Reptilian clinical pathology (Proceedings)

"Bad laboratory data are worse than no laboratory data at all." Considering the variety amongst our veterinary patients, I think that this old saying has the most significance in the practice of exotic animal medicine. There are certainly many clinical pathology laboratories around, and most of which do an outstanding job in veterinary laboratory analysis. But, just as is the case in the changing face of our profession, with every dog and cat practice now hanging out a shingle that claims expertise with exotics, almost all clinical laboratories are also claiming expertise in the non-domestic arena.

Especially with the birds and reptiles, animal groups claiming ownership of a unique leukocyte, called the heterophil, evaluation of the hemogram and differential can be very difficult, and requires substantial training and experience. As a case in point, take a single exotic blood sample, split it into five smears and send them to a small mammal laboratory. Two smears can be submitted on the first day, each with a different name, and the remaining three can each be submitted on subsequent days, also with different names. I can almost guarantee that you will have five different results!

Don't interpret this as arrogance on my part. I realize the difficulty of the task, and as a result, I always send my hematology samples to a trained exotic animal clinical pathologist. The morphology of the cells vary between animal groups (eg. birds - reptiles), and between species within a group (iguanas - chameleons). Unless the technician is thoroughly familiar with the normals for EACH species, there is no possible way they will be able to evaluate either normal or abnormal samples from clinical cases.

The laboratory that I use for my hematology samples has only one technician read ALL of the slides. This is very important since I know that when I am sending in serial samples, each subsequent slide is always being evaluated by the same individual. Many large commercial laboratories utilize the service of many technicians. There will inevitably be differences between their abilities. If you send in a sample and get a qualified technician you may get reliable results. However, you may also get one of the less experienced technicians (less experienced with non-domestic bloods), and the results may be worthless (or even potentially dangerous).

The most productive scenario is to either do all of your exotic hematology yourself, or have a trained and practiced technician do your lab work. A technician skilled at reading exotic hemograms is a valuable asset (and practically a necessity) in any non-domestic practice. There are a few books available on the subject, and for their price, are worth every penny.

Obtaining samples for hematology and clinical chemistry in reptiles can be a clinical challenge. Standard sampling techniques such as cardiocentesis, jugular, axillary, femoral, buccal and tail vein venipuncture can all be used. Each has its advantages and limitations, and with practice and experience each clinician will develop proficiency in the techniques that suits him or her best. Comprehensive descriptions of these techniques are beyond the scope of this paper, but accurate descriptions are available elsewhere.

Briefly, in snakes, either tail vein (especially in crotalids) or cardiocentesis are the best techniques for collecting blood. I routinely use cardiocentesis, and in twelve years and literally thousands of snakes, have never had a problem.

In lizards I prefer tail vein bleeding. Although, axillary is satisfactory in many cases. There is an increased chance of lymph contamination in the latter technique.

In chelonians either jugular vein bleeding, or collection of blood from either the axillary or femoral plexus can all be used. The former is the preferred method in gentle animals.

I do not encourage toe-nail bleeding. With all of the other convenient alternatives available I see no reason to take a perfectly normal tissue (the nail) and destroy it. Not only does this technique require more time, it yields a lymph contaminated sample and undoubtedly causes considerable pain to the patient. If you don't believe this, try clipping the tip of your finger off just under the nail bed!

Blood volumes in reptiles vary from five to eight percent of their total body weight. Of this amount, up to ten percent can be safely collected for analysis without harm to the patient. As a rough approximation, the sample size should never be larger than one percent of the animal's total body weight. For example, in a 450 g iguana, you can safely remove 4.5 cc's of blood. This is way more than required by even the most antiquated autoanalyzers.

The standard capillary tube holds 70 µl. That means that the volume of a single capillary tube is the maximum amount of blood that you would want to take from any patient weighing a minimum of seven grams. Even though this is a small sample, there is enough to yield some valuable diagnostic information.

The total (plasma) protein (assuming that you use a heparinized capillary tube), packed cell volume and icterus index are all easily measured from a single capillary tube. Microscopic analysis of the blood filled capillary tube may reveal microfilaria, and after centrifugation, will also give an estimated buffy coat size. If a thin blood smear was made an estimated white blood cell count with a differential can be performed, and the red cells can be evaluated for any abnormalities or hemoparasites. After the cells have been spun down the remaining plasma can be used for a limited number of specific chemistries.

In reality, with the very small patients, only a single drop of blood might be all that you will be able to safely collect. A thin, well prepared blood smear will always be of value as a diagnostic aide.

Blood smears for staining should always be prepared from fresh, whole blood. Heparin or EDTA anti-coagulated blood does not stain well, and EDTA may actually cause some of th RBCs to lyse. Blood can be collected in heparinized capillary tubes, or in heparinized microtainers. Every lab has their own method of preference for smear preparation. The consensus is to make coverslip smears. Because of the large cell size of both the avian and reptilian erythrocytes, there is less cell damage from this technique than dragging slides across each other.

