Session #3000

Abstract

Birds often are presented to veterinarians only when they are near-terminally ill. In these cases, a quick answer is needed. In addition, birds are limited in their range of expression of clinical signs. In avian medicine, it has become common to conduct exhaustive diagnostic tests on patients, often with minimal attention to a complete history and physical examination. Selection of diagnostic tests should be based on a solid understanding of the species in question, the results of a thorough history-taking and physical examination, and a shortened list of probable differential diagnoses based on this workup. While avian biochemistry analysis can provide insight into internal organ function and physiology, it does not provide a precise diagnosis. Plasma biochemistry analysis is just one tool to facilitate reaching a diagnosis. Understanding what causes changes in biochemistry profile patterns is the key to using this tool more effectively.

Introduction

Veterinarians who treat birds are faced with challenges that are often not encountered by their counterparts who treat dogs and cats. Many birds are presented to veterinarians only when near-terminally ill, and a quick answer is often the difference between life and death. Birds are often limited in their range of expression of clinical signs, and many clinicians, through no fault of their own, lack the experience to conduct a thorough physical examination. The combination of these factors has led to an increasing tendency in avian medicine to conduct exhaustive diagnostic tests on patients, often with scant attention paid to a complete history and a careful physical examination and with little attempt to refine or focus the diagnostic efforts. The selection of diagnostic tests should be based on a solid understanding of the species in question, the results of a thorough history-taking and physical examination, and a shortened list of probable differential diagnoses based on this workup.

Before proceeding with diagnostic tests, the clinician should first ask the following questions:

• Are the test(s) appropriate to the patient (species, age, sex, etc.) and its clinical signs?
• Has the test been validated to ensure that the result obtained is likely to be both accurate and meaningful?
• Are the physical risks to the patient and cost of the test(s) to the client justified by the likely clinical value of the results?

If the answer to these questions is yes, then diagnostic testing should proceed.

One of the most used diagnostic tests in avian medicine is the analysis of serum biochemistry analytes. This involves the measurement of specific groups of chemicals within the blood and the interpretation of the results obtained. These chemicals include the following:

• Metabolites, which are the chemicals that are produced as the end products of various metabolic processes within the body.
• Tissue enzymes, which catalyze chemical reactions within the body without being altered themselves.
• Electrolytes including sodium, potassium, and chloride.
• Minerals such as calcium, phosphorus, and magnesium.
• Bile acids, which are produced in the liver from cholesterol and used in the emulsification of dietary fats.

The levels of these chemicals in the blood can be influenced by either physiologic or pathologic processes. Physiologic variations can be due to age, sex, body fat to muscle ratio, nutritional status, reproductive status, and species; however, pathologic processes, including cellular damage or abnormal function of an organ system (or systems), often produce significant changes in blood levels.

Causes of Plasma Biochemistry Abnormalities

There are three major causes of abnormal clinical biochemistries: (1) normal variation between species and individuals, (2) artifacts, and (3) pathology.¹ These causes are discussed below.

Normal Variation Between Species and Individuals

There are over 9,000 species of birds, with major differences in anatomy, physiology, form, and function. Some are carnivorous, some are nectarivorous, some are granivorous, and some are omnivorous. It is unrealistic to expect that they would all conform to a relatively narrow spectrum of biochemical values.

Other variations arise between individuals of the same species. These variations occur because of differences in age, sex, diet, husbandry, physiologic events (such as reproduction), etc.

Postprandial elevation in uric acid is normal in carnivorous birds. It is not seen in granivorous birds.

For this reason, some clinicians recommend establishing a set of normal values for individual birds during annual health examinations and then using these values as a comparison should the bird become ill.

Artifacts

When interpreting a biochemistry analysis, care must be taken to distinguish between abnormal results due to disease and abnormal results due to other factors. These other factors, referred to as artifacts, can occur for a variety of reasons, including the following:

• Physiologic changes
• Previous therapy
• Clinical condition of the patient
• Collection method
• Storage and transport of the sample

Physiologic Changes

Stress due to transport and handling of the patient can lead to a release of endogenous corticosteroids, resulting in changes in the hemogram and in blood glucose.

