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ONKOLOŠKI INSTITUTE 
INŠTITUT OF ONCOLOGY 
LJUBLJANA LJUBLJANA 
Slovensko 
združenje za 
pretočno 
citometrijo 
DIAGNOSTIKAA KUTNE LIMFOBLASTNE LEVKEMIJE 
IN MINIMALNE REZIDUALNE BOLEZNI 
Z VEČ BARVNIM PRETOČNIM CITOMETROM 
DIAGNOSING ACUTE LEUKEMIA AND MINIMAL 
RESIDUAL DISEASE WITH MULTIPARAMETRIC 
FLOW CYTOMETER 
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CD19APC-A 
Onkološki inštitut Ljubljana 
30. november 2015
-1,664 
CD19APC-A 

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'- Strokovni in organizacijski odbor: 
doc. dr. Veronika Kloboves Prevodnik, dr.med. 
dr. Jaka Lavrenčak, univ.dipl.biol. 
Andreja Brožič, univ.dipl.biol.,prof.biol. 
Simon Buček, univ.dipl.biokem. 
Urednika zbornika: 
doc. dr. Veronika Kloboves Prevodnik, dr.med. 
Andreja Brožič, univ.dipl.biol.,prof.biol. 
Organizator in izdajatelj (založnik): 
Oddelek za citopatologijo, Onkološki inštitut, Ljubljana 
Slovensko združenje za pretočno citometrijo 
Ljubljana, 2015 

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Program 
14.00- 14.20 Flow cytometric immunophenotyping at the Institute 
of Oncology Ljubljana; historic overview (Veronika Kloboves 
Prevodnik, Institute of Onco/ogy Ljubljana, S/ovenia) 
14.20-14.40 One year of flow cytometric diagnostics of MRD-ALL 
at the Institute of Oncology Ljubljana (Andreja Brožič, Institute of 
Oncology Ljubljana Slovenia) 
14.40- 15.40 Eight- and 10-color flow cytometric diagnostics of 
acute leukemias and MRD in children, optimal panels of antibodies 
(Michael Dworzak, St. Anna Kinderspital, Vienna, Austria) 
15.40-16.00 Clinical importance of MRD-ALL at 15
th
and 33
th 
day 
and during the recurrent disease (Janez Jazbec, Pediatric clinic 
Ljubljana, Slovenia) 
16.00-16.20 Coffee break 
16.20-16.50 Differentiation between MRD-ALL and hematogones, 
difficulties and pitfalls (Angela Schumich, St. Anna Kinderspita/, 
Vienna, Austria) 
16.50-17 .1 O Set-up and compensation of 10-color flow cytometer 
in every day practice (Dieter Prinz, St. Anna Kinderspital, Vienna, 
Austria) 
17.10-17.40 Set-up and compensation of 8-color flow cytometer; 
EuroFlow view (Tomaš Kalina, Childhood Leukemia lnvestigation, 
Prague, Czech Republic) 
17 .40-18.1 O Set-up and compensation of 10-color flow cytometer; 
BO view (Jiri Sinkora, BD Biosciences) 

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ONK0L0SKI INSTITUTE. 
INŠTITUT 0F ONC0LOGY 
LJUBLJANA LJUBLJANA 
Flow cytometric immunophenotyping 
at the Institute of Oncology Ljubljana; 
historic overview 
Veronika Kloboves Prevodnik 
Department of Cytopathology 
Institute of Oncology 
Ljubljana, _lovenia 
O Ljubljana Flow Cytometrlc Meeting 
Flow Cytometry at the 
Institute of Oncology Ljubljana 
01• IS 
• DNA period
- Since 1988
- DNA ploidy and proliferative activity
• FCI period
- Since 1997
- Diagnosing lymphomas
O Ljubljana Flow Cy1ome111c Meeong 
, o • 
,O" 
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DNA analysis from 1988 to 2015 
•
1988: PAS 1 (Partec)
•
1991: PAS II (Partec)
•
2001: PAS (Partec)
•
2009: CyFlow Space (Partec)
• DNA ploidy/proliferative activity
- Research
• Monitoring response to treatment
• Prognostic value
- Diagnostic
• Neuroblastoma
• ALL
Q Ljubljana Flow Cytometnc MeeUng 
LJ 
PI\ $ 
-
C)IFlow Spar:e 
..................... _______ _ 
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..., .. ,,......, 
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FCI from 1997 to 2015 and beyond 
•
1997: BD FACSCallbur (from 2 to 4 colors)
- 1997: 2-color FCI, standard protocols for sample
preparation
- 2000: in-house protocol for sample preparation
- 2001: 3-color FCI
- 2005: 4-color FCI
- 2006: beginning of quantitative FCI
•
2007: BO FACSCanto II (6-colors)
- 2008: 5-color FCI
- 2009: bone marrow
- 2010: 6-color FCI
- 2014: MRD-ALL
•
2015: BD FACSCanto 10c (10-colurs)
- Setup and compensation
- Creating 8- and 10-color panels
O l.jublJana Flow Cytomeuic Meetlng 
BO FACSC \ 1.J.1 
BO FACSCanto 11 
BO FACSCamo 1 Oc 
Cytopathological diagnostics of lymphomas 
at the Institute of Oncology Ljubljana 
•
Based on cell morphology,
immunophenotype and
molecular genetic features
•
Well excepted method in
diagnostic evaluation in
lymphoma patients
•
Not always like that!
Cervlca
1 
lymphaoanopath v 
O Ljubljana Flow CylOmetric Mooong 
19 years ago 
• Many discordant cytopathological and
histopathological diagnoses
• Question
- should cytopathological examination still be used in
diagnostic evaluation of lymphoma patients?
• Solution - Prospective study
- Which method should be used for the
o
Llubljana FIOW Cytomelnc Mee11ng 
2 

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Conclusion 
•
Flow cytometric method more sensitive and
specific than immunocytochemical method.
•
Two-color flow cytometer was bought.
Q Ljubljana FloW Cytomeulc Meetlng 
1997: Beginnings of FCI by 
BO FACSCalibur 
• Problems
- Low cellularity of cytological samples
- lnterpretation of results
- Low sensitivity of lymphoma detection
O L1ubljana F1ow Cytometric Meebng 
Solutions 
1. lnternational collaboration
O Ljubr1ana Flow Cy1omotnc Moonng 
3 

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Solutions 
2. To improve sensitivity and specificity
•
ln-house protocol for cytological sample
preparation
- Prevents celi loss during sample preparation 
- 150 000 cells per tube
•
Upgrading cytometer with additional laser
- From 2-color to 4-color FCI
•
4 tube initial B-cell lymphoma panel
- 9 different antibodies
•
Gating strategy
- From FSC/SSC to SSC/ CD45 or CD19 
lncreased sensitivity but same specificity 
O
Lfubl)ana flow CyU>me111c MeeUng 
Sensitivity and specificity 
Author/year Sensltlvlty 
Dunphy and Ramos, 1997 0.80 
Young et al, 1998 0.80 
Jeffers et al, 1998 0.86 
Meda et al, 2000 0.95 
Dong et al, 2001 0.76 
Zeppa et al, 2004 0.93 
Bangerter et al, 2007 0.85 
Swart et al, 2007 0.97 
Zeppa et al, 201 O 0.95 
Institute of Oncology 0.93 
Speclflclty 
1.0 
1.0 
1.0 
0.85/1.00* 
/ 
1.0 
1.0 
0.87 
0.99 
0.99 
Numberof 
cases 
73 
100 
46 
290 
139 
307 
131 
124 
446 
144 
2007: 6-color flow cytometer (BO FAcscanto 11)
•
Bone marrow and peripheral blood FCI
- From 4-color to 6-color FCI
•
Retrospective quantification of antigen expression
•
Poorly cellular vitreous samples
O Ljubl1oma Flow Cytomelnc Meatmg 
4 

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Cytopathological examination and FCI 
of bone marrow 
• 2008 - international guidelines far bone
marrow examination
• •,',l"' LI 
'l'::_, • t.UOUtHY t!UfAlOL�Y 
ICSH guidelines for the standardi1ation of bone marrow 
specimens and reports 
S•H Ut", W N UIEll:
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,._ Suao,U�U.UO.IJ „ HOUIOI.Nf 
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2009 - implementation of guidelines at our
Institute
- Aspiration and trephine biopsy of bone marrow performed
simultaneously
- Multidisciplinary diagnostic approach
Q Ljubljana Flow Cy,omelric Mee!Jng 
Haematopathological conferences since 2011 
• Aim:
- Discuss difficult lymphoma cases from different
point of views to reach a more accurate diagnosis
of lymphoma
O LitJbljana Flow Cyrometr1c Meeting 
Quantification of antigen expression 
•
The level of CD20 expression in B-cell lymphoma
patients is crucial for planning the Rituximab
containing therapy
- immunohistochemical method
- quantitative flow cytometric method
1„ 
-�--
O L1ubllana FloW Cvtomeirlc Meeting 
Reg,esslon plot 
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lmmunohistochemical method 
•
generally used
•
probably not precise enough
- some patients with CD20 positive DLBCL do not
respond to the rituximab treatment
- when in this patients the level of CD20 expression
was determined by quantitative flow cytometric
method it was very low
- CD 20 expression and response to the rituximab
containing treatment are most probally connected
O Ljubljana Flow Cytomeulc Mee!lng 
Quantitative flow cytometric method; 
CD20 expression in different B-cell lymphomas 
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- Flow Cyrometric session with prof. Michael Dworzak
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- lmpressed by prof. Michael Dworzak lecture
O l,Jubljana Flow Cytometrlc Meebng 
6 

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Beginning of MRD-ALL project at the Institute 
of Oncology Ljubljana 
6-ALL bef0l8 trea1ment 
t 
6-ALL at 15� day of treatment 
"' 
Ljubljana Flow Cytometrlc MeeUng 
2015: FACSCanto 1 Oc and future 
development 
•
lmprovement of sensitivity and specificity
detection of lymphoma/leukaemia by FCI
•
8-1 O color FCI measurements
•
Experiments with 8 to 12 antibodies per tube
Flow team 
o 
INSTTTUTt: 
0FONC0LOGY 
LJUBLJANA 
O l)u011ana Ftow Cy1ometr1c Meeung 
Mariana Matič, tocnnloan 
Brigita Šturbej, tochniOan 
Jaka Lavrenčak, ""'og1st. PhO 
Andreja Brožič, -• PhO atudent 
Simon Buček, �-
'
" 
Ulrika Klopčič, e,<opatnologlSI 
Sandra Jezeršek, c,<cpalJ10log'<t 
Veronika Kloboves Prevodnik, .,,._""'°"'"· PhD 
7 

