Automation

Lesson Objectives
At the end of this lesson students should be able to:
• Impedance counters
• Explain the principle of electrical impedance counters
• Apply the principle of impedance counters
• Identify and correct errors associated with impedance counters

• Light Scattering counters

• Explain the principle of light scattering counters
• Apply he principle of light scattering counters
• Identify and correct errors associated with light scattering counters
Necessity for Automation
• Cell counts
• Diagnosis of haemoglobinopathies
• Immunophenotyping
• Diagnosis of leukaemia and lymphomas
• Coagulation abnormalities
Automation
Advantages
• Speed and efficient handling
• Accuracy and precision
• Multiple tests on a single platform
• Significant reduction in labour

Disadvantages
• Flagging
• RBC morphology
• Erroneous results
• Expensive
Types of Automated Hematology
Analyzers
Semi-automated analyzers
• Measures only few parameters
• Some steps like dilution of
blood is carried out manually

Fully automated analyzers

• Measures multiple parameters
• Requires only anticoagulated
blood samples
Wallace H. Coulter (1913-1998)
• Inventor of the first
automated analyzer for
counting and sizing
cells based on his
famous “coulter
principle”
 Components of a cell counter
HYDRAULICS
• Aspirating unit
• Dispensers
• Diluters
• Mixing chambers
• Aperture bath
• Haemoglobinometer
PNEUMATICS
• Vacuums & Pressures for
operating valves
ELECTRICALS
• Analyzers & Computing
circuitary
Principles of a working automated blood
analyzer
• Electrical Impedance
• Light Scatter
• Fluorescence
• Light Absorption
• Electrical Conductivity
 Electrical impedance
• Cell counting & sizing is based on the Coulter
principle - detection & measurement of
changes in electrical impedance (resistance)
produced by a blood cell as it passes through
an electrical field
• Blood cells are poor conductors of electricity
but are suspended in an electrically
conductive diluent
• 2 chambers filled with a conductive buffered
electrolyte solution separated by a glass tube
having a small aperture
• A DC current is generated between two
electrolytes
Electrical impedance
• As a cell passes through the aperture,
flow of current is impeded and a voltage
pulse is generated
• The no: of pulses indicate the no: of the
blood cells
• The amplitude (height) of each pulse is
proportional to the cell volume
• The requisite condition for cell counting
by this method is high dilution of sample
Optical light scatter
• Each cell flows in a single line through a flow cell
• A LASER device is focused on the flow cell
• As LASER light beam strikes a cell, it is scattered in various directions
• Photodetectors capture the light
• Forward Scatter Light (FALS) ∝ to cell size
• Side Scatter Light (SS) (90°) corresponds to nuclear complexity &
granularity of cytoplasm
• Used to distinguish between granulocytes, lymphocytes & monocytes
Data Analysis
• Data is collected and stored in the computer – can be displayed
in various formats
• Parameters
• Forward Scatter
• Side scatter
• emitted fluorescence
• Data plots
• Single Parameter – Histogram
• Two Parameters – Dot Plot
Gating
• A boundary that can be set to restrict the analysis to a specific
population within the sample
Could be
• Inclusive – Selection of events that fall within the boundary
• Exclusive - Selection of events that fall outside the boundary
• Data selected by the gate is then displayed in subsequent plots
Sorting
• Consists of collecting cells of interest (defined through criteria
of size and fluorescence) for further analysis (microscopy /
functional/ chemical analysis)