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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

8 Vet Clin Pathol 44/1 (2015) 8–25 ©2014 American Society for Veterinary Clinical Pathology
Braun et al The preanalytic phase

request, preparation and identification of the patient, Numerous preanalytic variations reported in animals are
collection of the primary sample(s), and transportation to available also in a search engine and in an open chapter of
and within the laboratory, and end when the analytic the teaching website of the Toulouse Veterinary School. 5,6
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. Categories of Preanalytic Factors of Variation
In human clinical pathology, blood sampling is
performed by specially trained and certified profes-sionals, Preanalytic factors can roughly be classified into 2 gen-eral
on patients duly prepared according to the required analyses, categories, (1) technical effects due to the sam-pling
such as by overnight fasting, and in a quiet isolated technique and specimen management before analysis, and
environment. However, even in environments with high (2) biologic factors inherent with the animal sampled.
standards, errors still may occur: the preanalytic phase is
reported to be respon-sible for the majority (ie, between 50% The first category includes effects such as the choice of
and 75%) of laboratory errors, and recent surveys in human anticoagulant, the sampling technique, and the stability of
labo-ratories show that most of these errors are not only the specimen during storage or ship-ping to a laboratory.
preventable2, but cause potential harm in 3–12% of all These effects are relatively easy to identify and to control
cases.3 with adequate standard procedures.

Importantly, the preanalytic phase in veterinary The second category is more diverse and comprises
medicine is often initiated remotely from an analytic such effects as fasting, stress, sedation, and exercise.
laboratory, either in an indoor or outdoor practice Physiologic effects due to age, sex, breed, pregnancy,
environment, making adherence to good preanalytic practice lactation, etc are more relevant in the field of reference
sometimes challenging. The quantitative importance of interval determination and will not be considered here. In
preanalytic errors in veterinary clinical pathology has been most cases, biologic factors cannot be con-trolled, but their
little investigated. To our knowledge, there is only one effects should be documented and taken into account in the
quantitative report by an Austrian laboratory where interpretation of laboratory results.
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 Available Information on Preanalytic
clinical pathology. In addition, in most cases the animals Variability in Veterinary Clinical Pathology
have not been prepared for blood analysis: they may have
been fed, and stressed by transport and a new clinical There are many textbooks on preanalytic variability in
environment. There-fore, it is not uncommon for colleagues human clinical pathology, including a very useful col-
in charge of clinical pathology laboratories to report that a lection based on thousands of original articles summa-rizing
nota-ble proportion of specimens submitted for analysis are most effects published up to 2007.7 Much of this
of poor quality to the degree that they have to be rejected for information can likely be adapted to veterinary clinical
the performance of the required lab-oratory analyses. pathology, but some caution is necessary when trans-lating
Alternatively, the analysis of flawed specimens results in information from people to animals, and confir-mation with
errors and misinterpre-tations, repeats of sampling and original data obtained in the species of interest is
analyses, and alto-gether the inefficient use of personnel and recommended.
financial resources. In veterinary clinical pathology, book chapters in
standard textbooks refer to preanalytic variability, but often
the provided information is not referenced to original
Therefore, it is necessary to be aware of the possi-ble articles. In addition, there are some review articles for
causes of preanalytic variability, be it to either pre-vent the domestic8–10 or laboratory animals11–13, and
interfering factors whenever possible, or to take them into recommendations with guidelines for quality con-trol of
account in the interpretation of results. This article reviews preanalytic, analytic, and postanalytic phases have been
the main causes of preanalytic variability in animals with published by the American Society of Veter-inary Clinical
select specific examples. Pathology (ASVCP).14–16 However, the provided
information is often general, and details can only be found
in original studies.

