cancer research center, university & university hospital of salamanca (spain)
DESCRIPTION
MONOCLONAL B-CELL LYMPHOCYTOSIS. CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) Multicolor Immunophenotyping: Standardization and Applications March 9-11, 2012 TMH, Mumbay (India). - PowerPoint PPT PresentationTRANSCRIPT
CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN)
Multicolor Immunophenotyping: Standardization and ApplicationsMarch 9-11, 2012 TMH, Mumbay (India)
MONOCLONAL B-CELL LYMPHOCYTOSIS
- Monoclonal B-cell Lymphocytosis (MBL) indicates the presence of <5x109 clonal B-cell/L in PB of otherwise healthy subjects, with or without lymphocytosis
MONOCLONAL B CELL LYMPHOCYTOSIS (MBL)
(Marti et al, Br J Haematol 2005)
- CLL-like MBL: <5x109 clonal CD5+ B-cells/L in PB of otherwise healthy subjects, with or without lymphocytosis
- Typical: CD5+, CD20lo, CD79lo and sIglo
- Atypical: CD5+ and CD20hi and/or CD79bhi and/or sIghi
- Non-CLL-like MBL: <5x109 clonal CD5- B-cells/L in PB of otherwise healthy subjects, with or without lymphocytosis.
CLASSIFICATION OF MONOCLONAL B CELL LYMPHOCYTOSIS (MBL)
(Marti et al, Br J Haematol 2005)
B CELL CHRONIC LYMPHOPROLIFERATIVE DISORDERSHeterogeneous group of diseases typically characterized by
a monoclonal expansion of a mature-appearing neoplastic B-lymphocyte
Mature/peripheral B cell chronic lymphoid leukemias: Chronic lymphocytic leukemia/Small B cell lymphocytic lymphoma Prolymphocytic leukemia Hairy cell leukemiaMature/pheripheral B-cell lymphomas: Lymphoplasmacytic lymphoma Splenic marginal zone lymphoma Extranodal marginal zone lymphoma (MALT-type) Nodal marginal zone lymphoma Follicular lymphoma Mantle cell lymphoma Diffuse large B-cell lymphoma Burkitt lymphomaPlasma cell neoplasias: Multiple myeloma/plasmacytoma
WHO CLASSIFICATION OF B-CLPD
IMMUNOPHENOTYPIC PATTERNS OF DIFFERENT
TYPES OF B-CLPD (Orfao et al, In: “B-CLL”.Humana Press, 2004) sIg CD5 CD10 CD20 CD11c CD23 CD24 CD25 CD38 CD43 CD79b CD103 FMC7
B-CLL d + - d -/+ ++ + + -/+ + d - -
PLL + -/+ - + -/+ -/+ + -/+ -/+ -/+ + - +
HCL + - - ++ ++ - -/+ ++ - - + + +
SMZL + -/+ - + + - + -/+ - - + -/+ +
LPL + - - + - - + + -/+ - + - -/+
MCL + + - + -/+ - + -/+ - + + - -/+
FL + - + + -/+ -/d + -/+ + - + - +
LDBCL + - - + -/+ - -/+ - + - + - +
BL -/+ - + + - - + - ++ -/+ -/+ - +
WHO: B-cell malignancies
Immunophenotype
CLLHCL
WHO: B-cell malignancies
Cytogenetics
MCLBL
Immunophenotype
CLLHCL
WHO: B-cell malignancies
Histology &cytology
DLBCLB-PLL
Cytogenetics
MCLBL
Immunophenotype
CLLHCL
WHO: B-cell malignancies
Histology &cytology
DLBCLB-PLL
Cytogenetics
MCLBL
Immunophenotype
CLLHCL
Clinic
MALT
- Chronic lymphocytic leukemia (CLL) is the most frequent subtype of leukemia in the Western World
- Diagnosis of CLL requires the presence of >5x109 clonal B-cells/L in PB with a CLL-like immunophenotype even if there are no symptoms.
MONOCLONAL B CELL LYMPHOCYTOSIS (MBL)
vs CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)
(Morton et al, Blood 2006; http://www.hmrn.org/)
- Between 5%-7% and 12% of healthy adults, display small clones of CLL-like and 2%-2.5% of healthy adults have small clones of non-CLL-like B-cells in peripheral blood.
