Transcript Clinical Chemistryx

Clinical Chemistry
Reference: Laboratory Procedures for
Veterinary Technicians 5th Ed.
(Hendrix & Sirois)
Sample Collection & Handling
Most chemical analyses require collection
and preparation of serum samples
 Whole blood or blood plasma used for
some test methods or with specific types
of equipment

– Do not use EDTA; heparin is a good choice for
clinical chemistry samples
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Most adverse influences on sample quality
can be avoided by careful consideration of
sample collection and handling
Sample Collection & Handling
Specific blood collection protocols vary
depending on patient species, volume of
blood needed, method of restraint, and
type of sample needed
 Collect blood samples for chemical testing
before beginning treatment.
 Preprandial samples are preferred;
postprandial samples may produce
erroneous results.
 Label sample tube with date and time of
collection, owner’s name, patient’s name,
and patient’s clinic identification number.
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Serum Sample Collection
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Blood should be collected from calm, fasted
animal when possible
Avoid hemolysis by selecting needles of the
correct size.
Place blood in a container that contains no
anticoagulant.
Allow blood to clot at room temperature for 20 to
30 minutes.
Gently separate clot by “rimming” with a wooden
applicator stick around the inside of the tube.
Replace top and centrifuge at 2000 to 3000 rpm
for 10 minutes.
Remove serum with a pipette and transfer to
appropriate container.
Factors Influencing Results
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Hemolysis: may result when a blood
sample is:
– drawn into a moist syringe
– mixed too vigorously after collection
– forced through a needle when being
transferred to a tube
– Frozen as a whole blood sample
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Hemolysis can also occur when excess
alcohol is used to clean the skin and not
allowed to dry prior to drawing blood.
Hemolysis
Fluid from hemolyzed blood cells can
dilute the sample, resulting in falsely
lower concentrations of constituents
present in the animal.
 Certain constituents, normally not found in
high concentrations in serum or plasma,
escape from ruptured blood cells, causing
falsely elevated concentrations in the
sample.
 Hemolysis may elevate levels of
potassium, organic phosphorus, and
certain enzymes in the blood
 Hemolysis also interferes with lipase
activity and bilirubin determinations.
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Factors Influencing Results
Chemical contamination: collection tubes
must be chemically pure
 Improper labeling: label all tubes properly.
 Patient influences: obtain samples from a
fasting animal
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– Postprandial samples may have increased
blood glucose levels and decreased inorganic
phosphorus.
– Lipemia results in turbid or cloudy serum
– Kidney assays affected due to increase in GFR
after eating.
Factors Influencing Results
 Improper
Sample Handling:
complete chemical analysis within 1
hour of sample collection.
– Do not allow samples to become too
warm.
– Thoroughly mix serum or plasma that
has been frozen after thawing to avoid
concentration gradients.
Reference Ranges
 Reference
ranges are a range of
values derived when a laboratory has
repeatedly assayed samples from a
significant number of clinically
normal animals of a given species
using specific test methods.
Protein Assays
Plasma proteins are produced primarily by
the liver, as well as reticuloendothelial
tissues, lymphoid tissues, and plasma cells
 Plasma proteins have many functions:
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– Form the structural matrix of all cells, organs,
and tissues
– Maintain osmotic pressure
– Serve as enzymes for biochemical reactions
– Act as buffers in acid-base balance
– Serve as hormones
– Function in blood coagulation
– Defend the body against pathogenic
microorganisms
– Serve as transport/carrier molecules for most
constituents of plasma
Protein Assays
 Total
Plasma Protein
 Total Serum Protein
 Albumin
 Globulins
 Albumin/Globulin Ratio
 Fibrinogen
Total Protein
Total plasma protein measurements
include fibrinogen values; total serum
protein determinations measure all the
protein fractions except fibrinogen.
 Total protein concentration may be
affected by altered hepatic synthesis,
altered protein distribution, and altered
protein breakdown or excretion, as well as
dehydration or overhydration.
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Total Protein
 Alterations
in plasma protein
concentrations occur in a variety of
disease conditions, especially disease
of liver and kidneys.
 Age-related changes in plasma
protein are also seen.
Determination of Total Protein Levels:
Refractometric & Biuret Photometric Methods
Refractometric method measures the
refractive index of serum or plasma with a
refractometer.
 Refractive index of the sample is a
function of the concentration of solid
particles in the sample. In plasma, the
primary solids are the proteins.
 Biuret method measures the number of
molecules containing more than three
peptide bonds in serum or plasma.
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Albumin
Albumin comprises 35% to 50% of the
total plasma protein in most animals.
 Significant hypoproteinemia is most likely
caused by albumin loss.
 Synthesized by hepatocytes
 Liver disease, renal disease, dietary
intake, and intestinal protein absorption
may influence the plasma albumin level.
 Major binding, transport protein in blood;
responsible for maintaining osmotic
pressure of plasma
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Globulins: Complex Group of
Proteins
Alpha globulins are synthesized in the
liver and primarily transport and bind
proteins (include HDL & VLDL).
 Beta globulins include complement,
transferrin, and ferritin
 Gamma globulins (immunobulins) are
synthesized by plasma cells
 Concentration estimated by determining
difference between total protein and
albumin concentrations.
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Albumin/globulin (A/G) ratio
 Alteration
in the normal ratio is
frequently the first indication of a
protein abnormality
 Determined by dividing the albumin
concentration by the globulin
