MERCK MANUAL Veterinary Manual

Veterinary / Clinical Pathology and Procedures /

Diagnostic Procedures for the Private Practice Laboratory

Clinical Biochemistry
By Trevor J. Whitbread, BSc, BVSc, MRCVS, DECVP, Abbey Veterinary
Services ; Karen W. Post, DVM, MS, DACVM, North Carolina Veterinary
Diagnostic Laboratory System, Consumer Services, Rollins Animal Disease,
Diagnostic Laboratory ; Charles M. Hendrix, DVM, PhD, Department of
Pathobiology, College of Veterinary Medicine, Auburn University

Clinical biochemistry refers to the analysis of the blood plasma (or serum) for a wide
variety of substancessubstrates, enzymes, hormones, etcand their use in 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 six tests that merely confirm or deny six possibilities, a well-chosen
group of six 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, because evaluation of both together is essential for optimal
recognition of many of the most characteristic disease patterns (see Clinical Hematology).

Before samples are collected, a list of differential diagnoses should already be established
based on the history and clinical examination. Then, additional appropriate tests can be
added to the basic panel below.

Making a diagnosis entails establishing a list of differential diagnoses based on the

history and clinical examination. Based on this list, tests can be selected to include or
exclude as many of the differentials as possible. Yet more tests may be necessary until
only one of the original list remains to determine the diagnosis. If all differentials are
excluded, then the list must be reevaluated. It is not good practice to order tests without a
sensible differential list unless the animal presents without definitive clinical signs.

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
two analytes), urea, creatinine, ALT, and alkaline phosphatase (ALP). In addition, a
yellow color seen in the plasma should be considered an indication to measure bilirubin.
This panel may be modified as appropriate for other species, eg, glutamate
dehydrogenase (GDH) and/or glutamyl transferase (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 level 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 level increases due to dehydration. It decreases due to the same factors as total
protein, plus liver failure.

Urea level 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. Urea measurement is used
especially to indicate renal disease and to a lesser extent liver dysfunction.

Creatinine level increases due to renal dysfunction, blocked urethra, and ruptured
bladder. It decreases due to sample deterioration. Animals with a high muscle mass have
high-normal creatinine concentrations, whereas animals with a low muscle mass have
low-normal creatinine concentrations. Creatinine measurement is used especially for
renal disease.

ALT is present in the cytoplasm and mitochondria of liver cells and, therefore, increases
due to hepatocellular damage. It has a half life of 24 hr and rises higher than AST but
recovers quicker. There are minor increases with muscle damage and hyperthyroidism.

ALP level increases due to increased bone deposition, liver damage, hyperthyroidism,
biliary tract disease, intestinal damage, hyperadrenocorticism, corticosteroid
administration, barbiturate administration, and generalized tissue damage (including
neoplasia). The most common causes for an increase is raised levels of circulating
steroids and biliary disease. The half-life is 72 hr in dogs but only 6 hr in cats. Levels in
the cat are generally much lower than in the dog, and any increase in cats is considered
significant. In dogs, ALP levels in the thousands of units are usually associated with
increased steroid levels. ALP and ALT levels rarely rise above 1,000 units, even in severe
liver disease.

GDH level increases in hepatocellular damage, particularly hepatic necrosis, in horses

and ruminants.

GT 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 level increases are reported in hypothyroidism. Only a very
small amount of muscle damage such as bruising or IM injections can result in high
serum CK levels. In dogs and cats, unless investigating specific muscle disease, increased
levels are generally of no clinical significance.

AST level increases in both muscle and liver damage but is of less value than ALT. The
half-life is 5 hr in dogs and 77 min in cats. It is also reported to increase in
hypothyroidism.

Most of the above parameters are associated with liver function/dysfunction and are
frequently overinterpreted. In small animals, increases in ALT and ALP levels can reach
four times normal and still be associated only with fatty change, a nonspecific finding and
not, in most cases, a primary liver problem. Some laboratories also frequently receive
liver biopsies from dogs that have significant increases in liver enzymes and bile acids
>80 but that have normal histologic morphology. The reason for this is unknown.

