The Preanalytical 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
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Abstract In recent years, considerable attention has been given to
The preanalytic phase is an important component the influence of preanalytic effects on laboratory results.
of total laboratory quality. A wide range of variables Compendia of preanalytic effects and books have addressed
that affect the result for a patient from whom a the effect of the preanalytic phase on laboratory results.1,2
specimen of blood or body fluid has been collected, Preanalytic variables can be grouped broadly under 3
including the procedure for collection, handling, and categories: physiologic, specimen collection, and influence or
processing before analysis, constitute the preanalytic interference factors. In this review, I address these variables
phase. Physiologic variables, such as lifestyle, age, and provide examples of their effects across a broad range of
and sex, and conditions such as pregnancy and laboratory disciplines. Examples of preanalytic variables
menstruation, are some of the preanalytic phase factors. affecting mainly clinical chemistry, hematology, and coagula-
Endogenous variables such as drugs or circulating tion tests are discussed.
antibodies might interact with a specific method to yield Admittedly, given the comprehensive range of subject
spurious analytic results. The preanalytic phase matter that spans the realm of the preanalytic phase, this
variables affect a wide range of laboratory disciplines. 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, homocys-
teine, 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 vari-
ables, 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, condi-
tions 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
changes in renal function solely on the basis of aging, the
The effect of age ❚Table 1❚ on laboratory results has overlap of disease processes, such as the presence of renal
been well recognized to the point that separate reference vascular disease in elderly patients, must be considered.
intervals have been used to distinguish pediatric, adolescent, Secondary risk factors, such as genetics and obesity, may
adult, and geriatric populations. Bone growth and develop- have a role in the decrease in glucose tolerance with aging.9
ment in a healthy growing child is characterized by increased Homeostatic control by the hypothalamic-pituitary-
activity of bone-forming cells, the osteoblasts, that secrete adrenal axis is compromised because of aging. Hence, there
the enzyme, alkaline phosphatase. Hence, the activity of this is an increase in plasma corticotropin and corticosteroid
enzyme is approximately 3 times higher in a growing child levels due to aging.10,11 Usually in young adults, the concen-
compared with the activity in a healthy adult.3,4 tration of the cytokine, interleukin (IL)-6, in serum is low
A substantial increase in the RBC count in the neonates and is undetectable in absence of inflammation.11 However,

compared with that in adults is the reason glucose is metabo- IL-6 levels are detectable readily during middle age. Also, in
lized very rapidly in neonates.2 Increased arterial oxygen healthy elderly adults, serum IL-6 levels are increased appar-
content within a few days of birth leads to an increase in ently due to the loss of the normal regulation of IL-6 levels.
hemoglobin levels owing to the destruction of the RBCs. The decrease in dehydroepiandrosterone sulfate with age,
Serum bilirubin levels then increase, because the immature which normally has a suppressive effect on IL-6, is related to
liver lacks enzymes to convert bilirubin to the water-soluble the increase in IL-6 levels.12 Increased IL-6 levels, in turn,
bilirubin diglucuronide.2 The total number of neutrophils are increase the percentage of cells bearing the receptor for the
at a maximum for 1 to 2 days after birth. In the neonate, the cytokine, transforming growth factor beta, a powerful
monocyte count is increased for up to 2 weeks after birth, immunosuppressive agent, thus providing an explanation for
while the eosinophil count stays elevated for up to 1 week the age-related immunosuppressive phenomena.13
after birth.5,6 The lymphocyte count is increased markedly at Increases in the level of plasma antidiuretic hormone (or
birth and remains elevated in children up to 4 years of age.6 arginine vasopressin) have been related to aging with levels
In contrast, the basophil count is elevated only transiently for significantly higher in 1 study of healthy subjects 53 to 87
up to 1 day after birth. A substantial decrease in serum uric years of age (4.7 – 0.6 pg/mL [4.4 – 0.6 pmol/L] in 68
acid levels is noted between days 1 and 6 after birth.2 healthy subjects) compared with subjects 21 to 51 years of
Age also affects renal function, as reflected by an effect age (2.1 – 0.2 pg/mL [1.9 – 0.19 pmol/L] in 45 healthy
on creatinine clearance, which decreases progressively with subjects).14
each decade.7 For example, the average value for creatinine Thyroid homeostasis apparently is affected during
clearance at age 30 years is 140 mL/min (2.33 mL/s) per aging. Thyrotropin levels in serum have been reported to be
1.73 m2 of body surface area. In contrast, at age 80 years, the 38% higher in an elderly group (mean age, 79.6 years)
creatinine clearance value decreases to 97 mL/min (1.62 compared with a younger group of subjects (mean age, 39.4
mL/s) per 1.73 m2 of body surface area.7 Beyond the age of years).15 In the same study, serum triiodothyronine levels
50 years, the decrease in creatinine clearance is also related were 11% lower in the older age group compared with levels
to a decrease in muscle mass.7,8 However, when interpreting in the younger group of subjects.
❚Table 1❚
Effect of Age on Laboratory Results
Age Group/Effects References
Neonate
Increased RBC count leading to increased glucose metabolism Guder et al2
Increased neutrophil, monocyte, eosinophil, and lymphocyte counts Monroe et al5; Weinberg et al6
Increased hemoglobin and unconjugated bilirubin values, decreased uric acid level Guder et al2
Lymphocyte count elevated up to 4 years of age Weinberg et al6
Growing child
3-fold increase in alkaline phosphatase level due to bone growth Narayanan3; McComb et al4
Progressive changes
Creatinine clearance decreases with each decade; age-related change in renal function; may Narayanan and Appleton7; Narayanan11
overlap with disease process in elderly persons
Decrease in glucose tolerance with age; overlaps with secondary risk factors, such as genetics Pacini et al9
and obesity
Homeostatic control by hypothalamic-pituitary-adrenal axis affected by aging; corticotropin and De Kloet10; Narayanan11
corticosteroid levels increase
Dehydroepiandrosterone sulfate levels decrease, interleukin-6 levels increase with age Daynes et al12
Antidiuretic hormone and thyrotropin levels increase, triiodothyronine levels decrease with age Crawford et al14; Harman et al15
430 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
which in turn affects the circulating level of various analytes, compared with those for male subjects, with the difference
one must also keep in perspective the overlapping effects due disappearing in subjects older than 65 years.2
to disease and dietary deficiencies of trace elements, such as Sex differences are seen for several analytes as can be
zinc and other nutrients.11 noted by reviewing a current list of reference laboratory
values.16 The differences for some of the analytes based on sex
Sex Differences can be explained as due to differences in muscle mass and to
Between 15 and 55 years of age, the levels of total and endocrine and organ-specific differences.
low-density lipoprotein cholesterol (LDL-C) increase
progressively, with levels slightly higher in premenopausal Time
females compared with the levels in males ❚Table 2❚. Thus, There are time-related fluctuations in the level of some

in female subjects, a change in cholesterol value from 174 analytes (Table 2). Circadian rhythm is responsible for the
mg/dL (4.50 mmol/L) at age 25 years to 213 mg/dL (5.51 diurnal changes seen in the circulating levels of some
mg/dL) at 55 years has been reported. 2 In the corre- analytes. A classic example of an analyte subject to diurnal
sponding age span for men, cholesterol values have been variation is cortisol, which generally peaks at around 6:00
reported to range from 166 mg/dL (4.29 mmol/L) at age 25 AM, with levels becoming lower toward the evening and
years to 232 mg/dL (6.00 mmol/L) at 55 years.2 In contrast, midnight.2 Because cortisol is a powerful immunosuppres-
the high-density lipoprotein cholesterol (HDL-C) level sant, its diurnal rhythm affects proinflammatory cytokine
does not fluctuate in either sex between the ages of 15 and production. Indeed the level of proinflammatory cytokines,
55 years; levels are slightly higher in females, presumably such as IL-1 alpha, IL-12, tumor necrosis factor alpha and
owing to the stimulating effect of estrogens.2 interferon gamma have been shown to be inversely corre-
Analytes with levels that depend on muscle mass, such lated with the level of plasma cortisol.17 The inverse rela-
as creatinine and creatine kinase (CK), are generally at a tionship with plasma cortisol levels extends to the number
higher concentration and activity, respectively, in males of circulating T lymphocytes, especially the CD4+ T cells,
compared with females. 2,7 However, when increased and to the urinary secretion of neopterin, which is a marker
muscle mass is acquired as a consequence of strenuous of cellular immune activity.18,19 Even routinely measured
athletic activities in female subjects, the sex difference in analytes such as glucose, potassium, and iron exhibit
levels of creatinine and CK is abolished.2 Serum iron levels diurnal variation.1,2
❚Table 2❚
Effect of Sex, Time, Season, and Altitude on Laboratory Results
Variable/Effects References
Sex
Between 15 and 55 years, progressive increase in total and LDL cholesterol levels; higher in Guder et al2
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 Guder et al2
Serum iron lower level in premenopausal females Guder et al2
Sex differences reflected in separate reference intervals Kratz and Lewandrowski16
Time
Cortisol peak, 6:00 AM; lower toward evening and midnight Guder et al2
Proinflammatory cytokines (IL-1 alpha, IL-12, TNF-alpha, INF-gamma, CD4+ T cells) and urinary Petrovsky et al17; Miyawaki
neopterin levels opposite the cortisol rhythm et al18; Auzeby et al19
Glucose tolerance test values higher in afternoon Guder et al2
LH, FSH, and testosterone released in short bursts Narayanan8
Potassium, iron, thyrotropin, and growth hormone subject to diurnal effects Young1; Guder et al2; Keffer20;
Narayanan21
Season
Summer
Vitamin D levels higher Narayanan3
Triiodothyronine levels 20% lower Guder et al2; Harrop et al25
Winter
Triglyceride level lower Narayanan3; Statland and Winkel24
Slight (2.5%) increase in total cholesterol level Cooper et al22; Narayanan23
Altitude
At 1,400 m, hematocrit and hemoglobin values 8% higher Young1; Guder et al2
At 3,600 m, 65% increase in C-reactive protein level Guder et al2
As altitude increases, levels of renin, transferrin, and estriol and creatinine clearance decrease Young1; 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.

Glucose values obtained during an oral glucose toler- hormone, luteinizing hormone, and progesterone are influ-
ance test tend to be higher when the test is performed in the enced by the stages of menstrual cycle (ie, follicular, midcycle,
afternoon than when the test is performed in the morning. and luteal phases) ❚Table 3❚.16
The cortisol rhythm apparently may be responsible for the Coincident with ovulation, serum cholesterol levels are
discrepancy noted in the results of an oral glucose tolerance lower than at any other phase of the menstrual cycle. 2
test performed in the afternoon.2 During midcycle or the luteal phase, the aldosterone
Thyrotropin levels peak during the late evening hours, concentration is approximately 2-fold higher compared
with the lowest values observed around midday.20,21 Growth with values during the follicular phase.1,2 Renin activity
hormone levels during waking hours are minimal, with may increase during the luteal phase of the cycle. 1 In
increases noted during sleep.1 contrast, serum phosphate and iron levels decrease during
Reproductive hormones, such as luteinizing hormone, menstruation.1,2

