iccs e-newsletter csi winter 2012
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ICCS e-Newsletter CSI Winter 2012. Julia Almeida, MD PhD Department of Medicine and Cancer Research Center University of Salamanca Salamanca, Spain. e-CSI – History and physical examination. - PowerPoint PPT PresentationTRANSCRIPT
ICCS e-Newsletter CSI Winter 2012
Julia Almeida, MD PhD
Department of Medicine and Cancer Research CenterUniversity of Salamanca
Salamanca, Spain
• A 77-year-old male with no significant past medical history was studied because of a lymphocytosis detected in a routine blood analysis
• Patient was asymptomatic
• Physical examination did not revealed any pathological sign (no organomegalies, no skin involvement)
e-CSI – History and physical examination
e-CSI – Peripheral blood cell counts
CBC Normal range
WBC: 14.6 x 109/l (4.5 – 10.8)
RBC: 4.35 x 1012/l (4.0 – 5.4)
Hgb: 13.0 g/dl (12.0 – 18.0)
Hct: 38.3 % (35.0 – 52.0)
MCV: 88.1 fl (80.0 – 99.0)
MCH: 29.9 pg (27.0 – 32.0)
MCHC: 33.9 g/dl (31.5 – 36.0)
RDW: 14.3% (10.5 – 14.5)
Plts: 346 x 109/l (150 – 450)
e-CSI – CBC differential
CBC differential(PB)
% from WBC
Absolute numbers (x109/l)
Reference range(Absolute #)
Neutrophils 27.2 3.97 1.4 - 6.5 x 109/l
Monocytes 3.7 0.54 0.3 – 0.9 x 109/l
Lymphocytes 67.8 9.90 1.2 – 3.5 x 109/l
Eosinophils 1 0.15 0 – 0.5 x 109/l
Basophils 0.3 0.04 0 – 0.1 x 109/l
PB: Peripheral blood
e-CSI – Biochemical parameters
Parameter Value Normal range
B2 microglobulin (mg/dl) 1.0 <2.5 mg/l
LDH (IU/l) 340 < 460 IU/l
• Peripheral Blood in EDTA was received for evaluation of lymphocytosis
• Flow cytometric immunophenotyping was performed on this sample, and the results from selected multiple-stained tubes are provided for review:
e-CSI – Work-up and evaluation
e-CSI – Flow cytometric (FCM) studies
Data acquisition was performed in a FACSCanto II (BDB); data analysis was done with the INFINICYT software (Cytognos SL)
Pac.B Pac.O FITC PE PerCP-Cy5.5 PECY7 APC APCH7
Step 1 (screening tube)
CD45 - CD8Anti-sIgk
CD56Anti-sIgl
CD4CD19
CD20 CD3 -
Step 2(T-cell clonality study)
CD4 - TCR-Vb8 +
TCR-Vb13.6
TCR-Vb13.1+
TCR-Vb13.6
CD3 - CD8 -
Step 3 (T-cell CLPD panel)
CD4CD4CD4CD4CD4
CD45CD45CD45CD45CD45
CD7CD27CD5
CD57cytPERF
CD26CD197CD25CD30
cytGRZ
CD3CD3CD3CD3CD3
CD2CD45ROHLADR
-CD16
CD28CD45RAcytTCL1CD11cCD94
CD8CD8CD8CD8CD8
Sequential FCM strategy and combinations of fluorochrome MAb used:
cytPERF: cytoplasmic perforine; cytGRZ: cytoplasmic granzyme B
94% CD4+ T cells(CD4/CD8 ratio: 15.5)
94% CD56+/CD4+
T cells
e-CSI – Flow cytometric (FCM) studiesStep 1: screening tube
Exclusion of debris
Selection of T cells
Refining selection of
T cells
Display only CD3+ T cells
CD3+ gated T cells CD4+ gated T cells CD4+ gated T cells CD4+ gated T cells
Most CD4+ T cells (94%) are CD56+. In addition, they show partial expression of CD8dim and higher SSC values in comparison to CD56- CD4+ T cells
These cells represent around 67% of total leukocytes
e-CSI – Flow cytometric (FCM) studiesStep 1: screening tube
ConclusionThe lymphocytosis is at expense of CD4+ T cells showing cytotoxic-related markers: expression of CD56, partial expression of CD8dim and high SSC values (67% of all WBC; 9.78 x 109 cells/l)
Are these expanded cells clonal?
