flow cytometry in transfusion medicine use

Flow cytometry is a lab test used to analyze characteristics of cells or particles. During the process, a sample of cells or particles is suspended in fluid and injected into a flow cytometer machine. Approximately 10,000 cells can be analyzed and processed by a computer in less than one minute. A flow cytometer with sample injection tube. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. Flow cytometers utilize lasers as light sources to produce both scattered and fluorescent light signals that are read by detectors such as photodiodes or photomultiplier tubes. Flow cytometry is a laser-based technique used to detect and analyze the chemical and physical characteristics of cells or particles. It is most commonly used to evaluate bone marrow, peripheral blood and other fluids in your body. Flow cytometry studies are used to identify and quantify immune cells and characterize hematological malignancies.Flow cytometry studies are used to identify and quantify immune cells and characterize hematological malignancies.

1. Flowcytometry Dr.Dnyaneshwar Patale SR PDCC Apheresis ILBS

2. Outline History Basics of Flow Def Fluidics Optics Electronics Data analysis Application

3. Historical Highlights Flow cytometry was initially conceived as a practical methodology to count blood cells. In 1879 Lord Rayleigh observed that fluid emerging from an orifice breaks into a series of droplets. Cell sorting is based on the physics of droplet formation. In 1934, A. Moldavan reported the development of the first device that could count red blood cells automatically while in flow. ( Science. 1934 Aug 24;80(2069):188-9 PHOTO-ELECTRIC TECHNIQUE FOR THE COUNTING OF MICROSCOPICAL CELLS) In 1949, Wallace Coulter filed a patent entitled, “Means for Counting Particles Suspended in a Fluid”. The patent was issued in 1953. This lead to the development of the “Model A” Coulter Counter. Today, clinical hematology laboratory instruments used to count blood cells employ the principles developed by Coulter.

4. In 1965, Mack J. Fulwyler reported the first flow cytometry instrument capable of sorting cells. He sorted cells based on their Coulter volume by using a Coulter cell sizing instrument and modifying the electrostatic ink jet droplet deflection technique developed by Richard G. Sweet. (Rev. Sci. Instrum . 36, 131 (1965); http://dx.doi.org/10.1063/1.1719502 High Frequency Recording with Electrostatically Deflected Ink Jets) In 1968 Wolfgang Göhde designed a fluorescence based flow cytometer (ICPII) in 1968 which was commercialized by Partec in 1969. In 2000 he turned his efforts toward developing a program to provide low cost CD4 tests for people with AIDS in Africa. Lou Kamentsky and Myron Melamed worked on distinguishing cancer cells from normal cells using differences in the absorption and scattering of light. Kamentsky designed the Rapid Cell Spectrophotometer (RCS), which measured nucleic acid content and cell size. Leonard Herzenberg , an immunologist at Stanford, used the RCS prototype realizing how useful this technology would be in cell biology , He coined the term ‘ FACS ’ – Fluorescence Activated Cell Sorter . Becton Dickinson (BD) owns the FACS trade name and launched the first commercial instrument, FACS-1 in the early 1970’s. Today, flow cytometers with 5 lasers capable of analyzing or sorting cells labeled with 18 fluorochromes is possible. Soon instruments capable of 50 parameters may be possible.

5. What Is Flow Cytometry? Flow ~ motion Cyto ~ cell Metry ~ measure

6. Flow Cytometry Background  A system that integrates electronics, fluidics, optics, laser technology and computer analysis in a single platform.  A llows for simultaneous multiparametric analysis of physical/chemical/biological characteristics of cells or particles at the single-cell level by detecting fluorescence intensity as they travel in suspension one by one past a sensing point What is it? Flow chamber and fluidics Emission light Optical components Detectors/amplifiers (PMT) Digital (computer/software) Excitation light Lasers Mixed cells with fluorescence label

7. Optics Fluidics Electronics Mechanics of a Flow Cytometer Cells in suspension are brought in single file past a focused laser where they scatter light and emit fluorescence that is filtered and collected then converted to digitized values that are stored in a file for analysis

8. The Fluidics System “ Cells in suspension flow single file” Cells must flow one-by-one into the cytometer to do single cell analysis Accomplished through a pressurized laminar flow system. The sample is injected into a sheath fluid as it passes through a small orifice (50um-300um)

9. Hydrodynamic Systems Sample in Sheath Sheath in Laser beam Piezoelectric crystal oscillator Fluorescence Sensors Scatter Sensor Core Sheath Signal direction Flow Chamber

10. 10 psi 10.2 psi 10 psi 10.4 psi 10 psi 10.8 psi Difference in pressure between sample and sheath This will control sample volume flow rate The greater the differential, the wider the sample core. If differential is too large, cells will no longer line up single file Results in wider CV’s and increase in multiple cells passing through the laser at once » » » Faster cell analysis BUT loss of resolution!! Fluidics- Sample Differential

11. Hydrodynamic Focusing V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3 Notice how the ink is focused into a tight stream as it is drawn into the tube under laminar flow conditions. PBS/Sheath Sample/cells/core Laminar flow Hydrodynamic Focusing Laminar flow occurs when a fluid flows in parallel layers, with no disruption between the layers

12. Incoming Laser Sample Sheath Sheath Sample Core Stream Low Differential High Differential or “turbulent flow Laser Focal Point

13. Sample Preparation Samples must be in single cell suspension Solid tissue requires mechanical dissociation and often enzymatic digestion Adherent cell lines require detachment from the culture dish and dissociation Cell aggregates must be filtered out Red cells should be lysed Well prepared single cell suspensions yield good data

14. 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.

