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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

A b s t r a c t The preanalytic phase is an important component

of total laboratory quality. A wide range of variables

that affect the result for a patient from whom a

specimen of blood or body fluid has been collected,

including the procedure for collection, handling, and

processing before analysis, constitute the preanalytic

phase. Physiologic variables, such as lifestyle, age,

and sex, and conditions such as pregnancy and

menstruation, are some of the preanalytic phase factors.

Endogenous variables such as drugs or circulating

antibodies might interact with a specific method to yield

spurious analytic results. The preanalytic phase

variables affect a wide range of laboratory disciplines.

In recent years, considerable attention has been given to

the influence of preanalytic effects on laboratory results.

Compendia of preanalytic effects and books have addressed

the effect of the preanalytic phase on laboratory results.1,2

Preanalytic variables can be grouped broadly under 3

categories: physiologic, specimen collection, and influence or

interference factors. In this review, I address these variables

and provide examples of their effects across a broad range of

laboratory disciplines. Examples of preanalytic variables

affecting mainly clinical chemistry, hematology, and coagula-

tion tests are discussed.

Admittedly, given the comprehensive range of subject

matter that spans the realm of the preanalytic phase, this

review must be selective. Hence, a mere passing mention is

made in the concluding section, “Summary Perspectives,” of

topics such as blood gases, molecular biology, tumor

markers, and problem analytes, such as cytokines, 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.

Am J Clin Pathol 2000;113:429-452 429© American Society of Clinical Pathologists



Narayanan / THE PREANALYTIC PHASE

Age

The effect of age ❚Table 1❚ on laboratory results has

been well recognized to the point that separate reference

intervals have been used to distinguish pediatric, adolescent,

adult, and geriatric populations. Bone growth and develop-

ment in a healthy growing child is characterized by increased

activity of bone-forming cells, the osteoblasts, that secrete

the enzyme, alkaline phosphatase. Hence, the activity of thisenzyme is approximately 3 times higher in a growing child

compared with the activity in a healthy adult.3,4

A substantial increase in the RBC count in the neonates

compared with that in adults is the reason glucose is metabo-

lized very rapidly in neonates.2 Increased arterial oxygen

content within a few days of birth leads to an increase in

hemoglobin levels owing to the destruction of the RBCs.

Serum bilirubin levels then increase, because the immature

liver lacks enzymes to convert bilirubin to the water-soluble

bilirubin diglucuronide.2 The total number of neutrophils are

at a maximum for 1 to 2 days after birth. In the neonate, the

monocyte count is increased for up to 2 weeks after birth,

while the eosinophil count stays elevated for up to 1 week

after birth.5,6 The lymphocyte count is increased markedly at

birth and remains elevated in children up to 4 years of age.6

In contrast, the basophil count is elevated only transiently for

up to 1 day after birth. A substantial decrease in serum uric

acid levels is noted between days 1 and 6 after birth.2

Age also affects renal function, as reflected by an effect

on creatinine clearance, which decreases progressively with

each decade.7 For example, the average value for creatinine

clearance at age 30 years is 140 mL/min (2.33 mL/s) per

1.73 m2 of body surface area. In contrast, at age 80 years, the

creatinine clearance value decreases to 97 mL/min (1.62

mL/s) per 1.73 m2 of body surface area.7 Beyond the age of

50 years, the decrease in creatinine clearance is also related

to a decrease in muscle mass.7,8 However, when interpreting

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

overlap of disease processes, such as the presence of renal

vascular disease in elderly patients, must be considered.

Secondary risk factors, such as genetics and obesity, may

have a role in the decrease in glucose tolerance with aging.9

Homeostatic control by the hypothalamic-pituitary-

adrenal axis is compromised because of aging. Hence, there

is an increase in plasma corticotropin and corticosteroidlevels due to aging.10,11 Usually in young adults, the concen-

tration of the cytokine, interleukin (IL)-6, in serum is low

and is undetectable in absence of inflammation.11 However,

IL-6 levels are detectable readily during middle age. Also, in

healthy elderly adults, serum IL-6 levels are increased appar-

ently due to the loss of the normal regulation of IL-6 levels.

The decrease in dehydroepiandrosterone sulfate with age,

which normally has a suppressive effect on IL-6, is related to

the increase in IL-6 levels.12 Increased IL-6 levels, in turn,

increase the percentage of cells bearing the receptor for the

cytokine, transforming growth factor beta, a powerful

immunosuppressive agent, thus providing an explanation for

the age-related immunosuppressive phenomena.13

Increases in the level of plasma antidiuretic hormone (or

arginine vasopressin) have been related to aging with levels

significantly higher in 1 study of healthy subjects 53 to 87

years of age (4.7 – 0.6 pg/mL [4.4 – 0.6 pmol/L] in 68

healthy subjects) compared with subjects 21 to 51 years of

age (2.1 – 0.2 pg/mL [1.9 – 0.19 pmol/L] in 45 healthy

subjects).14

Thyroid homeostasis apparently is affected during

aging. Thyrotropin levels in serum have been reported to be

38% higher in an elderly group (mean age, 79.6 years)

compared with a younger group of subjects (mean age, 39.4

years).15 In the same study, serum triiodothyronine levels

were 11% lower in the older age group compared with levels

in the younger group of subjects.

430 Am J Clin Pathol 2000;113:429-452 © American Society of Clinical Pathologists

❚Table 1❚Effect of Age on Laboratory Results

Age Group/Effects References

NeonateIncreased 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 al2Lymphocyte count elevated up to 4 years of age Weinberg et al6

Growing child3-fold increase in alkaline phosphatase level due to bone growth Narayanan3; McComb et al4

Progressive changesCreatinine clearance decreases with each decade; age-related change in renal function; may Narayanan and Appleton7; Narayanan11

overlap with disease process in elderly personsDecrease in glucose tolerance with age; overlaps with secondary risk factors, such as genetics Pacini et al9

and obesityHomeostatic control by hypothalamic-pituitary-adrenal axis affected by aging; corticotropin and De Kloet10; Narayanan11

corticosteroid levels increaseDehydroepiandrosterone 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



Clinical Chemistry / REVIEW ARTICLE

While changes in organ function occur due to aging,

which in turn affects the circulating level of various analytes,

one must also keep in perspective the overlapping effects due

to disease and dietary deficiencies of trace elements, such as

zinc and other nutrients.11

Sex Differences

Between 15 and 55 years of age, the levels of total andlow-density lipoprotein cholesterol (LDL-C) increase

progressively, with levels slightly higher in premenopausal

females compared with the levels in males ❚Table 2❚. Thus,

in female subjects, a change in cholesterol value from 174

mg/dL (4.50 mmol/L) at age 25 years to 213 mg/dL (5.51

mg/dL) at 55 years has been reported.2 In the corre-

sponding age span for men, cholesterol values have been

reported to range from 166 mg/dL (4.29 mmol/L) at age 25

years to 232 mg/dL (6.00 mmol/L) at 55 years.2 In contrast,

the high-density lipoprotein cholesterol (HDL-C) level

does not fluctuate in either sex between the ages of 15 and

55 years; levels are slightly higher in females, presumably

owing to the stimulating effect of estrogens.2

Analytes with levels that depend on muscle mass, such

as creatinine and creatine kinase (CK), are generally at a

higher concentration and activity, respectively, in males

compared with females.2, 7 However, when increased

muscle mass is acquired as a consequence of strenuous

athletic activities in female subjects, the sex difference in

levels of creatinine and CK is abolished.2 Serum iron levels

generally are lower in premenopausal female subjects

compared with those for male subjects, with the difference

disappearing in subjects older than 65 years.2

Sex differences are seen for several analytes as can be

noted by reviewing a current list of reference laboratory

values.16 The differences for some of the analytes based on sex

can be explained as due to differences in muscle mass and to

endocrine and organ-specific differences.

Time

There are time-related fluctuations in the level of some

analytes (Table 2). Circadian rhythm is responsible for the

diurnal changes seen in the circulating levels of some

analytes. A classic example of an analyte subject to diurnal

variation is cortisol, which generally peaks at around 6:00

AM, with levels becoming lower toward the evening and

midnight.2 Because cortisol is a powerful immunosuppres-

sant, its diurnal rhythm affects proinflammatory cytokine

production. Indeed the level of proinflammatory cytokines,

such as IL-1 alpha, IL-12, tumor necrosis factor alpha and

interferon gamma have been shown to be inversely corre-

lated with the level of plasma cortisol.17 The inverse rela-

tionship with plasma cortisol levels extends to the number

of circulating T lymphocytes, especially the CD4+ T cells,

and to the urinary secretion of neopterin, which is a marker

of cellular immune activity.18,19 Even routinely measured

analytes such as glucose, potassium, and iron exhibit

diurnal variation.1,2


❚Table 2❚

Effect of Sex, Time, Season, and Altitude on Laboratory Results

Variable/Effects References

SexBetween 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 femalesCreatinine 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

TimeCortisol 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; Miyawakineopterin 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

SeasonSummer

Vitamin D levels higher Narayanan3

Triiodothyronine levels 20% lower Guder et al2; Harrop et al25

WinterTriglyceride level lower Narayanan3; Statland and Winkel24

Slight (2.5%) increase in total cholesterol level Cooper et al22; Narayanan23

AltitudeAt 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-

ance test tend to be higher when the test is performed in the

afternoon than when the test is performed in the morning.

The cortisol rhythm apparently may be responsible for the

discrepancy noted in the results of an oral glucose tolerance

test performed in the afternoon.2

Thyrotropin levels peak during the late evening hours,

with the lowest values observed around midday.20,21 Growthhormone levels during waking hours are minimal, with

increases noted during sleep.1

Reproductive hormones, such as luteinizing hormone,

follicle-stimulating hormone, and testosterone, are released

in bursts lasting barely 2 minutes, making accurate assess-

ment of their concentration problematic.8

Seasonal Changes

Vitamin D levels tend to be higher during the summer

(Table 2), apparently due to prolonged exposure to

sunlight.3 A slight increase in the total cholesterol level

(average, 2.5%) has been observed during the winter

compared with values measured during the summer.22,23

The decrease in the level of triglycerides in serum from

summer to winter is more striking than the extent of decline

noted between spring and autumn months when the temper-

ature tends to be milder.3,24

The levels of the thyroid hormone, triiodothyronine, are

reported to be 20% lower during the summer compared with

during the winter.2,25

Altitude

Changes in levels of some of the constituents in blood

occur when measured at sea level as opposed to measure-

ment at a higher altitude (Table 2). For example, hematocrit

and hemoglobin levels can be up to 8% higher at an altitude

of 1,400 m.1,2 A 65% increase in C-reactive protein has been

reported at 3,600 m. Concentrations of some analytes, such

as plasma renin, serum transferrin, urinary creatinine, and

estriol, and the creatinine clearance rate decrease with

increasing altitude.1,2

Menstruation

At the onset of menstruation, a low estrogen level triggers

the release of follicle stimulating hormone from the pituitarygland. The ovaries thus are stimulated to produce estrogen,

and the level begins to increase noticeably from the sixth or

seventh day after menstruation; the peak level is reached on

approximately the 13th day. A day later, a burst of luteinizing

hormone released from the pituitary gland signals ovulation.

