ROLE OF FLOW CYTOMETRY IN LEUKEMIAS

ROLE OF FLOW CYTOMETRY IN LEUKEMIAS

1. ROLE OF FLOW CYTOMETRY IN LEUKEMIAS Presenter: dr. hajra k. mehdi Moderator: dr. CSBR PRASAD SRI DEVARAJ URS MEDICAL COLLEGE, KOLAR

2. INTRODUCTION Flow –cells in motion Cyto – cells Metry - measure Measuring multiple physical and chemical properties of cells while in fluid stream. Quantitative & qualitative analysis. Expensive & sophisticated technology used increasingly from research to clinical laboratories.

3. 1986 1995 2004

4. Principle An optical-to-electronic coupling system that records how a cell scatters incident laser light and emits fluorescence. This process is performed at a rate of thousands of cells (5000-10000) per second. It can detect the size of cell as small as 0.1um.

5. 5 MAIN COMPONENTS Flow cell Optical system Detector & Analogue to Digital conversion(ADC) Amplification system Computer software

6. Instrument Overview

7. FLOW CELL Transports cells in a fluid stream to laser beam for interrogation. Monodisperse suspension Cells flow in single file. SHEATH FLUID SAMPLE CORE STREAM LASER

8. Light scattering Due to laser light reflecting & refracting off the cells without alteration of wavelength. Forward scatter Side scatter

9. Y Laser Side Scatter (SSC) 90° deflection ~ Cell structures Forward Scatter (FSC) < 10° deflection ~ Cell size Fluorescence Intensity Antigen Density

10. Forward scatter Light scattered in forward direction. 1-10 degrees Proportional to size of cell Refractive index & absorptive properties. Larger cells scatter more light than smaller cells.

11. Forward scatter detector The magnitude of the voltage pulse recorded for each cell is proportional to the cell size.

12. Side Scatter Light reflected from internal structures of the cell. Correlates with granularity of the cell. The signals collected by the side-scatter detector located 90 degrees from the laser’s path.

13. FLUORESCENSE Fluorochrome – absorb light over a range of wavelength & emit light at longer wavelength (stoke’s shift). Absorption of light electron to be raised to a higher energy level. Excited electron quickly decays to its ground state emitting excess energy as photon of light. Transition energy termed as fluorescence.

14. Common Fluorochromes Phycoerythrin (PE) Fluorescein Isothiocyanate (FITC) Allophycocyanin (APC) Peridinin-Chlorophyll Protein ( PerCP ) Tandem Dyes PE-Texas Red (ECD) PE-Cy5 (PC5) PE-Cy5.5 (PC5.5) PE-Cy7 (PC7) APC-Cy7

15. Optical filters Direct specified wavelengths of light to designated optical detectors. Side scatter & fluorescent light collected together & then separated in order to detect them independently. LASER

16. A typical arrangement for measuring four components is shown:

17. Types of Optical Filters

18. Cellular Parameters Measured by Flow No reagents or probes required (Structural) Cell size (Forward Light Scatter) Cytoplasmic granularity (90 degree Light Scatter)

19. Cellular Parameters Measured by Flow Reagents are required. Structural DNA content DNA ratios RNA content Functional Surface and intracellular receptors. DNA synthesis DNA degradation (apoptosis) Cytoplasmic Ca++ Gene expression

20. Data display Once a data file has been saved, the data can be displayed in a number of different plots Histograms 2-D plot s 3-D plots -Scattergram - tomogram plot - Density plot - Contour plot

21. Dot plot Bivariate display/ "scattergram" / bitmap Plots one dot or point on the display for each cell which passes through the instrument.

22. Amount of Blue Markers Amount of Yellow Markers Remember this 1 2 3 4

23. Density plot Density plots simulate a three dimensional display of events . “Third" parameter being the number of events. Usually coloured Shades of grey, indicate the relative numbers of events.

24. Gating It is done to isolate cell subpopulations of interest. Eliminates results from unwanted particles/ cells. Eliminates the need to sort cells physically to study their characteristics. Done electronically or manually.

