Small Mammal Clinical Pathology: The Basics

Karen L. Rosenthal, DVM, MS, Diplomate ABVP-Avian
Director, Special Species Medicine
Clinical Studies-Philadelphia
University of Pennsylvania
School of Veterinary Medicine
Philadelphia, PA, 19104

The principles of proper clinical pathology transcend a particular species and what is appropriate for dogs and cats, is usually appropriate for small mammals. Following these principles gives one the best chance of obtaining interpretable results. It is true that incorrect results are worse than no results at all. Wrong results can lead one down the incorrect diagnostic path and the diagnosis may be missed and ultimately, the patient is not treated properly. In all species, venipuncture techniques are the key element in determining how meaningful the results will be. As it would be "bad form" to clip a dog's toenail and use that sample to determine the health of your patient, so it is bad form to do the same for the small mammal patient. There are good venipuncture methods for most of the small mammals seen in practice. Why is it worth the effort to perform venipuncture in these species? It is because toenail clips cause so much tissue damage that abnormalities cannot be discerned from artifacts. Toenail clips allow tissue "juices" to enter the sample. This fluid can dilute the sample or add electrolytes into the sample and cause difficulties in interpretation. The PCV is difficult to interpret due to possible dilution of the sample. Microclots enter the sample and they render the CBC and biochemistry panel results suspect. Red blood cells can lyse with this method. This will cause an artifactually low PCV and will also cause abnormalities in the biochemistry panel. Lysis of the red blood cell membrane can increase the phosphorus and potassium values. This can pose a problem when interpreting blood results when considering diseases such as renal failure and hypoadrenocorticism.

Always use blood tubes appropriate for the size of the specimen. Successful venipuncture and sample collection are wasted if blood is placed in a tube meant for a larger sample. In larger tubes, the anticoagulant amount can dilute a small sample. Place small blood samples in microtainers. Small blood samples are those under 2 ml of blood. There are three commonly used microtainers: those with no anticoagulant (serum microtainers), KEDTA anticoagulant, and heparin anticoagulant. Almost all biochemistry tests have the same results whether run on serum or plasma. The advantage of the use of plasma is that per volume of sample, more plasma than serum can be obtained from the same volume of blood. And, since in exotic animal medicine, small samples are the rule, it is preferable to use plasma rather than serum for biochemistry panels. Therefore, it is preferable to place biochemistry samples in heparin microtainers rather than serum microtainers. Most laboratories will supply you with the proper microtainers. Although, plasma can also be obtained from KEDTA tubes, if possible, only use KEDTA tubes for CBC results. The potassium in KEDTA tubes immediately exchanges with the calcium in the blood sample. This results in a biochemistry panel report of a very high potassium concentration (over 10 mEq/dl) and a very low calcium concentration (close to 0 mg/dL). It may seem convenient to use microhematocrit tubes to collect blood from nail clips or the controlled laceration of a vessel. The blood is allowed to drip into the tubes. The tubes are spun down, broken open and the plasma collected. This method usually results in a great amount of cell lysis. The plasma biochemistry results may not reflect what is actually found in the patient. With use of the proper blood collection techniques, microhematocrit tubes do not need to be used for biochemistry analysis.

