Clinical Flow Cytometry

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2 Clinical Flow Cytometry An Overview of Basics from Sample Prep to Case Studies Kimberly Wayman, B.S., QCym Flow Cytometry Manager LMC Pathology- An Aurora Diagnostics Partner

3 Objectives: What are the advantages of flow cytometry? Flow Cytometry defined Understand how samples are prepared for flow cytometric testing Learn the basic operating principals of a flow cytometer What information does flow cytometry give the pathologist? Leukemia/ Lymphoma Panels: Case Studies

4 Advantages: Accuracy: Several thousand cells can be analyzed in seconds. Very small sub-populations (<1% of the total) can be counted and characterized with statistical confidence. Speed: A typical leukemia workup can be completed within a few hours. Each tube takes from several seconds to a few minutes to run. Specificity: The antibodies used are specific to only one antigen, and therefore specific to only the cells that have that antigen.

5 Flow Cytometry Defined: It is the measurement of characteristics of single cells as they flow through a saline solution. Before analysis, the cells have been mixed with antibodies that are conjugated with fluorescent dyes called fluorochromes, or colors. The antibodies each bind very specifically to a particular antigen. Each fluorochrome used emits light at a specific frequency or wavelength range. As each cell passes through the laser beam, the cytometer records how it scatters laser light and emits fluorescence. The data gathered can be analyzed by flow cytometry software to gather information such as size, complexity and phenotype. The phenotype is basically the cell s profile.

6 Sample Preparation Usual sample types for flow: peripheral blood, bone marrow, tissue, body fluids, fine needle aspirates. White blood cells are obtained from the sample. The method of obtaining the cells depends on the sample type. Blood and bone marrow: lyse the red blood cells, leaving just the white cells. Tissues: mechanical disaggregation. The cells have to be alive no frozen or fixed samples. Once the white cells are isolated from the sample, they are suspended in a saline solution. Concentration of the cells is adjusted to ensure maximum antibody saturation

7 Antigens, Antibodies and Fluorescent Staining Antigens Molecules which are generally found on the surface of cells, although some are found inside. Human cell antigens are often referred to by their CD ( cluster designation or cluster of differentiation ) names. Each white cell type has specific, unique antigens it will display Antibodies Proteins that bind tightly to a specific antigen. There is a huge library of commercial antibodies to human antigens. Fluorochromes Molecules that absorb light of one wavelength (the light from the laser) and emit light of a different, very specific wavelength, or color. Within each reaction tube in a panel, there will be 100uL of cells, and antibodies of each color.

8 ANTIBODIES COMMONLY USED: B-CELLS CD19 Pan B Cell FMC7 Mature B Cells. Useful in differentiation. CD20 Pan B Cell Κ (Kappa) and λ (Lambda) light chain. CD22 CD23 Mature B Cells. Mature B Cells. Useful in differentiation Antibodies are found in B Cells displayed in the cell membrane. Antibodies consist of heavy chains and light chains, with the light chains being either Kappa or Lambda. In a normal sample, the Kappa to Lambda ratio should be somewhere around 2:1. B Cells belong to the group of white blood cells called lymphocytes. B Cells begin maturation in the bone marrow. Immature B Cells migrate to secondary lymphoid tissues where they are called transitional B Cells. Eventually they go to the periphery, become mature B Cells and, when stimulated by a particular antigen, differentiate into either plasma cells or memory B Cells

9 ANTIBODIES COMMONLY USED: T-CELLS/ NK CELLS CD2 Pan T Cell. Also found on some Natural Killer Cells. CD7 Pan T Cell CD3 Pan T Cell CD8 T Suppressor lymphocytes. Also found on some Natural Killer Cells. CD4 CD5 T Helper lymphocytes. Also found in small amounts on monocytes (dim expression) Pan T Cell. Also coexpressed on B Cells in most cases of chronic lymphocytic leukemia. CD16 CD56 Natural Killer Cells. Also found on granulocytes. Natural Killer Cells T Cells are also a type of lymphocyte. They are called T Cells because they mature in the thymus. There are different subsets of T Cells, each with a different function. Those subsets most focused on in flow cytometry are T Helper and T Suppressor cells. T cells originate from stem cells in the bone marrow, migrate to the thymus, where they become immature thymocytes. They then mature into either the helper or suppressor cells, at which point they are released into the periphery.

