Clinical biochemistry refers to the analysis of the blood plasma (or serum) for a wide variety of
ALT increases due to hepatocellular damage, muscle damage, and hyperthyroidism.
ALP increases due to increased bone depo...
alkalosis, vomiting (especially after eating), and in association with hyponatremia.
Total CO 2 (bicarbonate) increases ...
obstruction; very little increase is seen in feline infectious peritonitis or mild cases of hepatic lipidosis.
clot from adhering to the vessel walls.
Samples for biochemical analysis should be separated as soon as possible after c...
hydroxybutyrate are also available.
Triglycerides may be visualized in a plasma or serum sample as lipemia. If the milki...
hours and during holidays. The veterinarian in charge of the laboratory is responsible for all the results
issued and in...
Pancreatic lipase immunoreactivity (PLI) concentration measures the concentration of classical
pancreatic lipase in the ...
functional redundancy of the GI tract. At the same time, many dogs and cats with chronic diarrhea and
weight loss have m...
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roles of veterinary laborotary diagnostic with special reference to biochemistry


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roles of veterinary laborotary diagnostic with special reference to biochemistry

  1. 1. 1 Clinical biochemistry refers to the analysis of the blood plasma (or serum) for a wide variety of substances—substrates, enzymes, hormones, etc—and their use in the diagnosis and monitoring of disease. Analysis of other body fluids (eg, urine, ascitic fluids, CSF) is also included. One test is very seldom specific to one clinical condition, and basic checklists of factors affecting the most commonly- requested analytes are given below. Thus, rather than 6 tests that merely confirm or deny 6 possibilities, a well-chosen group of 6 tests can provide information pointing to a wide variety of different conditions by a process of pattern recognition. Biochemistry tests should be accompanied by full hematology, as evaluation of both together is essential for optimal recognition of many of the most characteristic disease patterns (see Diagnostic Procedures for the Private Practice Laboratory: Clinical Hematology). Basic Test Panel Most veterinary laboratories offer a basic panel of tests, which represents a minimal investigation applicable to most general situations. For small animals, a typical panel includes total protein, albumin, globulin (calculated as the difference between the first 2 analytes), urea, creatinine, ALT, and alkaline phosphatase (ALP). In addition, a yellow color seen in the plasma should be considered an indication for measuring bilirubin. This panel may be modified as appropriate for other species, eg, glutamate dehydrogenase (GDH) and/or γ glutamyltransferase (γ GT) are more appropriate ―liver enzymes‖ for horses and farm animals, or it may be more appropriate to concentrate primarily on muscle enzymes (CK and AST) in athletic animals. Total protein increases due to dehydration, chronic inflammation, and paraproteinemia. It decreases due to overhydration, severe congestive heart failure (with edema), protein-losing nephropathy, protein-losing enteropathy, hemorrhage, burns, dietary protein deficiency, malabsorption, and some viral conditions (especially in horses). Albumin increases due to dehydration. It decreases due to the same factors as total protein, plus liver failure. Urea increases due to excess dietary protein, poor quality dietary protein, carbohydrate deficiency, catabolic states, dehydration, congestive heart failure, renal failure, blocked urethra, and ruptured bladder. It decreases due to low dietary protein, gross sepsis, anabolic hormonal effects, liver failure, portosystemic shunts (congenital or acquired), and inborn errors of urea cycle metabolism. Creatinine increases due to renal dysfunction, blocked urethra, and ruptured bladder. It decreases due to sample deterioration. Patients with a high muscle mass have high-normal creatinine concentrations, while patients with a low muscle mass have low-normal creatinine concentrations.
