Flow cytometry: Principles and Applications

CME in Hematology 2014

Pune
Sumeet u!ral" M#
Pro$essor"
#epartment o$ Pat%ology"
&MH" Mum'ai
s(gu!ral)outloo*+com
#iagnosis o$ leu*emia , lymp%oma
FCM: principles and applications
FCM: -ssues and trou'les%oots
My talk
#iagnosis o$ leu*emia , lymp%oma
FCM: principles and applications
FCM: -ssues and trou'les%oots
#iagnosis o$ leu*emia , lymp%oma
&umor cells may 'e mature loo*ing
or o$ 'lastic morp%ology++
Some 'lasts are classical.+
li*e.
/t%ers are semi classical.+
MP/
0SE
And ot%ers could 'e complicated++
1eu*emia lymp%oma diagnosis

Morp%ology
1eu*emia lymp%oma diagnosis

Morp%ology

Ancillary tec%ni2ues
1eu*emia lymp%oma diagnosis

Morp%ology

Ancillary tec%ni2ues
- Immunophenotyping (IHC and FCI)
1eu*emia lymp%oma diagnosis
BCL2
Neutrophils
CD13+, CD16+
-mmunop%enotyping

Morp%ology

Ancillary tec%ni2ues
- Immunophenotyping
- Cytogenetics
1eu*emia lymp%oma diagnosis
t(9;22)
Interphase FISH: BCR/ABL fusion
Cytogenetics
PA Amare

Morp%ology

Ancillary tec%ni2ues
3 Immunophenotyping
3
Cytogenetics
3
Molecular diagnostics
1eu*emia lymp%oma diagnosis
Molecular #iagnostics
0E# 45lac* pea*6: 0PM gene
7 FAM 45lue pea*6: F1& 8 gene
FL!
Pea" #: !$9 %p
Pea" 2: $#2 %p
&ifferen'e: (! %p
NPM
169 bp
What is the role of ancillary techniques??
9ole o$ a Pat%ologist

#iagnostic la'el

#iagnostic la'el

Prognostic mar*er
9ole o$ a Pat%ologist

#iagnostic la'el

Prognostic mar*er

Predicti:e mar*ers
9ole o$ a Pat%ologist

#iagnostic la'el

Prognostic mar*er

Predicti:e mar*er

Minimal residual disease 4treatment

e$$ecti:eness6
9ole o$ a Pat%ologist
&%us all t%ese ancillary tec%ni2ues %elp in.

#iagnosis

Prognosis , 9is* strati$ication

Prediction

&reatment e$$ecti:eness 4M9# detection6

Most important.
Morp%ology ; -mmunop%enotyping
#iagnosis o$ leu*emia , lymp%oma
Flow cytometry principles and applications
-ssues and trou'les%oots
-P& %elps in su'typing t%e lineage
o$ t%e tumors.
What is Immunophenotyping?

Uses antibodies to identiy, lo!ate, and stain

spe!ii! protein "ole!ules in tissue or in luids#

$ea!tion %isuali&ed by a "arker 'luores!ent

dye, en&y"e, !olloidal (old et!)

Dia(nosis, sub*typin(, pro(nosti!atin( and as a

predi!tin( "arker o therapeuti! response#
Met%ods $or -P&.
-mmuno%istoc%emistry
Flow cytometry
-mmuno$luorescence
Immunohistochemistry:
Histopat%ology"
para$$in em'edded 'iopsy
Flow cytometry:
Perip%eral 'lood"
'one marrow aspirate"
'ody $luids
1ymp% node 'iopsy and
aspirate
FCM $luids
multicolor immunop%enotyping
I!C by "outine Microscopy
'iopsy
arc%itecture
cytology
mostly single color
1+ -HC
-HC

Popular in solid tissues

Su'type tumors

Arc%itecture ; cytology

5ut is mostly single color

+o"e "arkers best on ,-C like epithelial "arkers, $C. panels
/thers on 0CM like 0MC1, -CL "arkers
C2./34$5.,N
CD3
CD26
C3*it and imatini' in -S&
C3er'52 and Herceptin
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-HC is a must in lymp%oma diagnosis
E<ample 3 #15C1

Commonest type o$ lymp%oma

=0> get cured" rest =0> not.

