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. Why LBC (Liquid based cytology) has
been 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. 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. 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. What does 'Liquid Based Cytology'
mean?
• Literally it means
“cytology (the study of cells)
through a liquid medium.”
11. 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
13. 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.
14. 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.
15. 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.
33. 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 mix
800 g for 10 min Pap
35. 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.
36. 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
37. 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)
38. 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.
39. 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
45. 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
46. 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”
47. 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.
48.
49. 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.
50. 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.
52. 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
54. 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.
55.
56. 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
57.
58. 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
59. Tumor cells are
identified and
gated based
upon cytokeratin
staining.
To verify the morphology of the two
populations, cells can be restained
with Wright-Giemsa or H & E. Cells
from each region can be relocated
and visualized by the CompuSort™
process.
60. 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
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.
