CE U P D A T E — P H L E B O T O M Y I

Jane C. Dale, MD

Preanalytic Variables in
Laboratory Testing

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two chemistry laboratories2 in the United King-
ABSTRACT The total testing process consists of three dom in 1992 reported 50% of errors due to prob-
separate phases—the preanalytic, the analytic, and the lems in the preanalytic phase, as defined in Table
postanalytic. The accuracy and reliability of a laboratory 1. In a study of 49 primary care clinics, Nutting
result depend on the quality of each phase. In this article, and others3 reported 55.6% of detected errors
the common variables in the preanalytic phase and their were attributable to the preanalytic phase, 13.3%
effect on test result quality are reviewed. This article to the analytic phase, and 27.8% to the postana-
lytic phase. As laboratories look for ways to
explains the rationale for the specimen collection, improve performance, the preanalytic phase of
processing, and transportation requirements for commonly the testing process represents an important
ordered laboratory tests. opportunity. This article describes the common
This is the first article in a four-part series on phlebotomy. The series will discuss types of preanalytic variables in the testing
sensors for patient monitoring, phlebotomy safety devices, blood collection devices, process (Table 2).
and preanalytic variables in laboratory testing. On completion of this series, the
reader will be able to describe and compare the various types of patient blood Diet
monitors, phlebotomy safety devices, and blood collection devices. The reader also The subject of dietary effects on laboratory
will be able to explain the rationale for specimen collection, processing, and results is complex and relates not simply to the
transportation requirements for common laboratory tests.
distinction between fasting and nonfasting status,
From the
The total laboratory testing process can be but also to a number of other factors, including
Department of
Laboratory Medicine
divided into three phases—preanalytic, analytic, the type of diet (eg, high fat, low fat, vegetarian),
and Pathology, and postanalytic. Errors that may affect the accu- the length of time since the last meal (eg, 2 hours,
Mayo Clinic, racy and the reliability of results can occur in any 12 hours, 48 hours, or more), and a very long list
Rochester, Minn. of the phases. Typically it is the analytic type of of test-specific dietary concerns. While many
Reprint requests to errors that most frequently come to mind as the dietary issues are outside the control of the labo-
Dr Dale, 378 Hilton reason for an incorrect result. However, a number ratory and must be controlled by persons caring
Bldg, Dept of
of studies have shown that analytic errors are not for the patient, the laboratory is responsible for
Laboratory Medicine
and Pathology, always the problem. In a study of 363 errors providing the appropriate information about
Mayo Medical detected in the laboratory of a 570-bed hospital dietary requirements for each test offered.
Center, 200 First St in 1987, Boone 1 reported that only 7% of Just as the ingestion of food can cause sub-
SW, Rochester, M N detected errors were analytic, while 46% were stantial changes in the blood level of some ana-
55905; or e-mail:
preanalytic and 47% were postanalytic. A study of lytes, so too can a prolonged period of food
dale.ja ne @ mayo.edu
deprivation. The analytes listed in Table 3 have
Table 1. Total Testing Process
been shown to be affected by prolonged fasting.4
Preanalytic Analytic Postanalytic In addition to the known effects of food inges-
Ordering Testing Recording tion, another reason for collecting most speci-
mens in the fasting state is that normal value
Collecting Reporting
studies have traditionally been performed on
Processing Interpreting
Transporting

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 fasting patients. Accordingly, the established nor-
Table 2. Preanalytic Variables
mal ranges are typically fasting normal ranges.
For some analytes, the normal range would be Patient and
somewhat different if nonfasting patients were Specimen- Transportation, Storage
studied. Related Collection and Processing
Medications also cause a wide range of effects Diet Anticoagulants Centrifugation
on laboratory tests, including their intended Exercise Collection timing Evaporation
(therapeutic) effects, side effects, and analytic
Hemolysis Diurnal variation in Light
interferences. A discussion of drug effects on lab-
analyte concentrations