follicle-stimulating hormone, and testosterone, are released
in bursts lasting barely 2 minutes, making accurate assess- Pregnancy
ment of their concentration problematic.8 A dilutional effect is observed due to an increase in
the mean plasma volume, which in turn causes hemodilu-
Seasonal Changes tion (Table 3). 2,3 The effect is more pronounced when
Vitamin D levels tend to be higher during the summer measuring trace constituents such as trace elements in
(Table 2), apparently due to prolonged exposure to serum.3 During pregnancy, there is a considerable increase
sunlight.3 A slight increase in the total cholesterol level in the glomerular filtration rate. Hence, the creatinine
(average, 2.5%) has been observed during the winter clearance rate is increased by 50% or more over the
compared with values measured during the summer.22,23 normal rate.7 During the third trimester, the urine volume
The decrease in the level of triglycerides in serum from may increase by approximately 25%.
summer to winter is more striking than the extent of decline During the second half of pregnancy, the placenta
noted between spring and autumn months when the temper- progressively begins to produce hormones antagonistic to
ature tends to be milder.3,24 insulin. As a result, the levels of hormones, such as
The levels of the thyroid hormone, triiodothyronine, are estrogen, progesterone, and human placental lactogen (also
reported to be 20% lower during the summer compared with called chorionic somatomammotropin), increase, leading
during the winter.2,25 to the onset of gestational diabetes.26
The increased metabolic demand during pregnancy
Altitude causes an increased mobilization of lipids. As a result,
Changes in levels of some of the constituents in blood especially during the second and third trimesters, the
occur when measured at sea level as opposed to measure- serum levels of apolipoproteins AI, AII, and B, triglyc-
ment at a higher altitude (Table 2). For example, hematocrit erides, and total cholesterol (especially LDL-C) are
and hemoglobin levels can be up to 8% higher at an altitude increased substantially.22,23,27 The levels of these lipids in
of 1,400 m.1,2 A 65% increase in C-reactive protein has been serum return to normal within 10 weeks after delivery
reported at 3,600 m. Concentrations of some analytes, such unless the mother breast-feeds her infant.28
as plasma renin, serum transferrin, urinary creatinine, and Human chorionic gonadotropin elaborated by the
estriol, and the creatinine clearance rate decrease with placenta has weak thyrotropic activity. Placental human
increasing altitude.1,2 chorionic gonadotropin levels are at the highest concentra-
tion toward the end of the first trimester of pregnancy, thus
Menstruation suppressing serum thyrotropin levels accompanied by a
At the onset of menstruation, a low estrogen level triggers slight increase in the free thyroxine level.21,29 During the
the release of follicle stimulating hormone from the pituitary second and third trimesters of pregnancy, thyrotropin
gland. The ovaries thus are stimulated to produce estrogen, levels are more reliable. Hence, trimester-based reference
and the level begins to increase noticeably from the sixth or intervals for thyrotropin are needed in pregnancy.
seventh day after menstruation; the peak level is reached on Associated with an increase in the glomerular filtration
approximately the 13th day. A day later, a burst of luteinizing rate in pregnancy is an increase in the clearance of iodide
hormone released from the pituitary gland signals ovulation. and a transfer of iodide and iodothyronines to the fetus. As
With the onset of ovulation, the progesterone level continues a result, the inorganic iodide level in serum decreases to
to rise until it decreases together with the estrogen level, just such an extent that pregnant women with marginal iodine
before the commencement of the next menstrual cycle. Thus, intakes of less than 50 µg/d develop iodine deficiency and,
the reference intervals for estradiol, follicle stimulating 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 Kratz and Lewandrowski16
Cholesterol level lowest at ovulation Guder et al2
Mid or luteal phase, aldosterone 2-fold higher than in follicular phase Young1; Guder et al2
Renin increased in luteal phase Young1
Decrease in serum iron and phosphate Young1; Guder et al2
Pregnancy
Increase in mean plasma volume leads to hemodilution Guder et al2; Narayanan3

Creatinine clearance increased by 50% or more due to increase in glomerular filtration rate Narayanan and Appleton7
Increase in urine volume (25%) during third trimester Narayanan and Appleton7
Estrogens, progesterone, and human placental lactogen levels increase during second half of Narayanan26
pregnancy, all are antagonistic to insulin; gestational diabetes develops
Increased mobilization of lipids during second and third trimesters (increase levels of total Cooper et al22,27;
cholesterol, LDL-C, triglycerides, and apolipoproteins AI, AII, and B) Narayanan23
Thyrotropin levels suppressed owing to increased placental HCG at end of first trimester Narayanan21; Burrow et al29
Increased iodide clearance; with marginal iodide intake, can affect thyroid function Narayanan21; Burrow et al29
Estrogen effect increases levels of TBG, T3 and T4 Narayanan21
ESR increases 5-fold due to acute phase proteins Guder et al2
Increased levels of factor VII and PAI-2; decreased levels of iron and ferritin 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 vegetarian counterparts. Diets rich in saturated fatty acids in
pregnancy of the sialylated thyroxine-binding globulin, general are lipogenic; the effect, however, varies depending on
which is induced by estrogens, is decreased. Hence, the level the fatty acid. Thus, while stearic acid has little effect on LDL-
of thyroxine-binding globulin increases, leading to the well- C levels, palmitic acid substantially increases plasma choles-
known increases in serum thyroxine and triiodothyronine terol levels.23,27 Complex carbohydrates and monounsaturated
levels during pregnancy. and polyunsaturated fatty acids, when substituted for saturated
Other analytes with increased levels during pregnancy fatty acids, tend, however, to lower LDL-C levels.23,27
include the heat-stable alkaline phosphatase elaborated by A diet rich in fish oils lowers serum triglyceride and very-
the placenta, alpha-fetoprotein, copper binding proteins such low-density lipoprotein (VLDL) levels, presumably because of
as ceruloplasmin, and, in turn, copper, acute phase proteins, the ability of fish oils to inhibit the synthesis of VLDL triglyc-
and coagulation factor VII.2,8 The increased level of acute erides.23,32 Even among vegetarians, there is a difference in
phase proteins increases the erythrocyte sedimentation rate serum lipid levels. Among strict vegetarians, plasma LDL-C
approximately 5-fold.2 Plasma concentrations of the fibrinol- levels were reported to be 37% lower and HDL-C levels 12%
ysis inhibitor, plasminogen activator inhibitor type 2, are lower compared with a nonvegetarian control population. In
increased up to 2 µmol/L during the third trimester of preg- contrast, among lactovegetarians, LDL-C levels were 24%
nancy.30,31 An increased metabolic requirement during preg- higher, and HDL-C levels 7% higher compared with a group
nancy leads to a deficiency of iron, and the depletion of iron of strict vegetarians.23,33
stores is reflected by a decrease in ferritin.2 Consumption of caffeine influences the results of some
analytes (Table 4). Caffeine inhibits the enzyme phosphodi-
Lifestyle esterase, which normally regulates the levels of cellular
Diet influences the results obtained for some analytes cyclic adenosine monophosphate (c-AMP) by converting it
❚Table 4❚. Differences between vegetarians and nonvegetarians to the inactive product 5¢-AMP.2,3 The increase in c-AMP
consuming a high-protein or a high-purine diet are well levels due to the caffeine-induced effect promotes glycol-
known.2 Nonvegetarians tend to have increased blood levels of ysis coupled with energy production, and the subject feels
uric acid, urea, and ammonia compared with those for their alert, awake, and energetic.

❚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 Guder et al2
compared with vegetarians
Differences in lipogenicity of saturated fatty acids: stearic acid, less effect; palmitic acid, Narayanan23; Cooper et al27
substantial effect in raising LDL-C
Complex carbohydrates, monounsaturated and polyunsaturated fatty acids lower the levels of LDL-C Narayanan23; Cooper et al27
Fish oils lower the levels of triglycerides and VLDL Narayanan23; Harris et al32
Lipid profile (LDL-C, HDL-C) differences for lactovegetarians vs strict vegetarians Narayanan23; Sacks et al33
Caffeine
Inhibits phosphodiesterase; raises cAMP; activation of triglyceride lipase causes 3-fold Guder et al2; Narayanan3;
increase in free fatty acids Statland and Winkel24

Decreased pH; increased ionic calcium level; free drug or free hormone stripped from albumin Narayanan8
Plasma renin activity and catecholamine levels increase Guder et al2; Robertson et al34
Ethanol
Short- vs long-term effects Guder et al2
Increased levels of GGT, AST, and ALT subject to interindividual and intraindividual variation Freer and Statland36,37
Moderate intake increases levels of HDL-C and apolipoproteins AI and AII Narayanan23; Freer and
Statland37; Taskinin et al38
Smoking
Long-term smoking increases carboxyhemoglobin, hemoglobin, RBC, WBC and MCV values; Narayanan3,8; Statland and
WBC level related to number of packs smoked Winkel24
Cadmium levels higher Narayanan3,8
Reduction in HDL-C dependent on extent of smoking Narayanan23; Cooper et al27
Increased levels of plasma epinephrine, aldosterone, cortisol, free fatty acids, and free glycerol Young1; Guder et al2
within 1 h of smoking
ACE activity reduced owing to destruction of lung endothelial cells Guder et al2; Haboubi et al39
ACE, angiotensin-converting enzyme; ALT, alanine aminotransferase; AST, aspartate aminotransferase; cAMP, cyclic adenosine monophosphate; GGT, gamma-
glutamyltransferase; 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 increase owing to the metabolism of ethanol to acetaldehyde
3-fold increase in nonesterified or free fatty acid levels.2,3,24 and further to acetate. The net effect of lactate formation is
The ability of free fatty acids to compete for binding sites to cause metabolic acidosis owing to the consumption of
on the albumin molecule can lead to the displacement of a bicarbonate.2
drug or hormone bound to albumin, thus affecting the Sustained consumption of ethanol can, by enzyme
measurement of free drug or hormone levels.8 The increase induction, lead to the increase in the activity of liver
in free fatty acid levels results in a decrease in pH, which, in enzymes, such as gamma-glutamyltransferase. There are,
turn, can strip calcium from the binding protein. Conse- however, marked intraindividual and interindividual varia-
quently, a spurious increase in ionic calcium concentration tions in the level of serum gamma-glutamyltransferase, as
can occur.8 Plasma renin activity and catecholamine levels demonstrated in a study in which subjects were challenged
have been increased 3 hours after the consumption of 250 for 3 days with a 0.75-g of ethanol dose per kilogram of
mg of caffeine.2,34 body weight.36 Other liver enzymes that become elevated
The effect of caffeine on lipid levels has been contro- owing to the direct cytotoxic effect of ethanol on liver are
versial, and the results obtained seem to be influenced by aspartate aminotransferase (AST) and alanine aminotrans-
coffee-brewing methods.23 The finding in a double-blind ferase (ALT).2 Thus, consumption of 225 g of ethanol per
study replacing caffeine-containing coffee with decaf- day for 1 month caused skeletal muscle changes that led to
feinated coffee for a period of 6 weeks of an insignificant the release of liver cell enzymes into blood.37
change in serum triglyceride, total cholesterol, and HDL-C Depending on the extent of consumption, the effect of
levels suggests that caffeine is not the substance in coffee ethanol on serum lipid levels is variable.23 The effect of
that elevates the total cholesterol level.35 short-term alcohol intake, especially by subjects who
Consumption of ethanol can induce short- and long- usually do not consume alcohol, is to increase plasma
term effects altering the results of several analytes (Table triglyceride levels, especially the VLDL fraction.38
4).2 Short-term effects reflected rapidly within the first 2 to A moderate intake of ethanol (<40 g/d) is reported to
4 hours of consumption include a lowered blood glucose increase the plasma levels of HDL-C, HDL3-C, and the
level and an increased plasma lactate level.2 Uric acid levels apolipoproteins AI and AII. However, if alcohol intake

exceeds 80 g/d, the synthesis of VLDL is stimulated, other variables need to be delineated and standardized to
together with the activation of the enzyme lipoprotein minimize preanalytic error.
lipase. Lipoprotein lipase hydrolyzes VLDL triglycerides,
with the result that plasma VLDL levels will be apparently Duration of Fasting
normal even when there is increased VLDL synthesis.23,27,38 Ideally, subjects should be instructed to fast overnight
Long-term smoking induces alteration in both biochem- for at least 12 hours before specimen collection (Table
ical and cellular profiles (Table 4).3,8 Carboxyhemoglobin 5).2,23 The reason for the 12-hour stipulation is based on the
levels in blood are higher in long-term smokers since ciga- fact that an increase in the serum triglyceride level after a
rette smoke contains carbon monoxide, which has a much fatty meal can persist for up to 9 hours, but there is little
greater affinity for hemoglobin than does oxygen. As a effect on the levels of total cholesterol or apolipoproteins
consequence of long-term smoking, there is an increase in AI and AII.40