Vet Clin Pathol 44/1 (2015) 8–25 ©2014 American Society for Veterinary Clinical Pathology 9
The preanalytic phase Braun et al

It is not easy to identify original studies on preana-lytic blood21–23, a single report in cattle24, but none in other
effects in animals from data banks such as PubMed because species.
articles are rarely indexed to key words such as Even if such recommendations are based on expertise
“preanalytic” or “preanalytical”, and terms such as and common knowledge, they often lack sci-entific
“hemolysis” or “storage” will also retrieve many irrelevant documentation and thus should be confirmed by accepted
articles. A survey of PubMed performed in July 2013 with scientific and statistical study designs. In addition, older
the keywords “preanalytic” and “preanalytical” retrieved > reports dating back 10–20 years are somewhat limited by the
2000 ref-erences and included many references with no rele- profound change and pro-gress that has taken place in
vance to clinical pathology. When filtered for “other (than instrument technology, therefore such published
human) animal species”, < 150 references remained. observations should some-times be regarded with caution.
Furthermore, much information about sta-bility of analytes
or effects of anticoagulants, storage, drugs, etc can be found
On the other side, the amount of provided infor-mation
as integrated information in method validation studies, but
is not always proportional to the clinical rele-vance. For
as it is not listed in the keywords it will not be indexed. In
instance, cortisol, corticosterone and their catabolites have
addition, in such studies, the number of cases is often low
been studied in plasma, serum, urine, saliva, and feces of
and the infor-mation is not very detailed. Several aspects
almost all species from fish to people as stress markers.
deserve attention when screening such studies for
Nowadays, such analyses are not the most frequently
information pertaining to preanalytic variation. For instance,
required.
a statement that “all hematologic and biochemical val-ues
Caution is also advised when extrapolating infor-
were within reference ranges at all time points”17 suggests
mation from human studies to veterinary species.
that routine analytes were unchanged under the conditions Obviously, strictly technical preanalytic effects (such as
tested, but without a detailed list of vari-ables the
glycolysis in whole blood, or zinc leakage from rub-ber
information remains hypothetical. Another limitation is the
stoppers from vacuum tubes) are likely applicable across
use of inadequate methods, such as heterologous
species. However, true matrix effects can only be assessed
hemoglobin and albumin for the study of interference by
with species-specific validation studies. The following
hemolysis and hyperproteinemia in ion measurements in
review will provide a succinct overview on preanalytic
canine sera.18 Simultaneous test-ing of multiple co- variations in veterinary clinical pathology based on
variables also limits interpretation of the reported data, for published data.
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 Pre-preanalytic Subphase
the ana-lytic methods used. Therefore, preanalytic effects
observed with a wet chemistry method were not the same as There is no internationally accepted definition of this
with a dry chemistry system in a veterinary practice
subphase. The idea of its promoters25 was to clearly identify
setting.20 Finally, biologic effects such as bio-logic rhythms actions associated with the test selection by the clinicians
can represent a preanalytic factor and have been described from the actual activity of specimen collection and
in healthy or apparently healthy animals; however, no management. Other definitions include all “initial
reports are available for diseased animals. 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
Interestingly, “oral tradition” appears to be a common veterinary medicine, where specimen collection and
source of information in the laboratory world, rather than handling is performed outside the laboratory by
scientific information, as the origi-nal source of commonly nonlaboratory professionals.
accepted preanalytic effects is sometimes impossible to find In veterinary clinical pathology, often organ or patient
in the most fre-quently used databases. One such example is cohort specific tests such as a “liver panel” or “geriatric
the rec-ommendation by most academic and commercial profile” are offered for pets, based on an assumption that the
laboratories not to use gel separator serum tubes for the selected tests provide the best value for money for particular
measurement of progesterone in animal species. To our clinical settings, in gen-eral without scientific evidence. On
knowledge, only a few references document the reason for the basis of avail-able evidence, we postulate that the pre-
this recommendation in human preanalytic phase could and should be optimized in
veterinary clinical pathology.

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Braun et al The preanalytic phase