Lipemia, while occasionally seen in diseases of the liver and reproductive system, can also occur naturally in the reproductively active female. Regardless of the cause, lipemia can cause false elevations in bile acids, protein, calcium, phosphorus, and uric acid. It may also falsely decrease amylase. Postprandial lipemia is uncommon in pet birds, so fasting will not help; the clinician needs to check with the laboratory if the sample submitted was lipemic before interpreting these biochemistries.

Previous Therapy

Before interpreting biochemistries, the clinician should consider if any treatment given prior to the sample collection could have influenced the results. Therapy given by another veterinarian or to stabilize a crashing patient can have marked effects. Parenteral fluids can dilute biochemistries; exogenous corticosteroids can markedly elevate aspartate aminotransferase (AST), creatine kinase (CK), and lactate dehydrogenase (LDH); and intramuscular injections, particularly of irritant drugs, can do the same.

Clinical Condition of the Patient

Trauma, starvation, and dehydration can all have marked effects on biochemistries and need to be considered when interpreting results. Trauma can cause elevations in AST, CK, and possibly glucose; starvation can lower glucose and elevate AST and CK if protein catabolism has begun; and dehydration can elevate uric acid and urea.

Collection Method

Ideally, sample collection should be performed in such a manner that it has minimal impact on the patient while providing an artifact-free sample suitable for analysis. This usually requires venipuncture to be performed on a minimally stressed patient. Inexperienced clinicians may need to consider gaseous anesthesia to collect a good sample without the bird struggling.

Venipuncture should be performed on a large vein (e.g., the jugular) using a needle that is large enough to minimize hemolysis while being small enough to minimize trauma to the blood vessel wall. Hemolysis can cause elevations in bile acids, LDH, CK, alkaline phosphatase (ALP), potassium, and phosphorus. Glucose and albumin may be decreased. Calcium may be elevated or decreased, according to the methodology used.

Toenail clipping should be discouraged. Not only is it unduly painful but crush artifacts and contamination with uric acid and bacteria from droppings on the perch can cause elevations in uric acid and decreased glucose if testing is delayed long enough for bacterial growth to occur in the sample.

Storage and Transport of the Sample

Blood collected for biochemistry analysis should be placed immediately into a lithium heparin tube. Ideally, miniature tubes as used in medical pediatrics should be used. The sample should be gently rolled or rocked; clotting must be avoided but hemolysis must be as well. If the analysis is to be performed in-house, it should be processed immediately. If a delay is likely, or if the sample is to be shipped to an outside laboratory, the sample should be centrifuged and the plasma harvested. Sending whole blood to an outside laboratory can result in decreased glucose (as cell metabolism continues) and hemolysis.

EDTA tubes are unsuitable for biochemistry analysis but can be used for hematology, lead analysis, and fibrinogen determination.

Some Pearls to Help You Get the Best Possible Results

Interpretation of avian biochemistry results is only part of the art of using hematology and biochemistry to assess your patients. To get the most out of a submitted sample, it is important to provide your laboratory (external or in-house) with the best possible sample in the best possible condition. The following are some hints for doing that.

