Pre Analytic Phase

Clinical Chemistry / THE PREANALYTIC PHASE
The Preanalytic Phase

An Important Component of Laboratory Medicine
Sheshadri Narayanan, PhD*
Key Words: Preanalytic phase variables; Physiologic; Specimen collection and processing; Analyte stability;
Endogenous interference factors
Abstract
The preanalytic phase is an important component
of total laboratory quality. A wide range of variables
that affect the result for a patient from whom a
specimen of blood or body fluid has been collected,
including the procedure for collection, handling, and
processing before analysis, constitute the preanalytic
phase. Physiologic variables, such as lifestyle, age,
and sex, and conditions such as pregnancy and
menstruation, are some of the preanalytic phase factors.
Endogenous variables such as drugs or circulating
antibodies might interact with a specific method to yield
spurious analytic results. The preanalytic phase
variables affect a wide range of laboratory disciplines.
In recent years, considerable attention has been given to

the influence of preanalytic effects on laboratory results.
Compendia of preanalytic effects and books have addressed
the effect of the preanalytic phase on laboratory results.1,2
Preanalytic variables can be grouped broadly under 3
categories: physiologic, specimen collection, and influence or
interference factors. In this review, I address these variables
and provide examples of their effects across a broad range of
laboratory disciplines. Examples of preanalytic variables
affecting mainly clinical chemistry, hematology, and coagulation tests are discussed.
Admittedly, given the comprehensive range of subject
matter that spans the realm of the preanalytic phase, this
review must be selective. Hence, a mere passing mention is
made in the concluding section, Summary Perspectives, of
topics such as blood gases, molecular biology, tumor
markers, and problem analytes, such as cytokines, homocysteine, and fibrinolytic activators and inhibitors, all of which
could be the subject matter for more than 1 review article.
Even so, an attempt is made to provide a comprehensive
overview of physiologic variables, specimen collection variables, including a discussion of traditional anticoagulants and
newer stabilizing additives for newer problem analytes, and
clinically relevant endogenous interferences.
Physiologic Variables
The effects of age, sex, time, season, altitude, conditions such as menstruation and pregnancy, and lifestyle are
some of the physiologic variables that affect laboratory
results.
American Society of Clinical Pathologists
Am J Clin Pathol 2000;113:429-452 429
Narayanan / THE PREANALYTIC PHASE
Age
The effect of age Table 1 on laboratory results has
been well recognized to the point that separate reference
intervals have been used to distinguish pediatric, adolescent,
adult, and geriatric populations. Bone growth and development in a healthy growing child is characterized by increased
activity of bone-forming cells, the osteoblasts, that secrete
the enzyme, alkaline phosphatase. Hence, the activity of this
enzyme is approximately 3 times higher in a growing child
compared with the activity in a healthy adult.3,4
A substantial increase in the RBC count in the neonates
compared with that in adults is the reason glucose is metabolized very rapidly in neonates.2 Increased arterial oxygen
content within a few days of birth leads to an increase in
hemoglobin levels owing to the destruction of the RBCs.
Serum bilirubin levels then increase, because the immature
liver lacks enzymes to convert bilirubin to the water-soluble
bilirubin diglucuronide.2 The total number of neutrophils are
at a maximum for 1 to 2 days after birth. In the neonate, the
monocyte count is increased for up to 2 weeks after birth,
while the eosinophil count stays elevated for up to 1 week
after birth.5,6 The lymphocyte count is increased markedly at
birth and remains elevated in children up to 4 years of age.6
In contrast, the basophil count is elevated only transiently for
up to 1 day after birth. A substantial decrease in serum uric
acid levels is noted between days 1 and 6 after birth.2
Age also affects renal function, as reflected by an effect
on creatinine clearance, which decreases progressively with
each decade.7 For example, the average value for creatinine
clearance at age 30 years is 140 mL/min (2.33 mL/s) per
1.73 m2 of body surface area. In contrast, at age 80 years, the
creatinine clearance value decreases to 97 mL/min (1.62
mL/s) per 1.73 m2 of body surface area.7 Beyond the age of
50 years, the decrease in creatinine clearance is also related
to a decrease in muscle mass.7,8 However, when interpreting
changes in renal function solely on the basis of aging, the

overlap of disease processes, such as the presence of renal
vascular disease in elderly patients, must be considered.
Secondary risk factors, such as genetics and obesity, may
have a role in the decrease in glucose tolerance with aging.9
Homeostatic control by the hypothalamic-pituitaryadrenal axis is compromised because of aging. Hence, there
is an increase in plasma corticotropin and corticosteroid
levels due to aging.10,11 Usually in young adults, the concentration of the cytokine, interleukin (IL)-6, in serum is low
and is undetectable in absence of inflammation.11 However,
IL-6 levels are detectable readily during middle age. Also, in
healthy elderly adults, serum IL-6 levels are increased apparently due to the loss of the normal regulation of IL-6 levels.
The decrease in dehydroepiandrosterone sulfate with age,
which normally has a suppressive effect on IL-6, is related to
the increase in IL-6 levels.12 Increased IL-6 levels, in turn,
increase the percentage of cells bearing the receptor for the
cytokine, transforming growth factor beta, a powerful
immunosuppressive agent, thus providing an explanation for
the age-related immunosuppressive phenomena.13
Increases in the level of plasma antidiuretic hormone (or
arginine vasopressin) have been related to aging with levels
significantly higher in 1 study of healthy subjects 53 to 87
years of age (4.7 0.6 pg/mL [4.4 0.6 pmol/L] in 68
healthy subjects) compared with subjects 21 to 51 years of
age (2.1 0.2 pg/mL [1.9 0.19 pmol/L] in 45 healthy
subjects).14
Thyroid homeostasis apparently is affected during
aging. Thyrotropin levels in serum have been reported to be
38% higher in an elderly group (mean age, 79.6 years)
compared with a younger group of subjects (mean age, 39.4
years).15 In the same study, serum triiodothyronine levels
were 11% lower in the older age group compared with levels
in the younger group of subjects.
Table 1
Effect of Age on Laboratory Results
Age Group/Effects
Neonate
Increased RBC count leading to increased glucose metabolism
Increased neutrophil, monocyte, eosinophil, and lymphocyte counts
Increased hemoglobin and unconjugated bilirubin values, decreased uric acid level
Lymphocyte count elevated up to 4 years of age
Growing child
3-fold increase in alkaline phosphatase level due to bone growth
Progressive changes
Creatinine clearance decreases with each decade; age-related change in renal function; may
overlap with disease process in elderly persons
Decrease in glucose tolerance with age; overlaps with secondary risk factors, such as genetics
and obesity
Homeostatic control by hypothalamic-pituitary-adrenal axis affected by aging; corticotropin and
corticosteroid levels increase
Dehydroepiandrosterone sulfate levels decrease, interleukin-6 levels increase with age
Antidiuretic hormone and thyrotropin levels increase, triiodothyronine levels decrease with age
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References
Guder et al2
Monroe et al5; Weinberg et al6
Guder et al2
Weinberg et al6
Narayanan3; McComb et al4
Narayanan and Appleton7; Narayanan11
Pacini et al9
De Kloet10; Narayanan11
Daynes et al12
Crawford et al14; Harman et al15
Clinical Chemistry / REVIEW ARTICLE
While changes in organ function occur due to aging,

which in turn affects the circulating level of various analytes,
one must also keep in perspective the overlapping effects due
to disease and dietary deficiencies of trace elements, such as
zinc and other nutrients.11
Sex Differences
Between 15 and 55 years of age, the levels of total and
low-density lipoprotein cholesterol (LDL-C) increase
progressively, with levels slightly higher in premenopausal
females compared with the levels in males Table 2. Thus,
in female subjects, a change in cholesterol value from 174
mg/dL (4.50 mmol/L) at age 25 years to 213 mg/dL (5.51
mg/dL) at 55 years has been reported. 2 In the corresponding age span for men, cholesterol values have been
reported to range from 166 mg/dL (4.29 mmol/L) at age 25
years to 232 mg/dL (6.00 mmol/L) at 55 years.2 In contrast,
the high-density lipoprotein cholesterol (HDL-C) level
does not fluctuate in either sex between the ages of 15 and
55 years; levels are slightly higher in females, presumably
owing to the stimulating effect of estrogens.2
Analytes with levels that depend on muscle mass, such
as creatinine and creatine kinase (CK), are generally at a
higher concentration and activity, respectively, in males
compared with females. 2,7 However, when increased
muscle mass is acquired as a consequence of strenuous
athletic activities in female subjects, the sex difference in
levels of creatinine and CK is abolished.2 Serum iron levels
generally are lower in premenopausal female subjects