Technical Preanalytic Factors concentration is about 25% lower in serum than plasma of
ruminants, although the interindividual variability appears
Blood collection considerable.43–45 Furthermore, the delay caused by clot
formation can allow degradation, for example, of unstable
Choice of anticoagulant and tube peptides. Such analytes should therefore be determined in
plasma samples with addition of protease inhibitors such as
Many general recommendations have been published in aproti-
chapters of textbooks or specialized review articles for
Due to the interference of fibrinogen with
people, pets, exotic and laboratory animals.27–31
b-globulin migration, protein electrophoresis should be
Whole blood, plasma, and serum: Anticoagulated performed using serum. In dogs, fibrinogen can be
whole blood is the specimen of choice for most hema-tology precipitated with ethanol if only plasma is available, unless
variables. Textbooks generally recommend sodium or
potassium-EDTA in mammals, and heparin or heparin salts capillary electrophoresis can be substituted.47
When multiple blood tubes are collected from the same
in birds, reptiles, and some other spe-cies.14 In some
patient, carryover of anticoagulants or other additives should
mammals, particularly cats, EDTA often causes platelet
be avoided. The recommenda-tions in human clinical
aggregation and clumping, so additional compounds such as
pathology are to start with the citrate tube for coagulation,
prostaglandins or a mixture of cit-rate, theophylline,
followed by plain serum tube, followed by heparin, EDTA
adenosine, and dipyridamole (CTAD) have also been
tube and fluoride/oxalate tube.48 To our knowledge,
suggested to prevent platelet aggregation.32,33 Cell counts
obtained from specimens anticoagulated with EDTA, possible effects of a different order of sampling have not
citrate, or heparin are gen-erally comparable, including been studied in animals, but in some environments, citrate
canine and feline platelet and reticulocyte concentrations. tubes are preferably filled after the plain serum tube to avoid
However, validations can be indicated depending on the specimens with initiated clotting (personal communication).
analyzer and the technology and reagents used.34
Types of tubes: For safety and convenience, glass tubes
have been mostly replaced by plastic tubes. The latter have
Most textbooks recommend the use of serum or heparin been shown to yield comparable results for most analytes in
plasma for routine biochemistry, sodium cit-rate blood for
hematology, biochemistry49, endocri-nology50, and
coagulation, and sodium fluoride tubes for the measurement
of unstable molecules such as glucose or ketones. The coagulation51 with human blood, but have not been
results of biochemical analy-ses of heparin plasma and validated with animal specimens. In horses, glass syringes
serum are very similar for most analytes. Some of the permitted longer stability than
differences observed would have little or no effect on plastic syringes or plastic tubes for pO2, but not for
clinical interpreta-tion, such as for many canine and avian pH.52,53
variables.35– 37 However for some analytes, specific plasma Caution or better validation tests may be advised with
and serum reference intervals need to be established, for tubes that contain additives, such as surfactant, coating, and
instance, human bile acids are about 60% lower in heparin gel layers to optimize the recovery or separation of
specimens. Interferences have been observed for instance
plasma than in serum.38 In cattle and sheep, differences
between serum and citrate or EDTA 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
plasma are more numerous and relevant, for example, higher
most chemical analytes were not altered in lithium-heparin
AST and CK activities in serum.39,40 Point-of- vacuum tubes used for canine blood up to 11 months after
care systems and potentiometry analyzers usually require
anticoagulated whole blood. the expiration date.55
The interchangeability of serum and different plas-mas
for biochemistry profiles is somewhat surprising, as clot
Choice of the route and technique of specimen collection
formation results in the modification of the con-centration
of some analytes. For instance, intracellular molecules such In large animals, there is usually little difference in routine
as potassium can leak from cells, as a consequence, hematology and biochemistry data for samples collected
potassium concentration can be lower in plasma than in from different large veins, whereas in smaller species like
serum in species with high intracellular potassium levels or laboratory rodents or cats statistically significant differences
in cases with thrombocytosis.41,42 At the same time, clot have been reported.56,57 Samples collected from the tail
formation can also result in sequestration of analytes; for vessels in cows can
instance, mean copper

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The preanalytic phase Braun et al

