AUTOMATED

• Automated Blood Cell Analysis


  Process of using instruments to count and analyze different types of blood cells
• Electronic Impedance


  Principle of cell counting based on detection and measurement of changes in electrical resistance produced by cells as they traverse a small aperture
• Radiofrequency


  Modification of electronic impedance that uses a high-voltage electromagnetic current flowing between electrodes to measure cell interior density in addition to cell volume
• Optical Scatter


  Principle that uses a focused light source (e.g. laser) to detect light scattered by cells as they pass through a sensing zone, allowing enumeration and differentiation of cell types
• Since the 1980s, automated blood cell analysis has virtually replaced manual hemoglobin, hematocrit, and cell counting because of its greater accuracy and precision, with the possible exception of phase platelet counting in certain circumstances
• Hematology analyzers are marketed by multiple instrument manufacturers and typically provide the eight standard hematology parameters (complete blood count [CBC]), plus a three-part, five-part, or six-part differential leukocyte count in less than 1 minute on 200 μL or less of whole blood
• Automation allows more efficient workload management and more timely diagnosis and treatment of disease
• Cells suspended in an electrically conductive diluent such as saline are pulled through an aperture

  Electrical resistance between electrodes, or impedance in the current, occurs as the cells pass through the sensing aperture, causing voltage pulses that are measurable
• The number of pulses is proportional to the number of cells counted


  The height of the voltage pulse is directly proportional to the volume of the cell, which allows discrimination and counting of cells of specific volumes through the use of threshold circuits
• Volume distribution histogram


  Plot of relative number on the y-axis and volume (channel number equivalent to a specific volume) on the x-axis, depicting the volume distribution of the cells counted
• Hydrodynamic focusing


  Technique that surrounds the sample stream with a sheath fluid as it passes through the central axis of the aperture, allowing laminar flow and separation of cells into single file for passage through the sensing zone
• Radiofrequency (RF) resistance


  Measurement of cell interior density, nucleus-to-cytoplasm ratio, nuclear density, and cytoplasmic granulation, in addition to cell volume measured by direct current (DC) impedance
• Two-dimensional distribution scatterplot


  Plot of DC impedance (cell volume) versus RF resistance (cell interior density and complexity) to separate and identify different cell populations
• Optical scatter


  Interaction between light (e.g. laser) and cells, resulting in scattered light that can be detected and used to enumerate and differentiate cell types
• Optical scatter may be used as the primary methodology or in combination with other methods such as electronic impedance
• Laser light differs from brightfield light in its intensity, coherence, and low divergence, allowing detection of interference and enabling enumeration and differentiation of cell types
• Light scatter results from the interaction between absorption, diffraction, refraction, and reflection as cells pass through the sensing zone and interrupt the light beam
• The detection of scattered light and its conversion into electrical signals is accomplished by photodetectors
• Laser light


  Monochromatic light emitted at a single wavelength, differs from bright-field light in its intensity, coherence (i.e., it travels in phase), and low divergence or spread
• Laser light


  • Allows for the detection of interference in the laser beam and enables enumeration and differentiation of cell types
• Optical scatter


  Light scattered in all directions as cells pass through the sensing zone and interrupt the beam, resulting from the interaction between absorption, diffraction, refraction, and reflection
• Optical scatter detection


  1. Light scattered rays detected and converted into electrical signals by photodetectors (photodiodes and photomultiplier tubes) at specific angles

  2. Lenses with blocker bars collect the scattered light

  3. Filters and mirrors separate the varying wavelengths and present them to the photodetectors

  4. Photodiodes convert light photons to electronic signals

  5. Photomultiplier tubes collect weaker signals and multiply the photoelectrons into stronger, useful signals

  6. Analog-to-digital converters change the electronic pulses to digital signals for computer analysis
• Forward-angle light scatter (0 degrees)

  Correlates with cell volume, primarily because of diffraction of light
• Orthogonal light scatter (90 degrees)

  Results from refraction and reflection of light from larger structures inside the cell and correlates with degree of internal complexity
• Forward low-angle scatter (2 to 3 degrees) and forward high-angle scatter (5 to 15 degrees)


  Correlate with cell volume and refractive index or with internal complexity
• Differential scatter


  Combination of low-angle and high-angle forward light scatter, primarily used on Siemens systems for cellular analysis
• The angles of light scatter measured by the different flow cytometers are manufacturer and method specific
• Scatter properties at different angles

  May be plotted against each other to generate two-dimensional cytograms or scatterplots
• Optical scatter


  May also be plotted against absorption, as on the Siemens systems, or against volume, as on the larger Beckman Coulter systems
• Computer cluster analysis of the cytograms may yield quantitative and qualitative information
• Hematology blood cell analyzers manufacturers

  • Abbott Laboratories
  • HORIBA Medical
  • Siemens Healthcare Diagnostics
  • Beckman Coulter
  • Sysmex Corporation
• Hematology analyzers have some common basic components, including hydraulics, pneumatics, and electrical systems
• Hydraulics system


  Includes an aspirating unit, dispensers, diluters, mixing chambers, aperture baths or flow cells or both, and a hemoglobinometer
• Pneumatics system


  Generates the vacuums and pressures required for operating the valves and moving the sample through the hydraulics system
• Electrical system


  Controls operational sequences of the total system and includes electronic analyzers and computing circuitry for processing the data generated
• Some older-model instruments have oscilloscope screens that display the electrical pulses in real time as the cells are counted
• Data display unit


  Receives information from the analyzer and prints results, histograms, or cytograms
• Specimen handling


  Varies from instrument to instrument based on degree of automation, ranging from discrete analyzers to walkaway systems with front-end load capability
• Computer functions


  Vary, with the larger instruments having extensive microprocessor and data management capabilities, including automatic start-up and shutdown, quality control, patient data storage and retrieval, host query, analysis of animal specimens, and body fluid analysis
• Beckman Coulter instruments


  Typically have two measurement channels in the hydraulics system for determining the hemogram data, with the RBC and WBC counts and hemoglobin measured directly