Written by Mike Corcoran, DVM, DABVP (REP/AMPH), CERT AQV

“So, when did you first notice this lump?” 

“Oh, that was there when I got him 6 months ago. I assumed it was normal, Doc.” 

We’ve all been there — a mystery lump on our patient. Imagine the mass in question is on a snake. How do we know to suspect something abnormal? Well, similar rules apply across all species: asymmetry, inflammatory signs, changes in size, shape, or color are all indications for potential concern. So when you see something like one of the items listed above, sampling the tissue is important.  

Discuss your concerns with the owner, and while you’re getting some aspirates for that snake, follow these steps to make sure you’re following best practices when it comes to reptile cytology and submitting important samples to a veterinary clinical pathologist. 

Reptile Sample Collection

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Firstly, sample collection in reptile cytology is very similar to other animals, so you can carry out the process with confidence knowing that your preferred veterinary diagnostics laboratory should be able to run and analyze your reptile sample. Here are a few steps to begin.

Gather supplies 

• 3cc syringes
• 22 gauge needles 
• Microscope slides 
• If sampling fluid, get the correct tubes ready; a no-additive tube will work for most samples, but you should also have an anticoagulant tube ready. For reptiles, avoid the purple-top calcium EDTA (Camus 2019). These tubes will lyse the red blood cells of most reptiles. Lithium heparin (green top) tubes may be used instead. 
• If there’s any chance that the lesion is infectious, have access to culture swabs or sterile swabs for PCR submission.  K-EDTA and serum separator tubes have bacteriostatic properties, avoid these when culture is indicated.

Prepare the area for sampling

The skin should be prepared similarly to other species, though some additional care may be required to clean in the deeper grooves of scales. Both betadine and chlorhexidine are appropriate to use in reptiles. For coelomic masses, ultrasound guidance helps ensure you’re sampling from the best location and may help avoid iatrogenic trauma. Ultrasound isn’t a bad idea for more superficial lesions, but the sample may be obtained with palpation as a guide. 

Sample the area

I try to get each sample onto four slides. I apply some of the sample from the syringe to two of the slides. I then spread the cells flat using another clean slide, using the same technique as you would with any other animal. I do this so I have options for staining at least one slide in-house to check the sample without taking away from what I can submit to the pathologist. If there is a small volume of sample, you may only get two slides.

Check the sample

At this point, look at the unstained slides microscopically in house. If all appear to have a good sample, then select the worst of the batch and prepare it with Diff-Quik. You can examine that slide in-house. You may get the information you need on the spot, but if you don’t, then you can at least feel confident of having a good sample to submit to an animal diagnostics laboratory — there are still three unstained slides for the veterinary pathologist. Select the two best slides for submission to a veterinary diagnostic lab.  At Moichor, we accept 2 slides per site.

What to Look for in-house 

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When analyzing a cytology sample in-house, look for three specific features: obvious infection, signs of neoplasia, and good cellularity for sample submission.  

Infection

In cases of bacterial infection, expect to see primarily inflammatory cells. Depending on the species, you’ll see different ratios of heterophils or lymphocytes. Sometimes you can see intermittent mononuclear cells or other white blood cells. Look for bacteria to be intracellular to diagnose an active infection. With these samples, I recommend taking an additional slide and doing a gram stain to guide the initial antibiotic therapy while you wait for culture results. Keep in mind that abscesses in reptiles won’t exfoliate as well as they will with mammals. The purulent material is more solid in reptiles. Keep that in mind when evaluating the slide, as well as planning a procedure to remove the material and the surrounding capsule.  

Neoplasia

In addition to abscesses, look for signs of neoplasia. Mitosis, anisocytosis, anisokaryosis, prominent nucleoli, and abnormal cells are all signs of neoplasia. When you see signs that raise concern for neoplasia, you should be positive due to the serious nature of the diagnosis. It is important to remember that severe inflammation and low-grade neoplasia can be hard to distinguish in almost all species. The slides can be submitted to a pathologist and you’ll have confidence that the slides have good representative cells. The three unstained slides are free for the pathologist to do any additional staining required for a definitive diagnosis as in one case of a bearded dragon cited in literature (Anderson, 2018). You can have a conversation about likely next steps with the client so they have more time to prepare for potential biopsy, excision, CT, chemotherapy, radiation, or whatever may be indicated.  

Good cellularity

If you don’t see obvious infection or neoplasia, then you can use the first slide to verify that you have a good cellular sample before submitting it to a veterinary pathologist.  It is important to keep in mind that bloody slides may look cellular, but they may only contain peripheral blood and may be nondiagnostic. 

Sample Submission 

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Slides or fluid can be submitted to the pathologist. You can include the stained slide that you examined in-house along with the unstained slides. Alternatively, if you suspect something based on the slide you examined, you can keep the slide for yourself and re-examine it after you get results from the pathologist. It’s a great way to learn.  

Include the signalment and a detailed description of the lesion to help the pathologist give you a good diagnosis. The more information you can provide with the cytology sample, the better chance you’ll get accurate information back from the reference lab. Whenever possible, utilizing the same lab for culture and/or PCR of the samples makes it easier to consolidate information.  

Joint fluid should always be submitted to an animal diagnostics lab even if an abscess is diagnosed. The same joint may be damaged from gout, and the infection may be secondary. Uric acid crystals can be difficult to impossible to identify on standard light microscopy. Polarization or other specialized techniques are available to veterinary pathologists.

Even if an abscess is diagnosed in-house, it’s not a bad idea to submit the sample. Reptiles more frequently have complex lesions involving bacterial infection along with additional problems. Acid-fast stains can be performed in cases of suspected mycobacterial infections. Special stains also allow a pathologist to look for fungal elements in the case of a concurrent bacterial and fungal infection. 

When Not to Sample a Reptile for Cytology 

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Just like other animals, sampling through fine needle aspirate carries some risk of spreading bacteria or neoplastic cells from a well-encapsulated lesion into the coelomic cavity. Balance the relative risk and discuss with the owners prior to sampling.  

With bearded dragons, exercise extra caution with cutaneous lesions around the head. They have a predisposition to forming aneurysms, and the lesions are often located on the head. Prior to sampling any of their lesions by fine needle aspirate, be sure to check the area using ultrasound with color flow. If the lesion is hypoechoic and has movement on color flow, then penetrating the capsule with a needle runs the risk of uncontrolled hemorrhage. It’s best to proceed on the assumption that an aneurysm is present. 

Final Thoughts around Reptile Cytology Sampling 

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Cytology in reptiles doesn’t need to be any more intimidating than with patients of other species as  most of the principles are the same.  

And what about that snake from the intro? A sample from your snake revealed a fungal infection — with surgical debridement and anti-fungal treatment, you were able to get a full resolution.  

References 

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1. Anderson K, Meinkoth J, Hallman M, Bailey K, and Brandao J. Cytological Diagnosis of Gastric Neuroendocrine Carcinoma in a Pet Inland Bearded Dragon (Pogona vitticeps). 2018. JEPM 29; p.188-193.  
2. Barten S, Wyneken J, Mader D, and Garner M. Aneurysm in the Dorsolateral Neck of Two Bearded Dragons (Pogona vitticeps). Proceedings of the Association of Reptilian and Amphibian Veterinarians, 2006. P. 43-44.  
3. Camus M and Yeuroukis C. Chapter 38: Cytology. In Mader’s Reptile and Amphibian Medicine and Surgery, Third Edition. Divers S and Stahl S editors. 2019, Elsevier. 

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