?aste 'as*et

Can we di$$erentiate good ones $rom 'ad ones

C#20 and 9itu<ima'
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Hans Algorit%m 3 #15C1
5C17
C#10
Mum1
;
3
3
C5
C5
0onC5
0on C5
;
;
3
CD 26 CD 16
b!l6
Mib1+ 7869
bcl2
CD 3
Mu"1
Dia(nosis
A)*anta+es of IHC , ar!hite!tural relationships and
ability to dete!t s!anty tu"or !ells, as in -L or .C$BCL#
+o"e antibodies "ay be better e%aluated in para:n
tissue 'e(, C-'.in&#, CD1;, and the presen!e o B!l*2,
B!l*6, !y!lin D1, 5L3*1, and !ytoplas"i! kappa and
la"bda)#
Like<ise, so"e "arkers <ork better on 0C, 'CD13,
CD1=, CD1>, CD33, et!)# $are "arkers or B?DC5 et!#
0CM is i"portant or luids@@@@##
2+ FCM
$low ; cyto ; metry

1@=8 3 &%e $irst impedance3'ased $low cytometry

de:ice" using t%e coulter principle 4?allace A
Coulter6+

1@7A 3 &%e $irst $luorescence3'ased $low cytometry

de:ice 4-CP 116 'y ?ol$gang B%de" Cni:ersity o$
Munster
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 events
per second)
,t analyses
* physi!al, as <ell as
* !he"i!al properties 'i""unoluores!en!e)
Buantitati%e sin(le !ell analysis
?rin!iple
F.uores'ent )-es ma- %in) or inter!alate <ith dierent
!ellular !o"ponents su!h as DN5 or $N5#
A))itiona..-/ anti%o)ies 'on0u+ate) to f.uores'ent
)-es 'an %in) spe!ii! proteins on !ell "e"branes or
inside !ells#
Co""only used dyes in!lude ?,, ?4, 0,.C, althou(h
"any other dyes are a%ailable# .ande" dyes <ith
internal luores!en!e resonan!e ener(y transer !an
!reate e%en lon(er <a%elen(ths and "ore !olors#
Components o$ a Flow Cytometer
0luidi!sC a lo< !ell <ith sheath luid 'hydrodyna"i! o!ussin()
Components o$ a Flow Cytometer
0luidi!sC a lo< !ell <ith sheath luid 'hydrodyna"i! o!ussin()

/pti!sC L5+4$+, sin(le <a%elen(th, !oherent li(ht 'ho<e%er
in!oherent li(ht is o rando"phase %aryin( <ith ti"e and
position)
Components o$ a Flow Cytometer
0luidi!sC a lo< !ell <ith sheath luid 'hydrodyna"i! o!ussin()

/pti!sC L5+4$+, sin(le <a%elen(th, !oherent li(ht 'ho<e%er
in!oherent li(ht is o rando"phase %aryin( <ith ti"e and
position)
a dete!tor and 5nalo(ue*to*Di(ital Con%ersion '5DC) syste" *
<hi!h (enerates 0+C and ++C as <ell as luores!en!e si(nals
ro" li(ht into ele!tri!al si(nals that !an be pro!essed by a
!o"puter
Components o$ a Flow Cytometer
0luidi!sC a lo< !ell <ith sheath luid 'hydrodyna"i!
o!ussin()

/pti!sC L5+4$+, sin(le <a%elen(th, !oherent li(ht 'ho<e%er
in!oherent li(ht is o rando"phase %aryin( <ith ti"e and
position)
a dete!tor and 5nalo(ue*to*Di(ital Con%ersion '5DC) syste"
* <hi!h (enerates 0+C and ++C as <ell as luores!en!e
si(nals ro" li(ht into ele!tri!al si(nals that !an be pro!essed
by a !o"puter

an a"plii!ation syste" E linear or lo(arith"i!
Components o$ a Flow Cytometer
0luidi!sC a lo< !ell <ith sheath luid 'hydrodyna"i! o!ussin()

