flowcytometry principles
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Flow cytometry: Principles and ApplicationsCME in Hematology 2014
Pune
Sumeet u!ral" M#
Pro$essor"
#epartment o$ Pat%ology"
&MH" Mum'ai
s(gu!ral)outloo*+com
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#iagnosis o$ leu*emia , lymp%oma
FCM: principles and applicationsFCM: -ssues and trou'les%oots
My talk
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#iagnosis o$ leu*emia , lymp%oma
FCM: principles and applicationsFCM: -ssues and trou'les%oots
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#iagnosis o$ leu*emia , lymp%oma
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&umor cells may 'e mature loo*ing
or o$ 'lastic morp%ology++
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Some 'lasts are classical.+
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li*e.
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/t%ers are semi classical.+
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MP/
SE
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And ot%ers could 'e complicated++
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eu*emia lymp%oma diagnosis
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Morp%ology
eu*emia lymp%oma diagnosis
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Morp%ology
Ancillary tec%niues
eu*emia lymp%oma diagnosis
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Morp%ology
Ancillary tec%niues
- Immunophenotyping (IHC and FCI)
eu*emia lymp%oma diagnosis
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BCL2
Neutrophils
CD13+, CD16+
-mmunop%enotyping
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Morp%ology
Ancillary tec%niues
- Immunophenotyping- Cytogenetics
eu*emia lymp%oma diagnosis
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t(9;22)
Interphase FISH: BCR/ABL fusion
Cytogenetics
PA Amare
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Morp%ology
Ancillary tec%niues
3 Immunophenotyping3 Cytogenetics
3 Molecular diagnostics
eu*emia lymp%oma diagnosis
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Molecular #iagnostics
E# 5lac* pea*6: PM gene
7 FAM 5lue pea*6: F& 8 gene
FL!Pea" #: !$9 %p
Pea" 2: $#2 %p
&ifferen'e: ! %p
NPM169 bp
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What is the role of ancillary techniques??
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9ole o$ a Pat%ologist
#iagnostic la'el
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#iagnostic la'el
Prognostic mar*er
9ole o$ a Pat%ologist
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#iagnostic la'el
Prognostic mar*er
Predictie mar*ers
9ole o$ a Pat%ologist
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#iagnostic la'el
Prognostic mar*er
Predictie mar*er Minimal residual disease treatment
e$$ectieness6
9ole o$ a Pat%ologist
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&%us all t%ese ancillary tec%niues %elp in.
#iagnosis
Prognosis , 9is* strati$ication
Prediction &reatment e$$ectieness M9# detection6
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Most important.
Morp%ology ; -mmunop%enotyping
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#iagnosis o$ leu*emia , lymp%oma
Flow cytometry principles and applications-ssues and trou'les%oots
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-P& %elps in su'typing t%e lineage
o$ t%e tumors.
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What is Immunophenotyping?
Uses antibodies to identiy, lo!ate, and stainspe!ii! protein "ole!ules in tissue or in luids#
$ea!tion %isuali&ed by a "arker 'luores!entdye, en&y"e, !olloidal (old et!)
Dia(nosis, sub*typin(, pro(nosti!atin( and as apredi!tin( "arker o therapeuti! response#
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Met%ods $or -P&.
-mmuno%istoc%emistry
Flow cytometry
-mmuno$luorescence
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Immunohistochemistry:
Histopat%ology"
para$$in em'edded 'iopsy
Flow cytometry:
Perip%eral 'lood"
'one marrow aspirate"
'ody $luids
ymp% node 'iopsy and
aspirate
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FCM $luids
multicolor immunop%enotyping
I!C by "outine Microscopy
'iopsy
arc%itecturecytology
mostly single color
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1+ -HC
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-HC
Popular in solid tissues
Su'type tumors
Arc%itecture ; cytology
5ut is mostly single color
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o"e "arkers best on -C like epithelial "arkers, $C. panels
/thers on 0CM like 0MC, -CL "arkers
C./4$5.N
CD3
CD2
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C3*it and imatini' in -S&
C3er'52 and Herceptin
Predict
i:emar*e
rs
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-HC is a must in lymp%oma diagnosis
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E get cured" rest =0> not.
