Flow cytometry

flowcytometry, introduction, basics, case studies, CLPD, acute leukaemia, markers

1. FLOW CYTOMETRY Moderator: Dr. R.M. Jaiswal By: Dr. Megha Gupta & Dr. Tashi Agarwal

2. FLOW CYTOMETRY Definition: Measuring properties of cell as they flow in a fluid suspension across an illuminated light path.

3. Basic mechanism Biological sample Label it with a fluorescent marker Cells move in a linear stream through a focused light source (laser beam) F luorescent molecule gets activated and emits light that is filtered and detected by sensitive light detectors (usually a photomultiplier tube) C onversion of analog fluorescent signals to digital signals

4. Flow Cytometry This method allows the quantitative and qualitative analysis of several properties of cell populations from virtually any type of fresh unfixed tissue or body fluid. The properties measured include a particle’s related size, relative granularity or internal complexity, and relative fluorescence intensity Most commonly analyzed materials are: blood, bone marrow aspirate and lymph node suspensions.

5. Principle of Flow Cytometry Flow cytometer is composed of three main components: The Flow system (fluidics) Cells in suspension are brought in single file past The Optical system (light sensing) a focused laser which scatter light and emit fluorescence that is filtered and collected The Electronic system (signal processing) emitted light is converted to digitized values that are stored in a file for analysis

6. The F low System O ne of the fundamentals of flow cytometry is the ability to measure the properties of individual particles, which is managed by the fluidics system. W hen a sample is injected into a flow cytometer, it is ordered into a stream of single particles. T he fluidic system consists of a FLOW CELL (Quartz Chamber): Central channel/ core - through which the sample is injected. Outer sheath - contains faster flowing fluid k/a Sheath fluid ( 0.9% Saline / PBS ) , enclosing the central core.

7. Hydrodynamic Focusing Once the sample is injected into a stream of sheath fluid within the flow chamber, they are forced into the center of the stream forming a single file by the PRINCIPLE OF HYDRODYNAMIC FOCUSING. 'Only one cell or particle can pass through the laser beam at a given moment .'

8. The sample pressure is always higher than the sheath fluid pressure , ensuring a high flow rate allowing more cells to enter the stream at a given moment. High Flow Rate - I mmunophenotyping analysis of cells Low Flow Rate - DNA Analysis Sheath Tank Waste Tank Line Pressure Vacuum Sample Pressure (Variable) Sheath Pressure (Constant) Sample Tube

9. OPTICS A fter the cell delivery system, the need is to excite the cells using a light source. The light source used in a flow cytometer: Laser (more commonly) Arc lamp Why Lasers are more common? They are highly coherent and uniform. They can be easily focused on a very small area (like a sample stream). They are monochromatic, emitting single wavelengths of light. ARGON Lasers - 488nm wavelength (blue to blue green)

10. W hen a light intersects a laser beam at the so called ' interogation point' two events occur: a) light scattering b) emission of light (fluorescence ) Fluorescence is light emitted during decay of excited electron to its basal state.

11. OPTICS a) LIGHT SCATTER When light from a laser interrogates a cell, that cell scatters light in all directions. The scattered light can travel from the interrogation point down a path to a detector.

12. OPTICS - FORWARD SCATTER (FSC) Light that is scattered in the forward direction (along the same axis the laser is traveling) is detected in the Forward Scatter Channel . The intensity of this signal has been attributed to cell size , refractive index (membrane permeability ).

13. OPTICS - SIDE SCATTER (SSC) Laser light that is scattered at 90 degrees to the axis of the laser path is detected in the Side Scatter Channel . The intensity of this signal is proportional to the amount of cytosolic structure in the cell ( eg . granules, cell inclusions, etc.) Side scatter detector Measuring cell granularity

14. FSC Detector Collection Lens SSC Detector Laser Beam

15. FSC SSC Lymphocytes Monocytes Granulocytes RBCs, Debris, Dead Cells Study of FSC and SSC allows us to know the differentiation of different types of cells. Why FSC & SSC?

16. T he light scattered in the forward direction is proportional to the square of the radius of a sphere, and so to the size of the cell or particle. The cells are labelled with fluorochrome -linked antibodies or stained with fluorescent membrane, cytoplasmic or nuclear dye.

