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Liquid based cytologyLiquid based Cytology
Background
• The conventional pap smear has been the most successful screening test
• Screening every 3-5years has resulted in a 70% reduction in incidence
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.
Limitations Of Conventional Smear (From UK studies)
i False Negative Rate of up to 55%1
Sampling and interpretive errors
i Ambiguous reports of 6.4%2
70% are truly negative
30% represent more severe abnormality
i Inadequate specimens of 9.7% 2
1. Hutchinson et al., AJCP, Vol 101-2; 215-219, 2. DOH Statistical Bulletin 2000/2001
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
What does 'Liquid Based Cytology' mean?
• Literally it means
“cytology (the study of cells) through a liquid medium.”
Liquid-based cytology techniques
Technique of LBCStep : I Collection of sample
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
Thinprep Processor
Cell dispersion
Cell Collection
Cell Transfer
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.
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.
• 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.
Thin Prep processor(3) Cell Transfer
Summary
Advantages of LBC
Conventional smear
The prepared slide with the new ThinPrep
What we see under the microscope conventional smear
What we see under the microscope. Notice the clean back ground and how well the cells are dispersed rendering easier to interpretation.
Disadvantages of LBC
Disadvantages of LBC
Manual membrane based method of LBC
• Relatively inexpensive method• Equipments required- vortexer and
laboratory centrifuge
• 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
Procedure
Collection of sample
Specimen is vortex mixed
Centrifuge at 800 g for 10 min Vortex mix
Add 1-2 ml of polymer solution
to tube
Decant fixative and blot
excessive fixative
Allow 3-6 drops of suspension to
glass slide
Allow to dry
Stain with conv. Pap
Laser Scanning Cytometry
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.
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
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)
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.
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
Principle
Lasers
Lasers
Photomultipliers
Interpretation
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
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”
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.
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.
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.
Applications in Cytology and Pathology
In Cytology
• It negates disadvantages of
Requires sufficient amount
Verification /reproducibility not possible
Not for adherent cells
Flow cytmetry
Experience of cytologist needed
Image analysis
• Circulating tumor cells– Quantification monitoring for potential
metastasis/recurrence– Molecular studies therapeutic purposes
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.
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
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
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.
Bacterial detection
• detect live and dead bacteria,
• estimate cell numbers, and
• calculate live/dead ratios.
• The methods are easier and more accurate than traditional, manual counts.
propidium iodide (PI), unable to permeate the intact cell membrane of a living cell, but does label dead cells with red fluorescence. SYTO® 16(Molecular Probes) will label the nucleic acids of living cells with green fluorescence. Shown here are E. coli bacteria captured on a membrane and stained with PI and SYTO 16
Disadvantages
• Expensive instrumentation• Exact DNA content of paraffin block is
difficult to determine.
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