?aste 'as*et
Can we di$$erentiate good ones $rom 'ad ones
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C#20 and 9itu
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Hans Algorit%m 3 #5C
5C7
C#10Mum1
;
3
3
C5
C5 onC5
on C5
;
;
3
CD 2 CD 1CD 3
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b!l6
Mib1+ 789
bcl2
Mu"1
Dia(nosis
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A*anta+es of IHC , ar!hite!tural relationships andability to dete!t s!anty tu"or !ells, as in -L or .C$BCL#
o"e antibodies "ay be better e%aluated in para:ntissue 'e(, C-'.in, CD1;, and the presen!e o B!l*2,B!l*6, !y!lin D1, 5L*1, and !ytoplas"i! kappa andla"bda)#
Like
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2+ FCM
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$low ; cyto ; metry
1@=8 3 &%e $irst impedance3'ased $low cytometry
deice" using t%e coulter principle ?allace A
Coulter6+
1@7 3 &%e $irst $luorescence3'ased$low cytometry
deice -CP 116 'y ?ol$gang B%de" niersity o$
Munster
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Measure"ent o !ellular properties as the !ells 'or nu!lei,"i!roor(anis"s, !hro"oso"es, and lateA beads) "o%e in
a luid strea",past a stationary set of detectors (thousand eventsper second)
t analyses
* physi!al, as
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F.uores'ent -es ma- %in or inter!alate
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Components o$ a Flow Cytometer
0luidi!s a lo< !ell
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Components o$ a Flow Cytometer
0luidi!s a lo< !ell
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Components o$ a Flow Cytometer
0luidi!s a lo< !ell
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Components o$ a Flow Cytometer
0luidi!s a lo< !ell
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Components o$ a Flow Cytometer
0luidi!s a lo< !ell
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Mae!ker, Nature $e%ie
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Mae!ker, Nature $e%ie
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Mae!ker, Nature $e%ie
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Mae!ker, Nature $e%ie
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1+ P%ysical propertiesScatter pattern
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0or
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L54$ a"ple in a hydrodyna"i!ally o!used strea" Dete!tors
5"plii!ation and
!o"puter
Laser
Scatter only
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2+ C%emical propertiesFC-
Scatter plus FC-
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?M.
?M.
?M.
L54$ a"ple in a hydrodyna"i!ally o!used strea" Dete!tors
5"plii!ation and
!o"puter
Laser
Scatter plus FC-
?M.
?M.
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?M.
?M.
?M.
L54$ a"ple in a hydrodyna"i!ally o!used strea" Dete!tors
5"plii!ation and
!o"puter
Laser
?M.
?M.
?M.
Data
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#ata s%own eit%er as a3 single parameter %istograms" or
3 two parameter correlated plots
Data "ay be sho
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&1A p.oi-
Count
2;6
38=2;6128
128
38=
FF1 F2M
phase
Single
paramete
r4Histo
gram6
"dy
#0Ac
ontent"line
arscal
e
scale
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&wop
arame
ter4dotplo
t6"no
dye"lin
earsc
scale
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&wopar
amete
r4dot
plot6"dye"l
inear:
ersus
logsca
cale
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Multip
leparamete
r4dot
plots6"
multip
ledyes"FC
-"linea
r,logs
cal
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&ypes o$ FCM
*in(le Laser or Multiple Lasers'1 laser three !olor, = lasers 18 luores!en!e dete!tors)
*orter 'so as to puriy populations o interest )
*Laser s!annin( !yto"eters
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Adantages o$ a FCM
tudy o !ells, !hro"oso"es and parti!les 'analysis,!ountin( and sortin()
.housand o parti!les per se!ond
Multipara"etri! analysis at a sin(le !ell le%el
?attern studies
ortin(
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$esear!h 5ppli!ations
Auto$luorescent Proteins
Antigen or igand #ensity
Apoptosis
EnDyme actiity
#A" 9A content and c%anges in t%e cell cycle
Mem'rane Potential
Cyto*ine receptors and its synt%esis #rug upta*e and e$$lu1 ,NDUL5$ 'BM)#3
CD1> * ?4
++C*
-ei(ht
16
11
12
13
1=
2;6
;12
68
12=
C#1@ ating"
C" post treatment
C#1@ ating"
new case o$ C
. !ell ly"pho!ytosis
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. !ell ly"pho!ytosis
6*;9 o ly"phoid !ells in peripheral blood are .*!ells
0C %s C CD=; Fatin( C atin+
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0C %s C CD=; Fatin( C atin+
Cells o interest "ay be s!anty
CD * 233>1 ,NDUL5$ 'BM)#3
ht
12=
FSC vs SSC
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0+C * -ei(ht
+
+
C
*
-
ei(
2;6 ;12 68 12
2;6
;12
68
CD * 233>1 ,NDUL5$ 'BM)#3
CD1> * ?4
++C
*-ei(ht
16
11
12
13
1=
2;6
;12
68
12=CD * 233>1 ,NDUL5$ 'BM)#3
CD3 * ?erC?