With the onset of ovulation, the progesterone level continues

to rise until it decreases together with the estrogen level, just

before the commencement of the next menstrual cycle. Thus,

the reference intervals for estradiol, follicle stimulating

hormone, luteinizing hormone, and progesterone are influ-

enced by the stages of menstrual cycle (ie, follicular, midcycle,

and luteal phases) ❚Table 3❚.16

Coincident with ovulation, serum cholesterol levels are

lower than at any other phase of the menstrual cycle.2

During midcycle or the luteal phase, the aldosterone

concentration is approximately 2-fold higher compared

with values during the follicular phase.1,2 Renin activitymay increase during the luteal phase of the cycle. 1 In

contrast, serum phosphate and iron levels decrease during

menstruation.1,2

Pregnancy

A dilutional effect is observed due to an increase in

the mean plasma volume, which in turn causes hemodilu-

tion (Table 3).2,3 The effect is more pronounced when

measuring trace constituents such as trace elements in

serum.3 During pregnancy, there is a considerable increase

in the glomerular filtration rate. Hence, the creatinine

clearance rate is increased by 50% or more over the

normal rate.7 During the third trimester, the urine volume

may increase by approximately 25%.

During the second half of pregnancy, the placenta

progressively begins to produce hormones antagonistic to

insulin. As a result, the levels of hormones, such as

estrogen, progesterone, and human placental lactogen (also

called chorionic somatomammotropin), increase, leading

to the onset of gestational diabetes.26

The increased metabolic demand during pregnancy

causes an increased mobilization of lipids. As a result,

especially during the second and third trimesters, the

serum levels of apolipoproteins AI, AII, and B, triglyc-

erides, and total cholesterol (especially LDL-C) are

increased substantially.22,23,27 The levels of these lipids in

serum return to normal within 10 weeks after delivery

unless the mother breast-feeds her infant.28

Human chorionic gonadotropin elaborated by the

placenta has weak thyrotropic activity. Placental human

chorionic gonadotropin levels are at the highest concentra-

tion toward the end of the first trimester of pregnancy, thus

suppressing serum thyrotropin levels accompanied by a

slight increase in the free thyroxine level.21,29 During the

second and third trimesters of pregnancy, thyrotropinlevels are more reliable. Hence, trimester-based reference

intervals for thyrotropin are needed in pregnancy.

Associated with an increase in the glomerular filtration

rate in pregnancy is an increase in the clearance of iodide

and a transfer of iodide and iodothyronines to the fetus. As

a result, the inorganic iodide level in serum decreases to

such an extent that pregnant women with marginal iodine

intakes of less than 50 µg/d develop iodine deficiency and,

in turn, enlargement of the thyroid gland.21,29





The clearance by the liver during the first trimester of

pregnancy of the sialylated thyroxine-binding globulin,

which is induced by estrogens, is decreased. Hence, the level

of thyroxine-binding globulin increases, leading to the well-

known increases in serum thyroxine and triiodothyronine

levels during pregnancy.

Other analytes with increased levels during pregnancy

include the heat-stable alkaline phosphatase elaborated by

the placenta, alpha-fetoprotein, copper binding proteins such

as ceruloplasmin, and, in turn, copper, acute phase proteins,

and coagulation factor VII.2,8 The increased level of acute

phase proteins increases the erythrocyte sedimentation rate

approximately 5-fold.2 Plasma concentrations of the fibrinol-

ysis inhibitor, plasminogen activator inhibitor type 2, are

increased up to 2 µmol/L during the third trimester of preg-

nancy.30,31

An increased metabolic requirement during preg-nancy leads to a deficiency of iron, and the depletion of iron

stores is reflected by a decrease in ferritin.2

Lifestyle

Diet influences the results obtained for some analytes

❚Table 4❚. Differences between vegetarians and nonvegetarians

consuming a high-protein or a high-purine diet are well

known.2 Nonvegetarians tend to have increased blood levels of

uric acid, urea, and ammonia compared with those for their

vegetarian counterparts. Diets rich in saturated fatty acids in

general are lipogenic; the effect, however, varies depending on

the fatty acid. Thus, while stearic acid has little effect on LDL-

C levels, palmitic acid substantially increases plasma choles-

terol levels.23,27 Complex carbohydrates and monounsaturated

and polyunsaturated fatty acids, when substituted for saturated

fatty acids, tend, however, to lower LDL-C levels.23,27

A diet rich in fish oils lowers serum triglyceride and very-

low-density lipoprotein (VLDL) levels, presumably because of

the ability of fish oils to inhibit the synthesis of VLDL triglyc-

erides.23,32 Even among vegetarians, there is a difference in

serum lipid levels. Among strict vegetarians, plasma LDL-C

levels were reported to be 37% lower and HDL-C levels 12%

lower compared with a nonvegetarian control population. In

contrast, among lactovegetarians, LDL-C levels were 24%

higher, and HDL-C levels 7% higher compared with a groupof strict vegetarians.23,33

Consumption of caffeine influences the results of some

analytes (Table 4). Caffeine inhibits the enzyme phosphodi-

esterase, which normally regulates the levels of cellular

cyclic adenosine monophosphate (c-AMP) by converting it

to the inactive product 5¢-AMP.2,3 The increase in c-AMP

levels due to the caffeine-induced effect promotes glycol-

ysis coupled with energy production, and the subject feels

alert, awake, and energetic.


❚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.




Activation of triglyceride lipase by c-AMP results in a

3-fold increase in nonesterified or free fatty acid levels.2,3,24

The ability of free fatty acids to compete for binding sites

on the albumin molecule can lead to the displacement of a

drug or hormone bound to albumin, thus affecting the

measurement of free drug or hormone levels.8 The increase

in free fatty acid levels results in a decrease in pH, which, in

turn, can strip calcium from the binding protein. Conse-

quently, a spurious increase in ionic calcium concentration

can occur.8 Plasma renin activity and catecholamine levels

have been increased 3 hours after the consumption of 250

mg of caffeine.2,34

The effect of caffeine on lipid levels has been contro-

versial, and the results obtained seem to be influenced by

coffee-brewing methods.23 The finding in a double-blind

study replacing caffeine-containing coffee with decaf-feinated coffee for a period of 6 weeks of an insignificant

change in serum triglyceride, total cholesterol, and HDL-C

levels suggests that caffeine is not the substance in coffee

that elevates the total cholesterol level.35

Consumption of ethanol can induce short- and long-

term effects altering the results of several analytes (Table

4).2 Short-term effects reflected rapidly within the first 2 to

4 hours of consumption include a lowered blood glucose

level and an increased plasma lactate level.2 Uric acid levels

increase owing to the metabolism of ethanol to acetaldehyde

and further to acetate. The net effect of lactate formation is

to cause metabolic acidosis owing to the consumption of

bicarbonate.2

Sustained consumption of ethanol can, by enzyme

induction, lead to the increase in the activity of liver

enzymes, such as gamma-glutamyltransferase. There are,

however, marked intraindividual and interindividual varia-

tions in the level of serum gamma-glutamyltransferase, as

demonstrated in a study in which subjects were challenged

for 3 days with a 0.75-g of ethanol dose per kilogram of

body weight.36 Other liver enzymes that become elevated

owing to the direct cytotoxic effect of ethanol on liver are

aspartate aminotransferase (AST) and alanine aminotrans-

ferase (ALT).2 Thus, consumption of 225 g of ethanol per

day for 1 month caused skeletal muscle changes that led tothe release of liver cell enzymes into blood.37

Depending on the extent of consumption, the effect of

ethanol on serum lipid levels is variable.23 The effect of

short-term alcohol intake, especially by subjects who

usually do not consume alcohol, is to increase plasma

triglyceride levels, especially the VLDL fraction.38

A moderate intake of ethanol (<40 g/d) is reported to

increase the plasma levels of HDL-C, HDL3-C, and the

apolipoproteins AI and AII. However, if alcohol intake


❚Table 4❚Effect of Lifestyle on Laboratory Results

Lifestyle/Effects References

DietHigh-protein and high-purine diet increase levels of uric acid, urea, and ammonia in blood Guder et al2

compared with vegetariansDifferences 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

CaffeineInhibits 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

EthanolShort- 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 andStatland37; Taskinin et al38

SmokingLong-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 smokingACE 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.




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

together with the activation of the enzyme lipoprotein

lipase. Lipoprotein lipase hydrolyzes VLDL triglycerides,

with the result that plasma VLDL levels will be apparently

normal even when there is increased VLDL synthesis.23,27,38

Long-term smoking induces alteration in both biochem-

ical and cellular profiles (Table 4).3,8 Carboxyhemoglobin

levels in blood are higher in long-term smokers since ciga-rette smoke contains carbon monoxide, which has a much

greater affinity for hemoglobin than does oxygen. As a

consequence of long-term smoking, there is an increase in

hemoglobin levels and the RBC and WBC counts. The

RBCs become larger, thus increasing the mean corpuscular

volume (MCV). Striking differences in the WBC count have

been observed between healthy nonsmokers and healthy

long-term smokers. Healthy smokers who smoked 1 pack of

cigarettes per day for 1 year had a WBC count of approxi-

mately 1,000/µL (1 ·109 /L) higher than that for healthy

nonsmokers. Healthy smokers who smoked 2 packs of ciga-

rettes per day for 1 year had a WBC count of approximately

2,000/µL (2 · 10 9 /L ) hi gh er th an th at fo r heal thy

nonsmokers.24

Cadmium levels in blood are higher in healthy long-

term smokers compared with levels for healthy nonsmokers,

thus requiring separate reference intervals to distinguish the

2 populations.3,8

Smoking can reduce HDL-C levels in plasma; the

extent of reduction apparently is related to the number of

cigarettes smoked per day.23,27 Short-term changes occur-

ring within 1 hour of smoking 1 to 5 cigarettes include an

increase in the plasma levels of epinephrine, aldosterone,

cortisol, free fatty acids, and free glycerol.1,2

Angiotensin-converting enzyme (ACE) activity is

decreased in smokers, presumably owing to the destruction

of lung endothelial cells with the resultant decrease in the

release of ACE into the pulmonary circulation or, alterna-

tively, to inhibition of enzyme.2,39

The extent of smoking (moderate or heavy) parallels

the blood levels of thiocyanate and cotinine. Compared with

nicotine, which is the parent compound, cotinine has a

longer half-life, approximating 20 to 28 hours, making it an

ideal marker for the assessment of the extent of smoking.2

Specimen Collection Variables

The preparation of subjects for blood specimen collec-

tion, such as the duration of overnight fast, time of specimen

collection, and posture during blood sampling ❚Table 5❚; the

effects of the duration of tourniquet application, infusion,

and exercise (Table 5); the effects of anticoagulants ❚Table 6❚

and stabilizing additives ❚Table 7❚ used for blood collection;

the anticoagulant/blood ratio; specimen handling and

processing; and the relative merits of anticoagulants and

other variables need to be delineated and standardized to

minimize preanalytic error.