/pti!sC L5+4$+, sin(le <a%elen(th, !oherent li(ht 'ho<e%er
in!oherent li(ht is o rando"phase %aryin( <ith ti"e and
position)
a dete!tor and 5nalo(ue*to*Di(ital Con%ersion '5DC) syste" *
<hi!h (enerates 0+C and ++C as <ell as luores!en!e
si(nals ro" li(ht into ele!tri!al si(nals that !an be pro!essed
by a !o"puter

an a"plii!ation syste" E linear or lo(arith"i!
a !o"puter or analysis o the si(nals
Mae!ker, Nature $e%ie<s, 2612
Mae!ker, Nature $e%ie<s, 2612
Mae!ker, Nature $e%ie<s, 2612
Mae!ker, Nature $e%ie<s, 2612
1+ P%ysical properties
Scatter pattern
0or<ard s!atter +ide s!atter
+i&e Franularity
L5+4$
+a"ple in a hydrodyna"i!ally o!used strea"
Dete!tors
5"plii!ation and
!o"puter
Laser
Scatter only
2+ C%emical properties
FC-
?M.
?M.
?M.
L5+4$
+a"ple in a hydrodyna"i!ally o!used strea"
Dete!tors
5"plii!ation and
!o"puter
Laser
Scatter plus FC-
?M.
?M.
?M.
L5+4$
+a"ple in a hydrodyna"i!ally o!used strea"
Dete!tors
5"plii!ation and
!o"puter
Laser
?M.
?M.
?M.
?M.
#ata s%own eit%er as a
3 single parameter %istograms" or
3 two parameter correlated plots
Data "ay be sho<n as

Linear s!ale .he s!ale on <hi!h the output is dire!tly

proportional to the input#

Lo(arith"i! s!ale .he s!ale on <hi!h the %alues

in!rease lo(arith"i!ally#
Data
&1A p.oi)-
Count
2;6
38=
2;6
128
128
38=
F
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+ phase
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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
Ad:antages 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(
$esear!h 5ppli!ations
Auto$luorescent Proteins
Antigen or 1igand #ensity
Apoptosis
EnDyme acti:ity
#0A" 90A content and c%anges in t%e cell cycle
Mem'rane Potential
Cyto*ine receptors and itEs synt%esis
#rug upta*e and e$$lu<
P%agocytosis
Fia'ility
C%anges in -ntracellular pH
C%anges in -ntracellular calcium
C%anges in -ntracellular glutat%ione
C%anges in /<idati:e 5urst
#rug disco:ery and :accine de:elopment
1# Monitorin( 5,D+ patients
2# ,""unophenotypin(
3# Monitorin( M$D
=# CD3= !ounts
;# $eti!ulo!yte Counts
6# ?N-
1# DN5 analysis o +*phase ra!tion
8# ?latelet !ounts
Dia(nosti! 5ppli!ations
Start a clinical cytometry facility
#o we really need oneG CentraliDed la's
Hospital , institute 'ased or a stand alone la'
9eagents,maintenance e<pensi:e
Sta*e %olders:
Management support
Cytometrist!athologistScientist
"ncologistHematologist support
#endor support
Clinical cytometry
95Cs
?5Cs
Platelets
/t%ers
Cytoplasmic,nuclear c%aracters
1ysis o$ red cells
?5Cs
Ac2uire ?5Cs wit%out any anti'odies
FCM 3 5ased on scatter pattern