15. Interrogation Light source needs to be focused on the same point where cells are focused. Light source 99%=Lasers

16. Lasers L ight a mplification by s timulated e mission of r adiation Lasers provide a single wavelength of light (monochromatic) They can provide milliwatts to watts of power Low divergence Provide coherent light Gas, dye, or solid state Coherent: all emiting photons have same wavelength, phase and direction as stimulation photons

17. Light collection Collected photons are the product of laser light scattering or bouncing off cells 488nm Information associated with physical attributes of cells (size, granularity, refractive index) Scatter Fluorescence VS Collected photons are product of excitation with subsequent emission determined by fluorophore 350nm-800nm Readout of intrinsic ( autofluorescence ) or extrinsic fluorescence (intentional cell labeling)

18. Colors 488 nm wavelength is the most commonly used type of laser in Flow Cytometers Many 5 laser instruments have these additional lasers 355 nm UV 405 nm Violet 640 nm Red 561 nm Yellow-Green http://antonine-education.co.uk/

19. Forward Scatter FSC Detector Laser Beam Original from Purdue University Cytometry Laboratories

20. Different size cells Particle or cell size (log scale) FSC Number of events small large 0.1 1 10 100 1000 0.9 20 90 200 700 While forward light scatter is not always related to cell size, in The majority of cases between 1-20 microns, it is a reasonable estimate

21. Side Scatter FSC Detector Collection Lens SSC Detector Laser Beam Original from Purdue University Cytometry Laboratories

22. Side Scatter 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, drug delivery nanoparticles.) Side Scatter = SSC =RALS=90 degree Scatter

23. 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 heterogenous cell population FSC SSC Lymphocytes Monocytes Granulocytes RBCs, Debris, Dead Cells LIVE Dead

24. 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 Optical filters are designed such that they absorb or reflect some wavelengths of light, while transmitting others 3 types of filters Long Pass Short Pass Band Pass Dichroic

25. Optical Filters

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

27. IMMUNOPHENOTYPING ANALYSIS Requires Antibodies. Fluorochromes .

28. 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. ANTIBODY

29. 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.

30. Understanding Fluoroscence The fluorescent molecule is excited by the excitation source (laser). This imparts energy to electrons in the molecule which in then released as the molecule relaxes. The energy is released as light. e - E x cited R e sting e- e- e - e - state Mechanism of Fluorscence

31. Fluorescence Detection Fluorochromes on the cell surface or inside the cell are excited by the laser beam as the cell passes the interrogation point. These fluorochromes then release energy as they leave their excited state. The energy release is in the form of a photon with a specific wavelength, longer than the excitation wavelength. These photons of light are steered and collected by optical lens and filters at specific wavelengths.

32. Characteristics of fluorochromes commonly used in flow cytometry . FLUOROCHROMES CONJUGATED TO ANTIBODIES EXCITATION WAVELENGTH(NM) EMISSION WAVELENGTH(NM) Fluorescein isothiocyanate (FITC) 488 5 3 Phycoerythrin (PE) 488 580 PE-Texas Red 488 6 1 5 PE-Cy5 488 670 Peridinin chlorophyl protein(PerCP) 488 670 Allophycocyanin (APC) 633 670 APC-Cy7 633 7 6 7 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.

33. How do I choose my Fluorochromes ? Antibody availability Fluorochrome brightness Excitation source Emission filters Other fluorochromes/ Signals present in my sample (spectral overlap)

34. Electronics Detectors basically collect photons of light and convert them to an electrical current The “electronics” process light signal and convert the current to a digitized value that computer can graph

35. Detectors There are two main types of photo detectors used in flow cytometry Photodiodes Used for strong signals, when saturation is a potential problem ( eg . FSC detector) Photomultiplier tubes (PMT) Used for detecting small amounts of fluorescence emitted from fluorochromes . Incredible Gain (amplification-up to 10million times) Low noise

36. Fluorescent emissions are detected as a voltage pulse from photomultiplier tube (PMT) detectors The area , voltage and height of the voltage pulse is measured Voltage Laser Laser Laser time time time Voltage Voltage 1. 2. 3. Flow Cytometry : Signal Conversion in PMT Photon  Current  Voltage  Digital Signal

37. 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. Basic parameters and Windows of cell population

38. CD45 & SSC As the first step, it is most important to determine whether the cells of interest are hematopoietic. Generally speaking, all hematopoietic/lymphoid cells express CD45 antigens (CD45+). Thus, a histogram of CD45 on a logarithmic scale vs. SSC on a linear scale is indispensable as a starting point of flow cytometry analysis. Based on antigen expression, cells are divided into CD45+ and CD45– groups. Among the CD45+ group, the cells can further separated into subgroups based on expression levels of CD45 and intensity of cytoplasmic granularity.

