INVITEDREVIEW

The preanalytic phase in veterinary clinical pathology

Jean-Pierre Braun1, Nathalie Bourges-Abella2, Anne Geffre1, Didier Concordet3, Cathy Trumel1
1
Sciences cliniques, 2Sciences biologiques et fonctionnelles, Universite de Toulouse, UPS, INP, ENVT, UMS 0006, Toulouse, France; and
3
Universite de Toulouse, INP, ENVT, UMR 1331Toxalim, Toulouse, France

Key Words Abstract: This article presents the general causes of preanalytic variability with a few
Anticoagulant, circadian, handling, examples showing specialists and practitioners that special and improved care should
sampling, stress
be given to this too often neglected phase. The prean-alytic phase of clinical pathology
Correspondence
includes all the steps from specimen col-lection to analysis. It is the phase where most
laboratory errors occur in human, and probably also in veterinary clinical pathology.
J.P. Braun, ENVT, 23 Chemin des
Capelles, Toulouse Cedex 31076, France Numerous causes may affect the validity of the results, including technical factors, such
E-mail: jp.braun@envt.fr as the choice of anticoagulant, the blood vessel sampled, and the dura-tion and
conditions of specimen handling. While the latter factors can be defined, influence of
DOI:10.1111/vcp.12206 biologic and physiologic factors such as feeding and fasting, stress, and biologic and
endocrine rhythms can often not be con-trolled. Nevertheless, as many factors as
possible should at least be docu-mented. The importance of the preanalytic phase is
often not given the necessary attention, although the validity of the results and
consequent clinical decision making and medical management of animal patients would
likely be improved if the quality of specimens submitted to the labo-ratory was
optimized.

Table of Contents 5.3.1 Temperature

5.3.2 Centrifugation
1. Introduction 5.3.3 Final quality assessment of specimen before
2. Categories of preanalytic factors of variation analysis
3. Available information on preanalytic variability in
6. Biologic preanalytic factors of variation
veterinary clinical pathology
4. The pre-preanalytic subphase 6.1 Nutritional status and diet
5. Technical preanalytic factors 6.2 Effects of stress
6.3 Effects of drugs and pollutants
5.1 Blood collection
6.4 Biologic rhythms
5.1.1 Choice of anticoagulant and tube 6.5 Environment—Living conditions
5.1.2 Choice of the route and technique of speci-men 6.6 Exercise/sport
collection
7. Conclusion
5.1.3 Choice of needles and syringes
8. References
5.2 Other specimens
5.2.1 Urine
5.2.2 Saliva
5.2.3 Feces
Introduction
5.2.4 Cerebrospinal fluid
5.2.5 Other body fluids The preanalytic phase is defined by ISO 15189 as “The
processes that start, in chronological order, from the
5.3 Standard procedures at the laboratory clinician’s request, and include the examination

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Braun et al
request, preparation and identification of the patient,
collection of the primary sample(s), and transportation to
and within the laboratory, and end when the analytic
examination begins”.1 In routine veterinary clinical
pathology, the preanalytic phase deals mostly with blood
and urine specimens, and analyses of hematology,
coagulation, and biochemistry and cytol-ogy interpretations.
In human clinical pathology, blood sampling is
performed by specially trained and certified profes-sionals,
on patients duly prepared according to the required analyses,
such as by overnight fasting, and in a quiet isolated
environment. However, even in environments with high
standards, errors still may occur: the preanalytic phase is
reported to be respon-sible for the majority (ie, between 50%
and 75%) of laboratory errors, and recent surveys in human
labo-ratories show that most of these errors are not only
preventable2, but cause potential harm in 3–12% of all
cases.3
Importantly, the preanalytic phase in veterinary
medicine is often initiated remotely from an analytic
laboratory, either in an indoor or outdoor practice
environment, making adherence to good preanalytic practice
sometimes challenging. The quantitative importance of
preanalytic errors in veterinary clinical pathology has been
little investigated. To our knowledge, there is only one
quantitative report by an Austrian laboratory where
preanalytic errors accounted for about 2/3 of the errors
recorded.4 Except in animal hospitals and experimental set-
tings, sampling is performed by veterinarians or nurses, who
are skilled professionals but usually not specialists in
clinical pathology. In addition, in most cases the animals
have not been prepared for blood analysis: they may have
been fed, and stressed by transport and a new clinical
environment. There-fore, it is not uncommon for colleagues
in charge of clinical pathology laboratories to report that a
nota-ble proportion of specimens submitted for analysis are
of poor quality to the degree that they have to be rejected for
the performance of the required lab-oratory analyses.
Alternatively, the analysis of flawed specimens results in
errors and misinterpre-tations, repeats of sampling and
analyses, and alto-gether the inefficient use of personnel and
financial resources.
Therefore, it is necessary to be aware of the possi-ble
causes of preanalytic variability, be it to either pre-vent the
interfering factors whenever possible, or to take them into
account in the interpretation of results. This article reviews
the main causes of preanalytic variability in animals with
select specific examples.