1ymp%ocytes

Monocytes

0eutrop%ils
Scatter pattern, FSC vs SSC
Forward
Side
Cells o$ interest
Special gating procedures can %elp
separate tumor cells $rom normal
lymp%ocytes
C#4= gating $or 'lasts
C#1@ gating $or 5 cell lymp%omas
Cells o$ interest and di$$erent types o$ ating
$% FSC and SSC
&% C'() and SSC
*% C'$+ and SSC
(% C'* and SSC
)% others
0ormal Perip%eral 'lood
Forward :s side scatter
0ormal 5one marrow
Forward :s side scatter
FCC3A
FSC3H
SSC3H
FSC3A
FSC3A
SSC3A
SSC3A
Gating
Scatter pattern, FSC vs SSC
Forward
Side
Gating
Scatter pattern, FSC vs SSC
Forward
Side
Gating
Scatter pattern, FSC vs SSC
Forward
Side
What is an abnormal pattern??
What is an abnormal in this case?
Where do the blasts of acute leukemia
appear in scatter pattern plot??
Forward
Side
Where do the blasts of acute leukemia go??
Forward
Side
Lots of blasts??
Scant blasts??
Where do the tumor cells of !air cell leukemia go?
Where do the hair cell leukemia go in flow plots?
Forward
Side
Ho, to separate cells of interest%%
Special gating procedures can %elp
separate tumor cells $rom normal
lymp%ocytes
1ysis o$ red cells
5dd 5ntibodies
e#(#, CD=;
C# 4= gating $or 'lasts
Similarly C'$+ gating for HC- tumor cells
Special gating 'ased on C#4=++
?5Cs
0eutrop%ils
1ymp%ocytes
Monocytes
Eosinop%ils
5asop%ils
Fery $ew stem cells 4more in 5M6
Nor"al BM E CD=; (atin(
C#4= ating in 5M
+o "any !lusters
C#4= ating
C#4= ating
C#4= ating
C#4= ating
C#4= ating
C#4= ating
"

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C#4= ating
CD * 233>1 ,NDUL35$ 'BM)#663
CD1> * ?4
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2
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6
2;6
;12
168
162=
C#1@ ating"
C11" post treatment
C#1@ ating"
new case o$ C11
. !ell ly"pho!ytosis
66*1;9 o ly"phoid !ells in peripheral blood are .*!ells
0+C %s ++C CD=; Fatin( C&#9 2atin+
Cells o interest "ay be s!anty
CD * 233>1 ,NDUL35$ 'BM)#663
0+C * -ei(ht
+
+
C