Duration of Fasting

Ideally, subjects should be instructed to fast overnight

for at least 12 hours before specimen collection (Table

5).2,23 The reason for the 12-hour stipulation is based on the

fact that an increase in the serum triglyceride level after afatty meal can persist for up to 9 hours, but there is little

effect on the levels of total cholesterol or apolipoproteins

AI and AII.40

The effect of prolonged fasting, however, can affect

some laboratory results. Thus, during a 48-hour fast, the

hepatic clearance of bilirubin could be approximately

240% more than the nominal value.3,24 With prolonged

fasting, there is a decrease in the level of specific proteins,

such as the C3

component of complement, prealbumin, and

albumin. Normal levels of these proteins are restored

rapidly with protein supplementation.24

The effect of fasting, however, varies depending on

body mass.23 In a lean person, the use of fat as reflected by

an increase in acetoacetic acid concentration in blood is

minimal during a 1-day fast but increases rapidly with

prolonged fasting.41 In an obese person, however, the

acetoacetic acid level increases markedly during a 1-day

fast. In contrast, while lipolysis, as reflected by a 3-fold

increase in the serum glycerol concentration with 3 days of

fasting is observed in a lean person, little change is noted in

an obese person.23,41

Time of Specimen Collection

The section “Time” referred to diurnal changes seen in

the circulating level of some analytes. It is, therefore,

important that the time of specimen collection be kept

constant from day to day to rule out variations introduced

by diurnal effects (Table 5).

Specimens for therapeutic drug monitoring (TDM)

should be obtained after a steady therapeutic concentration

of drug or steady state is achieved in the blood. A blood

sample obtained just before administering a drug dose after

a steady-state level is reached will reflect the trough level

or the lowest concentration to be obtained with the estab-

lished drug dose. If only 1 blood sample is to be collectedfor TDM, it is preferred that it reflect the trough level.42

Blood samples obtained when the drug concentration

is at its maximum, will, of course, reflect the peak level.

Peak-level specimens need to be obtained within 1 to 2

hours after intramuscular administration of the drug, 15 to

30 minutes after intravenous administration, or 1 to 5

hours after oral administration.42 Timing of specimen

collection for TDM, however, depends on the rate of distri-

bution of the specific drug. If the drug is infused intra-





venously, approximately 1 to 2 hours after the completion

of infusion are needed for the distribution phase to be

completed, with exceptions such as digoxin and digitoxin,

which need 6 to 8 hours for the completion of the distribu-tion phase.2

In general, since the rate of oral drug absorption differs

from person to person, blood specimens obtained for TDM

usually are timed to reflect trough levels. For the moni-

toring of certain drugs, such as theophylline, antibiotics,

and antiarrhythmic drugs, it may be necessary and useful to

measure peak levels after intravenous administration.2,43

The time for collection of urine specimens depends on

the constituent being measured. For clearance measure-

ments or for the measurement of analytes that have diurnal

variation, a 24-hour urine sample is needed.7 A timed

overnight urine sample or the so-called first-morning spec-

imen, provides not only an assessment of the concentratingability of the kidney, but also sufficient time for formed

elements or sediment to be produced for easy enumeration.

While a random or spot sample may be suitable for

routine screening, such a specimen may have its limitations

because it may be too diluted to detect accurately slight

increases in the concentration of analytes such as glucose,

protein, cells, and casts. Thus, a spot or random sample

may be of limited value in the initial screening for microal-

buminuria.44 However, in a strategy designed to detect and


❚Table 5❚Effects of Specimen Collection Variables on Laboratory Results

Variable/Effects References

Fasting12-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 dilutionPosture

In general, 5%-15% increase for most cellular and large molecules (eg, albumin) in sitting posture Guder et al2; Narayanan8; Statlandand Winkel24

Increase in upright to supine has dilutional effect: 10% and 12% decreases in cholesterol and Narayanan8,23; Young41

triglyceride levels, respectivelyChange 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 andWinkel24; Young41

Decreased pH; increased potassium, lactate, ionized calcium, and magnesium levels with Narayanan8

increased application timeRepeated fist clenching can increase potassium by 1-2 mmol/L, up to 2.7 mmol/L Don et al47; Skinner et al48

InfusionSpuriously 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

ExerciseStrenuous 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 CKIncreased epinephrine, norepinephrine, glucagon, cortisol, corticotropin, and growth hormone levels; Guder et al2

decreased insulin levelIncreased 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.




differentiate prerenal, glomerular, tubular, and postrenalcauses of proteinuria by measurement of specific proteins,

a morning spot urine specimen was adequate.45

Posture During Blood Sampling

Changes in posture during blood sampling can affect

the concentration of several analytes measured in serum or

plasma (Table 5). Change in posture from a supine to the

erect or sitting position can result in a shift in body water

from the intravascular to the interstitial compartment. As a

result, the concentration of large molecules that are notfilterable is increased. Hence, albumin levels generally are

higher among healthy subjects attending an outpatient clinic

from whom blood specimens are obtained with the subjects

in a sitting position compared with levels among healthy

hospitalized subjects from whom blood specimens usually

are obtained with the subjects in a supine position.8,24

Small molecules, such as glucose, however, owing to

their ability to move freely between the interstitial space

and the circulation, are least affected by posture during


❚Table 6❚Anticoagulants for Blood Collection

Anticoagulant References

HeparinUnfractionated 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 andor 3.8% (0.129 mol/L) Hamasaki31; Narayanan65;

Adcock et al66

❚Table 7❚Stabilizing Additives

Additive References

Glycolytic inhibitorsSodium 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 glucoseCell stabilizers

Acid-citrate dextrose A and B formulations; B formulation has greater amounts of dextrose available Guder et al2

to metabolizing cellsCitrate, 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 inhibitorsEDTA (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 stabilizersAn 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 heparinFor 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 urineFibrinolytic 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.




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

as large as insulin can diffuse freely into and out of the

interstitial space and, thus, are not subject to variation due

to postural changes.8

While the free fraction of a metabolite, drug, hormone,

or metal ion is not subject to postural variation, the fraction

bound to protein, such as albumin, is affected by posture.

Thus, bilirubin bound to albumin and calcium bound toalbumin are affected by postural changes.2,8

In blood specimens obtained with subjects in the sitting

position, generally an increase in the range of 5% to 15% is

seen for most cellular and macromolecular analytes

compared with results when blood specimens were obtained

with subjects in the supine position.2

The effect of postural change from supine to erect is

accentuated in patients with a tendency to edema, such as

patients with cardiovascular insufficiency and cirrhosis of the

liver.2 In such patients, reduction in plasma volume in turn

causes increased secretion of hormones, such as aldosterone,

norepinephrine, and epinephrine, and the enzyme, renin.2 An

increased level of atrial natriuretic peptide (ANP) is measur-

able in plasma as a compensatory mechanism to correct the

decrease in blood pressure.2,46 Indeed, changing from supine

to erect can dramatically influence the concentration of

analytes such as renin and aldosterone, even in healthy

persons. Conversely, moving from upright to supine can have

a dilutional effect owing to an increase in plasma volume

resulting from the transfer of fluid from the extravascular to

the intravascular compartment.8 Thus, a change from upright

supine can reduce (after 5 minutes in the supine position) the

cholesterol level by 10% and triglyceride levels by 12%.23,41

If blood specimens are to be obtained with the subject in

a sitting position, such as in an outpatient setting, the sitting

posture should be standardized by instructing the subject to

remain seated for 15 minutes before specimen collection.

Duration of Tourniquet Application

The application of the tourniquet, by reducing the pres-

sure below the systolic pressure, maintains the effective

filtration pressure within the capillaries.2 As a result, small

molecules and fluid are transferred from the intravasal space

to the interstitium. Application of the tourniquet for longer

than 1 minute and up to 3 minutes can result in hemoconcen-tration, causing an increase in the concentration of large

molecules that are unable to penetrate the capillary wall

(Table 5).2,8,24,41

The venous stasis that results from prolonged tourniquet

application promotes anaerobic glycolysis with the accumu-

lation of plasma lactate and a reduction in blood pH.8 The

hypoxic effect drives potassium out of the cell causing a

spurious increase in the serum potassium level. This effect

can be accentuated if the phlebotomist requests the subject to

repeatedly clench the fist to permit visualization of the vein.

The contraction of forearm muscles during repeated fist

clenching causes release of potassium, since there is a reduc-

tion in the intracellular electronegativity during the depolar-

ization of muscle cells, which favors the release of potassium

rather than its uptake.47 Repeated fist clenching during phle-

botomy after application of the tourniquet can cause a 1- to

2-mEq/L (1- to 2-mmol/L) increase in potassium. In 1 study,an increase in the potassium level as much as 2.7 mEq/L (2.7

mmol/L) was noted in a healthy subject due to fist clenching

during phlebotomy.48 Application of the tourniquet for more

than 1 minute and up to 2 minutes can cause a 5% increase

in the total cholesterol level, which can increase by 10% to

15% if the tourniquet is left on for 5 minutes.22,23,41

In 1 study conducted on children, venostasis of 2 minutes

substantially decreased the blood pyruvate concentration by

approximately 18% yet had a negligible effect on the lactate

concentration, with a mean increase of just 2.2%.49

The reduction in blood pH during prolonged tourniquet

application can alter drug-protein or hormone-protein

binding, leading to an increase in the free drug or free

hormone concentration. Thus, at a low pH caused by anaer-

obic glycolysis during venous stasis, ionic calcium and

ionic magnesium levels increase since they are released

from albumin, the binding protein.8

To minimize the preanalytic effects of tourniquet appli-

cation time, the tourniquet should be released as soon as

the needle enters the vein.8 Avoidance of excessive fist

clenching during phlebotomy and maintaining tourniquet

application time to no more than 1 minute can minimize

preanalytic error.2

Effect of Infusion

For persons receiving an infusion, blood should not

be obtained proximal to the infusion site.2 Blood should

be obtained from the opposite arm. Very often, exception-

ally high glucose values are obtained by collecting blood

from the arm in which an infusion of glucose is being

administered (Table 5). Eight hours must elapse before

blood is obtained from a subject who has received a fat

emulsion. The waiting period for blood sampling from

persons receiving a carbohydrate-rich solution or amino

acid and protein hydrolysate or electrolytes is 1 hour aftercessation of the infusion.2 For subjects receiving blood

transfusions, the extent of hemolysis and, with it,

increased values for potassium, lactate dehydrogenase,

and free hemoglobin are related progressively to the age

of the transfused blood.2

Effect of Exercise

Exercise, such as running up and down several flights

of stairs, or strenuous activity the night before specimen





collection, such as a workout in a gymnasium or marathon

running, can affect the results obtained for several analytes

(Table 5).

Strenuous exercise depletes the energy-yielding

compound, adenosine triphosphate (ATP), from the muscle

cells. As a result, there is a change in cell membrane

permeability, and enzymes are released from the cells.

