1. ICCS e-Newsletter CSI April 2014 Z. Jenny Mao, MT (ASCP) Timothy P. Singleton, MD Department of Laboratory Medicine and Pathology, University of Minnesota

2. 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.

3. e-CSI – Laboratory Tests Complete blood counts were normal except for slight neutrophilia and slight monocytosis. Cardiac troponin levels were in the reference range.

4. 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.

5. 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.

6. e-CSI – Screening Panels Screening Panels with Fluorochromes TubeV450V500FITCPEPerCPPE-Cy7APCAPC-H7 BMLCD19CD45LambdaKappaCD14CD5CD10CD20 TubeV450V500FITCPEPerCP-Cy5.5PE-Cy7APCAPC-H7 TLNCD4CD45CD56CD7CD5CD2CD3CD8

7. 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

8. e-CSI – Screening Panel Analysis of TLN Tube Gate on T Cells Debris and cell doublets were excluded with light scatter.

9. e-CSI – Screening Panel Analysis of TLN Tube Stages of T-Cell Maturation Based on Density for CD45

10. e-CSI – Screening Panel Analysis of TLN Tube Stages of T-Cell Maturation Based on Density for CD45

11. e-CSI – Screening Panel Analysis of TLN Tube Stages of T-Cell Maturation

12. 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.

13. 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

14. e-CSI – Add-On 8-Color Panels Add-On 8-Color Panels TubeV450V500FITCPEPerCP-Cy5.5PE-Cy7APCAPC-H7 CD1a/ CD34 CD4CD45CD7CD1aCD5CD2CD34CD8 TubeV450V500FITCPEPerCP-Cy5.5PE-Cy7APCAPC-H7 CD10/ CD34 CD4CD45CD7CD34CD5CD2CD10CD8

15. e-CSI – Add-On 8-Color Panels CD1a, CD10, and CD34 on Stages of T-Cell Maturation

16. 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

17. e-CSI – Add-On 4-Color Panels Follow-up 4 color Panel TubeFITCPEPerCPAPC TAB Alpha-Beta T-Cell Receptor Gamma-Delta T-Cell Receptor CD45CD3 CD13/ CD33 CD34CD13CD45CD33 IC Ig for MPO IC IGG1 CD45 IC MPOIC CD3IC MPOIC 79aCD45 IC Ig for TdT IC IGG2CD7CD45CD3 IC TdT CD7CD45CD3

18. e-CSI – Add-On 4-Color Panels Tαβ, Tγδ, CD13, and CD33 on Stages of T-Cell Maturation

19. 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.

20. 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.

21. 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

22. e-CSI – Immunophenotypic Diagnosis Immunophenotype by flow cytometry is consistent with normal thymocyte maturation.

23. e-CSI – Follow-Up Patient had surgical resection of the mediastinal tumor. Final diagnosis was invasive thymoma, type B3.

24. 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.

25. e-CSI – Discussion The immunophenotype of the maturation sequence is more complicated than the three traditional stages: double- negative, double-positive, and single-positive T cells. There are transitional populations, such as the double- negative 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.

26. 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.

27. 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.

28. 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. 3 rd 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. 3 rd ed. Philadelphia (PA): Lippincott Williams and Wilkins; c2014.