Non-Gynae Cytology

Question 1

List the 3 main serous cavities from which serous effusions are taken.

Answer 1

1) . Pleural cavities
2) . Peritoneal cavities
3) . Pericardial cavity

Question 2

What are the sample requirement for a pleural effusion?

Answer 2

Need 20-25ml of fresh unfixed effusion in a plastic universal. Sample can be stored at 4 degrees Celsius overnight if required.

Fixative or culture media may be used in some centres where transportation to the laboratory is delayed.

Before beginning to process the sample, a description of its appearance should be noted. This helps the person examining the final prep to relate the cytological findings to the microscopic description.

Question 3

Describe how to prepare direct smears from serous effusion samples.

Answer 3

1) . Spin the sample in a 25ml universal and discard supernatant.
2) . Prepare direct smears from the deposit by either the ‘blood smear’ technique (air dry) or by the ‘squash’ technique (spray fix or place in fixative).
3) . Stain air-dried slides using Romanovsky stain. Stain fixed slides with Papanicolaou stain.

Alternatively, spin sample in a 25ml centrifuge tube then remove the buffy layer using a pipette and prepare and stain smears as above using sample from the buffy layer.

Question 4

Describe how general serous samples can be prepared using cyto centrifugation.

Answer 4

Cytospin preparations can be made from either fixed or unfixed samples using the same method.

1) . Spin sample in 25ml universal and discard supernatant.
2) . Assess the deposit and make a dilution by adding Cytospin fluid or a few drops of supernatant.
3) . Spin the dilution onto the slides in the cytocentrifuge.
4) . Remove slides and fix in alcohol before staining with Papanicolaou.

If you have used the supernatant as the diluent then the slides may be air dried and stained using Romanovsky stains.

Question 5

What is a benefit of using the cytocentrifugation method to prepare serous samples?

Answer 5

The benefit of cytocentrifugation is that the cells are concentrated onto a small area of the slide. This greatly speeds up the screening process.

Question 6

Describe how you might modify the cytocentrifugation serous effusion method if the sample contains high numbers of red blood cells.

Answer 6

If the sample contains a high number of red blood cells it is useful to remove these. This will concentrate the abnormal cells and prevent over-dilution. This can be achieved by lysine the red blood cells with a weak solution of acetic acid (1%).

1) . Add acetic acid to the spun deposit, mix well and spin again.
2) . Discard the supernatant and wash the deposit in isotonic saline solution. Spin once more and discard the supernatant.
3) . Make a suitable dilution using Cytospin fluid.
4) . Prepare slides in the cytocentrifuge and stain with Papanicolaou.

Question 7

What fixative would you use for processing a serous sample with SurePath?

Answer 7

Cytorich Red

Question 8

What fixative would you use for processing a serous sample with ThinPrep?

Answer 8

CytoLyt

Question 9

What downstream technique may benefit from you processing serous samples using LBC methods?

Answer 9

IHC.

Question 10

What is a cell block?

Answer 10

A cell block is a method of suspending cytology samples on a semi solid medium that can then be processed histologically. Uniform thin sections can then be cut which are ideal for ICC.

Question 11

List 4 commonly used cell block methods.

Answer 11

1) . Thrombin cell block
2) . Agar cell block
3) . Commercial kits
4) . Clots

Question 12

Describe the basic method of producing s thrombin cell block.

Answer 12

1) . Centrifuge the sample and discard the supernatant.
2) . Add a small volume of blood plasma and thrombin to the pellet.
3) . Agitate and within a few minutes a fibrin clot forms, trapping the cells.
4) . Fix the clot in formalin and process as a histological specimen.

Question 13

Describe the process for processing a sample in an agar cell block.

Answer 13

1) . Centrifuge the sample and discard the supernatant.
2) . Add molten agar to protect the pellet and allow to cool and solidify.
3) . Process the agar block histologically.

Question 14

What is the most widely used commercial cell block kit?

Answer 14

The Thermoscientific Shandon Cytoblock cell block preparation system. This kit consists of two reagents and the cassettes in which to make the blocks.

Question 15

Describe how clots may be processed.

Answer 15

Some fluids arrive in the lab containing large clots. These can be removed, fixed in formalin and processed histologically. The clots are best removed after centrifugation. Centrifugation removes the fluid from within the clots and makes them a more solid tissue.

Question 16

What is the role of cytology in respiratory pathology?

Answer 16

The role of cytology in respiratory pathology is primarily the identification of malignant cells but other lung diseases and infections may be identified.

Question 17

List the categories that respiratory samples may fall in to.

Answer 17

1) . Sputum
2) . Bronchial washings
3) . Bronchial alveolar lavage
4) . Bronchial brushings
5) . Transbronchial FNA (TBNA)
6) . Pleural effusions

Question 18

What 4 preparation techniques are commonly used for preparation of respiratory samples?

