1. Behzad Poopak, DCLS PhD bpoopak@yahoo.com
Payvand
Clinical Specialty Lab.
Flowcytometry
Behzad Poopak, DCLS PhD

2. What Is Flow Cytometry? Flow ~ cells in motion Cyto ~ cell
Metry ~ measure
Measuring properties of cells while in a fluid stream

4. Cytometry vs. Flow Cytometry
Localization of antigen is possible
Poor enumeration of cell subtypes
Limiting number of simultaneous measurements
Flow Cytometry.
Cannot tell you where antigen is.
Can analyze many cells in a short time frame.
Can look at numerous parameters at once.

5. Applications of Flow Cytometry.
Cell size.
Cytoplasmic granularity.
Cell surface antigens (Immunophenotyping).
Apoptosis.
Intracellular cytokine production.
Intracellular signalling.
Gene reporter (GFP).
Cell cycle, DNA content, composition, synthesis.
Bound and free calcium.
Cell proliferation
Cell sorting, single cell cloning

6. Flow cytometry & Hematopathology
1. Distinction between neoplastic and benign conditions, 2. Diagnosis and characterization of lymphomas and leukemias, 3. Assessment of other neoplastic and preneoplastic disorders such as plasma cell dyscrasias and MDS, 4. Detection of MRD in patients with acute leukemia or chronic lymphoid malignancies. 5. In some groups of lymphoid neoplasms, FCM study also provides prognostic information.

7. Principle of Flow Cytometry
Cells in suspension
Cells flow in single-file
Intercepted by light source(s) (laser)
Scatter light and emit fluorescence
Signal collected, filtered and
Converted to digital values
Storage on a computer
Fluidics
Optics
Electronics
Data display and analysis

8. Basic Principles of Flow Cytometry
Single cell or particle suspension
Fluorescent dyes or Abs that can be attached to an antigen or protein of interest
Flow cell, sheath fluid and a focused laser beam

9. The Flow Cell
The introduction of a large volume into a small volume in such a way that it becomes “focused” along an axis is called Hydrodynamic Focusing.
Sheath
Cell
Sample Stream

10. Low Differential High Differential Sample Sample Sheath Sheath Sheath
Core
Stream
Laser Focal Point
Incoming Laser
Low Differential
High Differential

11. Sample Differential Difference in pressure between sample and sheath
10 psi
10.2 psi
10 psi
10.4 psi
10 psi
10.8 psi
Difference in pressure between sample and sheath
This will control sample volume flow rate
The greater the differential, the wider the sample core.
If differential is too large, cells will no longer line up single file
Results in wider CV’s and increase in multiple cells passing through the laser at once. No more single cell analysis!

12. Basic Principles cont’d
Light is either scattered or absorbed when it strikes a cell
Light scatter is dependent on the internal structure, size and shape.
Forward scatter = size of the cell
Side Scatter = complexity of the cell

13. Forward Scatter
Laser Beam
FSC Detector

14. Side Scatter
Laser Beam
FSC Detector
Collection
Lens
SSC
Detector

15. Side scatter
Forward scatter
Granulocytes
Monocytes
Lymphocytes

16. Why Look at FSC v. SSC
Since FSC ~ size and SSC ~ internal structure, a correlated measurement between them can allow for differentiation of cell types in a heterogeneous cell population
FSC
SSC
Lymphocytes
Granulocytes
Monocytes
RBCs, Debris,
Dead Cells

17. Side scatter Forward scatter Low Medium High levels of forward scatter
----> increasing cell size

18. increasing cell granularity Increasing levels of side scatter ---->
Low
High
increasing cell granularity
Increasing levels of side scatter ---->
Medium
Side scatter
Forward scatter

19. BY “GATING” EACH OF THE AREAS IN
2 DIMENSIONS, YOU CAN ADAPT FLOW CYTO-
METRY TO PERFORM DIFFERENTIAL COUNTS!
Side scatter
Forward scatter
Lymphocytes
Monocytes
Granulocytes

20. FLOW CYTOMETRY - Cells are labeled with fluorescent antibodies
directed against cell surface molecules
- Using different color fluorochromes allows
counting of many markers simultaneously
and allows identification of several markers
on the same cell ( Multiparameter Flow)
- In the instrument, cells pass one-by-one past
a laser to excite the fluorochromes and
there are detectors for each type of
fluorochrome

21. - cells are labeled with fluoresence antibodies
Flouresent tag
Surface of a cell, e.g., a lymphocyte
(in solution)

22. Fluorescence
Photon emission as an electron returns from an excited state to ground state

23. What Happens in a Flow Cytometer (Simplified)

24. Fluorochrome

25. Basic Principles cont’d
Fluorescent dyes absorb light of a specific wavelength and reemit light of a different wavelength
Fluorescent signals are detected by PMT and amplified
Optical filters are used to steer light of specific wavelengths to the photo detector
Reflected
Dichroic Filter
Passed
Short or Long Pass Filter
Band Pass Filter
Adsorbed
Absorption Filter

