Flowcytometry Principles

8/12/2019 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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FCM: principles and applicationsFCM: -ssues and trou'les%oots


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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



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Morp%ology

Ancillary tec%niues



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Morp%ology

Ancillary tec%niues

- Immunophenotyping (IHC and FCI)



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BCL2

Neutrophils

CD13+, CD16+

-mmunop%enotyping


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Morp%ology

Ancillary tec%niues

- Immunophenotyping- Cytogenetics



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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



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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



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#iagnostic la'el

Prognostic mar*er

Predictie mar*ers



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#iagnostic la'el

Prognostic mar*er

Predictie mar*er Minimal residual disease treatment

e$$ectieness6



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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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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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0luidi!s a lo< !ell


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0luidi!s a lo< !ell


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0luidi!s a lo< !ell


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0luidi!s a lo< !ell


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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.


5"plii!ation and

!o"puter

Laser

Scatter plus FC-

?M.

?M.


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?M.

?M.

?M.


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


o si"ilar linea(e##

Four color panel


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C#8 3 F-&C

C#1@ 3 PE



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


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


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+#


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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Flowcytometry Principles

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Transcript of Flowcytometry Principles