hemoglobin levels and the RBC and WBC counts. The The effect of prolonged fasting, however, can affect
RBCs become larger, thus increasing the mean corpuscular some laboratory results. Thus, during a 48-hour fast, the
volume (MCV). Striking differences in the WBC count have hepatic clearance of bilirubin could be approximately
been observed between healthy nonsmokers and healthy 240% more than the nominal value.3,24 With prolonged
long-term smokers. Healthy smokers who smoked 1 pack of fasting, there is a decrease in the level of specific proteins,
cigarettes per day for 1 year had a WBC count of approxi- such as the C3 component of complement, prealbumin, and
mately 1,000/µL (1 · 109/L) higher than that for healthy albumin. Normal levels of these proteins are restored
nonsmokers. Healthy smokers who smoked 2 packs of ciga- rapidly with protein supplementation.24
rettes per day for 1 year had a WBC count of approximately The effect of fasting, however, varies depending on
2,000/µL (2 · 10 9 /L) higher than that for healthy body mass.23 In a lean person, the use of fat as reflected by
nonsmokers.24 an increase in acetoacetic acid concentration in blood is
Cadmium levels in blood are higher in healthy long- minimal during a 1-day fast but increases rapidly with
term smokers compared with levels for healthy nonsmokers, prolonged fasting. 41 In an obese person, however, the
thus requiring separate reference intervals to distinguish the acetoacetic acid level increases markedly during a 1-day
2 populations.3,8 fast. In contrast, while lipolysis, as reflected by a 3-fold
Smoking can reduce HDL-C levels in plasma; the increase in the serum glycerol concentration with 3 days of
extent of reduction apparently is related to the number of fasting is observed in a lean person, little change is noted in
cigarettes smoked per day.23,27 Short-term changes occur- an obese person.23,41
ring within 1 hour of smoking 1 to 5 cigarettes include an
increase in the plasma levels of epinephrine, aldosterone, Time of Specimen Collection
cortisol, free fatty acids, and free glycerol.1,2 The section “Time” referred to diurnal changes seen in
Angiotensin-converting enzyme (ACE) activity is the circulating level of some analytes. It is, therefore,
decreased in smokers, presumably owing to the destruction important that the time of specimen collection be kept
of lung endothelial cells with the resultant decrease in the constant from day to day to rule out variations introduced
release of ACE into the pulmonary circulation or, alterna- by diurnal effects (Table 5).
tively, to inhibition of enzyme.2,39 Specimens for therapeutic drug monitoring (TDM)
The extent of smoking (moderate or heavy) parallels should be obtained after a steady therapeutic concentration
the blood levels of thiocyanate and cotinine. Compared with of drug or steady state is achieved in the blood. A blood
nicotine, which is the parent compound, cotinine has a sample obtained just before administering a drug dose after
longer half-life, approximating 20 to 28 hours, making it an a steady-state level is reached will reflect the trough level
ideal marker for the assessment of the extent of smoking.2 or the lowest concentration to be obtained with the estab-
lished drug dose. If only 1 blood sample is to be collected
Specimen Collection Variables for TDM, it is preferred that it reflect the trough level.42
The preparation of subjects for blood specimen collec- Blood samples obtained when the drug concentration
tion, such as the duration of overnight fast, time of specimen is at its maximum, will, of course, reflect the peak level.
collection, and posture during blood sampling ❚Table 5❚; the Peak-level specimens need to be obtained within 1 to 2
effects of the duration of tourniquet application, infusion, hours after intramuscular administration of the drug, 15 to
and exercise (Table 5); the effects of anticoagulants ❚Table 6❚ 30 minutes after intravenous administration, or 1 to 5
and stabilizing additives ❚Table 7❚ used for blood collection; hours after oral administration. 42 Timing of specimen
the anticoagulant/blood ratio; specimen handling and collection for TDM, however, depends on the rate of distri-
processing; and the relative merits of anticoagulants and bution of the specific drug. If the drug is infused intra-

❚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 Guder et al2; Narayanan23
After 48-h fast, 240% increase in bilirubin level; decreased levels of prealbumin, albumin, and C3 Narayanan3; Statland and Winkel24
Effects dependent on body mass Narayanan23; Young41
Time
Standardize time to rule out diurnal effect Guder et al2
Therapeutic drug monitoring trough vs peak levels; timing regimens Guder et al2; Narayanan42;
Pippenger43
Urine, timed overnight or first morning specimen concentrated, ideal for sediment enumeration Narayanan44
24-hour sample for clearance measurements and for analytes with diurnal variation Narayanan and Appleton7
Random or spot urine generally suitable Guder et al45

But, sensitivity to detect slight increases in numbers of cells and casts and glucose and protein Narayanan44
levels affected by dilution
Posture
In general, 5%-15% increase for most cellular and large molecules (eg, albumin) in sitting posture Guder et al2; Narayanan8; Statland
and Winkel24
Increase in upright to supine has dilutional effect: 10% and 12% decreases in cholesterol and Narayanan8,23; Young41
triglyceride levels, respectively
Change in posture has no effect on free drug and small molecules Guder et al2; Narayanan8;
Young41
Postural effect substantial in patients with reduced plasma volume (eg, congestive heart Narayanan8
failure, cirrhosis)
Increased aldosterone, norepinephrine, epinephrine, renin, and atrial natriuretic peptide levels Guder et al2; Tan et al46
Tourniquet application time
>1 minute increase in concentration of large molecules; for total cholesterol level, 5% increase Guder et al2; Narayanan8,23;
at 2 minutes; up to 15% increase at 5 minutes Cooper et al22; Statland and
Winkel24; Young41
Decreased pH; increased potassium, lactate, ionized calcium, and magnesium levels with Narayanan8
increased application time
Repeated fist clenching can increase potassium by 1-2 mmol/L, up to 2.7 mmol/L Don et al47; Skinner et al48
Infusion
Spuriously increased blood glucose level if specimen obtained from arm receiving glucose infusion Guder et al2
Waiting times before blood specimen collection: subjects receiving fat emulsion, 8 h; carbohydrate, Guder et al2
protein, or electrolytes, 1 h Guder et al2
Extent of hemolysis related to age of transfused blood Guder et al2
Exercise
Strenuous exercise increases total CK and CK-MB; CK-MB as percentage of total CK normal; Narayanan8; Statland and
decreased haptoglobin value due to intravascular hemolysis Winkel24
Individual variability in CK related to extent of exercise training Guder et al2
Mitochondrial size and number increase with vigorous training; mitochondrial CK increases to >8% Guder et al2
of total CK
Increased epinephrine, norepinephrine, glucagon, cortisol, corticotropin, and growth hormone levels; Guder et al2
decreased insulin level
Increased serum and decreased urine uric acid levels due to increased lactate level Guder et al2
Increased apolipoprotein AI and HDL-C levels; decreased LDL-C, apolipoprotein B, and triglyceride Narayanan23; Cooper et al27;
levels with long-term strenuous exercise or brisk walking 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 ments or for the measurement of analytes that have diurnal
of infusion are needed for the distribution phase to be variation, a 24-hour urine sample is needed. 7 A timed
completed, with exceptions such as digoxin and digitoxin, overnight urine sample or the so-called first-morning spec-
which need 6 to 8 hours for the completion of the distribu- imen, provides not only an assessment of the concentrating
tion phase.2 ability of the kidney, but also sufficient time for formed
In general, since the rate of oral drug absorption differs elements or sediment to be produced for easy enumeration.
from person to person, blood specimens obtained for TDM While a random or spot sample may be suitable for
usually are timed to reflect trough levels. For the moni- routine screening, such a specimen may have its limitations
toring of certain drugs, such as theophylline, antibiotics, because it may be too diluted to detect accurately slight
and antiarrhythmic drugs, it may be necessary and useful to increases in the concentration of analytes such as glucose,
measure peak levels after intravenous administration.2,43 protein, cells, and casts. Thus, a spot or random sample
The time for collection of urine specimens depends on may be of limited value in the initial screening for microal-
the constituent being measured. For clearance measure- buminuria.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 Narayanan and Hamasaki31;
salts; lithium heparin preferred for plasma chemistry testing; nominal amount, 14.3 U/mL of blood Novotny et al51; Narayanan52,54;
Guder et al53; Berg et al55;
Doumas et al56
For ionic calcium, heparin in blood gas syringe can be calcium-titrated, electrolyte-balanced, or Narayanan52; Toffaletti et al57;
zinc lithium–titrated Toffaletti58; Landt et al59
EDTA: salts of EDTA generally used for routine hematology testing (dipotassium or tripotassium Narayanan52; Goosens et al62;
EDTA, disodium EDTA); nominal amount, EDTA, 1.5 mg/mL of blood Sacker63; Reardon et al64
Citrate: generally used for routine coagulation testing; trisodium citrate–dihydrate 3.2% (0.109 mol/L) Guder et al2; Narayanan and
or 3.8% (0.129 mol/L) 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]) Guder et al2; Chan et al67;
Sidebottom et al68
Lithium iodoacetate (0.5 mg/mL of blood) alone or in combination with lithium heparin Guder et al2; Chan et al67
(14.3 U/mL of blood) Sidebottom et al68
Sodium fluoride and lithium iodoacetate require 3 h to fully become effective Guder et al2
Adding mannose (3 mg/mL of blood) to sodium fluoride achieves efficient glycolytic inhibition Liss and Bechtel71;
Nakashima et al72
Mannose, however, has concentration-dependent interference Nakashima et al72; Ho et al73
Maintaining blood pH at 5.3-5.9 with mixture of citric acid, trisodium citrate, disodium EDTA, and Uchida et al70
0.2 mg sodium fluoride stabilizes glucose
Cell stabilizers
Acid-citrate dextrose A and B formulations; B formulation has greater amounts of dextrose available Guder et al2
to metabolizing cells
Citrate, theophylline, adenosine, dipyridamole mixture minimizes in vitro platelet Narayanan and Hamasaki31;
activation Contant et al74; Narayanan75;
Van Den Besselaar et al76;
Kuhne et al77
Proteolytic enzyme inhibitors
EDTA (1.5 mg/mL of blood) with aprotinin (500-2,000 KIU/mL) stabilizes several polypeptide hormones Narayanan52,79
Aprotinin also can be used with lithium heparin (14.3 U/mL) Narayanan52
A mixture of EDTA, aprotinin, leupeptin, and pepstatin stabilizes parathyroid hormone–related protein Pandian et al80; Narayanan81
Catecholamine stabilizers
An antioxidant such as glutathione, sodium metabisulfite, or ascorbic acid at a concentration of 1.5 Narayanan81
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 Boomsma et al82
each of EDTA and sodium metabisulfite per liter of urine
Fibrinolytic inhibitors
A mixture of thrombin and soybean trypsin or aprotinin Narayanan and Hamasaki31
Reptilase and aprotinin Connaghan et al83
Thrombin inhibitor: 5-mmol/L concentration of a phenylalanine, proline, arginine chloromethyl ketone Narayanan and Hamasaki31
mixture with 10-mmol/L concentration of citrate or 4.2-mmol/L concentration of EDTA Mohler et al84
KIU, kallikrein inhibitory unit.
differentiate prerenal, glomerular, tubular, and postrenal result, the concentration of large molecules that are not
causes of proteinuria by measurement of specific proteins, filterable is increased. Hence, albumin levels generally are
a morning spot urine specimen was adequate.45 higher among healthy subjects attending an outpatient clinic
from whom blood specimens are obtained with the subjects
Posture During Blood Sampling in a sitting position compared with levels among healthy
Changes in posture during blood sampling can affect hospitalized subjects from whom blood specimens usually
the concentration of several analytes measured in serum or are obtained with the subjects in a supine position.8,24
plasma (Table 5). Change in posture from a supine to the Small molecules, such as glucose, however, owing to
erect or sitting position can result in a shift in body water their ability to move freely between the interstitial space
from the intravascular to the interstitial compartment. As a and the circulation, are least affected by posture during