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oratory tests is beyond the scope of this article.
Interested readers are referred to a compendium Lipemia Hemolysis Specimen
of such information by Young.5 processing time
Intravenous solutions Temperature
Exercise
Order of draw
Exercise can result in transient or long-term
changes in a number of analytes. Some of the Posture of patient
more commonly described analytes affected by Specimen volume
exercise are listed in Table 4. The degree of change
depends on a number of factors, including the
person's fitness, amount of muscle mass, duration Table 3. Laboratory Value Changes Related to Prolonged Fasting
and intensity of the exercise, and amount of time
Increase Decrease
that has elapsed between the exercise event and
collection of the blood specimen. Amino acids Glucose
Exercise during specimen collection (ie, pump- Bilirubin High-density lipoprotein
ing of the fist) can induce local changes that may cholesterol
be manifested in the specimen collected from that Fatty acids Insulin
limb. Exercising muscle deprived of oxygen results
in increased lactic acid concentration and Glucagon Lactate dehydrogenase
decreased pH, which may, in turn, result in an Growth hormone Triiodothyronine (T3) c
0
increased level of ionized calcium and potassium.6 Ketones (0
Clenching the fist also may result in increased u
pressure in the veins, which may result in pres- Lactate c
sure-induced changes similar to those seen with Triglycerides £
E
tourniquet placement (see "Tourniquet" section). o
0

Posture Table 4. Laboratory Values That Increase

Posture can affect the concentration of a number
of analytes as a consequence of the normal phys-
iologic mechanisms responsible for maintaining
After Exercise

Aspartate aminotransferase
i
blood pressure. As the body changes from a lying Bilirubin
to a standing position, blood pressure-sensitive Creatine kinase
hormones increase in concentration to raise the !
High-density lipoprotein cholesterol
blood pressure to maintain adequate perfusion of
the brain. As the blood pressure increases, there is Hormones
an efflux of water from the intravascular space Lactate
into the interstitium, resulting, in turn, in a
Lactate dehydrogenase
decrease in blood volume and an increase in con-
centration of analytes that are too large to pass Neutrophils
through the vascular wall. In general, these are the Uric acid

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 Pressure-Induced Changes in Analyte Concentration

Vascular wall
• require outpatients to be seated for a time before
•
• blood specimens are collected. The Laboratory
Vascular wall
Standardization Panel on Blood Cholesterol Mea-
• ^V
f • surement of the National Institutes of Health rec-
• \
• J
/ * *
• -\ ommends that patients sit for 5 minutes before
collection of specimens for cholesterol and other
lipoprotein measurements to minimize this pos-
• •
• •1
I •' • / •*•>f •
ture-related change.8

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Tourniquet
• /
m Just as posture can cause pressure-induced
• changes, so can the application of a tourniquet.
Increase in pressure Tourniquets create an increase in pressure below
Interstitial space Interstitial space the tourniquet, which results in changes similar
to the postural changes already described. Pro-
1 Protein-bound analytes • Freely diffusable analytes tein-bound nondiffusible analytes increase in
concentration during the time of tourniquet
As a patient moves from a lying to a standing position, his or her blood application.6,9'10 There are two main schools of
pressure increases. Water flows from the intravascular space into the thought about how long a tourniquet should be
interstitium, resulting, in turn, in a decrease in blood volume and an increase left in place. Renoe and colleagues6 showed that
in serum concentration of analytes that are too large to pass through the
vascular wall. Generally these analytes are bound to large proteins.
the greatest changes in analyte concentration (eg,
total calcium levels increased by 8.4%, total pro-
tein levels by 12.4%) occurred at the time of the
Table 5. Laboratory Values Increased by Changing Position From
tourniquet release and, therefore, recommended
Lying to Standing
that samples be collected while the tourniquet
Pressure- and Volume •Sensitive Protein-Bound was in place or at least 1 minute after the tourni-
Hormonal Changes Nondiffusible Analyte: quet was released. The National Committee for
Aldosterone Albumin Clinical Laboratory Standards guideline for col-
Angiotensin Bilirubin lection of blood specimens by venipuncture states
that the tourniquet should be released as soon as
Antidiuretic hormone Calcium blood flow is established to minimize the time the
Catecholamines Cholesterol tourniquet is in place.11 Given these different rec-
Renin Drugs
ommendations, to minimize variations, each
institution should determine a standard
Enzymes* approach to be used for all collections. Occlusion
Total protein time should be minimized, and pumping of the
Triglycerides
fist should be avoided.
* Such as alkaline phosphatase and alanine aminotransferase.
Timing
analytes that are bound to large proteins (Figure, The timing of the collection is important for
Table 5). For example, cholesterol levels may some analytes. A number of analytes have been
change by up to 15%.7 shown to have predictable changes in concentra-
Note that most hospitalized patients are tion during a 24-hour period in response to a
supine when blood specimens are obtained, wide range of factors, including light and dark,
whereas most normal value studies are per- meals, sleep, posture, and stress changes that
formed on specimens from persons who have humans undergo during a typical day. Common
been walking around actively before being seated
for collection. These two populations may have
somewhat different normal ranges. Because of
these posture-related changes, some institutions