17. Commonly used Fluorochromes FLUOROCHROMES EMISSION MAXIMUM Fluorescein Isothiocynate (FITC) 530nm Phycoerythrin (PE) 576nm Peridin -chlorophyll alpha complex ( PerCP ) 680nm Allophycocyanin (APC) 660nm Texas red 620nm ECD( PE - Texas Red Tandem) 615nm PC5 (PE - cyanin 5 dye tandem) 667nm

18. Optics B) EMISSION OF FLUORESCENT LIGHT (FLUORESCENCE) As the fluorescent molecule present in or on the particle is interrogated by the laser light, it will absorb energy from the laser light and release the absorbed energy at longer wave length . Emitted photons pass through the collection lens and are split and steered down specific channels with the use of filters . Emitted fluorescence intensity is proportional to the amount of fluorescent compound on the particle.

19. Optics- Filters Different wavelengths of light are scattered simultaneously from a cell Need to split the light into its specific wavelengths in order to measure and quantify them independently. This is done with filters. The system of filters ensures that each photodetector receives light bands of various wavelengths. Optical filters are designed such that they absorb or reflect some wavelengths of light, while transmitting others. Types of filters 1. Long Pass 2. Short Pass 3 . Band Pass 4. Dichroic

20. Optics- Long Pass Filters Transmit all wavelengths greater than specified wavelength Example: 500LP will transmit all wavelengths greater than 500nm 400nm 500nm 600nm 700nm Transmittance Original from Cytomation Training Manual

21. Optics- Short Pass Filter Transmits all wavelengths less than specified wavelength Example: 600SP will transmit all wavelengths less than 600nm. 400nm 500nm 600nm 700nm Transmittance Original from Cytomation Training Manual

22. Optics- Band Pass Filter Transmits a specific band of wavelengths Example: 550/20BP Filter will transmit wavelengths of light between 540nm and 560nm (550/20 = 550+/-10, not 550+/-20) 400nm 500nm 600nm 700nm Transmittance Original from Cytomation Training Manual

23. Optics- Dichroic Filters Long pass or short pass filters Placed at a 45º angle of incidence Part of the light is reflected at 90º , and part of the light is transmitted and continues. Dichroic Filter Detector 1 Detector 2

24. OPTICS - DETECTORS The photodetectors convert the photons to electrical impulses. Two common types of detectors used in flow cytometry : Photodiode used for strong signals, when saturation is a potential problem ( eg , forward scatter detector). Photomultiplier tube (PMT) more sensitive than photodiode but can be destroyed by exposure to too much light. used for side scatter and fluorescent signols .

25. ELECTRONICS The electronic subsystem converts photons to photoelectrons. M easures amplitude, area and width of photoelectron pulse. I t amplifies pulse either linearly or logarithmically and then digitalizing the amplified pulse.

26. Time  Photons / Detector ( Voltage) Electronics- Creation of a Voltage Pulse

27. Data Analysis- Plot Types There are several plot choices: Single Color Histogram Fluorescence intensity (FI) versus the number of cells counted. Two Color Dot Plot FI of parameter 1 versus FI of Parameter 2 Two Color Contour Plot Concentric rings form around populations. The more dense the population, the closer the rings are to each other Two Color Density Plot Areas of higher density will have a different color than other areas

28. Plot Types Contour Plot Density Plot Greyscale Density Dot Plot www.treestar.com Histogram

29. DATA ANALYSIS - GATING Gating is in essence electronic window that sets upper and lower limits on the type and amount of material that passes through. Selection of only a certain population of cells for analysis on a plot. Allows the ability to look at parameters specific to only that subset.

30. Interpretation of Graphs An important tool for evaluating data is the dot plot . The instrument detects each cell as a point on an X-Y graph. This form of data presentation looks at two parameters of the sample at the same time .

31. Three common modes for dot plots are: Forward scatter (FSC) vs. side scatter (SSC) To look at the distribution of cells based upon size & granularity Single color vs. side scatter T o visualize the expression of the fluorescence of the cells Two-color fluorescence plot. To differentiate between those cells that express only one of the particular fluorescent markers, those that express neither, and those that express both . used to discriminate dead cells from the live ones that are expressing the desired fluorescence .