++C
*-ei(ht
1
11
12
13
1=
2;6
;12
68
12= tudy B*!ellstudy .*!ells
. and B !ells
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?%en to call it positieG
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?%at is positieG
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?%at is positieG
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Canwed
i$$erentiate
t%esed
ots.++
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gamma delta & cells
alp%a 'eta & cells
and monocytes
53cells
/t%er cells
Fluoroc%romes
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&-e 34'itation 3mission 5o.e'u.ar 6ei+ht0.C =88 n" ;2 n" 38> Da
?4 =88 n" ;8 n" 2= Da
4CD =88 n" 613 n" 2; Da
?C =88 n" 668 n" 1; Da
?erC? =88 n" 688 n" 3; Da
5?C 613 n" 66; n" 1; Da
4a!h antibody is ta((ed
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FCM 3 ApplicationsFCM 3 Applications
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1+ Multicolor -mmunop%enotyping1+ Multicolor -mmunop%enotyping
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?%at is Multicolor -P& G 8 or more colors
Single laser: 834 colors
&wo lasers: 7 colors
&%ree lasers: colors plus
More lasers - more colors. more anti/odies. more
fluorochromes. tandem dyes. /etter data. moreinformation. fle0i/ility. ho,e1er. more issues
Single color
means one anti'ody
&wo color means two
anti'odies
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means one anti'ody anti'odies
4 color wit% C#4= gatingC' as a trac2ing mar2er
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C%&'
Myeloid tube . !ell tube B !ell tube MD tube /ther tube
C%&' C%&' C%&'C%&'
Fourcolor
s
-mmunop%enotyping %elps in detecting:-mmunop%enotyping %elps in detecting:
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p yp g p g
-mmunop%enotyping %elps in detecting:-mmunop%enotyping %elps in detecting:
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p yp g p g
33 presence or a'sence o$ an antigenpresence or a'sence o$ an antigen
3 intensity o$ e
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B*!ell !lonality
Clonality o$ 5 cells
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Polyclonal5 cells
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Monoclonal5 cells
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& cell normal and a'normal
3ormal 4 cell su/sets0> o$ lymp%oid cells in perip%eral 'lood are & cells
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0> o$ lymp%oid cells in perip%eral 'lood are & cells
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Anti'odies used $or eu*,ymdiagnosis
3 Acute leu*emia3 ymp%oma,CP#s
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9eagents reuired in acute leu*emia
&3cell tu'e I C#4=" C#2" C#8" C#4" C#="C#" C#
53cell tu'e I C#4=" C#1@" C#20" C#10"
Myeloid cells I C#4=" C#18" C#88" C#11Myeloid tu'e I C#4=" C#14" C#17" C#74
Cytopl+ tu'e I C#4=" &dt" cC#8" cC#@a" cC#22" AntiMP/
/t%ers I C#4=" C#84" HA#9 and so on
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9eagents reuired in lymp%omas
Screening tu'e 3
53cell tu'e I C#4=" C#1@" C#20" C#10" *appa" lam'da
C tu'e I C#4=" C#1@" C#20" C#=" C#28&3cell tu'e I C#4=" C#2" C#8" C#4" C#="C#" C#
HC tu'e: C#4=" C#1@" C#11c" C#2=" C#108" C#128
J cell tu'e: C#4=" C#8" C#17" C#=7" C#
PC tu'e: C#4=" C#8" C#18" cytoJ" cyto" C#=7" C#1@/t%ers.+
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-ndian uidelines5pproach to 5cute eu2emia and C!'s
-KPM" 200
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How to ma*e panelsG
3 literature 'ased"
3 training 'ased"
3 trial or %unc% 'ased