25. Flow cytometry protocol SPECIMEN HANDLING & SAMPLE PREPARATION Specimen -single cell suspension -peripheral blood, bone marrow, effusions, CSF, urine, semen Blood & BM - EDTA/heparin - RBC lysis – ammonium chloride

26. METHODOLOGY Pipette 100 ul of specimen into a tube Add appropriate monoclonal antibody combination labelled with fluorescent dye (5-20 microl) Incubate at room temperature for 15 min Add 1 ml of lysing solution Centrifuge for 5 min at 2000 rpm & discard supernatant Add 2ml of PBS (Phosphated buffer saline) Centrifuge for 5 min at 2000 rpm & discard supernatant Repeat washing with PBS second time Re-suspend the cells in 0.2 to 0.5 ml of sheath fluid ( Isotoin ) Read on flow cytometer Collect ,store & analyze the data

27. Immunophenotyping A process used to identify cells, based on the types of antigens or markers on the surface of the cell. It is one of the application of flow cytometry The technique is called "immunophenotyping" for 2 reasons: 1) It is dependent on the activity of antibodies, which are immunological substances 2) It is used chiefly to identify lymphoid and hematopoietic cells, which are part of the immune system.

28. Because of being a fast, objective, and quantitative method, flow cytometry has now become the preferred method for : (1) lineage assignment. (2) maturational characterization of malignant cells. (3) detection of clonality . (4) heterogeneity and aberrant features of the malignant cell populations. (5) quantitation of hematopoietic cells. (6) To detect minimal residual disease.

29. USES Hematological malignancies (leukemias & lymphomas) The diagnosis & classification Assessment of biological parameters associated with prognosis Detection of antigen used as therapeutic targets Detection of residual neoplastic cells following therapy Plasma cell neoplasms Myelodysplastic syndrome & Myeloproliferative disorders Paroxysmal Nocturnal Hemoglobinuria

30. Immunophenotyping in leukemias Antigen expression in normal cells is tightly regulated process resulting in characteristic pattern of antigen acquisition & loss with maturation that is cell lineage specific. Gain of antigens not normally expressed by cell type or lineage - Aberrant expression. Abnormally increased or decreased levels of expression. Asynchronous antigen expression. Abnormal homogenous expression of one or more antigens.

31. Normal Pattern Of Antigen Expression All hematopoietic cells arise from hematopoietic stem cells. These stem cells can be identified due to expression of: Bright CD34, CD133 Intermediate CD45 Dim to absent CD38 Variable CD90 Dim CD123, CD117, HLA-DR, CD13, CD33 Maturation towards lineage commited progenitors is accompanied by: Slight decrease in CD34 & CD45 Loss of CD13 & CD90 Increase in CD38 & HLA-DR

32. CD Markers Lineage independent antigen Lineage specific/ Lineage associated CD34-Stem cell marker CD 19 CD 3 CD 13 HLA-DR CD 22 CD 7 CD 33 CD10(CALLA) CytoCD22 CD 5 CD 117(C-Kit) CD45Leucocyte common Ag Cyto 79 a Cyto CD3 CD 14 (Monocytic) CD4,CD8 B Cell T Cell Myeloid

33. Lineage assignment in Acute leukemia AML ALL-B cell ALL-T cell Definitive Cytoplasmic MPO Cytoplasmic CD79a Cytoplasmic CD22 CytoplasmicCD3 Surface CD3 Strongly associated CD117 CD 19 CD10 CD7 Moderately associated CD13 CD33 TdT CD5 CD2

34. FCM and common markers

35. Panels for Acute Leukemia A) Primary panel : B-cells - CD10, CD19 T-cells - CD3, CD7, CD4, CD8 Myeloid - CD13, CD33, CD117 Non-lineage - HLADR, CD34 Positive Control: CD45 (LCA) Negative Control: Isotype IgG1 B) Secondary panel: B-lineage specific - cytoCD22 / cytoCD79a T-lineage specific - cytoCD3 Myeloid lineage specific - Anti-MPO Other Markers – Tdt , CD99, CD41, CD61, SmIg & CD56

36. Principle Example Implementation At least one reagent for population identification CD45 for general cell type, Lineage associated antigen for specific cell lineage,e.g . CD19 for B cell Multiple antigens of same lineage & maturational stage to identify inappropriate expression levels Use of CD2,CD3.CD4,CD5,CD7,CD8 simultaneously to evaluate mature T-cells Multiple antigens of same lineage but different maturational stage to identify normal maturation & dyssynchronous expression Use of CD13 & CD16 simultaneously to demonstrate neutrophilic maturation PANEL DESIGN STRATEGIES