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The preanalytic phase
Numerous preanalytic variations reported in animals are
available also in a search engine and in an open chapter of
the teaching website of the Toulouse Veterinary School. 5,6
Categories of Preanalytic Factors of Variation
Preanalytic factors can roughly be classified into 2 gen-eral
categories, (1) technical effects due to the sam-pling
technique and specimen management before analysis, and
(2) biologic factors inherent with the animal sampled.
The first category includes effects such as the choice of
anticoagulant, the sampling technique, and the stability of
the specimen during storage or ship-ping to a laboratory.
These effects are relatively easy to identify and to control
with adequate standard procedures.
The second category is more diverse and comprises
such effects as fasting, stress, sedation, and exercise.
Physiologic effects due to age, sex, breed, pregnancy,
lactation, etc are more relevant in the field of reference
interval determination and will not be considered here. In
most cases, biologic factors cannot be con-trolled, but their
effects should be documented and taken into account in the
interpretation of laboratory results.
Available Information on Preanalytic
Variability in Veterinary Clinical Pathology
There are many textbooks on preanalytic variability in
human clinical pathology, including a very useful col-
lection based on thousands of original articles summa-rizing
most effects published up to 2007.7 Much of this
information can likely be adapted to veterinary clinical
pathology, but some caution is necessary when trans-lating
information from people to animals, and confir-mation with
original data obtained in the species of interest is
recommended.
In veterinary clinical pathology, book chapters in
standard textbooks refer to preanalytic variability, but often
the provided information is not referenced to original
articles. In addition, there are some review articles for
domestic8–10 or laboratory animals11–13, and
recommendations with guidelines for quality con-trol of
preanalytic, analytic, and postanalytic phases have been
published by the American Society of Veter-inary Clinical
Pathology (ASVCP).14–16 However, the provided
information is often general, and details can only be found
in original studies.
9
The preanalytic phase
It is not easy to identify original studies on preana-lytic
effects in animals from data banks such as PubMed because
articles are rarely indexed to key words such as
“preanalytic” or “preanalytical”, and terms such as
“hemolysis” or “storage” will also retrieve many irrelevant
articles. A survey of PubMed performed in July 2013 with
the keywords “preanalytic” and “preanalytical” retrieved >
2000 ref-erences and included many references with no rele-
vance to clinical pathology. When filtered for “other (than
human) animal species”, < 150 references remained.
Furthermore, much information about sta-bility of analytes
or effects of anticoagulants, storage, drugs, etc can be found
as integrated information in method validation studies, but
as it is not listed in the keywords it will not be indexed. In
addition, in such studies, the number of cases is often low
and the infor-mation is not very detailed. Several aspects
deserve attention when screening such studies for
information pertaining to preanalytic variation. For instance,
a statement that “all hematologic and biochemical val-ues
were within reference ranges at all time points”17 suggests
that routine analytes were unchanged under the conditions
tested, but without a detailed list of vari-ables the
information remains hypothetical. Another limitation is the
use of inadequate methods, such as heterologous
hemoglobin and albumin for the study of interference by
hemolysis and hyperproteinemia in ion measurements in
canine sera.18 Simultaneous test-ing of multiple co-
variables also limits interpretation of the reported data, for
example, reference intervals determined in wild animals
indiscriminately of age, methods of trapping or hunting, and
season.19 Of course, observed effects sometimes depend on
the ana-lytic methods used. Therefore, preanalytic effects
observed with a wet chemistry method were not the same as
with a dry chemistry system in a veterinary practice
setting.20 Finally, biologic effects such as bio-logic rhythms
can represent a preanalytic factor and have been described
in healthy or apparently healthy animals; however, no
reports are available for diseased animals.
Interestingly, “oral tradition” appears to be a common
source of information in the laboratory world, rather than
scientific information, as the origi-nal source of commonly
accepted preanalytic effects is sometimes impossible to find
in the most fre-quently used databases. One such example is
the rec-ommendation by most academic and commercial
laboratories not to use gel separator serum tubes for the
measurement of progesterone in animal species. To our
knowledge, only a few references document the reason for
this recommendation in human
10
Braun et al
blood21–23, a single report in cattle24, but none in other
species.
Even if such recommendations are based on expertise
and common knowledge, they often lack sci-entific
documentation and thus should be confirmed by accepted
scientific and statistical study designs. In addition, older
reports dating back 10–20 years are somewhat limited by the
profound change and pro-gress that has taken place in
instrument technology, therefore such published
observations should some-times be regarded with caution.
On the other side, the amount of provided infor-mation
is not always proportional to the clinical rele-vance. For
instance, cortisol, corticosterone and their catabolites have
been studied in plasma, serum, urine, saliva, and feces of
almost all species from fish to people as stress markers.
Nowadays, such analyses are not the most frequently
required.