blood specimen collection.8,41 In general, molecules even repeatedly clench the fist to permit visualization of the vein.
as large as insulin can diffuse freely into and out of the The contraction of forearm muscles during repeated fist
interstitial space and, thus, are not subject to variation due clenching causes release of potassium, since there is a reduc-
to postural changes.8 tion in the intracellular electronegativity during the depolar-
While the free fraction of a metabolite, drug, hormone, ization of muscle cells, which favors the release of potassium
or metal ion is not subject to postural variation, the fraction rather than its uptake.47 Repeated fist clenching during phle-
bound to protein, such as albumin, is affected by posture. botomy after application of the tourniquet can cause a 1- to
Thus, bilirubin bound to albumin and calcium bound to 2-mEq/L (1- to 2-mmol/L) increase in potassium. In 1 study,
albumin are affected by postural changes.2,8 an increase in the potassium level as much as 2.7 mEq/L (2.7
In blood specimens obtained with subjects in the sitting mmol/L) was noted in a healthy subject due to fist clenching
position, generally an increase in the range of 5% to 15% is during phlebotomy.48 Application of the tourniquet for more

seen for most cellular and macromolecular analytes than 1 minute and up to 2 minutes can cause a 5% increase
compared with results when blood specimens were obtained in the total cholesterol level, which can increase by 10% to
with subjects in the supine position.2 15% if the tourniquet is left on for 5 minutes.22,23,41
The effect of postural change from supine to erect is In 1 study conducted on children, venostasis of 2 minutes
accentuated in patients with a tendency to edema, such as substantially decreased the blood pyruvate concentration by
patients with cardiovascular insufficiency and cirrhosis of the approximately 18% yet had a negligible effect on the lactate
liver.2 In such patients, reduction in plasma volume in turn concentration, with a mean increase of just 2.2%.49
causes increased secretion of hormones, such as aldosterone, The reduction in blood pH during prolonged tourniquet
norepinephrine, and epinephrine, and the enzyme, renin.2 An application can alter drug-protein or hormone-protein
increased level of atrial natriuretic peptide (ANP) is measur- binding, leading to an increase in the free drug or free
able in plasma as a compensatory mechanism to correct the hormone concentration. Thus, at a low pH caused by anaer-
decrease in blood pressure.2,46 Indeed, changing from supine obic glycolysis during venous stasis, ionic calcium and
to erect can dramatically influence the concentration of ionic magnesium levels increase since they are released
analytes such as renin and aldosterone, even in healthy from albumin, the binding protein.8
persons. Conversely, moving from upright to supine can have To minimize the preanalytic effects of tourniquet appli-
a dilutional effect owing to an increase in plasma volume cation time, the tourniquet should be released as soon as
resulting from the transfer of fluid from the extravascular to the needle enters the vein.8 Avoidance of excessive fist
the intravascular compartment.8 Thus, a change from upright clenching during phlebotomy and maintaining tourniquet
supine can reduce (after 5 minutes in the supine position) the application time to no more than 1 minute can minimize
cholesterol level by 10% and triglyceride levels by 12%.23,41 preanalytic error.2
If blood specimens are to be obtained with the subject in
a sitting position, such as in an outpatient setting, the sitting Effect of Infusion
posture should be standardized by instructing the subject to For persons receiving an infusion, blood should not
remain seated for 15 minutes before specimen collection. be obtained proximal to the infusion site.2 Blood should
be obtained from the opposite arm. Very often, exception-
Duration of Tourniquet Application ally high glucose values are obtained by collecting blood
The application of the tourniquet, by reducing the pres- from the arm in which an infusion of glucose is being
sure below the systolic pressure, maintains the effective administered (Table 5). Eight hours must elapse before
filtration pressure within the capillaries.2 As a result, small blood is obtained from a subject who has received a fat
molecules and fluid are transferred from the intravasal space emulsion. The waiting period for blood sampling from
to the interstitium. Application of the tourniquet for longer persons receiving a carbohydrate-rich solution or amino
than 1 minute and up to 3 minutes can result in hemoconcen- acid and protein hydrolysate or electrolytes is 1 hour after
tration, causing an increase in the concentration of large cessation of the infusion.2 For subjects receiving blood
molecules that are unable to penetrate the capillary wall transfusions, the extent of hemolysis and, with it,
(Table 5).2,8,24,41 increased values for potassium, lactate dehydrogenase,
The venous stasis that results from prolonged tourniquet and free hemoglobin are related progressively to the age
application promotes anaerobic glycolysis with the accumu- of the transfused blood.2
lation of plasma lactate and a reduction in blood pH.8 The
hypoxic effect drives potassium out of the cell causing a Effect of Exercise
spurious increase in the serum potassium level. This effect Exercise, such as running up and down several flights
can be accentuated if the phlebotomist requests the subject to of stairs, or strenuous activity the night before specimen

Anticoagulants for Blood Collection

collection, such as a workout in a gymnasium or marathon
running, can affect the results obtained for several analytes Various salts of heparin, EDTA, and sodium citrate are
(Table 5). used widely in the clinical laboratory (Table 6).
Strenuous exercise depletes the energy-yielding
compound, adenosine triphosphate (ATP), from the muscle Heparin
cells. As a result, there is a change in cell membrane Heparin is the preferred anticoagulant for blood specimen
permeability, and enzymes are released from the cells. collection intended for determination of electrolyte levels and
Thus, CK levels can be very high after strenuous exercise other routine chemistry values. The standard commercial-
(levels approaching 1,000 U/L) with a release of the CK- grade heparin preparation, also called unfractionated heparin,
MB fraction, although CK-MB as a percentage of total CK with a molecular mass ranging from 3 to 30 kd (mean, 14 kd)
would be normal.8 exerts its anticoagulant effect by binding to antithrombin III.

A high degree of individual variability is seen in a This interaction causes a conformational change in the
hypoxia-mediated increase in CK, which depends on the antithrombin III molecule, thereby accelerating the inhibition
extent of exercise training.2 Mitochondrial size and number of coagulation factors Xa, IXa, and thrombin.31 The standard
are increased in persons who maintain a vigorous training commercial grade heparin also can inhibit thrombin with the
schedule, allowing their muscles to oxidatively metabolize aid of a plasma cofactor, called heparin cofactor II. In vivo,
glucose, fatty acids, and ketone bodies. As a result, even in heparin can cause the release of a potent coagulation inhibitor,
the absence of myocardial damage, mitochondrial CK-MB called tissue factor pathway inhibitor, from the endothelial
levels increase to greater than 8% of total CK activity.2 surface.31,51
Training increases muscle mass, thereby increasing the Various salts of heparin, such as lithium, sodium, and
concentration of creatinine in plasma and the urinary excre- ammonium, have been used as anticoagulants for the collec-
tion of creatinine and creatine, while decreasing the level of tion of blood specimens intended for routine clinical chemistry
plasma lactate compared with untrained persons who determinations.52 The nominal amount of heparin used in
indulge randomly in exercise. blood specimen collection tubes is 14.3 U/mL of blood,
During strenuous exercise, there is some amount of although a range of 12 to 30 U/mL of blood has been deemed
intravascular hemolysis. As a result, the level of plasma satisfactory.53
haptoglobin, an acute phase reactant, is reduced owing to Although all of the 3 salts of heparin give comparable
its “complexation” with free hemoglobin released during results for electrolyte determinations, lithium heparin has been
hemolysis and the subsequent clearance of the complex favored over the others, more because of the perception that
from circulation.8,24 sodium heparin might overestimate sodium levels and ammo-
As a result of exercise, the concentrations of hormones nium heparin might spuriously increase serum urea nitrogen
such as epinephrine, norepinephrine, glucagon, cortisol, concentrations when measured by the urease procedure.52,54
corticotropin, and growth hormone increase, while insulin Just how much sodium is present in a sodium heparin
levels decrease, thereby causing an increase in glucose blood collection tube depends not only on the nominal
levels because of the gluconeogenetic effect of hormones amounts of heparin present (14.3 U/mL of blood), but also on
antagonistic to insulin.2 other variables, such as the molecular weight of unfractionated
Volume shift from the intravasal to the interstitial heparin, the number of dissolvable milliequivalents of sodium
compartment or volume depletion due to sweating during per milligram of heparin, and the consistency of the heparin
exercise can increase serum albumin levels.2 Increased preparation from lot to lot. Indeed, in 1 study when blood was
lactate concentration due to anaerobic glycolysis during filled to half of the tube’s nominal volume, thereby increasing
exercise can decrease the urinary excretion of uric acid, 2-fold the nominal amounts of sodium heparin present in the
thereby increasing its serum concentration.2 There are, blood collection tube, plasma sodium values were not
however, beneficial effects of long-term strenuous exercise increased artifactually.54
or brisk walking in terms of reducing the levels of serum Obvious differences in results for certain analytes
LDL-C, apolipoprotein B, and triglycerides and increasing between serum and heparinized plasma are related to the
the levels of apolipoprotein AI and HDL-C.23,27,50 consumption of fibrinogen and the lysis of cellular elements
To minimize preanalytic variables introduced by exer- during the process of clotting. Thus, potassium values are
cise, subjects should be instructed to refrain from stren- higher in serum than in heparinized plasma, while in contrast,
uous activity at least the night before testing and to not total protein values are higher in plasma than in serum. The
exert themselves by walking a long distance or running or effect of heparinized plasma on some of the enzyme measure-
climbing stairs before blood specimen collection.8 ments may be related, in part, to the bias introduced by the
instrument-reagent combination.55,56

Since the commonly used anticoagulant, such as lant by chelating calcium ions, which are required for the
lithium or sodium heparin, in the syringe to obtain speci- clotting process.
mens for blood gas measurement binds to ionic calcium, Potassium and sodium salts of EDTA have been used
with the effect dependent on the concentration of heparin, for blood specimen collection. The solubility of potassium
attempts have been made to eliminate this bias. One salts is better than sodium salts of EDTA. The pH of EDTA
approach has been to use calcium-titrated heparin, in which varies depending on the salt. As more ions are added to the
heparin in an amount less than 50 U/mL of blood is titrated free acid, the acidity of EDTA decreases. Thus, while a satu-
to an ionized calcium concentration of 1.25 mmol/L.52 The rated solution of EDTA as free acid has a pH of 2.5 – 1.0, the
use of electrolyte-balanced heparin that involves the use of tripotassium salt (K3EDTA) as 1% solution has a pH of 7.5 –
dry heparin in the amount of 40 U/mL of blood balanced 1.0.52 The disodium (Na2EDTA) and dipotassium (K2EDTA)
with calcium, sodium, and potassium decreases bias to less salts of EDTA in a 1% solution have a lower pH. Na2EDTA