compared with those for male subjects, with the difference
disappearing in subjects older than 65 years.2
Sex differences are seen for several analytes as can be
noted by reviewing a current list of reference laboratory
values.16 The differences for some of the analytes based on sex
can be explained as due to differences in muscle mass and to
endocrine and organ-specific differences.
Time
There are time-related fluctuations in the level of some
analytes (Table 2). Circadian rhythm is responsible for the
diurnal changes seen in the circulating levels of some
analytes. A classic example of an analyte subject to diurnal
variation is cortisol, which generally peaks at around 6:00
AM, with levels becoming lower toward the evening and
midnight.2 Because cortisol is a powerful immunosuppressant, its diurnal rhythm affects proinflammatory cytokine
production. Indeed the level of proinflammatory cytokines,
such as IL-1 alpha, IL-12, tumor necrosis factor alpha and
interferon gamma have been shown to be inversely correlated with the level of plasma cortisol.17 The inverse relationship with plasma cortisol levels extends to the number
of circulating T lymphocytes, especially the CD4+ T cells,
and to the urinary secretion of neopterin, which is a marker
of cellular immune activity.18,19 Even routinely measured
analytes such as glucose, potassium, and iron exhibit
diurnal variation.1,2
Table 2
Effect of Sex, Time, Season, and Altitude on Laboratory Results
Variable/Effects
Sex
Between 15 and 55 years, progressive increase in total and LDL cholesterol levels; higher in
premenopausal females, while HDL level does not fluctuate; HDL level slightly higher in females
Creatinine and CK are muscle mass dependent; generally higher in males
Serum iron lower level in premenopausal females
Sex differences reflected in separate reference intervals
Time
Cortisol peak, 6:00 AM; lower toward evening and midnight
Proinflammatory cytokines (IL-1 alpha, IL-12, TNF-alpha, INF-gamma, CD4+ T cells) and urinary
neopterin levels opposite the cortisol rhythm
Glucose tolerance test values higher in afternoon
LH, FSH, and testosterone released in short bursts
Potassium, iron, thyrotropin, and growth hormone subject to diurnal effects
Season
Summer
Vitamin D levels higher
Triiodothyronine levels 20% lower
Winter
Triglyceride level lower
Slight (2.5%) increase in total cholesterol level
Altitude
At 1,400 m, hematocrit and hemoglobin values 8% higher
At 3,600 m, 65% increase in C-reactive protein level
As altitude increases, levels of renin, transferrin, and estriol and creatinine clearance decrease
References
Guder et al2
Guder et al2
Guder et al2
Kratz and Lewandrowski16
Guder et al2
Petrovsky et al17; Miyawaki
et al18; Auzeby et al19
Guder et al2
Narayanan8
Young1; Guder et al2; Keffer20;
Narayanan21
Narayanan3
Guder et al2; Harrop et al25
Narayanan3; Statland and Winkel24
Cooper et al22; Narayanan23
Young1; Guder et al2
Guder et al2
CK, creatine kinase; FSH, follicle stimulating hormone; HDL, high-density lipoprotein; IL, interleukin; INF, interferon; LDL, low-density lipoprotein; LH, luteinizing hormone;
TNF, tumor necrosis factor.
Am J Clin Pathol 2000;113:429-452
431
Glucose values obtained during an oral glucose tolerance test tend to be higher when the test is performed in the
afternoon than when the test is performed in the morning.
The cortisol rhythm apparently may be responsible for the
discrepancy noted in the results of an oral glucose tolerance
test performed in the afternoon.2
Thyrotropin levels peak during the late evening hours,
with the lowest values observed around midday.20,21 Growth
hormone levels during waking hours are minimal, with
increases noted during sleep.1
Reproductive hormones, such as luteinizing hormone,
follicle-stimulating hormone, and testosterone, are released
in bursts lasting barely 2 minutes, making accurate assessment of their concentration problematic.8
Seasonal Changes
Vitamin D levels tend to be higher during the summer
(Table 2), apparently due to prolonged exposure to
sunlight.3 A slight increase in the total cholesterol level
(average, 2.5%) has been observed during the winter
compared with values measured during the summer.22,23
The decrease in the level of triglycerides in serum from
summer to winter is more striking than the extent of decline
noted between spring and autumn months when the temperature tends to be milder.3,24
The levels of the thyroid hormone, triiodothyronine, are
reported to be 20% lower during the summer compared with
during the winter.2,25
Altitude
Changes in levels of some of the constituents in blood
occur when measured at sea level as opposed to measurement at a higher altitude (Table 2). For example, hematocrit
and hemoglobin levels can be up to 8% higher at an altitude
of 1,400 m.1,2 A 65% increase in C-reactive protein has been
reported at 3,600 m. Concentrations of some analytes, such
as plasma renin, serum transferrin, urinary creatinine, and
estriol, and the creatinine clearance rate decrease with
increasing altitude.1,2
Menstruation
At the onset of menstruation, a low estrogen level triggers
the release of follicle stimulating hormone from the pituitary
gland. The ovaries thus are stimulated to produce estrogen,
and the level begins to increase noticeably from the sixth or
seventh day after menstruation; the peak level is reached on
approximately the 13th day. A day later, a burst of luteinizing
hormone released from the pituitary gland signals ovulation.
With the onset of ovulation, the progesterone level continues
to rise until it decreases together with the estrogen level, just
before the commencement of the next menstrual cycle. Thus,
the reference intervals for estradiol, follicle stimulating
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hormone, luteinizing hormone, and progesterone are influenced by the stages of menstrual cycle (ie, follicular, midcycle,
and luteal phases) Table 3.16
Coincident with ovulation, serum cholesterol levels are
lower than at any other phase of the menstrual cycle. 2
During midcycle or the luteal phase, the aldosterone
concentration is approximately 2-fold higher compared
with values during the follicular phase.1,2 Renin activity
may increase during the luteal phase of the cycle. 1 In
contrast, serum phosphate and iron levels decrease during
menstruation.1,2
Pregnancy
A dilutional effect is observed due to an increase in
the mean plasma volume, which in turn causes hemodilution (Table 3). 2,3 The effect is more pronounced when
measuring trace constituents such as trace elements in
serum.3 During pregnancy, there is a considerable increase
in the glomerular filtration rate. Hence, the creatinine
clearance rate is increased by 50% or more over the
normal rate.7 During the third trimester, the urine volume
may increase by approximately 25%.
During the second half of pregnancy, the placenta
progressively begins to produce hormones antagonistic to
insulin. As a result, the levels of hormones, such as
estrogen, progesterone, and human placental lactogen (also
called chorionic somatomammotropin), increase, leading
to the onset of gestational diabetes.26
The increased metabolic demand during pregnancy
causes an increased mobilization of lipids. As a result,
especially during the second and third trimesters, the
serum levels of apolipoproteins AI, AII, and B, triglycerides, and total cholesterol (especially LDL-C) are
increased substantially.22,23,27 The levels of these lipids in
serum return to normal within 10 weeks after delivery
unless the mother breast-feeds her infant.28
Human chorionic gonadotropin elaborated by the
placenta has weak thyrotropic activity. Placental human
chorionic gonadotropin levels are at the highest concentration toward the end of the first trimester of pregnancy, thus
suppressing serum thyrotropin levels accompanied by a
slight increase in the free thyroxine level.21,29 During the
second and third trimesters of pregnancy, thyrotropin
levels are more reliable. Hence, trimester-based reference
intervals for thyrotropin are needed in pregnancy.
Associated with an increase in the glomerular filtration
rate in pregnancy is an increase in the clearance of iodide
and a transfer of iodide and iodothyronines to the fetus. As
a result, the inorganic iodide level in serum decreases to
such an extent that pregnant women with marginal iodine
intakes of less than 50 g/d develop iodine deficiency and,
in turn, enlargement of the thyroid gland.21,29
Table 3
Effect of Menstruation and Pregnancy on Laboratory Results
Condition/Effects
References
Menstruation
Estradiol, FSH, LH, progesterone levels depend on stages of cycle
Cholesterol level lowest at ovulation
Mid or luteal phase, aldosterone 2-fold higher than in follicular phase
Renin increased in luteal phase
Decrease in serum iron and phosphate
Pregnancy
Increase in mean plasma volume leads to hemodilution
Creatinine clearance increased by 50% or more due to increase in glomerular filtration rate
Increase in urine volume (25%) during third trimester
Estrogens, progesterone, and human placental lactogen levels increase during second half of
pregnancy, all are antagonistic to insulin; gestational diabetes develops
Increased mobilization of lipids during second and third trimesters (increase levels of total
cholesterol, LDL-C, triglycerides, and apolipoproteins AI, AII, and B)
Thyrotropin levels suppressed owing to increased placental HCG at end of first trimester
Increased iodide clearance; with marginal iodide intake, can affect thyroid function
Estrogen effect increases levels of TBG, T3 and T4
ESR increases 5-fold due to acute phase proteins
Increased levels of factor VII and PAI-2; decreased levels of iron and ferritin
Kratz and Lewandrowski16

Guder et al2
Young1
Guder et al2; Narayanan3
Narayanan and Appleton7
Narayanan26
Cooper et al22,27;
Narayanan23
Narayanan21; Burrow et al29
Narayanan21; Burrow et al29
Narayanan21
Guder et al2
Guder et al2; Narayanan8;
Sprengers and Kluft30;
Narayanan and Hamaski31
ESR, erythrocyte sedimentation rate; FSH, follicle stimulating hormone; HCG, human chorionic gonadotropin; LDL-C, low-density lipoprotein cholesterol; LH, luteinizing
hormone; PAI-2, plasminogen activator inhibitor type 2; T3, triiodothyronine; T4, thyroxine; TBG, thyroxine-binding globulin.
The clearance by the liver during the first trimester of

pregnancy of the sialylated thyroxine-binding globulin,
which is induced by estrogens, is decreased. Hence, the level
of thyroxine-binding globulin increases, leading to the wellknown increases in serum thyroxine and triiodothyronine
levels during pregnancy.
Other analytes with increased levels during pregnancy
include the heat-stable alkaline phosphatase elaborated by
the placenta, alpha-fetoprotein, copper binding proteins such
as ceruloplasmin, and, in turn, copper, acute phase proteins,
and coagulation factor VII.2,8 The increased level of acute
phase proteins increases the erythrocyte sedimentation rate
approximately 5-fold.2 Plasma concentrations of the fibrinolysis inhibitor, plasminogen activator inhibitor type 2, are
increased up to 2 mol/L during the third trimester of pregnancy.30,31 An increased metabolic requirement during pregnancy leads to a deficiency of iron, and the depletion of iron
stores is reflected by a decrease in ferritin.2
Lifestyle
Diet influences the results obtained for some analytes
Table 4. Differences between vegetarians and nonvegetarians
consuming a high-protein or a high-purine diet are well
known.2 Nonvegetarians tend to have increased blood levels of
uric acid, urea, and ammonia compared with those for their
vegetarian counterparts. Diets rich in saturated fatty acids in

general are lipogenic; the effect, however, varies depending on
the fatty acid. Thus, while stearic acid has little effect on LDLC levels, palmitic acid substantially increases plasma cholesterol levels.23,27 Complex carbohydrates and monounsaturated
and polyunsaturated fatty acids, when substituted for saturated
fatty acids, tend, however, to lower LDL-C levels.23,27
A diet rich in fish oils lowers serum triglyceride and verylow-density lipoprotein (VLDL) levels, presumably because of
the ability of fish oils to inhibit the synthesis of VLDL triglycerides.23,32 Even among vegetarians, there is a difference in
serum lipid levels. Among strict vegetarians, plasma LDL-C
levels were reported to be 37% lower and HDL-C levels 12%
lower compared with a nonvegetarian control population. In
contrast, among lactovegetarians, LDL-C levels were 24%
higher, and HDL-C levels 7% higher compared with a group
of strict vegetarians.23,33
Consumption of caffeine influences the results of some
analytes (Table 4). Caffeine inhibits the enzyme phosphodiesterase, which normally regulates the levels of cellular
cyclic adenosine monophosphate (c-AMP) by converting it
to the inactive product 5-AMP.2,3 The increase in c-AMP
levels due to the caffeine-induced effect promotes glycolysis coupled with energy production, and the subject feels
alert, awake, and energetic.
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433
Table 4
Effect of Lifestyle on Laboratory Results
Lifestyle/Effects
References
Diet
High-protein and high-purine diet increase levels of uric acid, urea, and ammonia in blood
compared with vegetarians
Differences in lipogenicity of saturated fatty acids: stearic acid, less effect; palmitic acid,
substantial effect in raising LDL-C
Complex carbohydrates, monounsaturated and polyunsaturated fatty acids lower the levels of LDL-C
Fish oils lower the levels of triglycerides and VLDL
Lipid profile (LDL-C, HDL-C) differences for lactovegetarians vs strict vegetarians
Caffeine
Inhibits phosphodiesterase; raises cAMP; activation of triglyceride lipase causes 3-fold
increase in free fatty acids
Decreased pH; increased ionic calcium level; free drug or free hormone stripped from albumin
Plasma renin activity and catecholamine levels increase
Ethanol
Short- vs long-term effects
Increased levels of GGT, AST, and ALT subject to interindividual and intraindividual variation
Moderate intake increases levels of HDL-C and apolipoproteins AI and AII
Smoking
Long-term smoking increases carboxyhemoglobin, hemoglobin, RBC, WBC and MCV values;
WBC level related to number of packs smoked
Cadmium levels higher
Reduction in HDL-C dependent on extent of smoking
Increased levels of plasma epinephrine, aldosterone, cortisol, free fatty acids, and free glycerol
within 1 h of smoking
ACE activity reduced owing to destruction of lung endothelial cells
Guder et al2
Narayanan23; Cooper et al27
Narayanan23; Harris et al32
Narayanan23; Sacks et al33
Statland and Winkel24
Narayanan8
Guder et al2; Robertson et al34
Guder et al2
Freer and Statland36,37
Narayanan23; Freer and
Statland37; Taskinin et al38
Narayanan3,8; Statland and
Winkel24
Narayanan3,8
Guder et al2; Haboubi et al39
ACE, angiotensin-converting enzyme; ALT, alanine aminotransferase; AST, aspartate aminotransferase; cAMP, cyclic adenosine monophosphate; GGT, gammaglutamyltransferase; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; MCV, mean corpuscular volume; VLDL, very-low-density
lipoprotein.
Activation of triglyceride lipase by c-AMP results in a

3-fold increase in nonesterified or free fatty acid levels.2,3,24
The ability of free fatty acids to compete for binding sites
on the albumin molecule can lead to the displacement of a
drug or hormone bound to albumin, thus affecting the
measurement of free drug or hormone levels.8 The increase
in free fatty acid levels results in a decrease in pH, which, in
turn, can strip calcium from the binding protein. Consequently, a spurious increase in ionic calcium concentration
can occur.8 Plasma renin activity and catecholamine levels
have been increased 3 hours after the consumption of 250
mg of caffeine.2,34
The effect of caffeine on lipid levels has been controversial, and the results obtained seem to be influenced by
coffee-brewing methods.23 The finding in a double-blind
study replacing caffeine-containing coffee with decaffeinated coffee for a period of 6 weeks of an insignificant
change in serum triglyceride, total cholesterol, and HDL-C
levels suggests that caffeine is not the substance in coffee
that elevates the total cholesterol level.35
Consumption of ethanol can induce short- and longterm effects altering the results of several analytes (Table
4).2 Short-term effects reflected rapidly within the first 2 to
4 hours of consumption include a lowered blood glucose
level and an increased plasma lactate level.2 Uric acid levels
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Am J Clin Pathol 2000;113:429-452
increase owing to the metabolism of ethanol to acetaldehyde

and further to acetate. The net effect of lactate formation is
to cause metabolic acidosis owing to the consumption of
bicarbonate.2
Sustained consumption of ethanol can, by enzyme
induction, lead to the increase in the activity of liver
enzymes, such as gamma-glutamyltransferase. There are,
however, marked intraindividual and interindividual variations in the level of serum gamma-glutamyltransferase, as
demonstrated in a study in which subjects were challenged
for 3 days with a 0.75-g of ethanol dose per kilogram of
body weight.36 Other liver enzymes that become elevated
owing to the direct cytotoxic effect of ethanol on liver are
aspartate aminotransferase (AST) and alanine aminotransferase (ALT).2 Thus, consumption of 225 g of ethanol per
day for 1 month caused skeletal muscle changes that led to
the release of liver cell enzymes into blood.37
Depending on the extent of consumption, the effect of
ethanol on serum lipid levels is variable.23 The effect of
short-term alcohol intake, especially by subjects who
usually do not consume alcohol, is to increase plasma
triglyceride levels, especially the VLDL fraction.38
A moderate intake of ethanol (<40 g/d) is reported to
increase the plasma levels of HDL-C, HDL3-C, and the
apolipoproteins AI and AII. However, if alcohol intake
exceeds 80 g/d, the synthesis of VLDL is stimulated,