26. Electronics
Electrical pulses are digitized, the data is stored (‘list mode data’), analysed and displayed through a computer system.
The end result is quantitative information about every cell analysed
Large numbers of cells can be processed quickly

27. Comprehensive antibody panels
The rationale :
The lineage of the cells of interest (e.g., myeloid, B-cell, T-cell),
Their maturity status,
The clonality, where appropriate,
The specific subtype of hematopoietic malignancy and
The status of the normal elements present.
Appropriate isotype controls are included in the panels. The evaluation of the FCM data also relies on internal controls, however (e.g., T-cells serve as internal control for B-cells and vice versa)

28. Abs Panel, the European Group for the Immunological Characterization of Leukemias (EGIL) for the diagnosis and classification of acute leukemia

29. Panel of antibodies recommended by the British Committee for Standards in Haematology (BCSH) for the diagnosis and classification of acute leukemia

30. Panel of antibodies recommended by the European LeukemiaNet, ELN for the diagnosis and classification of acute leukaemia

31. Panel of antibodies recommended by the US–Canadian Consensus Group for the diagnosis and classification of acute leukemia

32. Reactivity of mAbs vs. FAB-AML

33. Development of a FACS histogram
45%

34. Fluorescent intensity
Negative cells
Positive cells
Counts
Fluorescent intensity
Intensity scales
are logarithmic
Note: The operator can set the “gate” by visual inspection of
the histogram. The “gate” defines negative versus positive.

35. THE OPERATOR SETS “GATES” DEFINING
POSITIVE AND NEGATIVE FOR EACH MARKER.
CD19
CD3
CD19
CD3
CD CD19+
CD CD3 +
CD19- CD19-
CD3- CD3+
CD CD3+
Therefore, you can define each cell counted with regard to CD19 or CD3 positivity. Note that there are not normally cells in the circulation that express both T and B cell surface markers.

36. Below are the FCM results on a peripheral blood specimen studied) at a teaching hospital:
CD2 48% moderate CD19 47% moderate
CD3 45% moderate CD20 26% moderate
CD4 21% moderate CD22 47% moderate
CD7 47% moderate sIgM 48% moderate
CD8 20% moderate Kappa 3% moderate
CD13 3% moderate Lambda 2% moderate
CD33 1% moderate CD10 36% moderate
CD34 1% weak CD45 100% strong
TdT 55% moderate HLA-DR 55% moderate

37. Interpretation
The results indicated a proliferation of immature cells (TdT+). The case was interpreted as ALL with a mixed (B-cell and T-cell) lineage.
Because of the data-reporting format, it is unclear whether the immature cells are of B- or T-cell lineage, however. Although fluorescence intensities were mentioned, data interpretation in this particular laboratory was actually based on percent positive with an arbitrary 20% cutoff.
When proper visual data analysis was subsequently applied to the raw data, it became apparent that the blood sample contained a clearly identifiable neoplastic population of precursor B-ALL, admixed with a high number of normal T-cells.

38. Steps in Flowcytometry
Preanalytical (specimen handling and processing, including antibody staining),
Analytical (running the sample through the flow cytometer and acquiring data), and
Postanalytical (data analysis and interpretation).
Deficiencies such as suboptimal instrument performance, poor reagent quality (antibodies and/or fluorochromes), or poor specimen quality can all result in inadequate resolution of positive and negative immunofluorescence.

39. Preanalytical Phase
Little control over certain factors, eg. specimen collection and transportation, which can adversely affect the sample prior to its arrival.
Poor specimen collection
The time elapsed between specimen acquisition and delivery to the laboratory, and the environmental conditions during transport are critical factors
As a rule, specimens cannot be held for more than 48 hours in the fresh state after collection. This time window is much narrower for samples harboring a tumor with a high turnover rate (e.g., Burkitt lymphoma).
Exposure to extreme temperatures and the presence of blood clots (or gross hemolysis) are conditions that can render a blood or bone marrow specimen unacceptable for analysis.

40. Fresh specimens for FCM
Liquid samples (peripheral blood, bone marrow, body fluids) and
Solid tissue (lymph nodes, tonsils/ adenoids, spleen, bone marrow biopsies, and extranodal infiltrates).

41. Specimen Type PB & BMA can be collected in either EDTA or heparin.
The volume required depends on the WBC count; 10 mL of blood is adequate in most instances.
Store & transfer at RT (at RT in delay). Referred blood ,should be accompanied by a hemogram and a fresh blood smear
Approximately 3 to 5 mL of BMA is usually sufficient for a comprehensive FCM analysis,
Degenerative changes in BMA tend to occur more quickly than PB

42. Diagram of lymph node slicing and the allocation of the slices to different studies
F, FCM analysis; H, histology; I, immunohistochemistry;
M, molecular studies.
Each slice is less than 2 mm thick.

