Basics of Clinical Flow Cytometry

1. Basics of Clinical Flow Cytometry Jessica Hughes, QCYM (ASCP)

2. What is flow cytometry? • The measurement of cells in a flow stream, which delivers the cells in single file past a point of measurement

3. Basics of a flow cytometer • Fluidics – Cells in suspension move in single file through a flow chamber • Optics – Cells pass through a laser beam, scatter light, and emit fluorescence • Electronics – Light scatter and fluorescence are converted to digital values and stored on a computer

4. Forward scatter & side scatter • Forward scatter measures a cell’s size • The larger the cell, the greater the forward scatter • Side scatter measures a cell’s granularity • The more granular a cell, the greater the side scatter

5. Forward vs. side scatter plot Can you figure out the different cell populations? More granular cells  Larger cells 

6. Forward vs. Side Scatter Plot More granular cells  Larger cells 

7. Fluorescence and Fluorochromes • Antibodies are labeled with different fluorochromes • The antibody will bind to an antigen on the cell’s surface, if present • A fluorescent signal is generated if that specific marker is present on the cell’s surface

8. Examples of Fluorochromes

9. 2 Parameter Dot Plot PerCP positive population (pink) PerCP and PE positive population (yellow) PerCP and PE negative population (green) PE positive population (blue)

10. Some flow cytometry clinical applications • Lymphocyte subset analysis (T cells, B cells, NK cells) • Immune disorders • Leukemia/lymphoma immunophenotyping • Paroxysmal Nocturnal Hemoglobinuria • Reticulocyte analysis

11. Lymphocyte subset analysis • T cells • Originate in bone marrow • Mature in thymus • Responsible for immunological defense mechanism known as cell-mediated immunity • CD3 – pan T-cell marker • CD4 – helper T-cell marker • CD8 – cytotoxic T-cell marker

12. Lymphocyte subset analysis • B-cells • Originate in the bone marrow • Major role is to secrete immunoglobulins • Responsible for humoral immunity defense • CD19, CD20 - B-cell markers

13. Lymphocyte subset analysis • Natural Killer cells • Cytotoxic lymphocytes • Cells kill by releasing small cytoplasmic granules • CD16, CD56

14. Lymphocyte subset analysis T cells + B cells + NK cells = 100% of lymphocytes

15. Lymphocyte subset analysis

16. T cells + B cells + NK cells = 100% of lymphs 69 + 15 + 13 = 97% (close enough)

17. Myeloid & Lymphoid Differentiation

18. Leukemia/Lymphoma Immunophenotyping Normal peripheral blood CD45 vs. SS plot (common gating technique) CD45 – common leukocyte antigen RBCs are negative for CD45, therefore can be excluded from analysis.

19. Leukemia/Lymphoma Immunophenotyping Normal bone marrow Normal bone marrow shows immature and mature cells

20. Analysis and gating techniques A population can be gated, then displayed on its own in a separate plot. Here, we are looking at the maturation pattern of neutrophils.

21. Antibody combinations / panels • Monoclonal antibodies (CD markers) are generally grouped in lineage specific groups • Panels are lab-specific • Example: leukemia/lymphoma panels • Shotgun approach – uses a large number of antibodies and covers everything. Diagnosis can be made without having to go back and stain additional tubes. This can be more expensive. • Screening approach – uses a minimal set of antibodies. More tubes will be stained with additional antibodies if abnormalities are found. This can be more time-consuming.

22. Example of a panel

23. Workflow in a flow cytometry lab • Sample processing • Acquiring sample on flow cytometer • Data analysis • Interpretation given by Hematopathologist • Result entry into patient’s record

24. Flow case study #1 70 year old patient with a history of myelodysplastic syndrome and complex cytogenetic abnormalities.

25. Flow case study #1

26. Peripheral Blood Lab findings WBC 1.27 K/uL 32% blasts

27. Morphology – case study #1 Blast in peripheral blood smear

28. Morphology – case study #1 Blasts in the marrow CD34 staining (red) in the marrow

29. Flow case study #1 - Diagnosis Hypercellular bone marrow with multilineage dysplasia, increased blasts and marked fibrosis, consistent with myelodysplastic syndrome with fibrosis transforming to acute myeloid leukemia. Other notes: Blasts approach 20% in the marrow and exceed 20% in the peripheral blood, consistent with transformation to acute myeloid leukemia. Additionally, this case shows a unique pattern of segmental/focal severe fibrosis of the marrow. Moderate-severe bone marrow fibrosis has been reported in up to 10% of patients with MDS and is usually associated with multilineage dysplasia, profound cytopenias, and complex cytogenetic abnormalities.

30. Flow case study #2 6 year old with immunodeficiency, EBV viremia, and unknown genetic defect

31. Flow case study #2 B-cell maturation:

32. Flow case study #2

33. Flow case study #2 Gated on CD3+ cells (T-cells)

34. Flow case study #2 - Diagnosis Normocellular marrow with progressive trilineage hematopoiesis, B-cell lymphocytosis and decreased CD4-positive T-cells. No evidence of lymphoma. Rare EBV-positive cells identified. Other Notes: Flow cytometric analysis of the bone marrow aspirate reveals increased precursor B-cells / B-cells that are positive for CD20, CD19, CD10 and negative for CD34. T-cell analysis reveals inverted CD4/CD8 T-cell ratio and reduced CD4 positive T-cells.

35. Flow case study #3

36. Flow case study #3

37. Flow case study #3 TRAP staining - brown

38. Flow case study #3

39. Flow case study #3 - Diagnosis Variably hypocellular marrow involved by residual Hairy cell leukemia, compromising approximately 70% of marrow cellularity. Other notes: Flow cytometric analysis on marrow aspirate detected approximately 6% of the lymphoid cells (95% of B-cells) that were monoclonal B-cells expressing bright CD11c, CD19, bright CD20, bright CD22, CD103, and surface lambda light chain consistent with residual Hairy cell leukemia.