together with the activation of the enzyme lipoprotein
lipase. Lipoprotein lipase hydrolyzes VLDL triglycerides,
with the result that plasma VLDL levels will be apparently
normal even when there is increased VLDL synthesis.23,27,38
Long-term smoking induces alteration in both biochemical and cellular profiles (Table 4).3,8 Carboxyhemoglobin
levels in blood are higher in long-term smokers since cigarette smoke contains carbon monoxide, which has a much
greater affinity for hemoglobin than does oxygen. As a
consequence of long-term smoking, there is an increase in
hemoglobin levels and the RBC and WBC counts. The
RBCs become larger, thus increasing the mean corpuscular
volume (MCV). Striking differences in the WBC count have
been observed between healthy nonsmokers and healthy
long-term smokers. Healthy smokers who smoked 1 pack of
cigarettes per day for 1 year had a WBC count of approximately 1,000/L (1 109/L) higher than that for healthy
nonsmokers. Healthy smokers who smoked 2 packs of cigarettes per day for 1 year had a WBC count of approximately
2,000/L (2 10 9 /L) higher than that for healthy
nonsmokers.24
Cadmium levels in blood are higher in healthy longterm smokers compared with levels for healthy nonsmokers,
thus requiring separate reference intervals to distinguish the
2 populations.3,8
Smoking can reduce HDL-C levels in plasma; the
extent of reduction apparently is related to the number of
cigarettes smoked per day.23,27 Short-term changes occurring within 1 hour of smoking 1 to 5 cigarettes include an
increase in the plasma levels of epinephrine, aldosterone,
cortisol, free fatty acids, and free glycerol.1,2
Angiotensin-converting enzyme (ACE) activity is
decreased in smokers, presumably owing to the destruction
of lung endothelial cells with the resultant decrease in the
release of ACE into the pulmonary circulation or, alternatively, to inhibition of enzyme.2,39
The extent of smoking (moderate or heavy) parallels
the blood levels of thiocyanate and cotinine. Compared with
nicotine, which is the parent compound, cotinine has a
longer half-life, approximating 20 to 28 hours, making it an
ideal marker for the assessment of the extent of smoking.2
Specimen Collection Variables
The preparation of subjects for blood specimen collection, such as the duration of overnight fast, time of specimen
collection, and posture during blood sampling Table 5; the
effects of the duration of tourniquet application, infusion,
and exercise (Table 5); the effects of anticoagulants Table 6
and stabilizing additives Table 7 used for blood collection;
the anticoagulant/blood ratio; specimen handling and
processing; and the relative merits of anticoagulants and
other variables need to be delineated and standardized to

minimize preanalytic error.
Duration of Fasting
Ideally, subjects should be instructed to fast overnight
for at least 12 hours before specimen collection (Table
5).2,23 The reason for the 12-hour stipulation is based on the
fact that an increase in the serum triglyceride level after a
fatty meal can persist for up to 9 hours, but there is little
effect on the levels of total cholesterol or apolipoproteins
AI and AII.40
The effect of prolonged fasting, however, can affect
some laboratory results. Thus, during a 48-hour fast, the
hepatic clearance of bilirubin could be approximately
240% more than the nominal value.3,24 With prolonged
fasting, there is a decrease in the level of specific proteins,
such as the C3 component of complement, prealbumin, and
albumin. Normal levels of these proteins are restored
rapidly with protein supplementation.24
The effect of fasting, however, varies depending on
body mass.23 In a lean person, the use of fat as reflected by
an increase in acetoacetic acid concentration in blood is
minimal during a 1-day fast but increases rapidly with
prolonged fasting. 41 In an obese person, however, the
acetoacetic acid level increases markedly during a 1-day
fast. In contrast, while lipolysis, as reflected by a 3-fold
increase in the serum glycerol concentration with 3 days of
fasting is observed in a lean person, little change is noted in
an obese person.23,41
Time of Specimen Collection
The section Time referred to diurnal changes seen in
the circulating level of some analytes. It is, therefore,
important that the time of specimen collection be kept
constant from day to day to rule out variations introduced
by diurnal effects (Table 5).
Specimens for therapeutic drug monitoring (TDM)
should be obtained after a steady therapeutic concentration
of drug or steady state is achieved in the blood. A blood
sample obtained just before administering a drug dose after
a steady-state level is reached will reflect the trough level
or the lowest concentration to be obtained with the established drug dose. If only 1 blood sample is to be collected
for TDM, it is preferred that it reflect the trough level.42
Blood samples obtained when the drug concentration
is at its maximum, will, of course, reflect the peak level.
Peak-level specimens need to be obtained within 1 to 2
hours after intramuscular administration of the drug, 15 to
30 minutes after intravenous administration, or 1 to 5
hours after oral administration. 42 Timing of specimen
collection for TDM, however, depends on the rate of distribution of the specific drug. If the drug is infused intraAm J Clin Pathol 2000;113:429-452
435
Table 5
Effects of Specimen Collection Variables on Laboratory Results
Variable/Effects
References
Fasting
12-h overnight fast recommended, since increase in triglycerides persist up to 9 h after fatty meal
After 48-h fast, 240% increase in bilirubin level; decreased levels of prealbumin, albumin, and C3
Effects dependent on body mass
Time
Standardize time to rule out diurnal effect
Therapeutic drug monitoring trough vs peak levels; timing regimens
Urine, timed overnight or first morning specimen concentrated, ideal for sediment enumeration
24-hour sample for clearance measurements and for analytes with diurnal variation
Random or spot urine generally suitable
But, sensitivity to detect slight increases in numbers of cells and casts and glucose and protein
levels affected by dilution
Posture
In general, 5%-15% increase for most cellular and large molecules (eg, albumin) in sitting posture
Increase in upright to supine has dilutional effect: 10% and 12% decreases in cholesterol and
triglyceride levels, respectively
Change in posture has no effect on free drug and small molecules
Postural effect substantial in patients with reduced plasma volume (eg, congestive heart
failure, cirrhosis)
Increased aldosterone, norepinephrine, epinephrine, renin, and atrial natriuretic peptide levels
Tourniquet application time
>1 minute increase in concentration of large molecules; for total cholesterol level, 5% increase
at 2 minutes; up to 15% increase at 5 minutes
Decreased pH; increased potassium, lactate, ionized calcium, and magnesium levels with
increased application time
Repeated fist clenching can increase potassium by 1-2 mmol/L, up to 2.7 mmol/L
Infusion
Spuriously increased blood glucose level if specimen obtained from arm receiving glucose infusion
Waiting times before blood specimen collection: subjects receiving fat emulsion, 8 h; carbohydrate,
protein, or electrolytes, 1 h
Extent of hemolysis related to age of transfused blood
Exercise
Strenuous exercise increases total CK and CK-MB; CK-MB as percentage of total CK normal;
decreased haptoglobin value due to intravascular hemolysis
Individual variability in CK related to extent of exercise training
Mitochondrial size and number increase with vigorous training; mitochondrial CK increases to >8%
of total CK
Increased epinephrine, norepinephrine, glucagon, cortisol, corticotropin, and growth hormone levels;
decreased insulin level
Increased serum and decreased urine uric acid levels due to increased lactate level
Increased apolipoprotein AI and HDL-C levels; decreased LDL-C, apolipoprotein B, and triglyceride
levels with long-term strenuous exercise or brisk walking
Guder et al2; Narayanan23

Narayanan3; Statland and Winkel24
Narayanan23; Young41
Guder et al2
Pippenger43
Narayanan44
Guder et al45
Narayanan44
Guder et al2; Narayanan8; Statland
and Winkel24
Narayanan8,23; Young41
Young41
Narayanan8
Guder et al2; Tan et al46
Guder et al2; Narayanan8,23;
Cooper et al22; Statland and
Winkel24; Young41
Narayanan8
Don et al47; Skinner et al48
Guder et al2
Guder et al2
Guder et al2
Guder et al2
Narayanan8; Statland and
Winkel24
Guder et al2
Guder et al2
Guder et al2
Guder et al2
Narayanan23; Cooper et al27;
Hardman et al50
CK, creatine kinase; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol.
venously, approximately 1 to 2 hours after the completion

of infusion are needed for the distribution phase to be
completed, with exceptions such as digoxin and digitoxin,
which need 6 to 8 hours for the completion of the distribution phase.2
In general, since the rate of oral drug absorption differs
from person to person, blood specimens obtained for TDM
usually are timed to reflect trough levels. For the monitoring of certain drugs, such as theophylline, antibiotics,
and antiarrhythmic drugs, it may be necessary and useful to
measure peak levels after intravenous administration.2,43
The time for collection of urine specimens depends on
the constituent being measured. For clearance measure436
Am J Clin Pathol 2000;113:429-452
ments or for the measurement of analytes that have diurnal

variation, a 24-hour urine sample is needed. 7 A timed
overnight urine sample or the so-called first-morning specimen, provides not only an assessment of the concentrating
ability of the kidney, but also sufficient time for formed
elements or sediment to be produced for easy enumeration.
While a random or spot sample may be suitable for
routine screening, such a specimen may have its limitations
because it may be too diluted to detect accurately slight
increases in the concentration of analytes such as glucose,
protein, cells, and casts. Thus, a spot or random sample
may be of limited value in the initial screening for microalbuminuria.44 However, in a strategy designed to detect and
Table 6
Anticoagulants for Blood Collection
Anticoagulant
References
Heparin
Unfractionated heparin molecular mass 3-30 kd (mean, 14 kd), as lithium, sodium, or ammonium
salts; lithium heparin preferred for plasma chemistry testing; nominal amount, 14.3 U/mL of blood
For ionic calcium, heparin in blood gas syringe can be calcium-titrated, electrolyte-balanced, or
zinc lithiumtitrated
EDTA: salts of EDTA generally used for routine hematology testing (dipotassium or tripotassium
EDTA, disodium EDTA); nominal amount, EDTA, 1.5 mg/mL of blood
Citrate: generally used for routine coagulation testing; trisodium citratedihydrate 3.2% (0.109 mol/L)
or 3.8% (0.129 mol/L)
Narayanan and Hamasaki31;

Novotny et al51; Narayanan52,54;
Guder et al53; Berg et al55;
Doumas et al56
Narayanan52; Toffaletti et al57;
Toffaletti58; Landt et al59
Narayanan52; Goosens et al62;
Sacker63; Reardon et al64
Guder et al2; Narayanan and
Hamasaki31; Narayanan65;
Adcock et al66
Table 7
Stabilizing Additives
Additive
References
Glycolytic inhibitors
Sodium fluoride (2.5 mg/mL of blood with EDTA [1 mg/mL] or potassium oxalate [2 mg/mL])
Lithium iodoacetate (0.5 mg/mL of blood) alone or in combination with lithium heparin
(14.3 U/mL of blood)
Sodium fluoride and lithium iodoacetate require 3 h to fully become effective
Adding mannose (3 mg/mL of blood) to sodium fluoride achieves efficient glycolytic inhibition
Mannose, however, has concentration-dependent interference
Maintaining blood pH at 5.3-5.9 with mixture of citric acid, trisodium citrate, disodium EDTA, and
0.2 mg sodium fluoride stabilizes glucose
Cell stabilizers
Acid-citrate dextrose A and B formulations; B formulation has greater amounts of dextrose available
to metabolizing cells
Citrate, theophylline, adenosine, dipyridamole mixture minimizes in vitro platelet
activation
Proteolytic enzyme inhibitors
EDTA (1.5 mg/mL of blood) with aprotinin (500-2,000 KIU/mL) stabilizes several polypeptide hormones
Aprotinin also can be used with lithium heparin (14.3 U/mL)
A mixture of EDTA, aprotinin, leupeptin, and pepstatin stabilizes parathyroid hormonerelated protein
Catecholamine stabilizers
An antioxidant such as glutathione, sodium metabisulfite, or ascorbic acid at a concentration of 1.5
mg/mL is used with egtazic acid, EDTA, or heparin
For urine, use 5 mL of a 6-mol/L concentration of hydrochloric acid per liter of urine or 250 mg
each of EDTA and sodium metabisulfite per liter of urine
Fibrinolytic inhibitors
A mixture of thrombin and soybean trypsin or aprotinin
Reptilase and aprotinin
Thrombin inhibitor: 5-mmol/L concentration of a phenylalanine, proline, arginine chloromethyl ketone
mixture with 10-mmol/L concentration of citrate or 4.2-mmol/L concentration of EDTA
Guder et al2; Chan et al67;