sometimes yield a mixture of venous and arterial blood reported. In human clinical pathology, it is recom-mended
inappropriate for blood gas determinations58, and inor-ganic to use needle gauges large enough to avoid intense shearing
phosphate concentrations have been reported to be higher in of RBC and hemolysis, and, for the same reason, to exert
only moderate negative pressure when aspirating blood with
tail specimens than jugular vein speci-mens.59 In cats and
dogs, specimens collected from ear capillaries can notably syringes.48 In veterinary clinical pathology, there are
differ from those obtained from a large vein, for example, particular challenges when collecting blood from small
HCT and plasma proteins in cats60, and plasma lactate animals. For instance, the collection of small volumes can
be performed using capillary tubes, which appear to result
concentration in dogs.61 In rats, HCT, RBC, and WBC in no clinically relevant differences in routine
counts were reported to be higher in blood collected by
hematology and biochemistry variables in cats and
terminal heart puncture than by in life orbital sinus or tail
dogs.56,68,69,72,73 In zoo and wild animals, blood sam-
vein collection, whereas MCV and MCH were identical.62
pling can be particularly challenging as they are not used to
In general, in laboratory rodents, the quality of the specimen handling. An interesting alternative is the use
also depends on the sampling method.63 of blood-sucking beetles such as Dipetalogaster
Specimen collection from indwelling catheters is maximus.74,75 In rabbits, the results of about 50% of
routinely performed in human clinical pathology. After the measured variables (eg, HGB concentration, HCT,
proper complete removal of dead volume prior to actual reticulocyte count, concentrations of albumin, total protein,
sample collection, variables such as coagulation times in glucose, creatinine, urea, amylase and mag-nesium, and
dogs64 and routine biochemistry and hematology profiles in ALT activity) did not differ from those obtained from
horses65 were comparable to variables in specimens conventional specimens.74,75
obtained by direct venipuncture.
Skin cleansing is not a common practice in ani-mals,
and there are no reports on potential effects on laboratory Other specimens
analysis, such as reported in people. In con-trast, proper
technique, particularly in small animals is of utmost Information about preanalytic factors of variation for other
importance, as incorrect venipuncture causes discomfort, biologic specimens is more limited. Many recom-
hematomas or more extensive tis-sue damage. This is not mendations on sampling and specimen processing are found
only of potential relevance for animal welfare, but also of in chapters of cytology textbooks (see for instance 76–78),
concern for clotting initiation and spurious increase of but original studies are often lacking; for instance, rapid
enzyme activities (eg, increased CK activity due to muscle processing of cytology specimens is generally
damage).66 recommended with reference to human clin-ical pathology
and not to specific animal studies. The general consensus is
The volume of blood collected from an animal includes
that for most specimens a short transition time to the
consideration of the total body size and the respective total
laboratory and immediate analy-sis are the process of
blood volume, particularly in small animals such as
choice.
laboratory rats and mice, and of course the analytic needs.
The rule of thumb is that removal of < 7.5% of the total Urine
blood volume will not elicit regenerative effects on
hematology variables.67 However, if blood collection is In contrast to people, urine collection from animals can
require restraint or even sedation to perform cath-eterization
done directly with vacuum tubes, the volume can greatly
or cystocentesis, which can influence urine composition.
exceed the volume requirements of modern instru-ments,
Free catch samples can be contaminated with bacteria, such
resulting in wasted specimen. Therefore, in
as in horses.79
small animals such as cats and small dogs, microtu-bes are A spot urine can easily be obtained, but 24-hour urines
require the use of metabolic cages. For efficient use, the
appropriate.68,69 Blood collection into tubes
animals must previously be accustomed to metabolic cages
with anticoagulant must consider proper proportions
between blood and anticoagulant, as an excess of EDTA can for a few days, for example, 3–4 days in rats.80 Moreover, 2
main alterations can occur in metabolic cages: (1) possible
cause artifacts such as RBC shrinkage70, or an excess of
instability of the analyte;
citrate results in prolongation of coagulation times in canine
blood.71 (2) unsuspected loss of the analyte on the walls or in the
funnel of the cage; this requires proper rinsing of the cage
Choice of needles and syringes as, for example, for the determination of urine clearances in
dogs.81
Possible effects of needle gauge, free flow vs syringe vs
vacuum tube have not been scientifically studied and

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Braun et al The preanalytic phase

Saliva
Standard procedures at the laboratory
Saliva can be a good substitute for plasma or serum for the
Prior to analysis, gentle mixing on specialized rocking
determination of certain variables; however, collection and
platforms and avoidance of vigorous shaking are rec-
storage have only been described in connection with very
select analytes in few species. In dogs, cortisol concentration ommended.48 The most common unstable analyte is glucose
due to in vitro glycolysis. Sodium fluoride tubes are
did not notably differ with sampling conditions82, and
therefore recommended for reliable glucose determinations.
remained unchanged during the first 4 minutes of restraint.83 Alternatively, rapid processing of
blood collected in gel separator tubes is also recom-
Feces mended.112,113

