1. health and fitness
2. disease

SAMPLE COLLECTION GUIDELINES AND AVOIDING PITFALLS
Linda Shell, DVM, DACVIM (Neurology)
Veterinary Education and Consulting
I.
Fasting vs Non-fasting sampling and tips to minimize lipemia
a. Prefer fasting: less chance of lipemia
b. Lipemia = normal for up to 8-10 hours post meal
c. Lipemia = triglycerides; hypercholesterolemia does not produce lipemia
d. Lipemia can induce hemolysis
e. Some animals lipemic no matter how long they fast
f. Lipemia avoidance: heparin 100 IU/kg IV and collect blood 15 minutes
later
g. Labs may use clearing agents on lipemic serum
II.
Collection tips to minimize hemolysis
a. Fast the animal; lipemia induces hemolysis
b. Use minimum amount of alcohol to raise vein
c. Use large vein and large needle large needle
d. Use vacutainer
e. If using syringe/needle, avoid excessive back pressure on syringe
f. Remove needle from syringe and stopper from tube; allow blood to flow
from syringe into serum tube
g. Use appropriate amount of anticoagulant (excessive amount = hemolysis)
h. Mix without excess agitation
i. Prompt centrifugation once clot forms
j. Gelatinous serum: loss of AT III; presence of acute phase proteins like
fibrinogen. Common cause: didn’t let serum clot before spinning it.
k. Serum color: yellow=icterus; red=hemolysis,
white/cloudy=lipids/triglycerides
III.
Effects of Lipemia and Hemolysis on Tests
a. Often machine-dependent
b. Lipemia may elevate these: TP, TBilirubin, albumin, globulin, glucose
calcium, phosphorus, and bile acids.
c. Lipemia may decrease these: lipase, ALT, AST, AP, amylase
d. Hemolysis can cause leakage of RBC constituents into serum/plasma
(increased K, creatine kinase, AST, ALT, P), dilution of serum
constituents (decreased Na and Cl), color interference (variable effects on
bilirubin, TP, AST, creatine kinase, albumin, lipase, BA, AP, Ca,
creatinine), and chemical interactions (decreases in CO2, thyroxine, and
insulin).
e. Both lipemia and hemolysis can affect bile acids.
IV.
Blood collection: tube order and other points
a. If you use vacutainer, fill tubes as follows: blue top, non-additive red top,
serum separator tube, green top, purple top.
b. If you use needle/syringe, most labs recommend filling serum before
EDTA. If you do fill the EDTA tube first, be careful to avoid
contaminating other tubes filled afterwards. If EDTA contaminates serum
tube, the K and Ca results will be very abnormal looking (very high K and
very low Ca).
c. Red marble with serum separator gel: no anticoagulant; used mainly for
serum chemistries. Avoid using it to collect serum for endocrine testing,
drug levels.
d. Red tube: no anticoagulant; used for serum chemistries, endocrine tests,
and drug levels.
e. Blue: citrate as anticoagulant; used for coagulation studies
f. Green: heparin as anticoagulant; used for plasma/whole blood chemistries.
Plasma samples should be centrifuged to separate plasma from cells & put
into another tube.
g. Allow blood collected in red top or red marble top tubes to sit at room
temperature to allow clotting (approximately 15-30 minutes), and then
centrifuge to separate serum from other blood constituents.
h. Never store whole blood in the freezer, as it causes irreparable cell
damage. Serum and EDTA tubes can be refrigerated.
V.
Coagulogram and von Willebrand’s factor
a. Require blue top (citrate) tube
b. Imperative that blood comes in contact with citrate as soon as possible
(delay= inaccurate results)
c. Avoid trauma, clots, hemolysis
d. Get enough! Inappropriate amount of blood = false results
e. If no vacutainer, use syringe method but check with your lab for their
specific instructions: Draw an exact amount of citrate into a syringe as
follows:
i. 0.2ml citrate + 1.8ml of blood = 2.0 ml total sample
ii. 0.3ml citrate + 2.7ml blood = 3.0 ml total sample
iii. 0.4ml citrate + 3.6ml blood = 4.0 ml total sample
VI.
Drugs that affect tests
a. Bromide can falsely increase the measured chloride level
b. Ace inhibitors can increase potassium value
c. UD diet should result in very low BUN and urine specific gravity
d. Steroids, even topicals: increased alkaline phosphatase, SGGT
e. Oxyglobin®: Due to the dilutional effects of Oxyglobin®, PCV and RBC
count are not accurate measures of the degree of anemia for 24 hours
following administration. Prothrombin time (PT) and activated partial
thromboplastin time (aPTT) results are accurate if determined using
mechanical, magnetic and light scattering methods but inaccurate with
optical methods. Dose and which machine determines to some degree
what chemistry results are affected so it is very variable. Urine dipstick
measurements (i.e., pH, glucose, ketones, protein) are inaccurate while
gross discoloration of the urine is present.
f. Methimazole: Eosinophilia, leukopenia, and lymphocytosis may be noted
in approximately 15% of cats treated within the first 8 weeks of therapy
g. Prednisolone (but not dexamethasone) can affect cortisol value if
administered within 12-24 hours of collecting sample
h. Many drugs can lower T4 (NSAIDs, phenobarbital, sulfa drugs) and
sometimes even free T4 by ED
VII.
Breed differences and diseases
a. Greyhounds, sighthounds, Dachshunds: Higher PCV than normal
b. Some Yorkies can have a high BUN and normal creatinine without
evidence of renal disease. Unknown why. One theory: gi disease
c. Akita & Shib Inu: elevated K with hemolytic sample (RBCs have higher
K). One case report of SharPei too
d. Greyhounds: lower platelet and WBC counts, lower thyroid values, higher
creatinine values, lower globulins,
e. Cavalier King Charles spaniels: falsely low platelet count because of very
large platelets
f. Birman cat: Autosomal recessive granulation anomaly. Neutrophils have
fine eosinophilic or magenta colored cytoplasmic granules. Normal
function.
g. Foxhounds, Australian shepherd, Basenji, Boston terrier, Cocker spaniel,
& cats: Pelger Huet anomaly describes hyposegmented neutrophils that
may be reported as band forms. The total cell count is normal in these
patients and there are no signs of infection. Affected leukocytes function
normally.
h. Persians: Chediak-Higashi syndrome is an autosomal recessive trait that
causes abnormal granulation of white cells and melanocytes. Neutrophils
may be reduced in number and many will contain large fused pink or
eosinophilic granules within the cytoplasm.
i. Poodles: Bone marrow dyscrasia causing macrocytosis of red cells,
nucleated red cells, low normal or slighly low number of neutrophils, and
hypersegmentation of leukocytes.
VIII. Urine collection
a. Collection technique influences how the sample results are interpreted.
b. Which collection technique to use is determined you and what disease
process you needs information about.
c. If a free-catch sample is needed, often times, a first morning, mid-stream,
sample is preferred, as it tends to be the most concentrated and precedes
the morning meal. Random samples may be used for evaluation too.
d. A cystocentesis collection is often done for routine visits because of
convenience. It is also preferred for sick patients. In general, it is easier to
interpret results of a cystocentesis sample collection except in some cases
of hematuria.
e. A cystocentesis collection is preferred for urine culture/sensitivity.
f. Disadvantages to cystocentesis collections: hitting blood vessel which
contaminates sample with RBCs; hitting colon which contaminates sample
with bacteria.
g. If owner’s complaint is hematuria, a free catch sample may be preferred.
But sometimes in cases of hematuria, we want to look at both a
cystocentesis and a free catch sample.
h. Use a sterile, opaque, airtight, labeled container; ideally 6 ml minimum. If
culture/sensitivity is anticipated, put some aside in a sterile container
before you start analysis (to avoid contamination).
i. IF looking for crystals, confirm type on a freshly collected,
unrefrigerated sample within 60 minutes of collection
j. If sample isn’t analyzed within 60 minutes of collection, refrigerate it and
try to get it analyzed in next 12 hours.
k. Warm all refrigerated urine samples to room temperature prior to analysis
to minimize artifacts caused by cooling of the sample
l. Potential artifacts associated with refrigeration:
i. In vitro crystal formation (especially, calcium oxalate dihydrate)
increases with the duration of storage.
ii. A cold urine sample may inhibit enzymatic reactions in the
dipstick (e.g., glucose), leading to falsely decreased results
iii. Specific gravity of cold urine may be falsely increased because
cold urine is denser than room temperature urine
IX.
m. Potential artifacts associated with prolonged storage at room temperature
and their effects:
i. Bacterial overgrowth: increased urine turbidity, altered pH
(increased pH if urease-producing bacteria are present or decreased
pH if bacteria use glucose to form acidic metabolites), decreased
concentration of chemicals that may be metabolized by bacteria
(e.g., glucose, ketones), increased number of bacteria in urine
sediment and therefore altered urine culture results
ii. Increased urine pH: may occur due to loss of carbon dioxide or
bacterial overgrowth; can cause false positive dipstick protein
reaction, degeneration of cells and casts, and alteration of the type
and amount of crystals present.
Fecal collections and results
a. Always use fresh feces. Older feces may contain eggs, oocysts, and larvae
that have developed beyond their diagnostic stage.
b. Using 1 gram sample increases fecal egg recovery.
c. Freezing will kill or damage eggs/parasites.
d. Free living mites and grain mites: non-pathogenic
e. Rhabditiform larvae: free living nematode, old sample with larvated
hookworm eggs, strongyloides. Later two are pathogenic.
f. Coprophagy of other animals feces results in “parasites” that “pass
through” i.e. non-pathogenic for dog or cat so no treatment necessary: Eimeria
(coccidia of rabbits, rodents), Strongyle eggs (horses), Anoplocephala
(tapeworm of horses, deer, rabbits), Nematodirus (trichostrongyles of
ruminants).
Sensitivity and Specificity
a.
