Download your lab focus

YLF1_Jan03.qxd
12/4/02
10:47 AM
Page 49
your lab focus
CE Update [coagulation and hematology | phlebotomy]
Preanalytical Variables in the Coagulation
Laboratory
Jeffry B. Lawrence, MD
Medical and Scientific Affairs, BD Clinical Laboratory Solutions, Franklin Lakes, NJ
DOI: 10.1092/ER9P64EBMCFR47KY
After reading this article, the reader should understand how errors occur in the preanalytical phase of testing, and how efforts can
be made to correct these errors.
Hematology exam 0301 questions and the corresponding answer form are located after the “Your Lab Focus” section on p. 69.
왘 Errors in coagulation laboratory
왘
왘
results, whether in assays performed
for anticoagulant monitoring or for
screening and diagnostic testing for
hemorrhagic and thrombotic
disorders, can lead to clinical
mismanagement and significant risk
for the patient.
The majority of coagulation laboratory
errors arise in the preanalytical phase;
therefore, ensuring the quality of
citrated blood samples is critical for
accurate test results.
Understanding the sources of
preanalytical variability; educational
and quality improvement programs;
and standardization efforts directed at
optimizing sample quality provide the
most cost-effective opportunities for a
laboratory to improve the quality of
coagulation test data and enhance
clinical care.
Though cardiovascular disease
remains the most common cause of death
in Western countries, significant progress
has been made in the management of it
during the past 2 decades. Increased use
of anticoagulant therapy has played an
important role in the improved outlook for
patients with occlusive arterial or venous
thromboembolic disease. However, while
anticoagulant drugs reduce a patient’s risk
of heart attack and stroke by making the
blood less clottable, this same effect increases the patient’s risk of hemorrhage.
This necessitates laboratory monitoring of
the patient’s response to these drugs on
an ongoing basis. For instance, of the approximately 300 million coagulation tests
performed annually in the United States,
more than 40 million are prothrombin
times (PT) performed for monitoring of
warfarin therapy.
While monitoring of anticoagulant
therapy may have the most immediate
impact on patient management and outcome, clinicians also rely heavily on the
accuracy of screening and diagnostic PT
and activated partial thromboplastin
time (APTT) tests; platelet function
studies; work-ups to identify inherited
thrombotic predisposition; and measurement of coagulation factors and
inhibitors, D-dimer, and other coagulation analytes. Because of the
significantly improved instrumentation
and highly sensitive reagents available
in modern coagulation laboratories, preanalytical variability now represents the
most important source of errors that can
lead to inaccurate patient results, patient
mismanagement, and preventable hemorrhagic or thrombotic complications. This
article will review the importance of the
quality and integrity of the coagulation
©
laboratorymedicine> january 2003> number 1> volume 34
sample for the accuracy and precision of
patient test results, and discuss
approaches to improve coagulation laboratory performance by controlling preanalytical variability.
Laboratory Errors and Role
of Preanalytical Variables
Just as with all other laboratory testing, in the coagulation laboratory the ultimate goal is to reflect the patient’s actual
state of hemostatic function in vivo.
Whether testing is performed to diagnose
a bleeding or thrombotic disorder, to
screen a patient for surgery, or to monitor
coagulation factor replacement or
antithrombotic therapy, any factor that
causes the test to misrepresent the actual
state of coagulation function in the patient (whether in the preanalytical phase,
during the analysis, or in postanalytical
test reporting and interpretation given to
the clinician) can lead to adverse
outcomes for the patient.
Anticoagulant drugs, whether intravenous heparin or oral compounds such as
warfarin, interfere with several different
coagulation enzymes, resulting in inhibition of the coagulation system. Both warfarin and heparin have a narrow therapeutic
index. This means that there is a narrow
range between inadequate doses that can
lead to recurrent thrombosis and excessive
49
YLF1_Jan03.qxd
50
12/4/02
10:47 AM
Page 50
doses which predispose to hemorrhage.