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analytes that have been reported to exhibit diur-
nal variation are shown in Table 6.12 For some of
these analytes, laboratories may provide different
time-related normal ranges (eg, morning and
evening Cortisol levels). For other analytes this is
not die case, although significant differences have
been observed (eg, serum iron levels may be as
much as 30% lower in the evening than in the
morning). For these analytes, serial collections at
similar times of the day will minimize differences
owing to diurnal variations.
For some drug levels, timing of the collection is
crucial. This is especially true for drugs in which
the therapeutic window (ie, the difference between
subtherapeutic and toxic levels) is relatively small.
Trough drug levels are typically collected immedi-
ately before the next dose of medication is given.
Peak levels are drawn at a specific time after the last
dose, determined by the drug pharmacokinetics
and the route of administration.
The timing for collecting specimens for blood
cultures can be crucial. For some bloodstream
infections (eg, endocarditis) organisms typically
are shed into the blood continuously. For this
type of infection, timing is probably not critical.
But for other infections, shedding is irregular or
erratic. Shedding often precedes fever and chills
by up to 1 hour. Obviously, collection orders can-
not be written "collect 1 hour before fever spike."
But the temperature curve or fever pattern for a
particular patient may permit prediction of when
the next spike is likely to occur. In such cases, the
laboratory should collect the specimens at the
time specified by the physician in hopes of
obtaining the specimen around the time of bacte-
rial shedding. In other cases, when the fever curve
is unpredictable, collecting specimens on more
than one occasion is the best we can do. Most
would recommend at least two collections, sepa-
rated in time to maximize the laboratory's ability
to detect bacteremia.13
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Table 6. Common Analytes Exhibiting
Diurnal Variation
Acid phosphatase
Adrenocorticotropin
Aldosterone
Bilirubin
Blood urea nitrogen
Catecholamines
Cortisol
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Follicle-stimulating hormone
Growth hormone
Iron
Luteinizing hormone
Progesterone
Prolactin
Testosterone
Thyroid-stimulating hormone
Triglycerides
Uric acid
Anticoagulants
A number of anticoagulants have been developed
for laboratory use. Selecting the correct anticoag-
ulant is critical for many tests because anticoagu-
lants designed to optimize one test method may
interfere with another test method.14 For exam-
ple, anticoagulants containing fluoride, designed
for glucose measurements, may interfere with
hematologic and electrolyte studies by altering
blood cell membrane permeability and morpho-
logic features. EDTA designed for maintenance of
blood cell membranes chelates calcium and will
result in erroneous calcium levels. When per-
forming a multitube collection using evacuated
blood tubes, the potential for anticoagulants in
one evacuated blood tube to contaminate blood
collected into a subsequent evacuated blood tube
necessitates adherence to a standard order of col-
lection. In our facility we use the following order:
1. Tubes with sterilized stoppers (ie, blood culture
tubes), trace metal tubes, and stasis-sensitive
assays (eg, calcium) are collected first.
2. Nonadditive tubes (eg, plain red tubes)
3. Additive tubes in the following order: blue,
green, purple, and gray
SEPTEMBER 1998 VOLUME 29. NUMBER 9 LABORATO
 For specimens collected in tubes containing a
liquid anticoagulant, an adequate amount of
blood must be added to the tube to assure the
appropriate interaction between the blood and
the anticoagulant and to prevent dilution by the
anticoagulant itself.
S e p a r a t o r Gels
Serum or plasma separator tubes may be unaccept-
able for some analytes. 15~17 Manufacturers of evac-
uated blood collection tubes should provide lists of
analytes that have been shown to give comparable
results in serum or plasma obtained from tubes
containing separator gels compared with plain
tubes. When possible, in-house studies demon-
strating the suitability of gel-containing collection
tubes should be performed for analytic methods
not evaluated by the evacuated tube manufacturers
or described in the medical literature.
Intravenous Infusions