- Recent studies show a transformation rate of MBL into CLL (frequently assymptomatic) of around 1% per year, most CLL patients being preceeded by MBL.
Prediction of transformation of MBL into symptomatic remains a challenge
MONOCLONAL B LYMPHOCYTOSIS
(Rawstron et al, Blood 2002; Rawstron et al, NEJM 2008; Dagklis et al, Blood 2009, Nieto et al, Blood 2009, Almeida et al, Leukemia 2011)
(Rawstron et al, NEJM 2008; Landgren et al, NEJM 2009)
CLINICAL PROGRESSION OF A CLL-LIKE MBL CASE
Rawstron et al, Cytometry B, 2010
MBL cases presenting with or without lymphocytosis, is highly prevalent
among the general population
Increasing evidence suggests that this could represent a pre-leukemic condition, since CLL frequently
develops in individuals with prior history of MBL
MONOCLONAL B CELL LYMPHOCYTOSIS AND CHRONIC LYMPHOCYTIC LEUKEMIA
AIM:
1.- To determine the prevalence of CLL-like (and other B-CLPD) clonal PB B-cells in a groupd of healthy adults
from the Salamanca healthcare area
2.- To establish the immunophenotypic and genetic characteristics of each clonal B-cell population identified
(vs neoplastic B-cells from CLL and other B-CLPD.
3.- To investigate the potential clinical significance of the B-cell clones identified and its behaviour.
Randomly recruited
(Health Care Area of
Salamanca:
- Age:62±13years (range: 40-
97)
- Sex: (m/f): 46% / 54%
- N. of PB leucocytes:
6,3±1,6 x 109/L- N. of PB lymphocytes:
2,1±0,7 x 109/L- N. of PB B lymphocytes:
0,16±0,1 x109/L
- CLL:65 - Transf. CLL/PLL:3- MALT NHL:2 - FL:1 - MZL:1- Unclassifiable: 3
- Age: 67±13 years
(range: 35-91)
- Sex:(m/f): 57% / 43%
639 healthy adults > 40 years:
75 non-treated B-CLPD patients:
Healthy subjects and patients studiedMaterial & Methods
Methods: Immunophenotypic screening for aberrant PB B-cells
-8-color direct immunofluorescence technique:
PB PO FITC PE PerCP/Cy5.5 PECY7 APC AF700
CD20 CD45 CD8Anti-sIgl
CD56Anti-sIgk CD4 CD19 CD3 CD38
CD20 CD45 cyBcl2 CD23 CD19 CD10 CD5 CD38
- - Anti-sIgl Anti-sIgk CD19 CD10 CD5 -
Methods: Immunophenotypic screening for aberrant PB B-cell
populations
-Data acquisition: FACSCanto II flow cytometer: Window of analysis to simultaneously select for CD19+ and/or CD20+ events (B-cells), for > 5x106 leukocytes
CD
20 P
acif
ic b
lue
CD19 PerCP-Cy5.5 CD19 PerCP-Cy5.5
CD
20 P
acif
ic b
lue
FSC-AS
SC
-A
Frequency of PB B-cell clones in healthy adults
Found in 94/637 cases (14.8%)
No differences in distribution per sex group:
13.4% m /15.9% f ; p>0.05
637No. of individuals studied 150% of cases showing PB B-cell clones 10%
300
11.7%
400
14.2%
500
14.4% 14.8%
Negative PB Positive PB
Age (Mean ± 1SD) 61 ± 13 69 ± 11
Differences with age:
p<0.0001
Frequency along the recruitment for the study (18 months)
*0.35% of all B-cells & O.O3% of all leucocytes
Clonal CLL-like B-cells
Normal B lymphocyt
es
Immunophenotypic identification of PB B-cells with a CLL-like
phenotype
CD20 Pacific blue
CD
5 A
PC
CD20 Pacific blue
CD
5 A
PC
CD
23
PE
cyBCL2 FITC
CD
23
PE
cyBCL2 FITC
Lambda FITC
Ka
pp
a P
E
Lambda FITC
Ka
pp
a P
E
SS
C-A
CD19 PerCP-Cy5.5
SS
C-A
CD19 PerCP-Cy5.5
CD19 PerCP-Cy5.5
