Cytology: Sample Collection and Preparation for Diagnostic Interpretation

Sample Collection Methods in Cytology

Cytology samples can be obtained from solid masses on an animal’s body or during surgical procedures. Multiple techniques are available depending on the nature of the sample:

• Swab, Scrape, or Imprint Techniques: Commonly used for samples from solid masses.

• Fine Needle Biopsy (FNB): Used for both solid and fluid samples.

• Centesis: Specifically used for the collection of fluid from body cavities.

Swab Collection and Handling

Swabs are generally used only when imprints, scrapings, and aspirates cannot be performed. Common applications include collection from fistulous tracts and vaginal collections.

• Procedure for Collection:

  • A sterile cotton or rayon swab should be moistened with sterile isotonic fluid, such as $0.9\,\%\,saline$.

  • Moistening the swab helps to minimize cell damage during the collection process.

  • After the sample is collected, the swab must be gently rolled along the flat surface of a clean slide.

  • Rubbing the swab on the slide is prohibited as it causes significant cell damage.

• Ear Swabs:

  • Ear samples frequently contain excess wax. To minimize interference during evaluation, the slide can be gently heated to dissolve the wax.

  • Acceptable heating methods include passing the slide through a flame or using a hair dryer.

  • Care must be taken to avoid excessive heat, which may damage the cells.

  • Besides Gram staining, this is the only time it is acceptable to apply heat to a cytology slide.

Scraping Techniques

Scrapings are used to create smears from tissues collected during necropsy, surgery, or from external lesions.

• Advantages: This method allows for the collection of a large number of cells from firm lesions.

• Disadvantages:

  • It is more difficult to collect material via scraping compared to other methods.

  • Only superficial cells are collected.

  • The results may only reflect secondary bacterial infection or inflammation, which can hinder the diagnosis of an underlying neoplasia.

• Procedure:

  • The lesion must be localized, cleaned, and blotted.

  • A scalpel blade is held perpendicular to the lesion.

  • The blade is pulled across the lesion several times.

  • The collected material is transferred to a clean slide and spread using techniques similar to those for aspirate or solid mass preparations.

Imprint and Impression Smear Techniques

Impression smears can be prepared from external lesions or from tissue removed during surgery or necropsy. They are valued because they are easy to collect and require minimal restraint of the animal.

• Disadvantages of Imprints:

  • Fewer cells are typically collected compared to scrapings.

  • There is a large amount of potential contamination.

  • Samples may only reflect secondary inflammation or infection, potentially masking neoplasia.

• Tzanch Preparation: This is a specific procedure used for external lesions that requires at least $6$ slides:

  • Slide $1$: Imprint taken before the lesion is cleaned.

  • Cleaning: The lesion is cleaned with a saline-moistened surgical sponge.

  • Slide $2$: Imprint taken after cleaning.

  • Debriding: The lesion is debrided.

  • Slide $3$: Imprint taken after debridement.

  • Slide $4$: If a scab is present, an imprint is taken from the underside of the scab.

  • Slide $5$: Imprint taken of the exposed tissue.

• Tissues from Surgery or Necropsy:

  • Blood and tissue fluids must be removed by blotting the specimen first, as excessive fluid negatively impacts results.

  • If there is a delay in imprinting, a scalpel blade can be used to expose a fresh surface.

  • The tissue is touched to the middle of a slide.

  • Multiple slides should be produced if special stains are required.

Fine Needle Biopsy (FNB)

Fine needle biopsy is used for masses, including lymph nodes, nodular lesions, and internal organs. This technique minimizes superficial contamination from cutaneous lesions.

• Preparation:

  • If microbiological testing is required, surgical preparation of the site is mandatory.

  • For collections from body cavities (peritoneal, thoracic, joints), surgical prep is also necessary.

  • Otherwise, alcohol preparation similar to that used for vaccinations or venous puncture is sufficient.

• Equipment Selection:

  • Needle size: $21\text{- to }25\text{-gauge}$.

  • Syringe size: $3\text{- to }20\text{-mL}$.

  • Softer tissues require smaller needles and syringes. Large needles may result in tissue cores being aspirated rather than free cells, which leads to poor yield and greater blood contamination.

