Flow Cytometry Principles and Applications

Second Basic Hematopathology Course

TMH
June 111!" !#11
$r Sumeet %u&ral" M$
Associate Pro'essor
$epartment o' Pathology
Tata Memorial Hospital" Mum(ai
s)gu&ral*hotmail+com
,ole o' a Hematopathologist

-sta(lishing $iagnosis

Prognostication .su(classi'ication/

Prediction

-0aluation o' treatment e''ecti0eness .M,$/

1eu2emia31ymphoma $iagnosis

Clinical4 History and -5amination

,outine in0estigations

Morphology .smear" (iopsy/

Ancillary techni6ues
Ancillary techni6ues

7mmunohistochemistry"

Flow Cytometry"

Cytogenetics"

Molecular $iagnostics

$iagnostic

Prognostic

Predicti0e
100bp
ladder
1 2 3 4 5 6
RT-PCR (Reverse Transcriptase Polymerase Chain Reaction)
detects diagnostic translocation markers like BCR/ABL in CML -
BCR/ABL:
b3a2 437bp
Flow Cytometry
1ocali8ation o' antigens or proteins in cells using
la(eled anti(odies through antigenanti(ody
interactions"
,eaction 0isuali8ed (y a mar2er
.'luorescent dye" en8yme" colloidal gold etc/
7mmunophenotyping
Complementary
FCM
multicolor immunophenotyping
'luids
Immunoh!"o#hem!"r$
mostly single color
(iopsy
9ur practice
Flow cytometry 1i6uid and Solid
7mmunohistochemistry Solid and 1i6uid
'low : cyto : metry

1;<= The 'irst impedance(ased 'low cytometry

de0ice" using the coulter principle .>allace A
Coulter/

1;?@ The 'irst 'luorescence(ased 'low

cytometry de0ice .7CP 11/ (y >ol'gang %Ahde"
Bni0ersity o' Munster
7t is the measurement o' cellular properties as cells mo0e
in a 'luid stream .'low/" past a stationary set o' detectors
.thousand e0ents per second/
Techni6ue o' 6uantitati0e single cell analysis
Principle
7t analyses
physical" and
chemical properties .immuno'luorescence/ o' cell
Components o' a Flow Cytometer

Fluidics4 a %lo& #ell with sheath 'luid

.h$drod$nam# %o#u!!n'/

9ptics4 LA()R(" single wa0elength"

coherent light .howe0er incoherent light is o'
randomphase 0arying with time and
position/

a de"e#"or and Analogueto$igital

Con0ersion .A$C/ system which generates
FSC and SSC as well as 'luorescence
signals 'rom light into electrical signals that
can (e processed (y a computer

an ampl%#a"on !$!"em linear or

logarithmic

a #ompu"er 'or analysis o' the signals

Single or multiple 1asers
Sorter
Types
-
Single 1aser or Multiple 1asers
.1 laser three color" D lasers 1@ 'luorescence detectors/
-
Sorter .so as to puri'y populations o' interest /
-
1aser scanning cytometers
Ad0antages o' a FCM

Study o' cells" chromosomes and particles .analysis"

counting and sorting/

Thousand o' particles per second

Multiparametric analysis at a single cell le0el

Pattern studies

Sorting
,esearch Applications

Auto'luorescent Proteins

Antigen or 1igand $ensity

Apoptosis

-n8yme acti0ity

$EA" ,EA content and changes in the cell cycle

Mem(rane Potential

Cyto2ine receptors and itFs synthesis

$rug upta2e and e''lu5

Phagocytosis

Gia(ility

Changes in 7ntracellular pH

Changes in 7ntracellular calcium

Changes in 7ntracellular glutathione

Changes in 95idati0e Burst

$iagnostic Applications
1+ Monitoring A7$S patients
!+ 7mmunophenotyping4 $iagnosis" su(typing and
prognostication o' hematolymphoid malignancies
=+ Monitoring Minimal ,esidual$isease
D+ $etermining C$=D counts
<+ ,eticulocyte Counts
?+ $iagnosis o' PEH
H+ $EA analysis o' Sphase 'raction
@+ Platelet counts
7t re6uires a suspension o' single cells

Eo in'ormation on tissue architecture
-5pensi0e" maintenance
Shortcomings o' a FCM
7mmunophenotyping
Sample
Process
Ac6uire
Analysis
Final report
Flow cycle
,e'erring physician Sample collection3transportation
Preparation o' cells o' interest