concentration.
 In dogs and horses albumin>globulin
 In cats and cattle, albumin=/<globulin
Fibrinogen
 3%
to 6% of the total plasma protein
content
 Synthesized by hepatocytes
 Elevation occurs with acute
inflammation or tissue damage.
 Most common method of fibrinogen
evaluation is the heat precipitation
test (you can review this in chapter 2).
Hepatobiliary Assays
 Liver
functions include:
– metabolism of amino acids,
carbohydrates, and lipids
– synthesis of albumin, cholesterol,
plasma protein, and clotting factors
– digestion and absorption of nutrients
related to bile formation
– secretion of bilirubin, or bile
– elimination, such as detoxification of
toxins and catabolism of certain drugs
 These
functions are run by enzymatic
reactions.
Hepatobiliary Assays
The gallbladder is closely associated with
the liver, both anatomically and
functionally; its primary function is as a
storage site for bile.
 Malfunctions in the liver or gallbladder
result in CS such as jaundice,
hypoalbuminemia, problems with
hemostasis, hypoglycemia,
hyperlipoproteinemia, and
hepatoencephalopathy.
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Liver Disease vs. Liver Failure
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Liver disease includes any process resulting in
hepatocyte injury, cholestasis, or both.
These include hypoxia, metabolic diseases,
toxicoses, inflammation, neoplasia, trauma, and
bile duct blockage.
Liver failure usually results from some form of
liver disease and is recognized by both failure to
clear the blood of those substances normally
eliminated by the liver and by failure to
synthesize those substances normally produced
by the liver.
Liver Disease vs. Liver Failure
 Liver
disease does not always result
in liver failure.
 Hepatic cells are capable of
regeneration if damaged.
 70% to 80% of liver function must
be lost before liver failure occurs.
 Usually liver disease has to be
greatly progressed before clinical
signs appear.
Hepatobiliary Assays
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Tests for liver disease or failure fall into
three categories:
– Serum enzyme assays that detect
hepatocyte injury
– Serum enzyme assays that detect
cholestasis
– Tests that evaluate or are indicative of
liver functions.
Enzymes Released from
Damaged Hepatocytes
“Leakage Enzymes”
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Sorbitol dehydrogenase
Glutamate dehydrogenase
Alanine Aminotransferase (ALT)
Formerly known as serum glutamic
pyruvic transaminase (SGPT)
 Enzyme found free in the cytoplasm of
hepatocytes
 Considered a liver-specific enzyme in
dogs, cats, and primates
 Horses, ruminants, pigs, and birds do not
have enough ALT in the hepatocytes for
this enzyme to be considered liver specific
 Other sources of ALT are renal cells,
cardiac muscle, skeletal muscle, and
pancreas.
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ALT (cont’d)
Administration of corticosteroid or
anticonvulsant medications can lead to
increases in ALT
 Used as a screening test for liver disease
because it is not precise enough to
identify specific liver diseases
 Increases are usually seen within 12 hours
of hepatocyte damage and peak levels in
24 to 48 hours
 Serum levels will return to reference
ranges within a few weeks unless a
chronic liver insult is present.
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Aspartate Aminotransferase (AST)
Formerly known as serum glutamic
oxaloacetic transaminase (SGOT).
 Found free in the cytoplasm of
hepatocytes and bound to the
mitochondrial membrane.
 Levels tend to rise more slowly than ALT
and return to normal levels within a day if
chronic liver insult is not present
 Found in significant amounts in many
other tissues, including RBCs, cardiac
muscle, skeletal muscle, kidneys, and
pancreas
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AST (cont’d)
 Increased
blood level may indicate
nonspecific liver damage or be
caused by strenuous exercise or
intramuscular injection
 Assess creatine kinase activity to rule
out muscle damage before
attributing an AST increase to liver
damage.
Sorbitol Dehydrogenase (SDH)
Found in liver (primarily), kidney, small
intestine, skeletal muscle, and RBCs
 Especially useful for evaluating liver
damage in large animals such as sheep,
goats, swine, horses, and cattle.
 Plasma level rises quickly with
hepatocellular damage or necrosis.
 Assays can be used in all species to detect
hepatocellular damage or necrosis
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– Disadvantages: SDH is unstable in serum and
tests not readily available to average vet. lab.
Glutamate Dehydrogenase
(GLDH)
Mitochondrial-bound enzyme found in high
concentrations in the hepatocytes of
cattle, sheep, and goats
 Increase in this enzyme is indicative of
hepatocyte damage or necrosis.
 GLDH could be enzyme of choice to
evaluate liver function in ruminants and
avians but no standardized test method
has been developed for use in a veterinary
practice laboratory
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Enzymes Associated
with Cholestasis
“Induced Enzymes”
Alkaline phosphatase (AP)
Gamma glutamyltransferase (GGT)
Alkaline Phosphatase (AP)
Present as isoenzymes in osteoblasts in
bone, and as chondroblasts in cartilage,
intestine, placenta, and cells of the
hepatobilary system in the liver.
 Isoenzymes of AP remain in circulation for
approximately 2 to 3 days, with the
exception of intestinal isoenzyme, which
circulates for just a few hours.
 A corticosteroid isoenzyme of AP has been
identified in dogs with exposure to
increased endogenous or exogenous
glucocorticoids.
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AP (cont’d)
 Source
of an isoenzyme or location
of the damaged tissue is determined
by electrophoresis and other tests
performed in commercial or research
laboratories.
 In older animals, nearly all
circulating AP comes from the liver
as bone development stabilizes.
AP (cont’d)
 Assay
in a practice laboratory
determines the total blood
concentration of AP.
 Concentrations used to detect
cholestasis in adult dogs and cats
 Not a useful test for detecting
cholestasis in cattle and sheep
because of wide fluctuations in
normal blood levels of AP in these
species.
Gamma glutamyltransferase (GGT)
Also called gamma glutamyltranspeptidase
 Primary source is liver
 Also found in renal epithelium, mammary