  2. 2. 2 ALT increases due to hepatocellular damage, muscle damage, and hyperthyroidism. ALP increases due to increased bone deposition, liver damage, hyperthyroidism, biliary tract disease, intestinal damage, Cushing's disease, corticosteroid administration, barbiturate administration, and generalized tissue damage (including neoplasia). GDH increases in hepatocellular damage, particularly hepatic necrosis in horses and ruminants. γGT increases in longer-term liver damage; it is particularly useful in horses and ruminants. CK, the classic ―muscle enzyme,‖ increases markedly in rhabdomyolysis and aortic thromboembolism. Slight increases are reported in hypothyroidism. AST increases in both muscle and liver damage and is also reported to increase in hypothyroidism. In general, plasma enzymes decrease due to sample deterioration. Uncommonly, atrophy or fibrosis of an organ may result in unusually low plasma activities of the relevant enzymes. Additional Tests Further tests may be added to the basic panel, according to the principal presenting signs, in order to create panels for polydipsic patients, collapsing patients, etc. These panels are structured so that the patterns of abnormalities typical of all the likely differential diagnoses applicable to the situation can be discerned. For example, a polydipsia panel may add calcium, glucose, and cholesterol. Calcium allows recognition of hyperparathyroidism and other causes of hypercalcemia (which causes polydipsia and renal insufficiency), glucose picks up diabetes mellitus and contributes to the pattern characteristic of Cushing's disease, and cholesterol also adds to the appreciation of the ―Cushing's pattern.‖ Renal failure is covered by the tests already contained in the basic panel. In contrast, a ―collapsing animal‖ panel may add calcium and glucose to screen for hypocalcemia or hypoglycemia. Sodium and potassium are included to screen for Addison's disease or hypokalemia. Analytes that might be considered for incorporation in such expanded profiles are described below. Sodium increases due to Conn's syndrome (hyperaldosteronism), restricted water intake, vomiting, and dehydration due to most causes. It decreases due to Addison's disease, loss of any high-sodium fluid such as some forms of renal disease, and insufficient sodium provision during IV fluid therapy. Potassium increases due to Addison's disease and severe renal failure (especially terminal cases). It decreases due to Conn's syndrome, chronic renal dysfunction, vomiting, diarrhea, and insufficient potassium provision during intravenous fluid therapy. Congenital hypokalemia occurs in Burmese cats. Chloride increases in acidosis, and in parallel with increases in sodium concentration. It decreases in
  3. 3. 3 alkalosis, vomiting (especially after eating), and in association with hyponatremia. Total CO 2 (bicarbonate) increases in metabolic alkalosis and decreases in metabolic acidosis. It is less useful in assessing respiratory acid/base disturbances. Calcium increases due to dehydration (which is also associated with increased albumin), primary hyperparathyroidism (neoplasia of parathyroid gland), primary pseudohyperparathyroidism (neoplasms producing parathormone-related peptide or PRP, usually perianal adenocarcinoma or some form of lymphosarcoma), bone invasion of malignant neoplasms, thyrotoxicosis (uncommon), and overtreatment of parturient paresis. It decreases due to hypoalbuminemia, parturient paresis, oxalate poisoning, chronic renal failure (secondary renal hyperparathyroidism), acute pancreatitis (occasionally), surgical interference with parathyroid glands, and idiopathic (autoimmune) hypoparathyroidism. Phosphate increases due to renal failure (secondary renal hyperparathyroidism). Decreases are seen in some downer cows and as part of the stress pattern in horses and small animals. Magnesium increases are rarely seen, including during acute renal failure. It decreases in ruminants due to dietary deficiency, either acute (grass staggers) or chronic, and diarrhea (uncommon). Glucose increases due to high-carbohydrate meals, sprint exercise, stress or excitement (including handling and sampling stress), glucocorticoid therapy, Cushing's disease, overinfusion with glucose/dextrose-containing IV fluids, and diabetes mellitus. It decreases due to insulin overdose, insulinoma, islet cell hyperplasia (uncommon), acetonemia/pregnancy toxemia, acute febrile illness, and idiopathically (in certain dog breeds). β-Hydroxybutyrate increases in diabetic ketoacidosis, acetonemia/pregnancy toxemia, and extreme starvation. Bilirubin increases due to fasting (benign effect in horses and squirrel monkeys, may lead to hepatic lipidosis in cats), hemolytic disease (usually mild increase), liver dysfunction, and biliary obstruction (intra- or extrahepatic). Theoretically, hemolysis is characterized by an increase in unconjugated (indirect) bilirubin, while hepatic and post-hepatic disorders are characterized by an increase in conjugated (direct) bilirubin; however, in practice this differentiation is unsatisfactory. Better appreciation of the source of the jaundice is gained from bile acid measurements. Bile acids increase when hepatic anion transport is impaired, usually during liver dysfunction (bile acids are more sensitive than bilirubin to hepatic impairment), and in the presence of a portosystemic shunt (congenital or acquired). The latter condition is characterized by a marked increase in bile acid concentration after feeding, from a fasting concentration that may be normal. It also increases in bile duct