than 0.02 mmol/L of calcium in the analytic range of 0.9 to has a pH of 5.0 – 1.0, while K2EDTA has a pH of 4.8 – 1.0.
1.8 mmol/L.57 The use of dry heparin eliminates the dilu- This difference in pH affects the size of the RBC. Although
tion effect associated with liquid heparin. all salts of EDTA are hyperosmolar, causing the water to
Other approaches to minimize interference with leave the cells and the cells to shrink, this shrinking effect is
ionized calcium measurements have involved saturating the noticed only with K 3 EDTA and not with K 2 EDTA or
high-affinity binding sites on heparin to which divalent Na2EDTA.52 This is because although the cells shrink with
cations, such as zinc, can bind. Zinc heparin, however, in K2EDTA and Na2EDTA, the lower pH of the anticoagulant
the presence of excess zinc ions is reported to cause a posi- causes the cells to swell as well, thus balancing for the
tive interference in ionized calcium and total magnesium osmotically dependent shrinkage, and the cell size is not
measurements.58 One successful approach is to saturate altered. As a result, while the microhematocrit (centrifuged
high- and low-affinity binding sites on heparin, the former hematocrit) is lowered with K3EDTA, it is not affected by
with zinc and the latter with lithium. 59 This calcium- K2EDTA or Na2EDTA. The effect of dilution of the sample
neutralized lithium-zinc heparin has been satisfactory not in automated analysis restores cells to their native shape. At
only for ionized calcium measurements, but also for an optimum EDTA concentration of 1.5 mg/mL of blood, no
measurement of blood gases, total calcium, sodium, and differences in microhematocrit were noted between samples
potassium.59 collected in K2EDTA and K3EDTA and analyzed between 1
For TDM, the drug level in heparinized plasma would and 4 hours after collection.62
be expected to be higher than that in serum, especially if In the same study, the MCV measurement and the calcu-
the drug was bound to fibrinogen. In reality, the serum lated hematocrit values were influenced not only by the instru-
value for drugs such as amiodarone, desethylamiodarone, ment used, but also by the salt of EDTA (K2 or K3) used for
chloroquine, and desethylchloroquine was higher than the anticoagulation of the blood specimen. Thus, MCV values
plasma value, apparently due to the release of cell-bound were not affected with blood anticoagulated with K3EDTA,
drug during the clotting process. 52,60,61 Thus, results even at concentrations up to 10 times the nominal value. In
obtained for amiodarone were 11.5% to 13.5% lower in contrast, with 3 of the 4 instruments tested, increasing the
heparinized plasma than in serum, while results for concentration of K2EDTA beyond the nominal value of 1.5
desethylamiodarone were 4.4% to 8.5% lower in plasma mg/mL of blood resulted in an increase in MCV.62 One expla-
than in serum.60 nation may lie in the ready dissolution of liquid K3EDTA
The concentration of chloroquine was approximately compared with powdered K2EDTA, in which the effectiveness
2-fold higher in serum than in plasma, while the concentra- of mixing might become a variable, perhaps accounting for the
tion of its metabolite, desethylchloroquine, was approxi- discrepancy in results.
mately 4 times higher in serum than in plasma. These 2 The neutrophils are subjected to morphologic changes
drugs were present mainly in platelets and granulocytes. upon collection of blood in EDTA. These changes depend on
The increased levels of these 2 drugs noted in serum appar- the time the peripheral blood smear is prepared and the
ently were related to the release of these drugs from concentration of EDTA used for blood specimen collection.
platelets during the coagulation process. Thus, for some Minor changes begin to appear in the morphologic features
drugs, it is important to specify whether plasma or serum of neutrophils even at an optimal EDTA concentration of 1.5
was used for measurement.61 mg/mL of blood. These changes include swelling of the
neutrophils and loss of structure in the neutrophil lobes.
EDTA Thereafter, there is a loss of granulation in the cytoplasm and
EDTA is the commonly used anticoagulant for routine the appearance of vacuoles in the nucleus or cytoplasm of
hematologic determinations. It functions as an anticoagu- the cells.63 Between 1 and 3 hours after blood collection in

1.5 mg/mL of EDTA, the nucleus swells further, with a national normalized ratio (INR) units that are used to report
crossover of the nuclear chromatin. Beyond 3 hours, the the results of the PT.66 INR values generally are higher
morphologic features of the neutrophils become unclear as with a 0.129-mol/L concentration of sodium citrate than
the cell disintegrates. If a peripheral blood smear is made with a 0.109-mol/L concentration of sodium citrate, espe-
from blood anticoagulated with an EDTA concentration of cially when a responsive PT reagent, such as a recombinant
2.5 mg/mL of blood, approximating to a conventional blood thromboplastin that has a sensitivity similar to the World
collection tube filled to one half of its nominal volume, the Health Organization thromboplastin (with an international
morphologic features of the neutrophils are disintegrated sensitivity index [ISI] equal to 1), is used.66 The differences
totally. in INR between the 2 concentrations of citrate can vary
Similar time-related and EDTA concentration–related from 0.7 to 2.7 INR units.66
changes also occur in the mononuclear cells, although to a The INR will be in error when a laboratory that

lesser extent than in the neutrophils. Platelets also undergo routinely uses, for example, a 0.109-mol/L concentration of
changes, including swelling, giving rise to giant platelets citrate, occasionally analyzes a specimen collected with a
that ultimately disintegrate, producing fragments in the 0.129-mol/L concentration of citrate, but uses the mean of
same size range as the platelets that are mistakenly counted the normal range obtained with the 0.109-mol/L concentra-
as platelets by electronic counters, thus spuriously tion to calculate the PT ratio (the patients PT divided by
increasing the platelet count. mean of normal range for PT), which together with the ISI
Platelets rapidly change their shape from discoid to spher- are needed for the calculation of INR.66
ical when blood specimens are collected in EDTA, making
determinations of mean platelet volume unreliable. The Stabilizing Additives
limited stability of the WBC differential count (up to 6 hours Sodium fluoride and lithium iodoacetate have been used
at room temperature storage) achieved in most of the 5-part alone or in combination with an anticoagulant such as potas-
differential analyzers is another drawback with EDTA.64 sium oxalate, EDTA, citrate, or lithium heparin for blood
collection (Table 7).2 The concentration of sodium fluoride
Citrate when used alone to inhibit glycolysis is approximately 4.3
Traditionally, a 0.129-mol/L concentration or a 0.109- mg/mL of blood. In combination with an anticoagulant, such
mol/L concentration of trisodium citrate has been used as the as Na2EDTA or K2EDTA (1 mg/mL of blood) or potassium
anticoagulant for collection of blood specimens intended for oxalate (2 mg/mL of blood), the nominal concentration of
global coagulation tests, such as prothrombin time (PT) and fluoride that is used to inhibit glycolysis is 2.5 mg/mL of
activated partial thromboplastin time (aPTT).2,31 Citrate has blood (60 mmol/L).2,67
virtually replaced sodium oxalate as an anticoagulant for Similarly, lithium iodoacetate has been used alone (0.5
performing global coagulation tests, since factor V is more mg/mL of blood) or at that concentration in combination
stable in citrate than oxalate. Furthermore, citrate rapidly with lithium heparin (14.3 U/mL of blood).2 Even with the
complexes with calcium, forming a soluble complex in use of either of these glycolytic inhibitors, at least 3 hours
contrast with the slow formation of the insoluble complex of are needed for these inhibitors to become fully effective
calcium with oxalate.31 and stabilize glucose levels.2,67 During this 3-hour period in
The effective molarity of citrate depends on whether the healthy subjects, an approximately 9 mg/dL loss of glucose
dihydrate or the anhydrous salt has been chosen in the prepa- occurs. 2,67,68 However, in neonates or subjects with an
ration of the citrate solution. Nominally, a 3.2% solution of increased RBC, WBC, or platelet count, the decrease in
the dihydrate trisodium citrate salt corresponds to a 0.109- glucose during the 3-hour period, even in presence of
mol/L concentration, whereas a 3.8% solution of the same glycolytic inhibitors, is greater than in healthy subjects.67,69
dihydrate salt corresponds to a 0.129-mol/L concentration. Once the glycolytic inhibitors become fully effective after
However, a 3.2% solution of the anhydrous trisodium citrate the initial 3-hour period, glucose levels remain stable for at
solution would correspond to a 0.124-mol/L concentration, least 3 days.2,67,68
whereas a 3.8% anhydrous sodium citrate solution would In the absence of glycolytic inhibitors, a decrease in the
have a molarity of 0.147 mol/L.65 Hence, it is important that glucose level of as much as 24% can occur in 1 hour after
the dihydrate salt be used to prepare either the 3.2% or 3.8% blood collection in neonates in contrast with a 5% decrease
solution of trisodium citrate. in healthy adults when specimens are stored at room
A laboratory that has been using one of the concentra- temperature. By 5 hours after blood collection in specimens
tions (3.2% or 3.8%) to perform PT determinations for stored at room temperature, as much as 68% of glucose can
patients receiving oral anticoagulant therapy should not be consumed in specimens collected without glycolytic
interchange the formulations. Doing so will affect the inter- inhibitors from neonates with a high hematocrit.69

The reason that fluoride and iodoacetate take approxi- less interference noted at higher concentrations of hexoki-
mately 3 hours to stabilize glucose levels is that they do not nase used in the assay.73
act on the first enzyme in the glycolytic pathway, which is In practical terms, if plasma is separated from cells by
hexokinase. Instead, fluoride acts on enolase, which is the centrifugation promptly after blood specimen collection, the
eighth enzyme in the glycolytic pathway, while iodoacetate glucose level in plasma is expected to be stable, since the
acts on glyceraldehyde-3-phosphate dehydrogenase, which enzymes that metabolize glucose are in the cellular fraction.
is the fifth enzyme in the pathway in which glucose is Stabilization of RBCs by including a nutrient such as
metabolized.2 glucose together with an anticoagulant mixture is the basis
Potassium oxalate, which is used in combination with of 2 acid citrate dextrose (ACD) formulations. 2 These
fluoride, inhibits pyruvate kinase in the enzymatic step formulations, labeled ACD A and B, are similar in pH;
immediately after enolase in the glycolytic pathway and, ACD A has a pH of 5.05, and ACD B has a pH of 5.1. The

thus, can effectively retard lactate formation 1 hour after difference between the formulations is the additive/blood
blood collection. ratio; the A formulation has a ratio of 1:5.67. The B formu-
The drawback with both fluoride and iodoacetate is lation, however, with a ratio of 1:3, has relatively greater
that they promote hemolysis, which increases progressively amounts of dextrose available for the metabolizing RBCs
as the concentration of the inhibitor used and the time and, hence, can preserve them better. While ACD formula-
elapsed after blood specimen collection increases. Appar- tions can preserve RBCs for 21 days when blood is stored
ently both inhibitors deplete the organic phosphate (ATP) between 1 C and 6 C, their stability can be extended
content of the RBC, thus causing an efflux of potassium out further by including additives such as phosphate and
of the cell. adenine (citrate phosphate dextrose and citrate phosphate
A variety of approaches have been attempted to dextrose adenine, respectively).
improve the efficacy of glycolytic inhibition. In one The maintenance of increased levels of cyclic adenosine
approach, a mixture of citric acid, trisodium citrate, monophosphate (c-AMP) within the platelets is the basis of
Na2EDTA, and a very small concentration of sodium fluo- an additive mixture intended for blood specimen collection
ride (0.2 mg) effectively inhibits the enzymes in the to minimize in vitro platelet activation.74,75 The additive is a
glycolytic pathway by maintaining blood pH in the range mixture of citric acid, theophylline, adenosine, and dipyri-
of 5.3 to 5.9.70 With this additive mixture, glycolysis was damole with the final pH adjusted to 5.0.74 Adenosine acti-
inhibited for 8 hours in specimens stored at room tempera- vates the enzyme adenylate cyclase, thus increasing c-AMP
ture, while at 24 hours, a mean – SD decrease of 1.3 mg – levels within the platelets. Theophylline and, to a certain
1.1 mg/dL (0.07 – 0.06 mmol/L) of glucose was noted.70 extent, dipyridamole prevent the degradation of c-AMP by
Another strategy for achieving efficient glycolytic inhi- inhibiting the enzyme phosphodiesterase. Dipyridamole, in
bition is to include mannose in addition to fluoride.71 The addition, prevents the uptake of adenosine by the RBCs, thus
rationale is that mannose, which is an epimer of glucose effectively increasing the amount of adenosine available to
(differs structurally from glucose in the configuration around the platelets to activate adenylate cyclase and, in turn, the
just 1 carbon atom), will compete with glucose for hexoki- inhibition of platelet aggregation and release of contents of
nase, the first enzyme in the glycolytic pathway. Since platelet alpha granules. 31,74 The citrate in the additive
mannose is a competitive inhibitor, the inhibition is short- mixture functions as an anticoagulant by chelating calcium.
lived, lasting just 4 hours. However, by this time, fluoride, The citric acid, theophylline, adenosine, and dipyridamole
which is also in the additive mixture, would have become mixture is used for monitoring heparin therapy by aPTT or
effective thus achieving a better glucose stabilization.2,71 the chromogenic substrate assay and in measurement of
At a mannose concentration of 3 mg/mL of blood, no platelet activation markers, such as P-selectin (CD62), by
interference was noted in hexokinase and glucose oxidase flow cytometry.76,77
procedures for the measurement of glucose.72 However, Stabilizing additives to inhibit proteolytic enzymes are
when the mannose concentration was increased to 3 times needed for some analytes. Although EDTA itself is a metal-
the nominal concentration of 3 mg/mL of blood, an overes- loprotease inhibitor, sometimes EDTA alone is insufficient to
timation of glucose by 18 mg/dL (1.0 mmol/L) was inhibit proteolytic enzymes. For example, when a synthetic
observed.72 Some of the mannose preparations also may be protease inhibitor, such as nafamostat mesylate, is added to
contaminated with glucose. Negative interference has been EDTA, the mixture can substantially improve the stability of
reported in the hexokinase procedure when mannose complement components C3a, C4a, and C5a.52,78
concentrations exceed 3 times the nominal concentration.73 A proteinase inhibitor, aprotinin (Trasylol), when used
The extent of reduction in glucose levels depends on the in combination with an anticoagulant such as EDTA or
concentration of hexokinase used in the glucose assay, with heparin, has been useful for the stabilization of labile

