23_Biochemistry.qxd 8/23/2005 1:26 PM Page 611 CHAPTER 23 Diagnostic Value of Biochemistry KENDAL E. HARR, DVM, MS, D ipl ACVP Clinical chemistry, along with hematology and physical examination, is the cornerstone of medical diagnosis of disease in any species. Plasma biochemistry is especially important in avian species, which frequently show mini- mal overt clinical signs of disease, even when seriously ill. Veterinarians, therefore, need accurate and useful biochemical analysis to successfully diagnose and treat avian species. The Clinical Laboratory Improvement Amendments (CLIA) are the federal laws and regulations that govern human diagnostic laboratories. No such gov- erning policy exists in veterinary diagnostic medicine. This results in variable methodology, protocol, and most importantly, quality control, between veterinary labora- tories. It falls to the veterinary clinician to ensure that Fig 23.1 | A normal serum sample on the left and a lipemic the diagnostic laboratory being used maintains a high sample on right. standard of quality control and that results are accurate. The clinician should develop a working relationship with the laboratory of choice and must continually mon- itor results for possible inaccuracy and error. Feedback to the laboratory must occur to ensure that the labora- tory personnel are aware of any errors and can correct them. The laboratory used should be familiar with han- dling avian samples. Modified techniques in the labora- tory, such as the use of 10-µl microhematocrit tubes, pediatric sample cups and dilution, can extend the sam- ple and allow more data to be collected, especially from the smaller avian patient. When choosing a laboratory, the clinician also should consider its location, transport issues and delayed processing of the sample. All of these can cause artifactual change in the sample and decrease ability to diagnose disease. Concentrations of analytes represent a steady state of input, consumption and partitioning. The body is always in flux. Generally, pathologic conditions cause either an increased or decreased concentration of various ana- lytes. However, if both input and consumption are 23_Biochemistry.qxd 8/23/2005 1:26 PM Page 612 612 Clinical Avian Medicine - Volume II Reference intervals generated by a laboratory represent population reference intervals that are broader than the reference interval generated when repeatedly sampling an individual. Some individuals will regularly have con- centrations in the low end of the population’s range and some individuals will regularly have concentrations in the high end of the population’s range. Therefore, an individual may remain within the population reference range even though there are abnormalities in the patient. In addition to accurate normal reference intervals, the clinician must have knowledge of the sensitivity/speci- ficity and positive/negative predictive value of a test’s J. Harvey J. ability to diagnose diseases specific to that species. Fig 23.2 | 2.5% of the population is eliminated on each side of the curve to generate a 95% reference interval. This increases Although a great deal is now known regarding avian the likelihood that diseased patients are flagged as abnormal. It medicine, research into the diagnostic application, sensi- also means that it is quite likely that a healthy patient will have tivity, specificity, and positive and negative predictive val- one analyte that is mildly abnormal. ues of biochemical analytes is still needed. decreased or increased at the same time, the concentra- MEASURES OF THE tion may remain in the normal reference interval even ACCURACY OF A TEST though the patient is very ill. For example, the globulin Accuracy is how close the test approximates the true fraction may remain within the normal reference interval value in the body. Precision measures how far from the in a bird with marked enteritis because of increased pro- mean or average of replicate measurements a particular duction of acute phase inflammatory proteins and anti- measurement lies (Fig. 23.3). Most laboratory techniques bodies with concurrent loss of protein through a com- were designed for use in human medicine and are modi- promised gastrointestinal tract. Interpretation of clinical fied for use in birds. Assessment of analytic accuracy and chemistries must therefore be done on a case-by-case precision of a technique is very important when assess- basis with knowledge of species-specific physiology. ing different mammalian species, not to mention differ- ent kingdoms of animals. Any instrument error such as In comparison to domestic species, it can be a challenge old bulbs, slight variation in machine temperature, varia- to simply ascertain normal reference intervals for avian tion in reaction