Feces are sometimes the obligatory specimen for analyses Temperature


such as occult blood testing and endocrine markers in wild
Room temperature. There is no universal recommenda-
animals. Consideration should be given to potential
tion concerning the duration and temperature condi-tions,
interference of the carnivorous
nor is there an internationally accepted definition of the
diet with the analytic method, as shown for occult blood in stability of a variable. In general, it is recommended to
dogs and cats.84,85 The elimination of hor- process specimens within 2 hours from collection to obtain
mone catabolites in feces may be variable and not
optimal results114, however many analytes are stable for
reflect immediate effects due to digestive tract tran-sit.86,87 much longer periods, even at room temperature. In general,
In wild animals, feces are exposed to envi- hematology variables remain relatively stable in common
ronmental factors which may affect the concentration of the domestic species. In feline EDTA blood stored up to 48
variables of interest.88 hours, increased MCV, HCT, reticulocyte and eosinophil
Cerebrospinal fluid counts, and decreased MCHC and monocyte counts were
found. Effects were less pronounced when CTAD was
Stability of cells in cerebrospinal fluid (CSF) from dogs and added.115 In canine EDTA blood stored up to 48 hours,
cats can be improved by adding 10% serum to the increased MCV and HCT and decreased platelet and
specimen.89 Protein concentration in dogs and cats but not monocyte counts were measured.116–118 These changes can
horses is almost twice higher in CSF collected by lumbar also be observed on canine cell scatter-grams. In contrast, in
than by atlanto-occipital aspiration.90–92 In dogs or horses, sharks (Carcharhinus plumbeus),
the presence of intact or hemolyzed blood had no or very
marginal effects on protein concentration, CK activity, or WBC morphology was distorted after 3 hours storage at 4–
WBC counts.93–96 10°C.119
Likewise, many biochemical analytes are relatively
Other body fluids stable after 24–48 hours storage at room temperature,
nevertheless it is a standard procedure to collect and freeze
Synovia. In healthy horses, chemical composition and serum at 20°C in most labora-tories if storage is required. In
WBC count differed according to the joint sam-
addition, to prevent degradation of light-sensitive analytes
pled.97–99 Cell counts in repeated canine specimens did not such as biliru-bin or protoporphyrin specimens must be
differ.100 stored in the dark. For urine, little information is available.
Peritoneal fluid. In horses, protein content and cell In human urine, test strip analyses results were compa-rable
counts can be increased after laparotomy, castration, or
after 2 and 4 hours at room temperature.120
enterocentesis,101–103 but not after repeat samplings over 24
hours,104 blood contamination105, or after foaling in Refrigeration. Reports on effects of refrigeration on
mares.106
hematology results are controversial, for example, for WBC
Bronchoalveolar Lavage. For a review in cats and in dogs.34,118,121 The canine platelet count has
dogs, see107. In healthy dogs, the differential WBC count in been reported stable for 3 days122, however in canine blood,
bronchoalveolar lavage (BAL) did not differ between right platelet clumping was enhanced in refrigerated
and left lung108 and between the differ-ent lobes.109 specimens.123
Cytology results for BAL and tracheal wash differed in Unstable analytes such as catecholamines, ACTH, and
about 2/3 of canine cases.110 In cats, the differences other peptides require immediate refrigera-
observed between 3 consecutive lavages were slight.111 tion; however, reports are contradictory for
ammonium.124,125

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The preanalytic phase Braun et al

