1. ICCS e-Newsletter CSI Yao Schmidt, MD Department of Pathology
University of Texas Southwestern Medical Center
Dallas, Texas
03/23/2014

2. Clinical History
The patient is a 78 year-old female with anemia and neutropenia.
On physical exam, the patient was slightly pale, otherwise unremarkable without hepatosplenomegaly or lymphadenopathy

3. CBC Results CBC Result Normal Range WBC: 9 K/mm3 4.0-11.0 K/mm3
RBC: M/mm3
3.70 – 5.10 M/mm3
Hgb: g/dL
12.0 – 15.0 g/dL
HCT: % %
MCV: fl
80.0 – 98.0 fl
MCH: pg
27.0 – 33.0 pg
MCHC: g/dL
33.0 – 35.0 g/dL
RDW: %
11.3 – 15.1 %
Platelets: 189 K/mm3
150 – 450 K/mm3

4. Peripheral Blood Differential
11% Segmented Neutrophils
25% Lymphocytes
4% Monocytes
1% Eosinophils
1% Basophils
58% Atypical cells

5. A bone marrow specimen is received in flow lab with the indication of anemia and pancytopenia
A four color analysis was performed using a BD FACScalibur™ flow cytometer. An ungated, cluster analysis was performed with BD Paint-a-Gate™ software.
Files are in FCS2.0 format.

6. Selected tubes with the following antibody combinations are included for review.
FITC PE
PerCP
APC 1
CD8
CD5
CD3
CD4 2
CD7 3
CD2 4
CD57
CD45RO 5
TCR-αβ
TCR-γδ 6
CD16
CD11b 7
CD45RA 8
CD56
CD45

7. Tube 1
First CD3 is used to isolate all T lymphocytes, which is highlighted in green.
Second, the forward versus side scatter plot can be used to further define or “clean up” the population to exclude non-T lymphocytes, doublets and debris. Here, all events outside of the “lymphocytes gate” are highlighted in black.
A similar method can be used on all tubes to isolate T-cell singlets after the T-cells have been highlighted using CD3 or a combination of T-cell antigens
granulocytes
debris
doublets

8. Tube 1
On the CD3 versus CD5 plot, a unique subset of T lymphocytes is noted to be CD5 negative to dim positive. There is another distinct population which expresses CD5 at normal intensity. Here the CD5(- to dim +) population is highlighted red and the rest of the T lymphocytes remain highlighted in green.
On the CD4 versus CD8 plot, this unique subset of T lymphocytes is uniformly expressing CD8.

9. Tube 1 The aberrant T-cell population (red) is CD3(slightly bright +)
CD5(partial dim +)
CD8(+)
The green population is normal T lymphocytes

10. Tube 2 Tube 2 can be analyzed in a similar manner as tube 1.
The aberrant T-cell population (red) is found to be
CD7(partial dim +)
The green population is normal T lymphocytes

11. Tube 3 Tube 3 can be analyzed in a similar manner as tube 1.
The aberrant T-cell population (red) is found to be
CD2(slight dim+)
The green population is normal T lymphocytes

12. Tube 4 Tube 4 can be analyzed in a similar manner as tube 1.
The aberrant T-cell population (red) is
CD57(+)
CD45RO(-)
The green population is normal T lymphocytes

13. Tube 5
Since tube 5 lacks CD3, CD4 and CD8 can be used to isolate T-lymphocytes.
CD4(bright +) T-cells are colored green.
CD8(+) T-cells are colored red.
The T-cell singlets are then “cleaned up” to exclude non-lymphocytes, doublets and debris using forward and side scatter similar to tube 1.

14. Tube 5
Most of the CD8(+) T-lymphocytes (red) express TCR-αβ(+). Since the proportion of TCR-γδ T-cells is too small to account for the population of aberrant T-cells, these cells must be TCR-αβ(+).
The green population is CD4(+) T lymphocytes

15. Tube 6 Tube 6 can be analyzed in a similar manner as tube 1.
On previous tubes, the aberrant T-cell population was slightly bright for CD3. On this tube, the CD3(slightly bright +) population is also CD16(+).
The aberrant T-cell population (red) is
CD11b(partial dim +)
CD16(dim +)
The green population is normal T lymphocytes

16. Tube 7 Tube 7 can be analyzed in a similar manner as tube 1.
The aberrant T-cell population (red) is
CD45RA(+)
The green population is normal T lymphocytes

17. Tube 8
Since tube 8 lacks CD3, the population of interest is isolated by coloring the bright CD45 lymphocyte area.
The lymphocyte singlets are then “cleaned up” to exclude non-lymphocytes, doublets and debris using forward and side scatter similar to tube 1.
The aberrant T-cell population is CD16(+) on tube 6; thus, CD16 can be used to isolate this population.

