RECOMMENDATIONS FOR DELIVERING, CONSERVING AND TRANSPORTING SAMPLES.
TRANSPORT AND DELIVERY:
When extracting a blood sample, it is very important to apply not prolonged vein pressure, to
have the animal on an empty stomach (in order to prevent lipemic samples) and also to avoid
strong pressures.
Cells must be separated from serum or plasma by centrifugation. In order to properly obtain the
serum, blood should be allowed to coagulate by itself for 10-15 minutes; hence vials without any
anticoagulating agent must be used. When manipulating horse samples, more time should be
spent for coagulation. Then, samples should be centrifuged for 10-15 minutes (with horse
samples, 30 minutes or more) at 3500 rpm, then supernatant (serum) can be retrieved and will be
frozen in an eppendorf as soon as possible. To obtain plasma, blood should be introduced in vials
with anticoagulating agents (avoiding coagulation waiting time) and the same steps should be
performed.
IN ANY CASE, FREEZING, TRANSPORT, HEAVY CENTRIFUGATION OR DRAMATIC
CHANGES OF TEMPERATURE SHOULD BE APPLIED TO TOTAL BLOOD SAMPLES.
FURTHERMORE, SAMPLES SHOULD BE PROTECTED FROM LIGHT (BILIRRUBIN IS LIGHT
SENSITIVE).
Once samples have been obtained (serum or plasma), they should be sent to the Servei de
Bioquímica Clínica Veterinària (Facultat de Veterinària, Universitat Autònoma de Barcelona,
08193 – Bellaterra, Barcelona) with the following premises:
1.- Sample’s volume must be sufficient in order to perform all the determinations (please ask to
Servei’s staff).
2.- Samples must be delivered clearly identified, ordered and properly packaged, avoiding spills,
or rips
3.- During transport, freezing of samples must be assured.
4.- The external box must be resistant enough to keep on the transport.
Serum is a kind of sample recommended for biochemistry, specially with enzyme determination.
If necessary, the preferred anticoagulant reagent will be lithium heparin (see interferences).
The Fluoride/Oxalate (glycolysis inhibitor) is used where glucose determination and not being
able to separate cells from serum or plasma before an hour.
INTERFERENCES:
If it is the situation, specifying the treatment which is being performed on the animal is very
important, in order to realize possible interferences with medication.
For example, possible interferences by anticoagulant agents should be considered:
-
Heparin: it is the suggested anticoagulant because it shows a less number of
interferences. However, it should be considered the fact that depending on heparin’s salt
used, sodium, lithium or ammonium determination won’t be properly performed.
EDTA: Its anticoagulating effect is based on the chelant effect of calcium. It is also
chelant among other divalent ions such as magnesium. Therefore, ion determination
can’t be performed in plasma samples with EDTA, nor enzymes which require those ions
for its activity (like alkaline phosphatase, amylase o lypase).
Haemolytic samples:
It happens when eritrocites are ripped and part of its contents transfers to plasma or serum.
Hence, samples will display red colour because of haemoglobin.
Haemolysis happens because of a pathology or, in most cases, because a bad sample
processing.
A haemolytic sample will create the next alterations:
- The increase of parameters which are found in normal conditions to high concentrations inside
eritrocites: potassium, LDH and AST.
- The decrease of glucose concentration.
- Haemoglobin’s band shown in electrophoresis gel (between α-globulins’ and ß-globulins’).
- The haemoglobin produces a negative interference, decreasing α-amylase activity in a 10%,
when haemoglobin’s concentration of 6 g/L.
RECOMMENDATIONS FOR OBTAINING AND CONSERVING SAMPLES TO DETERMINE
ENZYMES:
The serum is the preferred sample used in enzyme determination.
ASPARTATE-AMINOTRANSFERASE (AST):
It is better to use non haemolytic serum. Plasma is not recommended. In absence of bacterial
contamination, AST keeps stable at 0-4°C for 7 days. Samples can be frozen in order to
conserve their enzymatic activity for longer time.
ALANINE-AMINOTRANSFERASE (ALT):
It is better to use non haemolytic serum. Plasma is not recommended. In absence of bacterial
contamination, ALT keeps stable at 0-4°C for 3 days. Samples can be frozen in order to conserve
their enzymatic activity for longer time.