43. Preparing nucleated cell suspensions
Cell yield and viability
Sample staining
Surface antigens, staining is performed on viable unfixed cells.
All staining is performed at 40C to minimize capping and antigen shedding.
Appropriate isotype controls are included.
The usual number of cells recommended for immunostaining is 106 cells (low cell yield, it is possible to perform the staining with as few as 1 × 105 to 2 × 105 cells/tube)
Intracellular antigens, the staining procedure is more laborious than cell surface antigen staining and calls for cell fixation and permeabilization

44. Case 0f ALL

46. Immunophenotype: Cytochemistry: Interpretation / Diagnosis:
Immunophenotyping of Bone marrow aspirate by flow cytometry shows predominant a B cell population (about 89% of the cells analyzed) The majority of these B cells show expression of CD10, CD19, HLA-DR. They were negative for CD2, CD5, CD7, CD13, CD33, CD20 and Tdt. Review of BMA smear shows a predominant lymphoblast population (65%). Dual Positive for CD10 / CD19.
Cytochemistry:
Myeloperoxidase: All leukemic blasts were MPX negative.
Interpretation / Diagnosis:
Immunophenotyping results, together with morphological findings and Cytochemistry of BMA, are consistent with B lymphoblastic leukemia (Early pre B-cell type).

47. AML-M3 , Classic or Hypergranular type

48. Immunophenotype: Cytochemistry:
Immunophenotyping of BMA by flow cytometry shows a predominant leukemic cell population (about 85% of the cells analyzed, Gated on region 1) that is positive for CD13, CD33 & CD117,CD45. The majority of gated cells were negative for CD2,CD3,CD4,CD5,CD7,CD8,CD10, CD11b, CD11c ,CD14,CD19, CD20,CD25,CD41, CD61, HLA-DR. These cells have intermediate granularity (based on side-scatter signal).
Cytochemistry:
All of the leukemic cells were intensely myeloperoxidase Positive.
Dr. Behzad Poopak, PhD (Hematologist)

49. Immunophenotyping results, together with morphological findings and Cytochemistry of BMA, are consistent with B lymphoblastic leukemia (Early pre B-cell type).

50. Patient Report Format

51. Patient Report-2 Format

52. Hematogone vs.Leukemic Lymphoblast

53. Diagnosing Multiple Myeloma
Three Diagnostic Criteria Required
for a Positive Diagnosis of Multiple Myeloma 1
Monoclonal plasma cells present in the bone marrow ≥10%
Presence of a documented plasmacytoma 2
Presence of M component in serum and/or urine* 3
One or more of the following (CRAB criteria):
Calcium elevation (serum calcium >11.5 mg/dL)
Renal insufficiency (serum creatinine >2 mg/dL)
Anemia (hemoglobin <10 g/dL or 2 g/dL <normal)
Bone disease (lytic lesions or osteopenia)
*Monoclonal M spike on electrophoresis IgG >3.5 g/dL, IgA >2 g/dL, light chain >1 g/dL in 24-hour urine sample.
Durie et al for the International Myeloma Working Group. Leukemia. 2006:1-7.

