Because diagnoses and treatment plans are made based on laboratory findings, it is imperative that the equipment utilized in the lab be in excellent working order, serviced at regular intervals, calibrated and cleaned as recommended by the manufacturer, and used properly. In addition to properly functioning equipment, there are things the technician can do to improve the accuracy of their test results:
- Follow manufacturer directions precisely.
- Become familiar with normal and abnormal findings.
- Log all activity of equipment, including daily, weekly, and monthly servicing.
- Save enough sample to perform tests more than once to verify accuracy of findings.
Remember, all laboratory equipment and its results are only as reliable as the human operating the equipment!
Automated hematology analyzers work on different principles:
- Electrical impedance
- Light scatter
- Fluorescence
- Light absorption
- Electrical conductivity.
Most analyzers are based on a combination of different principles.
(1) Electrical impedance: This is the classic and timetested technology for counting cellular elements of blood. As this method of cell counting was first developed by Coulter Electronics, it is also called as Coulter principle (see Figure 811.1). Two electrodes placed in isotonic solutions are separated by a glass tube having a small aperture. A vacuum is applied and as a cell passes through the aperture, flow of current is impeded and a voltage pulse is generated.
Figure 811.1 Coulter principle of electrical impedance
The requisite condition for cell counting by this method is high dilution of sample so that minimal numbers of cells pass through the aperture at one point of time. There are two electrodes on either side of the aperture; as the solution in which the cells are suspended is an electrolyte solution, an electric current is generated between the two electrodes. When a cell passes through this narrow aperture across which a current is flowing, change in electrical resistance (i.e. momentary interruption of electrical current between the two electrodes) occurs. A small pulse is generated due to a temporary increase in impedance. This pulse is amplified, measured, and counted. The height of the pulse is proportional to cell volume. The width of the pulse corresponds with the time required for the cell to traverse the aperture. Cells that do not pass through the center of the aperture generate a distorted pulse that is not representative of the cell volume. Some analyzers use hydrodynamic focusing to force the cells through the central path so that all cells take the same path for volume measurement.
An anticoagulated whole blood sample is aspirated into the system, divided into two portions, and mixed with a diluent. One dilution is passed to the red cell aperture bath (for red cell and platelet counting), and the other is delivered to the WBC aperture bath (where a reagent is added for lysis of red cells and release hemoglobin; this portion is used for leukocyte counting followed by estimation of hemoglobin). Particles between 2-20 fl are counted as platelets, while those between 36-360 fl are counted as red cells. Hemoglobin is estimated by light transmission at 535 nm.
(2) Light scatter: Each cell flows in a single line through a flow cell. A laser device is focused on the flow cell; as the laser light beam strikes a cell it is scattered in various directions. One detector captures the forward scatter light (forward angle light scatter or FALS) that is proportional to cell size and a second detector captures side scatter (SS) light (90°) that corresponds to the nuclear complexity and granularity of cytoplasm. This simultaneous measurement of light scattered in two directions is used for distinguishing between granulocytes, lymphocytes, and monocytes.
(3) Fluorescence: Cellular fluorescence is used to measure RNA (reticulocytes), DNA (nucleated red cells), and cell surface antigens.
(4) Light absorption: Concentration of hemoglobin is measured by absorption spectrophotometry, after conversion of hemoglobin to cyanmethemoglobin or some other compound. In some analyzers, peroxidase cytochemistry is used to classify leukocytes; the peroxidase activity is determined by absorbance.
(5) Electrical conductivity: Some analyzers use conductivity of high frequency current to determine physical and chemical composition of leucocytes for their classification.
Further Reading:
FLOW CYTOMETRY
Flow cytometry is a procedure used for measuring multiple cellular and fluorescent properties of cells when they flow as a single cell suspension through a laser beam by a specialized instrument called as a flow cytometer. Flow cytometry can analyze numerous cells in a short time and multiple parameters of a single cell can be analyzed simultaneously. From the measured parameters, specific cell populations are defined. Cells or particles with size 0.2-150 μm are suitable for flow cytometer analysis.Flow cytometry can provide following information about a cell (Box 807.1):
- Cell size (forward scatter)
- Internal complexity or granularity (side scatter)
- Relative fluorescence intensity.
A flow cytometer consists of three main components or systems: fluidics, optics, and electronics.