Sidebottom et al68
Guder et al2; Chan et al67
Sidebottom et al68
Guder et al2
Liss and Bechtel71;
Nakashima et al72
Nakashima et al72; Ho et al73
Uchida et al70
Guder et al2
Narayanan and Hamasaki31;
Contant et al74; Narayanan75;
Van Den Besselaar et al76;
Kuhne et al77
Narayanan52,79
Narayanan52
Pandian et al80; Narayanan81
Narayanan81
Boomsma et al82
Narayanan and Hamasaki31
Connaghan et al83
Mohler et al84
KIU, kallikrein inhibitory unit.
differentiate prerenal, glomerular, tubular, and postrenal

causes of proteinuria by measurement of specific proteins,
a morning spot urine specimen was adequate.45
Posture During Blood Sampling
Changes in posture during blood sampling can affect
the concentration of several analytes measured in serum or
plasma (Table 5). Change in posture from a supine to the
erect or sitting position can result in a shift in body water
from the intravascular to the interstitial compartment. As a
result, the concentration of large molecules that are not

filterable is increased. Hence, albumin levels generally are
higher among healthy subjects attending an outpatient clinic
from whom blood specimens are obtained with the subjects
in a sitting position compared with levels among healthy
hospitalized subjects from whom blood specimens usually
are obtained with the subjects in a supine position.8,24
Small molecules, such as glucose, however, owing to
their ability to move freely between the interstitial space
and the circulation, are least affected by posture during
Am J Clin Pathol 2000;113:429-452
437
blood specimen collection.8,41 In general, molecules even

as large as insulin can diffuse freely into and out of the
interstitial space and, thus, are not subject to variation due
to postural changes.8
While the free fraction of a metabolite, drug, hormone,
or metal ion is not subject to postural variation, the fraction
bound to protein, such as albumin, is affected by posture.
Thus, bilirubin bound to albumin and calcium bound to
albumin are affected by postural changes.2,8
In blood specimens obtained with subjects in the sitting
position, generally an increase in the range of 5% to 15% is
seen for most cellular and macromolecular analytes
compared with results when blood specimens were obtained
with subjects in the supine position.2
The effect of postural change from supine to erect is
accentuated in patients with a tendency to edema, such as
patients with cardiovascular insufficiency and cirrhosis of the
liver.2 In such patients, reduction in plasma volume in turn
causes increased secretion of hormones, such as aldosterone,
norepinephrine, and epinephrine, and the enzyme, renin.2 An
increased level of atrial natriuretic peptide (ANP) is measurable in plasma as a compensatory mechanism to correct the
decrease in blood pressure.2,46 Indeed, changing from supine
to erect can dramatically influence the concentration of
analytes such as renin and aldosterone, even in healthy
persons. Conversely, moving from upright to supine can have
a dilutional effect owing to an increase in plasma volume
resulting from the transfer of fluid from the extravascular to
the intravascular compartment.8 Thus, a change from upright
supine can reduce (after 5 minutes in the supine position) the
cholesterol level by 10% and triglyceride levels by 12%.23,41
If blood specimens are to be obtained with the subject in
a sitting position, such as in an outpatient setting, the sitting
posture should be standardized by instructing the subject to
remain seated for 15 minutes before specimen collection.
Duration of Tourniquet Application
The application of the tourniquet, by reducing the pressure below the systolic pressure, maintains the effective
filtration pressure within the capillaries.2 As a result, small
molecules and fluid are transferred from the intravasal space
to the interstitium. Application of the tourniquet for longer
than 1 minute and up to 3 minutes can result in hemoconcentration, causing an increase in the concentration of large
molecules that are unable to penetrate the capillary wall
(Table 5).2,8,24,41
The venous stasis that results from prolonged tourniquet
application promotes anaerobic glycolysis with the accumulation of plasma lactate and a reduction in blood pH.8 The
hypoxic effect drives potassium out of the cell causing a
spurious increase in the serum potassium level. This effect
can be accentuated if the phlebotomist requests the subject to
438
Am J Clin Pathol 2000;113:429-452
repeatedly clench the fist to permit visualization of the vein.

The contraction of forearm muscles during repeated fist
clenching causes release of potassium, since there is a reduction in the intracellular electronegativity during the depolarization of muscle cells, which favors the release of potassium
rather than its uptake.47 Repeated fist clenching during phlebotomy after application of the tourniquet can cause a 1- to
2-mEq/L (1- to 2-mmol/L) increase in potassium. In 1 study,
an increase in the potassium level as much as 2.7 mEq/L (2.7
mmol/L) was noted in a healthy subject due to fist clenching
during phlebotomy.48 Application of the tourniquet for more
than 1 minute and up to 2 minutes can cause a 5% increase
in the total cholesterol level, which can increase by 10% to
15% if the tourniquet is left on for 5 minutes.22,23,41
In 1 study conducted on children, venostasis of 2 minutes
substantially decreased the blood pyruvate concentration by
approximately 18% yet had a negligible effect on the lactate
concentration, with a mean increase of just 2.2%.49
The reduction in blood pH during prolonged tourniquet
application can alter drug-protein or hormone-protein
binding, leading to an increase in the free drug or free
hormone concentration. Thus, at a low pH caused by anaerobic glycolysis during venous stasis, ionic calcium and
ionic magnesium levels increase since they are released
from albumin, the binding protein.8
To minimize the preanalytic effects of tourniquet application time, the tourniquet should be released as soon as
the needle enters the vein.8 Avoidance of excessive fist
clenching during phlebotomy and maintaining tourniquet
application time to no more than 1 minute can minimize
preanalytic error.2
Effect of Infusion
For persons receiving an infusion, blood should not
be obtained proximal to the infusion site.2 Blood should
be obtained from the opposite arm. Very often, exceptionally high glucose values are obtained by collecting blood
from the arm in which an infusion of glucose is being
administered (Table 5). Eight hours must elapse before
blood is obtained from a subject who has received a fat
emulsion. The waiting period for blood sampling from
persons receiving a carbohydrate-rich solution or amino
acid and protein hydrolysate or electrolytes is 1 hour after
cessation of the infusion.2 For subjects receiving blood
transfusions, the extent of hemolysis and, with it,
increased values for potassium, lactate dehydrogenase,
and free hemoglobin are related progressively to the age
of the transfused blood.2
Effect of Exercise
Exercise, such as running up and down several flights
of stairs, or strenuous activity the night before specimen
collection, such as a workout in a gymnasium or marathon

running, can affect the results obtained for several analytes
(Table 5).
Strenuous exercise depletes the energy-yielding
compound, adenosine triphosphate (ATP), from the muscle
cells. As a result, there is a change in cell membrane
permeability, and enzymes are released from the cells.
Thus, CK levels can be very high after strenuous exercise
(levels approaching 1,000 U/L) with a release of the CKMB fraction, although CK-MB as a percentage of total CK
would be normal.8
A high degree of individual variability is seen in a
hypoxia-mediated increase in CK, which depends on the
extent of exercise training.2 Mitochondrial size and number
are increased in persons who maintain a vigorous training
schedule, allowing their muscles to oxidatively metabolize
glucose, fatty acids, and ketone bodies. As a result, even in
the absence of myocardial damage, mitochondrial CK-MB
levels increase to greater than 8% of total CK activity.2
Training increases muscle mass, thereby increasing the
concentration of creatinine in plasma and the urinary excretion of creatinine and creatine, while decreasing the level of
plasma lactate compared with untrained persons who
indulge randomly in exercise.
During strenuous exercise, there is some amount of
intravascular hemolysis. As a result, the level of plasma
haptoglobin, an acute phase reactant, is reduced owing to
its complexation with free hemoglobin released during
hemolysis and the subsequent clearance of the complex
from circulation.8,24
As a result of exercise, the concentrations of hormones
such as epinephrine, norepinephrine, glucagon, cortisol,
corticotropin, and growth hormone increase, while insulin
levels decrease, thereby causing an increase in glucose
levels because of the gluconeogenetic effect of hormones
antagonistic to insulin.2
Volume shift from the intravasal to the interstitial
compartment or volume depletion due to sweating during
exercise can increase serum albumin levels.2 Increased
lactate concentration due to anaerobic glycolysis during
exercise can decrease the urinary excretion of uric acid,
thereby increasing its serum concentration.2 There are,
however, beneficial effects of long-term strenuous exercise
or brisk walking in terms of reducing the levels of serum
LDL-C, apolipoprotein B, and triglycerides and increasing
the levels of apolipoprotein AI and HDL-C.23,27,50
To minimize preanalytic variables introduced by exercise, subjects should be instructed to refrain from strenuous activity at least the night before testing and to not
exert themselves by walking a long distance or running or
climbing stairs before blood specimen collection.8
Anticoagulants for Blood Collection

Various salts of heparin, EDTA, and sodium citrate are
used widely in the clinical laboratory (Table 6).
Heparin
Heparin is the preferred anticoagulant for blood specimen
collection intended for determination of electrolyte levels and
other routine chemistry values. The standard commercialgrade heparin preparation, also called unfractionated heparin,
with a molecular mass ranging from 3 to 30 kd (mean, 14 kd)
exerts its anticoagulant effect by binding to antithrombin III.
This interaction causes a conformational change in the
antithrombin III molecule, thereby accelerating the inhibition
of coagulation factors Xa, IXa, and thrombin.31 The standard
commercial grade heparin also can inhibit thrombin with the
aid of a plasma cofactor, called heparin cofactor II. In vivo,
heparin can cause the release of a potent coagulation inhibitor,
called tissue factor pathway inhibitor, from the endothelial
surface.31,51
Various salts of heparin, such as lithium, sodium, and
ammonium, have been used as anticoagulants for the collection of blood specimens intended for routine clinical chemistry
determinations.52 The nominal amount of heparin used in
blood specimen collection tubes is 14.3 U/mL of blood,
although a range of 12 to 30 U/mL of blood has been deemed
satisfactory.53
Although all of the 3 salts of heparin give comparable
results for electrolyte determinations, lithium heparin has been
favored over the others, more because of the perception that
sodium heparin might overestimate sodium levels and ammonium heparin might spuriously increase serum urea nitrogen
concentrations when measured by the urease procedure.52,54
Just how much sodium is present in a sodium heparin
blood collection tube depends not only on the nominal
amounts of heparin present (14.3 U/mL of blood), but also on
other variables, such as the molecular weight of unfractionated
heparin, the number of dissolvable milliequivalents of sodium
per milligram of heparin, and the consistency of the heparin
preparation from lot to lot. Indeed, in 1 study when blood was
filled to half of the tubes nominal volume, thereby increasing
2-fold the nominal amounts of sodium heparin present in the
blood collection tube, plasma sodium values were not
increased artifactually.54
Obvious differences in results for certain analytes
between serum and heparinized plasma are related to the
consumption of fibrinogen and the lysis of cellular elements
during the process of clotting. Thus, potassium values are
higher in serum than in heparinized plasma, while in contrast,
total protein values are higher in plasma than in serum. The
effect of heparinized plasma on some of the enzyme measurements may be related, in part, to the bias introduced by the
instrument-reagent combination.55,56
Am J Clin Pathol 2000;113:429-452
439
Since the commonly used anticoagulant, such as

lithium or sodium heparin, in the syringe to obtain specimens for blood gas measurement binds to ionic calcium,
with the effect dependent on the concentration of heparin,
attempts have been made to eliminate this bias. One
approach has been to use calcium-titrated heparin, in which
heparin in an amount less than 50 U/mL of blood is titrated
to an ionized calcium concentration of 1.25 mmol/L.52 The
use of electrolyte-balanced heparin that involves the use of
dry heparin in the amount of 40 U/mL of blood balanced
with calcium, sodium, and potassium decreases bias to less
than 0.02 mmol/L of calcium in the analytic range of 0.9 to
1.8 mmol/L.57 The use of dry heparin eliminates the dilution effect associated with liquid heparin.
Other approaches to minimize interference with
ionized calcium measurements have involved saturating the
high-affinity binding sites on heparin to which divalent
cations, such as zinc, can bind. Zinc heparin, however, in
the presence of excess zinc ions is reported to cause a positive interference in ionized calcium and total magnesium
measurements.58 One successful approach is to saturate
high- and low-affinity binding sites on heparin, the former
with zinc and the latter with lithium. 59 This calciumneutralized lithium-zinc heparin has been satisfactory not
only for ionized calcium measurements, but also for
measurement of blood gases, total calcium, sodium, and
potassium.59
For TDM, the drug level in heparinized plasma would
be expected to be higher than that in serum, especially if
the drug was bound to fibrinogen. In reality, the serum
value for drugs such as amiodarone, desethylamiodarone,
chloroquine, and desethylchloroquine was higher than the
plasma value, apparently due to the release of cell-bound
drug during the clotting process. 52,60,61 Thus, results
obtained for amiodarone were 11.5% to 13.5% lower in
heparinized plasma than in serum, while results for
desethylamiodarone were 4.4% to 8.5% lower in plasma
than in serum.60
The concentration of chloroquine was approximately
2-fold higher in serum than in plasma, while the concentration of its metabolite, desethylchloroquine, was approximately 4 times higher in serum than in plasma. These 2
drugs were present mainly in platelets and granulocytes.
The increased levels of these 2 drugs noted in serum apparently were related to the release of these drugs from
platelets during the coagulation process. Thus, for some
drugs, it is important to specify whether plasma or serum
was used for measurement.61
EDTA
EDTA is the commonly used anticoagulant for routine
hematologic determinations. It functions as an anticoagu440
Am J Clin Pathol 2000;113:429-452
lant by chelating calcium ions, which are required for the