epithelium, biliary epithelium, kidneys,
pancreas, intestine, and muscle cells
 Cattle, horses, sheep, goats, and birds
have higher blood activity than dogs and
cats
 Blood level is evaluated with liver disease,
especially obstructive.
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Hepatocyte Function Tests
Bilirubin
Bile acids
Cholesterol
Others (dye excretion, ammonia
tolerance, caffeine clearance)
Hepatocyte Function Tests
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Tests of liver function include measurement of
serum concentrations of substances that are
normally removed from the blood by the liver and
then metabolized and/or excreted via the biliary
system (bilirubin, bile acids, cholesterol,
ammonia).
In addition, these tests include measurement of
the serum concentrations of blood constituents
that are normally synthesized by the liver
(albumin, globulins, urea, cholesterol,
coagulation factors)
Abnormal concentrations + evidence of liver
injury = liver disease or liver failure
Bilirubin
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RBCs phagocytized and hemoglobin is dismantled
Heme portion split into iron and protoporphyrin
Protoporphyrin converted to biliverdin then
bilirubin
Bilirubin attached to protein (albumin or globulin)
and transported to liver
Bilirubin conjugated to water-soluble glucuronic
acid in liver (bilirubin glucuronide) and secreted
in bile
Bacteria in GI system act on bilirubin glucuronide
to produce urobilinogen which is broken down
and excreted in feces
Bilirubin (cont’d)
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Some bilirubin glucuronide is absorbed back in
bloodstream rather than excreted in bile and
excreted by kidneys.
Unconjugated (albumin-bound) bilirubin is less
water soluble and comprises ~2/3 of the total
bilirubin in serum.
Measurements of the circulating levels of these
various populations of bilirubin can help pinpoint
the cause of jaundice.
Assays can directly measure total bilirubin
(conjugated bilirubin plus unconjugated bilirubin)
and conjugated bilirubin
Bilirubin (cont’d)
Blood levels of conjugated (direct)
bilirubin are elevated with liver damage or
bile duct injury/obstruction
 Blood levels of unconjugated (indirect)
bilirubin are elevated with excessive
erythrocyte destruction or defects in the
transport mechanism that allow bilirubin
to enter hepatocytes for conjugation.
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Bile Acids
Aid in fat absorption and modulate
cholesterol levels
 Synthesized by hepatocytes from
cholesterol and conjugated with glycine or
taurine
 Conjugated bile acids are secreted across
the canalicular membrane and reach the
duodenum by the biliary system
 Gallbladder stores bile acids (except in the
horse) until contraction associated with
feeding.
 95% are reabsorbed and travel back to
liver and are reconjugated (these are what
are detected in serum tests)
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Bile Acids (cont’d)
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Any process that impairs the hepatocellular,
biliary, or portal enterohepatic circulation of bile
acids results in elevated serum levels.
Serum level is normally elevated after a meal
because the gallbladder has contracted and
released increased amounts of bile into the
duodenum.
Paired serum samples performed after 12hours of
fasting and 2 hours postprandial are needed to
perform the test.
– Difference in concentration of the samples is reported
– Only a single sample is tested in horses.
Bile Acids (cont’d)
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Inadequate fasting or spontaneous gallbladder
contraction can increase fasting bile acids;
prolonged fasting, diarrhea, and GI malabsorption
decreases bile acids
Bile acid levels are unspecific regarding the type of
liver problem that exists (used as a screening test)
May detect liver problems before other CS present
(icterus, jaundice, etc.)
Used to follow progress of liver disease during
treatment
Lipemia will interfere with chemical analysis via
spectrophotometry.
Bile acid test that uses immunologic methods
(ELISA) is available for use in the veterinary clinic.
Cholesterol
 Produced
primarily in the liver and
ingested in food.
 Some forms of hepatic failure =
decreased blood cholesterol
 Cholestasis causes an increase in
serum cholesterol in some species
because bile is a major route of
cholesterol excretion from the body.
 In some animals with liver failure,
the serum cholesterol concentrations
are normal.
Cholesterol (cont’d)
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Assay is sometimes used as a screening
test for hypothyroidism
– Thyroid hormone controls synthesis and
destruction of cholesterol in the body
Other diseases associated with
hypercholesterolemia include
hyperadrenocorticism, diabetes mellitus,
and nephrotic syndrome.
 Administration of corticosteroids may also
cause an elevated blood cholesterol
concentration.
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Other Tests of Liver Function
Dye excretion: anaphylactic reactions
have been observed in humans; therefore
dye excretion method not widely used.
Measurement of bile acid concentration =
more specific and easier to perform. Dye
excretion tests require injection of a dye.
 Ammonia tolerance: any condition that
reduces the uptake of ammonia or
conversion of ammonia to urea can lead to
increased plasma ammonia concentration.
 Caffeine clearance: test used in human
medicine; few experimental studies have
been performed in canine species.
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Kidney Assays
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Kidney functions:
– Conserve or eliminate water and electrolytes in
times imbalance.
– Excrete or conserve hydrogen ions to maintain
blood pH within normal limits.
– Conserve nutrients (eg. glucose and proteins)
– Remove end products of nitrogen metabolism
(urea, creatinine, allantoin)
– Produce renin, erythropoietin, and
prostaglandins
– Aid in regulation of body temperature and
platelet aggregation (prostaglandins)
– Aid in vitamin D activation
Kidney Assays
Kidneys receive blood from the renal
arteries; blood enters the glomerulus of
the nephrons where nearly all water and
small dissolved solutes pass into the
collecting tubules.
 Each nephron contains sections that
function to reabsorb or secrete specific
solutes.