Answer 18

1) . Direct smears
2) . Cytocentrifugation
3) . LBC
4) . Cell blocks

Question 19

What is the simples method of preparing a respiratory sample? What factors might you need to consider when preparing a respiratory sample using this method?

Answer 19

The direct smear is the simplest method of preparation. This involves selecting bloody or solid particles from the sample, smearing them onto a slide and then fixing in ethanol and staining by Papanicolaou.

This method will have relatively low diagnostic sensitivity, which may be increased by the use of mucolytic agents or by homogenisation of the sample. DTT is a common mucolytic substance.

Homogenisation of the sample can be achieved with the use of a blender or by ultrasonic disintegration. The sample is then processed by centrifugation and a direct smear is made of the deposit.

Question 20

How could you increase the diagnostic sensitivity of a direct smear from a respiratory sample?

Answer 20

Use mucolytic substances such as DTT or homogenise the sample and spin it down then make a smear using the deposit.

Question 21

How might you make a direct smear from a bronchial brush sample?

Answer 21

This can be done by rolling the brush directly onto a slide at the point of sampling. The slides can then be air dried or fixed using spray fixative.

Question 22

How might you make a direct smear from a TBNA sample?

Answer 22

This can be done at the point of sampling by depositing the sample from the needle onto a slide, then either spreading it out using the bloodsmear it squash technique.

Question 23

What bronchial samples might you prepare using cytocentrifugation?

Answer 23

Primarily TBNT samples received in Cytospin fluid but can also be used for bronchial washings and lavage samples.

Brushes received in cytospin fluid must be well agitated prior to processing to remove as many cells as possible.

Question 24

Briefly describe the Cytospin method of sample preparation.

Answer 24

1) . Spin the samples in 25ml universals and discard the supernatant.
2) . Resuspend the deposit in a small volume of Cytospin fluid.
3) . Load sample into a Cytospin funnel and spin in cytocentrifuge.
4) . Fix in alcohol and Papanicolaou stain.

Question 25

What are the 3 different types of urine that may be collected for cytological examination?

Answer 25

1) . Voided Urine - most common exfoliated (cells shed into fluid) sample type. Mainly mid stream urines (msu) will be received.
2) . Ileal conduit urine - an ileal conduit urinary diversion is a surgical technique used following bladder cancer, trauma or other irregularity of the bladder or urinary tract.
3) . Catheter urine.

Question 26

How are cerebro spinal fluid (CSF) samples collected?

Answer 26

Lumbar puncture.

Question 27

In what container should CSF samples be received?

Answer 27

A labelled plastic universal. If received in any other container deemed suitable the sample must be decanted into a universal.

Question 28

Why is it particularly important to get CSF samples to the lab quickly?

Answer 28

To ensure that the cells don’t deteriorate too much. CSF samples are particularly difficult to obtain and so it is even more important to make sure the sample isn’t wasted.

Question 29

What volume of urine will usually be received in the lab?

Answer 29

20-25ml of fresh sample (no fixative needed).

Question 30

What volume of CSF sample will usually be received in the lab?

Answer 30

Usually a very small amount of fluid (

Question 31

What is the preferred fixative for CSF samples?

Answer 31

If a fixative is to be used for CSF samples then Shandon Cytospin collection fluid is the preferred fixative.

Question 32

What is the preferred fixative for urine samples?

Answer 32

No fixative is needed.

Question 33

A). Describe how urine and CSF samples are prepared using cytocentrifugation.

B). What variations in preparation may occur between labs?

C). What is the desired outcome of this processing procedure?

Answer 33

A).
1). The samples are centrifuged at 2100RPM for 5 mins in a universal.

2) . The supernatant is discarded.
3) . The cell pellet consisting of the buffy layer and red blood cells is re suspended in Shandon Cytospin fluid.
4) . A Cytospin funnel containing an integrated filter card is paired to a labelled glass slide. The glass has been positively charged to aid adhesion. Place into a Cytospin clip and assemble into the Cytospin centrifuge.
5) . Place 0.5ml of sample into the samples designated Cytospin funnel chamber using a pipette and centrifuge for 4 mins at 1700RPM.
6) . Remove the used funnel and discard it to expose the slide. Place this slide in the appropriate slide holder with 99% alcohol to fix the sample and leave for about 15 mins for fixation to take place.
7) . Fixation in alcohol dehydrates the cell, removing approximately 80% of the water. It does not excessively shrink or swell the cells. It doesn’t distort or dissolve the cellular components. It preserves nuclear details and allows subsequent staining techniques to enhance the cell components.
8) . Stain the sample using Papanicolaou stain.

Following processing procedures samples are stored in a refrigerator for preservation and may be used for repeat tests provided sufficient sample remains. Samples are then disposed of following an agreed timescale decided locally.