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Ljubljana Flow Cytometric Meeting: 
Diagnosing acute leukemia and minimal resldual disease 
with multiparametric flow cytometer 
Ljubljana, 30 November 2015 
One year of flow cytometric 
diagnostics of MRD-ALL 
at the Institute of Oncology Ljubljana 
Andreja Broflč 
o 
ONKOtošKI INS!lTUT! 
• 
Slovensko zdruten1e 
INSTITUT OF0NCOlOGY 
lJUB!.jANA L1ueqANA 
za pretocno c1tomern10 
BEGINNING 
• Learn about:
leukemia immunophenotyping
MRD monitoring
• 4-color testing panels
2013 
Stoveman Soc1ety 
Fo, Ftow Cyromer,y 
M AU.ol dx � lknac 
(dkllltl'IC�«'C'UfTi'tlAlL) AU.dlhrT� 
CI>! 
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BEGINNING 
\UICG 
2013 
• measuring reactive bone marrow samples (normal) with
leukemia antibody panels 
• measuring leukemia bone marrow testing samples
• training in Vi�nna
• defining 6-color panels ,;
'l 
8 

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BEGINNING 2014 
• 6-color panels measuring leukemia samples stained
with leukemia antibody panel on BD FACSCanto II
• international collaboration
- lnternational Berlin-Frankfurt-MOnster Study
Group (1-BFM-SG)
- supervised by laboratory in Vienna
LEARNING 
•
introducing with MRD monitoring
- Presence of leukemia cel/s that have avoided the
action of antitumor drugs
- The longer the tumor cel/s are retained during
therapy, the worse the prognosis is
LEARNING 
•
ALL IC-BFM protocol
- cell input, staining, lysing, acquisition, ...
�-<» - 1 • _, .. 
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-
• 
1 
- FC MRD: at least 30 blasts in 300 000 cell: MRD +
<0,1% FLR 
>0,1 and <10 % FMR 
>10 % FHR 
9 

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LEARNING 
• Sensitivity of FC
1 leukemia celi in 10 000 to 100 000 cells 
•
How to flnd them
• lnterpretation of results
SURFACE 
1
. (045 
APC-CY7
DEFINING PANELS 
2. CD3 4 me /CD117 
P
E /CD33 PerCpcys,s /HLA_DR AJ>d'CD14 
PE-co/CD45 
APC·CV7
3. CD3 
FIT C /CD19 
P
E / CD5
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PC-07 
4. CD38 me /CD34
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C YTOPLA SMIC 
5 . CD45 
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6. c-TdT me /CD7 PE / c-CD3 
P
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pe.cvi CD45 APC·CV7
7. c-MPOmc /CD34
p
e / c-CD3 PerCpcy5 ,5 /CD10 
A
PC/ CD19
p
e.m/ CD45 APC-m
B· LL 
SURFACE 
DEFINING PANElS 
1. S YTO 16mc / CD34
pc 
/CD45 p., ,cpey5, s / CD19.,JCD10 Pt-cv
7 
/CD20 ••c-cv, (MRD- A LL) 
2. C DSB mc / CDll a
l'f 
/CD45 Pe.cpey5 .5/ CD19 • .J CD 10 pc.c:r, /CD20 ••c-m (MRD- ALLl 
CYTOPLASMIC 
3. c-kapa me /c--lamda PE /c-CD79a p.,,cpe,s,s /CD19 
A
PC / CD10 ,c-cv
7
'CD4S 
APC<V 
4. c-CD22mc /c-µ{lgM),c /c-CD79a .. ,c,,o,s. s /CD19 APC /CDlO PE •C'/7
/CD45 
A
PC
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6. CD45 APC-Cf'I 
7. kapa 
fJTC 
/lamda " /CD79a P,tCpCy
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APC 
/ CDlO pt-cv7/CD45 
A
PC
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T- LL 
SURFACE 
1. S YT016 mc / CD99
p
c /C 03 P , ,CpCy s, ,/ CD7 
APC/C DS 
PC -m /CD4S •Pc-m (MRD· ALL) 
2. C D4mc /CDB •t / CD3 ...cpe,s,s /CD7 •PC / CD5 pt.cv
7 /CD45 •PC·CY ' 
{MRD-T ALL) 
CYTOPLASMIC 
3. C· dT mc /c- D3 PE / CD3 , .. cpCyS, S /CD 7 APC / CDS
PC -cv,/CD45
APC .Cf'I ( MRD·T A LL) 
4. c-CD 3mc /CD99 PE / CD 3 ,.,c,,c,s.s /CD7 
A PC / CD16+56pc -cv
7
/CD4 S•PC-cn 
S. c-CD3mc /CD99 PE / CD3 ..,cpeys,s /C04APC / CDB
PC .c:n /CD45• •c= 
10 

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Diagnose: 
70,0% blasts 
Day 15: 
2 populatlons of blasts 
Shift In a expression 
Oay33: 
Same atypical 
population 
Shift in a expression 
B-ALL
� : �::., ,1 
1.:�J 
T-ALL
Day 15: differnet gating strategy; different fluorochromes of CD3; MRD+; 0,07
_
%
_
bl
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ast
_
s 
__ 
PITFALLS 
•
Problems with antibodies: CD3 APC-CY7, CD22, ...
skipping unnecessarily, change done, fluorochrome 
•
Atypical populations - blasts?
•
Down modulation - CD34
11 

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PITFALLS 
•
Dilution of antibodies: correct volu me cell - antibodies
Day15 
Day33 
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Usual cellularity Low cellularity 
High celi volume 
Not enough antibody 
Usual celi volume (titrated) 
Enough antibody 
•
Low cellularity sam ples - one tube
PITFALLS 
•
Syto16 - to much; wash step
overspill in red 
•
Number of cells
depends on MRD (if high-less if low-more) 
sensitivity 0,01 % {1 dot in 10 000) 
Day 15 
MRD+45% 
B-ALL
Day 15 
MRD+0,07% 
STATISTICS 
T-ALL
12 
■ FLR
■FMR
■ FHR

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FUTURE WORK 2015 -2016 11 
• Measuring bone marrow samples stained with leukemia
antibody panel on:
-BO FACSCanto II and
-BO FACSCanto 1O-color
• Oefining 8-color (1O-color) panels
• Collaboration
13 

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Multicolor flow cytometric diagnostics and MRD 
in acute leukemias in children 
Panel optimization and more ... 
Michael N. Dworzak 
St. nna Chlldren's Cancer Research Institute 
Vienna, Austria, EU 
• Technical issues and standardization
• Pre-analytics {sampling, transportation etc.)
• Pre-acquisition issues {sample preparation, staining & panel set-up)
• Acquisition issues (machine set-up and QC, acquisition standards)
• Post-acquisition issues {data analysis, software, gating strategy)
• Data interpretation and diagnostic rules {EGIL, WHO, LeukemiaNet, ... )
• Technical issues and standardization
• Pre-analytics {sampling, transportation etc.)
• Pre-acquisition issues (sample preparation, staining & panel set-up)
• Acquisition issues {machine set-up and QC, acquisition standards)
• Post-acquisition issues {data analysis, software, gating strategy)
• Data interpretation and diagnostic rules {EGIL, WHO, LeukemiaNet, ... )
14 

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L 
MPAL can be i<lentified using the recommcnded pand 
of the European LeukemiaNet � or other comprd1ensi, e 
combination s. 
3 
lmportantly, seYeral markers specific for
the myelo/monocytic lineage as well as for the B- and 
T-lymphoid lineages must be tested. exclu<ling a rcstric­
ti, e i.trakgy of quick orientation followed by i.elected
lineage-i. pecific marker s.
This proposal relied on 26 antibod­
ies to perfonn a proper score, yet published reports seldom 
applied such extensive panels. 
Am J Ciin PatllOI 2015;144:361-376 
.�nna /'c,n.-ir, Ml), Pltl),
1 
anJ Man,C B,;,d, l'h.am$ol), Phl
>
' 
---- ----- - -l- • .-
• . 
:• : ,:.: : / ? . -� ; > ) . .. . 
. , . : .' . 1 ·-�-.... -�•-. � : ··"· -·. • •• ••• ...... .. 1 • 1,,\1 ... """' 1 � .�1. 
WHO 2008 CIUTEltlA l'Olt MPAL DEl'INITION 
1 My,t lokl ...... , 
Myeloperoaic18se (1low cyt>melry, lmmUnol>islocllemia'y. o, cylOChemilllyJ 
« 
,- -, , 
,� --, 
Manrxyue cifer� (NSE( C011c C014 C084. al tflCIZYme >i r.,,... , 
....... _.,, , .... __ ... , 
---- -- --
C)olqllasmlc CD3 (low cylOme(ly wrfl al'llbocies III CD3 epslon cha,n, lmmunohislochemtsuy using pdydonel 
anl- CD3 anllbody may deleCI CD3 zeta chain which I• nol T-<:811 specllc) 
Surtace C03 (r8"1 In mbted phenolype leullaemlas) 
B-llneage(mulllplea......,......,.1'9d): ,---- ---.. 
SVong C019 w,th et le• 1 dihe ,-ing &Vong� .,..,.._ C079a{cytoptasmic CD22,):o 10 
� '�-------
W8ak co19""' at 1eas1 2 ol tht lollowing IIIOngly •� C079a. aytoptasmk: co _ :12. _ co _ ,o ____ ,. 
Note: Monocytlc dlfferentlatlon require.s positlvlty of l:2 of the.se antigens; 
The T-<:ell component ls r•cognl:zed by brlght expresslon of ICD3, elther on the entlr• blast 
poputetion or on a separate subpopufatlon of leukemk: cens ___ should be as bright or nearly as 
brlght as that of norma l residual T cells present In the sample. 
'_,I �- , • • •• .• -
•
. ... ,' -
• -
EarlyT-cell precursor leukaemia: a subtype of very high-risk 
acute lymphoblastic leukaem ia lancetOn,of 2009; 10: 147-56 
rbn,C..,...• ·�o../n G� M,llo,!aO,.., f< -Gll<l>n 5u!<nlClbrnoMl,ll<qlncJFIICll<l>j0..,IC""""9S.. 
J<tl,<),ERu...,�S-,,A ..... �°""9'1b1"'1,}aalntO.-.nsDonoC..,..,. 
Development of an ETP-ALL scoring system 
ETP-ALL shows a distinctive immature immunophenotype 
ch aracterized by lack of CD 1 a and CD8 expression, weak CDS 
expression with <75% positive blasts, and expression of one or 
more of the following myeloid or stem celi antigens on at least 
25% of lymphoblasts: CDI 17, CD34, HLA-DR, CDl3, CD33, 
(cDiLJjia nd/o (CD651 C o ustan-Smith e/ 11/, 2009). 
'�--;' '�--;' 

