Professional Documents
Culture Documents
-sta(lishing $iagnosis
Prognostication .su(classi'ication/
Prediction
,outine in0estigations
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
Pattern studies
Sorting
,esearch Applications
Auto'luorescent Proteins
Apoptosis
-n8yme acti0ity
Mem(rane Potential
Phagocytosis
Gia(ility
Changes in 7ntracellular pH
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FSC vs SSC
Study Bcells
Study Tcells
T and B cells
C$ !==;1 7E$B1JA, .BM/+##=
FSC Height
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%ating
Small cell 1ymphoma
>hat is C$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
Morphology
HN- stain Biopsy
%iemsa stain Aspirate
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$1a
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C$=
C$D3C$@
sur'ace
$PT
C$D:
C$@:
cytoplasmic
Myelodysplastic syndrome
M$S
Scatter pattern and maturation pattern
FSC
SSC
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%ate <
FCS Filename %ate S o' -0ents Q o' %ated
Cells
Q o' All
Cells
1"=a"1##)M,$
TBB-+'cs
Eone 1#DD?D 1##+# DD+H<
1"=a"1##)M,$
TBB-+'cs
%ate 1 1#DD?D 1##+# DD+H<
1"=a"1##)M,$
TBB-+'cs
%ate ! <@#D1 <<+<? !D+@?
1"=a"1##)M,$
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%ate= 11<H 1+11 #+<
1"=a"1##)M,$
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%ate < ?D@1 ?+! !+H@
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TBB-+'cs
%ate? 1!1#! 11+<@ <+1@
1 in 100 tumor cell dilution MRD Tube
MRD = $13!
Kuality control
Tandem dyes
Morphology
Cytogenetics
Molecular $iagnostics