(1) Fluidics: The function of the fluidics system is to transport cells in a stream to the laser beam for interrogation. Cells (fluorescence-tagged) are introduced into the cytometer (injected into the sheath fluid within the flow chamber) and made to flow in a single file past a laser (light amplification by stimulated emission of radiation) beam. The stream transporting the cells should be positioned in the center of the laser beam. The portion of the fluid stream where the cells are located is called as the sample core. Only a single cell or particle should pass through the laser beam at one time. Flow cytometers use the principle of hydrodynamic focusing (process of centering the sample core within the sheath fluid) for presenting cells to the laser.
(2) Optics: This system consists of lasers for illumination of cells in the sample, and filters to direct the generated light signals to the appropriate detectors.
The light source used in most flow cytometers is laser.
The laser most commonly used in flow cytometry is Argon-ion laser. The light signals are generated when the laser beam strikes the cell, which are then collected by appropriately positioned lenses. A system of optical mirrors and filters then directs the specified wavelengths of light to the designated detectors. Two types of light signals are generated when the laser beam strikes cells tagged with fluorescent molecules: fluorescence and light scatter. The cells tagged with fluorescence emit a momentary pulse of fluorescence; in addition, two types of light scatter are measured: forward scatter (proportional to cell diameter) and side scatter (proportional to granularity of cell).
(3) Electronics: The optical signals (photons) are converted to corresponding electronic signals (electrons) by the photodetectors (photodiodes and photomultiplier tubes). The electronic signal produced is proportional to the amount of light striking a cell. The electric current travels to the amplifier and is converted to a voltage pulse. The voltage pulse is assigned a digital value representing a channel by the Analog-to Digital Converter (ADC). The channel number is then transferred to the computer which displays it to the appropriate position on the data plot.
Further Reading:
Fluorescence
A fluorochrome absorbs light energy and emits excess energy in the form of photon light (fluorescence). Fluorescence is the property of molecules to absorb light at one wavelength and emit light at a longer wavelength. The fluorescent dyes commonly used in flow cytometry are fluorescein isothiocyanate (FITC) and phycoerythrin (PE). The fluorochrome-labeled antibodies are used for detection of antigenic markers on the surface of cells. A particular cell type can be identified on the basis of the antigenic profile expressed. Multiple fluorochromes can be used to identify different cell types in a mixed population of cells.
Light Scatter
Light is scattered when the incident light is deflected by a particle traversing through a beam of light. This depends on the physical properties of the cell. Two forms of light scatter are used to identify different cell types: forward scatter and side scatter. Forward scatter (light scattered in the same direction as the laser beam) is related to cell size. Side scatter (light scattered at a 90° angle to the laser beam) is related to internal granularity of the cell. Main subpopulations of leukocytes are identified on the basis of correlated measurements of forward and side scatters. When a cell passes through laser beam, side scatter and fluorescent signals that are emitted by the cell are directed to photomultiplier tubes, while the forward scatter signals are directed to a photodiode. Photomultiplier tubes and photodiodes are called as detectors. Optical filters are placed before the detectors that allow only a narrow range of wavelengths to reach the detectors (see Figure 806.1).
Figure 806.1 Principle of working of a flow cytometer
Data Analysis
The data collected and stored in the computer can be displayed in various formats. A parameter means forward scatter, or side scatter, or emitted fluorescence from a particle as it passes through a laser beam. A histogram is a data plot of a single parameter, with the parameter’s signal value in channel numbers or relative fluorescence intensity on X-axis (horizontal axis) and number of events on the Y-axis. A dot plot is a two parameter data graph in which each dot represents one event that the flow cytometer analyzed; one parameter is displayed on the X-axis and the other on the Y-axis. A 3-D plot represents one parameter on X-axis, another parameter on Y-axis, and number of events per channel on Z-axis.
Gating
A gate is a boundary that can be set to restrict the analysis to a specific population within the sample. For example, a gate boundary can be drawn on a dot plot or histogram to restrict the analysis only to cells with the size of lymphocytes. Gates can be inclusive (selection of events that fall within the boundary) or exclusive (selection of events that fall outside the boundary). Data selected by the gate is then displayed in subsequent plots.