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• Extensive single- latform panel for acute leukemia in children
Surface 
Optionlll / 
Morlu,,. (eacn comb,neclwr!h CO<I$) 
IC03. ICD22. IC079a, llgM (µ-dlaln� l.yscr,.,!le, IMPO 
C021. C03, COll,C07, C01 0. C019. C020: C011c. C011b,C01 3. 
C014,C015, C033, C064, C065', C01 17; C034, (C045), C056, 
HLA-DR 
WT .... LL co,, co.i. coe. TCRo�. TCR>/(1 
W 8-111 � •-Chal\ A-chaln (1<1r1ace stelning after p,e.washlng 
orlnttee-,) 
II cases· NG2', Clec12A"' 
tt BCP-N..L C011et, C022, C024, C038, C044, COM, C06ec, 
C0123l. CRI.F21' 
ReconYnended H--ALL C099, ITdT 
-WBAI.-� looo,,e,al....,. C024,ITdT 
•snb ·r •tands tor 1nttaua.ar •�nir'ii 
l��IIO(PE)tec­
•a,aillbleOff'f_wilh_oirlil-)'INllo(FITC) 
•done7.t.8ec�er,M11ml,,FLUSA 
�-•50C1.S.c.,.Ok-B/ooclfflcft(BD�San.JoM,CAUSA 
' -- 100-PE.elolegand. s.., DieQo. CA USA 
,qa;t,f!I, . ·:-.. -'.· . ' •• , �;. f,.. -
' 
·1•\'; :. · __ :{:_ .;_ --�)<", <��:: 
.. . '
.· 
�;�� i: � �' r :� !If'� ���-t�� l - �-� :./ - , _,· ( ► "-· . • • 
� • 
=--- � � 
compatible with 
WHO 2008 and 
EGIL score 
Dako (pc) Exbio (me) BD (me) 
� 
- -
�- �-
<· 
<) - <) -
◊-
� � 
� 
31„ 
<Š - �"-
g
„ 
.! 
,. 
�-
!! 
o 
-
� � 
. ,.,. 
o 10' 10' 
..... 
10' 10' 10' 
�" 
••' ... 10' 
CD45APC-Cy7-A CD45APC-Cy1-A CD45.APC-Cy1-A 
lmpact of methodology on MPAL-diagnosis? 
❖ WHO 2008 does not define weak/strong!
❖ What is positive? (EGIL rules date back to 1995)
❖ lmpact of procedura! peculiarities (permeabilization, choice of antibody clones)
❖ Adaptation to multi-color flow cytometry is lacking!
16 

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....... 
WHO 2008 CRITEIUA FOR MPAL DEFINITION 
Myelokl IMage: 
MyelopefOllidase (flow cytome"Y. immLWlOhistochemis"Y. cx cytochemiatry) 
or 
Monocyt ic dlflerentiallOfl (NSE. CD11c CD14. CD64. or .,.sozyme) 
T-lneage: 
Cytoplasmic CD3 (llow cy10metry wilh anlibod1es 10 CD3 epslon chain. immunohistochem,stry using polyclonal 
anti- CD3 antibody mey deled CO3 zeta chain. which is not T-<:ell specilc) 
or 
Surface CD3 (rare in mixed phenolype leukaemias) 
, .g:j..;_ --.l� p1e antigens ntqlll�------ .... ,
Slrong CD19 wil\i at lea&I 1 of the foU6w1ng slrong.,. exflltlsaed- CD79a. cytoplasmlc CD22, CD10 
(K I 
I 
1 
I 
\ 
, Wea.k CD19 Yf,111 at leasl 2 ol lhe lol
�
g strongly e� aed. C O79a . cytoplasmlc CD22. CD10 
---- � '�
-
---
-
-' 
--------� 
Note: Monocytlc dlfferentlat.lon requ ires posttlvlty of il:2 of these antigens; 
The T-cell component is recognized by brlght expresslon of ICD3, either on the entire blast 
population or on a sepanne subpopulatlon of leukemlc cells ... should be as bright or nearly as 
bright as that of normal residua l T ce lls pre-nt in the sampte. 
OPEN FORUM 
Proposals for the immunological classification of acute leukemias 
European Group for the lmmunological Characterization of Leukemias (ECIL): MC Bene'. G Castofdi
2
, W Knapp
>
, 
WD Ludwig", E Matutes•, A Orfao
6 
and MB van't Veer' 
There was general consensus on the cut-olf point to con­
sider a marker posftive and this was set up at 20% of ceffs 
stafned wilh the monocfonal antibody (MoAb) whe1her usfng 
fndireet immunofluorcscence wfth microscope or flow cy to­
metry or lmmunocytochemicaf techniques . An exceptron was 
made lor anti-MPO, CD3 and CD79a due to their high degree 
of specificity, as wefl as TdT, being the cut-off point for these 
markers set up to a minimum of t 0% of bfast cefls stained, 
provfding that confirmation of filasts stalned with the antibQdy 
is made .ID' light microscoQy exam ination . These cut-off poi nts 
are appficable to both diagnosis of the acute leukemias and 
classification of the various ALL and AML subtypes. 
There was concern on a number of technologica l aspects, 
eg different pattern oi staining when using direcdy co n juga ted 
phycoerythrin MoAb vs fluorescein conju_gated or unlabelfecl, 
adequate gating, quantification of the antigenic mofecules, 
cytopfasmic staining by flow cytometry. etc. However, alf 
these aspects were beyond the ain,s of the group and wilf 
fikefy be considered as a topic in the future. 
Leukemia (1995) 9, l 78�1786 
Flow cytometry thresholds of myeloperoxidase detection to 
discriminate between acute lymphoblastic or myeloblastic 
leukaemia 
Summary 
tbt Wotkt lfa:.hh \ltg,ln1;;.a1,on !0011 C Ll'll-1""�'11)n nnphiw-o myd°"'nAA-td.1""' 
Ml'C)) dc'l«tinn 111, �ffiocnf k,, .i.WJlliall 11 bb,, popul;111011 to tht m)TloiJ linir-, 
111>" 
,;;,; � ':':' -- •' 
" f fi,.. .._,..: .. rn•'ffl' 
l f 
" .,,,..._ i..mr,,11 lkn "-Y ,�tuJtcJ dw f<,..\.1-�IPO thtt,,holJ b) ,»m�t("' 
R'lh�thd ) ' l?tl M.1.Urr fi'mrhPhl.l\.lk. la1k«t111;., ,nJ � -.111rr lf'l)'liu.J ku�tllJ,lill 
• •
nhl:tUt nwur„11'-�, .ill � t.,, b,c,v.1,o.h�b.LW c.,1T.Knuni',t,y. A o-.. thrc-t.hold 
"'° fuunJ to lx-r"C'K°Y.im uMnJ .an M'4 �,rol n b.k • "'n,J-,c(uvn, • .- 1,c·mlt� 
in v� 1, "l'' 
Afrcr pcrmc.1bilization, the cclb 
wcrr suineJ wtlh S µl of ;UU1 Ml'O· tluorcS<cin i.sothio­
C)'iln1HC' (FITf"l•l., lxlf,: d mu1k)('l11nal omtiboJy (p.1k.l, or 
lm munvh. \. h) ur �l)1X' <·ontru:b (for 96/1.!8 ALL and 10 / 75 
AML �11 sampb). 
(E) 
111, e 
-
17 
Juli cn Guy, 
1 
IJl-ana Antony-lkbl'C.:u 
EJnm�l\ue.l �noyoun.:11 l�hclle Arnoux,• 
Chann,J Fossat,41 M.lg.aU Le:. Garff• 
'Tavernicr.
S 
Anna R.iUntxmh.3 M.ichč:le 
lmbcr1,2..J. Marc MaynndiiC',
1 
Fnnci..1 
l.-;11comOO," Maric C BinC'and Orio.nne 
\V;: ,gner•Ballon
.l.J 
on heh.tlf or 1he-GE.IL 
(Groupc-d'Elude lmmunol�iquc dcs 
Lcuccm.ic5) 

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\....., 
Flow cytometric detection of intracellular myeloperoxidase, 
CD3 and CD79a 
lnteraction between monoclonal antibody clones, fluorochromes 
and sample preparation protocols 
Jauos Kappellllayer•·•. Jan W. Gra1awa•. Eva Kaniszi'. Pablo Meueodez•.
Juana Ciudad
<
. Rosana Rivas
<
. Albeno Otfao•
Pauwise 
comparison of FITC and PE coDjugates of the same 
done revealed, that PE CO DJUgates yielded s1 �­
cantl higll, er MF1 Ihan FITC conJUg!tes (MPO- 7, 
P=0.01; H-43-5, P=0.009). Tbe percentages of 
MPO+ cells (expressed as a fraction of total 
leukocytes) in Donnal PB aru! BM samples, as well 
as in AML samples, did not differ significantly 
betweeD the five fixation-perme.ibilization systems 
(data not shown). Finalfy,_ DO false-posili\•e reactions 
of any of the antJ - MPO clones (irttspectJve of
fl uorochro me) was seen OD precursor 8-ALL and 
T- ALL blasts prepared with any fixation-peuneabih ­
zation lot except followang use of Cytofix/Cyto­
penn
,. 
(Table 2). where all tbree anti-MPO clones 
stained B-ALL and T- ALL blasts diJll!y. 
Comparative Analysis of Different Permeabilization 
Methods for the Flow Cytometry Measurement 
of Cytoplasmic Myeloperoxidase and Lysozyme 
in Normal and Leukemic Cells 
Francesco Lanu: An�J• Latorraca. Sabrina Moreni. Barbara Castagnarl. LulSa Ferrari.
and Cianlulgl Casroldl 
S«tloo o(l�. U.,h""'4.r ol "'"""• Fomn, lt,Jy 
used an FITC- conjugated anti- MPO McAb (done MP0 - 7. 
l gGl isoty e , from DAKO, Glos tru..11 Denmark). whereas 
only the F&P reagent was characterlzed by a good speclfi c -
lty and sensitlvity in detecting_}he two granule c onstitu· 
ents (MPO , 1 so�me) on leukocytes t.aken from healthy 
subjects. The remaining two permeabilizalion techniques 
(OPF and FLy) were characterlzed (at least under our 
experimental conditions) by a lower specificity in detect-
ing MPO and, to a lesser extent, lysozyme antigens; 
- -
trials dealing with leukemias. A standardization of 
cyto fluorimetric analysis of intracellular antlgens is needed 
in order to lmprove rhe reproduc lbillt and com arablll 
of results in multicenter studies. 
Arber et al / MYELOl'1:ROXIDAS1a·POSITIVE ACUTE LYMPHOBLASTIC UUKEMIA Am J Ciin Patho/ 2001 :116:25-33 
o , z 3 • s s 7 a ! 10 11 z t3 
Tim•IY•arsJ 
l f lJon, 11 Owr&II survMII from dote ol d,agn0$15 fn J)<ltients 
woth polyclonal myelope<oxklase-pos,tive o, pofyclonal 
myelope,oxid&se-negauve aeute lyml)hoblas tic leulcemia. P 
� .07 (log-rar,k IOSI). llo<tical marks represonl censored dota. 
The 19 pMPO-positive casei. were ali of precursor S­
celi linea ge (P = .06) and, therefore. had a higher frequency 
of CDI O. CD 19. and CD20 expression Ihan did the pMPO­
negative group ITublc 3 1. No dilference in aberrant CD33 
expression was identified between lhe 2 groups (P = 1.00). 
but the exprcssion of either CD 13 or CD 15 was incrcased 
significantly in the pMPO-posilive group (53% vs 21%: P 
=.01). lmmunocytochemical studies performed on cylocen­
trifugcd preparations of Frozen cells from 8 cases, including 
4 pMPO-positivc and 4 pMPO-ncgativc ALLs using a 1110110-
clonal MPO antibody. wcre negative in ali cases. 
studicd with a monoclonal MPO antibody (done MP0-7; 
1: 100 dilution: DAKO) on methanol-fixed cytoccntrifuged 
18 