In general, plasma enzyme levels 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,
to create panels for polydipsic animals, collapsing animals, 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 may indicate diabetes mellitus and contributes to the pattern
characteristic of hyperadrenocorticism, and cholesterol also adds to the appreciation of
the Cushing pattern. Renal failure is covered by the tests already included in the basic
panel. In contrast, in a panel for a "collapsing animal, calcium and glucose may be added
to screen for hypocalcemia or hypoglycemia. Sodium and potassium are included to
screen for hypoadrenocorticism or hypokalemia. Analytes that might be considered for
incorporation in such expanded profiles are described below.

Sodium level increases due to Conn syndrome (hyperaldosteronism), restricted water

intake, vomiting, and most causes of dehydration. It decreases due to
hypoadrenocorticism, loss of any high-sodium fluid such as some forms of renal disease,
and insufficient sodium provision during IV fluid therapy.

Potassium level increases due to hypoadrenocorticism and severe renal failure

(especially terminal cases). It decreases due to Conn syndrome, chronic renal
dysfunction, vomiting, diarrhea, and insufficient potassium provision during IV fluid
therapy. Congenital hypokalemia occurs in Burmese cats.

Chloride level increases in acidosis, and in parallel with increases in sodium

concentration. It decreases in alkalosis, vomiting (especially after eating), and in
association with hyponatremia.

Total CO2 (bicarbonate) level increases in metabolic alkalosis and decreases in

metabolic acidosis. It is less useful to assess respiratory acid/base disturbances.

Calcium level increases due to dehydration (which is also associated with increased
albumin), primary hyperparathyroidism (neoplasia of parathyroid gland), primary
pseudohyperparathyroidism (neoplasms producing parathormone-related peptide [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 level 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 level 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 level increases due to high-carbohydrate meals, sprint exercise, stress or

excitement (including handling and sampling stress), glucocorticoid therapy,
hyperadrenocorticism, 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).

-Hydroxybutyrate level increases in diabetes mellitus. It is a major component of

ketoacidosis and as such is also increased in acetonemia/pregnancy toxemia and extreme
starvation. It can be measured in both blood and urine.

Bilirubin level increases due to fasting (benign effect in horses and squirrel monkeys,
may be caused by 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, whereas
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 acid levels 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 obstruction; very
little increase is seen in feline infectious peritonitis or mild cases of hepatic lipidosis. Very
high levels can sometimes be seen without structural histologic changes. The reason for
this is not known.

Cholesterol level increases due to fatty meals, hepatic or biliary disease, protein-losing
nephropathy (and other protein-losing syndromes to some extent), diabetes mellitus,
hyperadrenocorticism, 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. It is therefore of very limited value in general clinical practice.

Sorbitol dehydrogenase level increases in acute hepatocellular damage in horses but

is a very labile analyte.

-Amylase level increases in acute pancreatitis but in dogs is also increased in chronic
renal dysfunction. It is therefore of limited use in the diagnosis of pancreatitis. Pancreatic
lipase immunoreactivity is now the test of choice for diagnosis of pancreatitis in dogs and
cats. Amylase is not a useful indicator of pancreatitis in cats.
Lipase level 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.

Immunoreactive trypsin (trypsin-like immunoreactivity) level decreases in

exocrine pancreatic insufficiency in dogs. It will also increase (irregularly) in pancreatitis.

Tests for Pancreatic Disease:

Pancreatitis:

Serum amylase and lipase activities have been used for several decades to diagnose
pancreatitis in both people and dogs. Unfortunately, neither 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 there are sources of
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 diagnosis of pancreatitis. Serum amylase and/or lipase activities that are 3
5 times the upper limit of the reference range, in animals with clinical signs 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 diagnosis of pancreatitis. Although cats
with experimental pancreatitis can show an increase 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 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 kidneys. In addition, any prematurely activated
trypsin is quickly removed by proteinase inhibitors, such as 1-proteinase inhibitor and
2-macroglobulin. In turn, 2-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 diagnosis of
pancreatitis in dogs and cats.

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 to measure PLI in canine (cPLI) and
feline (fPLI) serum have been developed and validated and are commercially available.
Serum PLI is highly specific for exocrine pancreatic function. Also, serum PLI is far more
sensitive for diagnosis of pancreatitis than any other diagnostic test currently available.

A patient-side semiquantitative assay for diagnosis of canine pancreatitis is also available.