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– Resorption of glucose occurs in the proximal
convoluted tubule
– Secretion and reabsorption of mineral salts
occurs in the ascending limb of the loop of
Henle and in the distal convoluted tubule.
Kidney Assays (cont’d)
 Nephron
has a specific resorptive
capability for each substance called
the renal threshold.
 Blood returns from the kidneys to
the rest of the body through the
renal veins, which connect to the
caudal vena cava.
 Urine and blood may be analyzed to
evaluate kidney function.
Kidney Assays (cont’d)
 Primary
serum chemistry tests for
kidney function: urea nitrogen and
creatinine.
 Other tests are designed to evaluate
the rate and efficiency of glomerular
filtration.
Blood Urea Nitrogen (BUN)
Also called serum urea nitrogen (SUN)
 Urea is the principal end product of amino
acid breakdown in mammals.
 Urea passes through the glomerulus and
enters the renal tubules
 Approximately half the urea is reabsorbed
in the tubules and the remainder excreted
in the urine
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– If the kidneys do not remove sufficient urea
from the plasma, BUN levels increase.
BUN (cont’d)
Several photometric tests are available to
measure urea nitrogen
 Chromatographic tests are available and
tend to be less accurate.
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– Use only as a quick screening test
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Contamination of the blood sample with
urease-producing bacteria may result in
decomposition of urea and decreased BUN
levels.
– Staphylococcus aureus, Proteus spp. and
Klebsiella spp
BUN (cont’d)
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Dehydration results in increased retention
of urea in the blood (azotemia)
– Urea is insoluble molecule; must be excreted
in a high volume of water
High-protein diets and strenuous exercise
may cause elevated BUN levels because of
increased amino acid breakdown (not
decreased glomerular filtration)
 Differences in rate of protein break-down
in male vs female animals as well as
young vs older animals also affect BUN
levels
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Serum Creatinine
Formed from creatine found in skeletal
muscle as part of muscle metabolism
 Creatine diffuses out of muscle cells and
into most body fluids, including blood
 Amount of creatine metabolized to
creatinine usually remains constant, as
does blood level of creatinine
 Total amount of creatinine is a function of
the animal’s total muscle mass.
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Creatinine (cont’d)
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Serum creatinine is filtered through the
glomeruli and eliminated in urine
– Any condition that alters glomerular filtration
rate alters serum creatinine level
Nearly 75% of kidney tissue must be
nonfunctional before blood creatinine
levels rise.
 Postprandial decreases in creatinine occur
from transient increase in glomerular
filtration rate after a meal.
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Creatinine (cont’d)
Increased serum creatinine levels are seen
when there is a lack of functional
glomeruli
 Serum creatinine concentrations are
influenced by:
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– Fluid and hydration levels
– Prerenal factors, such as shock
– Postrenal factors, such as bladder and urethral
obstructions
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Used to evaluate glomerular function
BUN/Creatinine Ratio
Both measurements have a wide range of
reference intervals
 Used in human medicine for diagnosis of
renal disease
 BUN and creatinine have an inverse
logarithmic relation
 A disproportionate increase in BUN can
indicate dehydration, dietary treatment
failure, or owner noncompliance with
treatment regimens.
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Urine Protein/Creatinine Ratio
Mathematical method that compares urine
protein level with urine creatinine levels in
a single urine sample
 Based on the concept that the tubular
concentration of urine increases urinary
protein and creatine concentrations
equally
 5 to 10 mL of urine collected via
cystocentesis
 Sample is centrifuged and supernatant
used to determine both concentrations for
each sample by photometric methods.
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Water Deprivation Test
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Urine concentration test performed to determine
if inappropriate diuresis is attributable to failure
of the neuroendocrine pathway that releases ADH
or if nephrons are not responding properly.
The patient is gradually deprived of water over 3
to 5 days until there is a stimulus for endogenous
ADH release. (This usually occurs at about 5%
weight loss)
Failure to concentrate urine over the duration of
the test is indicative of insufficient ADH or
unresponsive nephrons.
Contraindications: dehydration, azotemia
Pancreas Assays
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The pancreas has endocrine and exocrine
functions. Pancreatic endocrine function
involves production of glucagon and
insulin. Diabetes mellitus, or a deficiency
of insulin resulting in hyperglycemia, is
the most common endocrine disorder of
the pancreas. Pancreatic exocrine
function involves the production of
lipase, amylase, and trypsin. Most
pancreatic disturbances occur in the
exocrine function of the pancreas. Dogs
seem to have a greater incidence than
cats.
Pancreas Assays
Exocrine pancreas: also referred to as
the acinar pancreas.
 Secretes enzymes necessary for digestion
into the small intestine
 Primary pancreatic enzymes are trypsin,
amylase, and lipase
 Trauma to pancreatic tissue is often
associated with pancreatic duct
inflammation that results in a back-up of
digestive enzymes into peripheral
circulation.
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Pancreas Assays
Endocrine Pancreas: interspersed within
the exocrine pancreatic tissue are the
islets of Langerhans
 Four types of islet cells present;
designated as alpha, beta, delta, and PP
(pancreatic polypeptide) cells.
 Delta and PP cells comprise less than 1%
of the islet cells and secrete somatostatin
and pancreatic polypeptide, respectively.
 Beta cells comprise approximately 80% of
the islet cells and secrete insulin.
 20% consists of alpha cells that secrete
glucagon and somatostatin.