Thus, CK levels can be very high after strenuous exercise(levels approaching 1,000 U/L) with a release of the CK-

MB fraction, although CK-MB as a percentage of total CK

would be normal.8

A high degree of individual variability is seen in a

hypoxia-mediated increase in CK, which depends on the

extent of exercise training.2 Mitochondrial size and number

are increased in persons who maintain a vigorous training

schedule, allowing their muscles to oxidatively metabolize

glucose, fatty acids, and ketone bodies. As a result, even in

the absence of myocardial damage, mitochondrial CK-MB

levels increase to greater than 8% of total CK activity.2

Training increases muscle mass, thereby increasing the

concentration of creatinine in plasma and the urinary excre-

tion of creatinine and creatine, while decreasing the level of

plasma lactate compared with untrained persons who

indulge randomly in exercise.

During strenuous exercise, there is some amount of

intravascular hemolysis. As a result, the level of plasma

haptoglobin, an acute phase reactant, is reduced owing to

its “complexation” with free hemoglobin released during

hemolysis and the subsequent clearance of the complex

from circulation.8,24

As a result of exercise, the concentrations of hormones

such as epinephrine, norepinephrine, glucagon, cortisol,

corticotropin, and growth hormone increase, while insulin

levels decrease, thereby causing an increase in glucose

levels because of the gluconeogenetic effect of hormones

antagonistic to insulin.2

Volume shift from the intravasal to the interstitial

compartment or volume depletion due to sweating during

exercise can increase serum albumin levels.2 Increased

lactate concentration due to anaerobic glycolysis during

exercise can decrease the urinary excretion of uric acid,

thereby increasing its serum concentration.2 There are,

however, beneficial effects of long-term strenuous exerciseor brisk walking in terms of reducing the levels of serum

LDL-C, apolipoprotein B, and triglycerides and increasing

the levels of apolipoprotein AI and HDL-C.23,27,50

To minimize preanalytic variables introduced by exer-

cise, subjects should be instructed to refrain from stren-

uous activity at least the night before testing and to not

exert themselves by walking a long distance or running or

climbing stairs before blood specimen collection.8

Anticoagulants for Blood Collection

Various salts of heparin, EDTA, and sodium citrate are

used widely in the clinical laboratory (Table 6).

Heparin

Heparin is the preferred anticoagulant for blood specimen

collection intended for determination of electrolyte levels and

other routine chemistry values. The standard commercial-grade heparin preparation, also called unfractionated heparin,

with a molecular mass ranging from 3 to 30 kd (mean, 14 kd)

exerts its anticoagulant effect by binding to antithrombin III.

This interaction causes a conformational change in the

antithrombin III molecule, thereby accelerating the inhibition

of coagulation factors Xa, IXa, and thrombin.31 The standard

commercial grade heparin also can inhibit thrombin with the

aid of a plasma cofactor, called heparin cofactor II. In vivo,

heparin can cause the release of a potent coagulation inhibitor,

called tissue factor pathway inhibitor, from the endothelial

surface.31,51

Various salts of heparin, such as lithium, sodium, and

ammonium, have been used as anticoagulants for the collec-

tion of blood specimens intended for routine clinical chemistry

determinations.52 The nominal amount of heparin used in

blood specimen collection tubes is 14.3 U/mL of blood,

although a range of 12 to 30 U/mL of blood has been deemed

satisfactory.53

Although all of the 3 salts of heparin give comparable

results for electrolyte determinations, lithium heparin has been

favored over the others, more because of the perception that

sodium heparin might overestimate sodium levels and ammo-

nium heparin might spuriously increase serum urea nitrogen

concentrations when measured by the urease procedure.52,54

Just how much sodium is present in a sodium heparin

blood collection tube depends not only on the nominal

amounts of heparin present (14.3 U/mL of blood), but also on

other variables, such as the molecular weight of unfractionated

heparin, the number of dissolvable milliequivalents of sodium

per milligram of heparin, and the consistency of the heparin

preparation from lot to lot. Indeed, in 1 study when blood was

filled to half of the tube’s nominal volume, thereby increasing

2-fold the nominal amounts of sodium heparin present in the

blood collection tube, plasma sodium values were not

increased artifactually.54

Obvious differences in results for certain analytes

between serum and heparinized plasma are related to the

consumption of fibrinogen and the lysis of cellular elements

during the process of clotting. Thus, potassium values are

higher in serum than in heparinized plasma, while in contrast,

total protein values are higher in plasma than in serum. The

effect of heparinized plasma on some of the enzyme measure-

ments may be related, in part, to the bias introduced by the

instrument-reagent combination.55,56





Since the commonly used anticoagulant, such as

lithium or sodium heparin, in the syringe to obtain speci-

mens for blood gas measurement binds to ionic calcium,

with the effect dependent on the concentration of heparin,

attempts have been made to eliminate this bias. One

approach has been to use calcium-titrated heparin, in which

heparin in an amount less than 50 U/mL of blood is titrated

to an ionized calcium concentration of 1.25 mmol/L.52 Theuse of electrolyte-balanced heparin that involves the use of

dry heparin in the amount of 40 U/mL of blood balanced

with calcium, sodium, and potassium decreases bias to less

than 0.02 mmol/L of calcium in the analytic range of 0.9 to

1.8 mmol/L.57 The use of dry heparin eliminates the dilu-

tion effect associated with liquid heparin.

Other approaches to minimize interference with

ionized calcium measurements have involved saturating the

high-affinity binding sites on heparin to which divalent

cations, such as zinc, can bind. Zinc heparin, however, in

the presence of excess zinc ions is reported to cause a posi-

tive interference in ionized calcium and total magnesium

measurements.58 One successful approach is to saturate

high- and low-affinity binding sites on heparin, the former

with zinc and the latter with lithium.59 This calcium-

neutralized lithium-zinc heparin has been satisfactory not

only for ionized calcium measurements, but also for

measurement of blood gases, total calcium, sodium, and

potassium.59

For TDM, the drug level in heparinized plasma would

be expected to be higher than that in serum, especially if

the drug was bound to fibrinogen. In reality, the serum

value for drugs such as amiodarone, desethylamiodarone,

chloroquine, and desethylchloroquine was higher than the

plasma value, apparently due to the release of cell-bound

drug during the clotting process.52,60,61 Thus, results

obtained for amiodarone were 11.5% to 13.5% lower in

heparinized plasma than in serum, while results for

desethylamiodarone were 4.4% to 8.5% lower in plasma

than in serum.60

The concentration of chloroquine was approximately

2-fold higher in serum than in plasma, while the concentra-

tion of its metabolite, desethylchloroquine, was approxi-

mately 4 times higher in serum than in plasma. These 2

drugs were present mainly in platelets and granulocytes.The increased levels of these 2 drugs noted in serum appar-

ently were related to the release of these drugs from

platelets during the coagulation process. Thus, for some

drugs, it is important to specify whether plasma or serum

was used for measurement.61

EDTA

EDTA is the commonly used anticoagulant for routine

hematologic determinations. It functions as an anticoagu-

lant by chelating calcium ions, which are required for the

clotting process.

Potassium and sodium salts of EDTA have been used

for blood specimen collection. The solubility of potassium

salts is better than sodium salts of EDTA. The pH of EDTA

varies depending on the salt. As more ions are added to the

free acid, the acidity of EDTA decreases. Thus, while a satu-

rated solution of EDTA as free acid has a pH of 2.5 – 1.0, thetripotassium salt (K

3EDTA) as 1% solution has a pH of 7.5 –

1.0.52 The disodium (Na2EDTA) and dipotassium (K

2EDTA)

salts of EDTA in a 1% solution have a lower pH. Na2EDTA

has a pH of 5.0 – 1.0, while K2EDTA has a pH of 4.8 – 1.0.

This difference in pH affects the size of the RBC. Although

all salts of EDTA are hyperosmolar, causing the water to

leave the cells and the cells to shrink, this shrinking effect is

noticed only with K3EDTA and not with K

2EDTA or

Na2EDTA.52 This is because although the cells shrink with

K2EDTA and Na

2EDTA, the lower pH of the anticoagulant

causes the cells to swell as well, thus balancing for the

osmotically dependent shrinkage, and the cell size is not

altered. As a result, while the microhematocrit (centrifuged

hematocrit) is lowered with K3EDTA, it is not affected by

K2EDTA or Na

2EDTA. The effect of dilution of the sample

in automated analysis restores cells to their native shape. At

an optimum EDTA concentration of 1.5 mg/mL of blood, no

differences in microhematocrit were noted between samples

collected in K2EDTA and K

3EDTA and analyzed between 1

and 4 hours after collection.62

In the same study, the MCV measurement and the calcu-

lated hematocrit values were influenced not only by the instru-

ment used, but also by the salt of EDTA (K2

or K3

) used for

anticoagulation of the blood specimen. Thus, MCV values

were not affected with blood anticoagulated with K3EDTA,

even at concentrations up to 10 times the nominal value. In

contrast, with 3 of the 4 instruments tested, increasing the

concentration of K2EDTA beyond the nominal value of 1.5

mg/mL of blood resulted in an increase in MCV.62 One expla-

nation may lie in the ready dissolution of liquid K3EDTA

compared with powdered K2EDTA, in which the effectiveness

of mixing might become a variable, perhaps accounting for the

discrepancy in results.

The neutrophils are subjected to morphologic changes

upon collection of blood in EDTA. These changes depend onthe time the peripheral blood smear is prepared and the

concentration of EDTA used for blood specimen collection.

Minor changes begin to appear in the morphologic features

of neutrophils even at an optimal EDTA concentration of 1.5

mg/mL of blood. These changes include swelling of the

neutrophils and loss of structure in the neutrophil lobes.

Thereafter, there is a loss of granulation in the cytoplasm and

the appearance of vacuoles in the nucleus or cytoplasm of

the cells.63 Between 1 and 3 hours after blood collection in





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

crossover of the nuclear chromatin. Beyond 3 hours, the

morphologic features of the neutrophils become unclear as

the cell disintegrates. If a peripheral blood smear is made

from blood anticoagulated with an EDTA concentration of

2.5 mg/mL of blood, approximating to a conventional blood

collection tube filled to one half of its nominal volume, the

morphologic features of the neutrophils are disintegratedtotally.

Similar time-related and EDTA concentration–related

changes also occur in the mononuclear cells, although to a

lesser extent than in the neutrophils. Platelets also undergo

changes, including swelling, giving rise to giant platelets

that ultimately disintegrate, producing fragments in the

same size range as the platelets that are mistakenly counted

as platelets by electronic counters, thus spuriously

increasing the platelet count.