Sorting
Usually, when a cell passes through the laser beam, it is sent to waste. Sorting consists of collecting cells of interest (defined through criteria of size and fluorescence) for further analysis (such as microscopy or functional or chemical analysis). Sorting feature is available only in some flow cytometers.
- Leukemias and lympomas: Immunophenotyping (evaluation of cell surface markers), diagnosis, detection of minimal residual disease, and to identify prognostically important subgroups.
- Paroxysmal nocturnal hemoglobinuria: Deficiency of CD 55 and CD 59.
- Hematopoietic stem cell transplantation: Enumeration of CD34+ stem cells.
- Feto-maternal hemorrhage: Detection and quantitation of foetal hemoglobin in maternal blood sample.
- Anemias: Reticulocyte count.
- Human immunodeficiency virus infection: For enumeration of CD4+ lymphocytes.
- Histocompatibility cross matching.
The first fluorescence-based flow cytometry device (ICP 11) was developed in 1968 by Wolfgang Göhde from the University of Münster, Germany and first commercialized in 1968/69 by German developer and manufacturer Partec through Phywe AG in Göttingen. At that time, absorption methods were still widely favored by other scientists over fluorescence methods. The original name of the flow cytometry technology was pulse cytophotometry (German: Impulszytophotometrie). Only 10 years later in 1978, at the Conference of the American Engineering Foundation in Pensacola, Florida, the name was changed to flow cytometry, a term that quickly became popular. Soon after, flow cytometry instruments were developed, including the Cytofluorograph (1971) from Bio/Physics Systems Inc. (later: Ortho Diagnostics), the PAS 8000 (1973) from Partec, the first FACS instrument from Becton Dickinson (1974), the ICP 22 (1975) from Partec/Phywe and the Epics from Coulter (1977/78).
Principle of flow cytometry
A beam of light (usually laser light) of a single wavelength is directed onto a hydrodynamically-focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam: one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC) and one or more fluorescent detectors).
Each suspended particle from 0.2 to 150 micrometers passing through the beam scatters the light in some way, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a longer wavelength than the light source. This combination of scattered and fluorescent light is picked up by the detectors, and, by analyzing fluctuations in brightness at each detector (one for each fluorescent emission peak), it is then possible to derive various types of information about the physical and chemical structure of each individual particle.
Use the Reference manual for in-depth information on the principles of flow cytometry, information about what your instrument does, the methods it uses, its specifications, and information on installation, safety, and system options.
Use the Getting Started manual to become familiar with the controls and indicators for your system and to learn about protocols, regions, panels, and the basic skills you need to operate the system. This manual also has an overview of the software.
Use the Operator’s Guide for the day-to-day running of your instrument. Go through the detailed step-by-step procedures of startup, quality control (QC), running samples, analyzing data, printing reports, reviewing QC data, and shutdown.
Use the Data Management manual for instructions on how to export, save, copy, move, archive, and delete files. It also has information about the types of files your system creates and uses, instructions for working with QC features, and instructions for setting up the report template that you need to create your patient reports.
Use the Special Procedures and Troubleshooting manual to clean, replace, or adjust a component of the instrument. The Troubleshooting tables and error messages appear at the back of the manual.
Use the Operating Summary as a quick reference for basic procedures.
Use the Master Index to easily locate a topic in any of your manuals.
Use the User's Comment Card in the Reference manual to give us your comments about the manual and ways to improve it.
Use the Getting Started manual to become familiar with the controls and indicators for your system and to learn about protocols, regions, panels, and the basic skills you need to operate the system. This manual also has an overview of the software.
Use the Operator’s Guide for the day-to-day running of your instrument. Go through the detailed step-by-step procedures of startup, quality control (QC), running samples, analyzing data, printing reports, reviewing QC data, and shutdown.
Use the Data Management manual for instructions on how to export, save, copy, move, archive, and delete files. It also has information about the types of files your system creates and uses, instructions for working with QC features, and instructions for setting up the report template that you need to create your patient reports.
Use the Special Procedures and Troubleshooting manual to clean, replace, or adjust a component of the instrument. The Troubleshooting tables and error messages appear at the back of the manual.
Use the Operating Summary as a quick reference for basic procedures.
Use the Master Index to easily locate a topic in any of your manuals.
Use the User's Comment Card in the Reference manual to give us your comments about the manual and ways to improve it.
