Laser scanning cytometry and liquid based cytology


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  • Blood mucus overlap not preserved well
  • he TransCyt® filter has been plunged into the sample, it rotates at a high speed and facilitates cell and mucus dispersion. A vacuum is then applied to the filter, which collects cells on a 5 μm porosity membrane. A software program allows a homogeneous deposition of cells until saturation. The TransCyt filter is then inverted and a positive pressure allows cells to adhere to an electronegative slide. After insertion of another TransCyt filter and of another slide, the whole procedure may be repeated until the entire sample has been treated.
  • Image or scanning cytometry (IC) combines imaging and cytometric analysis in a single technology platform.Rather than randomly imaging an entire field (like a microscope does), it selects, images and measures cells that meet certain user-adjustable criterion (such as size or fluorescence). It is therefore not random, but event-based.It is closely related to conventional flow cytometry, which also analyzes individual cells that meet certain characteristics (and is also event-based). Both are therefore cytometric techniques Unlike conventional flow cytometry, IC usually analyzes cells fixed to a horizontal surface. However, the technology (light sources, detectors, etc.) is very analogous to traditional flow cytometry. With scanning cytometry, imagery becomes a parameter, and relates to the other parameters (scatter and fluorescence).
  • The microscope (Olympus Optical Co.) is the key part of the instrument, and it providesessential structural and optical components (Fig. 1). The specimen deposited on a micro-scope slide on the stage of the microscope is illuminated by laser beams that rapidly scan the slide. The beams from two lasers (argon ion and helium-neon) spatially merged by dichroic mirrors are directed onto the computer controlled oscillating (350 Hz) mirror, which reflects them through the epi-illumination port of the microscope and images through the objective lens onto the slide. The mirror oscillations cause the laser beams to sweep the area of micro-scope slide under the lens. The beam spot size varies depending on the lens magnification, from 2.5 (at 40x) to 10.0 m (at 10x). The slide, with its xy position monitored by sensors, is placed on the computer-controlled motorized microscope stage, which moves at 0.5 m steps per each laser scan, perpendicularly to the scan. Laser light scattered by the cells is imaged by the condenser lens and its intensity recorded by sensors. The specimen-emitted fluorescence is collected by the objective lens and part of it is directed to a charge-coupling device(CCD) camera for imaging. Another part is directed to the scanning mirror. Upon reflection,it passes through a series of dichroic mirrors and optical emission filters to reach one of the four photomultipliers. Each photomultipler records fluorescence at a specific wavelength range, defined by the combination of filters and dichroic mirrors. A light source, additional to the lasers, provides transmitted illumination to visualize the objects through an eyepiece or the CCD camera.
  • 1. Integrated fluorescence intensity representing the sum of intensities of all pixels (pic-ture elements) within the integration contour area. The latter may be adjusted to a de-sired width with respect to the threshold contour (Fig. 2).2. The maximal intensity of an individual pixel within this area (maximal pixel).3. The integration area, representing the number of pixels within the integration contour.4. The perimeter of the integration contour (in micrometers).5. The fluorescence intensity integrated over the area of a torus of desired width definedby the peripheral contour located around (outside) of the primary integration contour.For example, if the integration contour is set for the nucleus, based on red fluorescence(DNA stained by propidium iodide, PI), then the integrated (or maximal pixel) greenfluorescence of FITC (fluoresceinisothiocyanate)-stained cytoplasm can be measuredseparately, within the integration contour (i.e., over the nucleus) and within the periph-eral contour (i.e., over the rim of cytoplasm of desired width outside the nucleus). Allabove values of fluorescence (1,2,4) are automatically corrected for background,which is measured outside the cell, within the background contour (Fig. 2).6. The Xy coordinates of maximal pixel locating the measured object on of the microscopestage. 7. The computer clock time at the moment of measurement.The measurements by LSC are relatively rapid; having optimal cell density on the slide upto 5000 cells can be measured per minute. The accuracy and sensitivity of cell fluorescence measurements by LSC are comparable to the advanced flow cytometers
  • Intensity of maximal pixel of fluorescence is a sensitive marker of local hypo- or hyper-chromicity within the cell, reflecting low or high concentration (density) of the fluorescent probe. One of the early applications of LSC along this line was to detect chromatin conden-sation. Namely, DNA in condensed chromatin, such as mitotic or apoptotic cells, shows in-creased stainability (per unit area of chromatin image) with most fluorochromes. Thus, evenwhen integrated fluorescence of the analyzed cells (representing their DNA content) is the same, the intensity of maximal pixel of the cell with condensed chromatin is higher than that of the cell with more diffuse chromatin structure. Maximal pixel of the DNA-associated fluorescence was used as a marker to distinguish mitotic and immediately post-mitotic G 1 cells from interphase cells [5,6]. Although mitotic cells can be recognized by FC using a variety of markers [reviewed in 7], the advantage of this approach by LSC is that a single fluorochromeis used to discriminate between G 1 vs S vs G 2 vs M phase cells.