Freezing. Freezing is not an option for hematology more than 3% of specimens submitted.139,140 It is likely a
specimens, as the cells deteriorate due to the building of frequent preanalytic error in veterinary clinical pathology.
microcrystals, resulting in morphologic changes and leakage Free hemoglobin interferes with spectrometric
of cytoplasmic components. For plasma and serum, freezing measurements by absorbing light, so the degree of
is the standard procedure for long-term storage; however, interference of hemolysis depends on analyzers and methods
conditions of storage should be documented or validated for used.141 In some cases, interference is proportional to
each analyte in each spe-cies. Freezing can greatly alter the hemolysis intensity, permitting the use of correction
composition of urine. Freezing of centrifuged human urine equations, for instance, for haptoglobin in cattle and
led to the formation of precipitates, and lower calcium and sheep.142 Fur-thermore, hematology variables will be
pro-tein concentrations, unless the specimens were vigor- altered by hemolysis, and biochemistry profiles can be
ously shaken at room temperature and before analysis.126 In skewed after release of intracellular constituents, such as
dogs, urine protein-to-creatinine ratio ions (eg, potassium), enzymes (eg, ALT, AST), and proteins
(UPC), albumin, and cystatin C were stable up to 3 months (mainly HGB).
at 20°C.127–130 However, plasma enzyme
activities are generally stable for long periods at 20°C, urine Lipemia usually results in a whitish to milky opaci-
enzyme activities such as GGT in rats, rabbits, and horses, fication of serum or plasma, depending on the type and
and -N-acetylglucosaminidase amount of lipid present. Lipid droplet-related light scattering
(NAG) in dogs and cats are partially inactivated by interferes with hematologic measurements, as documented
freezing.131,132 for platelets in human samples.143 Likewise, there is
Serum and plasma specimens have a concentra-tion interference with many photometric measurements of
gradient of the analytes after thawing, requiring careful but biochemistry analytes including hemoglobin when
thorough homogenization.133 Repeat freeze-thawing cycles measured by the monochromatic method.144 Water
are usually not recommended for analytes such as hormones, displacement by lipids can result in skewed ion
cytokines, and enzymes, however, many routine chemical concentrations.145 In such specimens, ion measurements
analytes in canine plasma appeared unchanged even after 3 should be performed by direct rather than indirect
freeze-thaw cycles.134 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
Centrifugation specimens, for example, poly-ethylene glycol in canine
Relatively high speed centrifugation (eg, 2000g for 10 serum.146 Intense lipemia is often associated with increased
minutes) is the basic procedure to separate plasma and hemolysis in canine specimens.147
serum from the cellular components of blood.135 Likewise,
cytology samples are sometimes centrifuged at low speed to Icterus. Increased bilirubin concentrations or icteric
concentrate cells while preserving cell morphology. Urine plasma and serum can interfere with photometric readouts
sediment preparation by centrifu-gation appears to be in chemistry. For interferograms established
independent of speed for cell and crystal counts within a for canine, feline, bovine, and equine serum, see refer-
range of 400–3900g.136 ence148.
Final quality assessment of specimen before analysis Clots. The presence of small or large clots indi-cating
inadequate anticoagulation during blood col-lection is
Particularly in commercial reference laboratories, a careful probably an important cause for specimen rejection in
assessment of specimen quality is important, as such veterinary medicine. Microclots may be easily overlooked if
samples have usually undergone transport at more or less specimens are not carefully examined before processing.
optimal conditions. Standard monitoring includes the Microclots not only cause erroneous hematology cell counts
documentation of plasma and serum color. Visual estimation and coagu-lation results but they can also clog instrument
of color changes is relatively imprecise, even with color lines or contaminate sampling tubes. Microclots and
charts, and they should be microscopic platelet clumps occur in feline samples, and
quantified by objective techniques, as established for human less frequently in canine EDTA blood.32,149,150
specimens.137,138 Small white flakes sometimes can be observed in thawed
Hemolysis. A pink-to-red discoloration indicates heparin plasma. Their origin is not fully understood and their
hemolysis, the most frequent laboratory preanalytic possible effects on plasma ana-lytes have not been
interference in human clinical pathology, occurring in documented.151

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Braun et al The preanalytic phase