  4. 4. 4 obstruction; very little increase is seen in feline infectious peritonitis or mild cases of hepatic lipidosis. Cholesterol increases due to fatty meals, hepatic or biliary disease, protein-losing nephropathy (and other protein-losing syndromes to some extent), diabetes mellitus, Cushing's disease, and hypothyroidism. It decreases in some cases of severe liver dysfunction, and occasionally in hyperthyroidism. Lactate dehydrogenase is a ubiquitous enzyme with a number of isoenzymes; electrophoretic separation of isoenzymes is necessary to locate the source of increased activity. Sorbitol dehydrogenase increases in acute hepatocellular damage in horses but is a very labile analyte. α-Amylase increases in acute pancreatitis in dogs and in chronic renal dysfunction. Amylase is not a useful indicator of pancreatitis in cats. Lipase increases in acute pancreatitis in dogs (longer half-life than amylase) and also occasionally in chronic renal dysfunction. Lipase (routine assay) is not a useful indicator of pancreatitis in cats.Pancreatic-specific lipase is however a good indicator of pancreatitis in cats and dogs. Immunoreactive trypsin (trypsin-like immunoreactivity) decreases in exocrine pancreatic insufficiency in dogs. It will also increase (irregularly) in pancreatitis. (Also see Diagnostic Procedures for the Private Practice Laboratory: Tests for Pancreatic Disease.) Pattern Recognition The essence of pattern recognition is the identification of a single condition that will explain the totality of the findings—not only biochemical, but clinical and hematologic as well. Further specific investigations are then carried out to confirm or deny this hypothesis. If more than a single condition will explain all the findings, then these must be differentiated by further investigation. While the ―textbook‖ case of any condition is seldom encountered, a ―best-fit‖ approach is usually productive in identifying the most promising avenues of exploration. The postulation of 2 or more simultaneous diagnoses to account for all the abnormalities seen is usually counterproductive. Handling of Samples Most biochemistry tests can be performed on either serum or heparinized plasma. A few (eg, insulin) require serum, while potassium is best measured on heparin plasma separated immediately after collection. Glucose measurement requires fluoride/oxalate plasma. Suitable collection tubes with and without anticoagulant are available commercially. Plastic tubes are satisfactory for blood in anticoagulant, but clotted blood must be collected either into glass tubes or plastic tubes specially coated to prevent the
  5. 5. 5 clot from adhering to the vessel walls. Samples for biochemical analysis should be separated as soon as possible after collection to minimize artifacts caused by hemolysis and leakage of intracellular fluid components (eg, potassium) out of the cells. Samples in anticoagulant may be centrifuged immediately, but clotted samples need at least 30 min to allow the clot to form. Fluoride/oxalate samples hemolyze very readily because the cells can no longer respire, so timely separation is especially important. Proprietary gels or plastic beads assist with separation, and these may be incorporated into the collection tube or added before centrifugation. Larger bucket-type centrifuges will accept almost any type or size of tube, but the rotors require careful balancing. They should be spun at 3,000 rpm for 10 min. Dual-purpose, high-speed microhematocrit centrifuges are favored for in-practice use, as they separate samples more quickly and the same machine can be used for measuring PCV. However, they can handle only a limited range of small-volume tubes. Some ―gel-tube‖ products may provide a permanent separation of serum or plasma, otherwise this must be separated into a fresh tube. The new tube must be adequately labeled. Samples may then be sent to a professional laboratory or analyzed in the practice. In-practice Analysis A number of biochemical analytes may be estimated in the practice without the need for large analytical instruments. Total protein is measured by refractometry, using the same instrument as is used to measure urine specific gravity, provided that an instrument with a total protein scale is purchased. It is also valid for protein measurement of ascitic and pleural fluids. The readout may be in g/dL, in which case multiplying the result by 10 will yield the SI unit of g/L. Urea may be estimated by chromatographic reaction strips, which correlate well with standard laboratory methods. A rapid whole-blood color comparison strip is also available, but these only read up to ∼20 mmol/L and are thus of limited use. A dedicated reflectance meter for urea estimation is not available. Glucose meters for use on whole blood are widely available for home use by human diabetic patients. These yield acceptably accurate results on animal blood, although an unexpected hypoglycemia should be confirmed by a professional laboratory. Fresh whole blood may be used, but fluoride blood or plasma is the preferred sample if analysis is not immediate. Ketones may be estimated on either urine (preferred sample) or plasma/serum, using the ketone patch of a urine dipstick, giving a qualitative result. Whole-blood glucose meters with the capability of measuring β-
