ICCS e-Newsletter CSI
April 2014
Z. Jenny Mao, MT (ASCP)
Timothy P. Singleton, MD
Department of Laboratory Medicine and Pathology,
University of Minnesota
e-CSI – Clinical History and
Physical Examination
• A 39-year-old woman presented with
complaint of constant chest pressure for the
past 36 hours.
• Physical examination was unremarkable
except for tachycardia.
e-CSI – Laboratory Tests
• Complete blood counts were normal
except for slight neutrophilia and slight
monocytosis.
• Cardiac troponin levels were in the
reference range.
e-CSI – Other Tests
• EKG was normal except for sinus tachycardia.
• Chest X-ray was abnormal.
• Chest CT showed a 12 cm anterior
mediastinal tumor.
e-CSI – Specimen for Flow Cytometry
• Biopsy of the anterior mediastinal mass was
received in RPMI.
• Immunophenotyping was performed by flow
cytometry, and the results are shown for selected
4- and 8-color antibody panels.
• The flow cytometer was a FACSCanto II with
three lasers: blue (488 nm), red (HeNe, 633
nm), and violet (405 nm).
• The files were analyzed with the software
program Kaluza.
e-CSI – Screening Panels
Screening Panels with Fluorochromes
Tube
V450
V500
FITC
PE
PerCP
PE-Cy7
APC
APC-H7
BML
CD19
CD45
Lambda
Kappa
CD14
CD5
CD10
CD20
Tube
V450
V500
FITC
PE
PerCP-Cy5.5
PE-Cy7
APC
APC-H7
TLN
CD4
CD45
CD56
CD7
CD5
CD2
CD3
CD8
e-CSI – Screening Panel
Analysis of BML Tube
Polytypic B cells (1% of leukocytes) expressed CD19, CD20, CD45,
and kappa or lambda immunoglobulin light chains
e-CSI – Screening Panel
Analysis of TLN Tube
Gate on T Cells
Debris and cell doublets were excluded with light scatter.
e-CSI – Screening Panel
Analysis of TLN Tube
Stages of T-Cell Maturation Based on Density for CD45
e-CSI – Screening Panel
Analysis of TLN Tube
Stages of T-Cell Maturation Based on Density for CD45
e-CSI – Screening Panel
Analysis of TLN Tube
Stages of T-Cell Maturation
e-CSI – Screening Panel
Interpretation of TLN Tube
Stages of T-Cell Maturation
•
Normal thymocyte maturation proceeds through three traditional stages: from
double-negative thymocytes lacking both CD4 and CD8 to double-positive thymocytes
expressing both CD4 and CD8 to single-positive T cells expressing CD4 or CD8.
•
Since most clinical flow cytometry laboratories gate with CD45 rather than CD4 and
CD8 for routine analyses, the thymocyte maturation is displayed relative to the
density for CD45.
•
•
•
•
Immature thymocytes with the lowest-density CD45 include the double-negative
thymocytes and more mature cells that acquire CD4 before CD8.
The thymocytes with low-density CD45 are describe as double-positive since
essentially all of these cells express CD4 and CD8.
The T cells with the highest density CD45 are describe as single-positive since most
of these cells express CD4 or CD8.
An alternative analysis would be to put CD4 and CD8 in every T-cell panel and to gate
based on their expression. The alternative gating strategies could be explored with
the uploaded list-mode data files.
e-CSI – Screening Panel
Interpretation of TLN Tube
Stages of T-Cell Maturation
1.
Immature thymocytes
•
Expressed antigens: slightly low-density CD2, heterogeneous CD4, low-density
CD5, CD7, heterogeneous CD8 (predominantly negative), and lowest-density
CD45
•
Absent antigens: surface CD3 and CD56
2.
Double-positive thymocytes
•
Expressed antigens: CD2, heterogeneous surface CD3, slightly high-density CD4,
low-density CD5, low-density CD7, slightly low-density CD8, and low-density CD45
•
Absent antigens: CD56
3.
Single-positive T cells
•
Expressed antigens: CD2, CD3, CD5, CD7, and CD4 or CD8
•
Absent antigen: CD56
e-CSI – Add-On 8-Color Panels
Add-On 8-Color Panels
Tube
V450
V500
FITC
PE
PerCP-Cy5.5
PE-Cy7
APC
APC-H7
CD1a/
CD34
CD4
CD45
CD7
CD1a
CD5
CD2
CD34
CD8
Tube
V450
V500
FITC
PE
PerCP-Cy5.5
PE-Cy7
APC
APC-H7
CD10/
CD34
CD4
CD45
CD7
CD34
CD5
CD2
CD10
CD8
e-CSI – Add-On 8-Color Panels
CD1a, CD10, and CD34 on Stages of T-Cell Maturation
e-CSI – Screening Panel
Interpretation of Add-On 8-Color Tubes
Stages of T-Cell Maturation
1. Immature thymocytes
• Expressed antigens: CD1a, CD10, and heterogeneous CD34
2. Double-positive thymocytes
• Expressed antigens: CD1a and heterogeneous CD10
• Absent antigen: CD34
3. Single-positive T cells
• Absent antigens: CD1a, CD10, and CD34
e-CSI – Add-On 4-Color Panels
Follow-up 4 color Panel
Tube
FITC
PE
PerCP
APC
TAB
Alpha-Beta
T-Cell Receptor
Gamma-Delta
T-Cell Receptor
CD45
CD3
CD13/
CD33
CD34
CD13
CD45
CD33
IC Ig for
MPO
IC IGG1
IC IGG1
IC IGG1
CD45
IC MPO
IC CD3
IC MPO
IC 79a
CD45
IC Ig for
TdT
IC IGG2
CD7
CD45
CD3
IC TdT
IC TdT
CD7
CD45
CD3
e-CSI – Add-On 4-Color Panels
Tαβ, Tγδ, CD13, and CD33 on Stages of T-Cell Maturation
e-CSI – Add-On 4-Color Panels
Cytoplasmic CD3 and Cytoplasmic CD79a on Stages of T-Cell Maturation
Quadrants for cytoplasmic antigens were set
with immunoglobulin negative controls.