10 ANTIBODIES COMMONLY USED: MYELOID/ OTHER MYELOID SPECIFIC MARKERS: CD13 Monocytes and granulocytes CD33 Monocytes and granulocytes CD14 Monocytes CD64 Monocytes CD10 OTHER MARKERS COMMONLY USED: Lymphoid blasts and immature cells in bone marrow called hematagones. Expressed in most cases of acute lymphoblastic leukemia. CD45 Also called leukocyte common antigen. Found on the surface of all white blood cells. Used for gating purposes. CD11c Monocytes and neutrophils. Used to identify cases of Hairy Cell leukemia CD117 Also known as C Kit, found on the surface of hematopoietic stem cells CD34 Blast marker, found on all hematopoietic blasts CD138 Plasma cell marker CD38 Plasma cells tend to have bright CD38 expression. Also used as a prognostic marker in CLL.

11 A flow cytometer has 3 systems Fluidics System: Transports particles in a stream to a laser beam. Optics System: Consists of lasers to illuminate particles in the sample stream and optical filters to direct the signals to the appropriate detectors. Electronics system: Converts the light signals into electronic signals that can be processed by the computer.

12 Fluidics System The sample is injected into the center of a flow of saline solution. When the sample is injected, the cells shoot randomly into 3-D space. They must be organized into single file in order to be seen individually. The sample pressure is always greater than the sheath pressure, and the combined flow is reduced in diameter. In this way, cells are forced into the center of the stream single file as they reach the laser source. This process is called hydrodynamic focusing.

13 Optics system Scattered light: As the cell moves through the path of the laser beam, light is scattered in all directions. There are detectors that pick up and quantify the scattered light particles. In this way, the instrument can measure the cell s size, granularity and/or internal complexity. Fluorescent light: The laser causes the fluorochromeconjugated antibodies that have been attached to the cell surface to bnecome excited and emit light of another specific wavelegth, which is then converted into an electronic signal and sent to the computer to be visualized/analyzed.

14 Optics System: Scattered Light Forward scattered light is directly proportional to cell size. This light is picked up by a forward angle light (FAL) sensor. Laser Forward Angle Light Scatter FALS Sensor Side scattered light (SSC), also known as right-angle light scatter (or RALS) is proportional to cell granularity or internal complexity. SSC is collected at a 90 angle. Laser 90 Degree Light Scatter 90LS Sensor FALS Sensor

15 X Axis is forward scatter, or size. Lymphocytes are smaller than monocytes, which are smaller than granulocytes. Y Axis is side scatter, or internal complexity. Lymphocytes are much less internally complex than granulocytes.

16 Within each tube, you have antibodies conjugated to each of the different fluorchoromes available. The five fluorochromes above are what our particular lab uses.

17 Fluorescence Emitted fluorescence intensity is proportional to binding sites FITC FITC FITC FITC FITC Number of Events FITC FITC FITC FITC FITC Log scale of Fluorescent Intensity

18 A cell that has more of a particular antigen will result in a higher voltage signal, or pulse. In flow cytometry, when one cell has more of a particular antigen than another cell, we call that cell brighter.

19 Electronic System After passing through the series of filters, the photons of light are directed to detectors called photomultiplier tubes (PMTs) When the photons hit the PMT, they are converted to a proportional number of electrons, which creates an electric current. The electric current travels to an amplifier, which creates a voltage pulse. Additional voltage can be applied to the PMT to create larger, more detectable pulse.

20 PMT Schematic of a flow cytometer, showing how the fluorescent light is directed to each photmultiplier tube (PMT) by a series of mirrors and filters.

21 Signal Detection - PMTs Cathode Secondary emission Anode Photons in End Window Dynodes Amplified Signal Out Requires Current on dynodes Voltage applied increases the sensitivity of the PMT A low signal will require higher voltages applied to the PMT to measure the signal

22 Color Compensation Fluorochromes typically fluoresce over a large part of the spectrum. Depending on the filter arrangement, a detector may see fluorescence from more than one fluorochrome. You need to compensate for this overlap in wavelengths so that one detector reports signal from only one fluorochrome Specialized circuitry in the flow cytometer is used to subtract a portion of one detector's signal from another, leaving only the desired signal.

23 Color Compensation (cont.) When several colors are being detected at once, the instrument has to compensate for spectral overlap. The instrument is able to digitally subtract any interference between one color and the next.