CREATINE KINASE (CK):
It is better to use non haemolytic serum (in order to avoid interferences with eritrocite’s adenilatekinase). Plasma is not recommended. Physical exercise can increase CK activity, therefore it
should be avoided before extraction. CK’s stability in serum depends on isoenzymes proportion in
sample:
CK-MM (CK 3 or muscular): 2 weeks at 2-10°C.
Samples can be frozen in order to conserve their enzymatic activity for longer time.
L-LACTATE DESHIDROGENASE (LDH):
It is better to use non haemolytic serum. Plasma, because of its content in platelets, shows a
higher LDH concentration than serum.
Several anticoagulating agents inhibits LDH’s catalytic activity, like oxalates or fluorides. Some
opinions are formulated about heparin’s influence on LDH’s catalytic activity. Plasma obtained
with lithium iodumacetate is not recommended.
Because of Isoenzyme 1’s high concentraction (heart muscle and kidney) in eritrocites, a late
serum separation and haemolysis, high concentrations will result. Isoenzyme 5 (liver, bone
muscle and several neoplasia) is temperature sensitive, therefore freezing is not recommended if
a determination has to be performed.
LDH’s total catalytic concentration keeps stable during 24 hours at room temperature. After this
period of time, considerable loss of activity is detected, even keeping the sample frozen or at
room temperature.
GAMMA-GLUTAMILTRANSFERASE (GGT):
It is better to use non haemolytic serum. Plasma is not recommended because anticoagulating
agents interfere or generate problems of murkiness. GGT keeps its activity in serum for 5 days at
4°C or for 9 months at -80°C.
Several medicines like phenobarbital, diphenilehidantoine, barbiturates, oral contraceptives and
tolbutamide, induce hepatic synthesis of the enzyme, increasing concentration in serum.
PHOSPHATASE ALKALINE (ALP):
It is better to use non haemolytic serum. Chelant anticoagulating agents should not be used like
oxalate, citrate or EDTA, but heparin can be. Samples can be refrigerated for 5 days, but for
longer periods they must be frozen, leaving the specimen 1 hour at room temperature after
thawing and analyzing. Several medicines can interfere with the analysis. Aminofiline and teofiline
decrease ALP’s catalytic concentration. Biological interference is produced by a wide group of
medicines that produce hepathotoxicity, increasing enzyme concentration in serum.
α-AMILASE:
It is better to use non haemolytic serum. Chelant anticoagulating agents should not be used like
oxalate, citrate or EDTA, but heparin can be. α-amilase keeps its activity for a week at room
temperature and for 2 months when stored at 4°C.
LIPASE:
It is better to use non haemolytic serum. Only plasma with heparin can be used. Lipase is stable
for 7 days at 4°C. For longer periods, it can be frozen.
It is recommended to consult Young et al. table to know about interferences produced by
medicines when determining enzyme’s catalytic concentration: Young GS, Pestane LC,
Gibberman Y. Effects of drugs on clinical laboratory test. Clin Chem 1975;21:1D-432D.
RECOMMENDATIONS FOR OBTAINING AND CONSERVING SAMPLES TO DETERMINE
HORMONES:
In general, glass vials without any anticoagulating agent are recommended for sample extraction
and frozen serum must be kept in propylene eppendorfs. Most common hormones are:
- Cortisol: serum or plasma with heparin.
- Estradiol: serum.
- Insuline: serum or plasma with heparin (EDTA produces higher results).
- IGF-I: serum, plasma with heparin or EDTA.
- Progesterone: serum, plasma with heparin or EDTA.
RECOMMENDATIONS FOR PRINCIPAL METABOLITES AND ACUTE PHASE PROTEINS:
Serum
Haptoglobine
SAA
Bile Acids
Free Fatty Acids
Uric Acid
Direct Bilirrubin
Total Bilirrubin
Calcium
Clorides
Cholesterol
Creatinine
D-3- Hydroxybutyrate
Phosphorus
Glucose
Lactate
Magnesium
Potassium
Total Proteins
Sodium
Triglycerides
Urea
x
x
x
x
x
x
x
x
x
x
x
x
x
x
Plasma
heparin
with Plasma
EDTA
with Plasma
with
potassium
oxalate
or
sodium fluoride
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x