• Aspiration Procedure:

  • The mass must be held firmly to control needle direction and aid penetration.

  • The needle (attached to the syringe) is introduced into the center of the mass.

  • The plunger is drawn back to approximately three-fourths of the volume of the syringe.

  • The needle is redirected into different regions of the mass while maintaining negative pressure.

  • Negative pressure must be released before the needle is removed from the mass.

  • After removal, the needle is detached, air is drawn into the syringe, the needle is replaced, and the contents are expelled onto a slide.

• Nonaspirate Procedure (Capillary or Stab Technique):

  • This is easier to perform than aspiration as it does not require pulling back the plunger or redirecting the needle in the same way.

  • The mass is held firmly, and a $22\text{-gauge}$ needle (with or without a syringe for handling) is moved rapidly back and forth $5\text{ to }6\text{ times}$ along the same tract.

  • Cells are collected via shearing and capillary action.

  • The material is then rapidly expelled onto a slide using a syringe.

Tissue Biopsy Methods

Tissue biopsy involves sampling a piece of tissue from organs such as the kidney, liver, lung, lymph node, prostate, skin, spleen, or thyroid. Methods include abrasion with a blade, needle aspiration, excision, punch biopsy, and endoscope-guided biopsy.

• Preparation: Hair should be clipped carefully to avoid abrasion. There is no absolute need to cleanse the site unless specified.

• Wedge Biopsy:

  • Obtained using a scalpel.

  • Provides a large, variably sized specimen (either a portion or the entire lesion).

  • The specimen is trimmed on its long axis to provide a "transition zone" showing abnormal tissue, the transition, and normal tissue.

• Punch Biopsy:

  • Offers advantages in ease and speed over wedge biopsies.

  • Disposable skin biopsy punches are available in $3\text{-, }4\text{-, }6\text{- and }8\text{-mm}$ sizes.

  • $4\text{-mm}$ specimens typically do not require sutures, whereas $6\text{- or }8\text{-mm}$ specimens do.

  • The punch is rotated in one direction until the blade sections the tissue (rotating in one direction reduces tissue damage).

  • Specimens are fixed in $10\,\%\,neutral phosphate buffered formalin$ .

• Endoscopic Biopsy:

  • Specimens are gently flushed from the tip of the endoscope.

  • The specimen is blotted gently on a paper towel and placed on a small piece of tongue depressor to create a splint.

  • Once dried, it is placed in fixative.

Centesis Procedures

Centesis is the introduction of a needle into a body cavity to collect fluid. Types include:

• Abdominocentesis (also called paracentesis).

• Thoracocentesis.

• Cystocentesis.

• Arthrocentesis.

Collection Protocol:

• Requires aseptic preparation.

• Total volume collected and gross characteristics (color and turbidity) must be recorded.

• Equipment: Typically a $21\text{-gauge}$ needle and a $60\text{-ml}$ syringe.

• Preparation: Smears should be prepared quickly; a portion of the sample should be saved in an EDTA tube.

• Color and Turbidity: These are influenced by protein concentration and cell numbers. Discoloration increases with turbidity, often indicating peripheral blood contamination or inflammation.

Transtracheal and Bronchial Washes

These techniques assist in diagnosing pulmonary disease by evaluating samples from the trachea, bronchi, or bronchioles.

• Tracheal Wash Methods:

  • Passage of a catheter through an endotracheal tube in an anesthetized patient.

  • Passage through the nasal passages.

  • Transtracheal route: Performed through the skin and trachea; this route minimizes pharyngeal contamination.

• Percutaneous Technique:

  • An $18\text{- or }20\text{-gauge}$ through-the-needle catheter is used.

  • The laryngeal area is clipped and aseptically prepared.

  • Lidocaine is used for local anesthesia.

  • The needle is inserted into the trachea through the cricothyroid membrane.

  • Sterile saline is infused, and when the animal coughs, the plunger is pulled back to collect fluid.

• Orotracheal Technique:

  • The animal is anesthetized, and a sample is collected via an endotracheal tube.

  • Because the animal will not cough, the fluid must be withdrawn immediately after injection.

Fluid Concentration Techniques

Fluid samples with low cellularity require concentration to facilitate evaluation.