  • Laser scanning cytometry and liquid based cytology

    1. 1. Liquid based cytology Liquid based Cytology
    2. 2. Background • The conventional pap smear has been the most successful screening test • Screening every 3-5years has resulted in a 70% reduction in incidence
    3. 3. Why LBC (Liquid based cytology) hasbeen introduced ? • Continuing improvement to the Cervical screening Programme • Limitations of conventional cytology • Modernisation of the technique • Future benefits – Extra tests – HPV, chlamydia, Neiseria Gonorrhoea.
    4. 4. Limitations Of Conventional Smear (From UK studies) False Negative Rate of up to 55%1 Sampling and interpretive errors Ambiguous reports of 6.4%2 70% are truly negative 30% represent more severe abnormality Inadequate specimens of 9.7% 2 1. Hutchinson et al., AJCP, Vol 101-2; 215-219, 2. DOH Statistical Bulletin 2000/2001
    5. 5. Sources Of False Negatives • Sampling issues (70%) – cells not collected on the sampling device – cells collected, but not transferred to the slide • Thicker and thinner areas • Nuclear feathering artifact • Interpretive issues (30%) – abnormal cells present on slide but either not seen or misinterpreted • Blood/mucus • Air drying artifact
    6. 6. What does Liquid Based Cytologymean? • Literally it means “cytology (the study of cells) through a liquid medium.”
    7. 7. Liquid-based cytology techniques
    8. 8. Technique of LBCStep : I Collection of sample
    9. 9. Specimen Collection • PreservCyt Solution. • Capped, labeled, and sent to the laboratory equipped with a ThinPrep 2000 Processor . Composition • Buffered methanol • No active ingredient Storage • 15 to 30 C for 6 weeks
    10. 10. Thinprep Processor Cell Cell Cell dispersion Collection Transfer
    11. 11. Thin Prep processor (1)Cell dispersion • Swirling the sampling device in the preservation solution • Strong enough to separate debris and disperse mucus, but gentle enough to have no adverse effect on cell appearance.
    12. 12. Thin Prep processor (2) Cell Collection • A gentle vacuum is created within the ThinPrep Pap Test Filter, which collects cells on the exterior surface of the membrane. • Cell collection is controlled by the ThinPrep 2000 Processor’s software that monitors the rate of flow through the ThinPrep Pap Test Filter.
    13. 13. Thin Prep processor (3) Cell Transfer • After the cells are collected on the membrane, the ThinPrep Pap Test Filter is inverted and gently pressed against the ThinPrep Microscope Slide. • Natural attraction and slight positive air pressure cause the cells to adhere to the ThinPrep Microscope Slide resulting in an even distribution of cells in a defined circular area.
    14. 14. Summary
    15. 15. Advantages of LBC
    16. 16. Conventional smear
    17. 17. The prepared slide with the newThinPrep
    18. 18. What we see under the microscopeconventional smear
    19. 19. What we see under themicroscope. Notice the clean background and how well the cells aredispersed rendering easier to
    20. 20. Disadvantages of LBC
    21. 21. Disadvantages of LBC
    22. 22. Manual membrane based methodof LBC • Relatively inexpensive method • Equipments required- vortexer and laboratory centrifuge
    23. 23. • Solution required are; Fixative solution • Surepath preservation fluid • Lab prepared- water, NaCl, Na Citrate, 10% formalin,alcohol Polymer solution • Agarose, polyethylene glycol • Alcohol, poly-L-lysene
    24. 24. Procedure Decant fixative Allow 3-6 drops Collection of and blot of suspension to sample excessive glass slide fixative Add 1-2 ml of Specimen is polymer solution Allow to dry vortex mixed to tube Centrifuge at Stain with conv. Vortex mix800 g for 10 min Pap
    25. 25. Laser Scanning Cytometry
    26. 26. What is the need?• Our limited ability to undertake accurate, quantitative measurement of cellular and subcellular factors• Established technologies in clinical pathology, including conventional microscope- based histopathology and histochemistry, fluorescence microscopy, flow cytometry and computer-based image cytometry, all have limitations.