32. When to say an antigen is positive or negative? A sample that has some cells single positives for CD8 along the x-axis ( green arrow ) some single positives for CD4 along the y-axis ( red arrow ) . U pper right quadrant of the plot - cells positive for both fluorescent markers ( purple arrow ) . Lower left quadrant - cells negative for both markers ( orange arrow ).

33. How to differentiate dim & bright expression of an antigen? Dim : cells are present more towards the origin(0) on x( red ) - y axis ( pink ) Bright : cells are present away from the origin(0) on x( green ) & y( yellow ) axis. DIM BRIGHT Y-axis CD4 X-axis CD8

34. WHAT IS UNIQUE IN FLOWCYTOMETRY MULTIPARAMETRIC RAPID ANALYSIS OF LARGE NUMBER OF CELLS INFORMATION AT A SINGLE CELL LEVEL DETECTION OF RARE CELL POPULATIONS ALLOWS PHYSICAL ISOLATION OF CELLS OF INTEREST

35. USES OF FLOWCYTOMETRY

36. APPLICATIONS ANALYSIS Immunophenotyping Dyes that bind to nucleic acids (DNA, RNA) Functional assays CELL COUNTING CELL SORTING

37. CLINICAL APPLICATIONS

38. Cont..

39. CLPD on flowcytometry

40. Objectives Diagnosis of lymphoma Classification of lymphoma Ploidy analysis

41. Flow cytometric approach to the diagnosis and classification of B- cell lymphoid neoplasms . B Cell Lymphoma

42. B CELL DIFFERENTIATION

43. MONOCLONALITY CD 13, CD 33, CD 5 ON B CELLS

44. Normal, polyclonal B-cells are a mixture of kappa-B-cells and lambda-B-cells. A B-cell carries either kappa- or lambda-light chain on its surface. And normal polyclonal B-cells are a mixture of kappa-B-cells and lambda B-cells as can be seen in the left-hand figure. Monoclonal mature B-cells are either kappa or lambda.  If a malignant B-cell clone proliferates this will result in a B-cell population consisting of either only kappa- or only lambda-B-cells. The latter case (i.e. lambda-monoclonal B-cells) is symbolized in the left-hand figure. 

45. Expression of CD5 The arrow in the right panel points to the abnormal, strong expression of CD5 by B-cells. CD5 expression as strong as this can usually only be found on T-cells. Normal B-cells show no or only a weak expression of CD5 (left-hand panel) Weak expression of CD20 The B-cells in the right panel show only a weak expression of CD20 (arrow). For comparison: normal CD20 expression in the left-hand panel.

46. APPROACH TO B CELL LYMPHOMA CD5 POSITIVE NEGATIVE CD23 - FMC7 + CD23 + FMC7 - CD23 + FMC7 + MCL MARG : CD38-,CD23-, FMC7 + DLBCL: CD38+ LPL: CD38+ CLL PLL CD10 POSITIVE NEGATIVE FOLLICULAR: FMC7 + DLBCL BURKITT: CD23 - FMC7 + B-ALL: CD23 - FMC7 - CD103,CD25, CD123 POSITIVE-HCL NEGATIVE

47. Chronic lymphocytic leukemia Typical phenotype: CD20 (d), CD22 (d), sIg (d), CD23+ FMC-7- Characteristic morphology Testing for the prognostic markers CD38 and ZAP-70 can be considered

48. Mantle cell lymphoma Variable phenotype not typical for CLL; often CD20 ( i ), sIg ( i ), CD23-, FMC-7 + IHC : Cyclin-D1 FISH : t(11;14)/CCND1 rearrangement

49. Hairy cell lekaemia Typical pheotype : CD20 (b), CD22 (b), CD11c (b), CD25+, CD103+, sIg ( i ) Confirm characteristic morphology of a hairy cell and TRAP + A small subset of HCL are CD10+ but are morphologically similar to CD10- HCL.

50. Follicular lymphoma Usually bcl-2, CD43. Some follicular growth. t(14;18)/ BCL-2 rearrangement. D/D DLBCL : diffuse growth pattern against the nodular growth pattern in FL BL : morphologial (vacuoles), High S phase fraction.