Pancreas Assays
 Diseases
of the pancreas may result
in inflammation and cellular damage
that causes leakage of digestive
enzymes or insufficient production or
secretion of enzymes.
 Primary exocrine pancreas tests
are amylase and lipase; trypsinlike
immunoreactivity and pancreatic
lipase immunoreactivity
Amylase
Primary source is the pancreas, but also
produced in the salivary glands and small
intestine.
 Amylase functions to break down starches
and glycogen in sugars.
 Increases in serum amylase are nearly
always caused by pancreatic disease
(pancreatitis), especially when
accompanied by increased lipase levels
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Amylase (cont’d)
 Enteritis,
intestinal obstruction, or
intestinal perforation may also result
in increased serum amylase from
increased absorption of intestinal
amylase into bloodstream.
 Decrease in GFR for any reason can
lead to increased serum amylase
because amylase is excreted by the
kidneys.
Amylase (cont’d)
Animals have a greater serum amylase
activity level than humans (10 times
greater in dog and cat) so it is
recommended to dilute the serum with
normal saline before testing if using tests
designed for human samples.
 Lipemia, hemolysis, and calcium cheleating
anticoagulants will affect results.

Lipase
Nearly all serum lipase is derived from the
pancreas; function of lipase is to break down
fatty acids of lipids.
 Excess lipase is normally filtered through the
kidneys, so lipase levels tend to remain
normal in the early stages of pancreatic
disease.
 Lipase assay is more sensitive for detecting
pancreatitis than amylase assay.
 Increased lipase is also seen in renal failure,
hyperadrenocorticism, dexamethasone
treatment, and bile tract disease.
 Manual methods for testing are
cumbersome, easier to use automated or
SNAP test.
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Trypsinlike Immunoreactivity (TLI)
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Considered the test of choice, TLI is highly
specific and sensitive in detecting pancreatic
insufficiency in dogs.
Radioimmunoassay using antibodies to trypsin
that can detect both trypsinogen and trypsin
Antibodies are species specific
Trypsin and trypsinogen are produced only in the
pancreas
Serum TLI decreases in parallel with functional
pancreatic mass
Decreased glomerular filtration rate increases TLI
Important to fast animal for 12 hours prior to
collecting sample.
Serum Pancreatic Lipase
Immunoreactivity (PLI)
 Serum
feline PLI is specific for
pancreatitis and is recommended
instead of the previously validated
serum feline TLI to diagnose cats
with symptoms of pancreatitis.
 Cats must also be fasted for 12
hours prior to drawing blood sample.
Pancreas Assays
 Endocrine
Pancreas Tests
Primary test is blood glucose; others
include fructosamine, betahydroxybutyrate, glycosylated
hemoglobin, serum cholesterol, and
triglyceride
Glucose
Pancreatic islets respond directly to blood
glucose concentrations and release insulin
(from the beta cells) or glucagon (from
the alpha cells) as needed.
 Blood glucose level is used as an indicator
of carbohydrate metabolism in the body
and as a measure of endocrine function of
the pancreas.
 Some tests for blood glucose react with
only glucose, whereas others may quantify
all sugars in the blood.
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Glucose (cont’d)
Ideally, samples should be taken from an
animal that has been fasted for 16 to 24
hours (ruminants should not be fasted)
 Serum is preferred
 It is essential to centrifuge sample and
transfer serum to another tube
immediately because blood continues to
use glucose at a rate of 7% to 10% per
hour if allowed to remain in contact with
the serum or plasma.
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Glucose (cont’d)
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Hyperglycemia may result from diabetes
mellitus, or any of several nonpancreatic
causes such as stress and
hyperadrenocorticism (Cushing’s disease)
*Diagnosis of diabetes mellitus is not made
unless glycosuria accompanies hyperglycemia.