    27. 27. Introduction• Imaging + cytometric analysis• Not random, but event-based.• It is closely related to conventional flow cytometry, which also analyzes individual cells that meet certain characteristics (and is also event- based). Both are therefore cytometric techniques
    28. 28. Limitations of Flow cytometry 1. time-resolved events such as enzyme kinetics cannot be analyzed. 2. Simultaneous study of Morphology of the measured cell is not possible. 3. Cell analysis is at zero spatial resolution. 4. The cell once measured cannot be re- analyzed with another probe(s)
    29. 29. 5. Analysis of solid tissue requires cell or nucleus isolation, a procedure that may produce artifacts and loss of the information on tissue architecture.6.size samples such as fine needle aspirates, spinal fluid, thus, are seldom analyzed by FC.7. The measured sample cannot be stored for archival preservation.
    30. 30. Introduction• 2 manufacturers: – CompuCyte Corp. (Cambridge, MA) – Olympus Optical Co. (Tokyo), offers many advantages of flow cytometry but has no limitations listed above.• The analytical capabilities of LSC, therefore, complement these of FC, and extend the use of cytometry in many applications
    31. 31. Principle
    32. 32. Lasers LasersPhotomultipliers
    33. 33. Interpretation
    34. 34. Thresholding on flow cytometer • Setting the threshold (or discrimination) on the flow cytometer allows us to eliminate unwanted cells (like erythrocytes) from the saved analysis
    35. 35. Thresholding (or contouring) on the laser scanning cytometer• On the laser scanning cytometer, any event that is above the threshold (usually DNA fluorescence or sometimes forward scatter) is also considered a “cell” and is also displayed for all parameters.• The instrument marks such events with a red “contour”, which contains the amount of fluorescent signal above the threshold.• Thresholding on the LSC is therefore referred to as “contouring”
    36. 36. Event-based contouring on the iCys The red region is the event or thresholding contour. It is analogous to “thresholding” or “discrimination” on the flow cytometer. It defines the minimum signal intensity that defines a cell. Everything else below it is ignored. Like in cytometry, you need a universal parameter (like scatter or DNA luorescence) as the trigger for threshold contouring The green region is the inte grating contour. You can set this any number of pixels out from the thresholding contour and measure the brightest pixel (Max Pixel), or the total fluorescence (Integral) within the green region. The blue regions are the background contours. You can also set these any number of pixels out from the integrating contour, away from the cell. The signal between them is interpreted as the autofluorescence background, which is subtracted from the other signals.
    37. 37. Parameters studied by LSC 1. Integrated fluorescence intensity 2. The maximal intensity 3. The Integration area 4. The perimeter of the integration contour (in micrometers). 5. The fluorescence intensity integrated over the area of a torus of desired width defined by the peripheral contour located around (outside) of the primary integration contour.
    38. 38. 6. The X-Y coordinates of maximal pixel locating the measured object on of the microscope stage.7. The computer clock time at the moment of measurement.
    39. 39. Applications in Cytology andPathology
    40. 40. In Cytology• It negates disadvantages of Flow Image cytmetry analysis Requires Experience of sufficient cytologist amount needed Verification /reproducibility not possible Not for adherent cells
    41. 41. • Circulating tumor cells – Quantification monitoring for potential metastasis/recurrence – Molecular studies therapeutic purposes
    42. 42. Apoptosis studies• Characterized by certain morphological features e.g. nuclear fragmentation, nuclear condensation• Methods e.g. annexin V, loss of transmembrane potential in mitochondria, analysis of nuclear fragmentation, alteration of DNA condensation.
    43. 43. Immunophenotyping of leucocyte • Especially useful when amount of obtained sample is minimum e.g. neonate, critically ill patients • Upto 5 flurochromes in < 15 microlit of peripheral blood. • Analysis by 2 methods – Specific Immunostaining e.g. CD45 – DNA staining with 7-AAD- difference in intensity with different leucocytes
    44. 44. Ploidy and DNA index • Anueploid number characteristic of a malignant cell e.g. Gastric, colon, kidney, head and neck etc. • Prognostic marker in several tumors • LSC measures amount of DNA in cell
    45. 45. Tumor cells areidentified andgated basedupon cytokeratinstaining.To verify the morphology of the twopopulations, cells can be restainedwith Wright-Giemsa or H & E. Cellsfrom each region can be relocatedand visualized by the CompuSort™process.
    46. 46. Bacterial detection• detect live and dead bacteria,• estimate cell numbers, and• calculate live/dead ratios.• The methods are propidium iodide (PI), unable to permeate the intact cell membrane easier and more of a living cell, but does label dead accurate than cells with red fluorescence. SYTO® 16(Molecular Probes) will traditional, manual label the nucleic acids of living cells counts. with green fluorescence. Shown here are E. coli bacteria captured on a membrane and stained with PI and SYTO 16
    47. 47. Disadvantages• Expensive instrumentation• Exact DNA content of paraffin block is difficult to determine.
    48. 48. Thank You