polypeptide hormones and enzymes.52 Aprotinin inhibits be included in the blood specimen collection additive
specific proteases of kinin and coagulation and fibrinolytic mixture. 81 The specimen collected with this additive
enzyme systems, in addition to inhibiting the enzyme mixture should be centrifuged within 1 hour of collection. It
systems of leukocytes and damaged tissue cells. Thus, it is preferable, however, if possible to separate plasma within
inhibits kallikrein, trypsin, chymotrypsin, coagulation factors 15 minutes of specimen collection and store it in plastic
in the preliminary phase of blood clotting, the fibrinolytic tubes or vials at –20 C in case analysis is delayed. Prompt
enzyme plasmin, and lysosomal proteinases. The fact that processing of plasma obtained with an antioxidant-anticoag-
aprotinin inhibits kallikrein is the basis for the expression of ulant mixture stabilizes catecholamines in plasma for 1 day
its potency in terms of kallikrein inhibitory units (KIUs). at room temperature, for 2 days when stored at 4 C to 8 C,
Aprotinin has been used in combination with an anticoagu- and for 1 month at –20 C. Stability at –20 C can be
lant such as EDTA or heparin. A mixture of EDTA (1.5 extended to 6 months with added glutathione.2,82

mg/mL of blood) and aprotinin in the activity range of 500 to Apparently, at the higher concentrations of cate-
2,000 KIU/mL has been used to stabilize polypeptide cholamines present in urine compared with plasma, the
hormones such as glucagon and corticotropin, the enzyme hormones and their metabolites are stable for 4 days, even
renin, and gastrointestinal hormones, such as beta-endorphin, when stored at 20 C to 25 C without preservatives. 2,82
secretin, neurotensin, gut glucagon, somatostatin, vasoactive However, for long-term stabilization of catecholamines in
intestinal peptide, and peptide yy.52,79 urine specimens, collection is recommended in a container
In the absence of aprotinin, spuriously higher values with 5 mL of a 6-mol/L concentration of hydrochloric acid
may be obtained apparently because of the fragments of the per liter of urine or in a mixture of 250 mg each of EDTA
analyte produced by proteolytic enzyme degradation are and sodium metabisulfite per liter of urine, which will extend
measured as intact molecules by the polyclonal antibody stability to 3 weeks at 20 C to 25 C, and 4 months to 1 year
used in the assay. Thus, in a radioimmunoassay for glucagon, at 4 C to 8 C.2,82
a 26% spurious increase was noted in plasma obtained with The laboratory assessment of fibrinolysis by the
blood specimens collected with EDTA alone compared with measurement of the nonspecific fibrinogen-fibrin degrada-
plasma obtained with blood specimens collected in a mixture tion products requires inhibition of the in vitro generation of
of EDTA and aprotinin.52,79 the fibrinolytic enzyme plasmin with an inhibitor, such as
A mixture of lithium heparin (14.3 U/mL) and 1,000 soybean trypsin or aprotinin, and the conversion of residual
KIU/mL of aprotinin also has been used to stabilize fibrinogen to fibrin with thrombin.31 However, in patients
immunoreactive somatostatin, secretin, cholecystokinin receiving heparin therapy, the conversion of residual
(pancreozymin), glucagon, and C-peptide.52 fibrinogen to fibrin, even in presence of thrombin, will be
In addition to aprotinin and an anticoagulant such as slow, thus resulting in spuriously high fibrinogen degradation
EDTA, additional proteolytic enzyme inhibitors may be product values owing to the remaining unconverted
needed to stabilize a labile tumor marker, such as parathy- fibrinogen. In such cases, the addition of snake venom, also
roid hormone related protein (PTH-rP) that has been associ- known as reptilase, instead of thrombin to the additive
ated with the humoral hypercalcemia of malignant mixture, which also contains a plasmin inhibitor such as
neoplasm.80,81 Thus, with an additive mixture of EDTA (1.5 aprotinin, can result in the conversion of residual fibrinogen
mg/mL), aprotinin (500 KIU/mL), leupeptin (2.5 mg), and to fibrin, even in the presence of heparin.83
pepstatin (2.5 mg), PTH-rP levels were stable for 1 hour For monitoring fibrinolytic therapy, the additive used to
after blood specimen collection for specimens stored at inhibit plasminogen activation depends on the therapeutic
room temperature and for 24 hours, if specimens were agent administered. Thus, while aprotinin together with
refrigerated at 4 C.80 Even when blood is collected with this EDTA is effective for inhibiting plasminogen activation by
additive mixture, if the sample is stored at room temperature the therapeutic agent urokinase, it is ineffective for inhibiting
for 24 hours, as much as 60% of the PTH-rP activity can be the recombinant tissue plasminogen activator (rt-PA) from
lost.80 Without a stabilizing additive mixture, PTH-rP is so activating plasminogen.75 Specific synthetic peptides of argi-
unstable that as much as 60% of its activity can be lost nine chloromethyl ketone (PPACK) are needed to inhibit in
within 1 hour of specimen collection and storage at room vitro rt-PA activity. PPACK irreversibly inactivates rt-PA by
temperature. alkylating the active center amino acid histidine, in addition
Analytes that are susceptible to oxidative damage, such to functioning as a potent thrombin inhibitor.84 A 5-mmol/L
as catecholamines, require stabilization. Typically together concentration of PPACK combined with a 10-mmol/L
with an anticoagulant such as heparin, EDTA, or egtazic concentration of citrate or a 4.2-mmol/L concentration of
acid, an antioxidant such as glutathione, sodium metabisul- EDTA effectively inhibited rt-PA upon blood collection.75
fite, or ascorbic acid at a concentration of 1.5 mg/mL should However, since PPACK is unstable at neutral pH, blood upon

collection should be maintained at 4 C, centrifuged promptly anticoagulant/blood ratio to at least 90% of the nominal
to obtain plasma, and processed without delay or kept frozen ratio.87 In the aforementioned study,87 the need to fill blood
until analysis can be performed.31 collection tubes to the nominal capacity (1:9) was reinforced
As new diagnostic markers are proposed, the labile for the aPTT, since tubes filled to less than 90% of the nominal
nature of some poses challenges. A case in point is the capacity caused prolongation of the aPTT result.
measurement of ANP, which is a marker of chronic conges- In addition to maintaining the nominal anti-
tive heart failure. An additive mixture of EDTA and apro- coagulant/blood ratio (1:9), the amount of head space above
tinin is needed to stabilize ANP; the blood specimen the blood also has been reported to be a variable affecting the
collected with this mixture must be centrifuged immedi- aPTT result. Thus, collecting less blood (2.7 mL as opposed to
ately at 4 C and plasma maintained frozen at –20 C or the nominal 4.5 mL) but maintaining the anticoagulant/blood
below until ready for analysis.85 Furthermore, hemolysis ratio without decreasing the tube size effectively increases the

invalidates ANP measurements. Alternatively, the measure- head space above the blood. This, in turn, generally leads to a
ment of the N-terminal fragment of the prohormone pro- shortened aPTT in values outside the upper limit of the refer-
atrial natriuretic peptide (proANP) (which is formed when ence interval and in the therapeutic range for monitoring
proANP is split into equimolar amounts of ANP and the N- unfractionated heparin therapy.88 Thus, in the abnormal range,
terminal fragment) can be performed in blood specimens the mean value obtained with the partially filled tube was 11
collected in EDTA alone with storage at room temperature seconds shorter than the mean value obtained with the full
for up to 6 hours. At 4 C, N-terminal proANP is stable for tube.88 One possible explanation for the head space effect is
3 days and at –20 C for 4 weeks, with hemolysis not the increase in surface area relative to the volume of blood
markedly affecting the results.86 collected, favoring platelet activation and the subsequent
release of platelet factor 4, which in turn neutralizes some of
Anticoagulant/Blood Ratio the heparin, resulting in a shortened aPTT.88
The anticoagulant/blood ratio is critical for some labora- For polycythemic patients with hematocrit values above
tory tests. Obviously, if too little blood is obtained, osmotic 60% (0.60), the PT and the aPTT can be prolonged substan-
effects can be expected to result in cell shrinkage and, thus, to tially, even when the nominal 1:9 anticoagulant/blood ratio is
introduce dilutional changes in the concentration of extracel- maintained.89 This is because the plasma is overcitrated to the
lular analytes. extent that it complexes much of the calcium chloride added to
A well-known example of the critical effect of the antico- perform the PT and aPTT, effectively reducing the calcium
agulant/blood ratio is for the global coagulation test, aPTT. ions needed to clot the plasma and thereby prolonging the
Traditionally, a 1:9 ratio of anticoagulant (sodium citrate, a clotting time. This effect can be minimized by adjusting the
0.109-mol/L concentration or a 0.129-mol/L concentration) to citrate concentration in accordance with the hematocrit value
blood is used. However, if less blood is collected, simulating a by using empiric formulas or by using a 1:19
1:7 anticoagulant/blood ratio, there is a significant increase in anticoagulant/blood ratio.65,89 The latter ratio dilutes the citrate
the aPTT result compared with that obtained using the concentration to a point that results of PT and aPTT are not
nominal 1:9 ratio.31 In 1 study, a 2.4-second increase was affected across a spectrum of hematocrit values ranging from
observed at a 1:7 ratio compared with the aPTT result anemia to polycythemia.89
obtained at the nominal 1:9 ratio.65 In general, collecting blood specimens to less than the
However, for the global coagulation test, PT, the effect of nominal volume increases the effective molarity of the antico-
the anticoagulant/blood ratio becomes meaningful only when agulant and induces osmotic changes affecting cell morpho-
the ratio reaches 1:4.5, which would occur when the blood logic features as noted for the morphologic features of
collection tube is filled to just less than half of its nominal neutrophils when the EDTA concentration changes from the
volume.65 The anticoagulant/blood ratio also is influenced by nominal 1.5 mg/mL of blood to 2.5 mg/mL of blood. Further-
the sensitivity of the thromboplastin reagent that is used. In a more, the binding of analytes, such as ionic calcium or ionic
study of the effect of the anticoagulant/blood ratio using 3.2% magnesium, to heparin can be enhanced when the effective
(0.109-mol/L concentration) buffered citrate, the results for the concentration of unfractionated heparin increases beyond the
PT were valid even for specimen collection tubes that were nominal 14.3 U/mL of blood.52
filled to 65% of the nominal anticoagulant/blood ratio (1:9). Maintenance of the anticoagulant/blood ratio also is
However, filling the tube to at least 80% of the nominal ratio critical when the inhibitory effects of the anticoagulant are
was needed to obtain reliable results in the therapeutic range concentration-dependent. Thus, a 10% reduction in the
using a moderately sensitive thromboplastin reagent with an gentamicin level was noted in an enzyme-multiplied
ISI of 2.06. Using a highly sensitive thromboplastin reagent immunoassay technique when the concentration of heparin
with an ISI of 1.01 required maintaining the used for specimen collection increased to 25 U/mL of