37. Principle Example Implementation Separation of different cell lineage Use of CD11b & CD15 simultaneously to separate monocytic & neutrophilic maturation Demonstration of clonality Use of kappa & lambda in combination with a B cell lineage reagent,e.g.CD19 Identify frankly aberrent antigen expression Use of T or NK cell associated antigens such as CD7 / CD56 in combination with CD34 PANEL DESIGN STRATEGIES

38. Flow chart - Approach to acute leukemia

39. Provisional diagnosis: Acute leukemia

40. Acute Leukemia Auer Rods + ve Auer Rods - ve MPO and/or NSE + ve MPO / NSE - ve FCM AML

41. FCM with first line panel CD10, CD19, HLADR CD3 CD13, CD33, CD117 HLADR, CD34 Lineage established Blasts, no auer rods, MPO/NSE negative

42. However, if lineage not established FCM with second line panel cCD22 cCD3, CD4, CD8 anti MPO, CD41, CD61 Lineage established

43. FCM is a must - All cases of ALL, - AML M0/M7, and - Undifferentiated, Bi-phenotypic leukemia - MRD detection For diagnostic laboratories, at least three, preferably four color FCM is required

44. As bone marrow cells express CD45, when CD45 is combined with side scatter, which separates lineages based on cytoplasmic complexity, the bone marrow sample is readily separated into its cellular constituents. Infiltration of marrow by immature cells or blasts is more easily recognized on a CD45 versus side-scatter plot than on traditional forward side-scatter gating.

45. Leucocyte analysis Neutrophils Monocytes Basophils Lymphocytes RBC’s Myeloblasts

46. Acute myeloid leukemia with differentiation Blasts (red) showing abnormal expression of the myeloid associated antigens CD33 (dim) CD13 (bright) CD15 (dim partial) immature antigens CD34 and CD117. CD11b- Negative. The blasts do not express more mature neutrophilic antigens. There are background maturing granulocytes (green) that are normal and not part of the leukemic population

47. Acute promyelocytic leukemia The neoplastic cell population - promyelocytes (red): CD33 (bright) and CD13 (intermediate). High side scatter indicating abundant cytoplasmic granularity. Lacks expression of CD34 and HLA-DR and retains expression of CD117 as is seen on a subset of normal promyelocytes . However, in contrast to normal promyelocytes , the abnormal cells lack significant expression of CD15, a characteristic and common abnormality in this disorder.

48. Acute myelomonocytic leukemia. Blasts (red) with a larger population of cells showing monocytic differentiation (violet). The monocytic differentiation is reflected in the acquisition of early monocyte antigens CD64 (bright), and CD36 (intermediate to bright) along with other more mature myelomonocytic antigens CD15 (intermediate) and CD11b (low to intermediate) Without significant acquisition of the mature monocyte antigen CD14 (absent) or marked gain in the expression of CD45 as is seen in mature monocytes . This finding suggests differentiation to the promonocyte stage, a population usually included in morphologic blast counts. In addition, a lesser degree of neutrophilic differentiation (green) is present.

49. CD5+ CD5- CD23+ Cyclin D1+ CD10+ CD10- SLL MCL FL, ALL, BL MZL HCL CD25+ CD25- B-cells (CD19 +)

50. CLL/SLL The neoplastic cells show B cell antigens CD19 (intermediate) and CD20 (low) with surface lambda light chain expression (low), coexpression of CD5 and CD23 (intermediate). The combination of CD5 coexpression , low-level light chain restriction, low-level CD20 and CD23 without FMC7 is diagnostic for CLL. The important differential is with mantle cell lymphoma.

51. Plasma cell neoplasm The neoplastic cell population (green)with bright expression of CD38, but shows abnormal expression of CD45 (low), CD19 (absent) and cytoplasmic lambda light chain restriction with aberrant expression of CD56 (bright). This immunophenotype is characteristic of that seen in a variety of plasma cell neoplasms including multiple myeloma, plasmacytoma and monoclonal gammopathy of uncertain significance (MGUS). Definitive classification requires clinical and laboratory correlation.

52. Case 4 : 22 year old man with fever for past 3 months.

53. Scoring system (EGIL group) for acute biphenotypic leukemias (Bene et al) Score B-lymphoid T-lymphoid Myeloid 2 CD79a, cytoplasm CD22, cytoplasm IgM CD3, TCR- α / β , TCR- γ / δ Anti-MPO 1 CD19, CD20, CD10 CD2, CD5, CD8, CD10 CD117, CD13, CD33, CD65 0.5 TdT, CD24 TdT, CD7, CD1a CD14, CD15, CD64 Biphenotypic acute leukemia (EGIL) is defined when scores for the myeloid and one of the lymphoid lineages are > 2 points.

