Optical System and Measurement
Technology Presentation
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History of optical cellcounting
Original needs:
 Limited capabilities of volumetric impedance method: differentiation is
based on cell volume only
 Better leucocyte differentiation based on internal cell structures
 Recognition of atypical cells with clinical application: Nucleated RBCs, Reticulocytes, Immature WBC-s, Atypical Lymphocytes
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Optical methods
 Light Absorbance(combined with Volumetric Impedance or Radio
Frequency)
Laser Light Scattering (generally used in haematology, it some
cases combined with RF)
Fluorescence (used in immune-haematology and immunology)
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Forward Laser Light Scattering method:
Flow cell
Laser
Optics
Detectors
Low-, and high angle scatter lights from cells
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The optical head incorporates a solid-state laser light source
(wavelength: 635 nm, maximum power: 7mW),
the beam is focused by means of a lens assembly…
Lens assembly
(laser
aperture)
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…to the center of an optically clear, quartz-glass flow cell.
The channel located in the center is rectangular , its’ walls are parallel
with the outer walls of the flow cell to eliminate light diffraction.
Cross section for flow is 250 × 250 µm.
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Assembled Laser Head, side view
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Sample is injected from below of the flow-cell, in the sheath flow which
force the cells to pass through the flow-cell in the middle of
the sheath fluid one by one(laminar flow, hydrodynamic focusing).
„Laser” waste
Flow cell inner walls(counting channel)
Sensing zone(cross section of laser light)
Sample stream
Sample injection needle
Sheath inlet ports
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As a result an about 40 µm wide sample stream passes before the
sensing zone(an about 200 µm wide laser beam), the laser light is
scattered by the cells .
Scattered light signals are collected by an optical cable mounted
behind the flow-cell. The cable has two concentric zones divided by a
metal ring for low angle and high angle scattered light sensing.
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The other ends of the optical cable are
connected to the two PIN photodiodes
mounted on the Optosensor Board.
To avoid damage of the PIN photodiodes
direct laser light is filtered by a laser
dump mounted in front of the optical cable.
„Laser dump” plate
High angle zone
Focused laser beam
Metal ring (separates low
and high angle zones)
Low angle zone
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Cell membrane scatters the light in low angle, so low angle scatter
gives information about the size of the cells while higher angle scatter
comes from the internal structure thus provides information about the
complexity of cells.
High Angle Forward Scatter
(information about the
internal structure)
Low Angle Forward Scatter
(information about size)
Focused laser
beam
Direct Light
(goes through the cell, it is stopped
by the laser dump)
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With the measuring time fixed, from each sample around 4000 – 6.500
cells* are counted.
Each cell has scatter information recorded, and displayed on the
scattergram.
* in case of human samples with normal values and normal level control
blood
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Hemolyzer 5 performs 4Diff and BASO optical count from each
sample.
4Diff dilution:
- temperature controlled(TCU, 29°C)
- whole blood + Diluent + Lyse5P => mixing
- RBC’s, PLT’s are lysed
- Diff5P is added to avoid overlysing of WBC’s(the lysing
process is stopped)
- the 4Diff dilution is transferred to the optical head
4Diff = NEU, MON, LYM, EO populations
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„4 DIFF” Scattergram
NEU
COMPLEXITY
High angle scatter
EO
MONO
LYM
SIZE
RBC GHOST
Low angle scatter
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BASO measurement:
- in the 4Diff count the Basophile leukocytes cannot be clearly
differentiated from the other cells
- additonal optical BASO count is needed
- in the WBC/HGB dilution all WBC’s are (over)lysed except
the Basophiles (chem. behaviour like lyzer)
- after the impedance count the WBC/HGB dilution is
transferred to the optical head for optical BASO count
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„BASO” Scattergram
LYM,
MONO,
NEU,
EO
BASO
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Impedance measurements:
- RBC/PLT: aperture size: 70µm
- total WBC number(non differential measurement):
aperture size: 80µm
- photometric HGB determination
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THANK YOU FOR YOUR
KIND ATTENTION!
  
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