SS
C-A
CD19 PerCP-Cy5.5
CD
20
Pa
cif
ic b
lue
cyBCL2 FITC
CD
23
PE
Ka
pp
a P
E
Lambda FITC
Immunophenotypic identification of clonal B-cells in PB from healthy
subjects0.01% of leucocytes0.7% of B-cells CLL-like
phenotype
CLL-likephenotype
Non-CLL B-cellphenotype
0.0015% of leucocytes0.1% of B-cells
Nieto et al, Blood 2009
Frequency of clonal B cell populations in PB of healthy adults
En 95/639 casos (14,8%)
Per
cen
tag
e o
f M
BL
cas
es
40 45 50 55 60 65 70 75 80 85 90 95
Age (years)
0
10
20
30
40
50
60
70
80
90
100
5.7%
5.9%
18.5%
21.3%
26%
75%
Mean age: 70±11 years for MBL (vs. 61±13 years for non-MBL; p<0.05)
PB clonal B-cells in healthy adults: size of the B-cell
population% of PB leukocytes % of PB B-
lymphocytes
Median[range]
0.01%[0.0004%-14.8%]
0.53%[0.02%-96%]
£ 0.01% Clonal B-cells
>0.01-0.1% Clonal B-cells
>0.1-1% Clonal B-cells
>1% Clonal B-cells
54%
% of PB leucocytes
29%
8.5% 8.5%
P>0.05
Age group (years)
% in
filt
ració
n r
esp
ecto
al to
tal d
e leu
cocit
os
0,0
0,1
0,2
0,3
0,4
0,5
P>0.05
40-49 50-59 60-69 70-79 80-89 >90 %
of
PB
leu
kocyte
s
Salamanca(330.000 inhabitants)
Castilla y León(2.625.000 inhabitants)
The Salamanca Area within Spain
Frequency of biclonal cases among healthy individuals
Immunophenotypic features of PB clonal B-cells from healthy subjects
BICLONAL21% (n=20)
MONOCLONAL79% (n=74)
- Direct stain-lyse-wash immunofluoresce technique: Panel of 8-color MAb combinations for: CD11c,CD22,CD24,CD25,CD27,CD34,CD43,CD49d,CD103,CD79b, FMC7,IgM,CCR6,cyZAP70.
CLL: Trisomy 12, del(13q14), del(11q22-23) (ATM & MLL genes), del(17p13) & del(6q21); FL: t(14;18) (LSI IgH/bcl2 dual color); MCL: t(11;14) (LSI IgH/CCND1 dual color); DLBCL & BL: probes for 3q27 (BCL6 gene), t(8;14) (q24;q32), t(2;8) & t(8;22)
Purification of aberrant B-cells: FACSAria (purity: ≥98%)
Genetic analysis of purified B-cells by iFISH:
Immunophenotypic characterization of aberrant B-cells
Statistical methods: SPSS 15.0
Methods: Characterization of (purified) circulating PB B-cell clones poblaciones
Sequencing of IGH e IGK o IGL genes: Repertoire of the V, D, J regions of the IGH gene & VJ of the IGK & IGL genes IGH gene mutational status.
B-CLL
non-class NHL.
MCL
MALT
MZSL
Atypical B-CLL
MZSL or HCLv
MALT or MZSL
B-CLL+B-CLL
B-CLL + non-class. NHL
MZSL + non-class NHL
Non class. NHL + Non-class. NHL
MZSL or MALT + CLL
PB clonal B-cells from helthy subjects
MONOCLONAL
BICLONAL
Immunophenotypic features of the B-cell clones
Median CLL
count
13q14 deletion Trisomy 12 11q
deletion17p
deletion
CLL (17) >5,000/uL 54% (178/238)
16% (53/325)
18% (58/235) 7% (23/325)
Clinic(Mayo) (12,13)
2757 44% (56/126)
18% (21/126) 2% (2/126) 3% (4/126)
Clinic (Leeds) (8) 3141 58% (19/33) 21% (7/33) 6% (2/33) 3% (1/33)
Population (Leeds) (8) 9 39% (15/38) 18% (4/22) 0% (0/21) 0% (0/10)
Population (Salamanca)
(6)*0.5 36% (16/45) 8% (4/45) 0% (0/45) 0% (0/45)
Rawstron et al, Cytometry B, 2010
CLL vs CLL-LIKE MBL: Genetic features of clonal B-lymphocytes
SourceMedian CLL cell
count
CLL-like B-cells (median
%)
Cases with <98% IGHV
homology
Predominant CLL cell IGHV
geneSimilar to CLL?
