Flowcytometry 1

Flowcytometry Clinical Pathology

1. FLOW CYTOMETRY . SUNIL KUMAR.P Department of Clinical Pathology St.John’s Medical College Bangalore 10/13/2018 1 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

2. INTRODUCTION 10/13/2018 2 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

3. The concept of flow cytometry has been in existence for more than five decades. Flow cytometric immunophenotyping (FCI) first appeared in clinical laboratories in the 1980s, in the wake of the AIDS epidemic. Initially utilized to assess CD4 T-cells, the technique was soon applied to lymphoid and eventually myeloid neoplasms. INTRODUCTION 10/13/2018 3 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

4. Current flow cytometers have the capability of simultaneously measuring multiple parameters of individual cells in a cell suspension . Thus , a large number of cell specimens can be processed with a quick turnaround time . In addition, flow cytometry is also highly sensitive and can detect immunophenotype of cells in a specimen with thousands of cells. 10/13/2018 4 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

5. The parameters analyzed by flow cytometry include physical properties of cells; the size, cytoplasmic granularity, and amount of DNA contents; and cell antigens/markers (surface, cytoplasmic, and nuclear) that can be recognized by specific antibodies. 10/13/2018 5 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

6. By using appropriate antibody panels, flow cytometry can reveal the cell type (hematopoietic , lymphoid, or nonhematopoietic ), cell lineage (B- and T cells , natural killer cells, myeloid/ monocytic cells, neuro / neuroendocrine cells , and epithelial cells ), cell maturation stage (precursors vs. matured cells ) 10/13/2018 6 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

7. PRINCIPLES OF CYTOMETRY 10/13/2018 7 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

8. Flow cytometry involves the analysis of the optical and fluorescence characteristics of single particle (e.g. cells, nuclei, chromosomes) during their passage within a narrow, precisely defined liquid stream. 10/13/2018 8 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

9. COMPUTER SYSTEM ELECTRONIC SYSTEM OPTICAL SYSTEM FLOW SYSTEM For cell analysis, the basic components of a flow cytometer include:- 10/13/2018 9 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

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11. SCHEMATIC DIAGRAM OF A FLOW CYTOMETER PMT- photomultiplier tubes ADC-analogue-to-digital converter 10/13/2018 11 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

12. Physical properties, such as size (represented by forward angle light scatter) and internal complexity (represented by right-angle scatter) can resolve certain cell populations . CONCEPT OF SCATTERING 10/13/2018 12 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

13. FSC collects light at 180° from the point at which the laser beam intersects the cells, usually on a linear scale. It is correlated with cell size, and thus can distinguish normal lymphocytes (small ), monocytes (intermediate ), and neoplastic cells (generally they are large in size). SSC collects right-angle light at 90° and is correlated with cytoplasmic granularity and nuclear configuration . 10/13/2018 13 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

14. The combination of both FSC and SSC can distinguish normal lymphocytes, granulocytes, and monocytes. The detection of lymphocytes and monocytes provides a reliable internal control to evaluate the size of the cells of interest. 10/13/2018 14 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

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16. IMMUNOPHENOTYPING ANALYSIS Requires Antibodies. Fluorochromes . 10/13/2018 16 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

17. ANTIBODY Highly specific monoclonal antibodies are used that are produced by cloned antibody secreting cells. Antibodies are based on cluster of differentiation (CD)- a protocol used for identification and distinction of cell surface antigens. Using CD system we can identify cells by the presence or absence of particular surface markers for e.g. CD3+ or CD20- etc. 10/13/2018 17 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

18. FLUOROCHROMES Fluorochromes are substances that can be excited by certain light source (such as laser) and emit a fluorescent signal at a single wavelength. Fluorescent dyes can directly bind to certain cellular content, such as DNA and RNA, and allow us to perform quantitative analysis on individual cells. However, in most cases fluorochromes are conjugated with monoclonal antibodies, which specifically target cellular antigens/markers. 10/13/2018 18 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

19. Characteristics of fluorochromes commonly used in flow cytometry . FLUOROCHROMES CONJUGATED TO ANTIBODIES EXCITATION WAVELENGTH(NM) EMISSION WAVELENGTH(NM) Fluorescein isothiocyanate (FITC) 488 530 Phycoerythrin (PE) 488 580 PE-Texas Red 488 615 PE-Cy5 488 670 Peridinin chlorophyl protein( PerCP ) 488 670 Allophycocyanin (APC) 633 670 APC-Cy7 633 767 Interestingly, although some of them can be excited by the same light source, the different fluorochromes may emit fluorescent signals with different wavelengths/colors. Thus, multiple fluorochromes can be simultaneously excited by a light source and detected by their emission fluorescent signals with different wavelengths, respectively. 10/13/2018 19 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