Administration of these drugs results in
prolongation of the PT or APTT, respectively, and the magnitude of the prolongation is used to determine the optimal
dose of the drug for the patient. Thus,
these tests, in essence, serve as therapeutic drug monitoring bioassays that
directly determine drug dosage.
Erroneously prolonged PT or APTT results would mislead the clinician to believe that the patient has a greater
anticoagulant response to warfarin or
heparin in vivo than is actually the case,
leading to dose reductions and a corresponding risk of recurrent thrombosis.
Alternatively, an artifactually shortened
clotting time would typically cause the
physician to inappropriately increase the
anticoagulant dose, putting the patient at
risk for hemorrhage.
Similarly, inaccurate coagulation test
results performed to diagnose or screen
for potential hemorrhagic or bleeding disorders can either lead to the failure to detect a treatable life-threatening condition
or erroneously suggest the presence of a
disease when the patient is free from a
coagulation disorder. The latter often results in cancellation of needed invasive
procedures and/or unnecessary follow-up
diagnostic testing.
In an effort to better understand the
source of laboratory errors across disciplines, Plebani and Carraro1 analyzed
all 40,490 stat results reported over a 3month period in the University Hospital
of Padua to the Intensive Care Unit and
the inpatient Departments of Internal
Medicine, Surgery, and Nephrology.
They identified 189 errors (0.47% of all
results) and determined that 68% of the
errors originated in the preanalytical
phase, compared with 13% of errors
arising during analysis and 18% in the
postanalytical phase. In over one-fourth
of the cases, these laboratory errors resulted in unnecessary further investigations and/or inappropriate care or
changes in therapy. In this paper, we
define preanalytical variables as any influence before testing of the specimen
that causes the coagulation test result to
fail to reflect the patient’s in vivo hemostatic function.
Susceptibility of Tests to
Preanalytical Variability
Clinical chemistry, immunochemistry, hematology, and other laboratory
tests each show some dependence upon
the quality of the sample. However, coagulation tests are exquisitely susceptible to
error introduced by suboptimal specimen
quality. This is due to the fact that the
very act of obtaining a blood sample initiates the hemostatic response, the physiologic system that the testing is designed
to assess. Coagulation assays are highly
vulnerable to preanalytical variability because of a number of factors: (a) the complexity of the biochemical and cellular
reactions measured in assays such as the
PT and APTT; (b) the lability of several
coagulation proteins; (c) the calcium dependence of many of these reactions; and
(d) the highly excitable nature of blood
platelets—the key cellular “player” in
these assays. During the past 20 years,
ever more sensitive and reliable PT and
APTT reagent systems have been introduced into the laboratory, resulting from
an explosion in understanding the basic
biochemistry and physiology of the coagulation system. Furthermore, advances in
laboratory instrumentation and automation have dramatically reduced analytical
variability in coagulation analyzers.
While these developments have substantially improved the accuracy and precision of coagulation laboratory data,
paradoxically they have increased the
dependence of the test results on the
quality of the citrated blood sample.
Consequently, preanalytical variability is
more hazardous than ever, particularly
given the recent dramatically increased
use of anticoagulant therapy as well as
the clinical importance of diagnostic testing for von Willebrand’s disease,
prothrombotic disorders, disseminated
intravascular coagulation, and other hemostatic abnormalities.
It is impossible to completely reproduce physiologic conditions in the coagulation laboratory in vitro, since the act
of removing the blood from the patient
and putting it into a container causes initiation of clotting. However, calcium is
required for many of the biochemical
steps of the coagulation mechanism, and
laboratorymedicine> january 2003> number 1> volume 34
©
artificial removal of the calcium by an
additive in the coagulation tube (ie,
sodium citrate) prevents coagulation from
occurring. The citrated blood sample is
transported from the site of phlebotomy
to the laboratory and centrifuged, resulting in the removal of the vast majority of
blood cells and the remaining plasma is
the specimen to be tested. The PT, APTT,
or other coagulation reagent is incubated
with the plasma, and the time it takes for
a clot to form after the subsequent addition of calcium is inversely proportional
to the patient’s coagulation response. Coagulation testing is optimized when these
processes of phlebotomy, calcium chelation, sample transport, centrifugation, and
recalcification proceed under maximally
standardized and controlled conditions.
