When Specimens Arrive……• Check and Ensure:

– patient's identification on specimen tallies with that on its' request form (also known as requisition form) and the despatch form.

– Each case is assigned with a unique accession number and labelled clearly on the request forms as well as the specimens before processing.• For Conventional Slides CY-XXXX• For Liquid Based Preparation TP-XXXX

Conventional Smears– Specimen is smeared onto a

microscopic glass slide, spray fixed with alchohol, and sent to lab.

– Receive and stain the slides (using the Leica XL auto-stainer).

– Manual mount slides before despatching them out to the cytotechnologists on screening duties (also known as screeners)• who will screen the slides for any abnormalities

etc.

Leica XL auto-stainerTaken from:http://medequipsource.com/images/Leica%20Autostainer%20XL.jpg

Liquid Based Preparation

– Patient's gynaecological sample is collected by the clinician using a cervical sampling brush, immersed and rinsed into a vial containing PreservCyt solution.• PreservCyt Solution contains methanol, which

enhances cell preservation and nuclear morphology.

– Specimen vials will be sent to the lab. After samples are received in the lab, processing is done using the ThinPrep 2000 processor. • If samples received are too mucoid or bloody,

digestion must be done before running the samples using the processor.

ThinPrep 2000 ProcessorTaken from:http://websites.labx.com/rankin/pics/50949.jpg

Liquid Based Preparation ThinPrep 2000

– Make use of mechanical, pneumatic and fluidic principles for dispersion, cell collection, and cell transfer.

– Accessories:• Specimen Vial, ThinPrep Microscopic Glass Slide,

Fixative Vial, Transcyt membrane filter, filter cap.• The specimen vial, fixative vial and the

microscopic glass slide (which is a special coated glass slide for preparation of ThinPrep samples) is placed into the machine.The Transcyt membrane filter is fixed onto the filter cap before being placed into the machine.

Left: Brush for specimen collectionRight: Specimen Vial containing PreservCyt SolutionTaken from:http://www.imvs.sa.gov.au/tissuepath/graphics/cervex_thinprep.jpg

Left: Conventional Smear SlideRight: ThinPrep Slide (specimen collected at the centre circle area)

Taken from:http://cytologystuff.com/indexppt.htm?../powerpoint/morph1.htm

Liquid Based Preparation ThinPrep 2000

• Process: Fluid level detection Dispersion Filter wetting Cell Collection Waste Clearing Bubble Point Cell transfer.1. The filter assembly (filter cap +

Transcyt filter) will be lowered; specimen vial raised towards the assembly.

2. When the membrane filter detects/makes contact with the fluid in the specimen vial check if level is satisfactory before carrying on the process.

• If level of fluid is not satisfactory, it will halt the program.

Liquid Based Preparation ThinPrep 2000

3. Dispersion system activated; rotates the Transcyt filter assembly within the specimen(cell suspension) creating shear forces in the fluid that are strong enough to separate randomly joined material and disperse mucus.

4. Negative pressure will be applied to aid in the drawing of a small amount of fluid through the Transcyt filter to wet it.

Liquid Based Preparation ThinPrep 2000

5. System will gently blow out the liquid in the filter to clear any cellular material from the filter menbrane surface• membrane is actually a biologically neutral, flat,

smooth and porous surface which is capable of collecting cellular material.

6. Pneumatic system will apply positive and negative pressure to the filter in a series of pulses draw the specimen through the filter membrane and collect the suspended cellular material onto the membrane surface. • As material in the specimen vial collects onto the

filter mebrane, the pores of the membrane gets blocked, thereby triggering the processor to stop aspirating.

7. Collection ends transcyt filter is withdrawn from the specimen vial the filtrate is aspirated into the waste bottle as the filter is inverted; collected cells will remain of the filter due to negative pressure.

8. Excess fluid will be removed from the filter membrane before transferring the cells onto the glass slides to enhance cell adhesion to the slides.

Liquid Based Preparation ThinPrep 2000

9. The slide handler move the slide into contact with the transcyt filter natural adhesion property of the cells and electrochemical charge of the glass slide promote the transfer of cells from the filter membrane onto the slide.

• Cells have a higher affinity for the glass slide than the membrane.

• Slight positive pressure may be introduced to aid in the process.

Liquid Based Preparation ThinPrep 2000

10. Slide will then be dropped into the fixative vial PAP stained using auto-stainer (just like conventional smears) manual mount and dispatched to screeners for screening.

• Fixative vial contains 95% ethanol, which aid in fixing the cells onto the slides, and prevent cell drop (which may occur as slides are not necessarily immediately stained.)

Liquid Based Preparation ThinPrep 2000

Inside the processorTaken from:http://cyto.igabinet.pl/data/user_files/image/TP%20Processor%202.JPG

Slides placed (with label end facing down) here.

Filter Assembly

Filter Cap

Transcyt Filter

Specimen vial placed here.

Fixative vial placed here.

ThinPrep Process (simplified diagram)Taken from:http://cytologystuff.com/indexppt.htm?../powerpoint/morph1.htm

PAP staining

• Papanicolaou Staining– Show the differences in cellular morphology,

maturity and metabolic activity.– Fixation nuclear staining cytoplasmic

staining clearing

• Stains used:– Haematoxylin: Nuclear stain to demonstrate

the nuclear chromatin and nuclear membrane.

– Orange G: Cytoplasmic stain use to demonstrate keratin of squamous cells.

– Modified Eosin Azure: stains various cellular components.

Staining reagent Time

70% ethanol For loading of rack (with slides)

50% ethanol 10 sec

Wash 1 (distilled water) 30 sec

Haematoxylin 1 min 30 sec

Wash 2 1 min

0.5% HCL 1 sec

Wash 3 5 mins

1% Lithium Carbonate 10 sec

Wash 4 10 sec

50% ethanol 10 sec

70% ethanol 10 sec

PAP staining protocol

Staining reagent Time

95% ethanol 10 sec

Orange G 1 min

95% ethanol 10 sec

95% ethanol 10 sec

Modified Eosin Azure 1 min

95% ethanol 25 sec

95% ethanol 20 sec

100% ethanol 25 sec

100% ethanol 35 sec

Xylene 35 sec

Xylene 1 min

Xylene 1 min

Xylene Till ready to mount

PAP staining protocol (continue)