20. Antibodies conjugated to fluorescent dyes can bind specific proteins on cell membranes or inside cells. When labeled cells are passed by a light source, the fluorescent molecules are excited to a higher energy state. Upon returning to their resting states, the fluorochromes emit light energy at higher wavelengths. The use of multiple fluorochromes , each with similar excitation wavelengths and different emission wavelengths (or “colors”), allows several cell properties to be measured simultaneously. IMMUNOPHENOTYPING ANALYSIS 10/13/2018 20 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

21. Multiple cell antigens ( Ag ) are recognized by fluorochromeconjugated specific antibodies ( Ab ). Because different fluorochromes have different emission wavelengths/colors, they can be simultaneously detected by a flow cytometer . FITC fluorescein isothiocyanate ; PE phycoerythrin ; PerCP peridinin chlorophyll protein ; PE-T Red PE-Texas Red . Simultaneous detection of multiple cell antigens/markers. 10/13/2018 21 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

22. Abnormal/ aberrant antigenic expression can be grouped into four basic categories: Abnormally increased or decreased levels of antigenic expression (aberrant expression) • Gain of antigens not normally expressed in the cell type • Expression of antigens not synchronized with normal development and maturation stage of the cell type or lineage • Homogeneous expression of antigen(s) by a cell population that normally show more heterogeneous expression 10/13/2018 22 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

23. PROCESSING OF SPECIMEN FOR FLOW CYTOMETRY 10/13/2018 23 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

24. Theoretically, any specimens from which a single cell suspension can be generated are suitable for flow cytometry analysis. However, a lack of distinct antigens or markers in the cells of interest or tissues limits the diagnostic value of flow cytometry. SPECIMENS SUITABLE FOR FLOW CYTOMETRY 10/13/2018 24 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

25. Peripheral blood, Bone marrow, Body fluids, Cerebrospinal fluid, Lymph node (cells or fresh tissues), Any fine-needle aspirates, Fresh tissues suspicious for hematopoietic and lymphoid disorders. Common specimens suitable for flow cytometry analysis include 10/13/2018 25 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

26. For blood and bone marrow specimens, anticoagulants such as EDTA, heparin, or acid citrate dextrose are needed. Fresh tissue specimens are best transported and stored in sterile tissue culture medium. Although specimens may be stored at room temperature, refrigeration is preferred, particularly when there is a delay for flow cytometric analysis. For flow cytometry analysis, single-cell suspensions of the fresh tissues can be achieved by mechanical dissociation. SPECIMEN STORAGE 10/13/2018 26 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

27. General Notes On Cell Preparation 1.Single cell suspensions are required for optimal staining of samples for flow cytometry. 2. The narrow bores of the sample injection needle and tubing on a flow cytometer will be easily clogged by aggregated cells and debris. 3. Preparation of single cell suspensions from solid tissue requires mechanical dissociation and/or enzymatic digestion for optimal recovery of cells from the tissue. 10/13/2018 27 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

28. APPLICATIONS OF FLOW CYTOMETRY 10/13/2018 28 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

29. DNA content Analysis The measurement of cellular DNA content by flow cytometry uses fluorescent dyes, such as propidium iodide , that intercalate into the DNA helical structure. The fluorescent signal is directly proportional to the amount of DNA in the nucleus and can identify gross gains or losses in DNA. Abnormal DNA content, also known as “DNA content aneuploidy ” , can be determined in a tumor cell population. DNA aneuploidy generally is associated with malignancy 10/13/2018 29 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

30. Erythrocyte analysis Detection and quantification of fetal red cells in maternal blood . The use of flow cytometry for the detection of fetal cells is much more objective, reproducible, and sensitive than the Kleihauer-Betke test . 10/13/2018 30 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

31. Conventional laboratory tests for the diagnosis of PNH include the sugar water test and the Ham’s acid hemolysis test . Antibodies to CD55 and CD59 are specific for decay-accelerating factor and membrane-inhibitor of reactive lysis , respectively, and can be analyzed by flow cytometry to make a definitive diagnosis of PNH. Diagnosis of PNH 10/13/2018 31 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

