 - POSITIVE

CD10
FMC7 + CD23 + FMC7 +

NEGATIVE
MCL MARG: CD38-,CD23-,
CLL FMC7 +
CD103,CD25, CD123 DLBCL: CD38+
PLL
LPL: CD38+
POSITIVE-HCL
NEGATIVE
Chronic lymphocytic leukemia

Typical phenotype: CD20 (d), CD22 (d), sIg

(d), CD23+
FMC-7-
Characteristic morphology
Testing for the prognostic markers CD38 and
ZAP-70 can be considered
Mantle cell lymphoma
Variable phenotype not typical for CLL;
often CD20 (i), sIg (i), CD23-, FMC-7 +

IHC : Cyclin-D1
FISH : t(11;14)/CCND1 rearrangement
Hairy cell lekaemia

Typical pheotype: CD20 (b), CD22 (b), CD11c

(b),
CD25+, CD103+, sIg (i)
Confirm characteristic morphology of a hairy
cell and TRAP +

A small subset of HCL are CD10+ but are

morphologically similar to CD10- HCL.
Follicular lymphoma

Usually bcl-2, CD43.

Some follicular growth.
t(14;18)/BCL-2 rearrangement.

D/D
1. DLBCL : diffuse growth pattern against the
nodular growth pattern in FL
2. BL : morphologial (vacuoles), High S phase
fraction.
CASE

80/F
c/o cervical adenopathy

On CBC : an absolute
lymphocytosis 5
109/L;

PBF is flooded with

small mature
lymphocytes with
condensed chromatin
and scant cytoplasm
along with numerous
smudge cells.
 On flow

A diagnosis of CLL can be made. CD5+ CD23+

Not that simple

A certain immunophenotype may be typical but

is by no means obligatory.

The significance of one marker depends on

the expression of other markers.

The strength of antigen expression is

important.
T CELL LYMPHOMA
Flow cytometric approach to the diagnosis
and classification of T- cell lymphoid
neoplasms.
 Finding abnormal T/ NK cells.

1) Search for 2) T cells with aberrant

monoclonal T cells antigen expression.

Loss of CD3
CD4/CD8 Ratio Overexpression of CD5
Normally, the CD4/CD8-T-
Cell ratio in peripheral
blood is about 2:1.

In a T-lymphocytic leukemia
this ratio can shift
dramatically. Unfortunately,
this ratio may also be altered
by many non-malignant
diseases. eg viral infections.
Therefore, only extreme
alterations of this ratio can
be regarded as a sign for T-
lymphocytic malignancy.
CD4/CD8 coexpression
In the right-hand dot-plot you can
see cells that express both the CD4
and the CD8-antigen (arrow) which
is highly irregular. In addition both
antigens are expressed weakly
(compared to normal T-cells). Left-
hand panel shows a normal
situation.

Loss of CD3
Overexpression of CD5
In the right-hand dot-plot you can
see T-cells which overexpress CD5
while they lack CD3 (arrow). Only a
few normal T-cells are present.
(blue oval). Left-hand panel shows
a normal situation.
PROBLEMS IN DIAGNOSIS OF T-
CLPD

Relatively low incidence.

5-25% of all lympoid neoplasms.
Clinico-biological heterogeneity.
Lack of distinctive genetic markers.
T CELL DIFFERENTIATION
 T- CLPD BY
FLOWCYTOMETRY
CTCL/Szary syndrome

Often CD7-, CD26-, CD4+, CD25+/- (with

heterogeneous staining intensity).
Confirm characteristic morphology and clinical
presentation.
HTLV-1-
PLOIDY ANALYSIS
DNA PLOIDY
S PHASE FRACTION
Cell cycle analysis

Thepercentage of the cells in each region is

analyzed. In normal tissues
95% cells - G0/G1 phase
2.5% cells - S phase
2.5% cells - G2/M phase
Inneoplasm, percentage of cells in S and G2/M
phase increases which is expressed as S phase
fraction or the proliferation index
S Phase, synthesis phase.

It is the part of cell cycle in which DNA is

replicated occurring between G1phase and G2
phase.
S phase has strong correlation with
grading.
DNA ploidy has no correlation with
grading.
ADVANTAGES OVER IHC
Highest proliferative
activity: mean SPF,
35.3%

20.4%

6.5%

6.6%
DIAGNOSIS OF ACUTE
LEUKAEMIAS ON
FLOWCYTOMETRY
 STEPS

Finding the blast population

Defining the immunophenotype

Diagnosis
 A malignant blast population
may be detected because of

Increase of Abnormal marker

expression of
immature cells immature cells

CD38 / CD45 AGAINST CD19, 7 on non lymphoid

SSC cell
Finding immature
cells using CD45-
CD34 dot-plots

The arrows points at the

blast populations, which
is very conspicuous in
case AL 1 (upper right)
and AL 3 (lower right).
In case AL 2 (lower left),
the difference between
the normal picture is
more subtle and the
blasts may be missed
because in this case the
blasts are CD34
negative.
 NEUTROPHI
LS

MONOCYTE
S
Intermediate
RBCS AND CD45 and
DEBRIS low side
scatter
BLAST
B CELLS WINDOW
LYMPHOCYT
ES