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Mixed-phenotype acute leukemia: clinical and laboratory features and outcome in a!O"'---:--- ,
100 patients defined according to the WHO 2008 claSl.ification 
E.$1eHa Mn1utes ,
1 
W_,fried F. Ptclt l,
2 
Mars van't Ve«,
1 
Aic:ardo Morila.' John SWPnSlxay,' Herber1 Strotl4.' 
,Anchst,c, Attarbnsdw, • Georg Hop(,nrger • Sue k!htey.• Mane et,,.ane 8':tne.' AntWI Po;wrt • Alberto Oriao.• Petr LemSl-, '° 
Alchald Schabalh." and Wolt·01t1er LudWig" 
BLOOD, 17 MARCH 2011 • VOLUME 117, NUMBER 11 
Most cases were from United Kingdom (64), Austria (21), 
and Holland (8). and a minorily were from France (3), Sweden (2). Spain 
( 1 ). and CLech Republic ( 1 ). 
According to each center's protocols. 
mult iparamctcr immunostaining with nuorochrome direclly labeled mono­
clonal antibodies (MoAbs) was perfonned. 
Pediatric patients: antiMPO done 8E2 Caltag AdG 
No expressed information on the anti - MPO mAb done given In the following papers on MPAL & BAL: 
• Matutes et al., Blood 2011
• Gerr et al., BJH 2010
• AI Seraihy et al., Haematologica 2009
• Rubnitz et al., Blood 2009
• Aribi et al., BJH 2007
_4 , T -- --•� - - _z � • • 
- -
: 
CD45 
' . 
C045 CD45 
o Lineage assignment: complete concordance between panels
o COMMON- - anel is valid for lineage assignment of pediatric AL 
o AIEOP-BFM thresholds for weak/strong distinction (CD19, iCD3) are very well set 
o Subclassification (EGIL): high degree of concordance between panels 
o Few discordances related to ilgM readings
o COMMON- - anel is generally valid for subdassification of pediatric AL 
o BAI/MPAL: relevant divergences occurred!
o Discordances related to MPO and other myeloid marker readings
o Local panels seem to overestimate MPAL incidence: MPAL 6% vs 2%
CD45 FSC•W 
o lf MPAL/BAL assessment is relevant within a collaborative trial and/or for therapy assignment, ertain involved markers
and/or procedures should be standardlzed to avoid inter- - enter bias
o AIEOP-BFM dominant llneage strategy is VERY relevant to limit influence of fault- •rone myeloid marker „expression"
o ETP: all 4 cases correctly assigned by ali panels
o COMMON- anel seems valid for ETP assignment (limited evidence: few cases tested)
19 

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l., 
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\.._... 
o AIEOP-BFM thresholds for weak/stron1 dlstinction (CD19 , iCD3) are very well set
o ICD79a ls more rellable than ICD22
o Dlscordances In
► llgM: quite random - •eem related to severa! factors: sample quality, antibody and/or permeabilization
► iCD22: permeabilization dependent - nrocedure- -ptimization warranted !
sCD22 is frequently more easily detectable - hut - �an be weak/negative in BI w Mllr & BIV
► MPO: most probably antibody--e lated (clone)
► Myeloid markers : due to weak expressions and to higher background en o,..,.nceornnur •>
o lineage assignment: very high degree of full concordance between 8 centers (26/29)
o Subclassification (EGIL): relevant divergences occurred (in 11 of 29 cases; (�7 /8: S cases)
o Most discordances related to B-11 vs. B-111 (3 ca.ses), T-11 vs. T-I V (4 cases), MPAL (2 cases)
o BAL/MPAL/ETP/blast celi heterogeneity: re levant divergences occurred !
o Only 1 of 7 BAL cases judged fully or '?.7 /8 concordant
o None of 2 MPAL cases judged fully or '?.7 /8 concordant
o None of 2 ETP cases judged fully concordant (�7 /8 concordant: 1 ETP)
o None of 2 cases with >1 phenotypic blast clone judged fully or �7 /8 concordant
10) lMMUNOPHENOTYPIC HIETEROGE.NEJTY OF 8LAST SU8POPUUTION.S: 
Thls ls deflned by the exlstence of ?:2 lmmunoph enotyplcally dl.stlnct 
subpopulatfons of leukemlc cells In a slngle Jeukemla case. Cases whleh e-ontain 
more than one blast population should be clearly flagged in th• data bas• and the 
type of heterogenelty should be descrlbed In the descrlpUve summary of the 
cllnlcal report. 
o Cases fulfllllng crlterfa of dltferent lineeiges - In the sense of blllneel leukemla -
as per WHO Ml'AL or EOIL IIAL d efln lUons. 
o L•ukemla cases wlth maturatlon. l.e. classlcat admJxture of matur1ng eells to 
lmmature brasts (e. g. AML M2. M4). also fall lnto this utegory. 
o Also heterogenelty In bla.st appearance wlthin slngl••llneage cases of A.LL wHI 
be recorded. Such slgnlficzmt difference:S ere deflned by unamblguous partlal 
� (NOT by dim expresslon) among the total blast populatlon wtth at least 
one marKer of the rouowtng Whkh are e.a.s•ntlal for ALL•subtype deftnltlon : CD10, 
tlgM, CD1a , C03, and C05. Thls heterogenelty (In order to be counted u such) 
must lead to dlvergent subtype deslgnatlons (lncludlng ETP) when assesslng 
blast celi eubsets separ at ety . 

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l., 
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'-­
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l...., 
-
o Also heterogenelty In blast appearance w1 thln slngle-llneage cases of ALL wlll 
be recorde<I. Such slgnlflcant dlfferences are detlned by unamblguous 2!.!!!!!. 
� (NOT by dim expresslon) among the total blast populatlon wtth at least 
one marker of ttt. followtng whlch are essentlal for ALL-subtype deftnltion: C0 1 O, 
ilgM , CD1a, CD3. and CD5 . Thls hetar
o
ganelty (In order to be counted as suc . h) 
must lead to dlvergent subtype dulgnatlons (lnctudlng ETP) when ...... 1ng 
bta1-t cell subsets aeparately. 
D IO
Q 
10 10
1 
C03 PE• Tono Re<l-A 
10
:) 
10• 
TCRab FJTC-A 
o Also heterogeneity In blest •�•rane• wllhln slngle-llneage cases of ALL wlll 
be recorded. Such slgnlflcent dlfferences ue deflned by unamblguoua R.1!11.i 
posltlvlty lNOT by dim expresslon) among the total blest populatlon wtth at least 
ona marker of the followlng whlch ara essantlal tor ALL-subtype daflnltlon: CD10 , 
llgM, C01 a , CD 3 , and C0 5. Thls heterogenelty (In order to be counted as such) 
must lead to dlvergent subtype desl gnatlons (lncludlng ETP) whan assesslng 
blast call subsats saparately. 
TABI.E 4: THE AIEOP-BFM SU8Cl.ASSlflCATION OF ALL' 
Sub!YJ>e Discrim inators Remarks 
B-1 (pr o- 8) C010"'9 BCP-All lineage crttena fullilled 
e-1I (common) C01� 
8-111 (pre-!I) ilgMP<- C01()mt"'-.,.may OCQJfl 
B-IV ( malllfe B) K• or A-<:haln""' may occur with FAB L 1/L2 morph0io9Y" 
T-l(pro-T}' only 1 COJPO' and C07"'" T-All !Nage c:nle<la fulfoJled 
T-11 (pre-T) ;o 1 ot CD2.,., C[)5P<>o, CDS,- surtace (s) COJ""'l.,. allowed' 
T .ffl (Cortlcal D co1a, .. sCOJR"' may OC<:ll„ 
T-IV (mature T) C013"'9 and sCD� sCD3-. or sCD3_.,. wi1h TCRI"'" 
ETP 
C01a'"11. C08"'9 
Jcm;-,g"°".:t21""ofHLAOR, 
(only addillve to 
usually cm;, .... _.,.
C011b . 13, 33,34, 65 . 117 ; 
and „1.,.ol HLAOR, 
T-torT-11) 
CD11b, 13 , 33, 34, 65 . 1 17 
sCO:J-a poo mayoccu.-
•actapted from refs . 8& 9. 
1 C0 10---" S-lil is frequenlly aasocialed wilh Mll..-eam,ngements (12) . 
• light.ct,;,;n... ceses - FAB L3-morphology and wiOxxd MYC­
- on, eligiJlo tor conventlonol All. �fflert. end lhus must be 
._- from 8u1<itl.fype matu-e 8-AU. ( 40,,43 ) . 
1 
T-l le Ve<y ,are ond cen be reported logether - T-11 (09 T-1111) 
• 0m o, oven mo<e hequen41y partiol -- pc,oiiMty wilh C03 (e.g. in a 
mnor bloot SWl)OIXJlatioo) oc..,,. _,, l«llitive melllodology II uoed and 
shcx.ld noC mislead 10 ciognose ma1ure T-AU. in tho eboence ot TCR 
e,ipression . 
.... 
- . ' ·-•'i( .....--.f r--,,.
F
•l-�Y ........ . ,...� •• r••--... .,, .. . , 
. 
. - - - . � 
,. -
21 