54. Case 5 : 5 year old boy with fever – 1 month

55. MPO negative Acute Leukemia

56. Flow cytometer

57. Provisional diagnosis: Acute leukemia Case 6 : 45 male, bone marrow

58. CD13+, CD33+, CD117+, CD34+

59. Case 7 The patient is a 52 year old man who presented with cellulitis on his elbow and was noted to have a high white count. A bone marrow was done and sent to a local laboratory for phenotyping . When the laboratory called the result back to the oncologist who had sent the sample, they were told that there must be some mistake, and a second opinion was sought.

60. Case 8 A 1 year old child was brought to the emergency room by his parents who noticed that he was extremely irritable. A white blood count was found to be 90,000, and he was admitted to the hospital. A peripheral blood was sent for phenotyping , and a bone marrow examination was performed.

61. Case 9 The patient is a 64 year old female on methotrexate therapy for rheumatoid arthritis. She developed anemia and a decreasing platelet count which persisted following discontinuation of this therapy. A bone marrow examination was performed.

62. Diagnosis: Acute myeloid leukemia (FAB M1) Antigen Profile: Strongly positive for HLADR,CD13,CD34,CD38; dimly positive for CD33, CD71; partly positive for CD7,CD11b

63. Case 10 The patient is a 57 year old female who presented with fatigue. She had had rheumatoid arthritis for about 5 years, and also reported a recent upper respiratory tract infection for which she had taken antibiotics. No blood work had been done at that time, but she was now found to have a hemoglobin of 10g, a white blood count of 8000 with a relative lymphocytosis , and a platelet count of 95,000.

64. Case 11 The patient is a 60 year old man who had pancytopenia for several months before being diagnosed with myelodysplastic syndrome about one year ago. About 2 months prior to this, he presented with decreasing counts and was found to have transformed into acute leukemia. He was treated with induction chemotherapy, and this marrow was performed 6 weeks following this therapy.

65. Case 12 The patient is a 72 year old female with fatigue and anorexia for two weeks. She complained of a skin rash which she developed about a day prior to coming to the doctor, and on examination was found to have cellulitis on her left arm. A bone marrow examination was performed.

66. The patient is a 49 year old man who presented to the emergency room with confusion and disorientation. A CT scan showed evidence of an early intracerebral bleed, and laboratory studies showed thrombocytopenia and evidence of disseminated intravascular coagulation. Following heparinization and stabilization of the patient's CNS status, a bone marrow examination was performed. Case 13

67. Note that the light scatter pattern is very broad, and in particular a forward versus RALS display shows what appears to be very large granular cells. There are no consistent phenotypic differences between M3 and M3v, though as shown here in the isotype control there is often considerable FITC autofluorescence in classic M3. Diagnosis: Acute promyelocytic leukemia (FAB M3) Antigen Profile: Positive for CD71, CD33, CD9, CD13, myeloperoxidase ; partly positive for CD34, HLADR.

68. Case 14 A 34 year old man in previously good health came to the emergency room complaining of shortness of breath. His hemoglobin was 9 g/dl and his white blood count was 165,000/ul.

69. In many cases of M4 AML the FALS vs RALS pattern can be very characteristic. As shown here it frequently gives this "forked" picture. This particular case is an example of M4e. Eosinophils may be difficult to detect in flow samples. When they are numerous they have the high RALS signal of granulocytes, but a higher intensity of CD45 expression similar to monocytes . It may be that some of the non-colored cells "above" the monocytes represent eosinophils . Diagnosis: Acute myelomonocytic leukemia with eosinophilia (FAB-M4eo) Antigen Profile: Positive for CD33, HLADR and CD13; heterogeneous positivity for CD34, CD11b, CD15, CD14; dimly positive for CD2

70. Case 15 The patient is a 64 year old man with a known history of anemia for the past year. About a month before this visit he had had a viral illness, and had never fully recovered his strength from that episode. On this visit, both his hemoglobin and his platelet count were much lower than they had been. A bone marrow was performed and sent for phenotyping .

71. Thank You