Biologic Preanalytic Factors of Variation cholesterol concentration, but had no influence on tri-
glyceride, phospholipid, or FFA concentration.169 In
Biologic factors such as species, strain, sex, and age are just neonates, colostrum ingestion shortly after birth results in
a few and the best defined factors which can com-plicate increased plasma total proteins and immuno-globulin levels.
proper clinical interpretation of laboratory data. In addition, In ruminants, successful colostrum
physiologic aspects such as reproductive cycle, nutrition, ingestion can be demonstrated based on increased GGT or
climate, breed, and all circumstances involved with blood ALP activities in neonatal serum.170,171 In canine
sampling in wild animal species, such as trapping and urine, false-positive glucosaminoglycan results were
sedation can affect test results. More recently, a number of observed in a dog supplemented with an algal
studies have addressed intraindividual variation.152,153 preparation.172
In laboratory rodents and rabbits, special attention
needs to be addressed to physiologic coprophagia. The
Nutritional status and diet
plasma urea concentration was 20–40% higher in rats which
Fasting is mostly relevant in monogastric animals. Although were allowed to consume their feces,173 but the HCT was
generally accepted, presampling fasting is considered as a not affected. The general possible effects of physiologic
deviation from the normal state by some authors,154 and coprophagia on plasma chemistry variables have not been
does not improve the medical reported to our knowledge.
interpretation of certain tests such as blood lipids in people
with normal food intake.155,156 Overnight fast-
Effects of stress
ing is an adaptation from human medicine, decreasing the
incidence of postprandial lipemia. In animals, even brief The definition of stress is not straight forward, but it can
fasting periods can strongly modify the concen- include any effects resulting in an activation of the
tration of certain analytes, such as higher bilirubin and adrenomedullary (acute stress) or cortical (subacute, chronic
triglyceride concentrations in equids,157,158 and higher stress) cells. Depending on the species and individual
bilirubin in rats.159 The approximate 12 hour over-night conditioning, any activities including trans-port, restraint or
capture, work, environmental, social conditions, and time
fasting period is adequate for most analytes. For example, in
rats, sheep, and monkeys, most hematol-ogy and chemistry spent in a waiting room174 can result in stress.
variables were almost unchanged
up to 16 hours after food withdrawal.160–162 However in Stress-related blood sampling in cats can cause
healthy dogs, fasting urea163,164 or triglyceride hyperglycemia (up to 25 mmol/L), hyperlactatemia, and
concentrations165,166 may be clearly lower than nonfa- lymphocytosis.175 Stress due to capture and restraint of wild
sting levels. Interestingly, fasting does not lead to steady- animals should be avoided just for reasons of animal
state plasma concentration of some analytes, such as canine welfare. Captured animals should be allowed a recovery and
free fatty acids.167 adaptation period before sampling for laboratory purposes is
Postprandial increase in blood glucose in dogs was done. Handling and sampling can cause stress in small
reported very early. The possible effects of a meal result laboratory animals.176 Increased plasma CK activity in
from the absorption of digested food and metabolites, rabbits and higher plasma cortisol concentration in monkeys
secretions from the gastrointestinal tract (eg, gastric acid, have been reported with repeat sampling, therefore animal
pancreatic enzymes, hepatic bile acids), and hor-mone training and conditioning should be standard practice in
secretions (eg, insulin). In dogs and cats, the main changes research organizations.177 Duration of handling had greater
include increased plasma concentrations of urea, glucose, effects than the gentle or rough handling procedure used on
creatinine, ammonium, bile acids, trypsinogen, and increasing plasma corti-costerone and lactate concentrations
triglycerides, and lower concentra-tions of bicarbonate.168 in poultry.178 In cattle, handling as well as physical fatigue
Importantly, the intensity and duration of the observed and dehy-dration and undernutrition connected with
variations differ notably according to the type and amount transport have been reported to affect levels concentrations
of food ingested, as shown for increases of urea and in place of levels of stress indicators (eg, increased plasma
creatinine in dogs, for example, the latter being more catecholamines and glucocorticoid concentra-tions, total
markedly increased after ingestion of cooked than raw WBC and neutrophil counts, decreased T
meat.163
Most routine tests are not influenced by the lymphocyte count, increased activities of muscle enzymes
composition of the diet. However, in dogs, a diet change such as CK).179–183
caused a notable difference in fasting plasma

Vet Clin Pathol 44/1 (2015) 8–25 ©2014 American Society for Veterinary Clinical Pathology 15
The preanalytic phase Braun et al