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Editorial 
Editorial 
MPAL - BIL 
� .... 1 (CII"°" c,,,,m.,,yJ 1!&11 152-J&] (201A) 
Michael J. Bor ow itz * 
Pmfessor of Pathology and OncoloID ', 
Johns Hopkins Medica! lnstilllrions , 
Baltimore, Maryland Mixed Phenoty
p
e Acute Leukemia 
critcria for 
T/, \�doi<l MP , \L (mL,c<l J)hcnol)_
p
c acute leukemia) c..�m 
bc mt·t m one of two wa) s . 'lllt' critcrion mrn,1 are famil­
iar ,,ith rc ulrcs thc c.. ·xp rcs.sion of ,lic mn�, , pe cil k 
m:1ri.ers for cach lincagc' - lfl tlib Gi'c c ·tu lasmi<: CDj 
;in_d m 1...�l®t:CID1i� 
llowncr , lt--:.i, fn:qucntl) n:cog ­
nizcd 1!, tht: ac1 1 1:11 cxprcsi,ion oll lnt:� srx:cilic 
mari<er, oni) :1pplit:!. IC> the siruatio n in \\ hich lh< :n: is a 
si nglt: populacion uf blasts: critcria for idcncifying a nt)C­
lnill compont:nl .uc abo mt:t • ... wh en t 1crc are rn o or 
murt: diMinc..'t popu la1ions of lcukat:mic cdl:,, ont: or 
"h1t:h woulcJ mect immuno
p
hcnot
} p
u: criu:ria for :1cu1c 
mn:loid lcukacmia (with thc cxcc 1ion !hal thl, pula • 
lion nccd nor comprbc 10" .. o al nuck-atcd c..dG ) .. ." 
Mixed Pheootype Acute Leukemia 
Simple co-expressing MPAL 
Bi-lineal MPAL 
Michae l]. BorowilZ* 
l'rofessor of Pathology and Oncology, 
Johns Hopkins Medica! lnstitutions, 
Baltimore, Mar
y
land 
1:stah lbh ,1 diagno�i, of Ml'AL. ll m\.CH :r. wc would r1c jc1:1 
1hc ti;,l· of IO"l. ,ts a dcfini1hc critcrion Wc do not hdin ·c 
th:11 :111 pcn :cntagt: thrc:.hold b ,lll Jccur:11e mca:.urc ofj 
bio log� . Nine pcrcent MPO positive myelohlam With an 
ahermnt phenotype would ckarly establish an MPAL diag­
nosis. whilc 15% MPO posi1ive normal myclohlasts that 
lcukemia . l11e WIIO , -cihrnll)_ :tnd ddihcr:nd de,._
.
., 
nlll HII ,1 lo wt-r hmil on lht · numhcr of m dohl.i,1, th,11 
muM hi.' , ·wn1 to rcrmit a thas_no_,, o \1l'AI _ This is 
22 

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o .l.!li!ii! dlagnoses of truly bllineal acute leukemia with a non-lymphoblastlc
blast component (irrespective of clonal proportions), AML, AUL (according to
above mentioned criteria), or other unusual AL as above are NOT considered apt
for inclusion into study AIEOP-BFM ALL 2009 as protocol cases.
o In contrary, cases w1 th a single blast populatlon wlth ALL-type dominant
lmmunophenotype (according to the AIEOP-BFM lineage assignment criteria
adapted from Mejstrikova et al. 2010} but fulfilling MPAUBAL criteria (below)
are apt to be included lnto study AIEOP-BFM ALL 2009 as protocol pa!Jents.
AIEOP-BFM ALL FLOW-SG 
Consensus lineage assignment 
❖ Standardizing the most relevant issues of divergence ..... 
►
{
i)lgM (e.g. Exbio done CH2)
► iCD22 (e.g. lnvitrogen done RFB4
)
► iMPO 
(
mind done differences , e.g. lnvitrogen 8E2 vs. Dako MP0-7 or BD 588)
► Permeabilization (e.g. lntrasure
™
)
► Doublet exdusion
► Slast done heterogeneity (as distinct from an antigen expressed heterogeneously ... )
' . . - . 'i 
Switch pB-ALL 
23 

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LllW.�(101411•11 
CJ014�1\;bhlw.i.u..td Aillfigfl•�Nl:7.-.NrW 
ORIGINAL ARTICU 
CD2-positivc B-ccll prccursor acute lymphoblastic leukemia 
with an early switch to the monocytic lineage 
l SI.Ynor,,,1
1 
J SUitkov.l\ E Frankov�•. M Z.lov•' L A11nnkkov.' ,:W Ydll"I O�Ur'. E Vodkkov•'• J Vo&.jttl(ov . t\ Z Z�a•. 
ttJlo9«ov•'· G Qrio
1
, MAgwt0,•, r KMhw
1 
tt �' . JP Bourql.An'. 8 BomhM.ISer'. M 0woru11.•. J tun.'. J Trk.t'. JSt.lry
1
• O „vwk
1 
tindf�nkova
1 
DayO 
Swftdws ftom ttw � eo fl'J)'tb,d line• duM9 � p,ecursor :ac:\lllt � II'.,_. Cec.P-ALlJ walmtffl ..-e 
cOMlcltred ,_ancto,U$taw ha'te bMn Mt� W\MU.-(�ano,td ••��e.•�• � ICP-M.1. ��fflff'!J 
8CP'"1JJ. o, <WAI.L wNch dtn',on,;.t�itd monocyto,b � <M1nO t1t•menL �e chmr rnonocytic:: � � 
lt\Nf! � 9B"t tN1,�1 WUt.........,. •� AII ,wAtb cte�,aifti ICP•ALL Wlfh m2 � 
.ttd no MU. �•1:iOnf. Md Ule p,oponMll'I ot ,w.Al..b QWJ MnOl'9 ICP-NJ..I •• �f'Cly Nsli' ( ...... Tl\4' upreg""'1on ol 
CTIPo MIO d.,,....t,,,t,cklin oJ N CEBPA. gw,,e �-,,� Ml tasti.M c,.gnos,s. p,b" co \tlt 11mit Mlien most ol PIII: -swt1ffllng 
occun. �wmtdlit• �s btM'ftn C014"""(01rto� B � .nit CDl4"'col�l4....,. �· � 
d,ttta-,d,.a.ndchM'IOffll\dwtllPf:�of,�PUI.M-<StRGM<Sfft�od'-19gt,neac:<.atnp,INtdthfiiwfw:h.M111lltiom:ln 
� IIIMO,: ,a,,ld ERG gi,ne,wc;e fflOft' frtq� 1ft twAU. P41net\Ur; howeYer.. � weft �d lift o,vy a MiftOdty ol MAI..U. 
Mo,,e,cwe, IW'lll:(NnQ could �(fQ--� ih ll'tcro.nics In fflOUM •lfn09l.tlu..Ahhough ch�en w,:h ,wAU,rnponcf �to iirwaM 
ll>tl�, 11>1:-l»l<d o\U. th<o� -• ,,,. •••- ol<-. IOr "'AU. Swl\U. ,,_. ,,.., •--- ln<o -,� 
·-- _,11o11y _ .. ..., - CUi'• - - a» _ .. _ 
� �«K"•._. �don. 14 iMwiMy 201._ dott0.t0J.lllftL2013.JS4 
Jl:.,Wonb! �I �u.l clioe.W. Jln,N,g• t,WltCh; CCMT�ff Dh'dlit'9 p,o(ttfl alpha. lCUlt- �SIIC ltukeme,a; 
�=-
! � � 
� •
� i i 
... .. •' 
C:O101'1--CYr-A 
! 
.. 
i 
,I 
� �J�· ;·o, 
.. 
•. ... . •' ,;, .. •' 
CO:U►.Cp.c:ys.S-.t. CD?P� 
:ri1b 
-· .. •' ., .. 
CDUN.� (Dt4 41„nctiMlt-A 
• 
., :, ":-''"':-
co,.,...., .. ,. .. 
Dayl 
. 
.. 
l�J 
i" 
.. 
.. •' ..,. ... :CJ 
CDJl,.C,.('t,1",A ..o, • ..,..., .... ,� 
24 

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...--. ____, 
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�..:.. � 
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o s ,o 15 20 22 
day after treatment start 
D•BM DBBM 08PB 015BM 
Tim•polnt 
Slamova et al., Leukemia 2014 
B 
.. 
Oay of cullure 
Slamova et al., Leukemia 2014 
r��.-·�.--��, .... �=-i"-r� . .:.:-1r-1."- .... �..., r � •----.-.,.;---.,:� ..... -·r1>r• ..:. --r .,r.-r-..--- r 
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25 
D33BM 

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CD2 draw-backs: Weak expression in ~40% of swALL cases 
Positive not only in swALL cases 
-
:�. 
i 
i;,.. �-=-��,.!..�� .. l!::l ··· 
ETP and MPAL 
EarlyT-cell precursor leukaemia: a subtype of very high-risk 
acute lymphoblastic leukaemia 
EfmN"Cw1ton-Smtd\Clw.ft1GMulighanMihodoOnou-m6lllktGlkflmSu1DMC�OeqingMOien9Chfng,XIOOplnfSo 
/,ff,,yf """'1t,. Gi""Pf" bsa. And,m ..., .. Cl,/ng-Hon l¼ }a.,.. Q Oownlng. Oodo Compa,,o 
r 111,. , in thc hroau:,t wm,c. ETI' Ali. ,,. a kim.l o(
- T/ m)cloi<i- k-ub.cmia . From a delinitional pt:rspective,
however. MPO expression exclud es ETP ALI., while the
grcat majority of <.:ases of MPAL are M.
P
O po�iliw. In
addition, the T cdl compom:nt o m t· OJ cukcmia
requt·ntl} wou ld mcct n11cria for En> AL I.. l"hu�. thc,t.
t" u lcuJ..t·ntia� a 21>car m1>rl· allkc 111:111 diffcrcnt
although bccause of the central import ancc of MPO ro 
labcling someth.ing as mye::loid, and thc w,1, 1<:ukem i :1 
1rca1111t·n1 proltlc.:ol, are ,1ruc1urnf the1 .1n· l k:llh
lrt :atnl dtfll:rct11li l nfommatcb 1hi, m,11 mab.t: it diffi ­
cuh cn-r to unJcr, tand whctlwr t hc,t' do in fact consti · 
llllC dil"fl-rent lcub.cmic l"lll i tic, It Will be intCTl'Sting to 
see how this sin1ation will bc trc·atcd in the next itern­
tion of the WJIO dassification. 
Cytometry Part B (Clinical Cytometry) 86B:152-153 (2014) 
Michael J. Borowitz•
Prof es s or of l�ulmloi.,') ' ,md Oncolo1,,
y
, 
johns Hopkins M�dical lnstitutions, 
Baltimore, Maryland 