18. Tube 8 The aberrant T-cell population (red) is CD11b(partial dim +)
The green population is normal T lymphocytes

19. Remaining tubes (not shown) are analyzed in a similar manner
Remaining tubes (not shown) are analyzed in a similar manner. The immunophenotype of the aberrant T-cell population is as follows:
CD2(slightly dim +)
CD3(slightly bright +)
CD7(partial dim +)
CD8(+)
CD10(-)
CD11b(partial dim +)
CD16(dim +)
CD25(-)
CD45(+)
CD45RO(predominantly -)
CD45RA(+)
CD52(variably +)
CD56(-)
CD57(predominantly +)
TCR-αβ(+)
TCR-γδ(-)

20. Flow Cytometry Interpretation
T-lineage lymphoproliferative disorder with immunophenotypic features characteristic of T-cell large granular lymphocytic leukemia.

21. T-LGL Cells in Peripheral Blood

22. T-LGL Cell in Peripheral Blood

23. T-LGL cells In Bone Marrow

24. T-LGL cells In Bone Marrow

25. Bone marrow biopsy LGL leukemia infiltrates bone marrow in an interstitial and sinusoidal pattern. Notably, the easily identifiable large nodular lymphocytic aggregates seen here are non-neoplastic. They contain numerous B-cells surrounded by non-neoplastic T-cells. These aggregates are a common feature associated with bone marrow involvement by T-cell LGL leukemia.

26. Bone marrow biopsy CD20 highlighting B-cells
Non-neoplastic lymphoid aggregate

27. Non-neoplastic T-cells
Bone marrow biopsy CD3
T-LGL cells showing interstitial
and intrasinusoidal infiltration
Non-neoplastic T-cells
associated with
nodular aggregate

28. CD8 – Sinusoidal Pattern of Infiltration; T-LGL cells

29. Granzyme – Sinusoidal Pattern; T-LGL cells

30. TIA1 – Sinusoidal Pattern; T-LGL cells

31. T cell receptor (gamma) PCR
Positive for two clonal peaks. One peak (160 bp amplicon) is associated with the Vg10 region and the other peak (179 bp amplicon) is associated the Vg9 region.
Rearrangements in these regions can be observed in up to 16% of peripheral T-cell lymphoma, 10% T-ALL, 16% precursor B-ALL cases, T-large granular lymphocytic leukemia and other condition.

32. Final Diagnosis
T-cell large granular lymphocytic leukemia (10-20%) involving a normocellular bone marrow

33. T-LGL Leukemia
T-LGL leukemia is a clonal disorder most commonly composed of an expansion of cytotoxic CD8(+) T cells, although variant phenotypes do occur
The most frequent immunophenotype is:
CD2(+)
CD3(+)
CD4(-)
CD5(- to dim)
CD7(- to dim)
CD8(+)
CD16(+)
CD56 (-)
TCR-αβ(+) in 90% of cases
CD57(+)
Often show expression of KIR receptors in a restricted pattern

34. Differential Diagnoses
T-LGL Lymphocytosis, CD4(+) variant
CD16(-), CD56(+), CD57(+), low CD7, variable or low CD8
No cytopenia
Maybe associated with cytomegalovirus
Chronic lymphoproliferative disorders of NK-cells
Surface CD3(-), CD4(-), CD5(-), CD7(often -)
TCR-αβ(-) & TCR-γδ(-)
CD11b(+), CD16(+), CD56(+)
CD57 low or absent
Abnormal pattern of KIR expression
Exclude Aggressive NK-cell leukemia which is EBV+

35. T-LGL Leukemia
T-LGL is a heterogeneous clonal disorder characterized by a persistent (>6 months) increase in the number of peripheral blood large granular lymphocytes, usually between 2-20x109/L without a clear identified case.
Often associated with autoimmune disorder and immune-mediated cytopenias.
The extent of the BM involvement is variable and LGL usually comprises less than 50% of the cellularity.
Recent studies suggest
up to 40% patients harbor STAT3 gene mutation.
2% patients harbor STAT5b gene mutation.

36. Lastly… LGLs can be increased in a number of reactive conditions
Reactive LGLs show similar immunophenotypic features as LGL leukemia.
Reactive LGLs may be associated with T-cell clonal
A diagnosis of LGL cannot be established solely by flow cytometry. It requires correlation of clinical, morphologic and immunophenotypic data