Flow cytometry

Principle and applications in flow cytometry

1. Dr. POOSAPATI RATNA KUMARI PROFESSOR DEPT OF MICROBIOLOGY FLOW CYTOMETRY

2. INTRODUCTION Flow cytometry is the science of measuring physical and chemical properties of live cells or other biological particles as they pass in a fluid, single-cell stream through a measuring apparatus. Flow ~ cells in motion Cyto ~ cell Metry ~ measure Measuring properties of cells while in a fluid stream

3. HISTORY 16 th century - Leeuwenhoek (simple microscope) – Father of Cytometry 1742 - Lomonosov - dark field illumination and performed light scatter measurements 1934 – Moldavan - photoelectric technique for counting cells flowing through a capillary tube, and flow cytometry was born 1938 - Caspersson built a crude flow cytometer to measure cell properties in the UV and visible regions 1940 - Crosland and Taylor developed a blood cell counter using the sheath flow principle, light scatter and darkfield illumination

4. 1949 - Wallace Coulter patented the first non-optical electronic blood cell counter 1957 - The first Model A Coulter Counter was introduced primarily to count erythrocytes and leukocytes from blood 1973 - Crissman and Steinkamp introduced propidium iodide Mid 1970s - Leonard Herzenberg at Stanford coined the term, Fluorescence Activated Cell Sorter, or FACS

5. PURPOSE OF FLOW CYTOMETRY Flow cytometry detects and measures - a particle’s relative size , - relative granularity or internal complexity - relative fluorescence intensity These characteristics are determined using an optical-to-electronic coupling system that records how the cell or particle scatters incident laser light and emits fluorescence.

6. PRINCIPLE OF FLOW CYTOMETER A flow cytometer is made up of three main systems: Fluidics Optics Electronics

7. Fluidic system - transports particles in a stream to the laser beam for interrogation. Optics system consists of lasers to illuminate the particles in the sample stream and optical filters to direct the resulting light signals to the appropriate detectors. Electronics system converts the detected light signals into electronic signals that can be processed by the computer.

8. THE FLUIDICS – FLOW SYSTEM One of the fundamentals of flow cytometry is the ability to measure the properties of individual particles, which is managed by the fluidics system The fluidics system consists of FLOW CELL (Quartz chamber) Central channel / core – through which the sample is injected Outer sheath – contains faster flowing fluid known as Sheath fluid (0.9% Saline), enclosing the central core. As the sheath fluid moves, it creates a massive drag effect on the narrowing central chamber. Under optimal conditions (laminar flow) the fluid in the central chamber will not mix with the sheath fluid.

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

10. OPTICS After the cell delivery system, the need is to excite the cells using a light source. The light source used in a flow cytometer is Laser (most commonly) and Arc lamp Lasers are more commonly used as they are highly coherent and uniform and they are monochromatic. Eg. Argon laser-488nm wavelength (blue to blue green)

11. Forward scatter channel (FSC) - equates to the particle’s size and can also be used to distinguish between cellular debris and living cells. Side scatter channel (SSC) provides information about the granular content within a particle. Both FSC and SSC are unique for every particle, and a combination of the two may be used to differentiate different cell types in a heterogeneous sample.

12. Neutrophils and eosinophils produce a great deal of side scatter due to their cytoplasmic granules. Large cells such as monocytes and neutrophils produce more forward scatter than normal RBCs, and normal lymphocytes

13. 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 Monocytes Granulocytes RBCs, Debris, Dead Cells

14. FLUORESCENT ABs - Cells are labeled with fluorescent antibodies directed against cell surface molecules - Using different c o l o r 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

15. OPTICAL FILTERS Once the fluorescence light from a cell has been captured by the collection optics, the spectral component of interest for each stain must be separated spatially for detection. Optical filters are designed such that they absorb or reflect a wavelength of light, while transmitting other. 4 types of filters are used in flow cytometry Long Pass filter Short Pass filter Band Pass filter Dichoric filter

16. OPTICAL DETECTORS The two main photo detector types used in flow cytometry are silicon photodiodes - to detect forward scatter detection Photomultiplier tubes (PMTs ) - high sensitivity and are, therefore, assigned to side scatter and fluorescence detection.

17. FLUOROCHROMES Fluorochromes are essentially dyes, which accept light energy (e.g. from a laser) at a given wavelength and re-emit it at a longer wavelength. These two processes are called excitation and emission . The process of emission follows extremely rapidly, commonly in the order of nanoseconds, and is known as fluorescence.

18. GATING An important principle of flow cytometry data analysis is to selectively visualize the cells of interest while eliminating results from unwanted particles e.g. dead cells and debris. This procedure is called GATING . Cells have traditionally been gated according to physical characteristics. Lysed whole blood cell analysis is the most common application of gating.