• Submit the largest size sample you can without compromising your patient’s wellbeing. Generally, we can collect 10% of the patient’s blood volume (equal to 1% of its bodyweight), but the amount we collect in pediatric and anemic patients is less. It is always worth speaking to your lab to find out what size sample they need, but negotiate with them if they require volumes larger than 0.5 mL of whole blood.
• Avoid hemolysis by using an appropriate size needle (e.g., 23–27 gauge) and using gentle negative pressure on the plunger to prevent turbulence during collection. Remove the needle before placing the sample into the collection bottles.
• Make several blood smears before placing the sample into anticoagulant. Rather than using another microscope slide to push the blood along the slide, use a coverslip to drag the sample along the slide.
• Use BD Microtainers® for your sample, but don’t under or overfill the tubes. Underfilling the tube mixes excessive anticoagulant with the blood; overfilling may result in a clotted tube.
• Use lithium heparin tubes (green top) for biochemistry and Na EDTA tubes (purple top) for hematology.
• Ensure the blood and anticoagulant are mixed immediately by rolling the tube along the palm of your hand or gently inverting the tube 10 times. Do not shake the sample, as this may result in hemolysis.
• If sample processing is likely to be delayed more than a few hours, centrifuge the lithium sample, and separate the plasma from the red cells. Decant the plasma and place in another lithium heparin bottle. This prevents artifacts associated by prolonged contact time with the erythrocytes.
• Avian plasma samples frequently are yellow due to carotenoid pigments, not bilirubin. Pink or red plasma is usually indicative of hemolysis.
• Avoid overinterpretation of results. Look for significant elevations or decreases, not changes that could be within the range of error or normal variations for the machine or patient. Be aware of artifactual changes affecting some parameters (e.g., hyperkalemia due to hemolysis).
• Always treat your patient, not your test results!

Pathology

Liver Enzymes

Detection of liver disease through biochemistry is complicated by the fact that there are no specific liver enzymes that can be evaluated conclusively in every case. Typically, decreases in hepatic enzyme activities do not have clinical relevance except in the cases of end-stage liver disease, leaving elevated levels as the screening tool for liver disease.

Liver disease can be broadly classified into three conditions: hepatocellular rupture, decreased hepatic function, and cholestasis. These conditions can occur either separately or concurrently.

Hepatocellular Rupture

A hepatocellular rupture releases intracellular enzymes, which then reach elevated levels in the blood. These so-called leakage enzymes include:

• Aspartate aminotransferase (AST). This cytosolic enzyme is distributed in high concentrations throughout hepatic parenchyma, skeletal muscle, heart, brain, and renal parenchyma, but the highest concentrations are found in skeletal muscle and liver. Significant elevations, therefore, usually represent either muscular or hepatocellular damage. AST, therefore, must be interpreted alongside creatine kinase (CK), also known as creatine phosphokinase (CPK), released from damaged muscle, to distinguish between the two. CK is specific and sensitive for muscle damage in birds. In general, an elevated AST with a normal CK indicates hepatocellular rupture; however, CK has a much shorter half-life than AST; a single-point muscle injury (e.g., an injection) 4–7 hours before sample collection could duplicate this biochemistry pattern. Although AST has long been considered to be the most useful liver enzyme, it cannot be considered in isolation as an indicator of liver disease.
  A recent study² showed there was poor correlation between AST/CK and histopathology in diagnosing liver disease. Since there are many confounding influences on the AST levels, this commonly used screening test may not be as useful as previously thought.
• Glutamate dehydrogenase (GLDH). A mitochondrially bound enzyme, GLDH is both specific and sensitive for the detection of liver disease that involves hepatocellular rupture. GLDH is found in many tissues in the body, including hepatocytes, kidney, intestine, muscle, and salivary gland; however, most of serum GLDH originates from hepatocytes. As GLDH is a large molecule bound to hepatocyte mitochondria, practically none is liberated in generalized inflammatory diseases of the liver such as infectious diseases. Liver diseases in which necrosis of hepatocytes is the predominant event, such as toxic liver damage, are characterized by high serum GLDH levels. It therefore has a high specificity for liver disease but a low sensitivity (as damage must be severe before elevation occurs). This enzyme has an extremely short biologic half-life (<1 hour) in racing pigeons and up to 72 hours in raptors.³
• Sorbitol dehydrogenase (SDH). SDH is found in the highest concentration in the liver. It is a cytoplasmic enzyme that catalyzes the conversion of fructose to sorbitol. It is a very specific indicator of liver disease in all species, although increases can occur with primary or secondary liver disease. SDH activity has a short half-life (<12 hours) and an increase suggests acute hepatocellular injury.⁴
• Alanine aminotransferase (ALT) and alkaline phosphatase. These are not considered useful in detecting liver disease in birds.¹ ALT in birds is very nonspecific for the liver, and normal levels have been shown in cases with severe liver damage. ALP elevations are more commonly associated with osteoclastic conditions in birds (e.g., growth, trauma, repair, osteomyelitis, neoplasia, nutritional secondary hyperparathyroidism, and eggshell deposition).