Hypoglycemia may result from
malabsorption, severe liver disease, or
prolonged contact of the serum or plasma
with the cellular component of blood.
Fructosamine
 Represents
irreversible reaction of
glucose bound to protein, particularly
albumin.
 Increased fructosamine indicates
persistent hyperglycemia
 Indicates average serum glucose over
time period represented by the half-life
of that species’ serum protein.
 Serum fructosamine may be
artifactually reduced in patients with
hypoproteinemia.
Glucose Tolerance Test
 Challenge
the pancreas with a
glucose load and measure insulin’s
effect by blood or urine glucose
concentrations; used to rule out
diabetes mellitus
 IV test is preferred over oral because
oral test is affected by abnormal
intestinal function such as enteritis
or hypermotility, and excitement.
Glucose Tolerance Test (cont’d)
 Glucose
is injected after a 12- to 16hour fast (except in ruminants)
 Blood glucose is subsequently
checked and progress mapped as a
tolerance curve.
 Results are standardized as
disappearance half-lives or glucose
turnover rates expressed as
percent/minute
Insulin Tolerance Test
 Probes
causes of diabetes mellitus
 “Glucose Curve”
 Serum glucose levels are measured
in blood samples obtained before
insulin injection (fasting blood
glucose) and every 30 minutes after
injection for 3 hours.
Other Endocrine Pancreas Tests
 Glucagon
tolerance: indicated when
repeated normal or borderline results
are obtained.
 Insulin/glucose ratio: involves
simultaneous measurements in a
fasting animal.
 Miscellaneous tests of insulin
release: glucose, epinephrine,
leucine, tolbutamide, or calcium
challenges may be attempted.
Other Endocrine
System Assays
Adrenocortical Function Tests
Thyroid Assays
Pituitary Function Tests
Adrenocortical Function Tests
Brain or pituitary tumors leading to
secondary bilateral adrenal hyperplasia,
idiopathic adrenal hyperplasia, or
neoplasia may cause excessive cortisol
release and hyperadrenocorticism.
 Misuse of glucocorticoids is the most
common cause of cortisol excess.
 Hypoadrenocorticism (Addison’s disease)
includes mineralocorticoid deficiency,
which does not occur in iatrogenic disease
from rapid withdrawal of glucocorticoids.