51. 80/F c/o cervical adenopathy On CBC : an absolute lymphocytosis ≥5 × 10 9 /L; PBF is flooded with small mature lymphocytes with condensed chromatin and scant cytoplasm along with numerous smudge cells . CASE

52. On flow A diagnosis of CLL can be made. CD5+ CD23+

53. Not that simple A certain immunophenotype may be typical but is by no means obligatory. The significance of one marker depends on the expression of other markers.   The strength of antigen expression is important.

54. Flow cytometric approach to the diagnosis and classification of T- cell lymphoid neoplasms . T CELL LYMPHOMA

55. CD4/CD8 Ratio Loss of CD3 Overexpression of CD5

56. Normally, the CD4/CD8-T-Cell ratio in peripheral blood is about 2:1 . In a T-lymphocytic leukemia this ratio can shift dramatically. Unfortunately, this ratio may also be altered by many non-malignant diseases. eg viral infections. Therefore, only extreme alterations of this ratio can be regarded as a sign for T-lymphocytic malignancy.

57. CD4/CD8 coexpression In the right-hand dot-plot you can see cells that express both the CD4 and the CD8-antigen (arrow) which is highly irregular. In addition both antigens are expressed weakly (compared to normal T-cells). Left-hand panel shows a normal situation. Loss of CD3 Overexpression of CD5 In the right-hand dot-plot you can see T-cells which overexpress CD5 while they lack CD3 (arrow). Only a few normal T-cells are present. (blue oval). Left-hand panel shows a normal situation.

58. PROBLEMS IN DIAGNOSIS OF T-CLPD Relatively low incidence. 5-25% of all lympoid neoplasms . Clinico -biological heterogeneity. Lack of distinctive genetic markers.

59. T CELL DIFFERENTIATION

60. T- CLPD BY FLOWCYTOMETRY

61. CTCL/ Sézary syndrome Often CD7-, CD26-, CD4+, CD25+/- (with heterogeneous staining intensity). Confirm characteristic morphology and clinical presentation. HTLV-1-

62. DNA PLOIDY S PHASE FRACTION PLOIDY ANALYSIS

63. Cell cycle analysis The percentage of the cells in each region is analyzed. In normal tissues – 95% cells - G0/G1 phase 2.5% cells - S phase 2.5% cells - G2/M phase In neoplasm, percentage of cells in S and G2/M phase increases which is expressed as S phase fraction or the proliferation index

64. S Phase, synthesis phase. It is the part of cell cycle in which DNA is replicated occurring between G1phase and G2 phase.

65. S phase has strong correlation with grading. DNA ploidy has no correlation with grading. ADVANTAGES OVER IHC With IHC: cells in G1, S and G2 phases cannot be differentiated.

66. Highest proliferative activity: mean SPF, 35.3% 6.6% 6.5% 20.4%

67. Diagnosis of acute leukaemias on flowcytometry

68. STEPS Finding the blast population Defining the immunophenotype Diagnosis

69. CD38 / CD45 AGAINST SSC CD19, 7 on non lymphoid cell

70. Finding immature cells using CD45-CD34 dot-plots The arrows points at the blast populations, which is very conspicuous in case AL 1 (upper right) and AL 3 (lower right). In case AL 2 (lower left), the difference between the normal picture is more subtle and the blasts may be missed because in this case the blasts are CD34 negative.

71. Intermediate CD45 and low side scatter BLAST WINDOW NEUTROPHILS LYMPHOCYTES MONOCYTES RBC’S AND DEBRIS B CELLS CD45/SSC gating strategy is more sensitive than FSC/SSC gating and it dilineates the blasts well.

72. Finding immature cells using  CD45-Side Scatter dot-plots The three cases of acute leukemia: The arrows point to the blast populations which are clearly visible in all three cases.  Even the blasts of case AL 2 can easily be spotted.

73. BLAST WINDOW B CELLS MONOCYTES RBC’S AND DEBRIS LYMPHOCYTES NEUTROPHILS CD45/SSC gating strategy is more sensitive than FSC/SSC gating and it dilineates the blasts well.

74. Example of an abnormal antigen expression on myeloid blasts Compare the normal blasts (upper dot-plot, blue oval) with those of an acute myeloid leukemia (lower dot-plot, red oval)): the malignant blasts abnormally express CD15 and they show an increased expression of CD34. Note: CD34-negative cells have been removed for reason of clarity.