Platelets rapidly change their shape from discoid to spher-

ical when blood specimens are collected in EDTA, making

determinations of mean platelet volume unreliable. The

limited stability of the WBC differential count (up to 6 hours

at room temperature storage) achieved in most of the 5-part

differential analyzers is another drawback with EDTA.64

Citrate

Traditionally, a 0.129-mol/L concentration or a 0.109-

mol/L concentration of trisodium citrate has been used as the

anticoagulant for collection of blood specimens intended for

global coagulation tests, such as prothrombin time (PT) and

activated partial thromboplastin time (aPTT).2,31 Citrate has

virtually replaced sodium oxalate as an anticoagulant for

performing global coagulation tests, since factor V is more

stable in citrate than oxalate. Furthermore, citrate rapidly

complexes with calcium, forming a soluble complex in

contrast with the slow formation of the insoluble complex of

calcium with oxalate.31

The effective molarity of citrate depends on whether the

dihydrate or the anhydrous salt has been chosen in the prepa-

ration of the citrate solution. Nominally, a 3.2% solution of

the dihydrate trisodium citrate salt corresponds to a 0.109-

mol/L concentration, whereas a 3.8% solution of the same

dihydrate salt corresponds to a 0.129-mol/L concentration.

However, a 3.2% solution of the anhydrous trisodium citratesolution would correspond to a 0.124-mol/L concentration,

whereas a 3.8% anhydrous sodium citrate solution would

have a molarity of 0.147 mol/L.65 Hence, it is important that

the dihydrate salt be used to prepare either the 3.2% or 3.8%

solution of trisodium citrate.

A laboratory that has been using one of the concentra-

tions (3.2% or 3.8%) to perform PT determinations for

patients receiving oral anticoagulant therapy should not

interchange the formulations. Doing so will affect the inter-

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

the results of the PT.66 INR values generally are higher

with a 0.129-mol/L concentration of sodium citrate than

with a 0.109-mol/L concentration of sodium citrate, espe-

cially when a responsive PT reagent, such as a recombinant

thromboplastin that has a sensitivity similar to the World

Health Organization thromboplastin (with an international

sensitivity index [ISI] equal to 1), is used.66 The differencesin INR between the 2 concentrations of citrate can vary

from 0.7 to 2.7 INR units.66

The INR will be in error when a laboratory that

routinely uses, for example, a 0.109-mol/L concentration of

citrate, occasionally analyzes a specimen collected with a

0.129-mol/L concentration of citrate, but uses the mean of

the normal range obtained with the 0.109-mol/L concentra-

tion to calculate the PT ratio (the patients PT divided by

mean of normal range for PT), which together with the ISI

are needed for the calculation of INR.66

Stabilizing Additives

Sodium fluoride and lithium iodoacetate have been used

alone or in combination with an anticoagulant such as potas-

sium oxalate, EDTA, citrate, or lithium heparin for blood

collection (Table 7).2 The concentration of sodium fluoride

when used alone to inhibit glycolysis is approximately 4.3

mg/mL of blood. In combination with an anticoagulant, such

as Na2EDTA or K

2EDTA (1 mg/mL of blood) or potassium

oxalate (2 mg/mL of blood), the nominal concentration of

fluoride that is used to inhibit glycolysis is 2.5 mg/mL of

blood (60 mmol/L).2,67

Similarly, lithium iodoacetate has been used alone (0.5

mg/mL of blood) or at that concentration in combination

with lithium heparin (14.3 U/mL of blood).2 Even with the

use of either of these glycolytic inhibitors, at least 3 hours

are needed for these inhibitors to become fully effective

and stabilize glucose levels.2,67 During this 3-hour period in

healthy subjects, an approximately 9 mg/dL loss of glucose

occurs.2,67,68 However, in neonates or subjects with an

increased RBC, WBC, or platelet count, the decrease in

glucose during the 3-hour period, even in presence of

glycolytic inhibitors, is greater than in healthy subjects.67,69

Once the glycolytic inhibitors become fully effective after

the initial 3-hour period, glucose levels remain stable for atleast 3 days.2,67,68

In the absence of glycolytic inhibitors, a decrease in the

glucose level of as much as 24% can occur in 1 hour after

blood collection in neonates in contrast with a 5% decrease

in healthy adults when specimens are stored at room

temperature. By 5 hours after blood collection in specimens

stored at room temperature, as much as 68% of glucose can

be consumed in specimens collected without glycolytic

inhibitors from neonates with a high hematocrit.69





The reason that fluoride and iodoacetate take approxi-

mately 3 hours to stabilize glucose levels is that they do not

act on the first enzyme in the glycolytic pathway, which is

hexokinase. Instead, fluoride acts on enolase, which is the

eighth enzyme in the glycolytic pathway, while iodoacetate

acts on glyceraldehyde-3-phosphate dehydrogenase, which

is the fifth enzyme in the pathway in which glucose is

metabolized.2Potassium oxalate, which is used in combination with

fluoride, inhibits pyruvate kinase in the enzymatic step

immediately after enolase in the glycolytic pathway and,

thus, can effectively retard lactate formation 1 hour after

blood collection.

The drawback with both fluoride and iodoacetate is

that they promote hemolysis, which increases progressively

as the concentration of the inhibitor used and the time

elapsed after blood specimen collection increases. Appar-

ently both inhibitors deplete the organic phosphate (ATP)

content of the RBC, thus causing an efflux of potassium out

of the cell.

A variety of approaches have been attempted to

improve the efficacy of glycolytic inhibition. In one

approach, a mixture of citric acid, trisodium citrate,

Na2EDTA, and a very small concentration of sodium fluo-

ride (0.2 mg) effectively inhibits the enzymes in the

glycolytic pathway by maintaining blood pH in the range

of 5.3 to 5.9.70 With this additive mixture, glycolysis was

inhibited for 8 hours in specimens stored at room tempera-

ture, while at 24 hours, a mean – SD decrease of 1.3 mg –

1.1 mg/dL (0.07 – 0.06 mmol/L) of glucose was noted.70

Another strategy for achieving efficient glycolytic inhi-

bition is to include mannose in addition to fluoride.71 The

rationale is that mannose, which is an epimer of glucose

(differs structurally from glucose in the configuration around

just 1 carbon atom), will compete with glucose for hexoki-

nase, the first enzyme in the glycolytic pathway. Since

mannose is a competitive inhibitor, the inhibition is short-

lived, lasting just 4 hours. However, by this time, fluoride,

which is also in the additive mixture, would have become

effective thus achieving a better glucose stabilization.2,71

At a mannose concentration of 3 mg/mL of blood, no

interference was noted in hexokinase and glucose oxidase

procedures for the measurement of glucose.72

However,when the mannose concentration was increased to 3 times

the nominal concentration of 3 mg/mL of blood, an overes-

timation of glucose by 18 mg/dL (1.0 mmol/L) was

observed.72 Some of the mannose preparations also may be

contaminated with glucose. Negative interference has been

reported in the hexokinase procedure when mannose

concentrations exceed 3 times the nominal concentration.73

The extent of reduction in glucose levels depends on the

concentration of hexokinase used in the glucose assay, with

less interference noted at higher concentrations of hexoki-

nase used in the assay.73

In practical terms, if plasma is separated from cells by

centrifugation promptly after blood specimen collection, the

glucose level in plasma is expected to be stable, since the

enzymes that metabolize glucose are in the cellular fraction.

Stabilization of RBCs by including a nutrient such as

glucose together with an anticoagulant mixture is the basisof 2 acid citrate dextrose (ACD) formulations.2 These

formulations, labeled ACD A and B, are similar in pH;

ACD A has a pH of 5.05, and ACD B has a pH of 5.1. The

difference between the formulations is the additive/blood

ratio; the A formulation has a ratio of 1:5.67. The B formu-

lation, however, with a ratio of 1:3, has relatively greater

amounts of dextrose available for the metabolizing RBCs

and, hence, can preserve them better. While ACD formula-

tions can preserve RBCs for 21 days when blood is stored

between 1 C and 6 C, their stability can be extended

further by including additives such as phosphate and

adenine (citrate phosphate dextrose and citrate phosphate

dextrose adenine, respectively).

The maintenance of increased levels of cyclic adenosine

monophosphate (c-AMP) within the platelets is the basis of

an additive mixture intended for blood specimen collection

to minimize in vitro platelet activation.74,75 The additive is a

mixture of citric acid, theophylline, adenosine, and dipyri-

damole with the final pH adjusted to 5.0.74 Adenosine acti-

vates the enzyme adenylate cyclase, thus increasing c-AMP

levels within the platelets. Theophylline and, to a certain

extent, dipyridamole prevent the degradation of c-AMP by

inhibiting the enzyme phosphodiesterase. Dipyridamole, in

addition, prevents the uptake of adenosine by the RBCs, thus

effectively increasing the amount of adenosine available to

the platelets to activate adenylate cyclase and, in turn, the

inhibition of platelet aggregation and release of contents of

platelet alpha granules.31,74 The citrate in the additive

mixture functions as an anticoagulant by chelating calcium.

The citric acid, theophylline, adenosine, and dipyridamole

mixture is used for monitoring heparin therapy by aPTT or

the chromogenic substrate assay and in measurement of

platelet activation markers, such as P-selectin (CD62), by

flow cytometry.76,77

Stabilizing additives to inhibit proteolytic enzymes areneeded for some analytes. Although EDTA itself is a metal-

loprotease inhibitor, sometimes EDTA alone is insufficient to

inhibit proteolytic enzymes. For example, when a synthetic

protease inhibitor, such as nafamostat mesylate, is added to

EDTA, the mixture can substantially improve the stability of

complement components C3a, C4a, and C5a.52,78

A proteinase inhibitor, aprotinin (Trasylol), when used

in combination with an anticoagulant such as EDTA or

heparin, has been useful for the stabilization of labile





polypeptide hormones and enzymes.52 Aprotinin inhibits

specific proteases of kinin and coagulation and fibrinolytic

enzyme systems, in addition to inhibiting the enzyme

systems of leukocytes and damaged tissue cells. Thus, it

inhibits kallikrein, trypsin, chymotrypsin, coagulation factors

in the preliminary phase of blood clotting, the fibrinolytic

enzyme plasmin, and lysosomal proteinases. The fact that

aprotinin inhibits kallikrein is the basis for the expression of its potency in terms of kallikrein inhibitory units (KIUs).

Aprotinin has been used in combination with an anticoagu-

lant such as EDTA or heparin. A mixture of EDTA (1.5

mg/mL of blood) and aprotinin in the activity range of 500 to

2,000 KIU/mL has been used to stabilize polypeptide

hormones such as glucagon and corticotropin, the enzyme

renin, and gastrointestinal hormones, such as beta-endorphin,

secretin, neurotensin, gut glucagon, somatostatin, vasoactive

intestinal peptide, and peptide yy.52,79

In the absence of aprotinin, spuriously higher values

may be obtained apparently because of the fragments of the

analyte produced by proteolytic enzyme degradation are

measured as intact molecules by the polyclonal antibody

used in the assay. Thus, in a radioimmunoassay for glucagon,

a 26% spurious increase was noted in plasma obtained with

blood specimens collected with EDTA alone compared with

plasma obtained with blood specimens collected in a mixture

of EDTA and aprotinin.52,79

A mixture of lithium heparin (14.3 U/mL) and 1,000

KIU/mL of aprotinin also has been used to stabilize

immunoreactive somatostatin, secretin, cholecystokinin

(pancreozymin), glucagon, and C-peptide.52

In addition to aprotinin and an anticoagulant such as

EDTA, additional proteolytic enzyme inhibitors may be

needed to stabilize a labile tumor marker, such as parathy-

roid hormone related protein (PTH-rP) that has been associ-

ated with the humoral hypercalcemia of malignant

neoplasm.80,81 Thus, with an additive mixture of EDTA (1.5

mg/mL), aprotinin (500 KIU/mL), leupeptin (2.5 mg), and

pepstatin (2.5 mg), PTH-rP levels were stable for 1 hour

after blood specimen collection for specimens stored at

room temperature and for 24 hours, if specimens were

refrigerated at 4 C.80 Even when blood is collected with this

additive mixture, if the sample is stored at room temperature

for 24 hours, as much as 60% of the PTH-rP activity can belost.80 Without a stabilizing additive mixture, PTH-rP is so

unstable that as much as 60% of its activity can be lost

within 1 hour of specimen collection and storage at room

temperature.