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Panels are decided 'ased on morp%ology and clinical indications
43 color FC-
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et us construct a 4 color A panel
Four color A panel
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C#8 3 F-&C
C#1@ 3 PE
C#18 I PerCPC#4= I &9
a !o!ktail o antibodies
o dierent linea(es##
Four color A panel
C#8 3 F-&C
C#4 3 PE
C# I PerCPC#4= I &9
a !o!ktail o antibodies
o si"ilar linea(e##
Four color panel
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C#8 3 F-&C
C#1@ 3 PE
C#18 I PerCPC#4= I &9
a !o!ktail o antibodies
o dierent linea(es##
Four color panel
C#8 3 F-&C
C#4 3 PE
C# I PerCPC#4= I &9
.*!ell tube
Four color panel 4,= tu'es
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Four color panel 3 4,= tu'es
C#8 3 F-&C
C#4 3 PE
C# I PerCPC#4= I &9
.*!ell tube
C#1@ 3 F-&C
C#203 PE
C#103 PerCPC#4= I &9
B*!ell tube
C#18 3 F-&C
C#88 3 PE
C#11 I PerCPC#4= I &9
Myeloid* tube
HA#9 3 F-&C
C#84 3 PE
C# I PerCPC#4= I &9
Mis!ellaneous
u%e
1o7
8 Bri..iant 8io.et
$2#
FIC P3 PerCP,C-7 P3,C- APC APC,H
# Color $cute %eu&emia Panel
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1 CD=;
;6
-3
1 tests
5ouse I+#
CD123
;62;1
>0;
; tests
5ouse I+#
CD38
3=>2
-B
; tests
5ouse I+#
CD;8
M1=3
5CD;8
1 tests
5ouse I+2aBe'"man Cou.ter
CD3=
3=23
8F12
1 tests
5ouse I+#
CD1
3=1>2
-1a
1 tests
5ouse I+#
CD1>
;;83;
G2;C1
1 tests
5ouse I+#
CD2
6=13>6
L2
1 tests
5ouse I+#
2 CD=; CD11
;62=3=
B;#B8
1 tests5ouse I+#
CD16
3=;23
Leu*11a
1 tests5ouse I+#
CD11b
;;;388
C$0==
1 tests5ouse I+#
CD1;
;6828
->8
; tests5ouse I+5
CD13
338=2;
L138
1 tests5ouse I+#
CD3=
3===1
8F12
1 tests
5ouse I+#
-L5*D$
6=13>3
L2=3'F=6*6)
1 tests
3 CD=; CD36
;61;3;
CB38'NL*)
; tests
CD6=
;;;;2
1#1
1 tests
CD2
;;2=;
M$C /H*1=
1 tests5ouse I+#
CD=
3=16;=
3
; tests5ouse I+#
CD33
333>=6
?6#6
1 tests5ouse I+#
CD133
13*>*826
5C133
1 tests5ouse I+#
5i.ten-i %iote'h
CD1=
6=13>= I
;618
M/?>
1 tests5ouse I+#
= CD=; CD3
;62=26
UC-.1
1 tests5ouse I+#
CD1a
;;;86
-1=>
1 tests5ouse I+#
CD
3=;81
M*.1
; tests5ouse I+#
CD;6
;68=2
B1;>
; tests
5ouse I+#
CD;
3=8>
L1012
1 tests5ouse I+2a
CD=
;618=1
$?5*.=
; tests
5ouse I+#
CD8
6=1=
1
1 tests5ouse I+#
; CD=; !ytoCD3 5M?/ Cyto CD>a Cyto CD22
# Color $cute %eu&emia Panel
u%e
1o7
8 Bri..iant 8io.et
$2#
FIC P3 PerCP,C-7 P3,C- APC APC,H
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; CD=; !ytoCD3
;62=26
UC-.1
1 tests5ouse I+#
5M?/
3=;8
;B8
; tests5ouse I+#
Cyto CD>a
3=;>
-M=
; tests5ouse I+#
Cyto CD22
3=>33
*-CL*1
1 tests5ouse I+2%
6
. !ell
M$D
CD8
;6161
1
; tests
5ouse I+#
CytoCD3
;62=26
UC-.1
1 tests5ouse I+#
.dt CD= sura!eCD3 CD; CD
;616=
M*.1
; tests
5ouse I+#
CD=;
CD=;
>
MD
tube1
CD=; CD11
;62=3=
B;#B8
1 tests5ouse I+#
-L5*D$
3=363
F=6*6
1 tests5ouse I+2a
CD11b
;;;388
C$0==
1 tests5ouse I+#
CD=
3=16;=
3
; tests5ouse I+#
CD33
333>=6
?6#6
1 tests5ouse I+#
CD3=
3===1
8F12
1 tests5ouse I+#
CD1=
6=13>= I
;618
M/?>
1 tests5ouse I+#
MD
tube2
CD=; CD11
;62=3=
B;#B8
1 tests5ouse I+#
CD16
3=;23
Leu*11a
1 tests5ouse I+#
CD66b
;616;
F10;
; tests5ouse I+5
CD1;
;6828
->8
; tests5ouse I+5
CD13
338=2;
L138
1 tests5ouse I+#
CD11b
;;1>
C$0==
1 tests
5ouse I+#
CD6=
;611>
1#1
; tests
5ouse I+#
# Color $cute %eu&emia Panel
u%e 8 Bri..iant 8io.et FIC P3 PerCP,C-7 P3,C- APC APC,H
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1
5R&
tu%eBALL
CD=;
;6
-3
1 tests5ouse I+#
CD1>
;62==
-B1>
1 tests5ouse I+#
CD2
;;;>18
G=*11
1 tests5ouse I+2%
CD2
;;2=;
M$C /H*1=
1 tests5ouse I+#
CD3=
3=23
8F12
1 tests5ouse I+#
CD1
3=1>2
-1a
1 tests5ouse I+#
CD133
13*>*826
5C133
1 tests5ouse I+#
5i.ten-i %iote'h
CD2
6=13>6
L2
1 tests5ouse I+#
1o7 $2#
