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

2. 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

3. agile - affordable - accurate  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

4. agile - affordable - accurate Forward Laser Light Scattering method: LaserOptics Flow cell Detectors Low-, and high angle scatter lights from cells

5. agile - affordable - accurate 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)

6. agile - affordable - accurate …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.

7. agile - affordable - accurate Assembled Laser Head, side view

8. agile - affordable - accurate 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)

9. agile - affordable - accurate 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.

10. agile - affordable - accurate 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)

11. agile - affordable - accurate 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.

12. agile - affordable - accurate 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

13. agile - affordable - accurate 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

14. agile - affordable - accurate „4 DIFF” Scattergram MONO Low angle scatter High angle scatter S I Z E COMPLEXITY RBC GHOST EONEU LYM

15. agile - affordable - accurate 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

16. agile - affordable - accurate „BASO” Scattergram BASO LYM, MONO, NEU, EO

17. agile - affordable - accurate Impedance measurements: - RBC/PLT: aperture size: 70µm - total WBC number(non differential measurement): aperture size: 80µm - photometric HGB determination

18. agile - affordable - accurate THANK YOU FOR YOUR KIND ATTENTION!