Caution is also advised when extrapolating infor-
mation from human studies to veterinary species.
Obviously, strictly technical preanalytic effects (such as
glycolysis in whole blood, or zinc leakage from rub-ber
stoppers from vacuum tubes) are likely applicable across
species. However, true matrix effects can only be assessed
with species-specific validation studies. The following
review will provide a succinct overview on preanalytic
variations in veterinary clinical pathology based on
published data.
The Pre-preanalytic Subphase
There is no internationally accepted definition of this
subphase. The idea of its promoters25 was to clearly identify
actions associated with the test selection by the clinicians
from the actual activity of specimen collection and
management. Other definitions include all “initial
procedures not performed in the clinical laboratory and not
under the control of laboratory personnel”26, that is,
collection, identification, and transportation of the
specimen(s).3 This represents the situation often observed in
veterinary medicine, where specimen collection and
handling is performed outside the laboratory by
nonlaboratory professionals.
In veterinary clinical pathology, often organ or patient
cohort specific tests such as a “liver panel” or “geriatric
profile” are offered for pets, based on an assumption that the
selected tests provide the best value for money for particular
clinical settings, in gen-eral without scientific evidence. On
the basis of avail-able evidence, we postulate that the pre-
preanalytic phase could and should be optimized in
veterinary clinical pathology.
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Braun et al
Technical Preanalytic Factors
Blood collection
Choice of anticoagulant and tube
Many general recommendations have been published in
chapters of textbooks or specialized review articles for
people, pets, exotic and laboratory animals.27–31
Whole blood, plasma, and serum: Anticoagulated
whole blood is the specimen of choice for most hema-tology
variables. Textbooks generally recommend sodium or
potassium-EDTA in mammals, and heparin or heparin salts
in birds, reptiles, and some other spe-cies.14 In some
mammals, particularly cats, EDTA often causes platelet
aggregation and clumping, so additional compounds such as
prostaglandins or a mixture of cit-rate, theophylline,
adenosine, and dipyridamole (CTAD) have also been
suggested to prevent platelet aggregation.32,33 Cell counts
obtained from specimens anticoagulated with EDTA,
citrate, or heparin are gen-erally comparable, including
canine and feline platelet and reticulocyte concentrations.
However, validations can be indicated depending on the
analyzer and the technology and reagents used.34
Most textbooks recommend the use of serum or heparin
plasma for routine biochemistry, sodium cit-rate blood for
coagulation, and sodium fluoride tubes for the measurement
of unstable molecules such as glucose or ketones. The
results of biochemical analy-ses of heparin plasma and
serum are very similar for most analytes. Some of the
differences observed would have little or no effect on
clinical interpreta-tion, such as for many canine and avian
variables.35– 37 However for some analytes, specific plasma
and serum reference intervals need to be established, for
instance, human bile acids are about 60% lower in heparin
plasma than in serum.38 In cattle and sheep, differences
between serum and citrate or EDTA
plasma are more numerous and relevant, for example, higher
AST and CK activities in serum.39,40 Point-of-
care systems and potentiometry analyzers usually require
anticoagulated whole blood.
The interchangeability of serum and different plas-mas
for biochemistry profiles is somewhat surprising, as clot
formation results in the modification of the con-centration
of some analytes. For instance, intracellular molecules such
as potassium can leak from cells, as a consequence,
potassium concentration can be lower in plasma than in
serum in species with high intracellular potassium levels or
in cases with thrombocytosis.41,42 At the same time, clot
formation can also result in sequestration of analytes; for
instance, mean copper
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The preanalytic phase
concentration is about 25% lower in serum than plasma of
ruminants, although the interindividual variability appears
considerable.43–45 Furthermore, the delay caused by clot
formation can allow degradation, for example, of unstable
peptides. Such analytes should therefore be determined in
plasma samples with addition of protease inhibitors such as
aproti-
Due to the interference of fibrinogen with
b-globulin migration, protein electrophoresis should be
performed using serum. In dogs, fibrinogen can be
precipitated with ethanol if only plasma is available, unless
capillary electrophoresis can be substituted.47
When multiple blood tubes are collected from the same
patient, carryover of anticoagulants or other additives should
be avoided. The recommenda-tions in human clinical
pathology are to start with the citrate tube for coagulation,
followed by plain serum tube, followed by heparin, EDTA
tube and fluoride/oxalate tube.48 To our knowledge,
possible effects of a different order of sampling have not
been studied in animals, but in some environments, citrate
tubes are preferably filled after the plain serum tube to avoid
specimens with initiated clotting (personal communication).