Identification of cell types in the standard differential analysis has been described in detail elsewhere (with color pictures!). The following is a list of some of the more salient features of the cell types encountered in the reptilian differential:

• Erythrocyte - Nucleated. They have a lower PCV and Hemoglobin than mammalian cells. You will have RBC lysis in your sample as a result of heat, EDTA and alcohol. Hypochromic anemia is common in patients suffering from poor nutrition.

• Monocytes - Similar in appearance to the mammalian monocyte. They may contain granules. Numbers increase in chronic disease. Differential counts vary, but are generally in the range of 0.5 - 3%.

• Lymphocytes - Both large and small. Similar in appearance to mammalian lymphocytes. Like the monocytes, they may contain granules. These may increase in chronic and viral diseases. Differential counts vary, but the range is usually from 40 - 60%.

• Neutrophils - These have a non-segmented nucleus. The cytoplasm contains baso-, eosino- and azurophillic granules. Toxic neutrophils contain foamy cytoplasm and toxic looking granules. These cells are not commonly found, and their counts are generally in the 3 - 7% range. In my experience, the numbers are even lower that that.

• Heterophils - These are the classic reptilian inflammatory cells. The nucleus in not lobed in most reptiles, but may be lobed in many saurians. Reactive and toxic changes are reflected by changes in the pattern of the nucleus.

• Azurophils - Reactive/inflammatory cells. Deeply basophillic cytoplasm. May have pseudopodia. Unique to reptiles. There is considerable debate as to the significance of the azurophil, and to its origin. But, it is currently believed to be related to the monocyte.

• Eosinophils - The granules which are spherical to beadlike may degranulate with disease. It is believed that they will increase with intestinal parasitism, allergies and tissue damage. Very common in reptilian blood, with ranges from 7 - 20%.

• Basophils - Inflammatory cells. Intense blue spherical and rod shaped granules. Common in reptile blood. Numbers range from 10 - 25%, with chelonians often having even higher numbers (greater than 50%).

Very little has been reported on the significance of blood chemistries in reptilian medicine. Various papers have been published on the presence or the "normal" ranges of the various enzymes and electrolytes in reptiles, but interpreting the significance of changes in their values is almost non-existant. The following is a thumb-nail sketch of the plasma chemistries commonly evaluated in clinical cases at my hospital:

• Total (Plasma) Protein - Normal ranges vary (3 - 6 g/dl). Questionable accuracy using the total solids, or refractometer method. Not certain if you can accurately separate Albumin from Globulin. Total Protein levels increase in dehydration, chronic disease and lipemia. Decreases in starvation, chronic disease and parasitism. Increase in fibrinogin (acute phase proteins) is a good indicator of inflammation.

• Calcium - Can be falsly elevated in laboratory errors and with certain hematocrit clays. Physiological elevations can be seen with dehydration, hyperalbuminemia, neoplasia and iatrogenic causes. Many gravid animals will have calciums in the mid to upper 20's. Some clinically normal snakes have been reported to have calciums in the low 200's.

• Phosphorus - Most commonly elevated with renal disease. An inverted Ca:P ration (<1:1) is a good indication of renal disease.

• Renal parameters - B.U.N. and Creatinine - Neither of these parameters are reliable indicators of renal disease. Reptiles are uricotelic, not ureotelic, and as a result, produce variable amounts of B.U.N., and it also varies depending on the state of repletion and hydration. I had the opportunity to take a blood sample from a snake which had a bilateral nephrectomy. It had a BUN of 9.1, a creatinine of 1.4 and a uric acid of 182! In general, uric acid is the renal parameter of choice for evaluating renal disease. However, there must be at least 70% damage for the uric acid to elevate. I have found evaluating the Ca:P ratio to be much more rewarding.

• Liver Parameters - Bilirubin, LDH, SGOT (AST) - In reptiles biliverdin, not bilirubin, is the primary endproduct of heme catabolism. Birds (and reptiles???) lack biliverdin reductase. Bilirubin, then, has questionable value in evaluating hepatic disease in reptiles.

• LDH - Who know what this means? I do not have it on my panels.

• SGOT (AST) - Found in the liver, heart, lung, kidney and the blood. Values increase in rough handling of laboratory samples. Also, with disease of any of the above organs. Normal values may be more significant than elevations. SGOT is more liver specific than SGPT in some avian species.

It is well known that ALL clinical laboratory values (both hematology and plasma chemistry) change with season, age and sex in reptiles. Therefore, reporting normals is almost non-sensical. As a result, changes in serial samples from a given patient are probably more significant than individual values.

Substantially more work needs to be done in the evaluation of laboratory samples in reptile medicine. I encourage all reptile clinicians to keep thorough records and report values in the literature. Eventually, this data may actually be useful!

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