Add anti(odies tagged with 'luorochromes
Ac6uire .'low/ the cells 1AS-,
Ampli'ied signals $igiti8ed
Analy8e ,eport ,e'erring physician
Antigen e5pression4
,BCs" >BCs" Platelets" 9thers
C$=
Cyto C$=
Tdt
1ysis o' red cells
Add reagents
Process
7ntracellular staining
For 7ntracellular staining" cells are 'irst 'i5ed in
suspension and then permea(ilised (e'ore adding
the 'luorochrome
This allows pro(es to access intracellular
structures while lea0ing the morphological scatter
characteristics o' the cells intact
Commercial 2its37nhouse
Cells are 'i5ed permea(ilised
add anti(ody .F1/
C$=
Cyto C$=
Tdt
,esults
Physical Properties
Scatter pattern" FSC 0s SSC
Forward - Size
Side - Granularity
Eote4 no ,BCs
Chemical properties
7mmuno'luoresence
How do we select cells o' interestI
%ating
Scatter pattern" FSC 0s SSC
Forward - Size
Side - Granularity
Cells o' interest4 FSC 0s SSC
Forward - Size
Side - Granularity
FSC 0s SSC C$D< %ating C*1+ ,a"n'
Cells o' interest may (e scanty
C$ !==;1 7E$B1JA, .BM/+##=
FSC Height
S
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C$1; P-
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C$= PerCP
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FSC vs SSC
Study Bcells
Study Tcells
T and B cells
C$ !==;1 7E$B1JA, .BM/+##=
FSC Height
S
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%ating
Small cell 1ymphoma
>hat is C$D< gatingI
How is C$D< gating (etter than SSC3FSC
gating in Acute 1eu2emiaI
Importance of CD45 gating when lasts are few
%uess
C$D< gating means C$D< in each tu(e
CD45
Myeloid tu(e T cell tu(e B cell tu(e
M$S tu(e 9ther tu(e
CD45 CD45 CD45 CD45
%enerally =? tu(es"
How to select anti(ody com(inationsI
C$= F7TC
C$1; P-
C$1= PerCP
C$D< T,
Four color means a coc2tail o' 'our anti(odies
C$= F7TC
C$D P-
C$@ PerCP
C$D< T,
or o' same lineage++
o' di''erent lineages++
-5pression o' myeloid antigens ? colors
C$11HAPC
C$11(P-CyH
C$1<F7TC
C$D<APC CyH
C$1=P-
C$1?P-Cy<
C$1= F7TC
7ndian %uidelines" !##@
Most la(s still do =D colors
.single 1aser/
1# anti(odies plus controls 'or Acute 1eu2emia
; anti(odies plus controls 'or C1P$
Pu(lished minimal panels may (e 7EA$-KBAT-
,ecommendations
7JPM" !##@
All lymphoid cells C$D<: .1CA/
Bcells C$1;" C$1#" C$!#" C$!=" FMCH" 23l 1C" cC$!!
Tcells C$=" C$D" C$<" C$H" C$@" cC$=
Myeloid cells C$1=" C$==" C$11H" anti MP9
Mega2aryocytic C$D1" C$?1
Blasts C$=D" Tdt" C$;;" H1A$,
9thers4 C$1a" C$!" C$11c" C$1<" C$1?" C$!<" C$=@" C$D=" C$<?"
C$<<" C$<; C$?D" C$H;a" C$1#=" C$1=@" TC, alpha3(eta" TC,
gamma3delta" 7gM" 7g$
Cytometry B Clin Cytom+!##; MayLH?.=/41;;!#<
? color and more
C$!=APC
C$<P-CyH
1am(da F7TC
C$1;APC HH
Jappa P-
C$!#PerCpCy<+<
1am(da F7TC
Single color ? color" all B cell lineage
Eormal ? color pattern with T and B cell mar2ers
Multiple gates
Eormal ? color pattern with T and B cell mar2ers
Colors unstoppa(leM

-uro'low @ colors

Brentwood et al 1# colors

,oederer et al 1< colors

Most la(s do =D color 7PT

7n 7mmunophenotyping" FCM helps in detecting 'ollowing4
presence or a(sence o' an antigen
intensity
(lasts
Clonality4 1C," G(eta repertoire
scanty cells" Maturation patterns
C$= anti(ody (y FCM detects the 'ully assem(led TC,C$= comple5"
which is present on the sur'ace o' T cells only+
7n contrast" the C$= 7HC stain usually detects only the epsilon
component o' C$=" there'ore cannot distinguish (etween T N EJ cells+
$iagnosis o' 1eu2emia3lymphoma