CLL (25) >5,000 >95%534/927 (57.6%)
3-07, 1-69, 4-34, 3-23
-
Clinic (Leeds) (8)
3141 >95% 18/20 (90%) 3-07, 3-23, 4-34 Yes
Clinic (Mayo) (12,13)
2757 >95% 84/109 (77%)3-07, 1-69, 4-34,
3-23Yes
Familial (Duke) (24)
26 25% 12/16 (75%) 3-07, 4-34 Yes
Population (Leeds) (8)
9 80% 18/20 (90%) 3-07, 3-23, 4-34 Yes
Population (Italy) (4)
1.0 7% 36/51 (70%) 4-59/61 No
Population (Salamanca)
(6)0.5 0.4% 2/7 (29%)
No CLL-associated
No
CLL vs CLL-LIKE MBL: Biological features of clonal B-lymphocytes
Rawstron et al, Cytometry B, 2010
0
10
20
30
40
50
60
70
Perc
en
tag
e o
f cases
Normal* 13q- +12
17p-;11q-
MBL (n=35)
CLL (n=65)
P<0,0560%
25%
38%
58%
7%
16% 16%
Genetic alteration
MBL CLL
13q- 67%±30% 67%±31%
+12 50%±13% 76%±17%
Other (17p-/11q-)
- 65%±29%
% of altered cells
• No alterations detected by FISH with probes for 13q-,+12, 17p-,11q-
Cytogenetic patterns of clonal CLL tumor cells vs PB CLL-like B-cells from
healthy adults
CLL-like MBLs with genetic abnormalities have higher absolute
numbers of CLL-like B-cells
0
50
100
150
200
250
300
Trisomy 12 Del (13q) No genetic abnormalities
Ab
s. N
. C
LL-l
ike B
cells
(c
ells
/mL)
P=0.001
Almeida et al, Leukemia 2011
0/2 cases carrying trisomy 12 and only 2/12 cases with del(13q) showed less than 1 PB CLL-like B-cell/mL, while 11/21 cases without detectable genetic abnormalities were “low-count” MBL (<1 circulating CLL-like B-
cell/mL)
Baseline
0,0
0,1
+1 year
% o
f clo
nal B
-cells f
rom
leu
cocyte
s
0,2
0,3
0,4
0,5 p=0,04
BASAL +1 year %
of
clo
nal B
-cells
Fro
m a
ll B
cells
p=0,03
0
5
10
15
20
25
Baseline +1 yearN.
of
clo
nal B
cells (
x1
03/m
L)
0
3
6
9
12
15 p=0,045
MBL: CLINICAL SIGNIFICANCE
0
2
4
6
8
10
12
% o
f clo
nal B
cells f
rom
le
ucocyte
s
Baseline +1 year
p=0,03
0
20
40
60
80
100
% o
f clo
nal B
cells
Fro
m a
ll B
cells
p=0,2
Baseline +1 year
0
200
400
600
800
1,000
1,200
1,400p=0,003
N.
of
clo
nal B
cells (
x1
03/m
L)
Baseline +1 year
NON-CLL-like MBL (11/13)
CLL-like MBL (39/82)
Nieto et al, Clinical Cytometry 2010
• A significant proportion (14.8%) of healthy adults aged >40 years display circulating B-cell clones frequently (13%) showing a CLL-like phenotype.
• The frequency of such CLL-like (and other) circulating B-cell clones increase with age.
• The higher frequency of circulating CLL-like clones could reflect the greater sensitivity of the screening approach used, half of the cases showing clonal B-cell numbers below previously reported threshold levels ( 0,01%).
• The immunophenotypic, genetic and molecular features of circulating B-cell clones overlap with those of clinically evident CLL and other B-CLPD.
• In all tested cases circulating B-cell clones remain detectable after one year with minimal but significant changes (increase) in their distribution.
