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 andqualitative analysis of several properties of cellpopulations from virtually any type of freshunfixed tissue or body fluid.

The properties measured include a particle’srelated size, relative granularity or internalcomplexity, 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

central core.

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

Sheath

Tank

Waste

Tank

Line PressureVacuum

Sample

Pressure

(Variable)Sheath

Pressure

(Constant)

Sample

Tube

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

Collection

Lens

SSC

Detector

Laser Beam

FSC

SS

CLymphocytes

Monocytes

Granulocytes

RBCs, Debris,

Dead Cells

Study of FSC and SSC allows us to know the

differentiation of different types of cells.

Why FSC & SSC?

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

(PerCP)

680nm

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

amount of fluorescent compound on the particle.

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

400nm 500nm 600nm 700nm

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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.

400nm 500nm 600nm 700nm

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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)

400nm 500nm 600nm 700nm

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Original from Cytomation Training Manual

Optics- Dichroic Filters

Long pass or short pass filters

Placed at a 45º angle of incidence

Part of the light is reflected at 90º , and part of the light is

transmitted and continues.

Dichroic Filter

Detector 1Detector 2

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.

Time

Electronics- Creation of a Voltage Pulse

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

other areas

Plot Types

Contour Plot Density Plot

Greyscale Density Dot Plot

www.treestar.com

Histogram

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

present more towards

the origin(0) on x(red)

- y axis (pink)

Bright : cells are

present away from

the origin(0) on

x(green) & y(yellow)

axis.

DIM

BRIGHT

Y-axis

CD4

X-axis

CD8

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

• Diagnosis and Classification

• Detection of MRDHematological Malignancies

• DNA Ploidy

• S Phase fraction Solid Tumours

• TBNK

• Phagocytic function defect

Primary Immunodeficiency

disorders

Cont..

• Reticulocyte count

• PNH

• Osmotic fragility assay

Hemolytic anaemia

• Feto- maternal Hemorrhage

• treatment response in Sickle Cell AnemiaFetal Hb detection

• Platelet receptor assays (Platelet count, GT, BSS)

• Platelet function assay (CD62P, PAC-1)

Bleeding Disorders

• CD34 STEM CELL COUNTS

• Residual WBC count in leukodepleted blood packs

• Flow cytometry Crossmatch

Transfusion and Transplant

• Surface markers in PMN, Monocytes

• Cytokine responseHost Immune

response in Sepsis

CLPD ON

FLOWCYTOMETRY

Objectives

Diagnosis of lymphoma

Classification of lymphoma

Ploidy analysis

Flow cytometric approach to the diagnosis

and classification of B- cell lymphoid

neoplasms.

B Cell Lymphoma

B CELL DIFFERENTIATION

How to differentiate between

Normal and Neoplastic B cells

1) Imunoglobulin light chain class restriction.

2) Aberrant antigen 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

LYMPHOMACD5

POSITIVE NEGATIVE

CD23 -FMC7 +

CD23 + FMC7 -CD23 + FMC7 +

MCL MARG: CD38-,CD23-, FMC7 +

DLBCL: CD38+LPL: CD38+

CLLPLL

CD10 POSITIVE

NEGATIVE

1. FOLLICULAR: FMC7 +2. DLBCL3. BURKITT: CD23 - FMC7 +4. B-ALL: CD23 - FMC7 -

CD103,CD25, CD123

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.

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.

CASE

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.

Flow cytometric approach to the diagnosis

and classification of T- cell lymphoid

neoplasms.

T CELL LYMPHOMA

Finding abnormal T/ NK cells.

1) Search for monoclonal T cells

2) T cells with aberrant antigen expression.

CD4/CD8 Ratio Loss of CD3

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 coexpressionIn 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 CD5In 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/Sézary syndrome

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

heterogeneous staining intensity).

Confirm characteristic morphology and clinical

presentation.

HTLV-1-

• DNA PLOIDY

• S PHASE FRACTION

PLOIDY ANALYSIS

Cell cycle analysis

The percentage 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

In neoplasm, 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

With IHC: cells in G1, S and G2 phases cannot be

Highest proliferative

activity: mean SPF,

35.3%

6.6%

6.5%

20.4%

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

immature cells

Abnormal marker expression of immature cells

CD38 / CD45 AGAINST

SSC

CD19, 7 on non lymphoid

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.

Intermediate

CD45 and

low side

scatter

BLAST

WINDOW

NEUTROPHI

LS

LYMPHOCYT

ES

MONOCYTE

S

RBC’S AND

DEBRIS

B CELLS

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.

BLAST

WINDOW

B CELLS

MONOCYTES

RBC’S AND

DEBRIS

LYMPHOCYT

ESNEUTROPHIL

S

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. Henry’s, 22nd edition,

Chapter 34

Flow cytometric approach to the diagnosis

and classification of ALL.

ACUTE LYMPHOID

LEUKAEMIA

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

ALL

HLA-

DR

TdT CD 10 CD 19 SmIg CyCD79

a

Pro- B ALL +/- + - + - +

Common

ALL

+ + + + - +

Pre B ALL + - - + - +

Mature B

ALL

- - - + +K/L +

Precursor B cell lymphoblastic

leukemia/lymphoma

Flow cytometric approach to the diagnosis

and classification of AML.

ACUTE MYELOID

LEUKAEMIA

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

Marker

CD41, CD61

B-cells cyCD22, CD22, CD19, CD20, FMC7, CD23,

CD79a, CD79b, SmIg, IgM

T-cells cyCD3, CD3, CD2, CD5, CD7, CD8, TCR-α/β,

TCR-γ/δ

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+,

anti-MPO+

Clinical, Genetic, Morphologic

Erythroi

d

Megakaryocyti

cMyeloid Monocytic

41+

61+

71++

GlyA+

36+, 64+,

14+, 33++36+

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

CD34 APC CD15 FITC CD56 A488

CD45

CD4

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

cytoplasm, lobulated or

indented nucleus with

variable nucleoli).

DLC

Blasts40 P8 L10 Monocytoid41

E1

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

ACUTE MYELOMONOCYTIC LEUKEMIA

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

Hematogones

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 lineage

MPO

FC, IHC, Cytochemistry

Monocyticdiferentiation

Atleast 2: NSE, CD11c, CD14,

CD64, lyzozyme

T lineage

Cytoplasmic CD3

Surface CD3

B lineage

Strong CD19 with atleast 1 : CD79a,

cytoplasmicCD22, CD10

Weak CD19 with atleast 2 strongly

expressed CD79a,

cytoplasmicCD22, CD10

Or Or Or

EGIL scoring system

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

scoring system

Flowcytometry analysis in MRD detection

Minimal Residual Disease

detection

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

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. Henry’s, 22nd edition, Chapter 34

ATLAS AND TEXT OF HEMATOLOGY. Dr Tejindersingh

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 Hübl