Cytology

Question 1

What is Diagnostic cytology?

What is it used for?

The health and activity of the cells is determined by what?

Answer 1

Diagnostic cytology is the examination of cell matter.

It is used to help with diagnosing a disease, usually cancer

Determined by the examination of individual cells and their respective morphology, indicating the health and activity of the cells

Question 2

What are Gynecological Specimens?

Where do the most common gynecological specimens come from?

What is the term used to identify these specimens?

Who is it named after?

Answer 2

Specimens from female genital tract

Most common specimens come from cervix, endocervix, and vagina

“Pap smears” is term used to identify these specimens

Named after Dr. George Papanicolaou in the 1940’s

Question 3

Where do nongynecological specimens come from?

What are the most common nongynecological specimens?

Answer 3

These specimens come from all other body sites

Most common specimens include: urine, sputum, cerebral spinal fluid, bronchial/gastric/esophageal washings, pleural/ascites/pericardial fluids and fine needle aspirations

Question 4

How are gynecological specimens prepared?

How are liquid gynecological specimens collected?

Some techniques require the head of the brush to be left where?

Answer 4

Gynecological specimens are collected by a clinician, smeared on a slide, and rapidly fixed

Liquid specimens are collected with a “brush” or “broom” and rinsed in a vial of preservation liquid media

Some techniques require the head of the brush to be left in the solution with the sample.

Question 5

How should nongynecological specimens be collected?

How are body fluids collected?

Answer 5

• Most nongynecological specimens should be collected fresh and unfixed

◦ Body fluids: Pleural, peritoneal, pericardial and ascites fluid; entire amount brought to the lab with or without heparin (can make a good cell block)

Question 6

Who should prepare breast/nipple discharge?

What is the primary collection method for breast/nipple discharge?

Why should a prefixative be considered when collecting CSF?

Answer 6

Collection of specimens
◦Breast/Nipple discharge should be prepared by the clinician.

Smears are the primary method, using a circular motion the size of a nickel. Slides should be spray fixed immediately

◦ Cerebral Spinal fluid cells can deteriorate quickly, meaning a pre-fixative solution can be considered. Dilute the liquid sample with equal amount of Saccomanno fluid or alcoholic saline

Answer 7

Collection of specimens
◦Direct scrapings for Viral Lesions (Tzanck Smears) slides are prepared similar to breast discharge through a smear using a circular motion the size of a nickel by the clinician. These slides should be spray fixed immediately.

Answer 8

Collection of specimens
◦Direct scrapings for Viral Lesions (Tzanck Smears) slides are prepared similar to breast discharge through a smear using a circular motion the size of a nickel by the clinician. These slides should be spray fixed immediately.

Answer 9

Collection of specimens
◦Bronchial, esophageal and gastric washings do not need to be fixed, just placed in the refrigerator.

◦Bronchial, esophageal, and gastric brushings specimens are smeared at time of collection using the same circular motion mentioned previously. The brush may be submitted for processing as well. Never place in fixative as this could stop cells sticking to the slide

Answer 10

Collection of specimen
◦Urine should not be the first morning urine sample. The low pH causes urine cells to be sensitive and delays should be avoided. Pre-fixative could help with this problem, equal amounts of Saccomanno fluid or alcoholic saline can be used.

Answer 11

Collection of specimen
◦Urine should not be the first morning urine sample. The low pH causes urine cells to be sensitive and delays should be avoided. Pre-fixative could help with this problem, equal amounts of Saccomanno fluid or alcoholic saline can be used.

Answer 12

Collection of specimens
◦Fine needle aspirations can be smeared using one drop of specimen using the pull apart method. Never use the “feathered-edge” technique. Cellularity of specimen can be determined using Diff-Quik or toluidine blue wet film stains

Answer 13

Fixation
◦Rapid fixation is essential!!!!
◦Fixative of choice is 95% Ethanol
◦Formalin should be avoided because they create distinct chromatin patterns not suitable for cytology
◦Some spray fixatives contain polyethylene glycol to coat the specimen, this must be removed by 15minutes in alcohol prior to staining

Answer 14

Fixation
◦Substitution alcoholic solutions is based on cell shrinkage: Methanol causes less shrinkage than ethanol so 100% methanol can be substituted for 95% ethanol. Isopropanol causes more shrinkage, so 80% Isopropanol can be substituted for 95% ethanol.
◦Air drying cells causes nuclear swelling and distortion and should be avoided

Answer 15

Fixation
◦Pre-Fixatives are used mainly to transport tissues
◦Saccomanno Fluid: 50% Ethanol (980mL) with melted Carbowax (20mL)
◦Alcoholic Saline: 1:1 ratio of Saline and 50% Ethanol
◦Smears with bloody specimens can be put in coplin jars of Clark or Carnoy fixatives to lyse RBC’s.