54. Diagnostic Evaluation of Multiple Myeloma
Test
Finding(s) With Myeloma
CBC with differential counts
↓ Hgb, ↓ WBC, ↓ platelets
Electrolytes
↑ Creat, ↑ Ca+, ↑ Uric acid, ↓ Alb
Serum electrophoresis with quantitative immunoglobulins
↑ M protein in serum, may have ↓ levels of normal antibodies
Immunofixation
Identifies light/heavy chain types M protein
β2-microglobulin
↑ Levels (measure of tumor burden)
C-reactive protein
↑ Levels (marker for myeloma growth factor)
24-hour urine protein electrophoresis
↑ Monoclonal protein (Bence Jones)
Bone marrow biopsy
≥10% plasma cells
Skeletal imaging
Osteolytic lesions, osteoporosis
Serum free light chain
↑ Free light chains
MRI
Evaluation of involvement of disease
The early stages of multiple myeloma are often asymptomatic and the disease may be discovered on routine laboratory testing
When myeloma is suspected (ie, older patients with unexplained pain, fracture, anemia), the diagnostic evaluation should include:
Laboratory tests of both serum and urine
Radiographic imaging
Bone marrow biopsy
The alterations associated with myeloma are listed here
Quantitative immunoglobulins measure the antibodies IgG, IgA, IgM, IgE, and IgD with normal ranges: (IgG mg/dL; IgA ( mg/dL; IgM mg/dL; IgE IU/ml)
Beta 2 microglobulin is a standard measure of tumor burden for myeloma (normal range, 2.0–2.5 µg/mL)
C-reactive protein is a surrogate marker for IL-6, a growth factor for myeloma cells
A 24-hour urine protein electrophoresis (UPEP) measures the presence and amount of myeloma protein in the urine
A serum-based assay (Freelite™) detects and quantifies free light chains, which may help predict the risk of progression from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma. This test is also easier for patients to complete than a 24-hour urine collection.
Emerging evidence suggests that use of magnetic resonance imaging (MRI) is more sensitive and specific than x-ray and scintigraphy for detecting bone metastases in multiple myeloma patients. Thus, MRI may also be used for initial assessment of multiple myeloma.
References:
Abella HA. MR tops x-ray and scintigraphy for detecting bone mets. Oncology News International. 2007;16(12):27.
Barlogie B, Shaughnessy J, Epstein J, et al. Plasma cell myeloma. In: Lichtman MA, Beutler E, Kipps TJ, Seligsohn U, Kaushansky K, Prchal JT, eds. Williams Hematology. 7th ed. New York, NY: McGraw-Hill; 2006:
Durie BGM, Kyle RA, Belch A, et al. Myeloma management guidelines: a consensus report from the Scientific Advisors of the International Myeloma Foundation Hematol J. 2003;4: [erratum Hematol J. 2004;5:285].
Multiple Myeloma Research Foundation (MMRF). Multiple Myeloma: Disease Overview. Norwalk, CT: Multiple Myeloma Research Foundation; Available at: Accessed January 8, 2008.
Rajkumar SV, Kyle RA, Therneau TM, et al. Serum free light chain ratio is an independent risk factor for progression in monoclonal gammopathy of undetermined significance. Blood. 2005;106(3):
Weininger M, Lauterbach B, Knop S, et al. Whole-body MRI of multiple myeloma: comparison of different MRI sequences in assessment of different growth patterns. Eur J Radiol. December 2007 [Epub ahead of print; Doi: /j.ejrad ].
Alb = albumin; CBC = complete blood count; Creat = creatinine; Hgb = hemoglobin;
MRI = magnetic resonance imaging; WBC = white blood cell
Abella. Oncology News International. 2007;16:27; Barlogie et al. In: Williams Hematology. 7th ed. 2006:1501; Durie et al. Hematol J. 2003;4:379; MMRF. Multiple Myeloma: Disease Overview Rajkumar et al. Blood. 2005;106(3):812.

56. Peripheral blood - rouleaux
B.Poopak

57. Malignant Plasma Cells in Marrow

58. Myeloma: A Cancer of Plasma Cells in the Bone Marrow
The function of plasma cells is to produce antibodies. On bone marrow aspirate and biopsy, plasma cells are readily identifiable as the large cells in this photomicrograph with an eccentric nucleus and abundant blue cytoplasm. Typically, they have a lighter area in the cytoplasm that is adjacent to the nucleus (“perinuclear huff”). On electron microscopy, this clear area corresponds to the golgi apparatus. In people with myeloma, the percentage of plasma cells is increased in the bone marrow. When there is an excess of plasma cells, there is usually an increase in antibodies that can be detected in the blood. 58

59. Patients with multiple myeloma show a "spike" in special regions of the serum protein electrophoresis

61. Serum Protein Electrophoresis
Monoclonal Protein in Myeloma
Normal
Kyle RA and Rajkumar SV. Cecil Textbook of Medicine, 22nd Edition, 2004

63. Figure 8. Quantitative immunoglobulins were within normal limits
Maslak, P. ASH Image Bank 2001;2001:100211
Copyright ©2001 American Society of Hematology. Copyright restrictions may apply.

64. Lazarchick, J. ASH Image Bank 2001;2001:100185
Figure 8. Immunofixation electrophoresis showing a monoclonal IgA lambda light chain restricted band
Lazarchick, J. ASH Image Bank 2001;2001:100185
Copyright ©2001 American Society of Hematology. Copyright restrictions may apply.

65. Immunofixation to Determine Type of Monoclonal Protein
IgG kappa M protein
Lambda Light Chains
Kyle RA and Rajkumar SV. Cecil Textbook of Medicine, 22nd Edition, 2004

68. CASE STUDY – MULTIPLE MYELOMA
Serum free light chains
Free kappa – mg/L
Free lambda – mg/L
Ratio – 1.7
Interpretation
Free lambda light chain monoclonal gammopathy
Radiology
Diffuse osteolytic lesions in thoracic and lumbar regions with several compression fracturres

69. Normal serum Immunofixation Report: Polyclonal Pattern

70. serum Immunofixation Report: Mono-Clonal IgA – Kappa Pattern

71. serum Immunofixation Report: Mono-Clonal IgM – Kappa Pattern

72. Thank you, any question?