The presence of an intravenous (IV) infusion
often complicates the phlebotomy procedure. If
Test Your
possible, specimens should be collected from an
Knowledge
Look for the CE
alternative site or by capillary puncture. If that is
Update exam on not feasible, the IV infusion should be turned off
Phlebotomy (808) in for 2 minutes, a tourniquet applied, and the col-
the December issue lection should occur from a vein below the IV
of Laboratory site. To eliminate interference or dilution from
Medicine. Participants
will earn 4 CMLE
the IV solution, a volume of blood (eg, 5 mL)
credit hours. should be discarded before the collection of the
required specimens. Even larger discard volumes
may be necessary to eliminate contamination by
compounds contained in the IV solution (eg, glu-
cose, heparin).
S p e c i m e n Processing
Specimens should be promptly processed after
collection. Allowing the cells to remain in contact
with the plasma or serum allows a number of
changes to occur. The concentration of some ana-
lytes may increase (eg, creatine kinase, lactate,
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lactate dehydrogenase, phosphate, and ammo-
nia), whereas the concentration of others may
decrease on standing (eg, glucose, bicarbonate,
and acid phosphatase). As a general rule, plasma
from anticoagulated specimens should be sepa-
rated from the cells within 1 hour of collection,
and serum should be separated from cells within
2 hours of collection.
Serum specimens must be allowed to clot
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completely. In general, 20 to 30 minutes is
required for clotting for most specimens collected
in glass tubes before centrifugation. Specimens
collected in plastic tubes may take longer to clot.
Clotting time may be shorter in tubes containing
clot activators. Tubes should be held upright at
room temperature while clotting. Centrifugation
time and speed must be in compliance with the
evacuated tube manufacturer's recommendations
to assure adequate separation. Primary tubes with
serum separator gels should not be recentrifuged.
Temperature
The temperature at which a specimen is trans-
ported, processed, and stored can affect the
results. While refrigeration or freezing may reduce
time-related changes, placing specimens at inap-
propriate temperatures can cause unreliable
results. The temperature must be maintained
throughout the preanalytic phase. Common room
temperature-labile analytes include ammonia,
blood gases, acid phosphatase, lactate, pyruvate,
gastrin, renin, and parathyroid hormone.4
Freeze-thaw cycles also can introduce changes.
A problem can result when specimens are inad-
vertently frozen in transit or when frozen speci-
mens must be redivided into aliquots or
reanalyzed. Self-defrosting freezers also can cause
problems and should not be used for storing med-
ical materials. A freeze-thaw cycle often causes
degradation of analytes (eg, creatine kinase, lac-
tate dehydrogenase), but occasionally freeze-thaw
cycles can result in an apparent increase in con-
centration (eg, plasma renin activity).
 Hemolysis
The presence of hemolysis can cause erroneous
results, especially for analytes that are present
within RBCs in higher concentration than in
serum, such as lactate dehydrogenase, aspartate
aminotransferase, alanine aminotransferase, and
potassium.
Conclusion
A substantial portion of laboratory errors are the
result of problems that arise in the preanalytic
phase. Since monitoring efforts probably can
detect only a fraction of total errors that occur,
prevention is the key to reducing preanalytic
errors. Prevention of preanalytic errors requires
knowledge of the many preanalytic variables that
can adversely affect laboratory results. Ideally, the
"system" should include checks and controls to
minimize preanalytic errors. The phlebotomy
staff is a key component of the control process.
Phlebotomists must understand the rationale
behind collection and processing requirements
and must adhere to the requirements for all spec-
imens collected. Prevention also requires educa-
tion of and cooperation among physicians,
nurses, ward clerks, phlebotomists, and labora-
tory professionals to assure that the right speci-
men is drawn under the right set of
circumstances.®
Acknowledgment
The author thanks Sharon Preuss for her assistance in the
preparation of this manuscript.
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