Types of tubes: For safety and convenience, glass tubes
have been mostly replaced by plastic tubes. The latter have
been shown to yield comparable results for most analytes in
hematology, biochemistry49, endocri-nology50, and
coagulation51 with human blood, but have not been
validated with animal specimens. In horses, glass syringes
permitted longer stability than
plastic syringes or plastic tubes for pO2, but not for
pH.52,53
Caution or better validation tests may be advised with
tubes that contain additives, such as surfactant, coating, and
gel layers to optimize the recovery or separation of
specimens. Interferences have been observed for instance
for total triiodothyronine in people.54 Importantly, the
expiry date of tubes should be respected, although the
security margins are usually quite large. Concentrations of
most chemical analytes were not altered in lithium-heparin
vacuum tubes used for canine blood up to 11 months after
the expiration date.55
Choice of the route and technique of specimen collection
In large animals, there is usually little difference in routine
hematology and biochemistry data for samples collected
from different large veins, whereas in smaller species like
laboratory rodents or cats statistically significant differences
have been reported.56,57 Samples collected from the tail
vessels in cows can
11
The preanalytic phase
sometimes yield a mixture of venous and arterial blood
inappropriate for blood gas determinations58, and inor-ganic
phosphate concentrations have been reported to be higher in
tail specimens than jugular vein speci-mens.59 In cats and
dogs, specimens collected from ear capillaries can notably
differ from those obtained from a large vein, for example,
HCT and plasma proteins in cats60, and plasma lactate
concentration in dogs.61 In rats, HCT, RBC, and WBC
counts were reported to be higher in blood collected by
terminal heart puncture than by in life orbital sinus or tail
vein collection, whereas MCV and MCH were identical.62
In general, in laboratory rodents, the quality of the specimen
also depends on the sampling method.63
Specimen collection from indwelling catheters is
routinely performed in human clinical pathology. After
proper complete removal of dead volume prior to actual
sample collection, variables such as coagulation times in
dogs64 and routine biochemistry and hematology profiles in
horses65 were comparable to variables in specimens
obtained by direct venipuncture.
Skin cleansing is not a common practice in ani-mals,
and there are no reports on potential effects on laboratory
analysis, such as reported in people. In con-trast, proper
technique, particularly in small animals is of utmost
importance, as incorrect venipuncture causes discomfort,
hematomas or more extensive tis-sue damage. This is not
only of potential relevance for animal welfare, but also of
concern for clotting initiation and spurious increase of
enzyme activities (eg, increased CK activity due to muscle
damage).66
The volume of blood collected from an animal includes
consideration of the total body size and the respective total
blood volume, particularly in small animals such as
laboratory rats and mice, and of course the analytic needs.
The rule of thumb is that removal of < 7.5% of the total
blood volume will not elicit regenerative effects on
hematology variables.67 However, if blood collection is
done directly with vacuum tubes, the volume can greatly
exceed the volume requirements of modern instru-ments,
resulting in wasted specimen. Therefore, in
small animals such as cats and small dogs, microtu-bes are
appropriate.68,69 Blood collection into tubes
with anticoagulant must consider proper proportions
between blood and anticoagulant, as an excess of EDTA can
cause artifacts such as RBC shrinkage70, or an excess of
citrate results in prolongation of coagulation times in canine
blood.71
Choice of needles and syringes
Possible effects of needle gauge, free flow vs syringe vs
vacuum tube have not been scientifically studied and
12
Braun et al
reported. In human clinical pathology, it is recom-mended
to use needle gauges large enough to avoid intense shearing
of RBC and hemolysis, and, for the same reason, to exert
only moderate negative pressure when aspirating blood with
syringes.48 In veterinary clinical pathology, there are
particular challenges when collecting blood from small
animals. For instance, the collection of small volumes can
be performed using capillary tubes, which appear to result
in no clinically relevant differences in routine
hematology and biochemistry variables in cats and
dogs.56,68,69,72,73 In zoo and wild animals, blood sam-
pling can be particularly challenging as they are not used to
handling. An interesting alternative is the use
of blood-sucking beetles such as Dipetalogaster
maximus.74,75 In rabbits, the results of about 50% of
the measured variables (eg, HGB concentration, HCT,
reticulocyte count, concentrations of albumin, total protein,
glucose, creatinine, urea, amylase and mag-nesium, and
ALT activity) did not differ from those obtained from
conventional specimens.74,75
Other specimens
Information about preanalytic factors of variation for other
biologic specimens is more limited. Many recom-
mendations on sampling and specimen processing are found
in chapters of cytology textbooks (see for instance 76–78),
but original studies are often lacking; for instance, rapid
processing of cytology specimens is generally
recommended with reference to human clin-ical pathology
and not to specific animal studies. The general consensus is
that for most specimens a short transition time to the
laboratory and immediate analy-sis are the process of
choice.
Urine
In contrast to people, urine collection from animals can
require restraint or even sedation to perform cath-eterization
or cystocentesis, which can influence urine composition.