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Compre%ensie panel o$ anti'odiesused $or , diagnosis
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Case 1
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= year old 'oy wit% $eer I 1 mont%
Classi!al 3*= !olor
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%iagnosis
MP/ negatie SE negatie
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%iagnosis
Blasts only
ice
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(mmunophenotping
0C %s C
8colo
r-P&
ICom
monp
ractice
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Guess
C#10;" C#1@;" HA#9;
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0L,
DLBCLB !ell 5LL,
BurkittJs Ly"pho"a
C#10;" C#1@;" HA#9;
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0L,
DLBCL
-e"ato(ones
'.dt+I*)
B !ell 5LL,
BurkittJs Ly"pho"a
Thus morpholo%y adeuate panels !ith patterns
analysis cyto%enetics and clinical history
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Case 2
$& year old girl. presented ,ith fe1er for & ,ee2s%
!eripheral /lood smear re1eals high counts ,ith
/last li2e cells
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Acute leu*emia
cytochemical M!" negati1e
What ne0t?
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FCI done
6 cell tu/e 7 3egati1e
Myeloid tu/e 7 3egati1e
5ll 4 cell mar2ers including C'* 7 3egati1e
6lasts e0pressed only C'8
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&3cell mar*ers- C'*
- C'8
- C'&- C'
- C'C'9
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?%at do you ne
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'o cytoplasmic mar2ers
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#iagnosis&3cell A
3ote: 6lasts may /e surface C'* negati1e
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&issue $low
4= year old male"
l $ i l i %
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le$t cerical since 1 mont%
ymp% node FAC done
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4= year old male" 3 $nac
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-ssue
so$8
color-P
&
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ubtypin( o "all B !ell ly"pho"as
Nodular pattern
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Common 5 cell Hs e
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ymp%oma #iagnosis
;old standard is /iopsy plus IHC
FCM and lymp%oma diagnosis
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5est $or CP#s
Mostly 5 cell type and panels are well de$ined
Also used $or lymp% node 'iopsy" aspirate"
$luids and ot%er tissues
& cell lymp%omas are rareeed ela'orate panel
Altered e
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Altered e
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ymp%oma Screening &u'e
- $ anti/odies in one tu/e
- Mutually e0clusi1e 5/s
ymp%oma screening tu'e
1= color panel
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p
C#4= Ale
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Plasma cell eoplasms
color Myeloma panel
ating strategies
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ating strategies
First tu'e $or Myeloma" &MH
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C#8 F-&C
C#1@ 4=0
C#20 APCH
C#2 PE
C#2APC C#4= =00
C#11PEcy
C#=7 PerCP cy=+=
P Su'ramanian
Second myeloma tu'e" &MH
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Cyto am'da F-&C
Cyto Jappa PE
C#4= APC H
C#8 PerCP3Cy=+=
C#1@ PE3Cy
C#=7 APC
5lan* 5lan*
P Su'ramanian
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Case o$ plasma cell dyscrasia
eig%t color6
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