clotting process.
Potassium and sodium salts of EDTA have been used
for blood specimen collection. The solubility of potassium
salts is better than sodium salts of EDTA. The pH of EDTA
varies depending on the salt. As more ions are added to the
free acid, the acidity of EDTA decreases. Thus, while a saturated solution of EDTA as free acid has a pH of 2.5 1.0, the
tripotassium salt (K3EDTA) as 1% solution has a pH of 7.5
1.0.52 The disodium (Na2EDTA) and dipotassium (K2EDTA)
salts of EDTA in a 1% solution have a lower pH. Na2EDTA
has a pH of 5.0 1.0, while K2EDTA has a pH of 4.8 1.0.
This difference in pH affects the size of the RBC. Although
all salts of EDTA are hyperosmolar, causing the water to
leave the cells and the cells to shrink, this shrinking effect is
noticed only with K 3 EDTA and not with K 2 EDTA or
Na2EDTA.52 This is because although the cells shrink with
K2EDTA and Na2EDTA, the lower pH of the anticoagulant
causes the cells to swell as well, thus balancing for the
osmotically dependent shrinkage, and the cell size is not
altered. As a result, while the microhematocrit (centrifuged
hematocrit) is lowered with K3EDTA, it is not affected by
K2EDTA or Na2EDTA. The effect of dilution of the sample
in automated analysis restores cells to their native shape. At
an optimum EDTA concentration of 1.5 mg/mL of blood, no
differences in microhematocrit were noted between samples
collected in K2EDTA and K3EDTA and analyzed between 1
and 4 hours after collection.62
In the same study, the MCV measurement and the calculated hematocrit values were influenced not only by the instrument used, but also by the salt of EDTA (K2 or K3) used for
anticoagulation of the blood specimen. Thus, MCV values
were not affected with blood anticoagulated with K3EDTA,
even at concentrations up to 10 times the nominal value. In
contrast, with 3 of the 4 instruments tested, increasing the
concentration of K2EDTA beyond the nominal value of 1.5
mg/mL of blood resulted in an increase in MCV.62 One explanation may lie in the ready dissolution of liquid K3EDTA
compared with powdered K2EDTA, in which the effectiveness
of mixing might become a variable, perhaps accounting for the
discrepancy in results.
The neutrophils are subjected to morphologic changes
upon collection of blood in EDTA. These changes depend on
the time the peripheral blood smear is prepared and the
concentration of EDTA used for blood specimen collection.
Minor changes begin to appear in the morphologic features
of neutrophils even at an optimal EDTA concentration of 1.5
mg/mL of blood. These changes include swelling of the
neutrophils and loss of structure in the neutrophil lobes.
Thereafter, there is a loss of granulation in the cytoplasm and
the appearance of vacuoles in the nucleus or cytoplasm of
the cells.63 Between 1 and 3 hours after blood collection in
1.5 mg/mL of EDTA, the nucleus swells further, with a

crossover of the nuclear chromatin. Beyond 3 hours, the
morphologic features of the neutrophils become unclear as
the cell disintegrates. If a peripheral blood smear is made
from blood anticoagulated with an EDTA concentration of
2.5 mg/mL of blood, approximating to a conventional blood
collection tube filled to one half of its nominal volume, the
morphologic features of the neutrophils are disintegrated
totally.
Similar time-related and EDTA concentrationrelated
changes also occur in the mononuclear cells, although to a
lesser extent than in the neutrophils. Platelets also undergo
changes, including swelling, giving rise to giant platelets
that ultimately disintegrate, producing fragments in the
same size range as the platelets that are mistakenly counted
as platelets by electronic counters, thus spuriously
increasing the platelet count.
Platelets rapidly change their shape from discoid to spherical when blood specimens are collected in EDTA, making
determinations of mean platelet volume unreliable. The
limited stability of the WBC differential count (up to 6 hours
at room temperature storage) achieved in most of the 5-part
differential analyzers is another drawback with EDTA.64
Citrate
Traditionally, a 0.129-mol/L concentration or a 0.109mol/L concentration of trisodium citrate has been used as the
anticoagulant for collection of blood specimens intended for
global coagulation tests, such as prothrombin time (PT) and
activated partial thromboplastin time (aPTT).2,31 Citrate has
virtually replaced sodium oxalate as an anticoagulant for
performing global coagulation tests, since factor V is more
stable in citrate than oxalate. Furthermore, citrate rapidly
complexes with calcium, forming a soluble complex in
contrast with the slow formation of the insoluble complex of
calcium with oxalate.31
The effective molarity of citrate depends on whether the
dihydrate or the anhydrous salt has been chosen in the preparation of the citrate solution. Nominally, a 3.2% solution of
the dihydrate trisodium citrate salt corresponds to a 0.109mol/L concentration, whereas a 3.8% solution of the same
dihydrate salt corresponds to a 0.129-mol/L concentration.
However, a 3.2% solution of the anhydrous trisodium citrate
solution would correspond to a 0.124-mol/L concentration,
whereas a 3.8% anhydrous sodium citrate solution would
have a molarity of 0.147 mol/L.65 Hence, it is important that
the dihydrate salt be used to prepare either the 3.2% or 3.8%
solution of trisodium citrate.
A laboratory that has been using one of the concentrations (3.2% or 3.8%) to perform PT determinations for
patients receiving oral anticoagulant therapy should not
interchange the formulations. Doing so will affect the inter American Society of Clinical Pathologists
national normalized ratio (INR) units that are used to report

the results of the PT.66 INR values generally are higher
with a 0.129-mol/L concentration of sodium citrate than
with a 0.109-mol/L concentration of sodium citrate, especially when a responsive PT reagent, such as a recombinant
thromboplastin that has a sensitivity similar to the World
Health Organization thromboplastin (with an international
sensitivity index [ISI] equal to 1), is used.66 The differences
in INR between the 2 concentrations of citrate can vary
from 0.7 to 2.7 INR units.66
The INR will be in error when a laboratory that
routinely uses, for example, a 0.109-mol/L concentration of
citrate, occasionally analyzes a specimen collected with a
0.129-mol/L concentration of citrate, but uses the mean of
the normal range obtained with the 0.109-mol/L concentration to calculate the PT ratio (the patients PT divided by
mean of normal range for PT), which together with the ISI
are needed for the calculation of INR.66
Stabilizing Additives
Sodium fluoride and lithium iodoacetate have been used
alone or in combination with an anticoagulant such as potassium oxalate, EDTA, citrate, or lithium heparin for blood
collection (Table 7).2 The concentration of sodium fluoride
when used alone to inhibit glycolysis is approximately 4.3
mg/mL of blood. In combination with an anticoagulant, such
as Na2EDTA or K2EDTA (1 mg/mL of blood) or potassium
oxalate (2 mg/mL of blood), the nominal concentration of
fluoride that is used to inhibit glycolysis is 2.5 mg/mL of
blood (60 mmol/L).2,67
Similarly, lithium iodoacetate has been used alone (0.5
mg/mL of blood) or at that concentration in combination
with lithium heparin (14.3 U/mL of blood).2 Even with the
use of either of these glycolytic inhibitors, at least 3 hours
are needed for these inhibitors to become fully effective
and stabilize glucose levels.2,67 During this 3-hour period in
healthy subjects, an approximately 9 mg/dL loss of glucose
occurs. 2,67,68 However, in neonates or subjects with an
increased RBC, WBC, or platelet count, the decrease in
glucose during the 3-hour period, even in presence of
glycolytic inhibitors, is greater than in healthy subjects.67,69
Once the glycolytic inhibitors become fully effective after
the initial 3-hour period, glucose levels remain stable for at
least 3 days.2,67,68
In the absence of glycolytic inhibitors, a decrease in the
glucose level of as much as 24% can occur in 1 hour after
blood collection in neonates in contrast with a 5% decrease
in healthy adults when specimens are stored at room
temperature. By 5 hours after blood collection in specimens
stored at room temperature, as much as 68% of glucose can
be consumed in specimens collected without glycolytic
inhibitors from neonates with a high hematocrit.69
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The reason that fluoride and iodoacetate take approximately 3 hours to stabilize glucose levels is that they do not
act on the first enzyme in the glycolytic pathway, which is
hexokinase. Instead, fluoride acts on enolase, which is the
eighth enzyme in the glycolytic pathway, while iodoacetate
acts on glyceraldehyde-3-phosphate dehydrogenase, which
is the fifth enzyme in the pathway in which glucose is
metabolized.2
Potassium oxalate, which is used in combination with
fluoride, inhibits pyruvate kinase in the enzymatic step
immediately after enolase in the glycolytic pathway and,
thus, can effectively retard lactate formation 1 hour after
blood collection.
The drawback with both fluoride and iodoacetate is
that they promote hemolysis, which increases progressively
as the concentration of the inhibitor used and the time
elapsed after blood specimen collection increases. Apparently both inhibitors deplete the organic phosphate (ATP)
content of the RBC, thus causing an efflux of potassium out
of the cell.
A variety of approaches have been attempted to
improve the efficacy of glycolytic inhibition. In one
approach, a mixture of citric acid, trisodium citrate,
Na2EDTA, and a very small concentration of sodium fluoride (0.2 mg) effectively inhibits the enzymes in the
glycolytic pathway by maintaining blood pH in the range
of 5.3 to 5.9.70 With this additive mixture, glycolysis was
inhibited for 8 hours in specimens stored at room temperature, while at 24 hours, a mean SD decrease of 1.3 mg
1.1 mg/dL (0.07 0.06 mmol/L) of glucose was noted.70
Another strategy for achieving efficient glycolytic inhibition is to include mannose in addition to fluoride.71 The
rationale is that mannose, which is an epimer of glucose
(differs structurally from glucose in the configuration around
just 1 carbon atom), will compete with glucose for hexokinase, the first enzyme in the glycolytic pathway. Since
mannose is a competitive inhibitor, the inhibition is shortlived, lasting just 4 hours. However, by this time, fluoride,
which is also in the additive mixture, would have become
effective thus achieving a better glucose stabilization.2,71
At a mannose concentration of 3 mg/mL of blood, no
interference was noted in hexokinase and glucose oxidase
procedures for the measurement of glucose.72 However,
when the mannose concentration was increased to 3 times
the nominal concentration of 3 mg/mL of blood, an overestimation of glucose by 18 mg/dL (1.0 mmol/L) was
observed.72 Some of the mannose preparations also may be
contaminated with glucose. Negative interference has been
reported in the hexokinase procedure when mannose
concentrations exceed 3 times the nominal concentration.73
The extent of reduction in glucose levels depends on the
concentration of hexokinase used in the glucose assay, with
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less interference noted at higher concentrations of hexokinase used in the assay.73

In practical terms, if plasma is separated from cells by
centrifugation promptly after blood specimen collection, the
glucose level in plasma is expected to be stable, since the
enzymes that metabolize glucose are in the cellular fraction.
Stabilization of RBCs by including a nutrient such as
glucose together with an anticoagulant mixture is the basis
of 2 acid citrate dextrose (ACD) formulations. 2 These
formulations, labeled ACD A and B, are similar in pH;
ACD A has a pH of 5.05, and ACD B has a pH of 5.1. The
difference between the formulations is the additive/blood
ratio; the A formulation has a ratio of 1:5.67. The B formulation, however, with a ratio of 1:3, has relatively greater
amounts of dextrose available for the metabolizing RBCs
and, hence, can preserve them better. While ACD formulations can preserve RBCs for 21 days when blood is stored
between 1 C and 6 C, their stability can be extended
further by including additives such as phosphate and
adenine (citrate phosphate dextrose and citrate phosphate
dextrose adenine, respectively).
The maintenance of increased levels of cyclic adenosine
monophosphate (c-AMP) within the platelets is the basis of
an additive mixture intended for blood specimen collection
to minimize in vitro platelet activation.74,75 The additive is a
mixture of citric acid, theophylline, adenosine, and dipyridamole with the final pH adjusted to 5.0.74 Adenosine activates the enzyme adenylate cyclase, thus increasing c-AMP
levels within the platelets. Theophylline and, to a certain
extent, dipyridamole prevent the degradation of c-AMP by
inhibiting the enzyme phosphodiesterase. Dipyridamole, in
addition, prevents the uptake of adenosine by the RBCs, thus
effectively increasing the amount of adenosine available to
the platelets to activate adenylate cyclase and, in turn, the
inhibition of platelet aggregation and release of contents of
platelet alpha granules. 31,74 The citrate in the additive
mixture functions as an anticoagulant by chelating calcium.
The citric acid, theophylline, adenosine, and dipyridamole
mixture is used for monitoring heparin therapy by aPTT or
the chromogenic substrate assay and in measurement of
platelet activation markers, such as P-selectin (CD62), by
flow cytometry.76,77
Stabilizing additives to inhibit proteolytic enzymes are
needed for some analytes. Although EDTA itself is a metalloprotease inhibitor, sometimes EDTA alone is insufficient to
inhibit proteolytic enzymes. For example, when a synthetic
protease inhibitor, such as nafamostat mesylate, is added to
EDTA, the mixture can substantially improve the stability of
complement components C3a, C4a, and C5a.52,78
A proteinase inhibitor, aprotinin (Trasylol), when used
in combination with an anticoagulant such as EDTA or
heparin, has been useful for the stabilization of labile
polypeptide hormones and enzymes.52 Aprotinin inhibits