Sensitivity: Out of 100 truly infected animals, how many will test positive with
the test? If 98/100 test positive, the sensitivity is 98%
b.
Specificity: Out of 100 truly NOT infected animals, how many will test negative
with the test? If 98/100 test negative, the specificity is 98%.
c.
Ideal test has 100% sensitivity and 100% specificity: no ideal test exists.
d.
Good screening tests have very high sensitivity to allow you to detect all
diseased individuals. Downside: expect to see some false positives.
e.
Good definitive diagnosis test has a high specificity. Disease prevalence and the
population that you test are important considerations!
THE WHITE BLOOD CELL LINE
Linda Shell, DVM, DACVIM (Neurology)
Veterinary Education and Consulting
1. General
a. Base interpretation on absolute numbers not percentage
b. The significant white cell indicators of inflammation are a neutrophilic left
shift, monocytosis, and persistent eosinophilia, either alone or in any
combination. Typically, will see > 1000 bands if inflammation is clinically
significant.
c. Leukocytosis as a response to inflammation or infection is more dramatic
in the dog than in the cat
d. In the dog, neutrophil counts may exceed 40,000 cells /µl in severe disease,
whereas in cats, counts rarely exceed 30,000 cells / µl
2. Leukocytosis
a. Inflammation
i. Peracute: Leukopenia mostly because of neutropenia
ii. Acute: leukocytosis with a neutrophilia and increased numbers of
immature granulocytes.
1. Regenerative left shift: increased neutrophils and
immature granulocytes/bands in moderate numbers.
Generally regarded as a proper early response to an
inflammatory reaction. The extent of the left shift (how
immature the cells are) will indicate the severity of the
disease.
2. Degenerative left shift: occurs when immature
granulocytes/bands are nearly equal in number to
segmented neutrophils. WBC count may be normal or low.
3. Degenerative right shift: Immature granulocytes/bands
exceed segmented neutrophils. Total WBC count may be
normal or slightly increased. Need to rule out sepsis.
iii. Chronic: Normal total WBC count with monocytosis. As storage
pool in marrow becomes exhausted, there may be a leukopenia
with left shift (degenerative left shift)
b. Steroid Effects: neutrophilia, monocytosis, eosinopenia, lymphopenia
c. Physiologic leukocytosis: excitement, seizures, exercise, tachycardia;
persists only as long as the cause persists.
d. Leukemoid reaction: Reactive leukocytosis indicates extremely high
leukocyte count or an extreme left shift to the extent where it resembles a
leukemia. This change typically involves the neutrophils, but may on
occasion occur in other cell lines such as lymphocytes or eosinophils.
3. Neutrophilia
a. Any acute or chronic inflammation
b. Physiologic: stress, corticosteroids
4. Neutropenia
a. Sepsis or endotoxemia
b. Infections: ehrlichia, parvovirus, FIV, FeLV, panleukopenia, ICH
c. Cyclic hematopoiesis (collies)
d. Bone marrow disease: infections, toxins, neoplasia
e. Drugs: estrogens, phenobarbital, methimazole, TMS, many others
5. Lymphocytosis
a. Physiologic: cats that are afraid or excited
b. Reactive: viral and rickettsial infections (Ehrlichia esp.)
c. Chronic antigenic stimulation
d. Acute or chronic lymphoid leukemia
e. Post vaccinal in young dogs and cats
f. Addison’s disease
6. Lymphopenia
a. Indicator of stress or severe viral infections
b. Common to see this in old cats.
7. Monocytosis
a. Indicates demand for phagocytosis and a stimulus for macrophage
production
b. Common feature of chronic inflammatory conditions but can also occur
acutely
c. Corticosteroids
d. Fungal infections
e. Monocytic leukemia rare
8. Eosinophilia
a. Parasites: external or internal, esp. heartworms
b. Allergic or hypersensitivity conditions that are IgE mediated: feline
asthma, atopy, flea/food allergy
c. Inflammation in tissues that have lots of mast cells (lungs, skin, intestine,
uterus) Ex: eosinophilic IBD, pemphigus foliaceus, PIE
d. Eosinophilic leukemia
e. Hypereosinophilic syndrome in cats
f. Pulmonary infiltrate with eosinophilia (PIE) in dogs
g. Neoplasias
9. Basophilia
a. As for eosinophilia. Heartworms esp.
b. Systemic mast cell tumors
c. Basophilic leukemia (rare)
d. Neoplasia
10. WBC morphology
a. Atypical lymphocytes: Describes varied changes within lymphocytes e.g.
eosinophilic cytoplasm, changes in nuclear texture, angular nuclei,
prominent nucleoli, large abundant cytoplasm. Nonspecific indicator of
disease (seen in increased numbers in both infectious and noninfectious
conditions). Significance remains controversial.
b. Reactive lymphocytes: Cells with dark blue cytoplasm and darker nucleus.
Noted in chronic infection. Not abnormal at all, but simply reflect normal
function of the immunocytic system
c. Döhle bodies: Normal if seen in low numbers of neutrophils in cats.
Cytoplasmic inclusion of ribosomal matter. R/O chronic bacterial
infection and viral diseases. Associated with toxic neutrophils.
d. Toxic neutrophils: Dohle bodies, vacuoles, foamy cytoplasm
i. +1 toxicity: Döhle bodies
ii. +2 toxicity: Döhle bodies and diffuse cytoplasmic basophilia that
results from the retention of cytoplasmic RNA
iii. +3 toxicity: all of above in addition to foamy cytoplasmic
vacuolation
iv. +4 toxicity: all of above in addition to giantism and/or nuclear lysis
e. Hypersegmented neutrophils: 6+ lobes of nucleus; chronic infection,
steroids, pernicious anemia
f. Vacuolization: septicemia, toxic diseases, storage diseases, artifact if
sample storage is prolonged
g. Toxic granulation: large cytoplasmic basophilic granules; infectious
diseases
h. Sideroleukocytes: Neutrophils or monocytes with hemosiderin inclusions
or stainable iron in cytoplasm. Seen occasionally in dogs with IMHA or
intravascular hemolysis.
i. Smudge or basket cell: degenerative WBC's that have ruptured. May be an
artifact if blood is held too long before making the smear; also associated
with leukemia. Other degenerative changes: pyknosis, karyolysis and
karyorrhexis
11. Platelets
a. Adequate numbers of platelets essential for formation of hemostatic plug.
b. Life span: 5-7 days in dog; fewer days for cat
c. Clinical signs of low platelets: petechial or ecchymotic hemorrhage
d. Platelet number vs platelet estimate
e. Platelet clumping big problem in many samples. Gives rise to falsely low
platelet count (if lab reports clumping yet platelet estimate is okay, assume
platelet numbers are okay as long as there are no signs of bleeding).
f. Decreased platelets:
i. Decreased production: bone marrow problem
ii. Increased use: DIC, blood loss (usually mild thrombocytopenia)
iii. Increased destruction: Immune mediated (primary or secondary to
infectious agents, drugs, neoplasia, other immune mediated dz).
iv. Increased sequestration: Spleen and liver. Can occur in healthy
animals as well as when these organs are involved in diseases.
v. Drugs that can cause low platelets: many
vi. Sampling error: Decreased platelets can occur with overfilling of
EDTA tube and inadequate mixing.
g. Increased platelets:
i. Essential thrombocythemia: myeloproliferative disease; rare.
ii. Secondary (reactive) thrombocytosis: neoplasia, GI disease,
Immune mediated diseases, blood loss, fractures, drugs (steroids,
cancer drugs), post splenectomy
iii. Physiologic thrombocytosis: transient due to exercise or
epinephrine release
iv. Usually don’t get too concerned until platelet count reaches
800,000 and is repeatable: start looking for causes of secondary
thrombocytosis if no other cell line is abnormal and if platelets
appear normal in appearance.
THE RED BLOOD CELL LINE
Linda Shell, DVM, DACVIM (Neurology)
Normal PCV/hematocrit = 37-55 % (dog); 30-45 % (cat); some breed variation. Value
affected by RBC mass and intravascular fluid volume.
Anemia Approach
1. Goal: Identify anemia as either regenerative or non-regenerative.
2. Regenerative: Bone marrow is responding appropriately to a reduction in
RBCs.
a. Common causes: blood loss and hemolysis.
b. Bone marrow response may take 2-3 days in cats; 4-5 days in dogs
3. Non-regenerative: Bone marrow is not responding appropriately to reduction
in RBCs
a. Causes: primary bone marrow problem (pancytopenia may be present),
chronic blood loss (iron deficiency), anemia of chronic disease.
chronic renal failure.
b. Some hemolytic anemias remain non-regenerative
4. Clues to look for: total protein, MCV, RBC morphology (see below)
5. Reticulocyte count is best way to determine regeneration
a. Immature RBCs released from bone marrow in response to anemia
b. More accurate count with New Methylene Blue stain
c. Normal reticulocyte counts are between 1-2 % or less than 60,000 for
the dog or 45,000 for the cat.
d. Regenerative anemias have corrected retic count of more than 2% and
much greater than 60,000 for dogs or 45,000 for cats.
e. Cats have 2 forms of reticulocytes: aggregate (counted), punctate
f. Ways that reticulocyte counts are reported:
i. Total count: > 60,000 regenerative in dog; > 45,000 for cat
ii. Uncorrected percentage: Percentage of reticulocytes with no
regard for the severity of the anemia. Ex: a 3 % uncorrected
value is not regenerative for an anemia of 15 % but could be
slightly regenerative for anemia of 32 %.
iii. Corrected percentage: The more severe the anemia, the more
the regenerative the response should be; corrected reticulocyte
% is more accurate than uncorrected %. Formula to correct
count for degree of anemia: *normal PCV = 45 % dogs; 37 %
cats
Corrected retic % = uncorrected retic % x
patient's PCV
normal PCV *
6. Evaluate PCV in conjunction with total protein
Condition
PCV
Total protein
Dehydration or hemoconcentration
Hemolysis
Decreased RBC production
Blood loss
increased
decreased
decreased
no change for 8-12
hours; then decreased
increased
no change
no change
no change for 8-12
hours; then decreased
7. Look at MCV (mean corpuscular volume), indicator of RBC size/volume,
and MHHC (mean corpuscular hemoglobin concentration), indicator of
hemoglobin concentration
a. Macrocytosis (increased MCV)
i. Reticulocytosis=regenerative anemias
ii. FeLV associated in cats
iii. Congenital in poodles
iv. Folate or B 12 deficiency
b. Microcytosis (decreased MCV)
i. Iron deficiency (chronic blood loss)
ii. Portosystemic shunt
iii. Congenital in Akitas
c. Hypochromic (decreased MCHC)
i. Reticulocytosis
ii. Iron deficiency
d. Hyperchromic (increased MCHC) not significant
8. RBC morphology
a. Polychromasia (bluish tint) and anisocytosis (variation in RBC size),
stomatocytes (RBC with slit-like center opening): suggest regeneration
b. Howell Jolly bodies: Basophilic nuclear remnant. Common in feline
regenerative anemias.
c. Poikilocytes: generic name for any abnormally shaped cell. Could also
occur due to poor smear technique.
d. Acanthocytes: (Spur cell): irregular projections from surface of RBC.
Can be seen with hemangiosarcomas, liver disease, iron deficiency,
DIC, fat malabsorption, metabolic lipid disorders.
e. Echinocytes (Burr cell): regular projections from surface of RBC.
Could be due to drying artifact or too much anticoagulant (under-filled
EDTA tube)
f. Leptocytes: RBC with an increase in membrane surface relative to cell
volume; appear as target cells, codocytes. Can be associated with
g.
h.
i.
j.
k.
portosystemic shunts, splenectomy, iron deficiency, hypothyroidism,
liver disease.
Basophilic stippling: bluish granular bodies on the surface of the RBC;
seen in lead poisoning in small animals
Siderocytes: Iron precipitates in RBCs
Heinz bodies: Round structures within the RBC representing denatured
hemoglobin. Small number may be normally present in cats. Causes:
Oxidative anemias (onions, acetaminophen in cats, benzocaine, DL
methionine, phenacetin, methylene blue). May also find eccentrocytes
(damaged erythrocytes, which have a rim of membrane that has
fastened together). Cats form Heinz bodies and dogs form
eccentrocytes.
Ghost cells: RBC “ghosts”; suggest IMHA
Spherocytes: RBCs that have a smaller diameter and deeper color
because they are spherical and thicker.
i. Moderate to large number of true spherocytes = IMHA
ii. Cats: Normal RBCs lack central pallor and therefore no one
can consistently identify spherocytosis in the cat
9. Nucleated RBCs
a. Appropriate in severe anemia if accompanied by reticulocytes
b. Increased nRBCs without reticulocytes or in nonanemic states suggests
bone marrow disease or cardiopulmonary dysfunction
c. Are not appropriate in absence of reticulocytes and are not necessarily
a sign of regeneration
d. Causes: regenerative anemias, lead poisoning, extramedullary
hematopoiesis, and bone marrow disease.
e. Sources: bone marrow, spleen, lungs
10. Saline slide agglutination testing vs. Direct Coomb’s test
a. Saline slide agglutination: Autoagglutination is the equivalent of a
positive Coombs test indicating antibodies on the RBC
i. Put 1 drop of blood and 1-2 drops of saline on slide; mix well.
ii. Look for agglutination (clumping) grossly and microscopically.
iii. Rouleaux should disperse with saline; autoagglutination won’t
disperse with saline.
iv. Autoagglutination is estimated to occur in 50-65 % of cases
b. Direct Coomb’s test: A positive Coomb's is not diagnostic for IMHA
but in the cases of anemia, it provides evidence of IMHA.
i. Detects AB or complement bound to RBC membranes.
ii. Must use specific canine immunoglobulin.
iii. False negatives due to low AB levels, elution of AB from cell
membrane, technical errors, improper AG:AB ratio, or
glucocorticoid treatment of longer than 1 week duration.
iv. False positives can occur with nonspecific antibody coating on
RBC surface, prior blood transfusions, and in vitro binding of
complement
v. Sensitivity and specificity is highly variable. Sensitivity has
been reported to vary between 30-100 %.
11. Red blood cell parasites: Babesia (lightly basophilic), Mycoplasma or
Hemobartonella (basophilic rod to coccoid to chains), Cytauxzoon (blue rings,
matchstick, safety pin, Maltese cross pin), Ehrlichia, Anaplasma.
a. Make fresh blood smear and look for organisms
Polycythemia Approach
1. Strictly speaking polycythemia implies a rise of all blood cell counts above
normal. However the occurrence of leukocytosis and thrombocytosis along with
erythrocytosis is exceptionally rare in animals.
2. Forms of polycythemia
a. Relative: dehydration. PCV and TP drop with fluid therapy.
b. Absolute or true
i. Primary polycythemia or polycythemia vera: Erythropoietin
independent. Myeloproliferative disease.
ii. Secondary polycythemia
1. Appropriate: Secondary to hypoxia, cardiopulmonary
disease like right to left shunts, COPD
2. Inappropriate: Erythropoeitin producing tumors, renal
tumors
3. Hematocrit > 55%; true erythrocytosis/polycythemia is usually > 65 %.
4. Greyhounds, other sighthounds, and some Dachshunds normally have higher red
blood cell counts and hemoglobin levels than other dogs; and should not be
diagnosed as polycythemic until hematocrit (Hct) levels are greater than 65%.
5. Clinical signs: pu/pd, erythema, vomiting, diarrhea, seizure, organomegaly
6. Serum erythropoietin: Currently not available. Problem is that EPO
concentrations in polycythemic patients may overlap with the normal range and
there is lack of specific assays for dog/cat.
Polycythemia
Goes away with fluid therapy
Relative polycythemia
Does not go away with fluid therapy
Absolute polycythemia
Rule out secondary causes:
Cardiovascular dz
Pulmonary dz
Renal dz
Cushing’s dz
To get to primary cause of
polycythemia vera (myeloproliferative dz)
THE SERUM CHEMISTRIES
Linda Shell, DVM, DACVIM (Neurology)
I.
II.
III.
Young vs older patients
a. Young animals tend to have BUN and total protein values lower than
established norms and ALP, TBIL, and phosphorus values higher than
established norms.
b. Similarly, older animals tend to have physiological decreases in BUN,
GGT, creatinine, and albumin and increases in total protein and potassium
Albumin
a. Normal values
i. Healthy adult = 3.5 g/dL
ii. 1.5 g/dL or less = edema, ascites, and pleural effusion
iii. High normal or above normal values = dehydration, lipemia
iv. Trends in decreasing values are key to detecting causes of
hypoalbumemia early
b. Causes of low albumin
i. Decreased production by liver (70 + % of liver compromised)
1. Decreased or low normal cholesterol, BUN
2. Liver enzymes may be normal or below normal
3. MCV: sometimes microcytosis
4. Globulins: often normal to elevated
ii. Increased loss
1. Glomerular diseases: Glomerulonephritis, amyloidosis
a. May or may not have azotemia
b. May or may not have pu/pd or low urine specific
gravity
c. If proteinuria found, check urine protein creatinine
ratio
2. Gastrointestinal:
a. May or may not have GI signs.
b. Globulins may be decreased too.
c. Causes: inflammatory bowel disease (IBD),
neoplasia (particularly lymphosarcoma),
histoplasmosis, lymphangiectasia, GI parasites,
Addison’s disease, pythiosis
3. Third spaces: Vasculitis, serosal inflammation within
cavities
a. Addison’s disease: related to GI loss?
Globulins
a. Elevated: Protein electrophoresis
i. Monoclonal: lymphoma, multiple myeloma, ehrlichiosis
ii. Polyclonal: ehrlichiosis, FIP, many other infectious/inflammatory
diseases
b. Low: Immunodeficiency, GI loss concurrently with albumin
IV.
Calcium:
a. General information
i. Present in mainly 2 forms: 50 % ionized and 40 % protein bound
(90 % is bound to albumin)
ii. Low albumin = low calcium
iii. Adjust the serum calcium concentrations for hypoalbuminemia:
adjusted Ca = measured Ca - albumin + 3.5
iv. Adjustment formula may not be accurate
v. GI tract helps to regulate plasma Ca concentration
vi. Intestines can adapt to low or high calcium diets by increasing or
decreasing efficiency of absorption
vii. Hypo or hyper calcemia: First step is to repeat the test. Verify!
viii. If verified, do ionized calcium next (often done in conjunction with
PTH, PTHrp)
ix. iCa sample has to be collected anaerobically.
x. True hypercalcemia is associated with elevations in iCa levels.
Hypercalcemia without elevated iCA is not a significant clinical
concern
b. Hypocalemia with normal albumin (3.5): clinical signs can occur at values
< 6.5 mg/dl
i. Primary hypoparathyroidism is main differential diagnosis that
results in severe hypocalcemia
ii. Presumptive diagnosis of primary hypoparathyroidism based on
clinical signs, profound hypocalcemia with no other attributable
cause, and a favorable response to calcium and vitamin D
iii. Other less common causes: vitamin D deficiency, pancreatitis,
ethylene glycol toxicity, pseudo-hypoparathyroidism
c. Hypercalemia: > 12 mg/dl
i. Causes: dehydration, neoplasia, chronic renal failure,
hypoadrenocorticism, young animals (normal to a degree),
hypervitaminosis D (rodenticide, supplement), fungal diseases?
ii. If nothing obvious on examination: iCa, PTH PTHrp
Disease
Primary
Hyperparathyroidism
Renal secondary
hyperparathyroidism
Hypercalcemia of
malignancy
PTH
High normal or
increased
Increased
iCA
High
PTH rp
Normal/Low
Low
Normal/Low
Low
High
High
V.
Liver enzymes:
a. Serum hepatic enzyme activities do not reflect hepatic function. (Tests of
liver function: total bilirubin, bile acids, ammonia, albumin, globulin, and
cholesterol)
b. They do reflect either the integrity of the hepatocyte membrane or the
patency of the biliary system.
c. Severe hepatic dysfunction can occur in the face of normal enzyme
activities. Look for “quiet” liver enzymes as well as elevated ones!
d. Liver cells compartmentalize the work so that damage to one zone of the
liver may not impact all liver functions.
e. Leakage enzyme: ALT, AST, GLDH, and SDH
f. Cholestatic enzymes: ALP and GGT
g. GGT sources: pancreatitis, cholestasis, c-steroids, anticonvulsants.
VI.
Serum Alanine Aminotransferase (ALT)
a. Most liver-specific enzyme in the dog and cat, but can be elevated with
severe muscle disease
b. Found only in the cytoplasm of hepatocyte. Serum level increases when
there is increased permeability of the hepatocyte membrane, resulting in
leakage from the hepatocyte.
c. Mild to moderate increases seen in any disease that results in hypoxia
(right-sided heart failure, severe anemia, severe pulmonary disease,
endotoxemia, sepsis, status epilepticus).
VII.
Serum Aspartate Aminotransferase (AST)
a. High AST activity in skeletal muscle and red blood cells as well as liver
b. When AST is more elevated than ALT, consider muscle or RBC
isoenzymes rather than hepatic source.
VIII. Serum Alkaline Phosphatase (ALP)
a. Found in many tissues, but only the isoenzymes found in the liver and
bone are important.
b. Bone ALP increases with osteoblastic activity (thus elevated in young,
growing animals), bone tumors, osteomalacia, & hyperparathyroidism
c. Produced by cells lining the bile canaliculi, so production is induced by
biliary obstruction. Since there is inability to excrete the enzyme through
the biliary system, serum increase seen.
d. Often elevated in primary hepatocellular disease as a result of swelling of
hepatocytes and subsequent intrahepatic cholestasis.
e. Steroid-induced isoenzyme of ALP is produced in the liver, but is a
separate entity from that induced by biliary obstruction.
f. Cat: half-life of ALP is much shorter in the cat (6 hours versus 3 days).
Less ALP is produced in biliary obstruction because the cat liver contains
only one third the concentration of ALP per gram of liver than that of the
dog. Thus, even mild elevations of ALP activity in the cat are indicative
of marked hepatobiliary disease.
IX.
Serum Gamma Glutamyl Transpeptidase (GGT)
a. Parallels increases in ALP usually.
b. Seen with biliary stasis and steroid hepatopathy; drugs.
c. Serum activity of GGT may increase earlier in biliary disease than does
ALP activity.
d. Doesn’t seem to be very useful overall.
X.
Bilirubin (BR)
a. Formed from breakdown products of red blood cells, from other
hemoproteins, and from other enzymes such as the cytochromes
b. Indicator of liver dysfunction or hemolytic disease. Check PCV!
c. Must have considerable hepatocellular disease or increased BR load
(hemolysis) to result in hyperbilirubinemia because the liver's reserve
capacity for BR processing is up to 30 times the normal BR load.
d. Total bilirubin concentrations above 2 to 3 mg/dl are clinically detectable
as jaundice
e. Icteric serum and bilirubinuria is first detected when the serum bilirubin
concentration is between 0.6 and 1.0 mg/dl
f. Dog has a very low renal threshold for bilirubin excretion, so it’s common
to find+1 to +3 bilirubin in a concentrated urine.
g. Cat has a high renal threshold for bilirubin excretion, and any amount of
bilirubin in the urine is abnormal
XI.
Phosphorus
a. Decreased: Value probably not important until 1.0 or lower (may cause
RBC lysis, decreased WBC and platelet functions, CNS signs)
i. Causes: malabsorption, phosphate binders, renal tubular defects,
Cushing’s, mannitol, TPN, respiratory alkalosis, vitamin D
deficiency, eclampsia, Na bicarb
b. Increased:
i. Causes: postprandial, young dogs, healing bones, hemolysis, soft
tissue trauma, rhabdomyolysis, osteolysis, vitamin D toxicity,
azotemia, hypoparathyroidism, hyperthyroidism (cat), acromegaly,
tumor lysis
XII.
Potassium
a. Increased: drugs (NSAIDs, Beta blockers, ACE inhibitors, heparin,
potassium sparing diuretics, prostaglandin inhibitors), urinary outflow
obstruction, acidosis, whipworms, severe GI diseases in dogs, severe liver
disease.
b. Pseudohyperkalemia: thrombocytosis (usually 1 million +), leukocytosis
(usually 100,000 +) and hemolysis or RBCs
c. Breeds that have high K concentrations in RBCs: Akitas, English springer
spaniels, neonates
d. Decreased: prolonged anorexia, deficient diet, drugs (furosemide, thiazide
diuretics, penicillin derivatives, amphotericin B), urinary loss (CRF in cats,
post-obstructive diuresis, primary hyperaldosteronism, renal tubular
acidosis), GI losses via diarrhea or vomiting, alkalemia (esp. during
treatment of DKA), hypokalemic periodic paralysis (rare but reported in
Burmese cats)
e. If re-checking K, use plasma (heparinized sample) to avoid the clotting
process which might release K from cells.
XIII. Sodium
a. Hyponatremia: Addison's, diabetes, GI loss, third spacing, syndrome of
inappropriate ADH release, renal disease, nephrotic syndrome, edema,
psychogenic polydipsia, antidiuretic drugs, myxedema coma of
hypothyroidism, uroabdomen, pleural or peritoneal effusion, burns,
diuretics
b. In PU/PD cases: If polydipsia (from psychogenic polydipsia,
hypothalamic injury, salt intake, hypercalcemia, hyperreninemia,
excessive exercise, hot weather) is the cause of the polyuria, one would
expect to find normal to low serum sodium and low serum osmolality.
c. Hypernatremia:
i. Salt gain: ingestion of sea water, paint balls, home-made playdough that has a high sodium content, or other high salt containing
substances, use of saline emetics, IV hypertonic fluids or sodium
bicarbonate, hyperaldosteronism (expect hypokalemia &
hypertension), and hyperadrenocorticism
ii. Water loss: Pure water loss is associated with pituitary diabetes
insipidus, nephrogenic diabetes insipidus, heatstroke, fever,
inadequate access to water, burns, and essential hypernatremia in
which there is a failure of the hypothalamic osmoreceptors to
respond to an increase in serum osmolality. Hypotonic water loss
is associated with diarrhea, vomiting, oral hyperalimentation, or
osmotic diuresis (diabetes mellitus, diuretics, chronic and acute
renal failure, IV glucose, mannitol, or urea)
XIV. Cholesterol
a. Decreased: liver disease, maldigestion/malabsorption, PLE, azathioprine
b. Increased: hypothyroidism, hyperadrenocorticism, nephrotic syndrome,
diabetes mellitus, cholestatic disease, idiopathic hyperlipidemia
XV.
Triglycerides
a. Decreased: PLE, ascorbic acid
b. Increased: Post prandial, idiopathic hypertriglyceridemia, acute
pancreatitis, cholestasis, diabetes mellitus, hypothyroidism,
hyperadrenocorticism, nephrotic syndrome
XVI. Amylase and lipase
a. Amylase sources: salivary glands, small intestine, and pancreas
b. Lipase sources: pancreas, gastrointestinal tract. Excess lipase is easily
filtered through the kidneys so lipase levels tend to remain normal in early
stages of pancreatic disease.
c. Pancreatitis: The sensitivity/specificity for amylase and lipase have been
62%/57% and 73%/55% respectively. Possible explanations for the poor
sensitivity may be enzyme depletion or disruption of synthesis, which may
explain the lack of correlation with between severity of signs and enzyme
values. Possible explanations for the poor specificity include
extrapancreatic sources of the enzymes.
XVII. BUN
a. Renal azotemia does not occur until 75% of nephrons are no longer
functioning; therefore, by the time BUN is only mildly increased, there
has been a significant decline in renal function
b. Increased:
i. Pre-renal: dehydration (urea is insoluble and must be excreted in
large amount of water), high protein diet, gastrointestinal bleeding
ii. Renal: acute and chronic renal failure
iii. Post renal: urinary tract obstruction, uroabdomen
c. Decreased: neonates, diuresis, liver failure, low protein diet (UD), renal
medullary washout, urea cycle enzyme deficiency (rare)
XVIII. Creatinine
a. Formed during normal muscle metabolism; thus the amount of creatinine
is affected by the animal's total muscle mass.
b. Better indicator of GFR than urea nitrogen as it is not affected by renal
tubular reabsorption, diet, protein catabolism, or hepatic function
c. Decreased: poor body condition
d. Increased: Heavily muscled dogs; pre-renal azotemia, renal failure
XIX. BUN:Creatinine ratio
a. High BUN to creatinine ratio suggests an azotemia is pre-renal.
b. Ideally we want it to be about 10-15:1
c. May help in monitoring CRF patients.
d. If you have a stable CRF patient eating a certain diet, the BUN creatinine
ratio should stay pretty stable. If it increases despite no diet change, it
might suggest GI bleeding or dehydration.
THE URINALYSIS
Linda Shell, DVM, DACVIM (Neurology)
Urine Specific Gravity (SG)
1. Measure of the ratio of a volume of urine to the weight of the same volume of
distilled water at a constant temperature.
2. Indicator of concentration of dissolved materials in the urine
3. Indicator of kidney function. Alteration in the concentrating ability of the kidneys
is an early indicator of renal tubular damage.
4. Measure via refractometer, not dipstick pad
5. Always good idea to get urine sample at same time of chemistries/CBC.
Dipstik Information
1. Interpret in conjunction with urine sediment findings and collection method.
2. Note the expiration date and general condition of strips; use well-mixed, room
temperature urine samples
3. Leaking from one pad to another can be a problem
4. Protein:
a. The urine dipstik is the most readily available test of urine protein (detects
albumin and globulins) but is also the least reliable. Both false positives and
false negatives occur.
b. Sensitivity and specificity of the urine protein dipstik: as low as 54% & 69%,
respectively, in the dog and 60% & 31%, respectively, in the cat.
c. Normally present in very low quantities in the urine (at or below the limit of
sensitivity of the urine reagent strips).
d. A trace or 1+ reaction is considered normal with a specific gravity greater
than 1.035.
e. Highly alkaline urine (>8) can produce a false positive result
f. Transient, physiologic, or nonpathologic proteinuria has many causes:
strenuous exercise, fever, seizures, and venous congestion of the kidneys;
rarely of any significance.
g. Persistent proteinuria is usually due to renal glomerular disease:
glomerulonephritis or amyloidosis.
5. Glucose:
a. Glucose is freely filtered by the glomerulus and is reabsorbed in the proximal
tubules.
b. Renal threshold for exceeding the tubular reabsorption occurs at blood
glucose levels of 180 mg/dl in dogs and 280-300 mg/dl in cats.
c. Transient and stress-induced hyperglycemia in the cat can result in glucosuria.
d. Cats with cystitis may give a false positive reaction.
e. Glucosuria warrants automatic look at serum glucose because the most
common cause of glucosuria is hyperglycemia.
f. If the blood glucose level is normal, evaluate another urinalysis.
6.
7.
8.
9.
g. Causes of glucosuria with concurrent hyperglycemia: diabetes mellitus,
pancreatitis, hyperadrenocorticism
h. Causes of glucosuria without concurrent hyperglycemia: proximal renal
tubular dysfunction (e.g., Fanconi's syndrome), fluid therapy with dextrose,
epinephrine release, antibiotics (gentamicin, cephalosporins, amoxicillin,
outdated tetracycline), chemicals (lysol, maleic acid), heavy metals (lead,
mercury, cadium, uranium), xylazine in cats, phenothiazine, corticosteroid.
*Chicken jerky treats* made in China!
Ketonuria:
a. Ketonuria without glucosuria: excessive lipid catabolism (starvation, fasting,
anorexia), high fat diet, or possibly impaired liver function.
b. Ketonuria without glucosuria in an otherwise normal patient is not likely
significant.
c. The stix detects acetoacetate or a combination of acetoacetate and acetone,
depending on the brand.
Bilirubinuria:
a. The stix and tablet methods detect conjugated bilirubin. Test pad may give
false negative results if exposed to air or light for long periods of time or if
urine has dark color (hemoglobinuria) which masks color changes on stix.
b. Normal cats do not have bilirubinuria: a positive reaction is significant.
c. To be excreted into the urine, bilirubin must first be conjugated. This
normally occurs in the liver, but the dog’s renal tubular cells are also capable
of conjugating bilirubin.
d. Dogs have a low renal threshold. Therefore, trace, 1+ or 2+ results are
frequent in normal dogs with high urine specific gravities.
e. Common causes of bilirubinuria: hepatic disease, posthepatic bile duct
obstruction, and hemolytic diseases.
f. Cats: Bilirubinuria precedes bilirubinemia, and bilirubinuria is always
abnormal in cats.
Occult blood:
a. Detects myoglobin, hemoglobin, and/or intact red cells. Normally five or
fewer RBCs per hpf occur in normal urine (proestrus is exception).
b. Normally myoglobin and hemoglobin are not present in urine.
c. Hematuria is most common cause of positive occult blood and can be
confirmed if RBCs are found in urine sediment.
d. If no RBCs are found in sediment, look at the patient's hematocrit and plasma
color: if plasma is red or pink (and proper venipuncture technique was
performed), then hemoglobinuria is present and indicates hemolytic anemia.
e. Myoglobin is a protein found in muscle. Myoglobinuria is usually seen in
dogs with rhabdomyolysis. Serum creatinine kinase should be elevated.
pH: Normal 6.0-7.5; some list a wider range of 5.0-8.5
a. Run off from adjacent pads can affect pH results.
b. Alkaline urine: recent meal, ingestion of alkali (bicarbonate or citrate), UTI
with urease-producing bacteria, renal tubular acidosis, vegetable source diet,
alkaline fluid therapy, and metabolic and respiratory alkalosis.
c. Acid urine: meat/milk protein diets, catabolic state, acidic fluid therapy.
d. Heuter et al. (1998, JAVMA, 213:996-998) found that pH determinations by
urine dipsticks were not accurate enough to predict the likelihood of struvite
precipitation. In this study, a urine dipstick measurement of pH 7.0 was
associated with a pH meter measurements that ranged from 6.2 to 8.2. In cats,
the urine dipstick measurement was consistently lower than that of the pH
meter, while in dogs there was no consistent direction of error.
e. Use a pH meter if pH measurements are critical in the evaluation of a patient.
10. Urobilinogen: Useless in veterinary medicine because reagent strips can’t
accurately measure it. If bile flow is re-established after bile duct obstruction, a
marked increase in urine urobilinogen occurs.
11. Leukocytes:
a. Insensitive and produces false negative results in the presence of pyuria.
b. False positives are a frequent occurrence in feline urines.
12. Nitrite:
a. Designed to detect the presence of gram negative bacteria, but has very
limited value for detection of bacteriuria.
Sedimentation Information
1. Generalizations:
a. Sediment of urine collected by cystocentesis or catheterization may
contain RBCs from trauma
b. Voided samples often contain cells/bacteria from genital tract, caudal
urethra
c. A high urine SG will cause cells in urine to shrink while low SG can cause
cell swelling to occur.
d. Centrifuge 5-10 mls at low speed for about 5 minutes; pour off supernatant
except for about 1 ml
e. Resuspend sediment in 1 ml of supernatant. Put drop on microscope slide.
Cover slip. Exam on low power for casts and epithelial cells; high power
for cells and bacteria.
f. Casts and crystals are best visualized on the unstained preparation; cells,
organisms and sperm are best seen on the stained preparation.
2. Cells
a. RBCs: RBCs crenate (shrivel) in concentrated urine; swell & lyse
(appearing as colorless rings or ghost cells) in dilute urine.
b. WBCs: Greater than 5/hpf indicates inflammation. Degenerate in old urine
and may lyse in hypotonic or alkaline urine.
c. Squamous epithelial cells are common & are the largest of cells. Thin/flat
with angular borders & large nuclei. These originate in distal urethra,
vagina, vulva, or prepuce.
d. Transitional epithelial cells originate in the bladder, ureters, renal pelvis,
and proximal urethra. They may occur in clumps, especially if the urine
was collected by catheterization. Increased number suggests inflammation.
e. Renal epithelial cells originate in renal tubules; tubular degeneration.
3. Crystals: Can be within normal limits
a. Identify crystals in uncontaminated fresh urine sediment, preferably at
body temperature.
b. Formation of crystals depends on the amount of the substance in the urine,
the solubility of the particular crystal type, the pH & specific gravity.
c. Acidic urine crystals: Calcium Oxalate, Amorphous Urates, Sodium
Urates, Uric Acid, Calcium Sulfates, Cystine (Newfoundlands)
d. Alkaline urine crystals: amorphous phosphates or struvite
e. Struvite (triple phosphate): infection-induced (alkaline) vs sterile (slightly
acid or higher)
f. Calcium oxalate: check for hypercalcemia, steroids
g. Urate: Dalmations, liver disease
h. Bilirubin: Normal dogs can have slight bilirubinuria, especially in
concentrated urine
i. Medication crystals: sulfonamides
4. Casts:
a. Cylinders of material formed from protein matrix secreted by the renal
tubules.
b. Although a few casts may be seen in normal urine, the presence of casts in
urine samples usually indicates tubular damage.
c. Casts are fragile, easily destroyed with improper preparation of sample
d. Casts dissolve in alkaline urine, so identification and quantification is best
done with fresh urine samples.
e. Hyaline casts lack cells or cell debris; least indicative of renal tubular
damage.
f. Granular casts have granules of degenerate WBC, RBC, and/or epithelial
cells, described as coarse or fine. Indicate tubular degeneration & severe
kidney damage.
g. Waxy casts are wide and smooth, with sharp margins and blunt ends.
h. Fatty casts contain high amounts of lipid material incorporated into the
protein matrix of the cast.
5. Bacteria:
a. Bacturia without pyuria usually indicates some error.
b. The error could be contamination of an old urine sample with bacterial or
yeast etc. Overgrowth or growth of an organism in one's stain used for
urine sediment is common and can be checked by looking at only the stain.
Bacteria may also come from the surface of the vagina or prepuce.
c. Bacteria on a cystocentesis sample may come from puncturing the bowel.
These bacteria may not grow if sample is cultured because they die
quickly (need anaerobic environment). But these bacteria may be picked
up on a Gram stain.
FECAL TESTS
Linda Shell, DVM, DACVIM (Neurology)
a. Odd Fecal Results
i. Coprophagy of other animals feces results in “parasites” that “pass
through” i.e. non-pathogenic for dog or cat so no treatment
necessary: Eimeria (coccidia of rabbits, rodents), Strongyle eggs
(horses), Anoplocephala (tapeworm of horses, deer, rabbits),
Nematodirus (trichostrongyles of ruminants)
ii. Pathogenic uncommon parasites of dog or cat
1. Spirometra: tapeworm. Southeastern US
2. Alaria: intestinal fluke. Northern US.
3. Capillarid eggs: some capillaria had name change
a. Eucoleus aerophilia (C. aerophilia): bronchioles &
trachea of dogs/cats. Swallowed & passed in feces
b. Eucoleus boehm: nasal capillaria
b. Giardia ELISA
i. Vaccine doesn’t cross react on assays
ii. Gold standards: ELISA (antigen) and DIFA (cysts). If ELISA +
and IFA -, probable false + since IFA give a morphologic
identification.
iii. SNAP Giardia Test compares well with above
1. Detects Giardia cell wall antigens in both cat/dog.
2. IDEXX reports a 95% sensitivity and 99% specificity with
their SNAP Giardia test
3. Anecdotal evidence suggests that some successfully treated
dogs (based on clinical remission) can remain antigen
positive for several months after treatment.
4. "Effective" treatment has historically been measured by
suppression of cyst shedding and does not necessarily
indicate clearance of the organism from the intestinal tract.
It appears that in some pets, Giardia can become part of the
"normal" flora following treatment. Thus SNAP test may
not become negative.
5. Not recommended for follow up testing in asymptomatic
animals
6. Reasons for persistent positive SNAP test:
a. Organism not cleared: subclinical carrier
b. Patient is re-infected: short pre-patent period and
cysts persist in environment
c. False positive: If the probability of the patient
having the disease is virtually non-existent, then the
test result is likely a false positive.
iv. Zn sulfate flotation performs poorly in house and better in lab;
lower sensitivity than ELISA because of intermittent shedding.
Sensitivity improves with 3 samples
v. Giardia testing is currently recommended only for initial diagnosis
of giardiasis. Patients who have been successfully treated, with
resolution of clinical signs, do not need follow-up monitoring
tests unless the owner is immunocompromised and at risk for
zoonotic infection
c.
Tritrichomonas PCR on feces
i. 1 gram (lima bean size) feces (liquid sample preferred) in red top
tube filled half way with rubbing alcohol
ii. Animal must not be on antibiotics
iii. Appears superior to fecal culture: PouchTM TF (Biomed
Diagnostics, San Jose, CA). Pouches are inoculated with 0.05 g of
freshly passed or loop-collected feces; incubated in an upright
position at room temperature (25 C); examined eod using 20 or
40x objective. If no motile trophozoites are detected after 12 days,
the culture is discarded. This method has a detection limit of
>1000 trophozoites/0.05 g feces.
d. Alpha one protease inhibitor (alpha-1 PI) test
i. Plasma protein. Molecular weight similar to albumin
ii. Lost into the gastrointestinal (GI) tract, along with albumin, in
humans and animals with PLE.
iii. Unlike albumin however, alpha-1 PI is not degraded by enzymes in
the GI tract and is excreted in the feces essentially intact
iv. Species-specific assays necessary; canine specific ELISA available
v. Major use seems to be the hypoalbuminemic patient in which you
suspect PLE, but also has concurrent renal or liver disease.
vi. Requires collection of 3 separate, freshly passed fecal samples into
a special tube. Owner must store samples until all are collected.
vii. Does not provide an etiology and is not specific for PLE
viii. Not as nice & easy to interpret as we once thought; increased fecal
excretion of 51Cr is gold standard for diagnosis of GI protein loss
e. Fecal Occult Blood
i. Patient must be on a meat- free diet for 72 hrs prior to collection.
ii. Drugs such as cimetidine may cause false positive test results.
OTHER URINE TESTS
Linda Shell, DVM, DACVIM (Neurology)
1. Sulfosalicylic turbidmetric (SSA) test (also called bumin test)
a. More reliable than the urine dipstick for the detection of proteinuria (both
albumin and globulin).
b. Usually analyzed in a reference lab.
c. The amount of protein that is present in the urine of normal dogs and cats
is below the lower limit of detection for this test.
d. A positive result is interpreted in light of the urine specific gravity.
e. When both urine dipstik and SSA test results are available, the results of
the SSA test should be given greater consideration than those of the urine
dipstik.
2. Microalbuminuria
a. Microalbuminuria refers to increased urine albumin that remains beneath
the detection limit of the urine dipstik protein reaction (urine albumin 0.01
to 0.30 g/L).
b. Prevalence of microalbuminuria has been reported as 15 to 19% in
clinically normal dogs (Gary et al., 2004; Jensen et al., 2001) and 14% in
clinically normal cats.
c. In canine experimental models of progressive glomerular disease, the
prevalence of microalbuminuria was 75-76% and persistent
microalbuminuria preceded the development of overt proteinuria (Grauer
et al., 2002; Lees et al., 2002).
d. Detection of persistent (means multiple test samples are analyzed over
time) microalbuminuria in dogs may aid in early diagnosis of occult
glomerular disease prior to the development of a positive dipstik protein
reaction or increased urine protein-to-creatinine ratio.
e. ERD: species specific
f. Urine is diluted to a standard concentration (1.010) prior to the assay,
eliminating the need to consider urine specific gravity when interpreting
the test results.
3. Urine protein:creatinine ratio (UPC)
a. Quantitative test for total urine protein. Determines the magnitude and
therefore the significance of proteinuria.
b. Expressing the urine protein as a ratio to urine creatinine eliminates the
need to consider the urine specific gravity when interpreting the results of
this test.
c. In general, UPC values between 0.5 and 1.0 are borderline, as most normal
dogs and cats should have a UPC that is <0.5.
d. Dogs: Values of 0.5 to 1 repeatedly found on 3 or more specimens,
obtained 2 or more weeks apart, is evidence of persistent renal proteinuria.
e. Values greater than 1.0 should be considered abnormal if the urine
sediment is inactive.
f. Values greater than 2.0 means glomerular renal disease and treatment is
warranted.
g. How does hematuria affect UPC? Macroscopic hematuria (>250 RBC/hpf)
was reported to cause an increase in urinary albumin concentration, but
did not increase the UPC ratio in all dogs. However it is best not to
perform UPC on samples with macroscopic blood. (Vaden SL, et al:
Effects of urinary tract inflammation and sample blood contamination on
urine albumin and total protein concentrations in canine urine samples.
Vet Clin Pathol 2004; 14-9).
h. Does pyuria affect UPC? Albuminuria is associated with pyuria in some
dogs and is more common in dogs with bacteriuria. However, a large
percentage of dogs with pyuria do not have albuminuria. In one study of
70 samples with pyuria, 81% had normal UPC ratios. (Vaden SL, et al:
Effects of urinary tract inflammation and sample blood contamination on
urine albumin and total protein concentrations in canine urine samples.
Vet Clin Pathol 2004; 14-9). Still, it is best to perform UPC on a nonpyuric sample.
i. At room temperature the UPC ratio significantly increased at 12h and
continued to increase until 72h post collection. UPC ratios did not
significantly vary in refrigerated urine over a one month period. (Rossi G
et al: Analytical variability of urine protein-to-creatinine ratio
determination in dogs. 20th ECVIM-CA Congress, 2010)
j. To decrease the day-to-day variability in the UPC ratio, some clinicians
now recommend that owner collect 1 urine sample/day x 3 days, store
each one covered in refrigerator and bring the samples to the clinic where
1 ml from each is taken and put into a separate tube for the UPC
evaluation.
k. Summary for dogs: UPC < 0.5 is normal. UPC> 0.5 but < 2 warrants
investigation (urine culture, signs of Cushing’s, repeat test); UPC > 2
warrants treatment.
l. Cats: If more than 1.0, suspect glomerular disease.
4. Urine cortisol:creatinine ratio (UCC)
a. Highly sensitive in detecting hyperadrenocorticism in dogs since cortisol
is excreted in urine: screening test
b. Unfortunately not that specific: elevated level seen in many other diseases.
c. Morning urine cortisol level reflects cortisol release over several hours.
d. Concentration of cortisol in urine increases with increased plasma
concentrations.
e. Relating the urine cortisol concentration to urine creatinine concentration
provides a correction for any differences in urine concentration.
f. Collect sample at home: less stress
g. UCC ratio is determined by dividing the urine cortisol concentration (in
µmol/l) by the urine creatinine concentration (in µmol/l).
5. Bladder Tumor Antigen test
a. Rapid latex agglutination assay
b. Detects antigen (in urine) produced by transitional cell carcinoma (TCC)
of bladder
c. Designed as a screening test for early diagnosis of TCC. But there is low
incidence of bladder tumors in dogs: <2% of canine cancers.
d. High rate of false positives if hematuria, pyuria, 4+ glucosuria, or 4+
proteinuria are present. Therefore, not useful in patients with TCC tumor
that has progressed to cause hematuria.
e. To decrease the possibility of false positive reactions, the test should be
performed upon the supernatant of centrifuged urine.
f. Using the supernatant of centrifuged urine, the specificity of the test has
been reported as 46% in unhealthy patients with urinary tract disease other
than TCC. The specificity increased to approximately 86% in clinically
normal animals or in animals with non-urinary tract disease (Henry et al.,
2003).
g. Some investigators feel that the test has an unacceptable number of false
positive reactions, even in dogs with normal urine sediment.
h. May have some utility in select at-risk canine populations, such as obese,
geriatric dogs from urban areas with lower urinary tract signs and breed
predisposition for TCC (e.g., Scottish Terriers, Shetland Sheepdogs,
Beagles, Wire Fox Terriers, West Highland White Terriers) (Mutsaers et
al., 2003), when a negative result can be used to help exclude the
possibility of TCC in a high risk individual.
SOME OLDER AND SOME NEWER LAB TESTS
Linda Shell, DVM, DACVIM (Neurology)
I.
Serum Bile Acids
a. Bile acids (BA) are stored in the gall bladder & released into the intestinal
tract. Most BA are actively resorbed in the ileum and carried to the liver
where they are reconjugated and excreted as part of the enterohepatic
circulation of BA.
b. No need to run BA if there is elevated bilirubin
c. Ursodiol (Actigall), an unconjugated bile salt, might falsely elevate BA,
but may depend on what method is used (might affect spectrophotometric
methods including atomic absorption). Abraham et al (Aust Vet J 2004)
showed that ursodiol did not alter BA test in normal dogs, but no one has
looked at it in dogs with liver disease.
d. Samples should be non-lipemic and non-hemolyzed; 6-12 hour fast.
e. About 15-20% of dogs have a higher fasting than post-prandial BA value.
Using random BA sample or single BA sample isn’t optimal but if
resting value is high, you can confirm liver damage.
f. Normal BA results don’t necessarily rule out hepatic disease.
g. Elevated BA concentrations are not specific for the type of underlying
disease.
h. Dogs with portosystemic systemic shunt (PSS) often have at least one BA
value > 200 umol/L, but not always. Some microvascular dysplasia
(MVD) dogs can develop BA as high as 200 umol/L.
i. Increased BA concentrations can also result from extrahepatic diseases
(hyperadrenocorticism) that secondarily affect the liver.
j. Higher fasting result than post prandial result:
i. handling or misidentification error
ii. lab error
iii. sporadic contraction of gall bladder and small intestine in fasted
state
k. Both pre and post prandial values are low
i. Prolonged fasting
ii. Intestinal malabsorption
iii. Increased intestinal transit time through bowel (diarrhea)
II.
Urine Bile Acids
a. In theory the urine BA would be better test as they would be measuring
concentrations over a longer period of time and would not be subject to
pre and post sampling & gall bladder contraction
b. Dogs: Appear to be more specific (100 %) than serum BA (67 %
specificity) for detection of liver disease but less sensitive (61%) than
serum BA (78%). These figures were based on a 9 dog study.
c. Cats: Urine BA specificity is equal to that of serum BA (88 %); sensitivity
is 85 % for urine vs 87 % for serum.
d. Results within normal reference interval do not exclude underlying liver
insufficiency.
e. Collect 2 ml urine within 4-8 hours of eating (optimal)
III.
Protein C Activity: (PCA)
a. PC is a short-lived anticoagulant protein made by liver.
b. Modulates inflammation and apoptosis.
c. Significantly reduced in dogs with severe liver dysfunction including
shunts. Recent abstract showed < 70% activity PCA in 96% of confirmed
congenital PSS cases and 74% of acquired PSS cases. (Center SA,
Randolph JF, et al: Best Predictors of Protosystemic Shunting in 568 dogs:
post feeding bile acids and protein C; ACVIM 2011).
d. PCA in MVD cases was normal ( ≥ 70%) in 95% of the affected dogs and
below normal in 88% of the PSS affected dogs. (Toulza O, Center SA, et
al: Evaluation of plasma protein C activity for detection of hepatobiliary
disease and portosystemic shunting in dogs: JAVMA 2006; 1761).
e. Therefore low PC concentrations may offer a noninvasive method to
help differentiate PSVA from MVD in some dogs.
f. Sample is collected in a special citrate anticoagulated vial.
IV.
TLI: trypsin-like immunoreactivity (cTLI and fTLI)
a. Trypsinogen synthesized exclusively by the exocrine pancreas
b. Species-specific ELISA
c. Highly sensitive and specific for the diagnosis of EPI; reduced value in
dogs/cats with EPI
d. Elevated values may indicate pancreatitis (30 % sensitive in cat and 36 %
in dog) or gastrointestinal disease if renal function is okay. In one study,
specificity was 65% for pancreatitis cases.
e. 12 hour fasted sample
f. Not affected by pancreatic supplements
V.
PLI: Pancreatic Lipase Immunoreactivity
a. Serum lipase activity does not necessarily reflect changes in pancreatic
acinar cells and therefore is not specific for pancreatitis or (EPI)
b. Pancreatic lipase is a more specific marker for the pancreas
c. PLI is significantly decreased in dogs with EPI
d. PLI is increased in dogs (approximately 80% sensitive) and cats
(approximately 70% sensitive) with pancreatitis.
e. Specificity: 95% when cutoff value was < 200 ug/L and 97.5 % with a
cutoff value of 400 ug/L. These figures have generated debate and the test
may not be as specific as these numbers indicate. (Neilson-Carley SC, et al.
Specificity of a canine pancreas-specific lipase assay for diagnosing
pancreatitis in dogs without clinical or histiologic evidence of the disease:
Am J Vet Res 2011; 302-7) (Trivedi S, Marks SL, et al: Sensitivity and
specificity of canine pancreas-specific lipase (cPL) and other markers for
pancreatitis in 70 dogs with and without histopathologic evidence of
pancreatitis. J Vet Intern Med 2011; 1241-7)
f. Clinical signs of pancreatitis overlap with those of many GI diseases.
Some clinicians suspect that increased cPLI values do occur with other GI
diseases. Look at all your data and don’t rely on high cPLI value to
diagnose pancreatitis.
g. Need fasted serum sample
VI.
Serum B12 (cobalamin) and folate
a. Fasted sample required. Folate absorbed in duodenum; B12 in ileum
b. Low B12:
i. Dogs: EPI, severe intestinal disease, small intestinal bacterial
overgrowth (SIBO), congenital deficiency in Giant Schnauzers,
ileal disease
ii. Cats: EPI, intestinal (including lymphoma), pancreatic or hepatic
disease
c. Low folate:
i. Dogs: severe duodenal or jejunal disease; some Irish Setters with a
gluten-sensitive enteropathy
d. High folate: Experimentally induced SIBO, EPI, German Shepherds with
SIBO, some Irish setters with a gluten-responsive enteropathy
e. Combination of low B12 and high folate: more suggestive of idiopathic
"SIBO" than finding increased folate alone. In SIBO, bacteria
synthesize folate and bind B12.
f. Combination of high B12 and high folate: high dietary intake or
supplementation
SEROLOGY AND PCR FOR INFECTIOUS DISEASES
Linda Shell, DVM, DACVIM (Neurology)
I.
Serology: Detects Antibodies
a. Whole organisms or select antigens are used as targets for antibodies in
the assays
b. Positive result for a single sample indicates exposure but not necessarily
active infection for some agents.
c. Has been the mainstay of diagnostic testing for vector borne disease for
decades due to technical difficulties associated with isolating and culturing
these organisms from patient samples.
d. Serologic testing on a single sample may be negative in the acute phase of
disease, prior to the time when antibodies are formed. Clinical signs for
some diseases (R. rickettsii, E. canis, Babesia species, Leptospira species,
and A. phagocytophilum) can sometimes be present prior to
seroconversion. Convalescent testing documenting seroconversion or
PCR on acute samples may confirm active infection in these acutely
infected patients.
e. Different types of serologic assays testing for the same organism may
yield different results because of different antigens used in the assays. For
example, some dogs actively infected with E. canis test seropositive using
IFA and seronegative using ELISA.
f. Sometimes serologic testing is negative during chronic infections. In some
chronically infections with Babesia or Bartonella, antibodies have not
been detected even after seroconversion should have occurred; yet patients
were PCR positive.
II.
PCR testing: Detects DNA
a. Detects DNA of organisms themselves without the need for culture.
b. A positive PCR result indicates active infection or the presence of
circulating dead organisms.
c. Recent advances in molecular biology have made PCR panels easy to
perform and widely available. Can directly test for the presence of
multiple organisms in peripheral blood and other samples with high
sensitivity and specificity, assuming appropriate laboratory controls are in
place.
d. False negatives occur. Could be related to the sensitivity of the assay, the
primers may not be the ones of the infecting species, or the organism's
DNA might not be present in the test sample even if the organism is
present in peripheral blood in high numbers.
e. A negative PCR test does not eliminate infection.
f. False positives can occur with vaccines in some tests.
III.
Which test to run: Serology or PCR?
a. May need both.
b. Consider length of clinical signs & whether or not the organism could still
be circulating in the blood or tissue that you want to submit for PCR.
c. Consider whether antibiotic use will affect results (yes if PCR is used; no
if serology test)
d. Consider which organism you are most interested in and where it ‘resides’
in the body. For example: Blood is not a good sample to submit for PCR
for toxoplamosis or Borrelia in most cases. Another example: Since R.
rickettsii endotheliotropic organisms circulate in low numbers in
peripheral blood, PCR is not as sensitive as is demonstrating a four-fold
change in titer. However since Anaplasma phagocytophilum infected
granulocytes are present in peripheral blood in relatively high numbers
during the acute phase, PCR testing of peripheral blood is sensitive.
IV.
What about antigen testing?
a. Antigen tests are more specific for the disease state than are antibody tests.
b. However, they have a lower sensitivity than PCR.
c. Probably the best current way to detect some fungal diseases if the
organisms can’t be found via cytology or histopathology. Ex:
Cryptococcus serum antigen test, blastomycoses urine antigen test;
histoplasmosis urine antigen test
d. Parvo fecal antigen test is not as sensitive as PCR testing. (ACVIM 2012,
Nakayama, Lappin, Veir, et al: Comparison of ELISA, Conventional PCR,
and Quantitative PCR for Detection of Canine Parvovirus).
Leptospirosis
I.
Microscopic Agglutination Titer (MAT) (Serology)
a. Helpful to know when dog was last vaccinated for leptospirosis.
b. Cannot tell difference between real infection and vaccine response.
c. However it is rare that vaccine titers exceed 800 and most vaccine titers
don’t persist at this level for longer than 3 months.
d. Any titer > 800 in a dog that has not been vaccinated in past 2 months is
highly suspicious of current infection.
e. Titers could be negative in early acute infections and sequential titers may
be required to confirm infection.
f. Interlaboratory results vary widely so the same laboratory should be used
for any serial titers on the same animal
g. With the MAT titer, a fourfold change (increase or decrease) in titer is
classically required for the definitive confirmation of a self-limiting
infection such as leptospirosis. The titer fall may take up to 3 months
following treatment to reduce to levels of 200 or less.
II.
PCR testing
a. PCR methods offer the theoretical advantage of detecting infection in its
early stages and prior to antibody increases.
b. Genetic methods allow for more specific determination of the infecting
strains, which is not possible with the MAT.
c. Unfortunately, the method and quality control for PCR detection can vary
widely resulting in either false-positive or false-negative results.
d. Vaccines apparently do not interfere with PCR results (ACVIM 2010.
Midence JN, Chandler AM, Goldstein RE: Assessing the Effect of Recent
Leptospira Vaccination on Whole Blood Quantitative PCR Testing in
Dogs).
e. Urine will become positive 7–14 days after infection at which time
leptospires may or may not be detected in the blood.
f. A PCR test by Idexx (RealPCR Test) requires both whole blood and urine
samples collected prior to antibiotic administration. A positive result on
whole blood in the presence of a negative urine result can occur during the
first 7–14 days of infection. Negative urine samples can are explained by a
pre-leptospiruria sample or intermittent
shedding. Negative blood results with positive urine results suggest dog
was likely infected at least 2 weeks prior to sample collection.
Asymptomatic chronic carriers can also shed Leptospira in the urine
intermittently for weeks to months. Positive urine samples are considered
a source of infection for other animals and humans. Serovar- specific
results are not available with this test.
Canine distemper testing
I.
II.
III.
PCR testing is preferred over IgM/IgG and IFA
Vaccine interference has been a problem because distemper vaccine is a
MLV one.
MLV strains of vaccine cross react with most PCR tests currently. (This
may change)
IV.
Distinguishing between vaccine and infection: Idexx has developed a
quantitative real-time PCR test based on the P gene for phosphoprotein. Viral
loads were determined in vaccinated and wild-type infected animals and a
cutoff value was established to discriminate between vaccine interference and
wild-type infection. (Leutenegger CM, Crawford C, Levy J, et al; ACVIM
2011: Canine distemper virus quantification by real-time PCR allows to
differentiate vaccine virus interference and wild-type infection).
Fungal Disease Testing
I.
II.
III.
IV.
V.
Try to find organism via cytology or biopsy. If you can’t, see if an antigen
test exists for the particular fungal organism the patient might have. Presence
of antigen signifies disease whereas presence of antibodies signifies exposure
but not necessarily disease.
Blastomycosis Antigen Test (Miravista labs)
a. If diagnosis cannot be made by identification of organisms via cytology,
culture, or histopathology of suspected lesions, look for antigen, not
antibody, in serum & urine. Only about 30% of dogs with blastomycosis
have serum antibodies at time of diagnosis so antibody test is not preferred.
b. Urine is preferred sample for antigen testing but serum can be used.
c. Urine antigen concentrations were maintained at 30 days but declined after
60 days of treatment. Dramatic decline in serum antigen concentrations
were found 30 days after the initiation of treatment with itraconazole.
d. Assay is known to cross-react with other systemic mycoses in humans
including Histoplasma capsulatum (histoplasmosis), Coccidioides immitis
(Valley fever), and Penicillium marneffei.
Histoplasma Antigen Test (Miravista labs): Cross reacts with blastomyces.
Coccidioidomycosis Antigen Test (Miravista labs) has not been studied in
the dog. Because of this some experts recommend the antibody assay offered
by Dr. Pappagianis at the School of Medicine, University of California.
Aspergillus Antigen Test ( MiraVista Labs) for use in dogs, but it is
currently not validated for use in this species. Antibody-based tests are
available through commercial labs.
ENDOCRINOLOGY
Linda Shell, DVM, DACVIM (Neurology)
I.
Insulin growth factor (IGF-1)
a. Most acromegaly cats had value > 100 nmol/L
b. Diabetics can have high level but may not be insulin resistant
c. Rule out Cushing’s before diagnosing acromegaly
II.
Serum cortisol
a. Hydrocortisone and prednisolone will be directly measured on the cortisol
assay, so they should not be used within 24 hours before the test.
b. Dexamethasone and betamethasone aren't directly measured on the
cortisol assay but still affect the endocrine tests, especially if they have
been administered for days to months (will suppress cortisol levels).
c. Dexamethasone has a 24-48 hour duration of activity in healthy dogs,
while in a dog with PDH, the duration is only 3-6 hours. So it is unlikely
for dexamethasone administered a week prior to the LDDST to affect
cortisol, but betamethasone, a longer acting corticosteroid, could affect the
test.
III.
ACTH stimulation test
a. Advantages of ACTH test
i. Takes only one hour to perform.
ii. Fewer false positives than with LDDS.
iii. Can run other tests at the same time e.g. bile acids.
iv. Can also rule-out Addison's disease.
v. Only test for iatrogenic Cushing's.
vi. Only test to monitor response to therapy for HAC
b. Disadvantages of ACTH test
i. More expensive (due to cost of synthetic ACTH) than LDDST.
ii. More false negatives.
iii. More likely to miss adrenal tumor.
iv. Cannot discriminate between PDH and ADH
IV.
LDDS test
a. Interpretation guidelines
i. Look at 8 hour value first. If greater than normal, the diagnosis of
hyperadrenocorticism (either PDH or ADH) is made.
ii. Next look at the 4 hour value. If it is less than half of the baseline
or if it is less than 1.0 mg/dl, the diagnosis is PDH.
iii. If you can’t make a determination at the 4 hour value, look again at
the 8 hour value. If it is less than 50 % of baseline, the diagnosis is
PDH.
b. Advantages of LDDST
i. More reliable than the ACTH test in confirming HAC, since the
results are diagnostic in all ADH cases and in 90 to 95 per cent of
dogs with PDH.
ii. Fewer false negatives compared to the ACTH test.
iii. Less expensive than the ACTH test.
iv. Can sometimes aid in discriminating PDH from ADH
c. Disadvantages of LDDST
i. Affected by more variables than the ACTH test
ii. More false positives than the ACTH test. For example, recent
administration of corticosteroids can interfere with the test.
iii. If corticosteroids have been administered within the last month,
better to run ACTH test.
iv. Takes 8 hours to complete.
v. More false positives.
vi. Don’t stress patient during the test e.g. no ultrasound, radiographs,
etc should be done.
V.
Thyroid tests
a. Total T4:
i. Sensitivity: 89% Specificity: 82%
ii. In one study, 50–60% of normal dogs had a low serum total T4 at
some time during the day.
iii. What can lower T4? Estrus, pregnancy, obesity, malnutrition,
drugs (any form of glucocorticoid, phenobarbital, salicylates sulfaderived drugs, phenylbutazone), any systemic illness (esp.
hyperadrenocorticism, diabetes mellitus, hypoadrenocorticism,
renal failure, hepatic failure, infection).
iv. Potential mechanisms for decreased total T4 in sick euthyroid
dogs: changes in protein binding, decreased conversion of T4 to
T3, inhibition of TSH secretion, and inhibition of thyroid hormone
synthesis by the thyroid gland.
v. While it is nice that many labs offer the T4 on chemistry profiles,
T4 results should be used to confirm normal function, not to
confirm hypothyroidism. In a dog with no signs of hypothyroidism
(obesity as the only sign doesn’t count most of time in my opinion),
a low resting T4 isn’t likely due to hypothyroidism.
vi. If signs of hypothyroidism are present and total T4 is normal, there
is still a chance the dog is hypothyroid (sensitivity of 89%). Or if
the T4 was done by RIA, autoantibodies could be falsely pushing
the T4 into a normal range.
vii. If you still really think the dog could be hypothyroid, confirm with
fT4 by equilibrium dialysis and TSH.
viii. Rule outs for elevated T4: thyroid supplement, lab error, presence
of T4 autoantibodies falsely elevating T4 value, hyperthyroidism
b. Free T4 (fT4):
i. Is the unbound fraction of T4 and is the biologically active form as
it can enter cells and exert its effects.
ii. Correlates best with thyroid status at the tissue level and is less
affected by nonthyroidal illness and drugs than is total T4.
iii. However a recent study found 21% of ill dogs and 44% of dogs
with severe illness have a low fT4. Lesson to be learned: Don’t
test a systemically sick/ill dog for hypothyroidism
iv. Majority of hypothyroid dogs have fT4 concentrations below the
reference range.
v. Few normal and euthyroid animals have results consistent with
hypothyroidism.
vi. Sensitivity: 98%. Specificity: 93%
vii. Specificity suggests that false positive tests are seen in only about
7% of cases. However specificity is not as good if moderate to
severe non-thyroidal illness is present (previous comment).
viii. T4 autoantibodies don't interfere with dialysis because they can't
cross the dialysis membrane. They can interfere with any other
antibody based test.
c. TSH
i. Sensitivity: 76 %. Specificity: 93 %
ii. At least 1/4-1/3 of dogs with confirmed hypothyroidism have
serum TSH concentrations within reference limits.
iii. About 7 % (5-12 % range) of euthyroid dogs (healthy and sick
euthyroids) have an elevated TSH. We don't understand exactly
why this happen. Some may develop hypothyroidism but currently
numbers aren’t known.
d. T3
i. T3 concentrations are highly variable in both hypothyroid and nonhypothyroid dogs.
ii. There is little difference between measured T3 concentrations
between normal or hypothyroid dogs.
iii. T3 is mainly intracellular, so measurement of serum concentrations
is not an accurate reflection of body levels.
iv. Not of much use in the diagnosis of hypothyroidism.
e. Summary
i. Don’t try to diagnose hypothyroidism from T4 found on most
chemistry panels.
ii. Don’t diagnose hypothyroidism unless clinical signs are present.
iii. Don’t diagnose hypothyroidism in a sick dog.
iv. The best method of diagnosing hypothyroidism is to perform a T4,
fT4 (equilibrium dialysis) and TSH ONLY in dogs where there is
a true suspicion of hypothyroidism and there is no concurrent
illness or drug administration to confuse the picture.
v. When T4 (total or free) and TSH concentrations were evaluated
together, specificity was higher than when T4 or TSH
concentration was evaluated alone.