Analytes that are susceptible to oxidative damage, such

as catecholamines, require stabilization. Typically together

with an anticoagulant such as heparin, EDTA, or egtazic

acid, an antioxidant such as glutathione, sodium metabisul-

fite, or ascorbic acid at a concentration of 1.5 mg/mL should

be included in the blood specimen collection additive

mixture.81 The specimen collected with this additive

mixture should be centrifuged within 1 hour of collection. It

is preferable, however, if possible to separate plasma within

15 minutes of specimen collection and store it in plastic

tubes or vials at –20 C in case analysis is delayed. Prompt

processing of plasma obtained with an antioxidant-anticoag-

ulant mixture stabilizes catecholamines in plasma for 1 dayat room temperature, for 2 days when stored at 4 C to 8 C,

and for 1 month at –20 C. Stability at –20 C can be

extended to 6 months with added glutathione.2,82

Apparently, at the higher concentrations of cate-

cholamines present in urine compared with plasma, the

hormones and their metabolites are stable for 4 days, even

when stored at 20 C to 25 C without preservatives.2,82

However, for long-term stabilization of catecholamines in

urine specimens, collection is recommended in a container

with 5 mL of a 6-mol/L concentration of hydrochloric acid

per liter of urine or in a mixture of 250 mg each of EDTA

and sodium metabisulfite per liter of urine, which will extend

stability to 3 weeks at 20 C to 25 C, and 4 months to 1 year

at 4 C to 8 C.2,82

The laboratory assessment of fibrinolysis by the

measurement of the nonspecific fibrinogen-fibrin degrada-

tion products requires inhibition of the in vitro generation of

the fibrinolytic enzyme plasmin with an inhibitor, such as

soybean trypsin or aprotinin, and the conversion of residual

fibrinogen to fibrin with thrombin.31 However, in patients

receiving heparin therapy, the conversion of residual

fibrinogen to fibrin, even in presence of thrombin, will be

slow, thus resulting in spuriously high fibrinogen degradation

product values owing to the remaining unconverted

fibrinogen. In such cases, the addition of snake venom, also

known as reptilase, instead of thrombin to the additive

mixture, which also contains a plasmin inhibitor such as

aprotinin, can result in the conversion of residual fibrinogen

to fibrin, even in the presence of heparin.83

For monitoring fibrinolytic therapy, the additive used to

inhibit plasminogen activation depends on the therapeutic

agent administered. Thus, while aprotinin together with

EDTA is effective for inhibiting plasminogen activation by

the therapeutic agent urokinase, it is ineffective for inhibiting

the recombinant tissue plasminogen activator (rt-PA) fromactivating plasminogen.75 Specific synthetic peptides of argi-

nine chloromethyl ketone (PPACK) are needed to inhibit in

vitro rt-PA activity. PPACK irreversibly inactivates rt-PA by

alkylating the active center amino acid histidine, in addition

to functioning as a potent thrombin inhibitor.84 A 5-mmol/L

concentration of PPACK combined with a 10-mmol/L

concentration of citrate or a 4.2-mmol/L concentration of

EDTA effectively inhibited rt-PA upon blood collection.75

However, since PPACK is unstable at neutral pH, blood upon





collection should be maintained at 4 C, centrifuged promptly

to obtain plasma, and processed without delay or kept frozen

until analysis can be performed.31

As new diagnostic markers are proposed, the labile

nature of some poses challenges. A case in point is the

measurement of ANP, which is a marker of chronic conges-

tive heart failure. An additive mixture of EDTA and apro-

tinin is needed to stabilize ANP; the blood specimencollected with this mixture must be centrifuged immedi-

ately at 4 C and plasma maintained frozen at –20 C or

below until ready for analysis.85 Furthermore, hemolysis

invalidates ANP measurements. Alternatively, the measure-

ment of the N -terminal fragment of the prohormone pro-

atrial natriuretic peptide (proANP) (which is formed when

proANP is split into equimolar amounts of ANP and the N -

terminal fragment) can be performed in blood specimens

collected in EDTA alone with storage at room temperature

for up to 6 hours. At 4 C, N -terminal proANP is stable for

3 days and at –20 C for 4 weeks, with hemolysis not

markedly affecting the results.86

Anticoagulant/Blood Ratio

The anticoagulant/blood ratio is critical for some labora-

tory tests. Obviously, if too little blood is obtained, osmotic

effects can be expected to result in cell shrinkage and, thus, to

introduce dilutional changes in the concentration of extracel-

lular analytes.

A well-known example of the critical effect of the antico-

agulant/blood ratio is for the global coagulation test, aPTT.

Traditionally, a 1:9 ratio of anticoagulant (sodium citrate, a

0.109-mol/L concentration or a 0.129-mol/L concentration) to

blood is used. However, if less blood is collected, simulating a

1:7 anticoagulant/blood ratio, there is a significant increase in

the aPTT result compared with that obtained using the

nominal 1:9 ratio.31 In 1 study, a 2.4-second increase was

observed at a 1:7 ratio compared with the aPTT result

obtained at the nominal 1:9 ratio.65

However, for the global coagulation test, PT, the effect of

the anticoagulant/blood ratio becomes meaningful only when

the ratio reaches 1:4.5, which would occur when the blood

collection tube is filled to just less than half of its nominal

volume.65 The anticoagulant/blood ratio also is influenced by

the sensitivity of the thromboplastin reagent that is used. In astudy of the effect of the anticoagulant/blood ratio using 3.2%

(0.109-mol/L concentration) buffered citrate, the results for the

PT were valid even for specimen collection tubes that were

filled to 65% of the nominal anticoagulant/blood ratio (1:9).

However, filling the tube to at least 80% of the nominal ratio

was needed to obtain reliable results in the therapeutic range

using a moderately sensitive thromboplastin reagent with an

ISI of 2.06. Using a highly sensitive thromboplastin reagent

with an ISI of 1.01 required maintaining the

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

ratio.87 In the aforementioned study,87 the need to fill blood

collection tubes to the nominal capacity (1:9) was reinforced

for the aPTT, since tubes filled to less than 90% of the nominal

capacity caused prolongation of the aPTT result.

In addition to maintaining the nominal anti-

coagulant/blood ratio (1:9), the amount of head space above

the blood also has been reported to be a variable affecting theaPTT result. Thus, collecting less blood (2.7 mL as opposed to

the nominal 4.5 mL) but maintaining the anticoagulant/blood

ratio without decreasing the tube size effectively increases the

head space above the blood. This, in turn, generally leads to a

shortened aPTT in values outside the upper limit of the refer-

ence interval and in the therapeutic range for monitoring

unfractionated heparin therapy.88 Thus, in the abnormal range,

the mean value obtained with the partially filled tube was 11

seconds shorter than the mean value obtained with the full

tube.88 One possible explanation for the head space effect is

the increase in surface area relative to the volume of blood

collected, favoring platelet activation and the subsequent

release of platelet factor 4, which in turn neutralizes some of

the heparin, resulting in a shortened aPTT.88

For polycythemic patients with hematocrit values above

60% (0.60), the PT and the aPTT can be prolonged substan-

tially, even when the nominal 1:9 anticoagulant/blood ratio is

maintained.89 This is because the plasma is overcitrated to the

extent that it complexes much of the calcium chloride added to

perform the PT and aPTT, effectively reducing the calcium

ions needed to clot the plasma and thereby prolonging the

clotting time. This effect can be minimized by adjusting the

citrate concentration in accordance with the hematocrit value

by using empiric formulas or by using a 1:19

anticoagulant/blood ratio.65,89 The latter ratio dilutes the citrate

concentration to a point that results of PT and aPTT are not

affected across a spectrum of hematocrit values ranging from

anemia to polycythemia.89

In general, collecting blood specimens to less than the

nominal volume increases the effective molarity of the antico-

agulant and induces osmotic changes affecting cell morpho-

logic features as noted for the morphologic features of

neutrophils when the EDTA concentration changes from the

nominal 1.5 mg/mL of blood to 2.5 mg/mL of blood. Further-

more, the binding of analytes, such as ionic calcium or ionicmagnesium, to heparin can be enhanced when the effective

concentration of unfractionated heparin increases beyond the

nominal 14.3 U/mL of blood.52

Maintenance of the anticoagulant/blood ratio also is

critical when the inhibitory effects of the anticoagulant are

concentration-dependent. Thus, a 10% reduction in the

gentamicin level was noted in an enzyme-multiplied

immunoassay technique when the concentration of heparin

used for specimen collection increased to 25 U/mL of





blood, presumably because of the inhibition of the enzymes

used in the immunoassay procedure, as the concentration of

heparin increased from the nominal value.90

At the other extreme is exceeding the anticoagulant/blood

ratio to a point that the tube is overfilled with blood to such an

extent that proper mixing of anticoagulant with blood becomes

problematic. Thus, in 1 study, the hemoglobin values had

doubled compared with a value obtained a week before, andthe WBC and platelet counts were reduced drastically. Reana-

lyzing the specimen for the fourth time from the same tube

yielded values similar to values obtained initially the week

before. Apparently, proper mixing of the specimen on a

rocking mixer was not achieved since there was no head space

or bubble left in the tube to assist in the mixing of the spec-

imen. After a sufficient amount of blood was withdrawn by

repeating the analysis 4 times, there was sufficient space in the

tube for the air bubble to move and effect thorough mixing on

the rocking-type mixing device.91

For bacteriologic determinations, inhibition of the normal

bactericidal properties of blood requires appropriate dilution

of blood in broth. Incorporation of a 0.025% solution of

sodium polyanethole sulfonate in blood culture media is

intended to inhibit phagocytosis, complement, and lysozyme.