75. DIAGNOSIS WHICH ONES TO IMMUNOPHENOTYPE? Equivocal morphology Cytochemistry is noncontributory Specific subtypes LEUKAEMIA VS NON LEUKAEMIA Overlapping morphology. Eg : hematogones , viral infections . Partially treated acute leukaemia

76. PROGNOSTIFICATION CYTOGENIC AND MOLECULAR ABNORMALITIES Association with specific cytogenic abnormalities DNA ploidy RESIDUAL DISEASE MONITORING

77. CLASSIFICATION Acute leukaemia is classified on the basis of immunological markers into B lineage ALL T lineage ALL Acute myeloid leukaemia Acute leukaemia of ambiguous lineage

78. How to define the lineage of leukaemia THE FLOW CYTOMETRIC EVALUATION OF HEMATOPOIETIC NEOPLASIA Brent L. Wood, Michael J. Borowitz . Henry’s, 22 nd edition, Chapter 34

79. Flow cytometric approach to the diagnosis and classification of ALL. ACUTE LYMPHOID LEUKAEMIA

80. How to diff ALL from NHL CD 34 TdT Bcl2 CD99 NHL cases with spillover demonstrate bright CD45 expression while it is moderate in B ALL.

81. Subtypes of ALL Flow cytometric immunophenotyping does not provide a suitable surrogate tool for detection of these subtypes of ALL. Subtype ALL HLA-DR TdT CD 10 CD 19 SmIg CyCD79a Pro- B ALL +/- + - + - + Common ALL + + + + - + Pre B ALL + - - + - + Mature B ALL - - - + +K/L +

82. Precursor B cell lymphoblastic leukemia/lymphoma

83. Flow cytometric approach to the diagnosis and classification of AML. ACUTE MYELOID LEUKAEMIA

84. Diagnosis of AML Morphology Auer rod Cytochemistry >3% MPO positive Immunophenotyping CD33, CD13, CD117, anti-MPO Cytogenetics t(8;21), t(15;17), inv16, MLL, t(9;11), t(6;9), t(3;3), t(1;22)

85. CD markers used for hematolymphoid neoplasms All white cells CD 45 (LCA) Myeloid cells Anti- MPO, CD13, CD33, CD14, CD117 Monocytic Markers CD14, CD64 Megakaryocytic Marker CD41, CD61 B-cells cyCD22, CD22, CD19, CD20, FMC7, CD23, CD79a, CD79b, SmIg , IgM T-cells cyCD3, CD3, CD2, CD5, CD7, CD8, TCR-α/β, TCR- γ / δ NK cells CD16, CD56, CD57 Plasma cells CD38, CD138, Kappa & Lambda chains Blasts CD34, TdT Others HLA-DR, CD55, CD59, cyclin D1, glycophorin A

86. Myeloblast characterization 13+, 15+, 33+, anti-MPO+ Clinical, Genetic, Morphologic Erythroid Megakaryocytic Myeloid Monocytic 41+ 61+ 71++ GlyA + 36+, 64+, 14+, 33++ 36+

87. Classification - FAB M0 : AML-minimal differentiation M1 : AML-without maturation M2 : AML-with maturation (blast<80%) M3 : AML- promyelocytic M4 : AML- myelomonocytic (>20% monocytes) M5 : AML- monocytic M6 : AML- erythroid M7 : AML-megakaryocytic

88. AML- minimal differentiation (M0) M yeloblasts - < 3% positivity with SBB, MPO & PAS-, NSE- M yeloid antigens - CD13+, CD33+, CD117+, and/or MPO+ CD34 , CD38, HLA-DR, and TdT - often expressed

89. AML- Promyelocytic Leukemia (M3) Phenotype - CD13h+, CD33++, CD34-, HLA-DR- Diagnostic molecular alteration - PML/RARA t(15;17) translocation Strongly positive - MPO, SBB, PAS cytoplasmic positivity. Characteristic morphology D/D : AML- monocytic leukemia (M5) - HLA-DR+, CD11c+, CD14+ & CD64+

90. AML - Myelomonocytic Leukemia (M4) Phenotype: myeloid antigens - CD13+ & CD33+, HLA-DR+ monocytic markers: CD14+, CD4+, CD11b+, CD11c+, CD64+, CD36+, CD68+ Blasts >20% of marrow NEC Monocytic component >20% of NEC & monocytes in blood >5 x 10 9 /L