Free catch samples can be contaminated with bacteria, such
as in horses.79
A spot urine can easily be obtained, but 24-hour urines
require the use of metabolic cages. For efficient use, the
animals must previously be accustomed to metabolic cages
for a few days, for example, 3–4 days in rats.80 Moreover, 2
main alterations can occur in metabolic cages: (1) possible
instability of the analyte;
(2) unsuspected loss of the analyte on the walls or in the
funnel of the cage; this requires proper rinsing of the cage
as, for example, for the determination of urine clearances in
dogs.81
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Braun et al
Saliva
Saliva can be a good substitute for plasma or serum for the
determination of certain variables; however, collection and
storage have only been described in connection with very
select analytes in few species. In dogs, cortisol concentration
did not notably differ with sampling conditions82, and
remained unchanged during the first 4 minutes of restraint.83
Feces
Feces are sometimes the obligatory specimen for analyses
such as occult blood testing and endocrine markers in wild
animals. Consideration should be given to potential
interference of the carnivorous
diet with the analytic method, as shown for occult blood in
dogs and cats.84,85 The elimination of hor-
mone catabolites in feces may be variable and not
reflect immediate effects due to digestive tract tran-sit.86,87
In wild animals, feces are exposed to envi-
ronmental factors which may affect the concentration of the
variables of interest.88
Cerebrospinal fluid
Stability of cells in cerebrospinal fluid (CSF) from dogs and
cats can be improved by adding 10% serum to the
specimen.89 Protein concentration in dogs and cats but not
horses is almost twice higher in CSF collected by lumbar
than by atlanto-occipital aspiration.90–92 In dogs or horses,
the presence of intact or hemolyzed blood had no or very
marginal effects on protein concentration, CK activity, or
WBC counts.93–96
Other body fluids
Synovia. In healthy horses, chemical composition and
WBC count differed according to the joint sam-
pled.97–99 Cell counts in repeated canine specimens did not
differ.100
Peritoneal fluid. In horses, protein content and cell
counts can be increased after laparotomy, castration, or
enterocentesis,101–103 but not after repeat samplings over 24
hours,104 blood contamination105, or after foaling in
mares.106
Bronchoalveolar Lavage. For a review in cats and
dogs, see107. In healthy dogs, the differential WBC count in
bronchoalveolar lavage (BAL) did not differ between right
and left lung108 and between the differ-ent lobes.109
Cytology results for BAL and tracheal wash differed in
about 2/3 of canine cases.110 In cats, the differences
observed between 3 consecutive lavages were slight.111
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The preanalytic phase
Standard procedures at the laboratory
Prior to analysis, gentle mixing on specialized rocking
platforms and avoidance of vigorous shaking are rec-
ommended.48 The most common unstable analyte is glucose
due to in vitro glycolysis. Sodium fluoride tubes are
therefore recommended for reliable glucose determinations.
Alternatively, rapid processing of
blood collected in gel separator tubes is also recom-
mended.112,113
Temperature
Room temperature. There is no universal recommenda-
tion concerning the duration and temperature condi-tions,
nor is there an internationally accepted definition of the
stability of a variable. In general, it is recommended to
process specimens within 2 hours from collection to obtain
optimal results114, however many analytes are stable for
much longer periods, even at room temperature. In general,
hematology variables remain relatively stable in common
domestic species. In feline EDTA blood stored up to 48
hours, increased MCV, HCT, reticulocyte and eosinophil
counts, and decreased MCHC and monocyte counts were
found. Effects were less pronounced when CTAD was
added.115 In canine EDTA blood stored up to 48 hours,
increased MCV and HCT and decreased platelet and
monocyte counts were measured.116–118 These changes can
also be observed on canine cell scatter-grams. In contrast, in
sharks (Carcharhinus plumbeus),
WBC morphology was distorted after 3 hours storage at 4–
10°C.119
Likewise, many biochemical analytes are relatively
stable after 24–48 hours storage at room temperature,
nevertheless it is a standard procedure to collect and freeze
serum at 20°C in most labora-tories if storage is required. In
addition, to prevent degradation of light-sensitive analytes
such as biliru-bin or protoporphyrin specimens must be
stored in the dark. For urine, little information is available.