24 Data Display Scatter Plots and Gating CD45 v. SS plot typically used for gating purposes. FS v. SS plot of the same cells Lymphocytes blue Monocytes red Granulocytes green

25 Data Output/ Results Single parameter histograms Dual parameter histogram (Lymphocyte Gate)

26 CASE STUDY #1 Normal Lymphocytes Peripheral Blood

27 Gates: Gate A (blue): Lymphocytes Gate B (red): Monocytes Gate C (green): Granulocytes Gate D (brown): Dim CD45/ Blast Area Gate E (gray): Debris Area Granulocytes are usually predominant The area below the lymphocytes is the blast area or dim CD45 area. In a normal blood sample, there shouldn t be much there. The area at the bottom is where you find debris/ unlysed red cells and platelets

28 LYMPHOCYTE GATE CD4 (T Helpers): 62% CD8 (T Suppressors): 18% CD4 and CD8 do not co express The CD4/CD8 ratio is normal. There are more T Helpers than T Suppressors in a normal sample. CD7: 75% CD3: 75% CD3 and CD7 should be approximately equal and mostly co express, since they are both pan T Cell markers Total T Cells should be >60%

29 LYMPHOCYTE GATE CD5: 81% CD19: 8% CD5 is approximately the same as the CD3 and CD7 CD19 is usually < 20% CD19 and CD5 do not co express CD19: 8% CD20: 8% CD19 and CD20 should be approximately the same CD19 and CD20 should be dual staining (normal B Cells have both antigens)

30 LYMPHOCYTE GATE B CELL SURFACE LIGHT CHAIN: CD19/ Kappa: 3.9% CD19/ Lambda: 1.7% Kappa/ Lambda Ratio: 2.3 The Kappa to Lambda ratio should be somewhere between In normal B Cells, the amount of Kappa light chain is more than the amount of Lambda light chain The Kappa and Lambda should add up to approximately the same as the total CD19

31 LYMPHOCYTE GATE CD34: 0% CD13: 0% CD34 should be negative, as it is a blast marker CD13 should be negative, as it is a myeloid marker CD56: 11% CD16: 10% These are both natural killer cell markers, with a normal range of anywhere from 0 25%.

32 CASE STUDY #2 Chronic Lymphocytic Leukemia/ Small Lymphocytic Lymphoma Bone Marrow

33 PATIENT HISTORY The patient is a 52 year old male with a submitted clinical history of chronic lymphocytic leukemia. Examination of this marrow sample confirms a normocellular marrow partially effaced by neoplastic small lymphoid cells accounting for 40 50% of the total cellularity. Bone marrow core biopsy

34 FLOW CYTOMETRY STUDIES PANEL USED: TUBE 1 (ISOTYPE CONTROL): TUBE 4: TUBE 7: IgG1 FITC LAMBDA FITC CD14 FITC IgG1 PE CD19 PE CD117 PE IgG1 ECD CD10 ECD CD34 ECD IgG1 APC KAPPA APC CD33 APC CD45 PC7 CD45 PC7 CD45 PC7 TUBE 2: TUBE 5: TUBE 8: CD8 FITC FMC7 FITC CD22 FITC CD7 PE CD10 PE CD52 PE CD3 ECD CD19 ECD CD19 ECD CD4 APC CD38 APC HLADR APC CD45 PC7 CD45 PC7 CD45 PC7 TUBE 3: TUBE 6: CD20 FITC CD2 FITC CD23 PE CD34 PE CD5 ECD HLADR ECD CD19 APC CD13 APC CD45 PC7 CD45 PC7

35 FLOW CYTOMETRY STUDIES CD3: 4% CD7: 5% The percentage of T Cells is very low. Usual T Cell percentage is greater than 60% CD4: 4% CD8: 1% The percentage of T Cells is very low

36 FLOW CYTOMETRY STUDIES CD5: 94% CD19: 91% CD19 and CD5 co express. This is a hallmark of CLL CD19 and CD5 also co express in mantle cell lymphoma CD20: 84% CD20 can vary from dim to moderate in CLL

37 FLOW CYTOMETRY STUDIES B CELL SURFACE LIGHT CHAIN: CD19/ Kappa: 88% CD19/ Lambda: 1% Kappa/ Lambda Ratio: 88.0 The Kappa to Lambda ratio is high at 88.0, which indicates a clonal population

38 FLOW CYTOMETRY STUDIES CD23: 73% CD23 is helpful in the differentiation of CLL from mantle cell leukemia, which is CD23 negative. About 85% of CLL cases are CD23+ FMC7/CD19: 10% FMC7 expression is usually dim positive to negative in cases of CLL