32. Reticulocyte counts are based on identification of residual ribosomes and RNA in immature nonnucleated red blood cells by using supravital stain . The flow cytometric enumeration of reticulocytes uses fluorescent dyes that bind the residual RNA, such as thiazole orange . A region has been drawn on the red cells in the scatter plot. The other major cluster in the scatter plot are the platelets. The histogram was gated on the red cells and the regions on it delineate cells with high (H), medium (M) and low (L) fluorescence corresponding to increasing reticulocyte maturity. N marks nucleated red cells. Reticulocyte analysis 10/13/2018 32 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

33. In the blood bank , flow cytometry can be used as a complementary or replacement test for red cell immunology, including RBC-bound immunoglobulins and red cell antigens. Flow cytometry has been used to accurately identify and phenotype the recipient’s red cells. Flow cytometry is being used increasingly in the blood bank to assess leukocyte contamination in leukocyte-reduced blood products . 10/13/2018 33 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

34. Leukocyte Analysis Perhaps the best example of simultaneous analysis of multiple characteristics by flow cytometry involves the immunophenotyping of leukemias and lymphomas . WHO classification has divided non-Hodgkin lymphoma into B-cell and T/NK cell subtypes, which are further subclassified into precursor and peripheral lymphomas. Immunophenotyping by flow cytometry (FCM) is an essential aid for accurately diagnosing and prognosticating leukemia and lymphoma. 10/13/2018 34 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

35. The ability to analyze multiple cellular characteristics, along with new antibodies and gating strategies, has substantially enhanced the utility of flow cytometry in the diagnosis of leukemias and lymphomas. Different leukemias and lymphomas often have subtle differences in their antigen profiles that make them ideal for analysis by flow cytometry. B cell:  CD5, CD10, CD19, CD20, CD45, Kappa, Lambda;  T cell:  CD2, CD3, CD4, CD5, CD7, CD8, CD45, CD56;  Myelomonocytic :   CD7, CD11b, CD13, CD14, CD15, CD16, CD33, CD34, CD45, CD56, CD117, HLA-DR;  Plasma cell:   CD19, CD38, CD45, CD56, CD138 10/13/2018 35 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

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37. Immunologic monitoring of HIV-infected patients is a mainstay of the clinical flow cytometry and provides the best possible way for enumeration of CD4+ T lymphocytes and HIV viral load. 10/13/2018 37 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

38. Flow cytometry can be used for lymphoma phenotyping of fine needle aspirates , and is a powerful adjunct to cytologic diagnosis. 10/13/2018 38 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

39. Functional deficiencies of leukocytes can be assessed by flow cytometry. Assays for oxidative burst, phagocytosis , opsonization , adhesion, and structure are available. One of the clinical example is LAD type I is caused by a genetic deficiency of β2 - integrins , which are heterodimers of CD11 and CD18 . This deficiency leads to a loss of neutrophil and monocyte migration. 10/13/2018 39 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

40. The high sensitivity and capacity for simultaneous analysis of multiple characteristics make flow cytometry useful for the detection of minimal residual disease , especially if abnormal patterns of antigen expression are present. 10/13/2018 40 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

41. Platelet analysis Flow cytometry is an excellent method for direct analysis of platelet-bound antibodies , and it has also been shown to be of benefit in detection of free plasma antibodies in ITP. The reticulated platelet count can be quantified by flow cytometry in order to assess the rate of thrombopoiesis . This measurement can separate unexplained thrombocytopenias into those with increased destruction and those with defects in platelet production. 10/13/2018 41 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

42. The pathogenesis and molecular defects of many primary thrombocytopathies are well known and relate to defects in structural or functional glycoproteins , such as the abnormal expression of gpIIb / IIIa in Glanzmann thrombasthenia and gpIb in Bernard- Soulier disease. Flow cytometry is a rapid and useful method of obtaining a diagnosis. 10/13/2018 42 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

43. Other applications Flow cytometry is indicated in the evaluation of serous effusions and CSF, including aqueous or vitreous humor of patients with a history of hematolymphoid neoplasia . Flow cytometry assists in the differential diagnosis between plasma cell myeloma and monoclonal gammopathies of undetermined significance by determining the percentage of aberrant or clonal plasma cells of all bone marrow plasma cells. Flow cytometry is useful in diagnostic evaluation of unexplained marrow plasmacytosis by assessing phenotypically aberrant or clonal plasma cells and its ability to detect other underlying monoclonal B-cell process. 10/13/2018 43 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

44. Tissue-based lymphoid neoplasias commonly affect lymph nodes, spleen, mucosa-associated lymphoid tissue, skin, or nonlymphoid solid organs resulting in masses or organomegaly . Flow cytometry is extremely useful in the diagnosis and subclassification of tissue-based lymphoid neoplasias ,, organomegaly and tissue infiltrates. 10/13/2018 44 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

