How To Submit Samples For Cytopathology

The following FAQ pages are intended for veterinarians. For Pet Owner FAQ pages please visit the Pet Owners section.

Please follow these guidelines for shipping cytology samples via UPS or mail:

• Use top-loading plastic slide holders (hold 5 slides/box) taped shut to prevent accidental opening during shipping. Smears submitted with cardboard holders or side loading plastic slide holders are more likely to arrive broken. Make sure the smears are rapidly and completely dried (a blow dryer works great!).
• Fluid samples in glass tubes should not be submitted loose in plastic bags or UPS envelopes. They should be wrapped with tissue or gauze and placed in top-loading plastic slide holders taped shut. If the tubes are too large to fit into a slide holder, aliquot in smaller tubes.
• Slide holders and request forms from each individual patient should be placed in separate plastic specimen bags. It is important to label the slide holders or smears if smears from more than one lesion are submitted.
• Samples can be shipped via regular mail, UPS, FedEx, or other express delivery services.
• Results are faxed the day samples arrive (except on weekends).
• Cytopathologic interpretations and UPS charges are billed on a monthly basis.
• Please call our office or indicate on the request form if you need additional top-loading slide holders, request forms, or to set up a UPS account.

How do I collect cytology specimens? (Fine Needle Biopsy Technique)

Introduction

The technical aspects of obtaining and preparing samples for cytopathologic evaluation are important factors commonly overlooked by clinicians. One should always keep in mind that if the sample is hemodiluted, clotted, improperly prepared, or poorly representative, the cytopathologist may have insufficient information to provide a diagnosis.

In the last three years we have modified our fine needle biopsy techniques. These modifications have facilitated the procedure and provided highly cellular samples.

Non-aspiration fine needle biopsy technique

Lesions or tissues are localized using palpation, anatomic landmarks, radiographs, or ultrasound images. The biopsy sites and the least traumatic needle paths are then determined. The puncture sites are clipped and surgically prepared.

The decision to use sedation or general anesthesia is dictated by the location of the lesion or organ, the proximity of major blood vessels, potential coagulation defects, and the temperament and/or mental status of the animal.

We use 22 gauge needles of various length (2.5 to 8.9 cm), without stylet. The needle is connected to a 12 ml syringe via a 84 cm flexible extension set for I.V. lines and the syringe is pre-filled with 5-10 ml of air. The syringe, connected to the extension set, is hung around the neck and the needle is held like a pen to allow precise manipulations.

If possible, the tissue is immobilized with one hand. The distance between the "target" and the skin surface is determined, and the needle tip is positioned rapidly within the lesion. Once within the lesion or tissue, the needle tip is moved rapidly back and forth 8 to 10 times without changing its path. Long needle passes are preferable, but the needle tip must remain within the target tissue during this part of the procedure.

The needle is then rapidly withdrawn and the biopsy material contained within the needle is immediately expelled on glass slides using the syringe that had been prefilled with air.

In most cases, the sample is small and only one smear is made using either the standard blood smear technique or a gentle squash preparation. The smear is immediately air-dried, manually or using a blow dryer. If the sample is hemodiluted, only one or two smears are made and the remainder of the sample is discarded.

Three to five cellular biopsies of different areas of each mass or tissue are obtained.

Hints to Increase Diagnostic Yield

Sedation or Anesthesia: Sedation or general anesthesia allows better palpation and immobilization of masses or tissues and reduces the risks of lacerations.

No stylet: We never use stylets, even for deep lesions. They slow down the procedure and make it more cumbersome. Sample contamination by superficial tissues is rare and has never caused diagnostic problems in our hands.

Proceed rapidly: Time is of the essence during fine needle biopsies: tissue trauma activates coagulation which can rapidly plug the needle or degrade the quality of the smear. Spend as little time as possible between needle introduction and smear preparation.

Long and quick needle passes: A common mistake is to move the needle slowly or to use short needle passes. A timid back and forth motion does not detach a sufficient number of cells. The needle tip, however, must remain within the target tissue during this part of the procedure to avoid contamination by surrounding tissues.