e-CSI – Add-On 4-Color Panels
Nuclear TdT on Stages of T-Cell Maturation
Quadrants for nuclear TdT were set with immunoglobulin-negative controls.
e-CSI – Screening Panel
Interpretation of 4-Color Add-On Tubes
Stages of T-Cell Maturation
1.
Immature thymocytes
•
Expressed antigens: cytoplasmic CD3, heterogeneous cytoplasmic CD79a, and
nuclear terminal deoxynucleotidyl transferase
•
Absent antigens: CD33 and cytoplasmic myeloperoxidase
2.
Double-positive thymocytes
•
Expressed antigens: heterogeneous alpha-beta T-cell receptor, cytoplasmic
CD3, and nuclear terminal deoxynucleotidyl transferase
•
Absent antigens: CD13, CD33, cytoplasmic CD79a, and cytoplasmic
myeloperoxidase
3.
Single-positive T cells
•
Expressed antigens: alpha-beta T-cell receptor
•
Absent antigens: CD13, CD33, cytoplasmic myeloperoxidase, and nuclear
terminal deoxynucleotidyl transferase
e-CSI – Immunophenotypic Diagnosis
• Immunophenotype by flow cytometry is consistent
with normal thymocyte maturation.
e-CSI – Follow-Up
• Patient had surgical resection of the mediastinal
tumor.
• Final diagnosis was invasive thymoma, type B3.
e-CSI – Discussion
• Thymomas are epithelial neoplasms.
• In thymomas, the lymphoid cells are not neoplastic.
The lymphoid cells may be only mature lymphocytes
or thymocytes maturing to single-positive T cells.
e-CSI – Discussion
• The immunophenotype of the maturation sequence is more
complicated than the three traditional stages: doublenegative, double-positive, and single-positive T cells.
• There are transitional populations, such as the doublenegative thymocytes acquiring CD4 before CD8.
• Also, in the presented case, the immunophenotype is
described for populations based on the density for CD45, as is
usually done in clinical laboratories, rather than the pattern
for CD4 and CD8, as is usually written in textbooks.
• In contrast to prior reports, heterogeneous CD10 was detected
on the double-positive population when using the bright
fluorochrome APC.
e-CSI – Discussion
• Normal thymocyte maturation needs to be
distinguished from T-lymphoblastic
lymphoma/leukemia.
• Normal thymocyte maturation may be seen in the
thymus, in an ectopic thymus, in thymoma, or in an
anterior mediastinal tumor.
e-CSI – Discussion
• Normal thymocytes
• Thymocytes are present only in thymic tissue.
• The immunophenotype has a precise maturation sequence
through three stages.
• The predominant double-positive population expresses
CD1a and TdT.
• The small double-negative population also expresses
heterogeneous CD34.
• T-lymphoblastic lymphoma/leukemia
• There is a maturation arrest.
• There are aberrant patterns of antigenic expression.
• CD1a and TdT are not normally seen on T cells outside of
thymic tissue.
e-CSI – References
• Li, Juco, Manna, and Holden. Flow cytometry in the differential
diagnosis of lymphocyte-rich thymoma from precursor T-cell
acute lymphoblastic leukemia/lymphoblastic lymphoma. Am J Clin
Pathol 2004;121:268-274.
• Racke and Borowitz. Precursor B- and T-Cell Neoplasms. In: Jaffe,
Harris, Vardiman, Campo, Arber, editors. Hematopathology. St.
Louis (MO): Saunders; c2011.
• Cerutti, et al. The immune system: Structure and function. In:
Orazi, Knowles, Foucar, Weiss, editors. Knowles’ Neoplastic
Hematopathology. 3rd ed. Philadelphia (PA): Lippincott Williams
and Wilkins; c2014.
• Reichard and Kroft. Flow cytometry in the assessment of
hematologic disorders. In: Orazi, Knowles, Foucar, Weiss, editors.
Knowles’ Neoplastic Hematopathology. 3rd ed. Philadelphia (PA):
Lippincott Williams and Wilkins; c2014.