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Subtype 
ETP 
(only additlve to 
T-1 orT-11) 
Dltcr1mlnators Remar1tt 
if CD&",.. .,.: �2"'" of HLAOR . 
C011b.13.33.34.65.117: 
sCOO-,,.. may occut' 
, ! -
, 
§ � § 
!Qi] -- . iQE • ·: tQj, : ; 
1 1 1 f,i,,,,,,-�.....,..; 
... . �«-C-,-: .. . «,Id�·: 
- 1 (O,fl //K-C4� 
.. 
Subtype 
ETP 
(only addftlve to 
T-1 orT-11) 
0 1scr1minators 
C01!1""1,C08',.. 
usoaly CD5"'9..--poo 
and :1:1 ... of HLAOR, 
C011b, 13,33,34,65,117 
Re1nar1<s 
lfC05-.,.: �ofHLADR
. 
C011b,13,33.34,65,117: 
sCOO- ... may occur' 
corin s stem based on 11 marker ex ression 
Score -2 -1 
1 
+1 +2
CD5 �75% 
1
CD8 2:5% 
CD13 �25o/JI 
--
CD33 2:25% 
----
CD34 
2:2
5% 
HLA-DR 
2:2
5
0;
.
CD2 �75%. <20% 
CD3 2:75% 
<20
°
/
. 
CD4 2:75% <20o/. 
CD10 2:75% <20o/ ;it 
CD56 2:20% 
► New markers: e.g. CLL-
haematologlca 2015: 100: 
· Cruima B11g11rff1,' JOUm,ln Samo, 
1 
011nm Pal,11l' 
A,�L,, M11t'Jf1 Sth'n10� • Curlt'fl)' tt' K.n:,,m,r., • 
MKl,arl D,,
.
.-:,1k,' ,L,!',fln S/1111111<!1,' � •.,m B,,J,J,,,, ' C 
RL f 
2 i ) OS<J1t Jl/aglia,' Simmu, Sni• • 11- • ' 
f G H@- , 
Je 11"·P'· • 
CRLf 2
1 
1 
---
o, p2RY8- • � .... .,, .... : 
Alt. HO -,,v,ur" GKtwm11i Ca=:a;,�' mul c;,,se,111( CatJ'tf., 
1 
°'' l,,,
,
/,11/f o(tl>< /-BI-ti l s11idr !U""I' 
2:75% 
27 
Dgn.: T-ALL 
EGIL: TI/II 
ETP: yes 
MPAL: yes 
BAL: yes 
Slast clone heterogeneity: yes 
(0) _,._ _,._ 
10 14420 02411 10 
Dgn,: T-ALL 
EGIL: TI/II 
ETP: yes 
MPAL: yes 
BAL: yes 
TICIIIC.'0'9 
lnukai-•core 
ETI'.-.C 
• 
Blast done heterogeneity: yes 
(O) Mlll!lbad� �----
tO ll420 01,1•10 
lnukai- •core 
•

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► New markers: e.g. CLL- (CD371), CRLF2, ...
► New subclassifications, new entities: e.g. ETP, switch pB- LL
► MPAL- (cross- ineage) blast subclones and aberrant maturation
MRD 
FLOW-standardization: challenges and pitfalls 
1) Pre-analytical sample quality
2) Staining panel, procedure optimization, definitions
3) Marker (in)stability and background
4) Human factor (experience - standardization)
�------- ----- ---
28 

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• acquisition: minimum 3x 10
5 
cells of lnterest 
• gatlng hierarchy/calculatlon basis: 
1. 
ntact nu deated cells 
2. IN/SSC 
3. mmature UN/SSC 
(l. D19, CD7) 
(e.g. CD10) 
4. mmature LIN/aberratlon marker (e.g. CD10 vs CD45) 
• cluster gating 
• positivity criteria: repro du clbl
e 
cluster of 2: 10 cells 
with related c
h a
r acteristics 
• criteria for quantifiable positivity: cluster of :!:30 cells 
• target sensitivity: 1 in 10
4 
(i. . o in 3x 10
5 
cells!) 
j� .;-. 'iii-· 
• •..,� ;> ...,,,., • ..__ -,. 1rff'T" ,; - -�--. ..,. • �-, •• --.-..;. .-.z..-
--
1 • ' • 
Tirne polnt-dependent concordance of flow cytometry and real-tlme quantltative 
polymerase chaln reactlon for mlnlmal resldual dlsease detectlon In chlldhood 
acute lymphoblastlc leukemla 
Gfuseppe G8lpa,
1 
Gl<Wannl Cazzant�.• Marta Graz.l& Vutsecchl.
1 
Renate Panzer-GrOm$yer.
11 
Barbara Buldlnl,' 
Oanlel3 Sllvestrf,
st 
Leonid Karaw-aJew! os.car Ma&11a,• Richard Rate1.• Alessandra Bernneuo,• Simona S."'lla.' 
An,ela Schumlch,> And,e SChrauder,•Tlzlana Vllla .
1 
M;uinena Veltronl.' Wolf-Dteter ludWI.C,� VaJenuno Conte,.• 
Martin. SChrappe,• Andrea Bk>ncll , * Mlcheel N. Dwottak! and Giuseppe Bas.so
„ 
hlvtt-ruolutfon PLOW-MllD 
• 7- or 8-colors 
• hlgher celi lnput/tube 
• f-•r tubes 
• lncrused sensltlvlty 
- more events acqulred 
- hogher LAIP complexlty 
• reduced costs 
u 
z 
,. �RO •stJ..,..tu fCM 
NC vs.. MNC Hmpfe 
fCM on NC 
n•277 
Dllfer.t -oclology: NC i. lnput 3 .io• <• colon) 
MNC i. lnput 0.75 - 1 dOO 17 colon) 
Enhanced sensitivity of flow cytometry for 
routine assessment of mlnimal residual disease 
ftlltn Do,wu,JC-• • CmtnUt 1k,r• · ,Vfi� �11tlr< .. 
lkrrmbt, Ctrb fmn::..' }OM A.fthlrfW P,m11M 
'""' )1111,.., ,11(.11#' 
haematologica 1 201 O, 95(4) 
Thc overall correlation ol MRD cstim.Jtes by PCR and by 
fCM with both ceU prepar.:u,ons is sl,own in Figua 2. Thc 
concordancc betwcen Ule two fCM -ys w�, 96
"- o 
(2..'i . 51266) In posltivc-negativc corrclattons, and 91 % 
(2421266; figun, 2) usi11g the cut-0ff of 0.0 1 '•· Of 24 divcr­
g,,nt samplcs at thc 0.01
°
. cut-0ff, II samplcs wen, nega­
tiv<: by FCM" but pos,nv,: by fCM,.., mostly at ve,y low 
levds of MRD Whcn ilm,nng rhe FCM
"'" 
asscssmcnt to 
only 3x 10' cclls, as for thc FCM
"' 
ilSS,lY, sevcn of these 11 
samJ'!es were MRI) 111.-gativc. Honco mO>t t
,
f thc ,n-.ruo.-..: 
in sc-n:,.,n,'1_9 ' W.JS ccl.JtrJ tQ thr numbcr of cclb oK. uu't'd 
Th<: MRD kvels measurcd by FCM- had a 1.85 timt> 
h
,g he, mcan Ihan those obtaincd by fCM• (SO 1.86; 
among 86 palred positive samples) 
haematolo\Jocu 1 2012; 97( 10) 
In a recem paper by Bene and Kaeda, 
1 
rechnicaJ 
approachcs for minimal residual disease (MRD) asscss­
ment are exrensively reviewed. PCR-based studies have 
proved to be 1-log more sensitive titan 6ow cycometry 
(FC). For this reason, thcy are increasingly being prcfcm:d 
for MRD aMlysis, espccially at the end of therapy or post 
hcmatopolcric sr.em celi rransplantation.U h would bc 
valuablc to develop MRD 6ow cytome
try 
ass.tys with this 
level oE sensitivity that could be applied routinely. In the 
prescnr work, wc analyzed MRI)' samples with a level of 
in8Jrration below the limit of detection of routinc FC, 
wlttch i:. accepted as I O" .1nd comes Erom the standard 
a cqui� 1uon of 2-Sx I O leuk o cyt
es LII 
At lea�t 10- fo ld more 
lcuk�tcs must bc �quired to u1creai.e scnsiuvity by 1-
log;_ this largc num er o leuk
ocy tcs can be acquired easi­
ly in digital cytometers by acquiring severa! individual 
tubcs staincd with the same combination of monodonal 
antibodies, and putting them in a single filc. Bccause the 
time of acquisirion for each individuaJ tube is not 
lncrcased, no problems of ccllular amegation arise. 
In summary, acquiring 6 million leukocytes is feasible 
with a digital cytometer on a routine basis. Because detec­
tion of 50-60 malignant cells is required to get a CV less 
than 15%, a sensitivity of 1 xlO
.s 
is achieved. Being able to 
routinely apply MRD FC assays with h.igh sensitivity 
would be very valuable, especially in cases where molec­
ular techniques cannot be used. 
29 

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u;�-- .,.:<::..... -,- ---.-�
,.. ... 
FSC C::D19 CD34 
homogeneous apl)earance: not wlth all markersl 
• erythrocyte lysls
• staining panel: minimum 2 tubes per sample 
constant bad<bone of 2-3 markers 
tor at least 4-co l or flow cytomet . ry 
minimum dlfferent 6-8 markers In set-up 
• B-UN obUqatory markers: co10, 19, 20, 34, 38, 45, ss
optional markers: CD9, Ua , 66c, 123 , .. . 
• T-UN obllqatory markers: cyCD3, sCD3 , CDS, 7, ¼5, 99, TdT 
optlonal markers: CD1a, 4, 8, 10, 34. 56/16. 117 . . . 
• panel rules: co10 with stronq labef: PE, PE-Cy7 preferred 
cyCD3 and sCD3: same moAb done 
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from normal B precursors with (our color llow cy1omelry: implications for residual 
diseaR detedion 
30 
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pressed in up to 81.4% of ALL cases; 
expression of some markers was associ­
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malities. 
Minimal residual disease analysis by eight-<:olor flow cytometry 
in relapsed childhood acute lymphoblastic leukemia 
Leon.ki MMDWljew �tlHl Owolz-.l RkMN:S Ratet.• Pet« Rhein,• Giuseppe Gal.pa.• Barbara ButdlrM.• CiUMppe 
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accurate. 
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robust results 
Acquire& 
anatf:e by flow 
cytomelry 
Minimal residual disease-directed therapy for childhood acute 
myeloid leukaemia: results of the AML02 multicentre trial 
Jelfr,y E Rubnb. Hro<o lnabo. Ga,yDoh( Roul C Ribdro. Wl'w! -..,.fl/<lftey Tou b. Starkyf> ovnds . Boss,m/ Razzout. NamanJ lacayo. 
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32 
.BECKHAN 
COUIIER 