CD45/SSC gating strategy is more sensitive than FSC/SSC

gating and it dilineates the blasts well.
Finding immature
cells using CD45-
Side Scatter dot-plots

The three cases of

acute leukemia: The
arrows point to the
blast populations which
are clearly visible in all
three cases.
Even the blasts of case
AL 2 can easily be
spotted.
 MONOCYTES
LYMPHOCYT
ES
NEUTROPHIL
S

B CELLS BLAST
WINDOW

RBCS AND
DEBRIS

CD45/SSC gating strategy is more sensitive than FSC/SSC

gating and it dilineates the blasts well.
Example of an abnormal antigen
expression on myeloid blasts

Compare the normal blasts (upper

dot-plot, blue oval) with those of an
acute myeloid leukemia (lower dot-
plot, red oval)): the malignant
blasts abnormally express CD15
and they show an increased
expression of CD34.

Note: CD34-negative cells have

been removed for reason of clarity.
DIAGNOSIS
WHICH ONES TO IMMUNOPHENOTYPE?
1. Equivocal morphology
2. Cytochemistry is noncontributory
3. Specific subtypes

LEUKAEMIA VS NON LEUKAEMIA

1. Overlapping morphology. Eg: hematogones, viral
infections.
2. Partially treated acute leukaemia
PROGNOSTIFICATION

CYTOGENIC AND MOLECULAR

ABNORMALITIES
Association with specific cytogenic
abnormalities
DNA ploidy

RESIDUAL DISEASE MONITORING

CLASSIFICATION

Acute leukaemia is classified on the basis of

immunological markers into

B lineage ALL
T lineage ALL
Acute myeloid leukaemia
Acute leukaemia of ambiguous lineage
 How to define the lineage of
leukaemia

THE FLOW CYTOMETRIC EVALUATION OF HEMATOPOIETIC

NEOPLASIA Brent L. Wood, Michael J. Borowitz. Henrys, 22nd edition,
Chapter 34
ACUTE LYMPHOID
LEUKAEMIA
Flow cytometric approach to the diagnosis
and classification of ALL.
How to diff ALL from NHL

CD 34
TdT
Bcl2
CD99

NHL cases with spillover demonstrate bright

CD45 expression while it is moderate in B
ALL.
Subtypes of ALL
Flow cytometric immunophenotyping does not
provide a suitable surrogate tool for detection
of these subtypes of ALL.
Subtype HLA- TdT CD 10 CD 19 SmIg CyCD79
ALL DR a
Pro- B ALL +/- + - + - +

Common + + + + - +
ALL
Pre B ALL + - - + - +

Mature B - - - + +K/L +
ALL
Precursor B cell lymphoblastic
leukemia/lymphoma
ACUTE MYELOID
LEUKAEMIA
Flow cytometric approach to the diagnosis
and classification of AML.
Diagnosis of AML

Morphology Auer rod

Cytochemistry >3% MPO positive

Immunophenotyping CD33, CD13, CD117, anti-

MPO
Cytogenetics t(8;21), t(15;17), inv16,
MLL, t(9;11), t(6;9), t(3;3),
t(1;22)
CD markers used for
hematolymphoid neoplasms
All white cells CD 45 (LCA)
Myeloid cells Anti-MPO, CD13, CD33, CD14, CD117
Monocytic Markers CD14, CD64
Megakaryocytic CD41, CD61
Marker
B-cells cyCD22, CD22, CD19, CD20, FMC7, CD23,
CD79a, CD79b, SmIg, IgM
T-cells cyCD3, CD3, CD2, CD5, CD7, CD8, TCR-/,
TCR-/
NK cells CD16, CD56, CD57
Plasma cells CD38, CD138, Kappa & Lambda chains
Blasts CD34, TdT
Others HLA-DR, CD55, CD59, cyclin D1, glycophorin A
 Myeloblast characterization

13+, 15+, 33+, 36+, 64+,

36+
anti-MPO+ 14+, 33++

41+ 71++
61+ GlyA+

Megakaryocyti Erythroi
Myeloid Monocytic
c d

Clinical, Genetic, Morphologic

Classification - FAB
M0 : AML-minimal differentiation
M1 : AML-without maturation
M2 : AML-with maturation (blast<80%)
M3 : AML-promyelocytic
M4 : AML-myelomonocytic (>20% monocytes)
M5 : AML-monocytic
M6 : AML-erythroid
M7 : AML-megakaryocytic
AML- minimal differentiation
(M0)

Myeloblasts - < 3% positivity with SBB, MPO &

PAS-, NSE-
Myeloid antigens - CD13+, CD33+, CD117+,
and/or MPO+
CD34, CD38, HLA-DR, and TdT - often
expressed
AML- Promyelocytic Leukemia
(M3)
Phenotype - CD13h+, CD33++, CD34-, HLA-
DR-
Diagnostic molecular alteration - PML/RARA
t(15;17) translocation
Strongly positive - MPO, SBB, PAS
cytoplasmic positivity.
Characteristic morphology
D/D : AML-monocytic leukemia (M5) -
HLA-DR+, CD11c+, CD14+ & CD64+
AML - Myelomonocytic Leukemia
(M4)