CONCLUSIONS
100%
50%
0%10.10.01 5 10 20 40 5030
Whole series
10.10.01 5 10 20 40 5030
40 - 59 years
10.10.01 5 10 20 40 5030
60 - 69 years
100%
50%
0%
100%
50%
0%
100%
50%
0%
70 years
PB Volume (mL)10.10.01 10 20 40 5030
% o
f ca
ses
wit
h a
CLL
-lik
e c
lone
5
70%
100% 100%
18%
46%
32%
88%
62%
36%
100%100%100%
DETECTED FREQUENCY
FREQUENCY OF CLL-like MBL IN HEALTHY ADULTS
Almeida et al, Leukemia 2010
100%
50%
0%10.10.01 5 10 20 40 5030
Whole series
10.10.01 5 10 20 40 5030
40 - 59 years
10.10.01 5 10 20 40 5030
60 - 69 years
100%
50%
0%
100%
50%
0%
100%
50%
0%
70 years
PB Volume (mL)10.10.01 10 20 40 5030
% o
f ca
ses
wit
h a
CLL
-lik
e c
lone
5
70%
100% 100%
18%
46%
32%
88%
62%
36%
100%100%100%
CALCULATED FREQUENCYDETECTED FREQUENCY
FREQUENCY OF CLL-like MBL IN HEALTHY ADULTS
Statistical predictive model(power regression analysis)Almeida et al, Leukemia 2010
CD20 Pacific blueC
D5 A
PC
CD20 Pacific blue
CD
5 A
PC
CD20 Pacific blue
CD
5 A
PC
CD20 Pacific blue
CD
5 A
PC
1 mL 50 mL
Lambda PE
Kap
pa F
ITC
Lambda PE
Kap
pa F
ITC
Immunophenotypic identification of CLL-like clonal B-cells in 50 mL from healthy
adults
Almeida et al, Leukemia 2011
Case N.Age
(years)
Sex
N. of CLL-like B-cell population
s*
sIg light chain
restriction
Ratio /k l% of CLL-like B-cells from WBC (x10-3)
% of CLL-like B cells from all PB B-cells
N. of CLL-like B cells
/mL
Monoclonal cases
Case #1 78 M One l NA 0.8% 0.041% 0.066
Case #2 73 M One k NA 0.73% 0.05% 0.036
Case #3 77 M One k NA 1.3% 0.08% 0.112
Case #4 88 M One k NA 0.14% 0.013% 0.009
Multiclonal cases
Case #5 77 F Two kl 20
0.28%0.014%
0.002%0.001%
0.0018 0.0009
Case #6 72 F Two kl 0.72
0.08%0.11%
0.006%0.008%
0.0070.009
Case #7 82 F Two kl 0.18
0.18%0.95%
0.02%0.01%
0.0090.004
Case #8 73 M Two kl 1.8
0.49%0.27%
0.016%0.009%
0.0320.018
Case #9 87 M ND ND NA <0.0006% <1.1x10-6 <1x10-6
M: male; F: female; ND: Not detected. NA: Not applicable*CLL-like B-cells were identified as those cellular events expressing CD19+, CD5+, CD20+dim, CD79b+dim and surface immunoglobulin light chain Ig+dim.§ Restricted to CLL-like B-cells (considered to be altered when ratio k/l >3.1 or <1:3)
Characteristics of CLL-like B-cells identified in 50 mL of PB from 9 healthy subjects older than 70 years
Almeida et al, Leukemia 2011
B-CLPD: COMMON ABERRANT PHENOTYPES
Diagnostic group Aberrant phenotype % of cases
typCLL/SLL CD22-/+dCD5+ 97%CD22-/+dCD23+ 98%
PLL sIg +++ 60%
HCL CD11c+++ 69%CD19+++ 69%CD103+++ 92%FSC/SSC+++ 85%
LPL CD22-/+dCD10- 67%
FL BCL2+++CD10+ 85%
MCL CD22-/+dCD5+ 100%Sanchez et al, Leukemia, 2002
0
20
40
60
80
100
0.01 0.1 1 10 100 1000 10000
Absolute Number of Neoplastic B-cells
Cum
ulat
ive
Perc
enta
ge o
f Cas
es Population Studies (Normal Blood Count)
Hematology Clinic Series (Lymphocytosis)
0
20
40
60
80
100
0.01 0.1 1 10 100 1000 10000
Absolute Number of Neoplastic B-cells
Cum
ulat
ive
Perc
enta
ge o
f Cas
es
0
20
40
60
80
100
0.01 0.1 1 10 100 1000 10000
Absolute Number of Neoplastic B-cells
Cum
ulat
ive
Perc
enta
ge o
f Cas
es Population Studies (Normal Blood Count)
Hematology Clinic Series (Lymphocytosis)
CUMULATIVE FREQUENCY OF CLL-LIKE MBL IN POPULATION- VS HOSPITAL-BASED COHORTS
Rawstron et al, Cytometry B, 2010
Conclusions
• In healthy adults over 70 years,
emergence of one or more CLL-like cell
populations occurs whenever > 50mL
PB are screened, suggesting that these
clonal cells may more likely represent
the normal counterpart of CLL cells,
rather than a leukemic precursor
Neoplastic trasnformatio
n
Reactive
process
Malignant trasnformati
on
Treatment
?(Wait and see
vs early therapy)
Genetic instability
Genetic and Chromosomal instability
Wait and see
X
ONCOGENIC EVENTS
Genetic/environmental factors
Subclones blocked at different
maturation stages and clinical
progression
Clonal expansion
Identical primary mutation ?