A test spot that is lighter in color than the reference spot suggests that pancreatitis can be
excluded. A test spot darker in color than the reference spot raises the suspicion of
pancreatitis and should prompt the clinician to measure a serum cPLI concentration in
the laboratory.

Other tests for diagnosis of pancreatitis in dogs and cats have been evaluated, including
plasma trypsinogen activation peptide (TAP) concentration, urine TAP concentration,
urine TAP: creatinine ratio, serum 1-proteinase inhibitor trypsin complex concentration,
and serum 2-macroglobulin concentration. However, none has been shown to be
of clinical usefulness.

Exocrine Pancreatic Insufficiency:

In the past, several fecal tests have been used to diagnose exocrine pancreatic
insufficiency (E PI). 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 to diagnose E PI, microscopic fecal examination is no longer justified.
Fecal proteolytic activity had been used to diagnose E PI 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 and in areas where the more
accurate and sophisticated tests are not available.

Serum TLI concentration is the diagnostic test of choice for E PI in both dogs and
cats. Assays for TLI measure trypsinogen circulating in the vascular space. In healthy
animals, only a small amount of trypsinogen is present in serum. However, in dogs and
cats with E PI, the number of pancreatic acinar cells is severely decreased. Serum TLI
concentration decreases significantly and may even be undetectable. The reference range
for TLI in dogs is 5.745.2 mcg/L with a cut-off value of 2.5 mcg/L considered
diagnostic for E PI. Similarly, the reference range for TLI in cats is 1282 mcg/L, with a
cut-off value of 8 mcg/L considered diagnostic. Rarely, animals with serum TLI
concentrations below the cut-off value for E PI do not have clinical signs of E PI. This is
probably because of the 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 E PI.
If there is no evidence of small-intestinal disease in such patients, a trial therapy with
pancreatic enzymes and reevaluation of serum TLI concentration after 1 mo is indicated.

PLI is also highly specific for exocrine pancreatic function and could be used to diagnose
E PI. However, initial studies showed there is a small degree of overlap in serum PLI
concentrations between healthy dogs and dogs with E PI, making the measurement of PLI
slightly inferior to that of TLI for accurate diagnosis. In view of these findings, PLI assays
for both dogs and cats have been optimized toward higher concentrations, and the
current assays are no longer suitable for diagnosis of E PI in dogs or cats.
A fecal canine elastase concentration assay has been developed and validated but is
inferior to the widely used TLI measurement.

Handling of Samples:
Most biochemistry tests can be performed on either serum or heparinized plasma. A few
(eg, insulin) require serum, whereas 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
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, because they
separate samples more quickly and the same machine can be used to measure 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.

Point-of-Care Tests:
A number of biochemical analytes may be estimated in the practice without the need for
large analytical instruments.

Total protein level is measured by refractometry, using the same instrument as is used
to measure urine specific gravity, provided the instrument has a total protein scale. 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 level 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 read only 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.
Ketone levels may be estimated on either urine (preferred sample) or plasma/serum.
This can be achieved by using the ketone patch of a urine dipstick, giving a qualitative
result. However, there are a number of point-of-care instruments for measurement of
blood glucose and ketone levels, including specifically -hydroxybutyrate.

Triglyceride levels 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, the
milkiness is caused by triglycerides. This is a qualitative judgment but is nevertheless
useful, especially in equine patients.

Bilirubin level may also be appreciated by eye in most species. E quine and bovine
plasma is normally yellow, which makes determination problematic, but in other species,
any yellow color is abnormal and indicates an increased bilirubin level. Visual assessment
of the depth and shade of color may provide additional information.

Other point-of-care tests include C-reactive protein as a marker for inflammation and
cardiac troponin as a marker for cardiac muscle damage.

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.

The Practice Laboratory:

E xtending in-practice analysis beyond these emergency basics requires a dedicated
instrument capable of measuring multiple analytes. Two types are availablethose 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 done 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
laboratory evaluation of nonemergency patients is best referred to a professional
laboratory from the outset, for reasons of cost, accuracy, range of analytes available, and
the assistance of the clinical pathologist in interpretation of the results.

If in-clinic analysis is to be relied on for general case laboratory evaluation, 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; unless these are
within the tolerance limits, no patient samples should be tested. Participation in an
external quality assessment program is also strongly recommended. E mploying a trained
technician will address some of these issues but has implications for availability of results
during off hours and 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.

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