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6
2;6
;12
168
162=
CD * 233>1 ,NDUL35$ 'BM)#663
CD1> * ?4
+
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16
2
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3
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168
162=
CD * 233>1 ,NDUL35$ 'BM)#663
CD3 * ?erC?
+
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16
2
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3
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162=
FSC vs SSC
+tudy B*!ells
+tudy .*!ells
. and B !ells
?%en to call it positi:eG
?%at is positi:eG
?%at is positi:eG
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gamma delta & cells
gamma delta & cells
alp%a 'eta & cells
and monocytes
alp%a 'eta & cells
and monocytes
53cells
53cells
/t%er cells
/t%er cells
Fluoroc%romes
&-e 34'itation 3mission 5o.e'u.ar 6ei+ht
0,.C =88 n" ;26 n" 38> Da
?4 =88 n" ;18 n" 2=6 666 Da
4CD =88 n" 613 n" 2;6 666 Da
?C =88 n" 668 n" 16; 666 Da
?erC? =88 n" 688 n" 3; 666 Da
5?C 613 n" 66; n" 16; 666 Da
4a!h antibody is ta((ed <ith a dierent luoro!hro"e
.ande" dyes
FCM 3 Applications
FCM 3 Applications
1+ Multicolor -mmunop%enotyping
1+ Multicolor -mmunop%enotyping
?%at is Multicolor -P& G 8 or more colors
Single laser: 834 colors
&wo lasers: 7 colors
&%ree lasers: A colors plus
More lasers - more colors. more anti/odies. more
fluorochromes. tandem dyes. /etter data. more
information. fle0i/ility. ho,e1er. more issues
Single color
means one anti'ody
&wo color means two
anti'odies
4 color wit% C#4= gating
C'() as a trac2ing mar2er
C%&'
Myeloid tube . !ell tube B !ell tube
MD+ tube /ther tube
C%&' C%&' C%&' C%&'
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-mmunop%enotyping %elps in detecting:
-mmunop%enotyping %elps in detecting:
-mmunop%enotyping %elps in detecting:
-mmunop%enotyping %elps in detecting:
3
3
presence or a'sence o$ an antigen
presence or a'sence o$ an antigen
3 intensity o$ e<pression
3 intensity o$ e<pression
3 presence o$ 'lasts
3 presence o$ 'lasts
3 clonality: 1C9" F3'eta repertoire
3 clonality: 1C9" F3'eta repertoire
3
scanty cells
scanty cells
3
maturation patterns
maturation patterns
CD3 antibody by FCM detects the fully assembled TCR-CD3 complex
!hich is present on the surface of T cells only"
#n contrast the CD3 #$C stain usually detects only the epsilon
component of CD3 therefore cannot distin%uish bet!een T & '( cells"
B*!ell !lonality
Clonality o$ 5 cells
Polyclonal 5 cells
Monoclonal 5 cells
& cell normal and a'normal
3ormal 4 cell su/sets
H0> o$ lymp%oid cells in perip%eral 'lood are & cells
Anti'odies used $or 1eu*,1ym
diagnosis
3 Acute leu*emia
3 1ymp%oma,C1P#s
9eagents re2uired in acute leu*emia
&3cell tu'e I C#4=" C#2" C#8" C#4" C#="C#H" C#A
53cell tu'e I C#4=" C#1@" C#20" C#10"
Myeloid cells I C#4=" C#18" C#88" C#11H
Myeloid tu'e I C#4=" C#14" C#17" C#74
Cytopl+ tu'e I C#4=" &dt" cC#8" cC#H@a" cC#22" AntiMP/
/t%ers I C#4=" C#84" H1A#9 and so on
9eagents re2uired in lymp%omas
Screening tu'e 3
53cell tu'e I C#4=" C#1@" C#20" C#10" *appa" lam'da
C11 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#H" C#A
HC1 tu'e: C#4=" C#1@" C#11c" C#2=" C#108" C#128
0J cell tu'e: C#4=" C#8" C#17" C#=7" C#A
PC tu'e: C#4=" C#8A" C#18A" cytoJ" cyto1" C#=7" C#1@
/t%ers.+
-ndian uidelines
5pproach to 5cute -eu2emia and C-!'s
-KPM" 200A
How to ma*e panelsG
3 literature 'ased"
3 training 'ased"
3 trial or %unc% 'ased

