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Introduction

Karen E. Russell DVM, PhD, DACVP (Clinical Pathology)1 and J. Jill Heatley DVM, MS, DABVP (Avian, Reptilian, Amphibian), DACZM2

1 Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, Texas, USA

2 Department of Small Animal Clinical Sciences, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, Texas, USA

Introduction

Veterinary clinical pathology is a branch of laboratory medicine that focuses on the study of animal disease through the examination of blood, serum or plasma, urine, body fluids, and tissues. The discipline covers a wide range of laboratory assays and methods and is important for diagnoses, patient care, prevention of disease, and the quality and accuracy of the laboratory tests.

Obtaining the appropriate sample from exotic animals may be challenging because of widely varying physiology and anatomy. While appropriate references and excellent techniques and illustrations exist, it is difficult for the researcher, biologist, or clinician to find and access them from the literature. Hence, we review the biological, basic science, laboratory animal, and exotic animal references to create a single useful text which will serve as a quick and easy reference for the veterinarian, biologist, researcher, or technician in need of guidance regarding what is known and what is unknown in order to obtain, handle, and store diagnostic samples for exotic animals.

Interpretation of results from clinical pathologic testing of zoo, wildlife, and exotic animals may be just as challenging, if not more so, than obtaining the sample. In fact, some advocate foregoing many tests because of the challenge of interpretation. While physical examination should remain the bedrock of diagnosis in clinical exotic animal medicine, clinicopathologic testing is an extremely useful tool which should not be overlooked in the clinician's diagnostic arsenal. Additionally clinicopathologic testing may be more useful in these species than in others because of their instinctual stoicism. Many exotic animals are prey species, and occult disease is common; clinical signs may not be apparent until complete health decompensation occurs. Clinical pathological testing is one of many possibilities that offer the hope of diagnosis earlier in the disease process and the chance of a better prognosis for the animal based upon early diagnosis.

Much of the challenge of clinicopathologic interpretation of exotic animals is based on lack of data. Another issue is the availability of data from the extant literature which, for exotic animals, is found scattered among many disciplines and stretches through time back to early anatomical drawings of the late 1800s. If knowing is indeed half the battle, then this text provides what is known and what is not known in a format accessible to the busy clinician. In addition, references have been curated to those of most use and importance to the researcher and clinician. Most chapters make the assumption that the reader has standard baseline veterinary clinicopathologic knowledge and continue into detailed specifics regarding sample collection and preservation, and result interpretation for the species. For more basic information regarding standard-specific clinical pathologic methods, we recommend consultation of the basic veterinary and exotic animal clinicopathology textbooks listed in the references of this section [1-15].

Reference Intervals

An additional challenge for those faced with interpretation of clinicopathologic data in exotic animals is that few true reference intervals are available for exotic, zoo, or wildlife animals. Reference intervals are an important part of all laboratory results. They are designed to be used as guidelines for interpretation and to determine if a result is "normal" or "abnormal." Reference intervals are often referred to as reference range, reference value, "normals," normal range, or normal value; however, these terms are incorrect, and their use is strongly discouraged for several reasons. A reference interval represents a statistical calculation from a group of results obtained from a defined population of animals. In determining this population (often referred to as a reference population), defining what normal actually means is challenging. Furthermore, sick animals may have laboratory results that fall within the reference interval. Use of terms such as "normal range" or "normal values" is not recommended, and they should not be used because of the difficulty in defining "normal." Reference intervals often vary from laboratory to laboratory depending on the instrumentation and methodology.

Reference intervals are influenced by many factors, such as the species, breed, age, and sex of the animal. Results can be affected by diet, exercise, excitement, medications, time of day, or season. Collection and processing of the sample can also influence the result. When establishing reference intervals, the criteria for inclusion of an individual needs to be defined before sample collection begins. Reference intervals are based on the measurement of an analyte in a population of clinically healthy animals that meet the inclusion criteria. Obtaining many samples from a single kennel or herd is not recommended as animals may lack sufficient variation needed for the reference interval to be representative, resulting in an overly narrow reference interval.

In general, samples from at least 120 individuals are recommended to establish a reference interval. However, this is usually quite difficult in veterinary medicine because of the lack of availability of appropriate individuals and the expense of obtaining and analyzing samples. Some veterinary guidelines suggest that a more realistic number is at least 60 qualified individuals that meet the selection criteria. Statistical guidance for the creation of reference intervals is available for researchers creating useful data sets for future reference [16]. Many of the data tables provided in this text fail to meet aforementioned criteria are therefore labeled as reference ranges, but the data may still serve as a starting point for evaluation of the patient and are meant to represent the best extant data available for the species group.

Reference intervals are typically derived from the mean?±2 standard deviations of the mean of the values fitting a normal (Gaussian) distribution. This assumes that 95% of the healthy population will fall within the established reference interval. When data are not normally distributed, nonparametric analysis or data transformation is typically used to remove the top and bottom 2.5 percentiles, as 2.5% of "normal" animals have values outside the reference range on the basis of this statistical model. Often, the ideal reference values are previous values, often called healthy or baseline values, obtained from the individual patient. Figure 1.1 illustrates the likely minimum number needed to create a reference interval for base excess in passerine birds. By convention, for a normally distributed data set, the reference interval for a particular test include 95% of all values from the general (presumed healthy) population. Because 5% of results fall outside this interval, values that may actually be unremarkable or acceptable can therefore sometimes be outside this range. Additionally, normal distributions may be less common for many analytes in exotic animal medicine, making statistical assessment of values for the creation of reference intervals more challenging.

In human medicine, reference intervals for a single analyte are created for a single instrument and/or laboratory, and values from a sample of hundreds of apparently healthy people may be stratified on the basis of gender, age, race, size, or other factors. The results are then further statistically evaluated to create a reference interval (Figures 1.1 and 1.2). Again, seldom in zoo, exotic, or wildlife species do we have the necessary numbers to create reference intervals meeting these stringent definitions, which creates a further barrier to publication of the data which we do have, but also creates a problem in interpretation. The publication and use of data for very small numbers of animals, which are of questionable health can lead to erroneous interpretation of clinical pathology, possibly to the animal's detriment. The wise clinician remembers that clinical pathology is used to confirm and further define a diagnosis, seldom to discover one, and this should be particularly true of nontraditional animal medicine. This intent of this text is also to provide extant reference data and to provide guidelines for interpretation of these analytes on the basis of the species at hand. To accomplish these goals, we have asked authors to provide a relevant literature review combined with their experience in clinical medicine. Chapters are meant to provide a clinically useful overview with references available for additional in-depth consultation to the researcher and student.

Laboratory Choices: The Use of a Reference or an In-House Laboratory

The ability to perform or request various diagnostic assays is available to the clinician in a variety of settings. Choices include sending samples to diagnostic reference veterinary laboratories or laboratories at teaching universities, establishing an in-house laboratory,...