time or degraded reagents can cause patients. Reference intervals for biochemical analytes are decreased accuracy and precision. highly dependent on the machines, reagents and meth- Sensitivity, specificity and predictive values are the meas- ods used, and may vary significantly between different ures of diagnostic accuracy. In medicine, sensitivity is the laboratories. According to CLIA, each human diagnostic likelihood that a diseased patient will have a positive test laboratory must establish reference intervals for each result in a population of individuals with the disease. methodology validated within that laboratory in order to Sensitivity is a measure of false negative values. To help create a diagnostic range that can be used medically. A remember the relationships note that the letter “N” is minimum of 100 healthy individuals is sampled. A 95% present in sensitivity and false negative. Specificity is the reference interval is created for normally distributed likelihood that a patient without the disease has a test analytes using the formula, mean +/- 1.96 standard devi- value that remains within the reference interval in a pop- ations. Therefore, the normal medical reference interval ulation of healthy individuals. Specificity is a measure of used by clinicians excludes 2.5% of normal individuals at false positive values. Note that the letter “P” is present the high and low ends of the range (Fig. 23.2). in specificity and false positive. The predictive value of a Biochemical reference intervals for common species of test is determined by its measurement in a population of psittacines (parrots), passerines (canaries and finches), healthy and sick individuals. A positive test result is and galliformes (turkeys and chickens) have been estab- measured when the disease is present (positive predic- lished by specialized laboratories, eg, California Avian tive value) and a negative test result is measured when Laboratory, Citrus Heights, CA. Each laboratory analyzing the disease is not present (negative predictive value). avian samples should develop species-specific and These can be mathematically determined using the for- methodology-specific reference intervals. These are still mulas in Table 23.2. Tests that are highly sensitive fre- lacking in some university and private laboratories. quently have a low specificity and vice versa. This does 23_Biochemistry.qxd 8/23/2005 1:26 PM Page 613 Chapter 23 | DIAGNOSTIC VALUE OF BIOCHEMISTRY 613 Fig 23.3 | Accuracy vs. Precision. When values are both precise and accurate, they are a tight cluster in the bull's eye. The precise val- ues are all similar, but are some distance from the actual value. The accurate values are all within the third circle, with one almost approximating the actual value, but are scattered around the bull's eye. carotenoid pigments; rarely, in severe disease states, Table 23.1 | Statistical Analysis of a Diagnostic Test 39,49 Formulas for the calculation of diagnostic sensitivity, diagnos- avian plasma may be truly icteric from bilirubin. Pink tic specificity, positive predictive value and negative predictive or red plasma is usually indicative of hemolysis, though value. TP = true positive, TN = true negative, FP = false posi- dyes from food should be ruled out. Green-tinged tive, FN = false negative. plasma is rarely observed, may be caused by biliverdin TP 23,31 • Diagnostic sensitivity = TP+FN and is usually indicative of liver failure. When work- TN ing with smaller species of birds, tuberculin or insulin • Diagnostic specificity = TN+FP TP syringes are frequently used, however, not all of these • Positive predictive value = TP+FP syringes have detachable needles. Avian red blood cells TN • Negative predictive value = TN+FN are larger and deteriorate more quickly than mammalian erythrocytes. This can make accurate analysis difficult with ideal sample handling. Ejecting blood through a not mean that the test is worthless. It simply means that 25-gauge or smaller needle can cause moderate to it may have to be used with other tests to assess organ marked hemolysis that will invalidate many biochemical function and disease. analysis.48 To avert this, attached needles can easily be cut from the syringe using a pair of large veterinary nail clippers or scissors before expelling the blood from the Sample Handling syringe. HANDLING ANTICOAGULANT In the USA, many exotic animal practitioners perform Prior to collection, the appropriate sample container is venipuncture using a syringe that has been coated with labeled with the names of the owner, patient and the injectable sodium heparin to prevent clot formation. signalment. Color-coded, rubber stoppered, evacuated Experienced avian veterinarians working with experi- tubes are well standardized.