Panels are decided 'ased on morp%ology and clinical indications

43A color FC-

1et us construct a 4 color A1 panel
C#8 3 F-&C
C#1@ 3 PE
C#18 I PerCP
C#4= I &9
a !o!ktail o antibodies
o dierent linea(es##
Four color A1 panel
C#8 3 F-&C
C#4 3 PE
C#A I PerCP
C#4= I &9
a !o!ktail o antibodies
o si"ilar linea(e##
C#8 3 F-&C
C#1@ 3 PE
C#18 I PerCP
C#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#A I PerCP
C#4= I &9
.*!ell tube
Four color panel 3 4,= tu'es
C#8 3 F-&C
C#4 3 PE
C#A I PerCP
C#4= I &9
.*!ell tube
C#1@ 3 F-&C
C#203 PE
C#103 PerCP
C#4= I &9
B*!ell tube
C#18 3 F-&C
C#88 3 PE
C#11H I PerCP
C#4= I &9
Myeloid* tube
H1A#9 3 F-&C
C#84 3 PE
C#H I PerCP
C#4= I &9
Mis!ellaneous
u%e
1o7
89:: Bri..iant 8io.et
$2#
FIC P3 PerCP,C-979 P3,C-; APC APC,H;
1 CD=;
;66111
-,36
166 tests
5ouse I+2#
CD123
;62;11
>0;
;6 tests
5ouse I+2#
CD38
3=6>21
-B1
;6 tests
5ouse I+2#
CD;8
,M1=36
5,CD;8
166 tests
5ouse I+22a
Be'"man Cou.ter
CD3=
3=1263
8F12
166 tests
5ouse I+2#
CD16
3=16>2
-,16a
166 tests
5ouse I+2#
CD1>
;;183;
+G2;C1
166 tests
5ouse I+2#
CD26
6=13>6
L21
166 tests
5ouse I+2#
2 CD=; CD111
;62=3=
2B;#B8
166 tests
5ouse I+2#
CD16
3=1;23
Leu*11a
166 tests
5ouse I+2#
CD11b
;;;388
,C$0==
166 tests
5ouse I+2#
CD1;
;66828
-,>8
;6 tests
5ouse I+5
CD13
338=2;
L138
166 tests
5ouse I+2#
CD3=
3=6==1
8F12
166 tests
5ouse I+2#
-L5*D$
6=13>3
L2=3'F=6*6)
166 tests
3 CD=; CD36
;61;3;
CB38'NL*61)
;6 tests
CD6=
;;;;21
16#1
166 tests
CD266
;;2=1;
M$C /H*16=
166 tests
5ouse I+2#
CD=
3=16;=
+33
;6 tests
5ouse I+2#
CD33
333>=6
?61#6
166 tests
5ouse I+2#
CD133
136*6>6*826
5C133
166 tests
5ouse I+2#
5i.ten-i %iote'h
CD1=
6=13>= I
;66186
M/?>
166 tests
5ouse I+2#
= CD=; CD3
;62=26
UC-.1
166 tests
5ouse I+2#
CD1a
;;;866
-,1=>
166 tests
5ouse I+2#
CD1
3=6;81
M*.161
;6 tests
5ouse I+2#
CD;6
;668=2
B1;>
;6 tests
5ouse I+2#
CD;
3=81>6
L11012
166 tests
5ouse I+22a
CD=
;618=1
$?5*.=
;66 tests
5ouse I+2#
CD8
6=1=66
+31
166 tests
5ouse I+2#
# Color $cute %eu&emia Panel
; CD=; !ytoCD3
;62=26
UC-.1
166 tests
5ouse I+2#
5M?/
3=6;86
;B8
;6 tests
5ouse I+2#
Cyto CD1>a
3=6;1>
-M=1
;6 tests
5ouse I+2#
Cyto CD22
3=6>33
+*-CL*1
166 tests
5ouse I+22%
6
. !ell
M$D
CD8
;61611
+31
;6 tests
5ouse I+2#
CytoCD3
;62=26
UC-.1
166 tests
5ouse I+2#
.dt CD= sura!eCD3 CD; CD1
;6166=
M*.161
;6 tests
5ouse I+2#
CD=;
1 CD=;
>
MD+
tube1
CD=; CD111
;62=3=
2B;#B8
166 tests
5ouse I+2#
-L5*D$
3=1363
F=6*6
166 tests
5ouse I+22a
CD11b
;;;388
,C$0==
166 tests
5ouse I+2#
CD=
3=16;=
+33
;6 tests
5ouse I+2#
CD33
333>=6
?61#6
166 tests
5ouse I+2#
CD3=
3=6==1
8F12
166 tests
5ouse I+2#
CD1=
6=13>= I
;66186
M/?>
166 tests
5ouse I+2#
MD+
tube2
CD=; CD111
;62=3=
2B;#B8
166 tests
5ouse I+2#
CD16
3=1;23
Leu*11a
166 tests
5ouse I+2#
CD66b
;616;6
F160;
;6 tests
5ouse I+5
CD1;
;66828
-,>8
;6 tests
5ouse I+5
CD13
338=2;
L138
166 tests
5ouse I+2#
CD11b
;;661>
,C$0==
166 tests
5ouse I+2#
CD6=
;611>6
16#1
;6 tests
5ouse I+2#
# Color $cute %eu&emia Panel
u%e
1o7
89:: Bri..iant 8io.et
$2#
FIC P3 PerCP,C-979 P3,C-; APC APC,H;
1
5R&
tu%e
BALL
CD=;
;66111
-,36
166 tests
5ouse I+2#
CD1>
;62==6
-,B1>
166 tests
5ouse I+2#
CD12
;;;>18
G=*111
166 tests
5ouse I+22%
CD266
;;2=1;
M$C /H*16=
166 tests
5ouse I+2#
CD3=
3=1263
8F12
166 tests
5ouse I+2#
CD16
3=16>2
-,16a
166 tests
5ouse I+2#
CD133
136*6>6*826
5C133
166 tests
5ouse I+2#
5i.ten-i %iote'h
CD26
6=13>6
L21
166 tests
5ouse I+2#
# Color $cute %eu&emia Panel
u%e
1o7
89:: Bri..iant 8io.et
$2#
FIC P3 PerCP,C-979 P3,C-; APC APC,H;
Compre%ensi:e panel o$ anti'odies
used $or 1,1 diagnosis
Case 1
= year old 'oy wit% $e:er I 1 mont%
Classi!al 3*= !olor
%iagnosis
MP/ negati:e 0SE negati:e
%iagnosis
(mmunophenotping
0+C %s ++C
Blasts only
8