Even when blood culture is supplemented with sodium

polyanethole sulfonate, at least a 1:10 blood/broth ratio is

considered optimal.92 However, a blood/broth ratio less than

1:10 has been acceptable when at least 2 separate blood

cultures are obtained routinely or when a biphasic medium is

used to culture blood (a broth bottle to which an agar slant

with media is attached).93

Since spurious blood culture results can be obtained

for patients receiving antibiotics, a 1:10 dilution of blood in

broth dilutes many of the antibiotics in the blood to sub-

inhibitory levels. The incorporation of sodium polyanethole

sulfonate inactivates aminoglycosides. Alternatively,

however, devices containing antibiotic-adsorbent resins can

be used to neutralize antibiotics, and cell lysis followed by

filtration or centrifugation neutralizes the antimicrobial

properties or components of blood and provides a concen-

trate for culture on antibiotic-free media.93

Specimen Handling and Processing

The time and temperature for storage of the specimenand the processing steps in the preparation of serum or

plasma or cell separation using density-gradient techniques

can introduce a preanalytic variable. Analytes subject to

cellular metabolism need prompt separation from cells.

Storage of anticoagulated whole blood or clotted blood

in a refrigerator (4 C) inhibits the Na+,K+-ATPase pump,

resulting in potassium leaking out of the cells and causing a

spurious increase in levels measured in serum or plasma.

This effect is seen even at 4 hours of storage of clotted blood

at 4 C.2,8,94 Inhibition of the Na+,K+-ATPase pump also

drives sodium into the cell. Since sodium is mainly extracel-

lular, in contrast with potassium that is mainly intracellular,

the decrease in sodium becomes noticeable only when whole

or clotted blood is stored beyond 24 hours at 4 C. Ideally,

when separation of plasma or serum from cells is needed,

blood should be processed within 1 hour of collection to

obtain plasma or serum.2Storage of clotted blood beyond 8 hours at room

temperature (23 C) can cause an increase in inorganic phos-

phate owing to the hydrolysis of cellular organic phosphate

by the enzyme alkaline phosphatase; the effect is more

pronounced when clotted blood is stored at 30 C.2 Thus,

while a 10% increase was noted in the inorganic phosphate

value at 24 hours compared with the 8-hour value during

storage at 23 C, the increase was 120% at a storage tempera-

ture of 30 C.2

A time-dependent increase in ammonia is accentuated

in specimens with increased gamma-glutamyltransferase

activity.95 The time between collection of blood and its

storage temperature before processing to yield plasma can

introduce a variable. Thus, in the measurement of cate-

cholamine levels in specimens collected with a stabilizing

additive mixture and stored at 4 C, there is an increase in

the level of catecholamines owing to a delay in the

processing of plasma that exceeds 1 hour.81,82 A delay in

the preparation of plasma for 2 hours after blood collection

and storage at 4 C can increase the mean plasma norepi-

nephrine level by 13% (N = 8).82 This effect is due to the

lysis of some of the RBCs at 4 C, leading to a release of

catecholamines into plasma and to the slow reuptake of

these hormones by cells at 4 C. In addition, cryoprecipita-

tion of plasma proteins at 4 C also can contribute to the

increased level of catecholamines measured in plasma.81,82

In contrast, storage of specimens at 20 C and a delay

beyond 1 hour in the processing of plasma after specimen

collection can lower the catecholamine level measured in

plasma owing to the uptake of catecholamines by cells such

as RBCs or platelets.82 Thus, the mean plasma norepineph-

rine levels decreased by 14% (N = 8) from the initial value

if blood specimens kept at 20 C were centrifuged 2 hours

after collection, while mean plasma epinephrine values

decreased by 20%.82

The storage temperature of a specimen influences the

results of global coagulation tests such as PT and aPTT.

Since factor VII, which is measured by PT, is activated

during prolonged storage at 4 C, storage of plasma in

contact with cells beyond 7 hours shortens the PT.65,96,97

Indeed, when a blood collection tube is kept well stop-

pered and stored at room temperature (25 C), the PT is

stable for up to 48 hours.89,97 As is well known, the aPTT

should be performed preferably within 2 to 4 hours of





specimen collection, and some investigators maintain

plasma on ice (4 C).2 In 1 study, a 10% to 15% increase in

aPTT was observed after 24 hours in specimens that were

maintained well stoppered and at room temperature.89

In contrast with refrigerated temperatures, freezing

plasma at –20 C and –70 C did not activate factor VII. The

PT and aPTT results were stable in plasma maintained

frozen at –20 C for 10 days and at –70 C for 21 days.97

Factors II, V, VII, and X are stable in plasma maintained

under refrigeration for up to 6 hours. All except factor V had

stability for up to 14 days when frozen at –20 C or –70 C.97

During a 7-day period, 20% of factor V activity was lost in

plasma stored at –20 C, while in samples stored at –70 C for

the same period, 10% of the activity was lost.

Factor VIII is the most labile factor; 10% of the activity is

lost in 4 hours during storage at 4 C, while at –20 C for a 3 day

period, 20% of the activity was lost.97 Typically, clotted blood

or anticoagulated blood is centrifuged at 1,000g to 1,200g for

10 to 15 minutes. Insufficient centrifugation time and lower

centrifugal forces can result in a gradient of platelets in plasma

derived from heparinized blood, thus affecting some of the

laboratory results, such as potassium levels.2

While centrifugation at 2,000g for 15 minutes is

recommended for whole blood to obtain platelet-poor

plasma for global coagulation tests, at slightly lower speeds

(1,800g), some residual platelet contamination was

reported based on a shortened thrombin clotting time by

10% in plasma frozen for 48 hours and thawed to room

temperature before testing.97

Despite the use of robotics and improved pneumatic

tube delivery systems, and despite the fact that erstwhile

preanalytic problems such as hemolysis during specimen

transportation may have been overcome by improved design

of transport systems, the existence of preanalytic problems

should not be discounted.98,99 A case in point is the likeli-

hood of erroneous results for PO2if the blood has been cont-

aminated with air before pneumatic tube transportation.99

Relative Merits of Anticoagulants

Platelets rapidly change their shape from discoid to spher-

ical when blood is collected in EDTA, thus making the deter-

mination of mean platelet volume unreliable. Approaches to

minimize this artifact have been attempted by the use of analternative anticoagulant, which is a mixture of trisodium

citrate, pyridoxal phosphate and tris(hydroxymethyl)-

aminomethane.100 The rationale is that pyridoxal phosphate

is an effective platelet antiaggregant and disaggregant. Mean

platelet volume measurement using the aforementioned addi-

tive mixture was stable at room temperature for 24 hours.100

ACD, heparin, and EDTA have been used for the sepa-

ration of mononuclear cells. However, regardless of whether

ACD, EDTA, or heparin was used as the anticoagulant,

the lymphocyte fraction was contaminated with granulo-

cytes if the blood specimen was more than 24 hours old.52

RBC contamination is a problem with aged EDTA blood

specimens to the extent that 9 RBCs may be encountered

for every lymphocyte separated from a 2-day-old blood

specimen.101

For flow cytometric analysis, ACD and heparin are

superior to EDTA for maintaining viable WBCsovernight.102 However, if analysis is to be performed the

same day, EDTA, ACD, and heparin give equivalent results.

However, at 24 hours and beyond, a substantial decrease in

granulocyte viability may occur in blood collected in EDTA.

K3EDTA also has been reported to be associated with a loss

of function in lymphocyte-mitogen stimulation assays.102

Heparin has been reported to be problematic for use in

molecular biology procedures using restriction enzymes and

DNA amplification by the polymerase chain reaction

(PCR).103 While heparin inhibition can be overcome by a

variety of approaches (eg, treatment with heparinase, or

separation of leukocytes by centrifugation followed by a

minimum of 2 washings in a saline buffer or use of an appro-

priate buffer), many investigators prefer not to use heparin

for DNA amplification by PCR.103

In vitro platelet, monocyte, and neutrophil activation

studies are influenced by the anticoagulant used for blood

collection.104 Thus, monocyte activation as measured by

release of tissue factor and tumor necrosis factor activity was

lowest with EDTA compared with citrate, heparin, and

hirudin. Neutrophil activation as measured by lipopolysac-

charide-induced release of lactoferrin also was the lowest

with EDTA. EDTA seems to suppress platelet degranulation.

Platelet factor 4 levels as an indicator of platelet activation

was the lowest with EDTA–platelet-poor plasma (217

ng/mL) compared with platelet-poor plasma obtained with

citrate (440 ng/mL), hirudin (469 ng/mL), and unfractionated

heparin (1,180 ng/mL). A low-molecular-weight heparin

preparation, however, gave platelet factor 4 values compa-

rable to those obtained with hirudin.104

Endogenous Interferences and RelatedVariables

The effect of drugs and their metabolites on laboratory

tests is so extensive that compendia of interferences are

available for consultation.105 Several are discussed in the

following text ❚Table 8❚.

Although the subject of endogenous interferences is

broad, some mention should be made of interferences that can

affect clinical interpretation of laboratory data. In that context,

patients who are exposed to mouse immunoglobulins through

imaging or therapeutic techniques are prone to develop





antibodies to mouse immunoglobulins. These antibodies,

called human antimouse antibodies, or HAMA, can give rise

to false-positive or false-negative results in 2-site immuno-

metric assays using murine monoclonal antibodies as

reagents.81,106

An increased level of a chemical analyte can affect some

hematology determinations. Thus, glucose levels more than

600 mg/dL (33.3 mmol/L) can lead to a transient increase in

the MCV as water enters the RBC, as the RBC is placed in an

isotonic diluent, owing to the osmotic effect of glucose.107

However, as the glucose level slowly equilibrates, water exits

from the RBC along with glucose, thus restoring the original

MCV. This transient hypoglycemic effect can be overcome by

a microdilution of the blood sample and a 5-minute waiting

period for the equilibration to be complete before analysis.107

Triglyceride levels exceeding 1,000 mg/dL (11.29

mmol/L) and hyperlipidemic specimens can cause a spuri-

ously high hemoglobin value. This effect can be overcome by

resuspending the RBCs in saline before analysis or by making

corrections with a plasma hemoglobin blank.107

Turbidity resulting from the precipitation by lysis

reagents of high concentrations of monoclonal proteins seen

in patients with myeloma or macroglobulinemia may spuri-

ously elevate the hemoglobin level and WBC count.107

Endogenous antibodies can affect both coagulation and

hematology results. Circulating antibodies directed to platelet

membrane phosphatidylcholine or lecithin are referred to as

lupus anticoagulants. Prothrombin is the antigen for most

lupus anticoagulants and, as such, inhibits phospholipid-depen-

dent global coagulation assays.31 Antibodies directed to the

platelet membrane phospholipids phosphatidylserine and phos-

phatidylinositol, which are called anticardiolipin antibodies,

also inhibit phospholipid-dependent global coagulation tests.

An in vitro phenomenon of platelet agglutination seen in

blood collected in EDTA is due to the presence of antibodies

in blood that are reactive to platelets. These antibodies are

directed to antigens such as the glycoprotein IIb/IIIa complex

that is hidden within the platelet membrane. EDTA, upon

chelating calcium, exposes these antigens. The exposed

platelet antigen becomes modified at low and room tempera-

ture, permitting platelet antibodies to agglutinate platelets.