Decreased Liver Function

Decreased liver function can occur with any number of liver diseases, not all of which involve hepatocellular rupture. Chronic cirrhosis, amyloidosis, and hepatic lipidosis can all have an adverse effect on liver function without causing any cellular damage. In these cases, a liver function test is necessary to detect the problem.

• Bile acids are produced in the liver and are excreted in bile into the small intestine where they act to emulsify fat. Most of the bile acids are then resorbed in the small intestine, enter the portal system and are taken up by the liver to be recycled. Elevated levels occur when there is significant impairment of the liver’s ability to remove bile acids from the portal circulation. Normal bile acids may not reflect a lack of liver disease, as there must be a high enough reduction in functional hepatocytes to observe a significant increase in bile acid concentration. A 2–4-fold increase in bile acids indicates such a decrease in liver function.
• Total protein concentration is substantially lower than that of mammalian species. The total protein value determined using a refractometer is frequently inaccurate in birds due to interference by high concentrations of other refractive compounds in plasma, such as chromogens, lipids, and glucose. Biuret analysis of total protein is therefore recommended in birds.
• The BCG method for albumin determination has not been validated in birds caused, in part, by use of human albumin standards and controls, which have different binding affinity for the dye than does avian albumin. Gel electrophoresis is the recommended method of albumin determination in avian species.³

Cholestasis

Cholestasis occurs when the biliary system is partially or totally obstructed. This can be seen with biliary neoplasia, pancreatic disease, or diffuse swelling of the entire liver.

• Gamma glutamyl transferase (GGT) is an enzyme found in the epithelial cell membranes of the bile ducts and kidneys. GGT activity is more likely to be increased in cholestatic conditions and biliary epithelial disorders in birds as it is in mammals. GGT is not sensitive to hepatocellular damage alone. The clinical utility of GGT in the diagnosis of biliary conditions in birds has not been adequately evaluated but may be elevated in conditions such as biliary carcinomas.²
• Bilirubin is not produced in birds (therefore, they cannot become jaundiced); they utilize biliverdin instead. There are no commercial assays for biliverdin.

Kidney Function

The avian kidney filters a large volume of fluid (approximately 11 times the entire body water each day for a 100-g bird) and then reclaims most filtered water by tubular and cloacal reabsorption. The glomerular filtration rate decreases with dehydration and with injection of arginine vasotocin, the avian analogue of mammalian antidiuretic hormone. Avian kidneys shunt blood from reptilian to mammalian-type nephrons when GFR is decreased, thus increasing a bird’s ability to concentrate urine.³

• Uric acid is the end product of protein metabolism in birds and is an oxidized form of hypoxanthine. It is produced primarily in the liver from purine metabolism, enters the circulation, and is then secreted by renal tubules (>90%) or filtered in the glomerulus (<10%). Due to this active renal tubular secretion, blood uric acid levels are not notably affected by dehydration until GFR is decreased to the point that uric acid is not moved through the tubules, which may occur in severe dehydration. Raptors and other carnivores have higher reference intervals for uric acid and marked increases in plasma uric acid concentration may be observed postprandially. Significant loss of renal tubules will therefore result in elevations of uric acid.
  At first glance, it appears that uric acid offers a sensitive and specific test for renal disease; however, there are several confounding factors. First, species differences: carnivorous birds have higher normal uric acid levels than granivorous birds. Second, age: juvenile birds may have lower levels than adults. Third, although significant elevations usually indicate renal disease, normal levels do not mean the kidneys are normal; mild increases could indicate early renal disease or dehydration (or both). There must be severe renal damage before uric acid levels begin to rise.
  Because of this relative insensitivity of uric acid in detecting renal disease, levels are best interpreted alongside a determination of the bird’s water intake and loss and a physical examination. To distinguish renal disease from dehydration, the patient’s hematocrit, total protein, and blood urea nitrogen (BUN) should be evaluated concurrently. Dehydration can lead to decreased GFR, in turn leading to elevated levels of BUN. This same decrease in GFR can lead to elevations of uric acid without primary renal disease being present. It is therefore prudent, in cases of an elevated uric acid level, to rehydrate the patient over 2–3 days before definitively diagnosing renal disease. Persistent hyperuricemia after fluid therapy and with hematocrit, total protein, and BUN returning to normal confirms a diagnosis of renal disease.
• Creatinine levels are normally low in birds and may be below the minimum detectable limit of the assays in the laboratory. It is generally accepted as being of little or no value in evaluating renal function in birds.
• Phosphorus elevations are sometimes seen in birds with renal disease and should be compared with calcium (the Ca:P ratio).