blood, presumably because of the inhibition of the enzymes at 4 C.2,8,94 Inhibition of the Na+,K+-ATPase pump also
used in the immunoassay procedure, as the concentration of drives sodium into the cell. Since sodium is mainly extracel-
heparin increased from the nominal value.90 lular, in contrast with potassium that is mainly intracellular,
At the other extreme is exceeding the anticoagulant/blood the decrease in sodium becomes noticeable only when whole
ratio to a point that the tube is overfilled with blood to such an or clotted blood is stored beyond 24 hours at 4 C. Ideally,
extent that proper mixing of anticoagulant with blood becomes when separation of plasma or serum from cells is needed,
problematic. Thus, in 1 study, the hemoglobin values had blood should be processed within 1 hour of collection to
doubled compared with a value obtained a week before, and obtain plasma or serum.2
the WBC and platelet counts were reduced drastically. Reana- Storage of clotted blood beyond 8 hours at room
lyzing the specimen for the fourth time from the same tube temperature (23 C) can cause an increase in inorganic phos-
yielded values similar to values obtained initially the week phate owing to the hydrolysis of cellular organic phosphate

before. Apparently, proper mixing of the specimen on a by the enzyme alkaline phosphatase; the effect is more
rocking mixer was not achieved since there was no head space pronounced when clotted blood is stored at 30 C.2 Thus,
or bubble left in the tube to assist in the mixing of the spec- while a 10% increase was noted in the inorganic phosphate
imen. After a sufficient amount of blood was withdrawn by value at 24 hours compared with the 8-hour value during
repeating the analysis 4 times, there was sufficient space in the storage at 23 C, the increase was 120% at a storage tempera-
tube for the air bubble to move and effect thorough mixing on ture of 30 C.2
the rocking-type mixing device.91 A time-dependent increase in ammonia is accentuated
For bacteriologic determinations, inhibition of the normal in specimens with increased gamma-glutamyltransferase
bactericidal properties of blood requires appropriate dilution activity.95 The time between collection of blood and its
of blood in broth. Incorporation of a 0.025% solution of storage temperature before processing to yield plasma can
sodium polyanethole sulfonate in blood culture media is introduce a variable. Thus, in the measurement of cate-
intended to inhibit phagocytosis, complement, and lysozyme. cholamine levels in specimens collected with a stabilizing
Even when blood culture is supplemented with sodium additive mixture and stored at 4 C, there is an increase in
polyanethole sulfonate, at least a 1:10 blood/broth ratio is the level of catecholamines owing to a delay in the
considered optimal.92 However, a blood/broth ratio less than processing of plasma that exceeds 1 hour.81,82 A delay in
1:10 has been acceptable when at least 2 separate blood the preparation of plasma for 2 hours after blood collection
cultures are obtained routinely or when a biphasic medium is and storage at 4 C can increase the mean plasma norepi-
used to culture blood (a broth bottle to which an agar slant nephrine level by 13% (N = 8).82 This effect is due to the
with media is attached).93 lysis of some of the RBCs at 4 C, leading to a release of
Since spurious blood culture results can be obtained catecholamines into plasma and to the slow reuptake of
for patients receiving antibiotics, a 1:10 dilution of blood in these hormones by cells at 4 C. In addition, cryoprecipita-
broth dilutes many of the antibiotics in the blood to sub- tion of plasma proteins at 4 C also can contribute to the
inhibitory levels. The incorporation of sodium polyanethole increased level of catecholamines measured in plasma.81,82
sulfonate inactivates aminoglycosides. Alternatively, In contrast, storage of specimens at 20 C and a delay
however, devices containing antibiotic-adsorbent resins can beyond 1 hour in the processing of plasma after specimen
be used to neutralize antibiotics, and cell lysis followed by collection can lower the catecholamine level measured in
filtration or centrifugation neutralizes the antimicrobial plasma owing to the uptake of catecholamines by cells such
properties or components of blood and provides a concen- as RBCs or platelets.82 Thus, the mean plasma norepineph-
trate for culture on antibiotic-free media.93 rine levels decreased by 14% (N = 8) from the initial value
if blood specimens kept at 20 C were centrifuged 2 hours
Specimen Handling and Processing after collection, while mean plasma epinephrine values
The time and temperature for storage of the specimen decreased by 20%.82
and the processing steps in the preparation of serum or The storage temperature of a specimen influences the
plasma or cell separation using density-gradient techniques results of global coagulation tests such as PT and aPTT.
can introduce a preanalytic variable. Analytes subject to Since factor VII, which is measured by PT, is activated
cellular metabolism need prompt separation from cells. during prolonged storage at 4 C, storage of plasma in
Storage of anticoagulated whole blood or clotted blood contact with cells beyond 7 hours shortens the PT.65,96,97
in a refrigerator (4 C) inhibits the Na+,K+-ATPase pump, Indeed, when a blood collection tube is kept well stop-
resulting in potassium leaking out of the cells and causing a pered and stored at room temperature (25 C), the PT is
spurious increase in levels measured in serum or plasma. stable for up to 48 hours.89,97 As is well known, the aPTT
This effect is seen even at 4 hours of storage of clotted blood should be performed preferably within 2 to 4 hours of

specimen collection, and some investigators maintain the lymphocyte fraction was contaminated with granulo-
plasma on ice (4 C).2 In 1 study, a 10% to 15% increase in cytes if the blood specimen was more than 24 hours old.52
aPTT was observed after 24 hours in specimens that were RBC contamination is a problem with aged EDTA blood
maintained well stoppered and at room temperature.89 specimens to the extent that 9 RBCs may be encountered
In contrast with refrigerated temperatures, freezing for every lymphocyte separated from a 2-day-old blood
plasma at –20 C and –70 C did not activate factor VII. The specimen.101
PT and aPTT results were stable in plasma maintained For flow cytometric analysis, ACD and heparin are
frozen at –20 C for 10 days and at –70 C for 21 days.97 superior to EDTA for maintaining viable WBCs
Factors II, V, VII, and X are stable in plasma maintained overnight.102 However, if analysis is to be performed the
under refrigeration for up to 6 hours. All except factor V had same day, EDTA, ACD, and heparin give equivalent results.
stability for up to 14 days when frozen at –20 C or –70 C.97 However, at 24 hours and beyond, a substantial decrease in

During a 7-day period, 20% of factor V activity was lost in granulocyte viability may occur in blood collected in EDTA.
plasma stored at –20 C, while in samples stored at –70 C for K3EDTA also has been reported to be associated with a loss
the same period, 10% of the activity was lost. of function in lymphocyte-mitogen stimulation assays.102
Factor VIII is the most labile factor; 10% of the activity is Heparin has been reported to be problematic for use in
lost in 4 hours during storage at 4 C, while at –20 C for a 3 day molecular biology procedures using restriction enzymes and
period, 20% of the activity was lost.97 Typically, clotted blood DNA amplification by the polymerase chain reaction
or anticoagulated blood is centrifuged at 1,000g to 1,200g for (PCR).103 While heparin inhibition can be overcome by a
10 to 15 minutes. Insufficient centrifugation time and lower variety of approaches (eg, treatment with heparinase, or
centrifugal forces can result in a gradient of platelets in plasma separation of leukocytes by centrifugation followed by a
derived from heparinized blood, thus affecting some of the minimum of 2 washings in a saline buffer or use of an appro-
laboratory results, such as potassium levels.2 priate buffer), many investigators prefer not to use heparin
While centrifugation at 2,000g for 15 minutes is for DNA amplification by PCR.103
recommended for whole blood to obtain platelet-poor In vitro platelet, monocyte, and neutrophil activation
plasma for global coagulation tests, at slightly lower speeds studies are influenced by the anticoagulant used for blood
(1,800g), some residual platelet contamination was collection.104 Thus, monocyte activation as measured by
reported based on a shortened thrombin clotting time by release of tissue factor and tumor necrosis factor activity was
10% in plasma frozen for 48 hours and thawed to room lowest with EDTA compared with citrate, heparin, and
temperature before testing.97 hirudin. Neutrophil activation as measured by lipopolysac-
Despite the use of robotics and improved pneumatic charide-induced release of lactoferrin also was the lowest
tube delivery systems, and despite the fact that erstwhile with EDTA. EDTA seems to suppress platelet degranulation.
preanalytic problems such as hemolysis during specimen Platelet factor 4 levels as an indicator of platelet activation
transportation may have been overcome by improved design was the lowest with EDTA–platelet-poor plasma (217
of transport systems, the existence of preanalytic problems ng/mL) compared with platelet-poor plasma obtained with
should not be discounted.98,99 A case in point is the likeli- citrate (440 ng/mL), hirudin (469 ng/mL), and unfractionated
hood of erroneous results for PO2 if the blood has been cont- heparin (1,180 ng/mL). A low-molecular-weight heparin
aminated with air before pneumatic tube transportation.99 preparation, however, gave platelet factor 4 values compa-
rable to those obtained with hirudin.104
Relative Merits of Anticoagulants
Platelets rapidly change their shape from discoid to spher-
ical when blood is collected in EDTA, thus making the deter-
Endogenous Interferences and Related
mination of mean platelet volume unreliable. Approaches to
Variables
minimize this artifact have been attempted by the use of an
alternative anticoagulant, which is a mixture of trisodium The effect of drugs and their metabolites on laboratory
citrate, pyridoxal phosphate and tris(hydroxymethyl)- tests is so extensive that compendia of interferences are
aminomethane.100 The rationale is that pyridoxal phosphate available for consultation.105 Several are discussed in the
is an effective platelet antiaggregant and disaggregant. Mean following text ❚Table 8❚.
platelet volume measurement using the aforementioned addi- Although the subject of endogenous interferences is
tive mixture was stable at room temperature for 24 hours.100 broad, some mention should be made of interferences that can
ACD, heparin, and EDTA have been used for the sepa- affect clinical interpretation of laboratory data. In that context,
ration of mononuclear cells. However, regardless of whether patients who are exposed to mouse immunoglobulins through
ACD, EDTA, or heparin was used as the anticoagulant, 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 Narayanan81; Primus et al106
Antibodies to platelet membrane phospholipids (lupus anticoagulant, anticardiolipin antibodies) Narayanan and Hamasaki31
inhibit phospholipid-dependent global coagulation tests
Antibodies to glycoprotein IIb/IIIa complex in the platelet membrane; EDTA-induced platelet Fiorin et al108
agglutination at low or room temperature; PTCP; effect abolished at 37°C
EDTA-dependent IgM antibody causes leukocyte agglutination at room temperature; spuriously Hillyer et al109
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 Bizzaro et al111; Peters et al112
temperature in EDTA-anticoagulated blood; occasionally, also seen in heparinized or citrated blood;
severe platelet satellitism causes spurious PTCP; effect abolished at 37°C

