laser scanning cytometry and liquid based cytology

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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