Identical IGH/IGL VDJ/VJ gene rearrangement
Restricted expressiofn Ig (k or L) light chains
MBL AND B-CLL: HYPOTHETICAL ONTOGENIC MODEL
Evolution
Secondary genetic alterations
....
....
...
.........
CD5:APC
CD
10
:PE
Cy7
CD
20
:PB
CD10:PECy7
CD38:APC-H7
sIg
D:P
E
CD38:APC-H7
sIg
M:F
ITC
sIgM:FITC
sIg
D:P
E
B CELL MATURATION PATHWAYS IN NORMALHUMAN BONE MARROW (GATED ON CD19+)
Memory B-cells
Somatic Hypermutation/IgH Switch
Naïve B-cells
Immature B-cellsBM PB
MATURATION-ASSOCIATED NORMAL PB B-CELL SUBSETS
Plasmablasts/Plasma cells
CD38:AF700 LOGIC
AL
CD27:APC LOGICAL
CD
10:
PE
-Cy7
LO
GIC
AL
Immature
Naïve
Memory
Plasmablasts
Percentage Absolute count
(cells/ L)
Immature 5.4%3.7% (1.8%-4.5%)
99(3-11)
Naive 64%12%(57%-73%)
10157(61-130)
Memory 31%12%(21%-40%)
5239(26-65)
Plasmablasts/Plasma cells
2.5%1.4%(0.8%-2.3%)
3.0 6.8(1.0-3.1)
Distribution of B-cell maturation subsets in peripheral blood from normal individuals
(N=600)
Memory Plasmablasts/Plasma cells
sIgA+
(21% 9%)
sIgG+
(23% 10%)
sIgMD+
(52% 15%)
sIgG+
(13% 11%)
sIgM+
(18% 12%)
sIgneg
(14% 12%)sIgD+
(5% 5%)
sIgA+
(49% 18%)
Caraux A Haematologica 2010, Perez-Andres M Clinical Cytometry 2010
CD27 EXPRESSION BY NORMAL PB CD5+ B CELLS
Normal B-cell maturationstemcell
Lymphoidprogenitor
Progenitor-Bcell
Pre-B cell
ImmatureB-lymphocyte
MemoryB-cell
Plasma cell
GerminalcenterB-cell
MaturenaiveB-cell
Lee et al.
Bone marrow
Lymphoid tissue
Peripheral blood
MM
MZLLPLMCL
DLBCLBL, FL
ALL
CLL
CIC, Department of Medicine & Cytometry Service (USAL)
J Almeida
J Ciudad
A López Fernández
A Nieto
W Nieto
A Rodríguez Caballero
ML Sánchez-García
C Teodosio
AKNOwLEDGEMENTS
Primary Health Care Area of Salamanca (SACYL)
JA Romero Furones
P Fernández Navarro
Primary Health Care Group of Salamanca for the study of MBL (40 medical doctors from the primary health care area of
Salamanca )
University Hospital of Salamanca
MB Muñoz Criado (Microbiology)
A Balanzategui (Hematology)
M González Díaz (Hematology)
MB Vidriales (Hematology)
Regional Health Care Department of Castilla y
León
AT Vega Alonso (Epidemiology)
Universidad Federal de
Río de Janeiro (Brazil)
CE Pedreira (Mathematics)
THANK YOU