Once the blood sample is placed in the microtainer, the next thing you do may alter the results you get and significantly change the direction of your treatment. Ideally the plasma should be separated from the cells. Allow the blood tube to sit for a few minutes and then spin the sample. Once it is spun, decant off the plasma and place the plasma in a tube without anticoagulant. If the plasma is placed back in a tube with heparin, this may cause sample dilution. If the plasma is not separated from the blood cells and if it is sent to the lab unspun, at least three analytes of the chemistry panel will be affected, blood glucose, potassium and phosphorus. Blood glucose is decreased when the plasma sits on the blood cells. The active cells use the glucose in the plasma. The laboratory will read a blood glucose concentration lower than what was in the sample originally. In ferrets, when an insulinoma is suspected, this value is critical. If the sample cannot be separated, then measure blood glucose immediately at patient-side. Both potassium and phosphorus can elevate in samples when the red blood cells are in prolonged contact with the plasma. Damage to the red blood cell membrane causes potassium and phosphorus to leak through the membrane or leak around a broken membrane (hemolysis). Certain disease states increase the fragility of the red blood cell membrane and leakage may be unavoidable. Elevated potassium or phosphorus values may not reflect poor technique or handling, but rather a disease condition. That is why correct handling is critical for proper interpretation. If the results reveal an elevated potassium or phosphorus and proper technique was followed, one may then pursue a diagnosis characterized by increased fragility of red blood cell membranes. There are some drawbacks when separating the plasma from the cells in the hospital. It does take extra time and effort to spin and pipette the sample. With very small samples it is technically difficult to adequately retrieve all of the plasma unless micropipettes are used. If a very small blood sample is retrieved, it may best to leave the pipetting of the sample to the laboratory. Finally, if the same tube is used to evaluate the CBC and the biochemistry profile, then the tube cannot be spun as this will render the cells useless for the CBC.

Even before your sample is in the blood tube, the integrity of the RBC membrane can be compromised. It is important to realize that potassium and phosphorus concentrations will increase in plasma even without gross hemolysis; they leak through damaged membranes. Damage can happen from the venipuncture itself. In the smaller exotic patients, one must use small bore needles that are more prone to causing hemolysis than the larger gauge needles. If possible, remove the needle from the syringe when placing the sample into the tubes to decrease the chance of cell lysis. Many of the routine chemistry analytes will be effected by hemolysis but not as greatly as potassium and phosphorus. Both bilirubin and bile acids are falsely elevated with hemolysis. Bile acids elevate not only with hemolysis but also with lipemia. Lipemia, in exotic animals, is most commonly noted in birds but has been occasionally seen in ferrets. Lipemia can cause RBC membranes to rupture, even with the best technique. Lipemia will also effect every aspect of the CBC and biochemistry analysis. When the sample is lipemic, none of these values are reliable indicators of patient health. It is possible to overcome the effects of lipemia by using a machine known as a dry chemistry analyzer. Most commercial labs and veterinary hospitals use wet analyzers.

Clotting of the blood sample is a problem that veterinarians fear and often encounter during venipuncture of small mammal patients. It is not that these species clot faster, but the venipuncture technique most veterinarians use on these animals is not as "perfected" as their technique is in dogs and cats. Therefore, since it may take longer to get a sample, there is more of a chance of the blood clotting. To prevent clotting, the needle and syringe can be heparinized. With this method, there is the potential to change the staining characteristics of the cells or to dilute the sample with too much heparin. This is where experience is important. If you feel too many of your samples are clotted then it is worth the risk of over-heparinization of the sample. Clotting of blood samples changes both the biochemistry and CBC results. The CBC may show falsely elevated or depressed cell values. Also, platelets are decreased. Clotting causes hemolysis therefore altering the biochemistry values.

Samples for the complete blood count are submitted both in slide form and in microtainers. In all species, it is ideal to make two blood smears immediately after venipuncture. These slides are the best representation of the complete blood count of the patient at the time of the sample. This is even true in dogs and cats. In some exotic patients, there is not enough blood to make smears and also fill a microtainer. In these patients, the smears will be the laboratory's only measurement of the CBC. In patients where larger quantities of blood can be taken, some blood can be placed in KEDTA tubes for CBC analysis. In mammals, this blood can be analyzed through an automated system. The hematologist prefers both fresh blood smears and whole blood to analyze. That allows them to make their own smears if the submitted smears are not perfect. It appears that both heparin and KEDTA will preserve blood cells for later analysis. KEDTA will give less staining artifacts but may not preserve the integrity of the cells as long as heparin will. Heparin causes many staining artifacts but may preserve the cells for a longer amount of time.