Extension Set: The use of an extension set for intravenous lines serves several purposes. The procedure is less cumbersome because the needle is separated from the syringe. Thus, by being able to manipulate the needle more precisely, one can obtain more accurate samples.

By prefilling the syringe with air, the transfer of the sample onto glass slides is expedited, which reduces sample clotting. Because the procedure is short and less cumbersome, the risks of laceration are reduced, particularly when sampling deep tissues.

No aspiration: We no longer use negative pressure during fine needle biopsies (unless we need to counteract a negative intra-abdominal pressure created by positioning and/or inspiratory efforts). Apparently, the vigorous back and forth motion and the capillary pressure within the needle are sufficient to detach cells, create mild bleeding, and fill the lumen of the needle with a cellular slurry.

The sample volume is reduced, but representative cellularity is increased. Obtaining a sample with less hemodilution is important for many diagnoses, including qualitative and quantitative evaluation of inflammation.

Aspiration vs Nonaspiration: Previously published studies show superior results using a nonaspiration technique, but others are favoring an aspiration technique. This debate is likely to be endless because high diagnostic yields can be obtained with either technique. In addition, the tissue of origin and the type of lesions are likely to have an influence on results.

In our hands, the results are similar (both exceed 90%), but we find the nonaspiration technique faster, easier and more efficient for both clinicians and cytopathologists.

Multiple Biopsy Sites: An important point is to obtain three to five cellular biopsies of different areas because each mass or tissue may contain a mixture of necrotic, inflammed, hemorrhagic, neoplastic, and normal tissue.

Cellular Smears: With experience, one can identify cellular smears with the naked eye, particularly smears from the liver, spleen, prostate, and several masses. If the smear does not appear cellular, the biopsy should be repeated. If there is any doubt, it can be evaluated by microscopic examination.

Variations based on Biopsy Sites

When very small--or no--samples are obtained:

Occasionally, a very small sample (or no sample at all) is obtained using the nonaspiration technique. We believe this is due to negative intra-abdominal pressure created by positioning and/or inspiratory efforts. This hypothesis is supported by the accumulation of microbubbles along the needle tract or within hepatic veins during unsuccessful ultrasound-guided fine needle biopsies. In those cases, we use mild negative pressure. We call it the " 5-6-5 technique".

The syringe connected to the extension set is given to an assistant. The clinician holds the needle as for the nonaspiration technique and says "5". The assistant fills the syringe with 5 ml of air (neutral pressure). The clinician introduces the needle. Once within the lesion or tissue, the clinician says "6" and the assistant pulls the plunger back to 6 ml (mild negative pressure) while the clinician moves the needle rapidly back and forth 5 to 6 times without changing its path. Again, long needle passes are preferable but the needle tip must remain within the target tissue during this part of the procedure.

The clinician says "5" and the assistant pushes rapidly the plunger to 5 ml (neutral pressure). The needle is then rapidly withdrawn and the biopsy material contained within the needle is expelled on glass slides using the 5 ml of air left in the syringe.

Fine needle biopsies of Kidneys:

Kidneys are high-pressure vascular tissues. Therefore hemodilution occurs even with a nonaspiration technique. We try to limit this effect by introducing only 1 ml of air in the syringe prior to the biopsies and by performing the procedure as rapidly as possible.

If a glomerular disease is suspected (proteinuria), the needle passes should be tangential to the surface so that the needle tip remains within the cortex to collect glomeruli.

Fine needle biopsies of lytic bone lesions:

Most bone tumors exfoliate very well and fine needle biopsies can provide a very high diagnostic yield. The key is to get samples at the core of the lytic lesion(s) seen on radiographs.

Heavy sedation or general anesthesia is required. The technique can be done blind or with radiographic guidance (preferable if the lesion is small or difficult to localize with palpation). The best insertion site--the site with maximum cortical bone lysis--is identified on radiographs. A 22-18 gauge needle is inserted through the lytic cortex using a "drilling" motion. Once within the core of the lesion, the needle is moved back and forth.