Interestingly, the type of transport did not affect the subtle effects, such as shown for organohalogen com-
stress response as such, for example, the increase in plasma pounds in pups195,196 and in wild raptors.197,198
corticosterone concentration was similar (approximately 2–
3-fold) in mice transported to an
Biologic rhythms
adjacent room for euthanasia and in rats transported by
airplane.184,185 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,
Effects of drugs and pollutants
including reproductive cycles, and, predominantly in wild
Drugs and pollutants can either affect the molecule itself, or animals, behavior associated with climate and location,
its metabolites. An example for interference with the amount and type of food available, migration over large
analytic technique are false high plasma chlo-ride distances, or molting in birds, and many others. Careful
documentation of rel-evant parameters and variables will
concentrations in dogs treated with bromide.186 An example
for a pharmacologic effect is the increased activity of ALP help avoiding the drawing of erroneous conclusions. 199 For
due to induction of a specific isoenzyme in the presence of some ana-lytes, variability is very high throughout the
increased intrinsic or extrinsic adre-nocortical stimulation. different seasons, for example, Melanocyte Stimulating
A rare effect, so far only reported in people, is interference Hor-mone (a-MSH) in horses.200 Circadian rhythms of a
with the excipient of a dexamethasone preparation particular analyte can differ according to species; for
containing a high con-centration of creatinine.187 instance, plasma glucocorticoids peak in the morning
Presampling sedation or anesthesia is compulsory in in people and in the early evening in rats, while there is
many cases, either for compliance with animal wel-fare almost no change in dogs.201,202 Circadian fluctua-
legislation, or if the procedure is lasting longer than an tions of RBC and WBC counts have been described in
animals’ cooperation can be expected. Seda-tion effects are different mouse strains.203
usually minimal causing few potential clinical
misinterpretations. However, they differ nota-bly according
to the drugs, associations, and doses used, which means that Environment and living conditions
any new and nonstandard procedure must be validated Management and husbandry practices can also influ-ence
before interpreting results in sedated animals. In certain blood variables such as milking frequency altering
anesthetized people, body posi-tion causes water shifts with plasma free fatty acid (FFA) and b-hydroxybu-tyrate
corresponding changes in analyte concentrations.188 To our concentrations.204 Adaptation to high altitude
knowledge, the effect of body position has not been caused higher PCV, HGB concentration, and RBC count in
documented in animals, except for prolonged recumbency poultry205, horses206,207, or in wild animals
causing increased plasma activities of AST, LDH, and CK such as Sloth bears208 and otters.209
in downer cows with milk fever.189 In wild animals, environmental factors can nota-bly
affect some variables, and a careful distinction is advised
In general, potential adverse treatment-related effects between free-ranging animals and animals living in
of pharmaceutical compounds need to be addressed in captivity.210 Infestation by parasites, the nutritional status
animals by law, and compounds causing kidney or liver and the overall health assessment
damage and respective clinical pathol-ogy results are can vary significantly and must be documented very
sometimes published. An area that has been much carefully.211,212 In migratory birds, conditions can dif-
investigated are treatment-related effects due to fer from year to year, as observed in Sage Grouse. 213
glucocorticoids and nonsteroidal anti-inflam-matory drugs However, hematology and biochemistry variables of
(NSAIDS). Typically, leukocyte numbers increase due to Bottlenose dolphins were mostly stable for 7 years at 4
higher neutrophils, while lymphocytes different locations.214 Habitat restriction due to human
and eosinophils are lower. Increased ALP activity and iron settlement can represent result in increased fecal corti-
concentrations are commonly seen in dogs.190,191 costeroids, and higher CK and AST activities in wolves
Examples for effects by pollutants or environmen-tal living within a park or close to farmland.215,216 In labo-
toxicants are the inhibition of cholinesterases by ratory animals, initiatives for enrichment had no sig-nificant
organophosphates and carbamates192,193, and delta- effect on routine murine hematology results, with the
aminolevulinate dehydratase (and consequently exception of increased interindividual vari-ability.217
compromised hemoglobin synthesis) with lead intoxi- Interestingly, in untrained Cynomolgus monkeys, serum
cation.194 Other environmental pollutants have more thyroid hormone concentrations

16 Vet Clin Pathol 44/1 (2015) 8–25 ©2014 American Society for Veterinary Clinical Pathology
Braun et al The preanalytic phase

decreased with increasing length of lag time during and Competence. 3rd ed. Geneva, Switzerland: ISO,
218 2012.
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Disclosure: The authors have indicated that they have no
laboratory primates: a neglected variable in biomed-ical
affiliations or financial involvement with any orga-nization research. J Appl Anim Welf Sci. 2000;3:321–333.
or entity with a financial interest in, or in financial
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in this article. assurance guidelines: control of preanalytical and
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