specific proteases of kinin and coagulation and fibrinolytic
enzyme systems, in addition to inhibiting the enzyme
systems of leukocytes and damaged tissue cells. Thus, it
inhibits kallikrein, trypsin, chymotrypsin, coagulation factors
in the preliminary phase of blood clotting, the fibrinolytic
enzyme plasmin, and lysosomal proteinases. The fact that
aprotinin inhibits kallikrein is the basis for the expression of
its potency in terms of kallikrein inhibitory units (KIUs).
Aprotinin has been used in combination with an anticoagulant such as EDTA or heparin. A mixture of EDTA (1.5
mg/mL of blood) and aprotinin in the activity range of 500 to
2,000 KIU/mL has been used to stabilize polypeptide
hormones such as glucagon and corticotropin, the enzyme
renin, and gastrointestinal hormones, such as beta-endorphin,
secretin, neurotensin, gut glucagon, somatostatin, vasoactive
intestinal peptide, and peptide yy.52,79
In the absence of aprotinin, spuriously higher values
may be obtained apparently because of the fragments of the
analyte produced by proteolytic enzyme degradation are
measured as intact molecules by the polyclonal antibody
used in the assay. Thus, in a radioimmunoassay for glucagon,
a 26% spurious increase was noted in plasma obtained with
blood specimens collected with EDTA alone compared with
plasma obtained with blood specimens collected in a mixture
of EDTA and aprotinin.52,79
A mixture of lithium heparin (14.3 U/mL) and 1,000
KIU/mL of aprotinin also has been used to stabilize
immunoreactive somatostatin, secretin, cholecystokinin
(pancreozymin), glucagon, and C-peptide.52
In addition to aprotinin and an anticoagulant such as
EDTA, additional proteolytic enzyme inhibitors may be
needed to stabilize a labile tumor marker, such as parathyroid hormone related protein (PTH-rP) that has been associated with the humoral hypercalcemia of malignant
neoplasm.80,81 Thus, with an additive mixture of EDTA (1.5
mg/mL), aprotinin (500 KIU/mL), leupeptin (2.5 mg), and
pepstatin (2.5 mg), PTH-rP levels were stable for 1 hour
after blood specimen collection for specimens stored at
room temperature and for 24 hours, if specimens were
refrigerated at 4 C.80 Even when blood is collected with this
additive mixture, if the sample is stored at room temperature
for 24 hours, as much as 60% of the PTH-rP activity can be
lost.80 Without a stabilizing additive mixture, PTH-rP is so
unstable that as much as 60% of its activity can be lost
within 1 hour of specimen collection and storage at room
temperature.
Analytes that are susceptible to oxidative damage, such
as catecholamines, require stabilization. Typically together
with an anticoagulant such as heparin, EDTA, or egtazic
acid, an antioxidant such as glutathione, sodium metabisulfite, or ascorbic acid at a concentration of 1.5 mg/mL should
be included in the blood specimen collection additive

mixture. 81 The specimen collected with this additive
mixture should be centrifuged within 1 hour of collection. It
is preferable, however, if possible to separate plasma within
15 minutes of specimen collection and store it in plastic
tubes or vials at 20 C in case analysis is delayed. Prompt
processing of plasma obtained with an antioxidant-anticoagulant mixture stabilizes catecholamines in plasma for 1 day
at room temperature, for 2 days when stored at 4 C to 8 C,
and for 1 month at 20 C. Stability at 20 C can be
extended to 6 months with added glutathione.2,82
Apparently, at the higher concentrations of catecholamines present in urine compared with plasma, the
hormones and their metabolites are stable for 4 days, even
when stored at 20 C to 25 C without preservatives. 2,82
However, for long-term stabilization of catecholamines in
urine specimens, collection is recommended in a container
with 5 mL of a 6-mol/L concentration of hydrochloric acid
per liter of urine or in a mixture of 250 mg each of EDTA
and sodium metabisulfite per liter of urine, which will extend
stability to 3 weeks at 20 C to 25 C, and 4 months to 1 year
at 4 C to 8 C.2,82
The laboratory assessment of fibrinolysis by the
measurement of the nonspecific fibrinogen-fibrin degradation products requires inhibition of the in vitro generation of
the fibrinolytic enzyme plasmin with an inhibitor, such as
soybean trypsin or aprotinin, and the conversion of residual
fibrinogen to fibrin with thrombin.31 However, in patients
receiving heparin therapy, the conversion of residual
fibrinogen to fibrin, even in presence of thrombin, will be
slow, thus resulting in spuriously high fibrinogen degradation
product values owing to the remaining unconverted
fibrinogen. In such cases, the addition of snake venom, also
known as reptilase, instead of thrombin to the additive
mixture, which also contains a plasmin inhibitor such as
aprotinin, can result in the conversion of residual fibrinogen
to fibrin, even in the presence of heparin.83
For monitoring fibrinolytic therapy, the additive used to
inhibit plasminogen activation depends on the therapeutic
agent administered. Thus, while aprotinin together with
EDTA is effective for inhibiting plasminogen activation by
the therapeutic agent urokinase, it is ineffective for inhibiting
the recombinant tissue plasminogen activator (rt-PA) from
activating plasminogen.75 Specific synthetic peptides of arginine chloromethyl ketone (PPACK) are needed to inhibit in
vitro rt-PA activity. PPACK irreversibly inactivates rt-PA by
alkylating the active center amino acid histidine, in addition
to functioning as a potent thrombin inhibitor.84 A 5-mmol/L
concentration of PPACK combined with a 10-mmol/L
concentration of citrate or a 4.2-mmol/L concentration of
EDTA effectively inhibited rt-PA upon blood collection.75
However, since PPACK is unstable at neutral pH, blood upon
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443
collection should be maintained at 4 C, centrifuged promptly

to obtain plasma, and processed without delay or kept frozen
until analysis can be performed.31
As new diagnostic markers are proposed, the labile
nature of some poses challenges. A case in point is the
measurement of ANP, which is a marker of chronic congestive heart failure. An additive mixture of EDTA and aprotinin is needed to stabilize ANP; the blood specimen
collected with this mixture must be centrifuged immediately at 4 C and plasma maintained frozen at 20 C or
below until ready for analysis.85 Furthermore, hemolysis
invalidates ANP measurements. Alternatively, the measurement of the N-terminal fragment of the prohormone proatrial natriuretic peptide (proANP) (which is formed when
proANP is split into equimolar amounts of ANP and the Nterminal fragment) can be performed in blood specimens
collected in EDTA alone with storage at room temperature
for up to 6 hours. At 4 C, N-terminal proANP is stable for
3 days and at 20 C for 4 weeks, with hemolysis not
markedly affecting the results.86
Anticoagulant/Blood Ratio
The anticoagulant/blood ratio is critical for some laboratory tests. Obviously, if too little blood is obtained, osmotic
effects can be expected to result in cell shrinkage and, thus, to
introduce dilutional changes in the concentration of extracellular analytes.
A well-known example of the critical effect of the anticoagulant/blood ratio is for the global coagulation test, aPTT.
Traditionally, a 1:9 ratio of anticoagulant (sodium citrate, a
0.109-mol/L concentration or a 0.129-mol/L concentration) to
blood is used. However, if less blood is collected, simulating a
1:7 anticoagulant/blood ratio, there is a significant increase in
the aPTT result compared with that obtained using the
nominal 1:9 ratio.31 In 1 study, a 2.4-second increase was
observed at a 1:7 ratio compared with the aPTT result
obtained at the nominal 1:9 ratio.65
However, for the global coagulation test, PT, the effect of
the anticoagulant/blood ratio becomes meaningful only when
the ratio reaches 1:4.5, which would occur when the blood
collection tube is filled to just less than half of its nominal
volume.65 The anticoagulant/blood ratio also is influenced by
the sensitivity of the thromboplastin reagent that is used. In a
study of the effect of the anticoagulant/blood ratio using 3.2%
(0.109-mol/L concentration) buffered citrate, the results for the
PT were valid even for specimen collection tubes that were
filled to 65% of the nominal anticoagulant/blood ratio (1:9).
However, filling the tube to at least 80% of the nominal ratio
was needed to obtain reliable results in the therapeutic range
using a moderately sensitive thromboplastin reagent with an
ISI of 2.06. Using a highly sensitive thromboplastin reagent
with an ISI of 1.01 required maintaining the
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anticoagulant/blood ratio to at least 90% of the nominal

ratio.87 In the aforementioned study,87 the need to fill blood
collection tubes to the nominal capacity (1:9) was reinforced
for the aPTT, since tubes filled to less than 90% of the nominal
capacity caused prolongation of the aPTT result.
In addition to maintaining the nominal anticoagulant/blood ratio (1:9), the amount of head space above
the blood also has been reported to be a variable affecting the
aPTT result. Thus, collecting less blood (2.7 mL as opposed to
the nominal 4.5 mL) but maintaining the anticoagulant/blood
ratio without decreasing the tube size effectively increases the
head space above the blood. This, in turn, generally leads to a
shortened aPTT in values outside the upper limit of the reference interval and in the therapeutic range for monitoring
unfractionated heparin therapy.88 Thus, in the abnormal range,
the mean value obtained with the partially filled tube was 11
seconds shorter than the mean value obtained with the full
tube.88 One possible explanation for the head space effect is
the increase in surface area relative to the volume of blood
collected, favoring platelet activation and the subsequent
release of platelet factor 4, which in turn neutralizes some of
the heparin, resulting in a shortened aPTT.88
For polycythemic patients with hematocrit values above
60% (0.60), the PT and the aPTT can be prolonged substantially, even when the nominal 1:9 anticoagulant/blood ratio is
maintained.89 This is because the plasma is overcitrated to the
extent that it complexes much of the calcium chloride added to
perform the PT and aPTT, effectively reducing the calcium
ions needed to clot the plasma and thereby prolonging the
clotting time. This effect can be minimized by adjusting the
citrate concentration in accordance with the hematocrit value
by using empiric formulas or by using a 1:19
anticoagulant/blood ratio.65,89 The latter ratio dilutes the citrate
concentration to a point that results of PT and aPTT are not
affected across a spectrum of hematocrit values ranging from
anemia to polycythemia.89
In general, collecting blood specimens to less than the
nominal volume increases the effective molarity of the anticoagulant and induces osmotic changes affecting cell morphologic features as noted for the morphologic features of
neutrophils when the EDTA concentration changes from the
nominal 1.5 mg/mL of blood to 2.5 mg/mL of blood. Furthermore, the binding of analytes, such as ionic calcium or ionic
magnesium, to heparin can be enhanced when the effective
concentration of unfractionated heparin increases beyond the
nominal 14.3 U/mL of blood.52
Maintenance of the anticoagulant/blood ratio also is
critical when the inhibitory effects of the anticoagulant are
concentration-dependent. Thus, a 10% reduction in the
gentamicin level was noted in an enzyme-multiplied
immunoassay technique when the concentration of heparin
used for specimen collection increased to 25 U/mL of
blood, presumably because of the inhibition of the enzymes

used in the immunoassay procedure, as the concentration of
heparin increased from the nominal value.90
At the other extreme is exceeding the anticoagulant/blood
ratio to a point that the tube is overfilled with blood to such an
extent that proper mixing of anticoagulant with blood becomes
problematic. Thus, in 1 study, the hemoglobin values had
doubled compared with a value obtained a week before, and
the WBC and platelet counts were reduced drastically. Reanalyzing the specimen for the fourth time from the same tube
yielded values similar to values obtained initially the week
before. Apparently, proper mixing of the specimen on a
rocking mixer was not achieved since there was no head space
or bubble left in the tube to assist in the mixing of the specimen. After a sufficient amount of blood was withdrawn by
repeating the analysis 4 times, there was sufficient space in the
tube for the air bubble to move and effect thorough mixing on
the rocking-type mixing device.91
For bacteriologic determinations, inhibition of the normal
bactericidal properties of blood requires appropriate dilution
of blood in broth. Incorporation of a 0.025% solution of
sodium polyanethole sulfonate in blood culture media is
intended to inhibit phagocytosis, complement, and lysozyme.
Even when blood culture is supplemented with sodium
polyanethole sulfonate, at least a 1:10 blood/broth ratio is
considered optimal.92 However, a blood/broth ratio less than
1:10 has been acceptable when at least 2 separate blood
cultures are obtained routinely or when a biphasic medium is
used to culture blood (a broth bottle to which an agar slant
with media is attached).93
Since spurious blood culture results can be obtained
for patients receiving antibiotics, a 1:10 dilution of blood in
broth dilutes many of the antibiotics in the blood to subinhibitory levels. The incorporation of sodium polyanethole
sulfonate inactivates aminoglycosides. Alternatively,
however, devices containing antibiotic-adsorbent resins can
be used to neutralize antibiotics, and cell lysis followed by
filtration or centrifugation neutralizes the antimicrobial
properties or components of blood and provides a concentrate for culture on antibiotic-free media.93
Specimen Handling and Processing
The time and temperature for storage of the specimen
and the processing steps in the preparation of serum or
plasma or cell separation using density-gradient techniques
can introduce a preanalytic variable. Analytes subject to
cellular metabolism need prompt separation from cells.
Storage of anticoagulated whole blood or clotted blood
in a refrigerator (4 C) inhibits the Na+,K+-ATPase pump,
resulting in potassium leaking out of the cells and causing a
spurious increase in levels measured in serum or plasma.
This effect is seen even at 4 hours of storage of clotted blood
at 4 C.2,8,94 Inhibition of the Na+,K+-ATPase pump also