If the needle cannot be moved back and forth, use a 5-10 ml intermittent negative pressure. Stop as soon as you see a drop of blood in the hub of the needle. Then, withdraw the needle and make smears rapidly. We recommend to biopsy three to four sites.

If there is minimal cortical lysis and insertion of a regular needle is difficult, use a bone marrow biopsy needle. Once the tip is within the marrow cavity, confirm its position with radiographs. Remove the stylet and insert a 22 gauge 3 1/2 in. spinal needle using the bone marrow biopsy needle as a tunnel to get to the core of the lesion.

If there is a soft tissue mass surrounding the bone lesion, fine needle biopsies of the mass should also be obtained.

In our experience, and that of others, bone fine needle biopsies can be more rewarding than surgical biopsies.

Fine needle biopsies of thickened intestines:

This is the least invasive way to confirm chronic inflammatory bowel disease in cats. The technique can be performed blind or with ultrasound guidance.

The patient is anesthetized and the abdomen is clipped and scrubbed. A single small bowel loop is isolated by palpation and immobilized between the thumb and fingers. Firm digital pressure ensures that the bowel loop is empty and hypoperfused (which prevents aspiration of chyle and blood).

An assistant hands you the needle attached to the extension set. In this case, a strong negative pressure is needed to aspirate large mucosal fragments, therefore the syringe should not be filled with air prior to sampling.

The trick is to insert the needle into the lumen and keep it parallel to the long axis during the back and forth motion. It can be done blind but ultrasound guidance is helpful to confirm that the needle is indeed in the lumen.

The assistant creates a 8-10 ml negative pressure during sampling and returns to neutral pressure (0 ml) before the needle is withdrawn. The syringe is then detached, filled with air, reattached to the extension set and the sample (usually very thick) is expelled using positive pressure and spread using the squash smear technique.

Using this technique, several complete intestinal vili including mucosa and submucosa are often found on the smears.

Fine needle biopsies of the lungs:

The method is similar to the general nonaspiration technique. Heavy sedation or anesthesia is recommended to minimize risks of lacerations due to motion.

The biopsy site is localized based on radiographic findings. Introduce the needle into the lung rapidly (avoid keeping the needle tip in the pleural area) to reduce lacerations at the surface of the lung during respiratory motion.

The needle is then moved rapidly back and forth using long needle passes (away from major blood vessels) without any negative pressure.

Blind biopsies can be performed on large masses or when large areas of the lungs are affected but in many cases, radiographic guidance (or sonographic guidance if the mass or nodule or consolidated lobe is in contact with the thoracic wall) is very useful, if not necessary.

Risks of pneumothorax are significantly reduced if the patient is kept in lateral recumbency (biopsy side down) for 30-60 minutes following the procedure; this simple precaution takes advantage of recumbancy atelectasis to seal small lung perforations or lacerations and prevent air leakage.

Fine needle biopsies of nasal cavities and sinuses:

The technique is similar to the one described for bone lesions and requires general anesthesia. A 22 gauge needle is inserted using a drilling motion into the nasal cavity through the hard palate at the level of the lesion(s) identified on radiographs. Bone lysis facilitates needle insertion.

If the needle can not be moved back and forth after being inserted, use a 5-10 ml temporary negative pressure and stop as soon as you see a drop of blood in the hub of the needle. We recommend to biopsy three to four sites. Samples for culture and sensitivity tests can be obtained using the same technique.

Biopsies from the frontal sinuses can also be obtained by drilling a needle through the dorsal wall of the sinus using the same technique. Rostral intranasal lesions can also be approached through the nares using the general fine needle biopsy technique.

Conclusion:

Fine needle biopsy is a powerful diagnostic tool that has very good client (and patient) acceptance because it is minimally invasive. A good biopsy technique widens the applications and increases diagnostic yield. In our experience, the use of an extension set in combination with a nonaspiration technique provides multiple advantages including easier sampling and decreased specimen hemodilution.