Patient-Related Variables
Strictly speaking, patient variables
do not represent preanalytical variables as
defined before, since they affect not only
coagulation laboratory results but also
hemostatic function in vivo. However,
they are important to recognize for the
determination of appropriate reference
ranges and the evaluation of the significance of slight abnormalities detected in
coagulation tests performed on patients
suspected of having hemostatic
disorders.2-19 [T1] For instance, use of
age-specific reference ranges is critical
for interpretation of coagulation data in
newborns and infants,2 and we and others
have demonstrated that age and gender
influence platelet function as well as coagulation activation.3-6 Individuals with
blood type O have significantly lower
plasma von Willebrand factor and factor
VIII activity than subjects with other
blood types.7 It has been reported that
platelet activation, and the incidence of
myocardial infarction are highest in the
morning,8 and increased coagulation activation has been described in cold
weather, associated with an increased incidence of myocardial infarction and
stroke.9
Diet and alcohol intake have been
shown to affect coagulation activation in
people with diabetes10 and in other populations. Smoking elevates plasma fibrinogen, von Willebrand factor, other
YLF1_Jan03.qxd
12/4/02
10:47 AM
Page 51
coagulation factors, thrombin generation,
and platelet activation.11 Beyond the antioxidant and antithrombotic effect of red
wine,10 moderate ethanol intake inhibits
platelet reactivity,12 and this effect has
been linked to the reduction in coronary
heart disease associated with light to
moderate alcohol consumption. In addition to the well-known platelet inhibitory
effect of aspirin and non-steroidal antiinflammatory drugs, a number of other
medications, including hormone replacement therapy and selective estrogen receptor modifiers, alter coagulation and
platelet responsiveness.13 Oral contraceptives, pregnancy, menopause, and the different phases of the menstrual cycle are
also associated with changes in coagulation.14 Vigorous physical exercise leads to
coagulation activation,15 and psychological stress has been associated with
changes in both the coagulation and fibrinolytic systems.16 Disease states which
lead to anemia, polycythemia, or hemolysis can also interfere with coagulation
tests since anemia results in an effectively
lower plasma citrate concentration and
shortened clotting times while
polycythemia has the opposite effect.17
Because of this, the NCCLS coagulation
guideline document H21-A3 provides
recommendations for adjusting the
amount of anticoagulant and blood for
patients with hematocrit values >0.55.18
Hemolysis can both influence the
biochemistry of coagulation reactions and
interfere with clot detection in some analyzers. Furthermore, anemia itself can
prolong the bleeding time because of the
important rheologic role red cells play in
platelet adhesion in primary hemostasis.19
Phlebotomy Practices
Positive patient identification is critical for accurate test results in all
disciplines within the laboratory. Identification errors can occur either at the time
of phlebotomy or after centrifugation
when specimens are aliquotted. Another
source of error can be mistakes in transcribing the specific coagulation assays
requested by the clinician and selection of
inappropriate blood collection tubes (and
needles). Because recent patient exercise
can activate platelets and the coagulation
Patient Variables That Can Influence Coagulation Test Results
T1
· Age
· Gender
· Blood type
· Intraindividual variability, circadian, and seasonal rhythms
· Diet
· Smoking
· Alcohol intake
· Medications
· Menstrual cycle, pregnancy, menopausal status
· Physical exercise
· Emotional stress and psychiatric disorders
· Disease states associated with anemia, polycythemia, or hemolysis
system as noted,15 patients should rest
comfortably for 15 to 30 minutes prior to
phlebotomy. When patients move from
the supine to the upright position, water
moves from the intravascular compartment to the interstitium, reducing the
plasma volume by 12% on average. This
increases the apparent concentration of
cells, macromolecules, and protein-bound
small molecules, and consequently the
platelet count and coagulation factor levels may be elevated and the PT and
APTT shortened. Also, upon standing,
vasoactive hormones are released which
can result in platelet activation.