e-CSI – Flow cytometric (FCM) studiesStep 2: assessment of T-cell clonality
CD3+ live gate
CD3+ gated T cells
CD3+ live gate
Selection of T cells
Refining selection of
T cells
Display only CD3+ T cells
Selection of CD4+ T cells
CD4+ gated T cells
Around 93% of CD4+ T cells
express the same TCR-Vbeta region
(TCR-Vb13.1+)
This specific tube has been selected from the total panel of the TCR Vbeta Kit IOTest Beta Mark IM3497, which includes a total of 24 MAb against different TCR-Vb regions in a 8-tube format
e-CSI – Flow cytometric (FCM) studiesStep 2: assessment of T-cell clonality
Conclusion
YES, the expanded CD4+ T-cells are clonal, as
they express the same TCR-Vbeta region
assessed by FCM (TCR-Vb13.1+)
e-CSI – Flow cytometric (FCM) studiesStep 3: phenotypic characterization
After applying the same gating strategy as previously shown to identify CD4+ T cells, then we proceed to characterize them:
Pac.B Pac.O FITC PE PerCP-Cy5.5 PECY7 APC APCH7
Step 3: T-cell CLPD panel TUBE1 CD4 CD45 CD7 CD26 CD3 CD2 CD28 CD8
Normal residual CD4+ T cells Clonal
CD4+ T cells
Clonal CD4+ T cells are mostly CD7-, CD26- and CD28- and CD2hi
e-CSI – Flow cytometric (FCM) studiesStep 3: phenotypic characterization
Normal residual CD4+ T cells
Clonal CD4+ T cells
Clonal CD4+ T cells show a typical phenotype of effector cells: CD45RA+ / CD197 (CCR7)- / CD27- / CD45RO-/+dim
Pac.B Pac.O FITC PE PerCP-Cy5.5 PECY7 APC APCH7
Step 3: T-cell CLPD panel TUBE2 CD4 CD45 CD27 CD197 CD3 CD45RO CD45RA CD8
After applying the same gating strategy as previously shown to identify CD4+ T cells, then we proceed to characterize them:
e-CSI – Flow cytometric (FCM) studiesStep 3: phenotypic characterization
Normal residual CD4+ T cells
Clonal CD4+ T cells
Clonal CD4+ T cells are CD5+, CD8-/dim , cytTCL1- and HLADR+ heterogeneous
Pac.B Pac.O FITC PE PerCP-Cy5.5 PECY7 APC APCH7
Step 3: T-cell CLPD panel TUBE3 CD4 CD45 CD5 CD25 CD3 HLADR cytTCL1 CD8
After applying the same gating strategy as previously shown to identify CD4+ T cells, then we proceed to characterize them:
e-CSI – Flow cytometric (FCM) studiesStep 3: phenotypic characterization
Normal residual (+some clonal)
CD4+ T cells
Clonal CD4+ T cells
85% of total CD4+ T cells are CD57+. All CD4+ T cells are CD11c- and CD30-
Pac.B Pac.O FITC PE PerCP-Cy5.5 PECY7 APC APCH7
Step 3: T-cell CLPD panel TUBE4 CD4 CD45 CD57 CD30 CD3 - CD11c CD8
After applying the same gating strategy as previously shown to identify CD4+ T cells, then we proceed to characterize them:
e-CSI – Flow cytometric (FCM) studiesStep 3: phenotypic characterization
Normal residual CD4+ T cells
Clonal CD4+ T cells
Clonal CD4+ T cells express Perforine and Granzyme B at the cytoplasmic level and are mostly CD94- and CD16-
Pac.B Pac.O FITC PE PerCP-Cy5.5 PECY7 APC APCH7
Step 3: T-cell CLPD panel TUBE5 CD4 CD45 cytPERF cytGRZ CD3 CD16 CD94 CD8
After applying the same gating strategy as previously shown to identify CD4+ T cells, then we proceed to characterize them:
Large granular lymphocyte (LGL) leukemia is a well-
recognized disorder of clonal mature CD8+ T lymphocytes
or less frequently natural killer cells; in addition, it has
recently been shown that clonal LGL lymphocytosis /
leukemia may also derive from CD4+ LGL T cells
Large Granular Lymphocytosis / Leukemia
TCRab+/CD4+ Large Granular Lymphocytosis / Leukemia
Clinical characteristics• These cases usually display an indolent clinical course –although rare cases associated with aggressive disease have also been reported – associated with a significantly lower frequency of cytopenias than CD8+ LGL leukemias
• Accordingly, diagnosis is usually made from a lymphocytosis detected in a routine blood analysis (>80%)
• However, these patients frequently (30%) show associated neoplasias (particularly B-cell chronic leukemias/lymphomas), so clinical outcome is determined by the associated tumor
Lima et al, Am J Pathol 2003
TCRab+/CD4+ Large Granular Lymphocytosis / LeukemiaPhenotypic characteristics of clonal cells