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1) Equivalent panels are needed:
► 10 colour tube design
► Extenslve 8 colour back-bone (different-, rom-.. ormal detection)
► 2 variable slots (patienhpecific LAIPs)
► lnnovative markers in new combinations
► Stable cocktail formulations tor the consortium (e.g. Duraclone BC; possibly also from BO)
► Single batch antibodies to variable markers for the consortium (e.g. Exbio)
► Towards automated software support
2) QC program including NEOAS AML-MRD trials
Blast CeU Deftclency of COJ ta as A Mar ker of 
Acute Megakaryoblastic Leukemia and Transicnt 
Myeloprollferative Dlsease in Childrcn with and 
Without Oown Syndrome 
lk��=�'r ���•:�= -�::.�;!r: ���� •' 
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gene tM:onio UOtJI J7 i 1606-- 1610; dot:l0.I0l&lltu.2011.J?I 1�":' ��.'!�C.::".!=::.f 
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backbone - a universal marker combination for the detection 
of minimal residual disease in acute myeloid leukaemia 
British Joumal of Haematology, 2014, 164, 212-222 
Spik ing expcrimcnts rcvealed that thc 
assay could deteci MRD down to io -• in normal bone marrow with sensitivi­
ties equalling those of valid ated qPCR assays. Moreover, it provided at least 
one MFC MRD markcr in 62/69 patients (90%). High levels of hMICU 
CD 123 LAIPs al the post-induction time-poin t were a strong prognostic mar­
ker for rclapse in patients in haematological complcte rc:mission (P < 0·001 ). 
Finally, in post induction sampl es, hM ICUCD 123 LALPs wcre strongly corrc­
latcd (r = 0-676, P = 0-0008) to applied qPCR largets. We condudc the 
hM J CUCDJ23-based MFC assay is a promising MRD tool in AML 
iBFM/pan- U pedAML- RD 
10-color panel
1 
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hlftnatlOnl 
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Morocco 
34 
hMICL = CLL-1 
Anne S. Roug,' Hanne 0. Larsen. 
1 
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Tom Just,2 Gordon Brown,3 
Charlotte. G. Nyvold,
1 
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1 

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G. Basso, B. Buldini (Padova)
G. Gaipa, O. Maglia, S. Sala (Monza)
O. Hrusak, E. Mejstrikova (Prague)
L. Karawajew, R. Ratei, S. Groeneveld-Krentz, J. Hoffmann (Berlin)
35 
.BECKHAN 
COULIER 

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ALL IC 
strategy for patients with relapsed 
ALL 
Janez Jazbec 
UKC Ljubljana 
Children's Hospital 
• Currently achieved rates of event-free survival
(EFS) in 1st complete remission (CR) are
70-80% with multi-drug chemotherapy
VOLUME 32 NUMBER 3 • JANUARY 20 2014 
J0URNAL 0F CLINICAL ONCOLOGY 
ORIGINAL REPORT 
Intensive Chemotherapy for Childhood Acute 
Lymphoblastic Leukemia: Results ofthe Randomized 
Intercontinental Tria! ALL IC-BFM 2002 
Jan Storr, Mnrffn Zimmtnnafln, Myriam CAmpbdl. Luis Ourillo, Eduardo Dibor, Swtlano Donsla, 
IJ,ja,,d,o Gonmln, Shm !z,a,fi, Drag,,mr }anic, Jana Jiub«, }a,ip konjA. Emili,, ,:,.;,,,.,,,, /m.y K""""'2yl, 
c.bc, ,:.....,, Clt� lr""f U Edina MIAfY"""Y, ""'"'""" Popn, &tia Start Yahio Jaboli. /M T,b,, 
Ond,,j H-1. Hon,jo,i Ri<hm, Giu,,pp< MM<m. an,I Mnrti,, &J,,.pp, 
c ....... _,,,.. .......................... , ...... , ................ 1CC\ 
36 

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• ALL IC-BFM 2002 study enrolled 5,060 children
with ALL in 15 countries on 3 continents.
• is a good example of international collaboration
in pediatric oncology.
• A wide platform of countries able to run
randomized studies in ALL has been established.
• Alternative DI did not improve outcome
compared with standard treatment and the
overall results are worse than those achieved by
longer established leukemia groups, the national
results have generally improved
What about patients who relapsed on 
ALL-IC 2002 
• The question not a part of study
• No sistematic data collection
• In Prague 2013 BFM meeting - request for
common strategy for All-lC "countries"
Relapses in ALL IC 2002 study 
• 5060 patients in ALL IC 2002 study
• By november 2010- 830 relapses registered
• Great variability in data
37 

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• Mean tirne to relapse: 2,19 years (0,11 to
6,59)
• By the end of 2013: 289 (35%) alive
542 {65%) dead 
All.RE22002 
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00 
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Overall survival 
l..00 
Survival Function 
4 00 6.00 
relsurv 
8.00 10 . 00 
....r, SuN1Yoll Funolon 
+Ctnwrtd 
38 

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OS by BFM Rez protocol 
a) ALL-REZ BFM 83-90 vs 95196 and 2002 
1,0 
,6 
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v ... 
8 10 
AU,-REZ
BFM 
- 2002; n= 591;cens=940;pEFS=.50".0'l 
- 95196; n = 595; cens = 219; pEFS = .98 • .O'l 
- 90-90; n = 1042; cens = 298; pEFS = .29" .01 
P<.001 
Survival by the tirne to relapse 
o .• 
.oo 2.QO 
Survlval Functions 
4.00 6.00 
relsurv 
1.00 10.00 
OS by risk group 
b) ALL-REZ BFM 2002, S1-4 
1.0 
,6 
,4 
.2 
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O 2 4 6 
- Sl: n 34; cens 25; pEFS = .68 • .09 
S2: n 359; cens 248: pEFS = .60 • .03 
- $3: n 70; cens 28; pEFS = .31 • .07 
- 54: n 128; cens 39; pEFS = .28 • .04 
P .001 
8 
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39 

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Survival by country 
Survival Functions 
councry 
··-
00 1 00 .fOQ 1.00 ,oo 10 00 
retsurv 
ALL-IC options 
• Run your own trial {Argentina, Chile)
• Join existing (lntReALL??)
• New single arm registry trial
ALL-IC relapsed ALL protocol 
• Prospective non-randomised observation
study
• Goal is to set up a large international study
group platform allowing far optimization of
standard treatment strategies
• The main goal of this study is to improve the
outcome of children and adolescents with first
relapsed acute lymphoblastic leukemia.
40 

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The aim of the proposal 
• to develop a single arm treatment guidelines
for the treatment of children with relapsed
ALL
• employ the combination of drugs that are
already available
• NO randomization in the first stage
• to homogenize the diagnostic criteria and the
treatment
ALL REZ PINDA 2013 
Flow MRD 
• Flow-cytometric determination of minimal
residual disease was already an integral part
of ALL-IC 2002 and 2009 study
• it is proposed to use Flow-cytometry for
estimation of minimal residual disease at the
end of the fourth week of induction (after F2
block) with threshold point of 10-
3 
to identify
subgroup of patients with sub-optimal
response to induction treatment.
41 

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Problem 
• Aplastic 015 post F2 marrow
• The question of the timing of post-F2 bone
marrow aspiration has not been finalized yet.
Some argued for dlS sampling for easier
laboratory analysis and evaluation.
• Vaskar Saha and Arend von Stackelberg
advised for sampling at the tirne of bone
marrow recovery based on the similar timing
found prognostic with PCR M RD measurement
Cornelia Eckert: 
•
Within the ALL-REZ BFM 2002 trial we had it
sometimes, that two weeks after finishing F1/F2
induction treatment the BM was stil! aplastic,
therefore a second BM aspiration after F1/F2 was
performed before start of the new treatment.
•
Regarding indication for HSCT, we always used
the second BM aspiration. lnterestingly, in most
cases the PCR MRD results was concordant.
Other issues omments 
• 1) Nelarabine .It could be optional and in that
case we agree to uniffy how to use it.
• 2) TYA (teenage and young adult group). Can
we change the inclusion age criteria from
under 18 to under 24 or > under 30 years?
• 3) Do we involve relapsed lymphoblastic
lymphoma patients?
• 4} Tyrosine kinase inhibitors (TKls).
42 

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Angela Schumich 
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Ljubljana December 2015 
......... difficulties and pitfalls 
• Most frequent questions
• Most frequent failures
• BCP-ALL
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JJubliana/Oec:ember 2015 
Questlon 1 
Answer 1 : more lntact cells have 
hlgher DNA c:ontent and therefore 
staln hlgher posltlve wlth Syto and 
show len apoptotlc: features In FSC/SSC 
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Angela Schumlch 
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Questlon 2 
Answer 2 : Granulocytea may ataln unapeclflc poaltlve wlth any AS 
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JJublJana/Oecember 2015 Angela Schurnlch 
Fallure 1 and 2 
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BCP· nonnal BM 
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Angela Schurnlch 
Note Syto 1s not an ABI 
Control Syto compensatlon 
to next nelghbor In every 
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Angela Schumlch 
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Jjub1janal0ec:em�r 2015 Angela Schumlch 
We can detect ALL in pB ifwe stain aufficlent number of cells 
JJubllani/Oecember 201� Angeli Scliumleh 
47 
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r;J 
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Of note: no T-hematogones In BMI 
Majorlty ofT-ALL 1s CD5 posltlvel 
Jjubljana/Oa<:amber 2015 Angela Schumlctl 
In healthy BM CD99 correlates wlth CD:W 
CD99 ls posltlve on Band Myelold precursers 
JjublJana/Oecember 201, 
__ 
Angela Schumlch 
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49 
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Ljubljana, 2015 
Set-up and compensation of 8-color flow cytometer; 
EuroFlow view 
Tomaš Kalina 
on behalf of Euroflow 
Charles University, 2
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What is the key feature of FCS data? 
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► Sample preparation
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Flow cytometry data interpretation 
FCS data: 
• Pattern analysis
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• Comparison to reference
Can you interpret somebody else's FCS data? 
What is limiting inter-laboratory collaboration? 
Variability (pre-analytical and analytical) 
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55 

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(D Standardization - How to?
(1) Compensation
@ Remarks
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8-color flow cytometry
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EuroFlow 
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APCAx750 
59 
BV421 
BV510 

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Presentation content 
(D Standardization - How to?
@ Compensation
@ Remarks
Fluorochromes 
J 
Single fluorochrome dyes Tandem dyes 
Generic reagent independent 
FL compensation 
Reagent conjugate & lot 
speciflc needs 
Compensation 
3 ,_,_ fTaa3 mc.,uwa> 51!rm sil 
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•--l (PerCP .:) r„ P (mm .:J _...,.,,_ 
EuroFlow 
1111 ftl 
PEtrue = PEmeasured - (0.15) x FITCmeasured www.bdbiosciences.com/spectra / 
60 