Morphology
HN- stain Biopsy
%iemsa stain Aspirate

Cytochemistry .MP9" ES-/

7mmunohistochemistry" Flow cytometry

Cytogenetics" F7SH

Molecular methods
Case
<< year old male
Bilateral cer0ical and a5illary lymph
nodes
Eormal Complete Blood Counts
Pro0isional diagnosis4 1ymphoma
Ad0ise4
Biopsy diagnosis
FEAC and 'low
= color
panel
= color
panel
C$D<" C$!#" C$1;" C$<" C$!=" C$!!w2" C$!#" Jappa 1C" C$!##
7mpression4 Bcell lymphoma" S11
Prognostic mar2ers4 C$=@" OAPH#
= color
panel
C$1; gating
Same patient o' C11"
howe0er"
C$1; gating was done
D color
panel
BC1P$ ? color panel
$e(ris
BC1P$ ? color panel
C$D<" C$!#" C$1;" C$<"
C$!=" C$!!w2" C$!#" Jappa
1C" C$!##
7mpression4 Bcell
lymphoma" S11
Su(typing o' Small B cell lymphomas
Patterns recognition

M$S

Hematogones
Eormal BM
Maturation patterns o' myeloid 3 monocytic cells
Eeutrophils
C$1=:" C$1?:
Eeutrophils
C$11(:"
C$11H
Bcell maturation
Cyto mu
hea0y chain
7gM:" 7g$:
C$<:
7gM
7g% 7gA
C$1#" BC1?:
Pa5< .My:/
C$H;a .T:/
7g%"A"
M+$+-
7gM
7gM
BC,
Tcell maturation
Prothymocyte Su(capsular Th Cortical Th Medullary Th PTC
C$H
Tdt
C$1a
C$!3C$<
C$=
C$D3C$@
sur'ace
$PT
C$D:
C$@:
cytoplasmic
Myelodysplastic syndrome
M$S
Scatter pattern and maturation pattern

FSC 0ersus SSC

Myeloid cells4 C$11c" C$1=" C$1<" C$1?

Monocytic4 C$==" C$<?" C$1D

-rythroid4 C$!=<" C$H1

Mega2aryocytic4 C$D1" C$?1" Morphology

Eormal M$S
Patterns o' antigen e5pression %ranulocytic maturation

FSC 0s SSC
Patterns o' antigen e5pression %ranulocytic maturation

C$D< 0s SSC
Eormal M$S
C$1= C$1?
C$11c C$1?
Eormally Maturing %ranulocytes
(#-le/Con#a.e pa""ern /a.e pa""ern
%ranulocytic maturation in M$S .C$1= 0s C$1?/
01RMAL M*(
Eormal M$S
%ranulocytic maturation in M$S .C$11C 0s C$1?/
01RMAL M*(
Eormal M$S
Hematogones
Hematogones
medium si8ed lymphoid cells"
scant cytoplasm"
round to irregular nuclei"
dense homogeneous chromatin
no 0ery small nucleoli
Bone marrow 'rom a =yearold (oy
with mega2aryocytic throm(ocytopenia+
Hematogones (ear close resem(lance
to the neoplastic lympho(lasts
Eumerous lympho(lasts are present in
this (one marrow smear 'rom a <year
old girl with precursor BA11
Hematogones B-cell ALL
Another case
C$1;:" C$1#:" FMCH:" C$H;(:" C$=@:" 11C:
C$=" C$<" C$!=" J1C
$iagnosis4 High grade B cell lymphoma
Pleural 'luid" lymphoid rich e''usion
C$1#:" C$1;:" H1A$,:
B cell A11"
Bur2ittPs 1ymphoma
C$1#:" C$1;:" H1A$,:
F1"
$1BC1
B cell A11"
Bur2ittPs 1ymphoma
C$1#:" C$1;:" H1A$,:
F1"
$1BC1
Hematogones
.Tdt:3/
B cell A11"
Bur2ittPs 1ymphoma
1ymph node FEAC and FCM
1ymph node Biopsy and FCM
Primary 'ollicles
7gM: 7g$:
Secondary 'ollicles" 7g$
Mantle 8one
7gM: 7g$:
B cell clonality
Small B cell lymphomas
CD!"#
Brent >ood et al
II ,ole o' FCM
Follicular lymphoma " %rade 1 Follicular hyperplasia
F1 .%rade =/ ,eacti0e
Bcl! might not help
'low 'low 'low
1ight chain restriction
C$!# and lam(da
Kuantitation (y FCM
Platelet counts
Garious method
7nternational Flow ,e'erence Method
RBC/Platelet Ratio Method
(Dual Platform Method)
Absolute Platelet Count=
RBCevents X RBC count
Platelet events (Automated Cell Analyzer)