45. FLOW CYTOMETRY INTERPRETATION 10/13/2018 45 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

46. Basic parameters and Windows of cell population Forward light scatter (FSC) and side light scatter (SSC) . FSC collects light at 180° from the point at which the laser beam intersects the cells .It is correlated with cell size.and thus can distinguish normal lymphocytes (small), monocytes (intermediate), and neoplastic cells (generally they are large in size). SSC collects right-angle light at 90° and is correlated with cytoplasmic granularity and nuclear configuration. The combination of both FSC and SSC can distinguish normal lymphocytes, granulocytes, and monocytes. The detection of lymphocytes and monocytes provides a reliable internal control to evaluate the size of the cells of interest. 10/13/2018 46 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

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48. Concept of Gating in brief Gating is the most important first step in immunophenotyping analysis. It is critical particularly in a specimen that contains mixed cell populations, such as bone marrow aspirate. Gating sets upper and lower limits on the type and amount of material that passes through. It is used to separate a sub-population from heterogeneous population. It permits very specific questions to be asked about a particular population. 10/13/2018 48 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

49. 1. By cell distribution in the CD45 vs. SSC . This is most useful in a specimen containing mixed cell populations . The grouped cells in individual windows represent different cell lineages. 2. By cell size : In FSC vs. SSC histograms, neoplastic cells (usually large in size) can be gated by using lymphocytes (small) and monocytes (intermediate) as an internal size control . Once the cells of interest are gated, further analysis of cell lineage can be performed. 3. By cell lineage-specific antigens (immunophenotype): If cells are CD45+ but do not fit into particular windows in the CD45 vs. SSC histogram, identification of lineage-specific antigen expression is needed Types of Gating 10/13/2018 49 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

50. Gating the Lymphocytes A region, R1, has been drawn around the lymphocytes ( A ). In B , the lymphocytes are coloured red. In C a gate has been set to show only the cells in R1 10/13/2018 50 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

51. A region, R2, has been drawn around the monocytes ( A ). In B , the monocytes are coloured blue. In C a gate has been set to show only the cells in R2 Gating the Monocytes 10/13/2018 51 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

52. Gating the T-Lymphocytes 10/13/2018 52 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

53. Quadrant regions showing the percentage of cells in each sub-population 10/13/2018 53 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

54. There are several kinds of quality controls. First the flow cytometer itself must be evaluated for proper function.This is usually accomplished with standardized fluorescent beads. These give very precise, reproducible patterns, which quickly assess instrument function. A second quality control material is used to set up the appropriate instrument settings for the type of staining used. These can be beads or antibody-stained cells. The third level of quality control is a control substance that mimics actual specimens. These controls are available commercially and usually consist of stabilized blood, sometimes with added tissue-culture cells that mimic a specific cancer cell. QUALITY CONTROL IN FLOW CYTOMETER 10/13/2018 54 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

55. Comparison of immunophenotypic techniques . FLOW CYTOMETRY IMMUNOHISTOCHEMISTRY Shorter turnaround time (minutes to hours) Longer turnaround time (hours to days) Less subjective result interpretation Subjective result interpretation Quantitative results Semiquantitative results Multiple antibodies/ fluorochromes per test Usually limited to a single antibody per slide Greater antibody selection Fewer antibodies available Data/results can be electronically transferred Slides can be shipped by mail or courier service Need fresh cells or tissue Can use fixed/archived tissue Limited morphologic correlation Architectural and cytologiccorrelation Cannot assess nonviable cells Can assess nonviable “ghost” cells 10/13/2018 55 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

56. CONCLUSION Flow cytometry is a powerful technique for correlating multiple characteristics on single cells. This qualitative and quantitative technique has made the transition from a research tool to standard clinical testing. Smaller, less expensive instruments and an increasing number of clinically useful antibodies are creating more opportunities for routine clinical laboratories to use flow cytometry in the diagnosis and management of disease And last but not the least, keeping pace with scientific and clinical advancements is the need of hour. 10/13/2018 56 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

57. REFERENCE 1.Text book of haematology Wintrobes 10 th edition 2.Textbook of haematology Williams. 3.Manual of Flowcytometry BD FACS calibaur. 10/13/2018 57 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE

58. THANK YOU 10/13/2018 58 SUNIL KUMAR. P ST.JOHN'S MEDICAL COLLEGE