Increased level of chemical analytes affecting hematology test results
Glucose level >600 mg/dL causes transient increase in MCV; as glucose equilibrates, water leaves Cornbleet107
RBC restoring original MCV
Triglyceride level >1,000 mg/dL causes spuriously high hemoglobin value; washing and resuspending Cornbleet107
RBCs in saline overcomes effect
MCV, mean corpuscular volume; PTCP, pseudothrombocytopenia.
antibodies to mouse immunoglobulins. These antibodies, An in vitro phenomenon of platelet agglutination seen in
called human antimouse antibodies, or HAMA, can give rise blood collected in EDTA is due to the presence of antibodies
to false-positive or false-negative results in 2-site immuno- in blood that are reactive to platelets. These antibodies are
metric assays using murine monoclonal antibodies as directed to antigens such as the glycoprotein IIb/IIIa complex
reagents.81,106 that is hidden within the platelet membrane. EDTA, upon
An increased level of a chemical analyte can affect some chelating calcium, exposes these antigens. The exposed
hematology determinations. Thus, glucose levels more than platelet antigen becomes modified at low and room tempera-
600 mg/dL (33.3 mmol/L) can lead to a transient increase in ture, permitting platelet antibodies to agglutinate platelets.
the MCV as water enters the RBC, as the RBC is placed in an This EDTA-induced pseudothrombocytopenia is, however, not
isotonic diluent, owing to the osmotic effect of glucose.107 seen when blood specimens are warmed to 37 C, since the
However, as the glucose level slowly equilibrates, water exits glycoprotein IIb/IIIa complex is dissociated at the higher
from the RBC along with glucose, thus restoring the original temperature. EDTA-induced pseudothrombocytopenia at 4 C
MCV. This transient hypoglycemic effect can be overcome by or room temperature can cause a spuriously low platelet count
a microdilution of the blood sample and a 5-minute waiting and a spuriously increased WBC count if the platelet clumps
period for the equilibration to be complete before analysis.107 are in the same size range as WBCs. Apparently an epitope on
Triglyceride levels exceeding 1,000 mg/dL (11.29 platelet membrane glycoprotein IIb is recognized by EDTA-
mmol/L) and hyperlipidemic specimens can cause a spuri- dependent IgG antibodies, causing pseudothrombocy-
ously high hemoglobin value. This effect can be overcome by topenia.108
resuspending the RBCs in saline before analysis or by making EDTA-dependent IgM antibody, most active at room
corrections with a plasma hemoglobin blank.107 temperature and of low titer, has been reported to cause leuko-
Turbidity resulting from the precipitation by lysis cyte agglutination noticeable on a peripheral blood smear but
reagents of high concentrations of monoclonal proteins seen to provide a spuriously low automated WBC count.109 This
in patients with myeloma or macroglobulinemia may spuri- phenomenon was not seen when the same patient’s specimen
ously elevate the hemoglobin level and WBC count.107 was obtained in a tube with heparin or citrate.
Endogenous antibodies can affect both coagulation and Both pseudothrombocytopenia and pseudoleukopenia
hematology results. Circulating antibodies directed to platelet have been reported at room temperature owing to the EDTA
membrane phosphatidylcholine or lecithin are referred to as antibody–induced platelet aggregates not being counted as
lupus anticoagulants. Prothrombin is the antigen for most platelets and to large neutrophil-platelet clumps falling outside
lupus anticoagulants and, as such, inhibits phospholipid-depen- the size range for WBCs.110 This phenomenon was abolished
dent global coagulation assays.31 Antibodies directed to the by warming the specimen to 37 C.
platelet membrane phospholipids phosphatidylserine and phos- An in vitro phenomenon that is referred to as platelet satel-
phatidylinositol, which are called anticardiolipin antibodies, litism is observed when the platelets surround the neutrophils
also inhibit phospholipid-dependent global coagulation tests. owing to EDTA dependent IgG autoantibodies that are directed

to the glycoprotein IIb/IIIa complex in the platelet membrane a mean – SD free hemoglobin value of 4.8 – 3.2 mg/dL, and
and neutrophil FC-gamma receptor III of neutrophils (CD16).111 moderately hemolyzed serum samples had a value of 43.5 –
This phenomenon, which is seen at room temperature, is not 13.9 mg/dL. Even slight hemolysis invalidated the results for
seen when blood is exposed to a temperature of 37 C or periph- LDH, total acid phosphatase, prostatic acid phosphatase, and
eral blood smears are made immediately with EDTA-anticoagu- potassium, and such specimens should be rejected.115 That the
lated or capillary blood. Occasionally, the phenomenon also has effect of hemolysis is method-dependent was demonstrated by
been noticed with citrated blood, as well as with heparinized the absence of statistically significant differences for the levels
blood. 112 Severe platelet satellitism can cause spurious of AST, ALT, inorganic phosphate, glucose, bilirubin, total
pseudothrombocytopenia. protein and albumin.115
A discussion of endogenous interferences would not be Adenylate kinase released from RBCs can spuriously
complete without discussing the most common interfer- increase CK activity measurements unless a sufficient

ences, such as hemolysis and turbidity due to hyperlipi- amount of inhibitors of adenylate kinase are incorporated in
demia. Apart from a few analytes, there are conflicting data the assay mixture.2
in scientific literature on the effect of hemolysis on a wide The peroxidative effect of heme can cause a positive and
range of analytes. The discrepancy is related not only to negative interference with various diazotization methods used
differences in methods for the same analyte, but also to for the measurement of bilirubin, depending on the regents
differences in instrumentation. While to some extent instru- used in the assay system.116 In classic diazotization procedures
ments that can subtract or blank the hemoglobin interfer- for the measurement of bilirubin, hemoglobin in the
ence by taking spectrophotometric measurements at the hemolyzed sample competes with nitrite for the sulfanilic acid
wavelength of light where hemoglobin absorbs, in addition reagent, leading to spuriously lower results.115
to the wavelength at which the analyte absorbs (bichromatic Depressed glucose values (values lower than the true
measurements), can minimize the interference, the hemo- value) have been reported with the glucose oxidase–peroxi-
globin interference is not eliminated totally, especially when dase procedure, which apparently is related to the reagent
the reagent system used is affected by hemoglobin. composition. The negative interference has been explained as
In general, slight hemolysis would have a negligible resulting from premature decomposition by hemoglobin of the
effect on many of the routine clinical chemistry laboratory hydrogen peroxide generated in the glucose oxidase reaction
procedures. Severe hemolysis, while having a substantial or from the dilution by cellular components.117
effect on constituents that are at a much higher concentration The effect of hemolysis on glucose oxidase procedures
within the RBC than in plasma, could induce a slightly dilu- depends not only on the composition of the glucose oxidase-
tional effect on the constituents present at a lower concentra- peroxidase reagent system, but also on how the assay is
tion in the RBC than in plasma. performed. Thus, differences on the effect of hemolysis have
Typical of the enzymes present within the RBC, the been reported when using a kinetic assay in which the color
concentration of lactate dehydrogenase (LDH) is 160-fold development is monitored between 60 and 120 seconds after
greater than that present in the plasma, acid phosphatase is 68- initiating the reaction as opposed to a bichromatic assay in
fold greater, and AST is 6.7-fold greater than the plasma which color is monitored at 2 different wavelengths; the addi-
levels.113 The concentration of potassium inside the RBC is tional wavelength is used to blank out the color resulting from
approximately 23 times as much as that found in plasma. substances other than glucose. For example, 2 grossly
Naturally one would expect the aforementioned tests to be hemolyzed pediatric samples with a low true glucose value
invalidated when the specimen is hemolyzed severely. were diagnosed correctly by the kinetic procedure that yielded
The visual evidence of hemolysis is provided when the glucose values of 29 and 27 mg/dL (1.6 and 1.5 mmol/L;
hemoglobin concentration exceeds 20 mg/dL. It has been reference range, 70-105 mg/dL [3.9-5.8 mmol/L]). The
reported that the appearance of serum when 0.1% of the bichromatic procedure, on the other hand, overestimated the
RBCs are lysed was virtually identical to that of glucose values on the 2 pediatric samples by reporting glucose
nonhemolyzed serum and, consequently, would go unde- values of 65 and 68 mg/dL (3.6 and 3.8 mmol/L).117
tected by laboratory personnel looking for hemolyzed spec- Hemolysis also interferes with the hexokinase procedure
imens.114 However, the appearance of serum when 1.0% of for the measurement of glucose.118 However, owing to the
RBCs were lysed was clear and cherry red. Such a spec- multiplicity of instrumentation and variations in methods for
imen would be characterized as having moderate hemol- the measurement of glucose, the magnitude of the effect of
ysis. Hemolysis of 1% of the RBCs affected the measure- hemolysis on each glucose procedure is unpredictable. As
ment of LDH, potassium, AST, and ALT.114 such, for accurate glucose measurements, it is desirable to
Hemolysis also has been classified based on measurement avoid hemolysis or, at the very least, not exceed levels that
of free hemoglobin levels.115 The nonhemolyzed samples had would make hemolysis visually apparent in serum.

While total acid phosphatase activity was, as expected, labile tumor markers, including handling of tumor tissue for
affected by hemolysis due to the release of RBC acid phos- the processing of labile hormone receptors, half-life, circadian
phatase, even the prostatic tartrate–sensitive fraction has been rhythm, distribution in cellular fractions, the effect of exercise
reported to be affected by hemolysis.118 Among the hormones on markers such as prostate-specific antigen, amplification by
very sensitive to hemolysis is insulin.2 Apparently the release molecular methods, and sample quality for cytogenetics and
of proteolytic enzymes during hemolysis degrades insulin.41 flow cytometry assessment of DNA histograms are just some
While some analytes clearly are affected by hemolysis, in of the many issues that come under the preanalytic umbrella.81
general, method-dependent variations should be considered As new markers are proposed, preanalytic problems
when assessing the influence of hemolysis. associated with their measurements also surface. For
Hyperlipidemia, by introducing turbidity, can be a source example, homocysteine has received considerable attention
of interference that also is method-dependent.119 Interference as a marker of cardiovascular risk. Yet the measurement of

could be due to a variety of mechanisms, such as inhomo- this amino acid presents challenges since it continues to be
geneity of the sample, displacement of water, and adsorption formed by cellular enzymes in vitro, thus making the
of lipophilic constituents.2 A visibly hyperlipidemic sample prompt separation of cells from plasma or serum manda-
can be expected to interfere with the measurement of total tory. Alternatively, measures should be taken to inhibit
protein and electrophoretic and chromatographic procedures.2 homocysteine generation and converting enzymes immedi-
ately after blood specimen collection.123
With advances in coagulation and fibrinolysis, including
increasing use of molecular methods to study mutations that
Summary Perspectives
predispose a person to hypercoagulability, preanalytic vari-
So much knowledge on the preanalytic phase has accumu- ables affecting these measurements need to be delineated and
lated during recent years that this review can only highlight key controlled. Some physiologic variables that affect the
issues affecting several contemporary topics. measurement of fibrinolytic activators and inhibitors,
On the subject of blood gases issues such as maintaining a including the extent of diurnal variation during a 24-hour
steady state of ventilation with the patient in a supine position period, the effects of smoking and alcohol need to be recog-
before and during arterial blood collection, the effect of the nized. Challenges in measurement of free t-PA in plasma by
container material (eg, glass vs plastic syringe), dry vs liquid sample treatment to dissociate the t-PA-plasminogen activator
heparin as the anticoagulant, leakage of gases owing to syringe inhibitor I complex should not be underestimated.31
material and conditions of storage, and time between collection Finally, preanalytic problems that are unique to a method
and analysis need to be recognized and standardized.120,121 should be kept in perspective as we begin to better understand
Molecular biology is another area in which information the complexities of the effect of new technology and therapy,
on the preanalytic phase is accumulating constantly. The type inasmuch as they affect the preanalytic phase.
of detergent used for cell lysis, its effect on DNA amplification
by PCR, the effect of RBC contamination due to the effect of From the Department of Pathology, New York Medical
hematin on the DNA polymerase enzyme, taq polymerase, College–Metropolitan Hospital Center, New York, NY.
used in PCR, isolation of RNA including steps to eliminate Address reprint requests to Dr Narayanan: Dept of
RNase contamination, treatment of tissue, the effect of fixa- Pathology, New York Medical College–Metropolitan Hospital
tives and duration of fixation, and protection of viral HIV Center, 1901, First Ave, New York, NY 10029.
*Dr Narayanan is a Becton Dickinson Fellow at Becton
RNA load in plasma from the inhibiting effect of neutrophils Dickinson, Franklin Lakes, NJ.
by prompt separation of plasma from cells after blood collec- Acknowledgments: I thank Carol Perello for secretarial
tion are just a few of a wide range of preanalytic variables that support and preparation of the tables.
merit consideration.103
As our knowledge of cell function advances and
studies on stimulation of cells to secrete cytokines become
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Age Group/Effects References

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KIU, kallikrein inhibitory unit.

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Anticoagulants for Blood Collection

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MCV, mean corpuscular volume; PTCP, pseudothrombocytopenia.

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