  6. 6. 6 hydroxybutyrate are also available. Triglycerides may be visualized in a plasma or serum sample as lipemia. If the milkiness rises to the top of the tube on storage, chylomicrons are present. Otherwise, it is triglycerides. This is a qualitative judgment but is nevertheless useful, especially in equine patients. Bilirubin may also be appreciated by eye in most species. Equine and bovine plasma is normally yellow, which makes determination problematic, but in other species, any yellow color is abnormal and indicates an increased bilirubin. Visual assessment of the depth and shade of color may provide additional information. For emergency in-clinic use, the most important analytes beyond these simple basics are sodium and potassium. A dedicated ion-specific electrode meter is the best way to measure these. Instruments are available that can analyze whole blood, although great care must be taken to avoid artifacts due to unappreciated hemolysis. Critical care meters are also available that can estimate a variety of analytes including glucose, urea, and electrolytes; however, these have not been extensively validated on nonhuman blood, and results should be interpreted with caution. Extending in-practice analysis beyond these emergency basics requires a dedicated instrument capable of measuring multiple analytes. Two types are available—those based on transmission/absorbance photometry (wet chemistry), and those based on reflectance photometry (dry-reagent chemistry). Transmission/absorbance photometry is the reference method on which all reference values and interpretive guidelines are based. Reflectance photometry methods do not always compare well with the reference method, and are best confined to simpler tests such as glucose and urea. For wider applications, such as enzyme analysis, wet chemistry instruments are preferred. In-clinic analysis is inevitably more expensive than the same investigations carried out by a professional laboratory, and the range of analytes available is more restricted. Additionally, the level of accuracy or reliability is likely to be lower. Therefore, it is still best practice to regard in-practice analysis as an interim emergency investigation, with the results to be confirmed as appropriate by a professional laboratory. Detailed case work-up of non-emergency patients is best referred to a professional laboratory from the outset, for reasons of cost, accuracy, range of analytes available, and the additional assistance of the clinical pathologist in the interpretation of the results. If in-clinic analysis is to be relied on for general case work-up, meticulous attention must be paid to quality assurance. Samples of known composition must be run at least daily for each analyte, in both normal and pathologic ranges, and no patient results should be accepted unless these are within the tolerance limits. Participation in an external quality assessment program is also strongly recommended. Employing a trained technician will address some of these issues but has implications for availability of results out-of-
  7. 7. 7 hours and during holidays. The veterinarian in charge of the laboratory is responsible for all the results issued and incurs a legal liability to prove accuracy and reliability. If these cannot be guaranteed to the same standard as a referral laboratory, then results should not be relied on without external confirmation. TESTS FOR PANCREATIC DISEASE Pancreatitis Serum amylase and lipase activities have been used for several decades to diagnose pancreatitis in both humans and dogs. Unfortunately, neither one of these tests is both sensitive and specific for pancreatitis in dogs. In one study, significant serum amylase and lipase activities remained after total pancreatectomy, indicating that there are sources for serum amylase and lipase activity other than the exocrine pancreas. Also, clinical data suggest a specificity for pancreatitis of only ∼50% for both of these markers. Many nonpancreatic diseases, such as renal, hepatic, intestinal, and neoplastic diseases, can lead to increases in serum amylase and lipase activities. Steroid administration can also increase serum lipase activity and cause variable responses in serum amylase activity. Thus, in dogs, measurement of serum amylase and lipase activities are of limited usefulness for the diagnosis of pancreatitis. Serum amylase and/or lipase activities that are 3–5 times the upper limit of the reference range, in patients with clinical signs that are consistent with pancreatitis, are suggestive of such a diagnosis. However, it is important to note that ∼50% of dogs that fulfill these criteria do not have pancreatitis. In cats, serum amylase and lipase activities are of no clinical value for the diagnosis of pancreatitis. While cats with experimental pancreatitis can show increases in serum lipase activity and a decrease in serum amylase activity, these changes are not consistent in cats with spontaneous pancreatitis. In one study of 12 cats with severe forms of pancreatitis, not a single cat had serum lipase or amylase activity above the upper limit of the reference range. Serum trypsin-like immunoreactivity (TLI) concentration measures mainly trypsinogen, the only form of trypsin that is circulating in the vascular space of healthy individuals. However, trypsin, if present in the serum, is also detected by these assays. Serum TLI concentrations can be measured by species-specific assays that have been developed and validated for both dogs and cats. In healthy animals, serum TLI is low, but during pancreatitis an increased amount of trypsinogen leaks into the vascular space, which can lead to an increase in serum TLI concentration. Trypsin that has been prematurely activated may also contribute to this increase. However, both trypsinogen and trypsin are quickly cleared by the kidney. In addition, any prematurely activated trypsin is quickly removed by proteinase inhibitors, such as α1- proteinase inhibitor and α2-macroglobulin. In turn, α2-macroglobulin-trypsin complexes are removed by the reticuloendothelial system. Thus, the serum half-life for TLI is short, and a significant degree of active inflammation is required to increase serum TLI concentration. Because of the limited sensitivity of serum cTLI and fTLI concentrations for canine and feline pancreatitis, respectively, and because only a limited number of laboratories measure these assays routinely, serum TLI concentration is of limited usefulness for the diagnosis of pancreatitis in both dogs and cats.