B). Some labs may air dry CSF samples. In this case a fixation fluid would not be added to the specimen prior to cytocentrifugation. The slide would then be stained using the Romanowsky staining technique (the most popular being the May-Grunwald-Giemsa (MSG) method).

Some labs may resuspend the samples produced after cytocentrifugation with the samples own supernatant rather than Cytospin fluid. The end outcome will be comparable.

C). The desired outcome is to concentrate cells in the samples which tend to be low in numbers and to do this whilst a,so preserving methodology.

We also want to ensure adequate material adheres to the glass slide and to deposit cells in a targeted area of the slide in order to aid screening.

We want to be able to subsequently stain the cells using Papanicolaou or Romanowsky staining techniques.

Question 34

Serous effusions are used to diagnose malignancy. Why do we need to concentrate serous effusions onto a small area of the slide?

Answer 34

Because serous effusions contain relatively few malignant cells.

Question 35

What is involved in FNA cytology?

Answer 35

FNA is the aspiration of cells or tissue fragments using a 22/23/25 gauge needle (external diameter 0.6mm-1mm).

The needle is usually attached to a syringe that is placed under negative pressure.

The diagnostic material is collected within the needle itself, not the syringe.

Question 36

List 5 applications of FNA cytology.

Answer 36

1) . Investigation of lumps and tissue masses of uncertain nature (e.g. Simple cysts).
2) . Assist in determining the extent of tumours and help in their staging (i.e. Lymph node sampling).
3) . Investigation of suspected cancer reoccurrence.
4) . Confirmation of inoperable or locally advanced cancer.
5) . Preoperative confirmation of clinically suspected cancer.

Question 37

How is FNA usually performed? What assistance may be needed?

Answer 37

FNA can be performed manually for simple sites.

Guided FNAs include CT/X-Ray/US.

The procedure is usually performed by a clinician/physician/radiologist.

Sample preparation may be done by the person performing the procedure, staff assisting the person performing the procedure, pathologist, or cytology technical staff.

Question 38

What 4 fundamental requirements must be met for FNA cytology to be successful?

Answer 38

1) . Samples must be representative of the lesion investigated.
2) . Samples must be adequate in terms of cells and other tissue components.
3) . Samples must be correctly prepared and processed.
4) . The sample must be accompanied by appropriate clinical/radiological information.

Question 39

How accurate is FNA cytology?

Answer 39

The accuracy of FNA cytology is generally very good.

Liver approximately 96% accuracy,
Lung approximately 85%,
Bone approximately 90%,
Breast approximately 93%,
Thyroid approximately 75%

Question 40

Describe some of the key advantages of FNA cytology.

Answer 40

1) . Fast procedure with early diagnosis.
2) . Less pain and less trauma than biopsy.
3) . No anaesthesia required.
4) . Acceptably to patients and doctors.
5) . Accurate.
6) . Minimally invasive.
7) . Shortens or avoids hospital admission.
8) . Inexpensive.
9) . Equal diagnostic accuracy when compared to core biopsies in thyroid.
10) . Ideal for confirming metastasis from a clinically or radio logically suspected primary site.
11) . Ideal for distinguishing between limited alternatives such as small cell or non small cell lung cancer.
12) . Mediastinal staging of lung cancers.

Question 41

What are the key limitations of FNA cytology?

Answer 41

1) . There is a possibility of needle track seeding.
2) . May get the formation of haematoma.
3) . May develop pneumothorax.
4) . Samples suffer from the loss of tissue architecture.
5) . Inability to diagnose invasive disease.
6) . Gives minimal indication of grade and subtype.
7) . Less accurate in comparison to core biopsies in breast.
8) . Requires experienced operator to perform procedure in order to obtain an adequate sample.
9) . A core biopsy is more appropriate when there is no indication of primary site.

Question 42

Why is it preferable to receive FNA samples in fixative rather than as prepared slides?

Answer 42

Prepared slides do not allow for additional ancillary testing to be performed.

FNAs received in a fixative are favoured as this ensures the most optimal sample is received by the lab. It also allows for the production of multiple slides that can be variously stained.

Immunohistochemistry can also be performed which in many malignant cases is extremely useful for not only differentiating between reactivity versus malignancy but also in determining likely primary sites of origin of tumours.

Question 43

What is the main limitation of FNA?

Answer 43

It is limited by the lack of tissue structure in the sample that makes it impossible to diagnose invasive disease.

Question 44

What are the main 3 advantages of FNA cytology?

Answer 44

It is minimally invasive, fast and accurate.

Question 45

Give some examples of techniques where you can use FNA to sample deep seated masses?

Answer 45

Endobronchial Ultrasound-guided transbronchial FNA (EBUS-TBNA).

Endoscopic Ultrasound-guided FNA (EUS-FNA).

These techniques allow sampling of mediastinal, subaortic and paraaortic lymph nodes.