SF RAB

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.