drives sodium into the cell. Since sodium is mainly extracellular, in contrast with potassium that is mainly intracellular,
the decrease in sodium becomes noticeable only when whole
or clotted blood is stored beyond 24 hours at 4 C. Ideally,
when separation of plasma or serum from cells is needed,
blood should be processed within 1 hour of collection to
obtain plasma or serum.2
Storage of clotted blood beyond 8 hours at room
temperature (23 C) can cause an increase in inorganic phosphate owing to the hydrolysis of cellular organic phosphate
by the enzyme alkaline phosphatase; the effect is more
pronounced when clotted blood is stored at 30 C.2 Thus,
while a 10% increase was noted in the inorganic phosphate
value at 24 hours compared with the 8-hour value during
storage at 23 C, the increase was 120% at a storage temperature of 30 C.2
A time-dependent increase in ammonia is accentuated
in specimens with increased gamma-glutamyltransferase
activity.95 The time between collection of blood and its
storage temperature before processing to yield plasma can
introduce a variable. Thus, in the measurement of catecholamine levels in specimens collected with a stabilizing
additive mixture and stored at 4 C, there is an increase in
the level of catecholamines owing to a delay in the
processing of plasma that exceeds 1 hour.81,82 A delay in
the preparation of plasma for 2 hours after blood collection
and storage at 4 C can increase the mean plasma norepinephrine level by 13% (N = 8).82 This effect is due to the
lysis of some of the RBCs at 4 C, leading to a release of
catecholamines into plasma and to the slow reuptake of
these hormones by cells at 4 C. In addition, cryoprecipitation of plasma proteins at 4 C also can contribute to the
increased level of catecholamines measured in plasma.81,82
In contrast, storage of specimens at 20 C and a delay
beyond 1 hour in the processing of plasma after specimen
collection can lower the catecholamine level measured in
plasma owing to the uptake of catecholamines by cells such
as RBCs or platelets.82 Thus, the mean plasma norepinephrine levels decreased by 14% (N = 8) from the initial value
if blood specimens kept at 20 C were centrifuged 2 hours
after collection, while mean plasma epinephrine values
decreased by 20%.82
The storage temperature of a specimen influences the
results of global coagulation tests such as PT and aPTT.
Since factor VII, which is measured by PT, is activated
during prolonged storage at 4 C, storage of plasma in
contact with cells beyond 7 hours shortens the PT.65,96,97
Indeed, when a blood collection tube is kept well stoppered and stored at room temperature (25 C), the PT is
stable for up to 48 hours.89,97 As is well known, the aPTT
should be performed preferably within 2 to 4 hours of
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445
specimen collection, and some investigators maintain

plasma on ice (4 C).2 In 1 study, a 10% to 15% increase in
aPTT was observed after 24 hours in specimens that were
maintained well stoppered and at room temperature.89
In contrast with refrigerated temperatures, freezing
plasma at 20 C and 70 C did not activate factor VII. The
PT and aPTT results were stable in plasma maintained
frozen at 20 C for 10 days and at 70 C for 21 days.97
Factors II, V, VII, and X are stable in plasma maintained
under refrigeration for up to 6 hours. All except factor V had
stability for up to 14 days when frozen at 20 C or 70 C.97
During a 7-day period, 20% of factor V activity was lost in
plasma stored at 20 C, while in samples stored at 70 C for
the same period, 10% of the activity was lost.
Factor VIII is the most labile factor; 10% of the activity is
lost in 4 hours during storage at 4 C, while at 20 C for a 3 day
period, 20% of the activity was lost.97 Typically, clotted blood
or anticoagulated blood is centrifuged at 1,000g to 1,200g for
10 to 15 minutes. Insufficient centrifugation time and lower
centrifugal forces can result in a gradient of platelets in plasma
derived from heparinized blood, thus affecting some of the
laboratory results, such as potassium levels.2
While centrifugation at 2,000g for 15 minutes is
recommended for whole blood to obtain platelet-poor
plasma for global coagulation tests, at slightly lower speeds
(1,800g), some residual platelet contamination was
reported based on a shortened thrombin clotting time by
10% in plasma frozen for 48 hours and thawed to room
temperature before testing.97
Despite the use of robotics and improved pneumatic
tube delivery systems, and despite the fact that erstwhile
preanalytic problems such as hemolysis during specimen
transportation may have been overcome by improved design
of transport systems, the existence of preanalytic problems
should not be discounted.98,99 A case in point is the likelihood of erroneous results for PO2 if the blood has been contaminated with air before pneumatic tube transportation.99
Relative Merits of Anticoagulants
Platelets rapidly change their shape from discoid to spherical when blood is collected in EDTA, thus making the determination of mean platelet volume unreliable. Approaches to
minimize this artifact have been attempted by the use of an
alternative anticoagulant, which is a mixture of trisodium
citrate, pyridoxal phosphate and tris(hydroxymethyl)aminomethane.100 The rationale is that pyridoxal phosphate
is an effective platelet antiaggregant and disaggregant. Mean
platelet volume measurement using the aforementioned additive mixture was stable at room temperature for 24 hours.100
ACD, heparin, and EDTA have been used for the separation of mononuclear cells. However, regardless of whether
ACD, EDTA, or heparin was used as the anticoagulant,
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the lymphocyte fraction was contaminated with granulocytes if the blood specimen was more than 24 hours old.52
RBC contamination is a problem with aged EDTA blood
specimens to the extent that 9 RBCs may be encountered
for every lymphocyte separated from a 2-day-old blood
specimen.101
For flow cytometric analysis, ACD and heparin are
superior to EDTA for maintaining viable WBCs
overnight.102 However, if analysis is to be performed the
same day, EDTA, ACD, and heparin give equivalent results.
However, at 24 hours and beyond, a substantial decrease in
granulocyte viability may occur in blood collected in EDTA.
K3EDTA also has been reported to be associated with a loss
of function in lymphocyte-mitogen stimulation assays.102
Heparin has been reported to be problematic for use in
molecular biology procedures using restriction enzymes and
DNA amplification by the polymerase chain reaction
(PCR).103 While heparin inhibition can be overcome by a
variety of approaches (eg, treatment with heparinase, or
separation of leukocytes by centrifugation followed by a
minimum of 2 washings in a saline buffer or use of an appropriate buffer), many investigators prefer not to use heparin
for DNA amplification by PCR.103
In vitro platelet, monocyte, and neutrophil activation
studies are influenced by the anticoagulant used for blood
collection.104 Thus, monocyte activation as measured by
release of tissue factor and tumor necrosis factor activity was
lowest with EDTA compared with citrate, heparin, and
hirudin. Neutrophil activation as measured by lipopolysaccharide-induced release of lactoferrin also was the lowest
with EDTA. EDTA seems to suppress platelet degranulation.
Platelet factor 4 levels as an indicator of platelet activation
was the lowest with EDTAplatelet-poor plasma (217
ng/mL) compared with platelet-poor plasma obtained with
citrate (440 ng/mL), hirudin (469 ng/mL), and unfractionated
heparin (1,180 ng/mL). A low-molecular-weight heparin
preparation, however, gave platelet factor 4 values comparable to those obtained with hirudin.104
Endogenous Interferences and Related

Variables
The effect of drugs and their metabolites on laboratory
tests is so extensive that compendia of interferences are
available for consultation.105 Several are discussed in the
following text Table 8.
Although the subject of endogenous interferences is
broad, some mention should be made of interferences that can
affect clinical interpretation of laboratory data. In that context,
patients who are exposed to mouse immunoglobulins through
imaging or therapeutic techniques are prone to develop
Table 8
Selected Endogenous Interferences
Interference
References
Circulating antibodies
Two-site immunometric assays; positive and negative interferences have been reported
Antibodies to platelet membrane phospholipids (lupus anticoagulant, anticardiolipin antibodies)
inhibit phospholipid-dependent global coagulation tests
Antibodies to glycoprotein IIb/IIIa complex in the platelet membrane; EDTA-induced platelet
agglutination at low or room temperature; PTCP; effect abolished at 37C
EDTA-dependent IgM antibody causes leukocyte agglutination at room temperature; spuriously
low automated WBC count not seen in specimens collected in heparin or citrate
Antibodies to platelet membrane and neutrophil receptors cause platelet satellitism seen at room
temperature in EDTA-anticoagulated blood; occasionally, also seen in heparinized or citrated blood;
severe platelet satellitism causes spurious PTCP; effect abolished at 37C
Increased level of chemical analytes affecting hematology test results
Glucose level >600 mg/dL causes transient increase in MCV; as glucose equilibrates, water leaves
RBC restoring original MCV
Triglyceride level >1,000 mg/dL causes spuriously high hemoglobin value; washing and resuspending
RBCs in saline overcomes effect
Narayanan81; Primus et al106

Fiorin et al108
Hillyer et al109
Bizzaro et al111; Peters et al112
Cornbleet107
Cornbleet107
MCV, mean corpuscular volume; PTCP, pseudothrombocytopenia.
antibodies to mouse immunoglobulins. These antibodies,

called human antimouse antibodies, or HAMA, can give rise
to false-positive or false-negative results in 2-site immunometric assays using murine monoclonal antibodies as
reagents.81,106
An increased level of a chemical analyte can affect some
hematology determinations. Thus, glucose levels more than
600 mg/dL (33.3 mmol/L) can lead to a transient increase in
the MCV as water enters the RBC, as the RBC is placed in an
isotonic diluent, owing to the osmotic effect of glucose.107
However, as the glucose level slowly equilibrates, water exits
from the RBC along with glucose, thus restoring the original
MCV. This transient hypoglycemic effect can be overcome by
a microdilution of the blood sample and a 5-minute waiting
period for the equilibration to be complete before analysis.107
Triglyceride levels exceeding 1,000 mg/dL (11.29
mmol/L) and hyperlipidemic specimens can cause a spuriously high hemoglobin value. This effect can be overcome by
resuspending the RBCs in saline before analysis or by making
corrections with a plasma hemoglobin blank.107
Turbidity resulting from the precipitation by lysis
reagents of high concentrations of monoclonal proteins seen
in patients with myeloma or macroglobulinemia may spuriously elevate the hemoglobin level and WBC count.107
Endogenous antibodies can affect both coagulation and
hematology results. Circulating antibodies directed to platelet
membrane phosphatidylcholine or lecithin are referred to as
lupus anticoagulants. Prothrombin is the antigen for most
lupus anticoagulants and, as such, inhibits phospholipid-dependent global coagulation assays.31 Antibodies directed to the
platelet membrane phospholipids phosphatidylserine and phosphatidylinositol, which are called anticardiolipin antibodies,
also inhibit phospholipid-dependent global coagulation tests.
An in vitro phenomenon of platelet agglutination seen in

blood collected in EDTA is due to the presence of antibodies
in blood that are reactive to platelets. These antibodies are
directed to antigens such as the glycoprotein IIb/IIIa complex
that is hidden within the platelet membrane. EDTA, upon
chelating calcium, exposes these antigens. The exposed
platelet antigen becomes modified at low and room temperature, permitting platelet antibodies to agglutinate platelets.
This EDTA-induced pseudothrombocytopenia is, however, not
seen when blood specimens are warmed to 37 C, since the
glycoprotein IIb/IIIa complex is dissociated at the higher
temperature. EDTA-induced pseudothrombocytopenia at 4 C
or room temperature can cause a spuriously low platelet count
and a spuriously increased WBC count if the platelet clumps
are in the same size range as WBCs. Apparently an epitope on
platelet membrane glycoprotein IIb is recognized by EDTAdependent IgG antibodies, causing pseudothrombocytopenia.108
EDTA-dependent IgM antibody, most active at room
temperature and of low titer, has been reported to cause leukocyte agglutination noticeable on a peripheral blood smear but
to provide a spuriously low automated WBC count.109 This
phenomenon was not seen when the same patients specimen
was obtained in a tube with heparin or citrate.
Both pseudothrombocytopenia and pseudoleukopenia
have been reported at room temperature owing to the EDTA
antibodyinduced platelet aggregates not being counted as
platelets and to large neutrophil-platelet clumps falling outside
the size range for WBCs.110 This phenomenon was abolished
by warming the specimen to 37 C.
An in vitro phenomenon that is referred to as platelet satellitism is observed when the platelets surround the neutrophils
owing to EDTA dependent IgG autoantibodies that are directed
Am J Clin Pathol 2000;113:429-452
447
to the glycoprotein IIb/IIIa complex in the platelet membrane