c
o
l
o
r

-
P
&

I

C
o
m
m
o
n

p
r
a
c
t
i
c
e
Guess
C#10;" C#1@;" H1A#9;
0L,
DLBCL
B !ell 5LL,
BurkittJs Ly"pho"a
C#10;" C#1@;" H1A#9;
0L,
DLBCL
-e"ato(ones
'.dt+I*)
B !ell 5LL,
BurkittJs Ly"pho"a
Thus morpholo%y ade)uate panels !ith patterns
analysis cyto%enetics and clinical history
Case 2
$& year old girl. presented ,ith fe1er for & ,ee2s%
!eripheral /lood smear re1eals high counts ,ith
/last li2e cells
Acute leu*emia
cytochemical M!" negati1e
What ne0t?
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
&3cell mar*ers
- C'*
- C'8
- C'&
- C')
- C'(C'9
?%at do you ne<tG
'o cytoplasmic mar2ers
#iagnosis
&3cell A11
3ote: 6lasts may /e surface C'* negati1e
&issue $low
4= year old male"
le$t cer:ical 10 since 1 mont%
1ymp% node F0AC done
4= year old male" 10 3 $nac
-
s
s
u
e
s

o
$

8

c
o
l
o
r

-
P
&
+ubtypin( o +"all B !ell ly"pho"as
Nodular pattern
Common 5 cell 0H1s e<pressing C#= 4&3cell mar*er6
1+C11 I e<press C#28
2+ MC1 I e<press cyclin #1 4not 'y $low6
1ymp%oma #iagnosis
;old standard is /iopsy plus IHC
FCM and lymp%oma diagnosis

5est $or C1P#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 rare
0eed ela'orate panel

Altered e<pression o$ pan & cell mar*ers li*e C#2" C#8" C#= and
C#H 4also seen in -M" CMF6

Su'set restriction o$ C#4 or C#A 4also seen in -M" H-F" A-#6

-ncreased e<pression o$ mar*ers li*e C#2= wit%,wit%out C#4

A'errant e<pression o$ C#10" C#80" C#108" &dt" Al*1 etc+

A'errant e<pression o$ C#17" C#=7

F 'eta repertoire

H&1F31 serology" PC9 $or &C9 gene rearrangements

1ymp%oma Screening &u'e
- $) anti/odies in one tu/e
- Mutually e0clusi1e 5/s
1ymp%oma screening tu'e
1= color panel
C#4= Ale<a H00
C#8A
C#1@;&C9# F421
C#8;C#14 F=00
C#= PCH
C#4; C#20
C#H;Cyto lam'da PE
C#A; cyto Jappa F-&C
C#10 APC
C#84 EC#
C#8A Ale<aH=0
Plasma cell 0eoplasms
A color Myeloma panel
ating strategies
First tu'e $or Myeloma" &MH

C#8A F-&C

C#1@ F4=0

C#20 APCHH

C#2H PE

C#2AAPC

C#4= F=00

C#11HPEcyH

C#=7 PerCP cy=+=

P Su'ramanian
Second myeloma tu'e" &MH

Cyto 1am'da F-&C

Cyto Jappa PE

C#4= APC HH

C#8A PerCP3Cy=+=

C#1@ PE3CyH

C#=7 APC

5lan*

5lan*
P Su'ramanian
Case o$ plasma cell dyscrasia
4eig%t color6
A color -P&