In human urine, test strip analyses results were compa-rable
after 2 and 4 hours at room temperature.120
Refrigeration. Reports on effects of refrigeration on
hematology results are controversial, for example, for WBC
in dogs.34,118,121 The canine platelet count has
been reported stable for 3 days122, however in canine blood,
platelet clumping was enhanced in refrigerated
specimens.123
Unstable analytes such as catecholamines, ACTH, and
other peptides require immediate refrigera-
tion; however, reports are contradictory for
ammonium.124,125
13
The preanalytic phase
Freezing. Freezing is not an option for hematology
specimens, as the cells deteriorate due to the building of
microcrystals, resulting in morphologic changes and leakage
of cytoplasmic components. For plasma and serum, freezing
is the standard procedure for long-term storage; however,
conditions of storage should be documented or validated for
each analyte in each spe-cies. Freezing can greatly alter the
composition of urine. Freezing of centrifuged human urine
led to the formation of precipitates, and lower calcium and
pro-tein concentrations, unless the specimens were vigor-
ously shaken at room temperature and before analysis.126 In
dogs, urine protein-to-creatinine ratio
(UPC), albumin, and cystatin C were stable up to 3 months
at 20°C.127–130 However, plasma enzyme
activities are generally stable for long periods at 20°C, urine
enzyme activities such as GGT in rats, rabbits, and horses,
and -N-acetylglucosaminidase
(NAG) in dogs and cats are partially inactivated by
freezing.131,132
Serum and plasma specimens have a concentra-tion
gradient of the analytes after thawing, requiring careful but
thorough homogenization.133 Repeat freeze-thawing cycles
are usually not recommended for analytes such as hormones,
cytokines, and enzymes, however, many routine chemical
analytes in canine plasma appeared unchanged even after 3
freeze-thaw cycles.134
Centrifugation
Relatively high speed centrifugation (eg, 2000g for 10
minutes) is the basic procedure to separate plasma and
serum from the cellular components of blood.135 Likewise,
cytology samples are sometimes centrifuged at low speed to
concentrate cells while preserving cell morphology. Urine
sediment preparation by centrifu-gation appears to be
independent of speed for cell and crystal counts within a
range of 400–3900g.136
Final quality assessment of specimen before analysis
Particularly in commercial reference laboratories, a careful
assessment of specimen quality is important, as such
samples have usually undergone transport at more or less
optimal conditions. Standard monitoring includes the
documentation of plasma and serum color. Visual estimation
of color changes is relatively imprecise, even with color
charts, and they should be
quantified by objective techniques, as established for human
specimens.137,138
Hemolysis. A pink-to-red discoloration indicates
hemolysis, the most frequent laboratory preanalytic
interference in human clinical pathology, occurring in
14
Braun et al
more than 3% of specimens submitted.139,140 It is likely a
frequent preanalytic error in veterinary clinical pathology.
Free hemoglobin interferes with spectrometric
measurements by absorbing light, so the degree of
interference of hemolysis depends on analyzers and methods
used.141 In some cases, interference is proportional to
hemolysis intensity, permitting the use of correction
equations, for instance, for haptoglobin in cattle and
sheep.142 Fur-thermore, hematology variables will be
altered by hemolysis, and biochemistry profiles can be
skewed after release of intracellular constituents, such as
ions (eg, potassium), enzymes (eg, ALT, AST), and proteins
(mainly HGB).
Lipemia usually results in a whitish to milky opaci-
fication of serum or plasma, depending on the type and
amount of lipid present. Lipid droplet-related light scattering
interferes with hematologic measurements, as documented
for platelets in human samples.143 Likewise, there is
interference with many photometric measurements of
biochemistry analytes including hemoglobin when
measured by the monochromatic method.144 Water
displacement by lipids can result in skewed ion
concentrations.145 In such specimens, ion measurements
should be performed by direct rather than indirect
potentiometry. Lipid interference can be reduced by
ultracentrifugation, refrigeration, or by “clearing agents”.
Some of the latter have been vali-dated for use in animal
specimens, for example, poly-ethylene glycol in canine
serum.146 Intense lipemia is often associated with increased
hemolysis in canine specimens.147
Icterus. Increased bilirubin concentrations or icteric
plasma and serum can interfere with photometric readouts
in chemistry. For interferograms established
for canine, feline, bovine, and equine serum, see refer-
ence148.
Clots. The presence of small or large clots indi-cating
inadequate anticoagulation during blood col-lection is
probably an important cause for specimen rejection in
veterinary medicine. Microclots may be easily overlooked if
specimens are not carefully examined before processing.