Blood Glucose

Glucose is an essential energy source for nearly every cell in the body. Blood levels are governed by its intake, absorption, the interactions of hormones controlling carbohydrate metabolism (insulin, glucagon, and somatostatin), the body’s metabolism, its ability to store glucose, and its excretion. As disorders of glucose metabolism involve so many organ systems, it is treated here as a separate entity.

Hyperglycemia may be a normal physiologic process (e.g., in juvenile birds); however, elevated levels are usually related to increased production or release (e.g., stress) or failure of tissues to take it up out of the blood (diabetes mellitus). Iatrogenic hyperglycemia occurs when corticosteroids are administered or intravenous dextrose is given. Female reproductive disease may also elevate blood glucose, but this may be an indirect result due to inflammation affecting the endocrine pancreas.

Although a positive urine glucose test is an indicator of hyperglycemia, stress hyperglycemia may cause up to 3+ glucosuria on a urine dipstick. Since avian urine/urates are retro-pulsed into the colorectum, false-positive glucosuria and proteinuria may occur due to fecal contamination, especially in frugivorous birds. Results of clinical chemistry tests, including repeated blood glucose, glucagon, and insulin concentrations; urinalysis; and clinical signs all should be considered prior to making a diagnosis of diabetes mellitus.

Hypoglycemia may result from poor handling of blood samples (i.e., artifactual rather than pathologic), with decreased food intake (starvation, anorexia), increased glucose usage (septicemias, neoplasia, and multiorgan failure), or decreased production (liver disease). Carnivorous birds can maintain fasting blood glucose levels much longer than granivorous species. Smaller granivorous species may become hypoglycemic after a 12-hour fast, especially if debilitated.

Lipids

Cholesterol and triglycerides are lipids synthesized in the body and used for a variety of metabolic processes. As they are insoluble, they are transported through the body bound to lipoproteins: high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL).⁵

Cholesterol, synthesized in the liver, plays a major role in the production of bile acids, steroid hormones, and vitamin D. It also provides stability and flexibility to cellular plasma membranes. Triglycerides, fatty acids attached to glycerol, are synthesized in the liver and intestinal mucosa and stored in fat. They are used as an energy source for metabolic processes.

VLDLs transport cholesterol, triglycerides, and protein around the body. Once in circulation, triglycerides are released to cells for energy. As the triglycerides are removed, the cholesterol component increases until it is higher than the triglyceride component, forming LDL. This is then taken up by cells where the contents are either stored, used for cell membrane structure, or converted into other products such as steroid hormones or bile acids. Excess cholesterol in the cells and blood vessels is taken up by HDL, transporting it back to the liver for subsequent conversion to bile acids.

While cholesterol and triglycerides are necessary for normal body functions, high levels (associated with high-fat diets, lack of exercise, obesity, and reproductive activity) may predispose birds to disease.

Elevations in cholesterol concentrations have been reported to be associated with a variety of disease conditions in birds, including hepatic lipidosis, atherosclerosis, hypothyroidism, bile duct obstruction, and diabetes mellitus. Decreased plasma cholesterol concentrations have been associated with some cases of hepatic disease (such as aflatoxicosis), decreased dietary fat, E. coli endotoxemia, and spirochetosis.

Elevated triglycerides are frequently associated with reproductive activity in female birds. Very high levels that are persistently elevated have been associated with ovarian pathology.