This EDTA-induced pseudothrombocytopenia is, however, not

seen when blood specimens are warmed to 37 C, since the

glycoprotein IIb/IIIa complex is dissociated at the higher

temperature. EDTA-induced pseudothrombocytopenia at 4 C

or room temperature can cause a spuriously low platelet count

and a spuriously increased WBC count if the platelet clumps

are in the same size range as WBCs. Apparently an epitope on

platelet membrane glycoprotein IIb is recognized by EDTA-

dependent IgG antibodies, causing pseudothrombocy-

topenia.108

EDTA-dependent IgM antibody, most active at room

temperature and of low titer, has been reported to cause leuko-

cyte agglutination noticeable on a peripheral blood smear but

to provide a spuriously low automated WBC count.109 This

phenomenon was not seen when the same patient’s specimen

was obtained in a tube with heparin or citrate.Both pseudothrombocytopenia and pseudoleukopenia

have been reported at room temperature owing to the EDTA

antibody–induced platelet aggregates not being counted as

platelets and to large neutrophil-platelet clumps falling outside

the size range for WBCs.110 This phenomenon was abolished

by warming the specimen to 37 C.

An in vitro phenomenon that is referred to as platelet satel-

litism is observed when the platelets surround the neutrophils

owing to EDTA dependent IgG autoantibodies that are directed


❚Table 8❚Selected Endogenous Interferences

Interference References

Circulating antibodiesTwo-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 testsAntibodies 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°CEDTA-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 citrateAntibodies 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 resultsGlucose level >600 mg/dL causes transient increase in MCV; as glucose equilibrates, water leaves Cornbleet107

RBC restoring original MCVTriglyceride 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.




to the glycoprotein IIb/IIIa complex in the platelet membrane

and neutrophil FC-gamma receptor III of neutrophils (CD16).111

This phenomenon, which is seen at room temperature, is not

seen when blood is exposed to a temperature of 37 C or periph-

eral blood smears are made immediately with EDTA-anticoagu-

lated or capillary blood. Occasionally, the phenomenon also has

been noticed with citrated blood, as well as with heparinized

blood.112 Severe platelet satellitism can cause spuriouspseudothrombocytopenia.

A discussion of endogenous interferences would not be

complete without discussing the most common interfer-

ences, such as hemolysis and turbidity due to hyperlipi-

demia. Apart from a few analytes, there are conflicting data

in scientific literature on the effect of hemolysis on a wide

range of analytes. The discrepancy is related not only to

differences in methods for the same analyte, but also to

differences in instrumentation. While to some extent instru-

ments that can subtract or blank the hemoglobin interfer-

ence by taking spectrophotometric measurements at the

wavelength of light where hemoglobin absorbs, in addition

to the wavelength at which the analyte absorbs (bichromatic

measurements), can minimize the interference, the hemo-

globin interference is not eliminated totally, especially when

the reagent system used is affected by hemoglobin.

In general, slight hemolysis would have a negligible

effect on many of the routine clinical chemistry laboratory

procedures. Severe hemolysis, while having a substantial

effect on constituents that are at a much higher concentration

within the RBC than in plasma, could induce a slightly dilu-

tional effect on the constituents present at a lower concentra-

tion in the RBC than in plasma.

Typical of the enzymes present within the RBC, the

concentration of lactate dehydrogenase (LDH) is 160-fold

greater than that present in the plasma, acid phosphatase is 68-

fold greater, and AST is 6.7-fold greater than the plasma

levels.113 The concentration of potassium inside the RBC is

approximately 23 times as much as that found in plasma.

Naturally one would expect the aforementioned tests to be

invalidated when the specimen is hemolyzed severely.

The visual evidence of hemolysis is provided when the

hemoglobin concentration exceeds 20 mg/dL. It has been

reported that the appearance of serum when 0.1% of the

RBCs are lysed was virtually identical to that of nonhemolyzed serum and, consequently, would go unde-

tected by laboratory personnel looking for hemolyzed spec-

imens.114 However, the appearance of serum when 1.0% of

RBCs were lysed was clear and cherry red. Such a spec-

imen would be characterized as having moderate hemol-

ysis. Hemolysis of 1% of the RBCs affected the measure-

ment of LDH, potassium, AST, and ALT.114

Hemolysis also has been classified based on measurement

of free hemoglobin levels.115 The nonhemolyzed samples had

a mean – SD free hemoglobin value of 4.8 – 3.2 mg/dL, and

moderately hemolyzed serum samples had a value of 43.5 –

13.9 mg/dL. Even slight hemolysis invalidated the results for

LDH, total acid phosphatase, prostatic acid phosphatase, and

potassium, and such specimens should be rejected.115 That the

effect of hemolysis is method-dependent was demonstrated by

the absence of statistically significant differences for the levels

of AST, ALT, inorganic phosphate, glucose, bilirubin, totalprotein and albumin.115

Adenylate kinase released from RBCs can spuriously

increase CK activity measurements unless a sufficient

amount of inhibitors of adenylate kinase are incorporated in

the assay mixture.2

The peroxidative effect of heme can cause a positive and

negative interference with various diazotization methods used

for the measurement of bilirubin, depending on the regents

used in the assay system.116 In classic diazotization procedures

for the measurement of bilirubin, hemoglobin in the

hemolyzed sample competes with nitrite for the sulfanilic acid

reagent, leading to spuriously lower results.115

Depressed glucose values (values lower than the true

value) have been reported with the glucose oxidase–peroxi-

dase procedure, which apparently is related to the reagent

composition. The negative interference has been explained as

resulting from premature decomposition by hemoglobin of the

hydrogen peroxide generated in the glucose oxidase reaction

or from the dilution by cellular components.117

The effect of hemolysis on glucose oxidase procedures

depends not only on the composition of the glucose oxidase-

peroxidase reagent system, but also on how the assay is

performed. Thus, differences on the effect of hemolysis have

been reported when using a kinetic assay in which the color

development is monitored between 60 and 120 seconds after

initiating the reaction as opposed to a bichromatic assay in

which color is monitored at 2 different wavelengths; the addi-

tional wavelength is used to blank out the color resulting from

substances other than glucose. For example, 2 grossly

hemolyzed pediatric samples with a low true glucose value

were diagnosed correctly by the kinetic procedure that yielded

glucose values of 29 and 27 mg/dL (1.6 and 1.5 mmol/L;

reference range, 70-105 mg/dL [3.9-5.8 mmol/L]). The

bichromatic procedure, on the other hand, overestimated the

glucose values on the 2 pediatric samples by reporting glucosevalues of 65 and 68 mg/dL (3.6 and 3.8 mmol/L).117

Hemolysis also interferes with the hexokinase procedure

for the measurement of glucose.118 However, owing to the

multiplicity of instrumentation and variations in methods for

the measurement of glucose, the magnitude of the effect of

hemolysis on each glucose procedure is unpredictable. As

such, for accurate glucose measurements, it is desirable to

avoid hemolysis or, at the very least, not exceed levels that

would make hemolysis visually apparent in serum.





While total acid phosphatase activity was, as expected,

affected by hemolysis due to the release of RBC acid phos-

phatase, even the prostatic tartrate–sensitive fraction has been

reported to be affected by hemolysis.118 Among the hormones

very sensitive to hemolysis is insulin.2 Apparently the release

of proteolytic enzymes during hemolysis degrades insulin.41

While some analytes clearly are affected by hemolysis, in

general, method-dependent variations should be consideredwhen assessing the influence of hemolysis.

Hyperlipidemia, by introducing turbidity, can be a source

of interference that also is method-dependent.119 Interference

could be due to a variety of mechanisms, such as inhomo-

geneity of the sample, displacement of water, and adsorption

of lipophilic constituents.2 A visibly hyperlipidemic sample

can be expected to interfere with the measurement of total

protein and electrophoretic and chromatographic procedures.2

Summary PerspectivesSo much knowledge on the preanalytic phase has accumu-

lated during recent years that this review can only highlight key

issues affecting several contemporary topics.

On the subject of blood gases issues such as maintaining a

steady state of ventilation with the patient in a supine position

before and during arterial blood collection, the effect of the

container material (eg, glass vs plastic syringe), dry vs liquid

heparin as the anticoagulant, leakage of gases owing to syringe

material and conditions of storage, and time between collection

and analysis need to be recognized and standardized.120,121

Molecular biology is another area in which information

on the preanalytic phase is accumulating constantly. The type

of detergent used for cell lysis, its effect on DNA amplification

by PCR, the effect of RBC contamination due to the effect of

hematin on the DNA polymerase enzyme, taq polymerase,

used in PCR, isolation of RNA including steps to eliminate

RNase contamination, treatment of tissue, the effect of fixa-

tives and duration of fixation, and protection of viral HIV

RNA load in plasma from the inhibiting effect of neutrophils

by prompt separation of plasma from cells after blood collec-

tion are just a few of a wide range of preanalytic variables that

merit consideration.103

As our knowledge of cell function advances andstudies on stimulation of cells to secrete cytokines become

diagnostically important, one must ensure that there is no

artifactual effect induced by the anticoagulant used for

blood collection. A case in point is the endotoxin- or

pyrogen-contaminated heparin stimulating monocytes to

secrete tumor necrosis factor, thereby invalidating the

measurement of this cytokine.122

Information related to the preanalytic phase of tumor

marker measurements is voluminous. The preservation of

labile tumor markers, including handling of tumor tissue for

the processing of labile hormone receptors, half-life, circadian

rhythm, distribution in cellular fractions, the effect of exercise

on markers such as prostate-specific antigen, amplification by

molecular methods, and sample quality for cytogenetics and

flow cytometry assessment of DNA histograms are just some

of the many issues that come under the preanalytic umbrella.81

As new markers are proposed, preanalytic problemsassociated with their measurements also surface. For

example, homocysteine has received considerable attention

as a marker of cardiovascular risk. Yet the measurement of

this amino acid presents challenges since it continues to be

formed by cellular enzymes in vitro, thus making the

prompt separation of cells from plasma or serum manda-

tory. Alternatively, measures should be taken to inhibit

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

predispose a person to hypercoagulability, preanalytic vari-

ables affecting these measurements need to be delineated and

controlled. Some physiologic variables that affect the

measurement of fibrinolytic activators and inhibitors,

including the extent of diurnal variation during a 24-hour

period, the effects of smoking and alcohol need to be recog-

nized. Challenges in measurement of free t-PA in plasma by

sample treatment to dissociate the t-PA-plasminogen activator

inhibitor I complex should not be underestimated.31

Finally, preanalytic problems that are unique to a method

should be kept in perspective as we begin to better understand

the complexities of the effect of new technology and therapy,

inasmuch as they affect the preanalytic phase.

From the Department of Pathology, New York Medical

College–Metropolitan Hospital Center, New York, NY.

Address reprint requests to Dr Narayanan: Dept of

Pathology, New York Medical College–Metropolitan Hospital

Center, 1901, First Ave, New York, NY 10029.

*Dr Narayanan is a Becton Dickinson Fellow at Becton

Dickinson, Franklin Lakes, NJ.

Acknowledgments: I thank Carol Perello for secretarial

support and preparation of the tables.

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