Adrenocortical Function Tests
(cont’d)
Addison’s disease also may result from
Lysodren (a medicaiton for adrenal
hyperplasia) or from idiopathic causes.
 Dogs with nonadrenal disease such as
diabetes mellitus, liver disease, or renal
disease may have false-positive results
 Adrenocorticotropic hormone (ACTH) and
cortisol concentrations may be a helpful
diagnostic aid in differentiating primary
(adrenal-dependent) from secondary
(pituitary-dependent) hypoadrenocorticism.

Adrenocortical Function Tests
(cont’d)
Measurements taken as baseline data and
compared with data obtained from
challenge to the adrenal gland with ACTH
or dexamethasone.
 Low to undetectable ACTH concentrations
occur in secondary Addison’s disease,
whereas normal (or increased)
concentrations are expected in primary
Addison’s disease.
 Refer to pp 100-101 in your textbook for
step-by-step instructions on performing
ACTH stim. and Dex. suppression tests!

Thyroid Assays
Baseline thyroxine concentrations are
used diagnostically, but normal values
vary dramatically
 Semiquantitative immunologic tests are
available to measure T4 concentrations
 TSH response test is used on small
animals (except hyperthyroid cats) and
horses and provides a reliable diagnostic
separation of patients with normal versus
abnormal thyroid function.

Thyroid Assays (cont’d)
Free T4 test measures the fraction of
thyroxine not bound to protein; levels are
less influenced by nonthyroidal diseases or
drugs than are total T4 concentrations.
 Triiodothyronine suppression test: based
on the expected negative feedback
regulation of TSH; induced by high
concentrations of circulating thyroid
hormone.

Pituitary Function Tests
 Diagnosis
of canine acromegaly may
be based on documentation of
elevated growth hormone (GH).
Electrolyte Assays
Electrolytes: negative ions, or anions,
and positive ions, or cations, of elements
found in all body fluids of all organisms.
 Functions of electrolytes include
maintenance of water balance, fluid
osmotic pressure, and normal muscular
and nervous functions.
 Also function in the maintenance and
activation of several enzyme systems and
in acid-base regulation
 Acid-base status depends on electrolytes
and should be interpreted together.

