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agile - affordable - accurate Optical System and Measurement Technology Presentation
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Optical System and Measurement Technology Presentation

Feb 23, 2016

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Optical System and Measurement Technology Presentation. 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 - PowerPoint PPT Presentation
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Page 1: Optical  System and  Measurement Technology Presentation

agile - affordable - accurate

Optical System and MeasurementTechnology Presentation

Page 2: Optical  System and  Measurement Technology Presentation

agile - affordable - accurate

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 RBC-s, Reticulocytes, Immature WBC-s, Atypical Lymphocytes

Page 3: Optical  System and  Measurement Technology Presentation

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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)

Optical methods

Page 4: Optical  System and  Measurement Technology Presentation

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Forward Laser Light Scattering method:

Laser Optics

Flow cell

Detectors

Low-, and high angle scatter lights from cells

Page 5: Optical  System and  Measurement Technology Presentation

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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)

Page 6: Optical  System and  Measurement Technology Presentation

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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.

Page 7: Optical  System and  Measurement Technology Presentation

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Assembled Laser Head, side view

Page 8: Optical  System and  Measurement Technology Presentation

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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

Sensing zone(cross section of laser light)

Sample stream

Sample injection needle

Sheath inlet ports

Flow cell inner walls(counting channel)

Page 9: Optical  System and  Measurement Technology Presentation

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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.

Page 10: Optical  System and  Measurement Technology Presentation

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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.

High angle zone

Low angle zone

„Laser dump” plate

Focused laser beam

Metal ring (separates low and high angle zones)

Page 11: Optical  System and  Measurement Technology Presentation

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Low Angle Forward Scatter(information about size)

High Angle Forward Scatter (information about the internal structure)

Direct Light (goes through the cell, it is stopped by the laser dump)

Focused laser beam

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.

Page 12: Optical  System and  Measurement Technology Presentation

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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

Page 13: Optical  System and  Measurement Technology Presentation

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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

Page 14: Optical  System and  Measurement Technology Presentation

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„4 DIFF” Scattergram

MONO

Low angle scatter

Hig

h an

gle

scat

ter

S I Z E

CO

MPL

EXIT

Y

RBC GHOST

EO NEU

LYM

Page 15: Optical  System and  Measurement Technology Presentation

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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

Page 16: Optical  System and  Measurement Technology Presentation

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„BASO” Scattergram

BASO

LYM, MONO,NEU, EO

Page 17: Optical  System and  Measurement Technology Presentation

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Impedance measurements:

- RBC/PLT: aperture size: 70µm

- total WBC number(non differential measurement): aperture size: 80µm

- photometric HGB determination

Page 18: Optical  System and  Measurement Technology Presentation

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THANK YOU FOR YOUR KIND ATTENTION!