Microclots not only cause erroneous hematology cell counts
and coagu-lation results but they can also clog instrument
lines or contaminate sampling tubes. Microclots and
microscopic platelet clumps occur in feline samples, and
less frequently in canine EDTA blood.32,149,150
Small white flakes sometimes can be observed in thawed
heparin plasma. Their origin is not fully understood and their
possible effects on plasma ana-lytes have not been
documented.151
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Braun et al
Biologic Preanalytic Factors of Variation
Biologic factors such as species, strain, sex, and age are just
a few and the best defined factors which can com-plicate
proper clinical interpretation of laboratory data. In addition,
physiologic aspects such as reproductive cycle, nutrition,
climate, breed, and all circumstances involved with blood
sampling in wild animal species, such as trapping and
sedation can affect test results. More recently, a number of
studies have addressed intraindividual variation.152,153
Nutritional status and diet
Fasting is mostly relevant in monogastric animals. Although
generally accepted, presampling fasting is considered as a
deviation from the normal state by some authors,154 and
does not improve the medical
interpretation of certain tests such as blood lipids in people
with normal food intake.155,156 Overnight fast-
ing is an adaptation from human medicine, decreasing the
incidence of postprandial lipemia. In animals, even brief
fasting periods can strongly modify the concen-
tration of certain analytes, such as higher bilirubin and
triglyceride concentrations in equids,157,158 and higher
bilirubin in rats.159 The approximate 12 hour over-night
fasting period is adequate for most analytes. For example, in
rats, sheep, and monkeys, most hematol-ogy and chemistry
variables were almost unchanged
up to 16 hours after food withdrawal.160–162 However in
healthy dogs, fasting urea163,164 or triglyceride
concentrations165,166 may be clearly lower than nonfa-
sting levels. Interestingly, fasting does not lead to steady-
state plasma concentration of some analytes, such as canine
free fatty acids.167
Postprandial increase in blood glucose in dogs was
reported very early. The possible effects of a meal result
from the absorption of digested food and metabolites,
secretions from the gastrointestinal tract (eg, gastric acid,
pancreatic enzymes, hepatic bile acids), and hor-mone
secretions (eg, insulin). In dogs and cats, the main changes
include increased plasma concentrations of urea, glucose,
creatinine, ammonium, bile acids, trypsinogen, and
triglycerides, and lower concentra-tions of bicarbonate.168
Importantly, the intensity and duration of the observed
variations differ notably according to the type and amount
of food ingested, as shown for increases of urea and
creatinine in dogs, for example, the latter being more
markedly increased after ingestion of cooked than raw
meat.163
Most routine tests are not influenced by the
composition of the diet. However, in dogs, a diet change
caused a notable difference in fasting plasma
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The preanalytic phase
cholesterol concentration, but had no influence on tri-
glyceride, phospholipid, or FFA concentration.169 In
neonates, colostrum ingestion shortly after birth results in
increased plasma total proteins and immuno-globulin levels.
In ruminants, successful colostrum
ingestion can be demonstrated based on increased GGT or
ALP activities in neonatal serum.170,171 In canine
urine, false-positive glucosaminoglycan results were
observed in a dog supplemented with an algal
preparation.172
In laboratory rodents and rabbits, special attention
needs to be addressed to physiologic coprophagia. The
plasma urea concentration was 20–40% higher in rats which
were allowed to consume their feces,173 but the HCT was
not affected. The general possible effects of physiologic
coprophagia on plasma chemistry variables have not been
reported to our knowledge.
Effects of stress
The definition of stress is not straight forward, but it can
include any effects resulting in an activation of the
adrenomedullary (acute stress) or cortical (subacute, chronic
stress) cells. Depending on the species and individual
conditioning, any activities including trans-port, restraint or
capture, work, environmental, social conditions, and time
spent in a waiting room174 can result in stress.
Stress-related blood sampling in cats can cause
hyperglycemia (up to 25 mmol/L), hyperlactatemia, and
lymphocytosis.175 Stress due to capture and restraint of wild
animals should be avoided just for reasons of animal
welfare. Captured animals should be allowed a recovery and
adaptation period before sampling for laboratory purposes is
done. Handling and sampling can cause stress in small
laboratory animals.176 Increased plasma CK activity in
rabbits and higher plasma cortisol concentration in monkeys
have been reported with repeat sampling, therefore animal
training and conditioning should be standard practice in
research organizations.177 Duration of handling had greater
effects than the gentle or rough handling procedure used on
increasing plasma corti-costerone and lactate concentrations
in poultry.178 In cattle, handling as well as physical fatigue
and dehy-dration and undernutrition connected with
transport have been reported to affect levels concentrations
in place of levels of stress indicators (eg, increased plasma
catecholamines and glucocorticoid concentra-tions, total
WBC and neutrophil counts, decreased T
lymphocyte count, increased activities of muscle enzymes
such as CK).179–183
15
The preanalytic phase
Interestingly, the type of transport did not affect the
stress response as such, for example, the increase in plasma
corticosterone concentration was similar (approximately 2–
3-fold) in mice transported to an
adjacent room for euthanasia and in rats transported by
airplane.184,185
Effects of drugs and pollutants
Drugs and pollutants can either affect the molecule itself, or
its metabolites. An example for interference with the
analytic technique are false high plasma chlo-ride
concentrations in dogs treated with bromide.186 An example
for a pharmacologic effect is the increased activity of ALP
due to induction of a specific isoenzyme in the presence of
increased intrinsic or extrinsic adre-nocortical stimulation.