Electrolyte Assays
 Sodium,
potassium, chloride, and
bicarbonate are the four electrolytes
in plasma.
 Minerals of importance are calcium,
phosphate, and magnesium
 These two groups together are often
simply called “electrolytes”
Electrolyte Assays
Calcium
Inorganic Phosphorus
Sodium
Potassium
Magnesium
Chloride
Bicarbonate
Calcium
Do not use EDTA, oxalate, or citrate
anticoagulants to collect calcium samples
for testing because they bind with calcium
and make it unavailable for assay.
 99% of the body’s calcium is in bone
 Remaining calcium maintains
neuromuscular excitability and tone, acts
as an enzyme activator, plays a role in
coagulation, and helps in transport of ions
across cell membranes.
 Serum calcium levels vary with serum
protein and albumin levels (these should
be elevated with increased serum calcium)

Calcium (cont’d)
 Hypercalcemia
seen with
hyperparathyroidism, excessive
vitamin D intake, bony metastases
 Hypocalcemia seen in
malabsorption, eclampsia, pancreatic
necrosis, hypoalbuminemia,
gastrointestinal stasis or blockage in
ruminants, postparturient lactation in
cow, bitch, ewe, and mare,
hypoparathyroidism
Inorganic Phosphorus




Most phosphorus in whole blood is found within
the RBCs as organic phosphorus
Phosphorus in plasma and serum is inorganic
phosphorus and is the phosphorus assayed in the
laboratory
Plasma or serum phosphorus and calcium
concentrations are inversely related: as
phosphorus concentrations decrease, calcium
concentrations increase
Hemolyzed samples should not be used because
organic phosphorus liberated form ruptured RBCs
may be hydrolyzed to inorganic phosphorus,
which results in a falsely elevated inorganic
phosphorus concentration.
Inorganic Phosphorus
 Hyperphosphatemia
may be seen
in renal failure, anuria, excessive
vitamin D intake, ethylene glycol
poisoning, and hypoparathyroidism.
 Hypophosphatemia may occur in
primary hyperparathyroidism,
malabsorption, inadequate intake,
hyperinsulinism, diabetes mellitus,
lymphosarcoma,
hyperadrenocorticism
Sodium
 Most
abundant extracellular cation
that plays a major role in the
distribution of water and the
maintenance of osmotic pressure of
fluids in the body.
 If sodium is retained, water is
retained.
 Heparin sodium should not be used
as an anticoagulant because it may
falsely elevate results.
Sodium (cont’d)
 Hypernatremia
is rare unless the
animal is deprived of water.
 Hyponatremia is quite common and
is seen in such conditions as renal
failure, vomiting, or diarrhea; use of
diuretics; excessive ADH; congestive
heart failure; water toxicity; or
excessive administration of fluids.
Potassium
Major intracelular cation; important for
normal muscular function, nerve impulse
transmission, and carbohydrate
metabolism.
 Serum levels are so low that
measurement of serum potassium does
not give much information about the
body’s potassium levels.
 Plasma is the preferred sample because
platelets may release potassium during
the clotting process (elevating K+ levels).
 Hemolysis releases potassium into plasma
(elevating K+ levels).

Potassium (cont’d)
 Hyperkalemia
will be seen in
adrenal cortical hypofunction,
acidosis, or late-stage renal failure.
 Hypokalemia will be seen in
alkalosis, insulin therapy, or excess
fluid loss due to diuretics, vomiting,
and diarrhea.
Magnesium
Functions to activate enzyme systems and
involved in production and decomposition
of acetylcholine
 Cattle and sheep are the only domestic
animals that show clinical signs related to
magnesium deficiencies.
 Imbalance in calcium-magnesium ratio
can lead to muscle tetany in cattle and
sheep
 Anticoagulants other than heparin may
artificially decrease results
 Hemolysis may elevate the results through
liberation of magnesium from RBCs

Chloride
Predominant extracellular ion.
 Functions in maintenance of water
distribution, osmotic pressure, and the
normal anion/cation ratio.
 Concentration is regulated by the kidneys
 There is a close relationship between
sodium and chloride levels
 Hemolysis may affect test results by
diluting the sample with RBC fluid

Chloride (cont’d)
 Hyperchloremia
may be due to
metabolic acidosis or renal tubular
acidosis
 Hypochloremia may be due to
excessive vomiting, anorexia,
malnutrition, or diabetes insipidus,
or may accompany hypokalemia.
Bicarbonate
Second most common anion of plasma.
 Functions in the bicarbonate/carbonic acid
buffer system and aids in the transport of
carbon dioxide from the tissues to the
lungs.
 Kidney regulates bicarbonate levels in the
body by excreting excesses after it has
resorbed all that it needed.
 Levels are frequently estimated from
blood carbon dioxide levels (arterial blood
= best)
 Bicarbonate level is approximately 95% of
the total carbon dioxide measured.