and neutrophil FC-gamma receptor III of neutrophils (CD16).111
This phenomenon, which is seen at room temperature, is not
seen when blood is exposed to a temperature of 37 C or peripheral blood smears are made immediately with EDTA-anticoagulated or capillary blood. Occasionally, the phenomenon also has
been noticed with citrated blood, as well as with heparinized
blood. 112 Severe platelet satellitism can cause spurious
pseudothrombocytopenia.
A discussion of endogenous interferences would not be
complete without discussing the most common interferences, such as hemolysis and turbidity due to hyperlipidemia. Apart from a few analytes, there are conflicting data
in scientific literature on the effect of hemolysis on a wide
range of analytes. The discrepancy is related not only to
differences in methods for the same analyte, but also to
differences in instrumentation. While to some extent instruments that can subtract or blank the hemoglobin interference by taking spectrophotometric measurements at the
wavelength of light where hemoglobin absorbs, in addition
to the wavelength at which the analyte absorbs (bichromatic
measurements), can minimize the interference, the hemoglobin interference is not eliminated totally, especially when
the reagent system used is affected by hemoglobin.
In general, slight hemolysis would have a negligible
effect on many of the routine clinical chemistry laboratory
procedures. Severe hemolysis, while having a substantial
effect on constituents that are at a much higher concentration
within the RBC than in plasma, could induce a slightly dilutional effect on the constituents present at a lower concentration in the RBC than in plasma.
Typical of the enzymes present within the RBC, the
concentration of lactate dehydrogenase (LDH) is 160-fold
greater than that present in the plasma, acid phosphatase is 68fold greater, and AST is 6.7-fold greater than the plasma
levels.113 The concentration of potassium inside the RBC is
approximately 23 times as much as that found in plasma.
Naturally one would expect the aforementioned tests to be
invalidated when the specimen is hemolyzed severely.
The visual evidence of hemolysis is provided when the
hemoglobin concentration exceeds 20 mg/dL. It has been
reported that the appearance of serum when 0.1% of the
RBCs are lysed was virtually identical to that of
nonhemolyzed serum and, consequently, would go undetected by laboratory personnel looking for hemolyzed specimens.114 However, the appearance of serum when 1.0% of
RBCs were lysed was clear and cherry red. Such a specimen would be characterized as having moderate hemolysis. Hemolysis of 1% of the RBCs affected the measurement of LDH, potassium, AST, and ALT.114
Hemolysis also has been classified based on measurement
of free hemoglobin levels.115 The nonhemolyzed samples had
448
Am J Clin Pathol 2000;113:429-452
a mean SD free hemoglobin value of 4.8 3.2 mg/dL, and

moderately hemolyzed serum samples had a value of 43.5
13.9 mg/dL. Even slight hemolysis invalidated the results for
LDH, total acid phosphatase, prostatic acid phosphatase, and
potassium, and such specimens should be rejected.115 That the
effect of hemolysis is method-dependent was demonstrated by
the absence of statistically significant differences for the levels
of AST, ALT, inorganic phosphate, glucose, bilirubin, total
protein and albumin.115
Adenylate kinase released from RBCs can spuriously
increase CK activity measurements unless a sufficient
amount of inhibitors of adenylate kinase are incorporated in
the assay mixture.2
The peroxidative effect of heme can cause a positive and
negative interference with various diazotization methods used
for the measurement of bilirubin, depending on the regents
used in the assay system.116 In classic diazotization procedures
for the measurement of bilirubin, hemoglobin in the
hemolyzed sample competes with nitrite for the sulfanilic acid
reagent, leading to spuriously lower results.115
Depressed glucose values (values lower than the true
value) have been reported with the glucose oxidaseperoxidase procedure, which apparently is related to the reagent
composition. The negative interference has been explained as
resulting from premature decomposition by hemoglobin of the
hydrogen peroxide generated in the glucose oxidase reaction
or from the dilution by cellular components.117
The effect of hemolysis on glucose oxidase procedures
depends not only on the composition of the glucose oxidaseperoxidase reagent system, but also on how the assay is
performed. Thus, differences on the effect of hemolysis have
been reported when using a kinetic assay in which the color
development is monitored between 60 and 120 seconds after
initiating the reaction as opposed to a bichromatic assay in
which color is monitored at 2 different wavelengths; the additional wavelength is used to blank out the color resulting from
substances other than glucose. For example, 2 grossly
hemolyzed pediatric samples with a low true glucose value
were diagnosed correctly by the kinetic procedure that yielded
glucose values of 29 and 27 mg/dL (1.6 and 1.5 mmol/L;
reference range, 70-105 mg/dL [3.9-5.8 mmol/L]). The
bichromatic procedure, on the other hand, overestimated the
glucose values on the 2 pediatric samples by reporting glucose
values of 65 and 68 mg/dL (3.6 and 3.8 mmol/L).117
Hemolysis also interferes with the hexokinase procedure
for the measurement of glucose.118 However, owing to the
multiplicity of instrumentation and variations in methods for
the measurement of glucose, the magnitude of the effect of
hemolysis on each glucose procedure is unpredictable. As
such, for accurate glucose measurements, it is desirable to
avoid hemolysis or, at the very least, not exceed levels that
would make hemolysis visually apparent in serum.
While total acid phosphatase activity was, as expected,

affected by hemolysis due to the release of RBC acid phosphatase, even the prostatic tartratesensitive fraction has been
reported to be affected by hemolysis.118 Among the hormones
very sensitive to hemolysis is insulin.2 Apparently the release
of proteolytic enzymes during hemolysis degrades insulin.41
While some analytes clearly are affected by hemolysis, in
general, method-dependent variations should be considered
when assessing the influence of hemolysis.
Hyperlipidemia, by introducing turbidity, can be a source
of interference that also is method-dependent.119 Interference
could be due to a variety of mechanisms, such as inhomogeneity of the sample, displacement of water, and adsorption
of lipophilic constituents.2 A visibly hyperlipidemic sample
can be expected to interfere with the measurement of total
protein and electrophoretic and chromatographic procedures.2
Summary Perspectives
So much knowledge on the preanalytic phase has accumulated during recent years that this review can only highlight key
issues affecting several contemporary topics.
On the subject of blood gases issues such as maintaining a
steady state of ventilation with the patient in a supine position
before and during arterial blood collection, the effect of the
container material (eg, glass vs plastic syringe), dry vs liquid
heparin as the anticoagulant, leakage of gases owing to syringe
material and conditions of storage, and time between collection
and analysis need to be recognized and standardized.120,121
Molecular biology is another area in which information
on the preanalytic phase is accumulating constantly. The type
of detergent used for cell lysis, its effect on DNA amplification
by PCR, the effect of RBC contamination due to the effect of
hematin on the DNA polymerase enzyme, taq polymerase,
used in PCR, isolation of RNA including steps to eliminate
RNase contamination, treatment of tissue, the effect of fixatives and duration of fixation, and protection of viral HIV
RNA load in plasma from the inhibiting effect of neutrophils
by prompt separation of plasma from cells after blood collection are just a few of a wide range of preanalytic variables that
merit consideration.103
As our knowledge of cell function advances and
studies on stimulation of cells to secrete cytokines become
diagnostically important, one must ensure that there is no
artifactual effect induced by the anticoagulant used for
blood collection. A case in point is the endotoxin- or
pyrogen-contaminated heparin stimulating monocytes to
secrete tumor necrosis factor, thereby invalidating the
measurement of this cytokine.122
Information related to the preanalytic phase of tumor
marker measurements is voluminous. The preservation of
labile tumor markers, including handling of tumor tissue for

the processing of labile hormone receptors, half-life, circadian
rhythm, distribution in cellular fractions, the effect of exercise
on markers such as prostate-specific antigen, amplification by
molecular methods, and sample quality for cytogenetics and
flow cytometry assessment of DNA histograms are just some
of the many issues that come under the preanalytic umbrella.81
As new markers are proposed, preanalytic problems
associated with their measurements also surface. For
example, homocysteine has received considerable attention
as a marker of cardiovascular risk. Yet the measurement of
this amino acid presents challenges since it continues to be
formed by cellular enzymes in vitro, thus making the
prompt separation of cells from plasma or serum mandatory. Alternatively, measures should be taken to inhibit
homocysteine generation and converting enzymes immediately after blood specimen collection.123
With advances in coagulation and fibrinolysis, including
increasing use of molecular methods to study mutations that
predispose a person to hypercoagulability, preanalytic variables affecting these measurements need to be delineated and
controlled. Some physiologic variables that affect the
measurement of fibrinolytic activators and inhibitors,
including the extent of diurnal variation during a 24-hour
period, the effects of smoking and alcohol need to be recognized. Challenges in measurement of free t-PA in plasma by
sample treatment to dissociate the t-PA-plasminogen activator
inhibitor I complex should not be underestimated.31
Finally, preanalytic problems that are unique to a method
should be kept in perspective as we begin to better understand
the complexities of the effect of new technology and therapy,
inasmuch as they affect the preanalytic phase.
From the Department of Pathology, New York Medical
CollegeMetropolitan Hospital Center, New York, NY.
Address reprint requests to Dr Narayanan: Dept of
Pathology, New York Medical CollegeMetropolitan Hospital
Center, 1901, First Ave, New York, NY 10029.
*Dr Narayanan is a Becton Dickinson Fellow at Becton
Dickinson, Franklin Lakes, NJ.
Acknowledgments: I thank Carol Perello for secretarial
support and preparation of the tables.
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Pre Analytic Phase

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Clinical Chemistry / THE PREANALYTIC PHASE

The Preanalytic Phase

In recent years, considerable attention has been given to

American Society of Clinical Pathologists

Am J Clin Pathol 2000;113:429-452 429

Narayanan / THE PREANALYTIC PHASE

changes in renal function solely on the basis of aging, the

Am J Clin Pathol 2000;113:429-452

American Society of Clinical Pathologists

Clinical Chemistry / REVIEW ARTICLE

While changes in organ function occur due to aging,

generally are lower in premenopausal female subjects

American Society of Clinical Pathologists

Am J Clin Pathol 2000;113:429-452

Narayanan / THE PREANALYTIC PHASE

Am J Clin Pathol 2000;113:429-452

Clinical Chemistry / REVIEW ARTICLE

Kratz and Lewandrowski16

The clearance by the liver during the first trimester of

vegetarian counterparts. Diets rich in saturated fatty acids in

Narayanan / THE PREANALYTIC PHASE

Activation of triglyceride lipase by c-AMP results in a

Am J Clin Pathol 2000;113:429-452

increase owing to the metabolism of ethanol to acetaldehyde

Clinical Chemistry / REVIEW ARTICLE

exceeds 80 g/d, the synthesis of VLDL is stimulated,

other variables need to be delineated and standardized to

Narayanan / THE PREANALYTIC PHASE

Guder et al2; Narayanan23

venously, approximately 1 to 2 hours after the completion

Am J Clin Pathol 2000;113:429-452

ments or for the measurement of analytes that have diurnal

Clinical Chemistry / REVIEW ARTICLE

Narayanan and Hamasaki31;

Guder et al2; Chan et al67;

KIU, kallikrein inhibitory unit.

differentiate prerenal, glomerular, tubular, and postrenal

result, the concentration of large molecules that are not

Narayanan / THE PREANALYTIC PHASE

blood specimen collection.8,41 In general, molecules even

Am J Clin Pathol 2000;113:429-452

repeatedly clench the fist to permit visualization of the vein.

Clinical Chemistry / REVIEW ARTICLE

collection, such as a workout in a gymnasium or marathon

American Society of Clinical Pathologists

Anticoagulants for Blood Collection

Narayanan / THE PREANALYTIC PHASE

Since the commonly used anticoagulant, such as

Am J Clin Pathol 2000;113:429-452

lant by chelating calcium ions, which are required for the

Clinical Chemistry / REVIEW ARTICLE

1.5 mg/mL of EDTA, the nucleus swells further, with a

national normalized ratio (INR) units that are used to report

Narayanan / THE PREANALYTIC PHASE

Am J Clin Pathol 2000;113:429-452

less interference noted at higher concentrations of hexokinase used in the assay.73

Clinical Chemistry / REVIEW ARTICLE

polypeptide hormones and enzymes.52 Aprotinin inhibits

be included in the blood specimen collection additive

Narayanan / THE PREANALYTIC PHASE

collection should be maintained at 4 C, centrifuged promptly

Am J Clin Pathol 2000;113:429-452

anticoagulant/blood ratio to at least 90% of the nominal