A rare effect, so far only reported in people, is interference
with the excipient of a dexamethasone preparation
containing a high con-centration of creatinine.187
Presampling sedation or anesthesia is compulsory in
many cases, either for compliance with animal wel-fare
legislation, or if the procedure is lasting longer than an
animals’ cooperation can be expected. Seda-tion effects are
usually minimal causing few potential clinical
misinterpretations. However, they differ nota-bly according
to the drugs, associations, and doses used, which means that
any new and nonstandard procedure must be validated
before interpreting results in sedated animals. In
anesthetized people, body posi-tion causes water shifts with
corresponding changes in analyte concentrations.188 To our
knowledge, the effect of body position has not been
documented in animals, except for prolonged recumbency
causing increased plasma activities of AST, LDH, and CK
in downer cows with milk fever.189
In general, potential adverse treatment-related effects
of pharmaceutical compounds need to be addressed in
animals by law, and compounds causing kidney or liver
damage and respective clinical pathol-ogy results are
sometimes published. An area that has been much
investigated are treatment-related effects due to
glucocorticoids and nonsteroidal anti-inflam-matory drugs
(NSAIDS). Typically, leukocyte numbers increase due to
higher neutrophils, while lymphocytes
and eosinophils are lower. Increased ALP activity and iron
concentrations are commonly seen in dogs.190,191
Examples for effects by pollutants or environmen-tal
toxicants are the inhibition of cholinesterases by
organophosphates and carbamates192,193, and delta-
aminolevulinate dehydratase (and consequently
compromised hemoglobin synthesis) with lead intoxi-
cation.194 Other environmental pollutants have more
16
Braun et al
subtle effects, such as shown for organohalogen com-
pounds in pups195,196 and in wild raptors.197,198
Biologic rhythms
While very stringent experimental designs in short-term
studies can be considered representative, labora-tory results
can be influenced by biologic rhythms or endocrine cycling,
including reproductive cycles, and, predominantly in wild
animals, behavior associated with climate and location,
amount and type of food available, migration over large
distances, or molting in birds, and many others. Careful
documentation of rel-evant parameters and variables will
help avoiding the drawing of erroneous conclusions. 199 For
some ana-lytes, variability is very high throughout the
different seasons, for example, Melanocyte Stimulating
Hor-mone (a-MSH) in horses.200 Circadian rhythms of a
particular analyte can differ according to species; for
instance, plasma glucocorticoids peak in the morning
in people and in the early evening in rats, while there is
almost no change in dogs.201,202 Circadian fluctua-
tions of RBC and WBC counts have been described in
different mouse strains.203
Environment and living conditions
Management and husbandry practices can also influ-ence
certain blood variables such as milking frequency altering
plasma free fatty acid (FFA) and b-hydroxybu-tyrate
concentrations.204 Adaptation to high altitude
caused higher PCV, HGB concentration, and RBC count in
poultry205, horses206,207, or in wild animals
such as Sloth bears208 and otters.209
In wild animals, environmental factors can nota-bly
affect some variables, and a careful distinction is advised
between free-ranging animals and animals living in
captivity.210 Infestation by parasites, the nutritional status
and the overall health assessment
can vary significantly and must be documented very
carefully.211,212 In migratory birds, conditions can dif-
fer from year to year, as observed in Sage Grouse. 213
However, hematology and biochemistry variables of
Bottlenose dolphins were mostly stable for 7 years at 4
different locations.214 Habitat restriction due to human
settlement can represent result in increased fecal corti-
costeroids, and higher CK and AST activities in wolves
living within a park or close to farmland.215,216 In labo-
ratory animals, initiatives for enrichment had no sig-nificant
effect on routine murine hematology results, with the
exception of increased interindividual vari-ability.217
Interestingly, in untrained Cynomolgus monkeys, serum
thyroid hormone concentrations
Vet Clin Pathol 44/1 (2015) 8–25 ©2014 American Society for Veterinary Clinical Pathology
Braun et al
decreased with increasing length of lag time during
218
which the monkeys could observe their mates being bled.
Exercise/sport
Not much literature is available on training effects, such as
absence of increased cardiac Troponin I con-centration in
Thoroughbred horses after intense train-ing.219 Importantly,
training effects may be dependent on the level of fitness of
the tested individuals, for example, changes may not be
identical in well-trained and maladapted horses.220 Working
Border Collies had lower plasma cholesterol and
triglyceride concentra-tions than family dogs.221 In sled
dogs, PCV, RBC, and
creatinine progressively decreased with training, whereas
WBC counts slightly increased. 222,223
Physical effort-related effects differ considerably with
the type of effort. In dogs, the most notable changes are
moderate increases in PCV and marked
increases in plasma lactate and ammonium concentra-
tion.224,225 As the intensity and duration of changes in
laboratory variables depend on intensity and duration of
physical exercise, blood sampling shortly after exer-cise
should be avoided, as the values may not represent the
“normal” or “healthy” situation.
Conclusion
The preanalytic factors possibly affecting the results of
laboratory analyses are numerous and can have cumu-lative
or compensatory effects. Although it is impossi-ble to
control all preanalytic factors, the implementation of well-
defined standard practices can help avoid many of them.
Well-recognized preanalytic influences, and careful and
detailed documentation will contribute to further
improvement of overall ana-lytic performance in veterinary
clinical pathology, as recommended by the ECVCP and
AVCP with SOPs required in accredited laboratories.
Disclosure: The authors have indicated that they have no
affiliations or financial involvement with any orga-nization
or entity with a financial interest in, or in financial
competition with, the subject matter or mate-rials discussed
in this article.
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