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Optical System and Measurement Technology Presentation

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

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Optical System and Measurement Technology Presentation

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  1. Optical System and Measurement TechnologyPresentation

  2. History of opticalcellcounting Originalneeds: • 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. Optical methods • Light Absorbance(combined with VolumetricImpedance or Radio Frequency) • Laser Light Scattering (generally used in haematology, it some cases combined with RF) • Fluorescence (used in immune-haematology and immunology)

  4. Flow cell Laser Optics Detectors Low-, and highanglescatterlightsfromcells • Forward Laser Light Scattering method:

  5. The optical head incorporates a solid-statelaserlightsource(wavelength: 635 nm, maximum power: 7mW), thebeam is focusedbymeans of a lens assembly… Lens assembly (laseraperture)

  6. …tothe center of an opticallyclear, quartz-glass flow cell. The channellocatedinthe center is rectangular , its’ wallsare parallel withtheouterwalls of the flow celltoeliminatelightdiffraction. Crosssectionfor flow is 250 × 250µm.

  7. Assembled Laser Head, side view

  8. Sample is injected from below of the flow-cell, in the sheathflow 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 cellinnerwalls(countingchannel) Sensingzone(crosssection of laserlight) Samplestream Sampleinjectionneedle Sheathinletports

  9. As a result an about 40 µmwidesamplestreampassesbeforethesensingzone(an about 200 µmwidelaserbeam), thelaserlight is scatteredbythecells . Scatteredlightsignalsarecollectedby an opticalcablemountedbehindtheflow-cell. The cable has twoconcentriczonesdividedby a metal ring forlowangle and highanglescatteredlightsensing.

  10. The otherends of theopticalcableare connectedtothetwo PIN photodiodes mountedontheOptosensorBoard. Toavoiddamage of the PIN photodiodes directlaserlight is filtered by a laser dumpmountedin front of theopticalcable. Highanglezone „Laser dump” plate Focusedlaserbeam Metal ring (separates low and high angle zones) Lowanglezone

  11. Cellmembranescattersthelightinlowangle, solowanglescattergivesinformationaboutthesize of thecellswhilehigheranglescattercomesfromtheinternalstructurethusprovidesinformationaboutthecomplexityofcells. HighAngleForwardScatter(informationabouttheinternalstructure) LowAngleForwardScatter (informationaboutsize) DirectLight (goesthroughthecell, it is stoppedbythelaserdump) Focusedlaserbeam

  12. Withthemeasuringtime fixed, fromeachsamplearound 4000 – 6.500 cells* arecounted. • Eachcell has scatterinformationrecorded, and displayed onthescattergram. • * incase of human sampleswithnormalvalues and normallevelcontrolblood

  13. Hemolyzer 5 performs 4Diff and BASO optical count from each sample. • 4Diff dilution: • - temperaturecontrolled(TCU, 29°C) • - wholeblood + Diluent + Lyse5P => mixing • - RBC’s, PLT’sarelysed • - Diff5P is addedtoavoidoverlysing of WBC’s(thelysingprocess is stopped) • - the 4Diff dilution is transferredtotheopticalhead • 4Diff = NEU, MON, LYM, EO populations

  14. „4 DIFF” Scattergram EO NEU Highanglescatter COMPLEXITY MONO LYM S I Z E Lowanglescatter RBC GHOST

  15. BASO measurement: - inthe 4Diff counttheBasophileleukocytescannot be clearlydifferentiatedfromtheothercells - additonaloptical BASO count is needed - in the WBC/HGB dilution all WBC’s are (over)lysed except the Basophiles (chem. behaviourlikelyzer) - aftertheimpedancecountthe WBC/HGB dilution is transferredtotheopticalheadforoptical BASO count

  16. „BASO” Scattergram LYM, MONO, NEU, EO BASO

  17. Impedancemeasurements: - RBC/PLT: aperture size: 70µm - total WBC number(non differential measurement):aperturesize: 80µm - photometric HGB determination

  18. THANK YOU FOR YOUR KIND ATTENTION!   

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