Phenotype:
myeloid antigens - CD13+ & CD33+, HLA-DR+
monocytic
markers: CD14+, CD4+, CD11b+,
CD11c+, CD64+, CD36+, CD68+
Blasts >20% of marrow NEC
Monocytic component >20% of NEC &
monocytes in blood >5 x 109/L
AML - Monocytic Leukemia
(M5)

Phenotype: CD33 (b), CD13+, HLA-DR+

Characteristic CD14+, CD11b+, CD11c+,
CD64+, CD68+
Cytochemistry : NSE +
M5a : Acute monoblastic leukemia
M5b : Acute monocytic leukemia
CD45

CD4

CD34 APC CD15 FITC CD56 A488

AML - Megakaryocytic leukemia
(M7)

CD41+, CD61+, CD13+, CD33+

CD34, HLA-DR - Negative

 CASE
29yrs/ F
O/E : Fever, Pallor, Gum
hyperplasia,
Hepatosplenomegaly.
CBC : Hb-5.2 g%, Plt-
19,000/cu.mm
PBF : shows blasts and dual
differentiation to
granulocytes and
monocytoid cells (large cells,
abundant pale blue DLC
cytoplasm, lobulated or Blasts40 P8 L10 Monocytoid41
indented nucleus with E1
variable nucleoli).
ACUTE MYELOMONOCYTIC LEUKEM

red - dim CD45and low

side scatter.
Positive for - CD13,
cyMPO, CD34, HLA-
DR, CD33, CD11c
Negative for - CD10,
CD19, CD3, CD79a.

Blue - bright CD45 &

moderate side scatter.
Positive for - CD14,
CD11c, CD13.
Negative for - CD34,
cyMPO, CD3, CD10,
CD19
Hematogones
Physiologic precursors of maturing B-cells.
Confused with neoplastic immature lymphoid
cells of B lymphoblastic leukemia/ lymphoma
or B-ALL.
Increased in:
Autoimmune or congenital cytopenias
Solid organ tumors e.g. neuroblastoma

AIDS

NHL

Post-chemotherapy and after BMT

Copper deficiency
 Morphologically, hematogones resemble
lymphoblasts.

Hematogones can be differentiated from

lymphoblasts by
Unique Immunophenotypic pattern :
CD34 < TdT < CD20 < PAX5
Variable CD10 & CD20
"J shaped trail pattern" : on CD10/20 Dot plot
 Lymphocytes

Hematogone

Immunophenotypic analysis of hematogones in 662 consecutive bone marrow

specimens by 4-color flow cytometry. Mckenna et al, BLOOD, 15 OCTOBER 2001
Acute leukaemia of ambiguous
lineage

Mixed phenotype acute leukaemia

Acute undifferentiated leukaemia

NK/plasmacytoid dendritic cell leukaemia

MPAL WHO 2008

Myeloid
T lineage B lineage
lineage

MPO Strong CD19 with

atleast 1 : CD79a,
FC, IHC, Cytoplasmic CD3
cytoplasmic
Cytochemistry CD22, CD10

Or Or Or
Monocytic Weak CD19 with
diferentiation atleast 2 strongly
expressed
Atleast 2: NSE, Surface CD3 CD79a,
CD11c, CD14, cytoplasmic
CD64, lyzozyme CD22, CD10
 EGIL scoring system

The European group for the Immunological Classification of Leukaemias (EGIL)

scoring system
Minimal Residual Disease
detection
Flowcytometry analysis in MRD detection
Introduction

At diagnosis the tumour burden is

approximately 10^12 leukaemic cells.
Induction chemothereapy achieves a 3 log cell
kill bringing it down to 10^9 leukaemic cells.
Light microscopy of BMA can detect leukaemia
only when there are more than 5 blasts/ 100
nucleated cells. Anything less than that is
termed remission.
Introduction

What is Minimal residual disease or MRD?

It is that submicroscopic disease that cannot be
detected by conventional light microscopic
examination of the BMA.

It could be as high as 1 billion leukaemic cells.

It can be performed by two techniques: FCM &

PCR.
Used mainly in -

1. Acute leukaemia for guiding thereapy as well

as prognostic purposes.
2. Patients with low grade B cell malignancies
undergoing high dose chemotherapy.
3. Post stem cell transplant and
immunothereapy.
4. Lymphoma spillover.
REFERENCES

THE FLOW CYTOMETRIC EVALUATION OF

HEMATOPOIETIC NEOPLASIA Brent L. Wood, Michael
J. Borowitz. Henrys, 22nd edition, Chapter 34
ATLAS AND TEXT OF HEMATOLOGY. Dr Tejinder
singh
Manual: 6th Advanced TCS Flowcytometry workshop on
hematological malignancies.
Flow Cytometry in Hematopathology. A visual approach
to data analysis and interpretation. Doyen, Lawrence
and Raul.
Flow Cytometric Analysis of Leukemia and Lymphoma -
The Basics Univ.Doz.Dr.med. Wolfgang Hbl