39. Compensation Fluorochromes typically fluoresce over a large part of the spectrum (100nm or more) A detector may “see” fluorescence from more than one fluorochrome . (referred to as bleed over) You need to “compensate” for this bleed over so that ONE detector reports signal from only ONE fluorochrome

40. Histograms Flow cytometry data can be plotted in several different ways: the axes of the graphs represent fluorescence intensity data, usually plotted on a log scale for histograms, the y axis is cell number Flow Cytometry : Data Analysis

41. 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. 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. 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

42. APPLICATION Applications in Clinical Laboratories Immunophenotyping (HIV) CD4 absolute counts Leukemia and lymphoma immunophenotyping Cell cycle and ploidy analysis of tumors Reticulocyte enumeration Flow cross-matching (organ transplantation) Stem cell enumeration Residual white blood cell detection (QC platelet, red blood cells)

43. Applications in Blood Banking Fetomaternal Hemorrhage Testing Flow in AIHA Associated with a Negative DAT HLA Antibody Detection CD34+ cell enumeration and viability Platelet receptor defects (Bernard Soulier sndrome and Glanzmann ) Storage lesion studies Pretransfusion compatibility testing Reticulocyte analysis

44. Platelet analysis by flow… Identification of inherited disorders Monitoring of anti-platelet therapy Monitoring of clinical course of disease Monitoring of platelet production in thrombocytopenia Identification of patients at risk of thrombosis Diagnosis of HIT

45. APPLICATION Research Laboratories Immune function studies Hematopoietic stem cells Multi-drug resistance studies (cancer) Kinetics studies (cell function) Platelet analysis (coronary disease) Environmental sample analysis Flow and FISH

46. APPLICATION Immunophenotyping Is the term used in the identification of cells by labeling with monoclonal antibodies identified as cluster designations ( CD ) Used to determine Cell lineage Activation status Adhesion migration and homing capacity of cells Ability to respond to stimuli Ability to respond to stimuli

47. Propidium iodide ( PI) - Cell Viability How the assay works: PI cannot normally cross the cell membrane If the PI penetrates the cell membrane, it is assumed to be damaged Cells that are brightly fluorescent with the PI are damaged or dead PI PI PI PI PI PI PI PI PI PI PI PI PI PI Viable Cell Damaged Cell

48. Minimum Residual Disease Patients that appear to be in complete remission can be found to have residual tumors cells that are too few in number to be counted by standard techniques. Flow cytometry can detect these rare cells before they proliferate and cause the patient to relapse.

49. Flow Cytometric Crossmatch (FCXM) Flow cytometry has become a valuable tool to assess potential solid organ allograph recipients. It is now recognized as the laboratory procedure of choice. Circulating alloantibodies at levels too low to be detected by standard methods can be detected by a flow cytometric crossmatch (FCXM). This means that transplants done based on a negative FCXM are more successful.

50. Diagnosis of PNH 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.

51. DNA studies These assays involve measurements of fluoroscence associated with DNA to determine Stage of cell cycle Apoptosis Gene Transfection Chromosomal Aberrations Endoreduplication Doubling Time

52. Leukemia and Lymphoma Lymphomas are tumors of the immune system, primarily in the lymph nodes, spleen, and bone marrow. Flow Cytometry has been used since the late 1970's to diagnose and classify human lymphomas. A standard panel of fluorescent labeled monoclonal antibodies is used for this purpose.

53. Fluorescence-Activated Cell Sorting (FACS) v laser PMT Mixture of cells to be sorted New drop empty drop + + + + + + + + + _ _ _ _ _ _ _ _ + + + _ _ _ FACS: a specialized type of flow cytometry to sort a heterogeneous mixture of cell suspension Features Sort up to 4 populations of interests 15 fluorescence color simultaneous on the same cell Sort different types cells Primary BM, PBMC, mouse splenocytes Any types of cell lines Large fragile cells like activated neutrophiles , lung DCs Sticky and hard to sort cells (e.g. solid tumor cells, neuron cells) Multi-purpose sorting 7ml round bottom tube, 15ml conical tubes. Tissue culture plates, 96 well PCR plates Microscope slides including multiwell chamber slides Single cell sorting Different modes to maximize sort purity (99% for qPCR ) or recovery (for assays requiring large number of cells sterile sorting, sample agitation, temperature control nozzle

54. 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. Each instrument sub system –Fluidics, optics ,electronics play critical role in accuracy of data that is collected 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