39 FLOW CYTOMETRY STUDIES CD38/ CD19:41% CD38 is useful as a prognostic marker. CD38+ CLL cases are associated with a poor prognosis. CD52/ CD19:71% CD52 is used to aid in decisions regarding drug therapy The drug Campath (alemtuzumab) targets the CD52 molecule

40 FLOW CYTOMETRY STUDIES CD22/ CD19:35% CD22 dimly positive in about 1/3 of CLL cases

41 ADDITIONAL STUDIES/ CASE SUMMARY The overall morphologic, immunophenotypic and cytogenetic findings are diagnostic of marrow involvement by chronic lymphocytic leukemia with mixed nodular and interstitial pattern. The presence of deletion involving the ATM gene, and unmutated IgVH, is associated with a more aggressive clinical course. FISH STUDIES: Deletion of the ATM gene observed (11q) Deletion of ATM is associated with poor prognosis PCR: PCR showed an unmutated IgVH gene Unmutated IgVH gene is associated with more aggressive disease and shorter survival FINAL DIAGNOSIS: CHRONIC LYMPHOCYTIC LEUKEMIA, UNFAVORABLE PROGNOSTIC GROUP

42 CASE STUDY #3 Plasma Cell Myeloma Bone Marrow

43 PATIENT HISTORY The patient is a 61 year old male with a clinical history of plasma cell myeloma. Examination of this marrow sample confirms marrow effacement by plasma cells consistent with persistent plasma cell myeloma. Overall, plasma cells account for approximately 80% of the total marrow cellularity. Bone marrow core biopsy Core Biopsy CD138+ Plasma Cells

44 FLOW CYTOMETRY STUDIES PANEL USED: TUBE 1 (ISOTYPE CONTROL): TUBE 4: TUBE 7: IgG1 FITC LAMBDA FITC CD14 FITC IgG1 PE CD19 PE CD117 PE IgG1 ECD CD10 ECD CD34 ECD IgG1 APC KAPPA APC CD33 APC CD45 PC7 CD45 PC7 CD45 PC7 TUBE 2: TUBE 5: TUBE 8: CD8 FITC FMC7 FITC CD38 FITC CD7 PE CD10 PE CD117 PE CD3 ECD CD19 ECD CD56 ECD CD4 APC CD38 APC CD138 APC CD45 PC7 CD45 PC7 CD45 PC7 TUBE 3: TUBE 6: TUBE 9: CD20 FITC CD2 FITC CyLambda FITC CD23 PE CD34 PE CD38 PE CD5 ECD HLADR ECD CyKappa APC CD19 APC CD13 APC CD45 PC7 CD45 PC7 CD45 PC7

45 PLASMA CELL IDENTIFICATION AND GATING Two different histograms showing the same populations. Plasma cells are usually very large cells, as can be seen from the population in brown in the FS/SS histogram.

46 FLOW CYTOMETRY STUDIES Gating performed on the plasma cells only by identifying the cells coexpressing bright CD38 and CD138 97% of the plasma cells co express CD56 Approximately 70% of all plasma cell myelomas co express CD56

47 FLOW CYTOMETRY STUDIES Surface Kappa and Lambda light chain are negative CD19 is negative

48 FLOW CYTOMETRY STUDIES Gating is performed on the plasma cell population Cytoplasmic staining is performed using fixation and permeabilization reagents Kappa clonality is determined

49 ADDITIONAL STUDIES/ CASE SUMMARY FISH STUDIES: Gain of 1q and trisomies of chromosomes 5, 9 and 15 FINAL DIAGNOSIS: PERSISTENT PLASMA CELL MYELOMA Examination of this marrow sample confirms marrow effacement by plasma cells consistent with persistent plasma cell myeloma. This impression is supported by flow cytometric analysis of this marrow sample which confirms the presence of a monoclonal plasma cell population with the following phenotype: CD45+, CD38+, CD56+ and CD138+ with cytoplasmic kappa light chain restriction. Molecular studies show gains of 1q and trisomies of 5, 9 and 15.The overall findings are diagnostic of persistent plasma cell myeloma.

50 In Conclusion: Flow cytometric analysis is an accurate way to determine a lot of information about cells in a short amount of time. By analyzing light scatter properties of each individual cell in solution, the instrument can determine size, complexity and the phenotype of the cells in a sample with statistical accuracy. The pathologist correlates flow cytometry findings with morphologic and sometimes cytogenetic/ molecular findings BD Biosciences has a great poster of current CD markers: