Laboratory
The lab is used as an adjunct to good animal medical care to
assist the veterinarian on making a diagnosis. Some important
things to understand are:
General principals of specimen collection
1) Blood
2) When we obtain a specimen for analysis it is critical to
remember that the results and therefore diagnosis and
treatment of the animal all depend on the quality of the
initial specimen
3) We are drawing blood through a needle into a tube. The
first thing to do is remember we need to maintain the
integrity of the cellular components of that blood
specimen.
4) Simple language:
Blood is composed of cells and fluid components. The
main cells are red blood cells (RBCs) and white blood
cells (WBCs). If the Red blood cells are broken or
damaged during specimen collection the results may be
effected. How do you prevent damage to RBCs?
1) Bore (size ) of the needle
2) Speed of the draw
3) Tube specimen drawn into
4) Order of tubes drawn
The following charts and information sheets are from a
veterinary diagnostic lab in Calgary BC, and are to be used for
informational purposes to familiarize yourself with types of
Vacutainers available and possible tests run as send outs as
well as some in-house tests.
Again this is informational educational material and does not
necessarily represent test we run.
Remember with today’s machines the best recommendation is
to read the manual that came with the analyzer to understand
how to obtain best specimen for analysis for that machine. The
manufacturers Web site should also have info and training
tools to assist you.
RED STOPPERED (RT AND SST)
RT - available as 3 & 10 mL, SST as 3.5 and 5 mL sizes and 600 µL
microtainers.
These tubes contain no anticoagulant. Best results are obtained if the tube is
kept upright at room temperature for 30 minutes so that the blood can clot. The
tube is then centrifuged and the serum removed and placed into another tube.
The SST tubes have a barrier gel that separates the rbcs from the serum after
the tube is centrifuged. There is always less hemolysis if the tubes are spun prior
to submitting.
Remember, certain tests such as phenobarbital cannot be submitted in an SST
tube. The gel is capable of interfering with immunologic based tests.
LAVENDER STOPPERED (LT)
Available as 7, 3, 2 mL & 500µl volumes. These tubes contain anticoagulant and
are used for CBC's and differentials. NEVER USE EDTA FOR AVIAN BLOOD
AS THERE WILL BE SEVERE HEMOLYSIS. The RBC morphology is best
preserved when the ratio of EDTA to blood volume is correct. Always fill these
tubes appropriately. Mix these tubes after collection by inverting 5 to 10 times. All
fluid cytology should be submitted in EDTA tubes.
GREEN STOPPERED (GRNT)
Available as a 3 mL tube.
These contain sodium heparin and are often used for special tests, although both
chemistry and hematology tests can be run off this tube. The cells are poorly
preserved for differentials so fresh slides must be submitted. These tubes are
recommended for all avian blood. The tubes should be mixed well after collection
by inverting 5 to 10 times. Some tests will require plasma to be harvested from
these tubes. Please check the manual for specific tests.
DARK BLUE STOPPERED (DBT)
These tubes are available as 7 mL draw and they are used for mineral testing
(trace elements) as the stoppers and glass are specially treated to stop
contamination of the sample - use for zinc, etc.
BLUE STOPPERED (BT)
These are available as a 2.7 mL draw, and have a light blue cap. These contain
3.2% sodium citrate and must be well mixed. This tube is required for all
coagulation tests. They must be filled correctly, spun and the plasma removed
(use a plastic transfer pipette) and placed in either a plastic tube or another
sodium citrate tube that has had the sodium citrate removed. Non-siliconized
glass activates coagulation factors.
GREY STOPPERED (GT)
Available as a 3 mL tube. These contain potassium oxalate and sodium fluoride
to inhibit glycolysis. Mix well and then spin immediately and remove plasma into
another empty tube. Transferring the plasma to another tube will prevent
hemolysis.
GREY STOPPERED FOR ACT
Available as a 5 mL tube, which contains diatomaceous earth. This is used as an
in house screen test for coagulation disorders.
PROCEDURE
Place 2 mL of freshly drawn blood into the tube, invert gently 5 to 10 times and
keep the tube warm (under arm, in incubator, etc;) and check every 30 seconds
for clot formation. Normal horses clot in 2 - 2½ minutes, dogs in 2 minutes and
cats in 1½-2 minutes. This screen should be done prior to submitting a
coagulation panel. The tubes can be purchased through the laboratory for
$4.00/each.
A minimum order of 5 tubes is required.
NOTE:
The date on the VACUTAINER® Tube refers to the vacuum and is not an
indication of an expired product. Tubes may be used after the expiration date as
long as a vacuum is not needed.
SAMPLE COLLECTION PROBLEMS:
****ALWAYS CHECK THE MANUAL PRIOR TO SAMPLE COLLECTION TO
IDENTIFY THE CORRECT CONTAINERS.****
1. Lipemia: Animals should be fasted for 12 hours prior to blood
sample collection. Animals with endocrinopathies, pancreatitis, and
hyperlipoproteinemias may have a persistent lipemia that will not
disappear with fasting. Fasting should be at least 24 hours for
animals with endocrinopathies.
2. Hemolysis: This can occur for a number of reasons:
a. Difficulty in drawing the sample. The longer it takes to collect
the blood the more chance there is of hemolysis.
b. If the needles are too small, then not only will time be a
factor, but the red cells can be damaged in the needle itself.
c. If the serum samples are not allowed to clot; this takes at
least 30 minutes at room temperature.
d. If the serum samples are not separated after clotting.
e. If Grey top plasma is not separated after spinning.
f. Freezing
g. Heating of the sample, especially in the summer, but also
from heaters in cars and trucks.
h. Lipemia
i. If none of the above applies and you still have a problem with
hemolysis, please call the laboratory and talk to a
technologist who will attempt to identify your problem.
3. Never use SST tubes for phenobarbital. There is a nonlinear
decrease in phenobarbital values with this gel.
4. If samples have been collected but were not picked up or have
been forgotten for a few days, remember the following:
a. If the serum has been separated, the serum should still be
adequate for routine chemistries even if held at room
temperature for 24 hours.
b. The EDTA sample will have artifactual changes if held at
room temperature for 24 hours. However if a fresh smear has
been made then an accurate differential can be performed
and the automated parameters assessed in light of the
differential.
c. Culturettes are only good for preserving the specimen for 48
hours. Also, our culturettes have rayon not cotton tips.
Clostridium perfringens and Clostridium difficile toxin media is supplied
in small eppendorf tubes and must be used when submitting feces to test
for either toxin.
Vacutainer is a registered trademark of Becton, Dickinson and Company
Hematology Testing
BUFFY COAT SMEARS
SPECIMEN REQUIRED
1 mL of EDTA blood.
PURPOSE
To determine if circulating mast cells are present. The presence of mast cells in
animals with mast cell tumors may be predictive of metastasis. But the test is
neither sensitive nor specific, therefore should be interpreted with caution.
CBC AND DIFF
SPECIMEN REQUIRED
Minimum of 0.5 mL of EDTA blood, can be done on Heparinized whole blood.
PURPOSE
Gives the automated parameters plus a differential. Technicians will evaluate all
blood smears and provide morphology, WBC morphology and platelet
information as comments on the differential. If the automated counts fall within
our selected parameters the automated differential will be used instead of the
manual differential. All automated differential results are in SI units.
LIMITATIONS
Fresh smears made at the time of collection will avoid artifacts that can be
induced in cells by EDTA.
CBC ONLY
SPECIMEN REQUIRED
0.5 mL of EDTA blood.
PURPOSE
This will give all the machine-derived parameters listed above.
LIMITATIONS
Without a differential the parameters may not allow meaningful interpretations.
May be useful for monitoring previously diagnosed conditions
CBC'S
CBC refers to a complete blood count and in this lab includes WBC, RBC, Hgb,
PCV, MCV, MCH, MCHC and RDW; in most species except cats a platelet count
will also be included. RDW is a measure of the red cell distribution width and
helps to determine if more than one population of rbcs is present. This lab uses a
CellDyn 3500 to perform the tests. This is a laser and impedance counter and
can do accurate differentials on some samples. The Coulter-S-Plus4 serves as a
backup instrument as it performs better on some samples than the CellDyn.
TECHNICAL INFORMATION
CV; within run and run to run are less than 1.5% for all parameters.
CBC/DIFF WITH FIBRINOGEN and PLASMA PROTEIN
(EQUINE)
SPECIMEN REQUIRED
0.5 mL of EDTA whole blood. Test can be run on heparinized whole blood.
PURPOSE
Fibrinogen is a better indicator of inflammation than the CBC and differentials in
all large animals: equines, ruminants, etc.
LIMITATIONS
Hemolysis may make this difficult to interpret. This is a semi-quantitative
methodology for fibrinogen
CELL COUNT-RBC/WBC
SPECIMEN REQUIRED
Minimum of 250 µL of any anticoagulated fluid e.g. EDTA/Heparin etc.
PURPOSE
To count cells including white, red, or even platelets within a fluid specimen.
LIMITATIONS
The CellDyn is capable of counting very low cell counts and most fluids other
than CSF can be processed through this machine
HEMOBARTONELLA
SPECIMEN REQUIRED
Air-dried smears or 1 mL EDTA
PURPOSE
To determine the presence of the blood parasite H.canis or H.felis. This is
routinely evaluated on all blood smears submitted. Acridine orange can also be
used.
LIMITATIONS
Hemobartonella are present in varying numbers and at variable times. Therefore,
they may not be seen on any one smear. Also, EDTA will result in the parasite
detaching from the cell surface. This is a time-related phenomenon. PLEASE
NOTE: Active infection is associated with hemolytic anemia, as parasitized cells
are removed by the spleen. Latent infections are not associated with parasitemia
or anemia and can only be reliably determined by splenectomy or the use of
PCR. Therefore if the animal is not anemic it is unlikely the parasite will be
present on the rbc's. PCR tests are available as a send out test.
FELINE SCREEN
SPECIMEN REQUIRED
0.5 mL of serum, 1 mL EDTA, and 1 mL Grey Top. There are different
combinations included with the Feline Screen. Can be ordered with T4, or with
T4 and UA.
PURPOSE
This test includes FeLV, FIV, and CBC/diff with chemistry screen and is an
especially useful database in young cats. Mycoplasma felis is always evaluated
on differentials and reported when it is present.
LIMITATIONS
This screen does not include pancreatic enzymes or bile acids, but these tests
may be added onto the panel.
Liver Function Test
ALBUMIN, SERUM
SPECIMEN REQUIRED
0.2 mL of serum. Can be run on heparinized or sodium fluoride plasma.
PURPOSE
Measures the main protein component of serum. This is produced by the liver
and has a half-life, which varies between species, anywhere from 9 to 21 days.
This protein may be lost through the glomerulus when there is glomerular injury,
and levels may decline when there is poor intestinal absorption or leakage of fluid
and protein into the intestinal tract. Low levels may also be indicative of chronic
liver disease. Low levels may occur as the result of acute exudation into a body
cavity or through burn sites. Elevated levels usually reflect dehydration or
interference with the sample.
LIMITATIONS
Hemolysis will increase levels, lipemia will decrease levels but hemolysis and
lipemia together will cause a modest increase in albumin levels.
TECHNICAL INFORMATION
CV, within run <1.0%, run to run 1.2%.
ALKALINE PHOSPHATASE
SPECIMEN REQUIRED
0.2 mL of serum or heparinized plasma.
PURPOSE
This test measures an enzyme that is produced primarily by bile duct epithelial
and canalicular cells. In dogs the enzyme is induced by corticosteroids, NSAIDS,
antiepileptic medications and also by pressure around the biliary tree. Nonspecific increases may also occur with neoplasia. Alkaline phosphatase is
produced by bone cells although serum elevations from this source are not
usually as high as from other sources. The exception is young animals which
may have alkaline phosphatase levels 3x that of the adults. The half-life of ALP is
variable but considered to be 1 to 5 days in most species.
LIMITATIONS
Hemolysis may cause values to decline especially in the feline. Severe lipemia
may cause an increase.
TECHNICAL INFORMATION
CV; within run 3%, run to run 3.5%.
ALT [SGPT]
SPECIMEN REQUIRED
0.2 mL of serum or heparinized plasma.
PURPOSE
This enzyme is an indicator of hepatocyte injury in small animals and many of the
exotic pets.
LIMITATIONS
Significant numbers of hepatocytes must be injured for this enzyme to increase.
Values can increase with hemolysis/lipemia in cats and horses, and are
significantly increased with combined hemolysis and lipemia in those species,
with only a modest change in dogs related to lipemia.
TECHNICAL INFORMATION
CV; within run 1.8%, run to run 2.4%.
AMINO ACID PROFILE
SPECIMEN REQUIRED
A minimum of 1 mL of serum.
PURPOSE
This test measures the different amino acids present in plasma and can be used
to categorize the type of liver dysfunction. This may be helpful in the
hepatocutaneous syndrome and may be of benefit in determining replacement
therapy.
LIMITATIONS
This should be a fasting sample and should not be hemolyzed. The patterns for
different diseases are not yet completely worked out.
AST [SGOT]
SPECIMEN REQUIRED
0.2 mL of serum.
PURPOSE
This enzyme is helpful in large animals as an indicator of muscle damage, and in
some species especially avian, as an indicator of hepatocyte injury. In cats this
enzyme is primarily localized to the liver rather than muscle.
LIMITATIONS
Must distinguish between liver or muscle origin in most species. Values will
increase with hemolysis and only modest increases may occur with lipemia.
TECHNICAL INFORMATION
CV; within run 2.4%, run to run 2.3%.
BETA HYDROXYBUTYRATE
SPECIMEN REQUIRED
100 µL of serum.
PURPOSE
This test measures the most significant ketone in the blood. Elevations will occur
with prolonged fasting, cachexia, endocrine disorders (diabetes mellitus), ketosis
and lipidosis. This test is very helpful in measuring therapy control in diabetes
mellitus.
LIMITATIONS
More experimental work is required to determine duration of elevations and
rapidity of responses to therapy.
TECHNICAL INFORMATION
CV within run 1.8%
REFERENCE RANGE
0.0-0.32 mmol/L; canine
0.0-0.21 mmol/L; feline
0.32-0.88 mmol/L; bovine
BILE ACID PANEL
This includes a fasted serum sample [minimum 12 hour fast] followed by a good
quality meal [pd/cd] and then a 2-hour post prandial sample. The meal should be
equal to a normal size feeding, gall bladder contraction may not occur with small
amounts of food. Gall bladder contraction can also occur with stress, excitement
and with food smells. Post-prandial values in normal dogs and cats are usually
less than 22 µmol/L. Gall bladder contraction occurs in association with eating or
even with food odors or sometimes with stress, and if contraction occurs prior to
the test the results may appear reversed.
TECHNICAL INFORMATION
CV; within run 1.9%, run to run 2.5%.
BILE ACIDS
SPECIMEN REQUIRED
0.5 mL of serum for each tube. Heparinized plasma may be used.
PURPOSE
Bile acids are produced by the liver, excreted in the bile and then reabsorbed
from the ileum. The test is a measure of liver function and portal circulatory
integrity. Bile acids will be elevated with hepatic dysfunction, especially when
chronic or when there are vascular anomalies. This is helpful when there is
minimal or no change in liver enzymes but the albumin is low.
LIMITATIONS
Fasted samples may be within the normal range even though there is significant
hepatic dysfunction. Therefore we recommend that a bile acid panel be run to
eliminate occult liver disease. Steroid induced liver diseases are usually
associated with elevated bile acids. With the new test methodology, there is little
interference with hemolysis and lipemia. This test has largely replaced blood
ammonia (technically difficult) and BSP
BILIRUBIN DIRECT (conjugated)
SPECIMEN REQUIRED
0.2 mL of serum.
PURPOSE
This analyte is increased when serum total bilirubin levels are elevated. There is
a disproportionate increase when there is damage to the liver as opposed to
hemolysis. This disproportionate change will only be present early in the course
of disease.
LIMITATIONS
The amount of direct bilirubin in serum is a reflection of timing. The test cannot
be used to distinguish between hemolysis and primary liver damage
BILIRUBIN TOTAL
SPECIMEN REQUIRED
0.2 mL of serum.
PURPOSE
Bilirubin is produced by the liver from the breakdown products of hemoglobin,
cytochromes, etc. Therefore it is a measure of hepatic function. Elevations will
occur when the liver cannot excrete this in the bile, or when overwhelmed by
breakdown products or a combination of both conditions.
LIMITATIONS
Hemolysis may falsely decrease values while lipemia will falsely elevate these
levels.
TECHNICAL INFORMATION
CV; within run 2.1%; run to run 2.6%
CHOLESTEROL
SPECIMEN REQUIRED
0.5 mL of serum, fasted samples are preferred as this test is very influenced by
food ingestion.
PURPOSE
Cholesterol is increased after eating, and with endocrine disorders such as
hyperadrenalism and hypothyroidism. Values will also increase in the nephrotic
syndrome. Values may be increased or decreased with liver disease depending
on the type of hepatic condition, e.g. decreased with portosystemic shunts.
Values may be decreased with intestinal malabsorption.
LIMITATIONS
This test is of no value if the sample is not from a fasted animal. Values may be
slightly decreased with hemolysis.
TECHNICAL INFORMATION
CV; within run 0.7%, run to run 1.2%.
GGT [GAMMA GLUTAMYL TRANSFERASE]
SPECIMEN REQUIRED
0.2 mL of serum, or heparinized plasma.
PURPOSE
This enzyme is from the biliary parenchyma. Also elevated with steroids/NSAIDS
etc. Very high elevations occur in foals and calves if they ingest colostrum. The
test is now part of all chemistry screens.
LIMITATIONS
The enzyme does not elevate with bone lesions but in dogs is increased with
antiepileptic medications and glucocorticoids. This enzyme is induced by many
medications; elevations should be interpreted with caution. Values decline with
hemolysis, and lipemia.
TECHNICAL INFORMATION
CV; within run 1.7%, run to run 2.3%.