91. AML - M onocytic Leukemia (M5) Phenotype: CD33 (b), CD13+, HLA-DR+ Characteristic CD14+, CD11b+, CD11c+, CD64+, CD68+ Cytochemistry : NSE + M5a : Acute monoblastic leukemia M5b : Acute monocytic leukemia

92. CD34 APC CD15 FITC CD56 A488 CD45 CD4

93. AML - Megakaryocytic leukemia (M7) CD41+, CD61+, CD13+, CD33+ CD34, HLA-DR - Negative

94. CASE 29yrs/ F O/E : Fever, Pallor, Gum hyperplasia, Hepatosplenomegaly . CBC : Hb-5.2 g%, Plt - 19,000/ cu.mm PBF : shows blasts and dual differentiation to granulocytes and monocytoid cells (large cells, abundant pale blue cytoplasm, lobulated or indented nucleus with variable nucleoli). DLC B lasts 40 P 8 L 10 Monocytoid 41 E 1

95. red - dim CD45and low side scatter. P ositive for - CD13, cyMPO , CD34, HLA-DR, CD33, CD11c Negative for - CD10, CD19, CD3, CD79a. Blue - bright CD45 & moderate side scatter. P ositive for - CD14, CD11c, CD13. Negative for - CD34, cyMPO , CD3, CD10, CD19 ACUTE MYELOMONOCYTIC LEUKEMIA

96. Hematogones P hysiologic precursors of maturing B-cells. Confused with neoplastic immature lymphoid cells of B lymphoblastic leukemia/ lymphoma or B-ALL. Increased in: Autoimmune or congenital cytopenias Solid organ tumors e.g. neuroblastoma AIDS NHL Post-chemotherapy and after BMT Copper deficiency

97. Morphologically, hematogones resemble lymphoblasts. Hematogones can be differentiated from lymphoblasts by Unique Immunophenotypic pattern :  CD34 < TdT < CD20 < PAX5  V ariable CD10 & CD20 " J shaped trail pattern " : on CD10/20 Dot plot

98. Lymphocytes Hematogones Immunophenotypic analysis of hematogones in 662 consecutive bone marrow specimens by 4-color flow cytometry . Mckenna et al, BLOOD, 15 OCTOBER 2001

99. Acute leukaemia of ambiguous lineage Mixed phenotype acute leukaemia Acute undifferentiated leukaemia NK/ plasmacytoid dendritic cell leukaemia

100. MPAL – WHO 2008 Or Or Or

101. EGIL scoring system The European group for the Immunological Classification of Leukaemias (EGIL) scoring system

102. Flowcytometry analysis in MRD detection Minimal Residual Disease detection

103. At diagnosis the tumour burden is approximately 10^12 leukaemic cells. Induction chemothereapy achieves a 3 log cell kill bringing it down to 10^9 leukaemic cells. Light microscopy of BMA can detect leukaemia only when there are more than 5 blasts/ 100 nucleated cells. Anything less than that is termed remission. Introduction

104. Introduction What is Minimal residual disease or MRD? It is that submicroscopic disease that cannot be detected by conventional light microscopic examination of the BMA. It could be as high as 1 billion leukaemic cells. It can be performed by two techniques: FCM & PCR.

105. Used mainly in - Acute leukaemia for guiding thereapy as well as prognostic purposes. Patients with low grade B cell malignancies undergoing high dose chemotherapy. Post stem cell transplant and immunothereapy . Lymphoma spillover.

106. REFERENCES THE FLOW CYTOMETRIC EVALUATION OF HEMATOPOIETIC NEOPLASIA Brent L. Wood, Michael J. Borowitz . Henry’s, 22 nd edition, Chapter 34 ATLAS AND TEXT OF HEMATOLOGY. Dr Tejinder singh Manual: 6 th Advanced TCS Flowcytometry workshop on hematological malignancies. Flow Cytometry in Hematopathology . A visual approach to data analysis and interpretation. Doyen, Lawrence and Raul. Flow Cytometric   Analysis of Leukemia and Lymphoma - The Basics Univ.Doz.Dr.med . Wolfgang Hübl