7S1H Tas2 Force" Am J Clin Pathol 11<" D?#D?D+.!##1/
C*
41/61
RBC
P
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Single Plat'orm 7mmunoplatelet method
Single Platform Bead Assay
Absolute platelet count =
Gated Plt events X Bead count
Gated bead events (fixed value
P
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B
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Sehgal J" Cytomerty B" Clinical Cytometry" !#1#
1ymphocyte su(set analysis
C$D3C$@ Counts
1ymphocyte su(set
analysis
Eormal peripheral (lood
C$D P-
C$@
F7TC
PEH studies
.>BCs and ,BCs/
7t is a rare clonal stem cell disorder o' hematopoietic cells
that is associated with intra0ascular hemolysis" 0enous
throm(osis" and cytopenias related to (one marrow
aplasia+
Somatic mutations o' P7%A gene
1oss o' %P7 .C$<<" C$<;/
F1A-, test4 7n 0itro (inding o' a 'luorescentla(eled
modi'ied to5in that is dependent on the presence o' %P7
anchors+
Eormal Eeutrophils
A(normal Eeutrophils
C$=D stem cells enumeration
7SHA%- protocol
$ual Plat'orm 1yse wash method
Per'ormed in duplicate
Ac6uire at least 1## C$=D: e0ents 'or an intra assay C+G o' 1#Q
Four parameters are used

FSC

SSC

7ntensity o' C$=D staining

7ntensity o' C$D< staining

All initial ungated e0ents must (e ac6uired and isotype controls
are not re6uired
Then analy8ed in a se6uential manner .B991-AE gating/
as per the 7SHA%- protocol
7sotype controls are not re6uired with 7SHA%- gating system
CD3 ! ="#"$ %$&&
Absolute CD3=
CD3! X 'BC
$&&
ERC$=D: e0ents in ,D
D=CD() events *n R$
7SHA%- Single Plat'orm
+ubtract ,AAD ) cells
from R$
R,=bead events
-yse no .ash /rocess*n0
Reverse /*/ett*n0 *s essent*al
,eticulocyte counts
$EA Ploidy
FCM studies cell sur'ace antigens .phenotype/" $EA content .ploidy/ .$EA inde5/ and
$EA 'low cytometric proli'eration analysis.Sphase 'raction or QSphase/+
Minimal residual disease in A11
.eg+" M,$ 1ite/
<yearold (oy presented with 'e0er 1 month
9n e5amination4 Pallor" Hepatosplenomegaly
Complete Blood Counts
H( ?gmQ
T1C DD"###3cmm
Platelets 1+!<la2h3cmm
Peripheral Blood Smear -5amination
$iagnosis
MP9" ES- negati0e
MP9 negati0e ES- negati0e
Diagnosis
= Color FCM
FSC 0s SSC
Blasts only
CD$%& CD$"& '()D* positive lasts
CD+4 negative
Diagnosis, --cell )((
Morphological di''erentials
C$1;" C$1#" H1A$, positi0e (lasts
1+ Bcell A11
Tdt" C$=D
!+ Bur2ittPs 1ymphoma
Jappa" 1am(da
1ymphocytosis
C$1#:" C$1;:" H1A$,:" Tdt:
B cell A11 Hematogones
$iagnosis4 Tdt : Bcell A11
Prognostication4 Cytogenetic studies
Chemotherapy4 MCP @D1 protocol
-0aluation o' Treatment4 $ay 1@ BM
Morphology
M,$ 1ite (y 'low
$ay 1@ post induction
M,$ lite

Hypothesis4 $ay 1@ post induction" no

hematogones in BM

C$1;" H1A$," C$=D" syto 1=

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FCS Filename %ate S o' -0ents Q o' %ated
Cells
Q o' All
Cells
1"=a"1##)M,$
TBB-+'cs
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1"=a"1##)M,$
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%ate? 1!1#! 11+<@ <+1@
1 in 100 tumor cell dilution MRD Tube
MRD = $13!
Kuality control

7nstrument 6uality control

-5periment setup includes4
PMT Goltage setup
Compensation setup and
Titration o' Anti(odies

,eagent3Anti(ody 6uality control

Process 6uality control

Tandem dyes

Trained sta''" CM-s

For ,e'erring 9ncologist3Pathologist
7ndications o' FCM" short cuts
Transportation30ia(ility o' sample
Multicolor analysis and reading the report
KC Final comprehensi0e report

Morphology

Flow cytometry 3 7HC

Cytogenetics

Molecular $iagnostics