T-cell related antigens:
CD3+ TCRab+
CD4++CD8-/+
CD5+CD7-/+
CD2++
NK/cytotoxic-cell associated (Nka) markers:
CD56+ CD57+cyGranzyme B+cyPerforine +
CD11b-/+CD11c-CD16-
CD94-CD161-
CD158a-NKB1-
Cytokine-receptors and activation-associated markers:
CD25- CD122- CD38- HLADR+
Maturation-associated antigens:
CD197 (CCR7) - CD45RA+CD45RO+
CD28-CD26-
CD27-CD62L-
CD4+/NKa+/CD8-/+dim T cells display relatively high FSC/SSC values and frequent dim reactivity for CD8, and show a phenotype of activated (CD7-/dim/CD2h/HLADR+) peripheral memory or effector
(CD26-/CD27-/CD28-/CCR7- with frequent coexpression of CD45RA and RO) cytotoxic (usually CD57+/CD56+ and granzyme B + /perforine + ) T cells in the absence of other Nka markers (i.e. CD16, CD94, CD161)
Lima et al, Am J Pathol 2003
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Analysis of the TCR-Vb repertoire by immunophenotype
TCRab+/CD4+ Large Granular Lymphocytosis / Leukemia
Normal CD4+ T cells
Clonal CD4+ LGL T cells
Perc
enta
ge o
f cas
es
TCRab+/CD4+ Large Granular Lymphocytosis / Leukemia have a restricted TCR-Vb repertoire with a preferential usage of a few TCR-Vb families; notably,
in more than 40% of cases clonal cells are TCR-Vb 13.1+Lima et al, Am J Pathol 2003
TCRab+/CD4+ Large Granular Lymphocytosis / Leukemia
In addition to the restricted TCR-Vb repertoire, other evidences strongly support the fact of the existence of a
common antigen-driven origin
There is a clear association between the TCR-Vb repertoire and the HLA genotype: all TCR-Vb13.1+ cases are HLADR*0701
HLADR7/TCRVb13.1+ cases show a highly homogeneous and strikingly similar TCR (high homology in their CDR3)
¿What is the nature of the antigen?
Clonal CD4+ LGL T cells show functional response to hCMVRodriguez-Caballero et al, Blood 2008
Garrido et al, Blood 2007
Patients with CD4+/NKa+/CD8-/+dim T-LGL lymphocytosis / leukemia show an indolent course of the disease; however, they frequently have a second neoplasia and clinical outcome is usually determined by the associated tumor Clonal CD4+/NKa+/CD8-/+dim T-cells show a typically activated, cytotoxic phenotype of effector T-LGL cells and a restricted TCR-Vb repertoire, with a preferential usage of a few TCR-Vb familiesTCR-Vb13.1+ patients display a common HLA-DRB1*0701 genotype and clonal cells express identical motifs in their CDR3-TCR-V sequences, supporting a common antigen-driven originClonal T cells usually display response to hCMV, suggesting the potential involvement of hCMV in the ontogeny of CD4+/NKa+/CD8-/+dim T-LGL lymphocytosis / leukemia
TCRab+/CD4+ Large Granular Lymphocytosis / Leukemia
Summary-
-
-
-
TCRab+/CD4+ Large Granular Lymphocytosis / Leukemia
References1. Lima M, Almeida J, Dos Anjos Teixeira M, et al. TCRalphabeta+/CD4+ large granular lymphocytosis
: a new clonal T-cell lymphoproliferative disorder. Am J Pathol. 2003 Aug;163(2):763-71.
2. Garrido P, Ruiz-Cabello F, Bárcena P, et al. Monoclonal TCR-Vbeta13.1+/CD4+/NKa+/CD8-/+dim T-LGL lymphocytosis: evidence for an antigen-driven chronic T-cell stimulation origin. Blood. 2007 Jun 1;109(11):4890-8.
3. Ghia P, Prato G, Stella S, Scielzo C, Geuna M, Caligaris-Cappio F. Age-dependent accumulation of monoclonal CD4+CD8+ double positive T lymphocytes in the peripheral blood of the elderly. Br J Haematol. 2007 Dec;139(5):780-90.
4. Rodríguez-Caballero A, García-Montero AC, Bárcena P, et al. Expanded cells in monoclonal TCR- alphabeta+/CD4+/NKa+/CD8-/+dim T-LGL lymphocytosis recognize hCMV antigens. Blood. 2008 Dec 1;112(12):4609-16.
5. Olteanu H, Karandikar NJ, Eshoa C, Kroft SH. Laboratory findings in CD4(+) large granular lymphocytoses. Int J Lab Hematol. 2010 Feb;32(1 Pt 1):e9-16.
6. Sáez-Borderías A, Romo N, Ruiz-Cabello F, et al. Natural killer cell receptor expression reflects the role of human cytomegalovirus in the pathogenesis of a subset of CD4+ T-cell large granular lymphocytosis. Hum Immunol. 2011 Mar;72(3):226-8.