19. DETECTION OF FLUOROCHROMES 1. Laser beam excites fluorochrome attached to particle at one wavelength 2. Fluorochrome emits light at different wavelength 3. Light is directed to the photomultiplier tube that detects and quantifies the light. 4. Computer software analyzes data and generates scatterplot Photomultiplier Tube CPU Cell

20. ELECTRONICS As a particle of interest passes through the focus, fluoresces and is detected by a photo detector, an electrical pulse is generated and presented to the signal processing electronics. The instrument is triggered when this signal exceeds a predefined threshold level. 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

21. ELECTRONICS

22. QUALITY CONTROL IN FLOWCYTOMETRY Divided into 2 parts Internal quality control External quality control

23. INTERNAL QUALITY CONTROL IN FLOWCYTOMETRY Internal quality control consists of a series of activities that are performed by the laboratory to ensure the instrument , reagents and staff are performing within the limits set by the laboratory.

24. Quality control of the instrument can be divided into three separate procedures First procedure is usually carried out once or twice a year by qualified service personnel who check the performance of components such as the lasers, photomultiplier tubes (pmts), optical filters, and log and linear amplifiers. Second procedure is performed by the operator with each start-up of the instrument. Using appropriate reference microbeads with dedicated plots and instrument settings, parameters such as cvs and mean or median channel number can be recorded. Third procedure consists of the calibration of the fluorescence channels

25. EXTERNAL QUALITY CONTROL IN FLOWCYTOMETRY Samples, either fresh or stabilized, are distributed on a regular basis, and the laboratory is asked to process them as they would any other sample. Results are submitted back to a central agency for analysis, and a report indicating the relative performance of each participating laboratory is issued. External quality control schemes check the complete process, from the technical side of sample preparation (staining, data analysis, interpretation)

26. ADVANTAGES DISADVANTAGES High speed analyses depending on the flow rate; Measures single cells and a large number of cells; Simultaneous analysis multiple parameters; Identifies small populations; Quantification of florescence intensities; Sorting of predefined cells populations (up to 70.000/s); Portable equipment's. Very expensive and sophisticated instruments; Requires management by a highly trained specialist and on-going maintenance by service engineers. Complex instruments are prone to problems with the microfluidics system (blockages) and also require warm-up, laser calibration and cleaning for each use. Needs single cell particle; Little information on intra-cellular distributions

27. Common specimens suitable for flow cytometry analysis include  Peripheral blood,  Bone marrow,  Body fluids,  Cerebrospinal fluid,  Urine,  Lymph node (cells or fresh tissues),  Any fine-needle aspirates,  Fresh tissues suspicious for hematopoietic and lymphoid disorders.

28. APPLICATIONS OF FLOW CYTOMETRY

29. FLUORESCENCE-ACTIVATED CELL SORTING (FACS) Fluorescence-activated cell sorting (FACS) is a development of flow cytometry that enables sorting of a mixture of cells into two or more fractions, cell-by-cell, utilising the scatter and fluorescence signals of each cell.

30. APPLICATIONS IN CLINICAL MICROBIOLOGY DETECTION OF BACTERIA Flow cytometry is a sensitive analytical technique that can be readily applied to the enumeration of viable bacteria in a biological sample. The possibility of using fluorochrome-labeled antibodies to specific antigens render them one of the most powerful tools in the identification of pathogens. Flow Cytometry in conjunction with fluorescent antibodies has been used to detect surface antigens in - Hemophilus, Salmonella, Mycobacterium, Brucella, Mycoplasma, Pseudomonas, Bacteroides and Legionella

31. DETECTION OF FUNGI Surface antigens of Candida albicans can be detected by flow cytometry in conjunction with available antibodies. Using FCM and antibodies directed against yeasts , Pierard et al identified fungal pathogens and differentiated them from non-pathogenic ones.

32. DETECTION OF PARASITES FCM has been used to detect intracellular parasites such as Plasmodium . The absence of DNA in erythrocytes has enabled this intracellular parasite’s DNA to be stained with specific fluorochromes and detected by FCM. The multiparameter analysis permitted by FCM can be used to study other characteristics, such as parasite antigens expressed by erythrocyte or viability state of parasitized cell.

33. DETECTION OF VIRUSES FCM can detect viral antigens either on the surface of or within infected cells. It can rapidly detect and quantify virus infected cells using antibodies that specifically recognise surface or internal antigens.

34. Leukocyte Analysis CD3 A normal person has a significant proportion of CD3-positive lymphocytes. In the patient with leukaemia, staining for CD3 is absent. CD20 In the leukaemia patient there are a large number of cells staining positive for CD20. In the healthy person only a few stain positive. HLA-DR The leukaemia patient is HLA-DR-positive. In the normal person only a small number of cells stain positive. Being CD3-negative, CD20-positive and HLA-DR-positive, a clinician could diagnose with certainty that this patient is suffering from a B cell lineage leukaemia or lymphoma

35. DNA analysis (Malignancy) DNA aneuploidy generally is associated with malignancy; often correlates with many types of cancer such as multiple myeloma, and childhood acute lymphoblastic leukemia (ALL). Flow Cytometry used to differentiate malignant cells from their normal counterparts. The distinction between normal and leukemic bone marrow precursors is essential for the diagnosis and treatment monitoring of acute lymphoblastic leukemia. Although conventional cytogenetics can detect smaller DNA content differences, flow cytometry allows more rapid analysis of a larger number of cells.