Answer 16

Smear Preparation
◦Many different techniques are used, you must be able to identify which technique could hinder or obscure your examination of the cells
◦The goal, similar to histotechnology, should be to get a monolayer of cells while preserving morphological detail

Answer 17

Smear Preparation
◦Direct smears include scraping, brushings, Tzank smears, and viral inclusion smears

Nickel Method: easy and quick. Helps reduce air drying avoid using cotton swabs

Answer 18

Fluids are usually concentrated with a centrifuge at 2000RPM for 10 minutes.
◦Pull-apart method is simple and gives a good mono layer of cells

Answer 19

Smear Preparation
◦Crosshatch method is a good choice for fluids containing blood. Larger cells for diagnostic markers tend to be at the ends of the “arms” which help locate them easier.

Answer 20

•Specimen preparation
◦Mucoid specimens include sputum and thick broncial washings

•Crush method involves crushing the specimen inbetween two slides to break up the mucus.

Answer 21

Specimen preparation
◦Sparsely cellular specimens must be condensed a bit as methods previously mentioned rarely give adequate amounts of cells on the slide for examination

◦Cytocentrifugation preparations must be prepared. Method involves centrifuging the specimen, then running through a cytocentrifuge system to capture cells

Answer 22

Specimen Preparation
◦Fine needle asparations come from superficial and deep organs; breast and thyroid, lung liver and pancreas. Often done in radiology. Drops used for cytology and needle rinsed in specimen preparation fluid/fixative.
•NOTE: Not same as core needle biopsy.

Answer 23

Problems related to cytology

◦Bloody specimens can compromise cell morphology. Cross hatch method can overcome this a bit, and use of Clark and Carnoy fixative can help lyse some red blood cells.

Answer 24

Problems related to cytology

◦Poorly adhesive specimens such as breast fluid and urine are lost during staining. Spray fixing instead of liquid fixing can help. Cytocentrifugation instead of direct smears can help. Using positively charged slides all help.

Answer 25

Cell blocks

◦Method of preparing cytology material so it can be routing processed, embedded, cut, and stained. Makes special stains easier (IHC). Specific methods have been used in the past depending on the type of cellular sample

Answer 26

Cell block

◦Specimens with spontaneous clots; liquid should be squeezed out and placed in cassette

◦Cellular material trapped inside mucus, tissue should be fixed with alcohol or NBF and centrifuged

◦Loose cellular material using albumin can be used with 95% ethanol (to fix) to create a soft ball.

Answer 27

Cytology staining

◦Special stains can be done as well as IHC, as long as the deparaffinization step is omitted and slides can be briefly air dried.

Answer 28

Cytology Staining

◦Hematoxylin is used to stain nuclei. Papanicolaou also uses either Harris or Gill hematoxylin.

◦OG-6 is the first counterstain, an alcoholic solution of orange G (gelb), with the mordant phosphatungstic acid. Can stain keratin tonofiliments to show keratinizing neoplasms. Glacial acetic acid can be used to intensify staining

Answer 29

Cytology Staining

◦EA is a polychrome stain containing eosin Y, light green SF yellowish, Bismark brown, and phosphotungstic acid. The acid help allow eosin and light green to stain differently. Light sensitive and can be made without unstable bismark brown

Answer 30

Papanicolaou stain
◦Purpose: distinguish cellular components through chromatin, counterstaining, cytoplasmic transparency
◦Fixative: any alcohol-fixed cytologic specimen
◦Results: Chromatin-blue
•Keratin-orange
•Superficial squamous cells-variable shades of pink
•Nucleoili, cilia, RBCs-variable shades of pink

Answer 31

Toluiding blue wet film
◦Purpose: rapid stain showing cellularity, metachromatic staining
◦Results: shows different shades of blue

Answer 32

Cytology cross contamination

◦ Due to the loose cellular nature of cytology specimens, cells can detach from slide and possibly attach to another, or float out in liquid solutions to be picked up on another specimen slide. Solutions should be filtered between batches and stained separately from nongynecological specimens.