  8. 8. 8 Pancreatic lipase immunoreactivity (PLI) concentration measures the concentration of classical pancreatic lipase in the serum. This is in contrast to serum lipase activity, which measures the enzymatic activity of all triglyceridases present in the serum, regardless of their cellular origin. Assays for the measurement of PLI in canine (cPLI) and feline (fPLI) serum have recently been developed and validated. Serum PLI is highly specific for exocrine pancreatic function. Also, serum PLI is far more sensitive for the diagnosis of pancreatitis than any other diagnostic test currently available. Assays for the measurement of serum PLI concentration in dogs and cats, Spec cPL® and Spec fPL®, respectively, have been developed and are commercially available. Also, a patient-side semi-quantitative assay for the diagnosis of canine pancreatitis, SNAP cPL has been released. A test spot that is lighter in color than the reference spot suggests that pancreatitis can be ruled out. A test spot darker in color than the reference spot raises the suspicion for pancreatitis and should prompt the clinician to measure a serum Spec cPL concentration in the laboratory. Other tests for the diagnosis of pancreatitis in dogs and cats have been evaluated. However, plasma trypsinogen activation peptide (TAP) concentration, urine TAP concentration, urine TAP:creatinine ratio, serum α1-proteinase inhibitor trypsin complex concentration, and serum α2-macroglobulin concentration have all been shown to be of little clinical usefulness for the diagnosis of spontaneous pancreatitis in dogs or cats. Exocrine Pancreatic Insufficiency In the past, several fecal tests have been used to diagnose exocrine pancreatic insufficiency (EPI). Microscopic fecal examination for fat and/or undigested starch or muscle fibers are at best useful to suggest maldigestion. However, in light of wide availability of tests for diagnosing EPI, microscopic fecal examination can no longer be justified. Fecal proteolytic activity had been used to diagnose EPI in small animals for several decades. Most of these methods, particularly the radiographic film clearance test, are unreliable. One method, which uses pre-made tablets to pour a gelatin agar, is considered most reliable. However, false positive as well as false negative results have been reported. The clinical use of fecal proteolytic activity is limited to species for which more specific assays to estimate pancreatic function are not available. Serum TLI concentration is the diagnostic test of choice for EPI in both dogs and cats. Assays for TLI measure trypsinogen circulating in the vascular space. In healthy individuals, only a small amount of trypsinogen is present in serum. However, in dogs and cats with EPI, the number of pancreatic acinar cells is severely decreased. Serum TLI concentration decreases significantly and may even be undetectable. The reference range for canine TLI is 5.7–45.2 μg/L with a cut-off value of ≤2.5 μg/L considered diagnostic for EPI. Similarly, the reference range for feline serum TLI is 12–82 μg/L, with a cut-off value of ≤8 μg/L considered diagnostic for feline EPI. Rarely, animals with serum TLI concentrations below the cut-off value for EPI do not have clinical signs of EPI. This is probably due to the
  9. 9. 9 functional redundancy of the GI tract. At the same time, many dogs and cats with chronic diarrhea and weight loss have mild decreases in serum TLI concentration. Most of these animals have chronic small- intestinal disease and should be investigated accordingly. However, a small number of these dogs and cats may have EPI. If there is no evidence of small-intestinal disease in such patients, a trial therapy with pancreatic enzymes and re-evaluation of serum TLI concentration after 1 mo is indicated. PLI is also highly specific for exocrine pancreatic function and could be used to diagnose EPI. However, initial studies showed that there is a small degree of overlap in serum PLI concentrations between normal dogs and dogs with EPI, making the measurement of PLI slightly inferior to TLI for accurate diagnosis. In response to these findings, both canine and feline PLI assays have been optimized towards higher concentrations and the current assays are no longer suitable for diagnosis of EPI in dogs or cats. A fecal canine elastase concentration assay has recently been developed and validated. This assay has been shown to be inferior to serum cTLI measurement and leads to many false-positive test results. The assay is also more cumbersome and more expensive than measurement of serum cTLI concentration. Fecal elastase concentration might be useful for the diagnosis of EPI due to obstruction of the pancreatic duct. However, this condition is extremely rare in both dogs and cats. If fecal elastase concentration is to be used as a screening test for EPI, each positive test (ie, an elastase concentration of <10 μg/g feces) must be confirmed by measurement of serum cTLI concentration.