36. Enzymatic deficiencies Gaucher disease is caused by a deficiency of the enzyme glucocerebrosidase. Macrophages transform into pathogenic Gaucher cells following the phagocytosis of red blood cells (RBCs) and subsequent accumulation of glucosylceramide. Flow cytometry is a tool for measuring Bglucocerebrosidase activity in Gauchers disease and to study the abnormalities RBCs shape.

37. IMMUNOLOGY DIAGNOSIS AND MONITORING OF HIV PROGRESSION Immunologic monitoring of HIV-infected patients is a mainstay of the clinical flow cytometry laboratory. HIV infects helper/inducer T lymphocytes via the CD4 antigen. As HIV disease progresses, CD4-positive T lymphocytes decrease in total number. The absolute CD4 count provides a powerful laboratory measurement for predicting, staging, and monitoring disease progression and response to treatment in HIV-infected individuals

38. Immunophenotyping Applications Flow cytometry is used in “Immunophenotyping” - Immunophenotyping is a technique used to study the protein expressed by cells such as erythrocytes, leukocytes, and platelets. Example: Immunophenotyping for HLA-B27.HLA-B27 is one of the multiple major histocompatibility complex (MHC) class I-specific antigens. Detection of HLA-B27 (positive) may associate the patient with several other disorders, such as Reiter’s syndrome, psoriatic arthritis, and inflammatory bowel disease. Immunophenotyping of leukemias and lymphomas by differentiating their antigen profiles. Flow cytometry can also be used to identify leukemias that may be resistant to therapy . Flow cytometry not only is can detect the presence or absence of antigens but also the strength of antigen expression which can also aid in diagnosis.

39. Genetic disease and carriage Flow cytometry can be useful in discriminating heterozygous gene mutations from normal phenotypes. It may also identify heterozygous family members who are carriers of a mutation for the purpose of genetic counseling.

40. APPLICATIONS IN HEMATOLOGY Detection of fetal Hb The use of flow cytometry for the detection of fetal cells is much more objective, reproducible, and sensitive than the Kleihauer-Betke test . Fluorescently labelled antibodies to the rhesus (D) antigen can be used, or more recently, antibodies directed against haemoglobin F This method has the ability to distinguish fetal cells from F-cells (adult red cells with small amounts of haemoglobin F).

41. PAROXYSMAL NOCTURNAL HEMOGLOBINURIA Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder that leads to intravascular hemolysis with associated thrombotic and infectious complications. Antibodies to CD55 and CD59 are specific for decay-accelerating factor and membrane-inhibitor of reactive lysis, respectively, and can be analysed by flow cytometry to make a definitive diagnosis of PNH

42. BIOLOGICAL APPLICATIONS FOR CELL SORTING Protein Engineering & Development Screening of peptide libraries for binding Selecting antibody mutants Screening for enzymatic activity Screening for over-producing cells Cell Engineering Disease Identification/Characterization Isolating cells to characterize them based on multiple modalities Nucleic acid Protein expression Cellular function

43. FUTURE DIRECTIONS-NEW TESTS

44. ICP 11 (1969) Distributed by Phywe , Göttingen The first commercial flow cytometer PDP 11 computer TPS 1974 - 1979, Designed by Bob Auer Epics II 1975, Designed by Mack Fulwyler and Jim Corell Delivered to NCI/NIH Early instruments

45. Modern Instruments

46. POLYCHROMATIC FLOW CYTOMETRY In many instances the small volume of a sample obtained from the patient is a limiting factor for the number of tests that can be performed . Hence the need for development of polychromatic flow cytometric tests.(upto 6 fluorochromes) This will reduce the requirement for large volume of specimen ,the time needed for sample processing and total no. of antibodies needed for analysis.

47. APPLICATIONS IN DIAGNOSIS OF ALLERGY FCM is used to assess the reaction of basophils to allergens . CD63 molecules in resting basophils are located in the membranes of intracellular granules and are not detectable on the cell surface. If basophils are sensitized in vivo with allergen specific IgE , then exposure to allergen in vitro will result in their degranulation, and therefore the expression of CD63 on the cell surface of activated basophils will increase in less than 10min.

48. ANTIFUNGAL SUSCEPTIBILITY TESTING One advantage of FCM AST over conventional methods is the 4 –hour incubation time compared with current methods that require an incubation time of 24 to 48 hrs. After incubation with antifungal agent, the fungal cells are stained with a specific fluorescent dye and the changes in fluorescence intensity of stained cells due to efforts of antifungal agents is measured by FCM. Fungal cells become more fluorescent if they are susceptible to the antifungal agent

49. C0NCLUSION  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.