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Compensation - what you need 
► CAVEAT: Stili the most frequent error in execution of
standardized protocol
► Understand the compensation theory and the DiVa
software
► Diva Manual
► EuroFlow SOP
► Single stained tubes
► Common sense and practice
Three Rules for Compensation Controls 
First and foremost, there must be a single stained control for every 
parameter in the experiment! 
In addition, there are three rules for "good" compensation controls: 
1) Controls need to be at least as bright or brighter than any sample the
compensation will be applied to
2) Background fluorescence should be the same for the positive and
negative control
Lympocytes T' beads � monocytes 
3) Compensation controls MUST match the exact experimental
fluorochrome
Pacific Orange � HV-500 
http://flawio.typepad.com/the_da11y_dong1e/2011/09/lhttt-tules•for-c0f'l"lperuatton-controls.htm1 
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#; Negative CompBaad used as negative reference population 
": Artificlally co14· ff10110C:Y1as c,eale<I by ·appendlng' 5000 evenls from lhe unstained tube to thll tube 
ecqullltlon _______________________ _ 
EuroFlow SOP 
61 

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Compensation - Automatic Method 
Automatic compensation in the Diva software offers a fast, easy and reliable method 
to set the correct compensation. 
• First, elect .Create Compensation Tubes· from the Instrument Menu:
1n s1r11 ment conngut111on. 
tns1r11ment Name 
Enab1e Test Features 
Gre• Compensation Tubes 
i9 fi 
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SelUp Catalog. 
Compensation - Automatic Method 
The software automatically creates a 
list of single color tubes, based on your 
instrument setting. 
Reagent specific controls for tandem 
dyes 
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over 20 
panels 
(all panels use names matching with catalog} 
62 

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(D Standardization - How to?
0 Compensation
® Remarks
Problems 
•
Reagent ageing
- > Use as few tandems as possible, use them up quick
- > Use stable lyo or dried reagents
•
Errors in compensation experiment
-Mostly minor overcompensation in non-overlapping
channels (PacB vs APC-H7), often single stained tube
handling issues, gating problems.
-> Manual check (not in SOP, hard to objectively setup)
-> Automated compensation check (generic min-max values,
constrains by past experiments)
Future directions 
•
lf signal is kept standardized (Rainbow or CS& T)
- Re-compensation is not necessary due to setup
But: 
Reagents must be identical 
Batch to batch variation (improving, but in the hands of 
companies) 
Stability over tirne (improving, lyo or dried reagents) 
Reagent control instead of re-compensation? 
-> reagent QC tool l nfinicyt 
63 

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More colors, more instruments 
• 16 color setup / Target values EuroFlow members
only
Difficulties: 
AII emission filters must be identical 
(or spectrally matched beads should be used) 
• 8 color setup for Navios / Rainbow Targets given
Difficulties: 
Using Navios filters Targets are valid for PacB 
and OC515 for whoch it was developed 
(or spectrally matched beads should be used) 
Anticipated questions 
Do you really see no need to adjust compensation a little bit for each particular sample? 
How good is good enough and how much we pay for perfection? 
lf the small imperfection is not changing my interpretation 
(e.g. negative becomes dim) -> 1 don't care 
lf it does -> 1 should change my panel becuase it is not robust 
Why don't you use CS&T instead of Rainbow? 
We work with Rainbow beads for ages, CS&T only simplifies it, 
the improvement of the data 1s not visible (CS& T are equally good though 1) 
EuroFlow 2006 - 2015 .. tlV\,d gof.V\,g OV\-

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Set-up and Compensation of 10-color Flow Cytometer 
BD view 
Jirl Šinkora, BO Biosciences 
Ljubljana Flow Cytometric Meeting 
30
th 
November and 1
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December 2015 
lnst.itute of Oncology, Ljubljana 
Goal of Standardization: 
To get consistent, reproducible results 
on samples 
stained with Standard reagents 
using Standard protocols 
Lyse / Wasb (LW) 
Lyse No Wasb (LNW) 
EuroFlow SOP 
on Standardized flow cytometers 
"ldentical" results 
across multiple users and days 
on every single cytometer 
Day 1 
User 1, 2, 3 .... 
Day 2 
User 1 
Another day
(
s
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User-independent 
data consistency 
In tirne 
Standardized flow cytometer 
65 

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across cytometers and sites 
Site 1 
Cytometer 1 
lnter-instrument 
data consistency 
Site 1 
Cytometer 2 
Site 2 
Any cytometer 
Standardized flow cytometers 
Do you need it? 
How to do it? 
"ldentical" (as similar as possible) results 
across cytometers and sites 
on different days 
for all users
-�---
Standardized flow cytometers 
How to do it? 
By bringing the selected celi population 
(e.g. lymphocytes in PBL preparations) 
to the same FSC and SSC intensity channels. 
By bringing brightly fluorescent objects 
(e.g. polychromatic beads or cel/s stained with anti-CD8 conjugates) 
to the same intensity channels in all fluorescence detectors 
Standardized flow cytometers 
66 

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By bringing the selected cell population 
(e.g. lymphocytes in PBL preparations) 
to the same FSC and SSC intensity channels. 
FSC„w-55 000 
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Example: EuroFlow 
By bringing brightly fluorescent objects 
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Example: EuroFlow 
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86,615 
183,654 
23,343 
150,263 
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231,265 
59,574 
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216,064 
27,462 
176,780 
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67 
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By bringing the selected celi population 
(e.g. lymphocytes in PBL preparations) 
to the same FSC and SSC intensity channels. 
By bringing brightly fluorescent objects 
(e.g. polychromatic beads or ce
l
/s stained with CD8-conjugates) 
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Manually Automatically 
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Benefits of Standardization 
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Laser power was lntentionally decreased 
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Decreased sensitivity 
on detuned cytometers 
affects low-end resolution 
However, 
intensity patterns remain similar 
II 
Benefits of Standardization 
Instruments equipped with lasers with different 
Power 
Wavelength 
provide results that are "as similar as possible" 
68 

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.. ... .. ....... .................... ... ., ................ .
1 
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Q,A 1ay 
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Standard patterns = identical compensation values 
! ! ! until optical characteristics of the cytometer cbange ! U 
AII Flow Cytometers 
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How many peaks are needed? 
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Median (Brlght) 1 Median (Medlum) • con1t 
How many peaks are needed? 
3 intensities: Dim, Medium, High + 2 sizes 
69 
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UGHT OETECTION EFFICIENCY (Qr) FOR EVERY SINGLE OETECTOR 
OPTICAL BACKGROUNO (Br) FOR ALL DETECTORS 
COEFFICIENT OF VARIATION (rCV) FOR ALL BEAOS ANO ALL DETECTORS 
TARGET VALU ES FOR BRIGHT CS& T BEADS FOR ALL OETECTORS 
SIGNAL LINEARITY FOR ALL OETECTORS ANO THEIR ELECTRONICS 
ELECTRONIC NOISE FOR ALL OETECTORS ANO THEIR ELECTRONICS 
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PQC 
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Performance Quality Control (PQC) 
BD FACSuite and CS& T 
,� .. 1. 
eeu MFI BeadMFI 
CQC 
Setup FL pattern for cells At the same settings 
Run CS& T beads = Get TTV 
lntegrated Reproducibility (Tube Target Values, TTV) 
BD FACSuite and CS& T 
Doy 1 
: 660V 
0.l'/2 VOll.19<1S 
: 670V 
650V - 570V 
Oay 1 A Oay2 
lntegrated Reproducibility (Tube Target Values, TTV) 
76 

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Progress in Standardization: 
FACSDiva: 
User settings (Applications Settings) are calculated from 
results of measurements of CS& T beads in the CS& T 
module under standard (Baseline defined conditions). 
Compensation matrix is duplicated (unchanged). 
PMTV increment = min. 1 V 
FACSuite 
Tube settings (TTV ) are measured using CS&T beads ofr 
every signle settings. Compensation matrix is 
recalculated for ali smallest imprecisssions. 
PMTV inc
r
ement = min. 0.1 V 
PMTV 
BO Lyse Wash (LW) and Lyse No Wash (LNW) avallable In 
6 colors in FACSDiva 
8 colors in FACSuite 
FITC, PE, PerCP(-Cy5.5), PE-Cy7, APC, APC-Cy7 (or H7), V450, V500c 
using Calibration beads (7�olor Setup Beads or CS&T beads) 
l7 
For special applications, expert (group) Target Values (TV) using recommended 
setup materlals are followed: EuroFlow setup (8 colors) 
For ad hoc settlngs (e.g. 8 color EuroFlow and9
th 
and 10
th 
color on FACSCanto) 
the 2.5 x SDEN rule is recommended 
while paying attention to balanclng detector PMTVs 
(CS&T module provides 10x SDEN values) 
lf possible, one set of PMTV per "Experlment" In FACSDiva 
Threshold 
On analyzers, the Threshold value should be set to include all relevant events 
In analysls and to see the "closest" part of debrls/irrelevant events as well. 
lf fully quantltative (peak Area) measurement ls done, signal intensity depends 
on appropriate Threshold and Window Gate Extension (WE) selection. 
The most recent recommendatlons are: 
FSC Threshold - 5% of the scale (13 000) and WE • 3 
77 

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FACSDiva: 
Automatic Compensation at the beglnning 
(Link and Save, Compensatlon Setup Catalogue} 
Repeated monthly 
Manual Compensation 
(visual or by comparing median FL of posltlve and negative events} 
between the automated procedure 
narnely for tandem conjugates (labels): perlodlcally or wlth a new lot 
FACSuite: 
Adding fluorochromes and updatlng compensation for label-speciflc reagents 
(tandem conjugates) when needed. 
On standardized instruments : 
Compensation matrix does not significantly change. Withln the tlme, only 
Spectral characteristics of a cytometer (optical fllters} play a significant role 
in compensation matrix changes, such processes normally take years before 
any significant dlfference is observed. 
Practical Approach 
FACSDiva: 
The most conveneient procedure 
of setting up 1 O color FACSCanto 
and maintaining it standardized 
(keeping Target Values and Maintalning Compensatlon} 
will be demonstrated during the practical part on day 2 
8D OneFlow 
Instrument Setup (EuroFlow TV) 
1 peak 
Very fastTV 
setup and maintenance 
Target values for 
montbly (+/-2%) 
daily (+/- 15%) 
setup 
CE-IVD template 
in FACSDiva 8.0.1 
78 
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BO OneFlow 
Multicolor cocktails 
8D OneFlow 
Multicolor cocktails 
Eilll • Clll CID C ( 
All components included 
No pipetting 
Controls for S months (or more) 
Highly reproducible 
No label-specific controls 
for OneFlow diagnostics 
Generic single-stained control 
candidate when OneFlow 
is used in combination 
with other cocktails 
43 
8�0/or (12 antibodies) lST Tube: 
FITC CD8, Lambda 
Pf. CDS6, Kappa 
PcrCP- yS.5 CDS 
Pl1:-Cy7 CD19, TCRgd 
APC 
CD3 
APC-H7 CD38 
V4S0 CD4. CD20 
VS00c CD45 
AH (dry) mAbs included 
= no pipetting error 
Storage at RT 
No expiration mismatch 
Highly reproducible 
.. 
No label-specific controls needed 
FulJ CE-IVD compliance 
in FACSDiva 8.0.1 
More is coming ..... 
79