• Low-Speed Centrifugation:

  • Centrifuged for $5\,\text{minutes}$ at $165\,\text{g}$ to $360\,\text{g}$ ($1000$ to $1500\,\text{rpm}$).

  • The supernatant is separated from the sediment and its total protein is measured.

  • The sediment is resuspended to make a smear using blood smear or compression techniques.

• Gravitational Sedimentation: Used primarily for cerebrospinal fluid (CSF).

  • A glass cylinder is attached to a slide with paraffin wax.

  • $1\,\text{mL}$ of CSF settles for $30\,\text{minutes}$.

  • The supernatant is removed, and the slide is air-dried and stained.

• Membrane Filtration: Used for alcohol-diluted CSF.

  • Pore size: $5\,\mu\text{m}$.

  • CSF gravity feeds through or is gently injected; the filter must stay horizontal.

  • Filter is fixed with $95\,\%\,ethanol$ for at least $30\,\text{minutes}$. A Trichrome-type stain is required.

• Cytocentrifugation: Specialized equipment used for low-cellularity fluids. It concentrates cells into a small circular area on the slide.

Smear Preparation from Solid Masses

Multiple techniques exist for preparing smears from solid tissue.

• Compression Preparation (Squash Prep):

  • Expel the aspirate onto the middle of a slide.

  • Place a second slide (spreader) at a right angle over the first.

  • Smoothly slide the spreader slide across the prep slide without applying downward pressure. Downward pressure causes excessive cell rupturing.

  • Modification: Rotate the second slide $45\text{-degrees}$ and lift upward.

• Combination Technique:

  • Place the aspirate in the middle of a slide.

  • Pull a spreader slide backward at a $45\text{-degree}$ angle to contact the back third of the sample, then move it forward smoothly.

  • Next, place the spreader slide horizontally over the back third of the sample and slide it across. This produces three distinct areas for evaluation.

• Starfish Smear:

  • The aspirate is dragged peripherally in multiple directions using the point of a needle.

  • This is ideal for viscous samples and tends not to damage cells, though it leaves a thicker layer.

Smear Preparation from Fluid Samples

Fluid smears should be prepared immediately after collection, preferably using EDTA-treated samples.

• Line Smear: Used for low cellularity or small volumes.

  • A drop of fluid is placed on the slide.

  • A blood smear technique is used, but the spreader slide is raised directly upward about three-fourths of the way through the smear to create a concentrated line of cells.

• Wedge Smear (Blood Smear):

  • Place a drop of fluid $1\text{ to }1.5\,\text{cm}$ from the end of the slide.

  • A second slide (spreader) is pulled backward at a $30\text{- to }40\text{-degree}$ angle into the drop.

  • Once the fluid spreads across the spreader, it is pushed forward smoothly to create a feathered edge.

Fixation and Staining of Cytology Samples

Proper fixation and staining are critical for diagnostic quality.

• Fixation: The preferred fixative is $95\,\%\,methanol$. Slides should remain in fresh, uncontaminated fixative for $2\text{ to }5\,\text{minutes}$.

• Romanowsky Stains: Includes Wright, Giemsa, Diff-Quik, and DipStat.

  • These are inexpensive and easy to use.

  • They stain organisms and cell cytoplasm.

  • Slides must be air-dried before staining to fix the cells to the slide.

• New Methylene Blue (NMB): A useful adjunct that provides excellent nuclear detail while staining cytoplasm weakly. Red blood cells (RBCs) do not stain with NMB.

• Papanicolaou Stains: Provide excellent nuclear detail and delicate cytoplasmic detail.

  • They do not stain cytoplasm well and do not demonstrate bacteria.

  • The process is time-consuming and involves many steps, making it difficult for standard practice use.

Submission of Cytologic Preparations

When in-house evaluation is insufficient, samples are submitted to clinical pathologists or cytologists.

• Submission Requirements:

  • $2\text{ to }3$ air-dried unstained smears.

  • $2\text{ to }3$ Romanowsky-stained smears.

  • For fluids: submit direct smears, concentrated smears, an EDTA tube, and a red-top tube.

• Safety Precautions:

  • Protect slides from breakage during mailing.

  • Do not mail unfixed slides in the same container as formalin-containing samples. Formalin fumes alter staining characteristics, and water causes cell lysis.