Elevated VLDL, LDL, and HDL concentrations, especially when combined with elevated cholesterol and triglycerides, are considered to be likely markers for atherosclerosis and hepatic lipidosis. More research needs to be done in this field. Most birds transport cholesterol mainly in the form of HDL (unlike humans, where LDL is predominant). Therefore, higher HDL values may not be associated with decreased risks of developing atherosclerosis as it is in humans; in fact, the converse may be true.

Amylase and Lipase

Amylase and lipase have been proposed as useful parameters in the detection of pancreatic disease.¹ There is still considerable discussion of the incidence of pancreatic disease and the specificity of these enzymes. Significant elevations of these enzymes, when accompanied by clinical signs of gastrointestinal dysfunction (e.g., vomiting, ileus, diarrhea, coelomic pain), should lead the clinician to consider pancreatic disease as a differential diagnosis; however, normal levels do not preclude a diagnosis of pancreatic disease, nor do abnormal levels confirm such a diagnosis.

Electrolytes

The predominant intracellular and extracellular anions and cations in birds are similar to those in mammals.³

Sodium

Sodium must be interpreted with the knowledge of the patient’s hydration status. It may be elevated with decreased water intake or dehydration through renal disease, vomiting, or diarrhea. Sodium may also be lost through the gastrointestinal tract or the kidneys. Other causes of hyponatremia include overhydration, end-stage liver disease, and congestive heart failure.

Chloride

Chloride is interpreted alongside sodium. It may be elevated with vomiting or regurgitation, although this is uncommon. Low levels are usually associated with regurgitation or vomiting, renal disease, congestive heart failure, and other conditions which cause water retention.

Potassium

Potassium may be decreased with vomiting and diarrhea and elevated with dehydration, hemolysis, tissue damage, or poor sample handling. Renal disease in birds has been associated with hyperphosphatemia and hyperkalemia; however, hypophosphatemia and hypokalemia also have been reported. The degree of artifactual change in potassium values appears to be species-specific and warrants immediate separation of plasma from RBCs in all avian samples.

Calcium

Avian total calcium values can be much higher under normal physiologic circumstances than would be tolerated by a mammal. Dramatic increases in plasma total calcium concentration are seen in reproductive, oviparous females due to estrogen-induced transport of calcium-bound yolk proteins to the ovary. Reproductive pathology such as egg binding and egg-yolk coelomitis also can result in marked total hypercalcemia. Marked hypocalcemia may be caused by malnutrition or reproductive abnormalities such as chronic egg laying.

Conclusions

While avian biochemistry analysis can provide an overview on the function of internal organs and physiology, it cannot provide a precise diagnosis. As such, plasma biochemistry analysis should be regarded as just one tool in the clinician’s arsenal in reaching a diagnosis. Understanding what causes the change in pattern in biochemistry profiles is the key to using this tool more effectively.

References

1.  Doneley B. Avian Medicine and Surgery in Practice: Companion and Aviary Birds. 2nd ed. Boca Raton, FL: CRC Press; 2016.

2.  Hung CSY, Sladakovic I, Divers SJ. Diagnostic value of plasma biochemistry, haematology, radiography and endoscopic visualisation for hepatic disease in psittacine birds. Vet Rec. 2020;186(17):563. https://doi.org/10.1136/vr.105214.

3.  Harr KE. Clinical chemistry of companion avian species: a review. Vet Clin Pathol. 2002;31(3):140–151. https://doi.org/10.1111/j.1939-165X.2002.tb00295.x.

4.  Williams SM, Holthaus L, Barron HW, et al. Improved clinicopathologic assessments of acute liver damage due to trauma in Indian ring-necked parakeets (Psittacula krameri manillensis). J Avian Med Surg. 2012;26(2):67–75. www.jstor.org/stable/41682462.

5.  Beaufrère H, Cray C, Ammersbach M, Tully TN. Association of plasma lipid levels with atherosclerosis prevalence in psittaciformes. J Avian Med Surg. 2014;28(3):225–231. www.jstor.org\stable\24624173.