Therefore, it is desirable to standardize
patient position during phlebotomy.
A broad category of variables related
to phlebotomy technique and practices
can introduce preanalytical variability.
Placement of the tourniquet for longer
than 1 minute causes migration of fluid
and low molecular weight molecules
from the intravascular to the interstitial
space, resulting in elevation in cells and
coagulation proteins. This can also lead to
coagulation activation. Traumatic and/or
prolonged phlebotomy accentuates the
hemostatic activation that is inevitable in
blood collection. This can produce an
artificially short PT and/or APTT, if excessive vascular injury results in slight to
moderate activation of coagulation factors
and platelets. Alternatively, with greater
difficulty in the phlebotomy, marked coagulation activation can lead to depletion
of fibrinogen and other factors, resulting
in prolonged clotting times. Underfilling
or overfilling of the citrate tube, with resulting imbalances in the blood/additive
©
laboratorymedicine> january 2003> number 1> volume 34
ratio, can produce artificially prolonged
or shortened clotting times,
respectively.18,20 Finally, it is important to
avoid obtaining blood samples from a site
near infusion of intravenous fluids (particularly heparin).
In the NCCLS guideline H21-A3, it
is recommended to use evacuated blood
collection systems into a tube containing
a specified amount of sodium citrate.18
The use of a needle and syringe to draw
blood samples for coagulation testing introduces a number of uncontrolled preanalytical variables. This is made worse if
“homemade” citrate additives are used as
anticoagulants to be added to glass or
plastic tubes. This technique also
increases risk of health care worker injury
from bloodborne pathogen exposure. Particularly with larger syringes, there is a
significant risk that clotting may occur.
The volume of blood drawn is critical for
accurate coagulation results. The
blood:citrate ratio of 9:1 must be maintained. Use of a syringe makes it more
difficult to consistently obtain the desired
blood volume draw. Syringe draws also
produce variability in the shear forces to
which platelets are exposed, depending
upon technique. This is particularly important for heparin monitoring because
shear force activates platelets, and this
can introduce clinically significant shortening of APTT results. Hemolysis is also
significantly more common with use of
needle and syringe compared with evacuated systems.
The NCCLS recommended order of
draw for obtaining multiple blood collection tubes is shown in T2.21 Citrate tubes
51
YLF1_Jan03.qxd
12/4/02
10:47 AM
Page 52
NCCLS Recommended Order of Draw21
T2
Blood culture tubes
Plain serum glass tubes
Sodium citrate tubes
Gel separator tubes/plain serum plastic tubes
Heparin tubes/heparin gel separator tubes
EDTA tubes
Glucose preservative tubes
52
should be drawn after blood culture tubes
and serum tubes lacking clot activator,
but before other additive tubes. If only a
coagulation sample is to be drawn, discard tubes are not necessary unless a
winged blood collection set is to be
used.18,22 In this case, a discard should be
drawn before filling the citrate tube in
order to compensate for the dead space in
the tubing of the blood collection set.
Prothrombin time, APTT, and other
coagulation assays require a specified
plasma concentration of sodium citrate,
which in turn controls the amount of ionized calcium available for clotting. As
described in the discussion of anemia and
polycythemia, if the citrate tube is overfilled with blood, the blood:citrate ratio
will be elevated, resulting in excessive
ionized calcium availability and
artificially short clotting times.
Conversely, in an underfilled tube, the
blood:citrate ratio is decreased, reducing
the availability of ionized calcium and
causing artifactually prolonged clotting
times. The latter may be accentuated by
dilution of the plasma by the liquid anticoagulant solution. Similarly, the citrate
concentration in the tube itself can influence PT and APTT results because less
ionized calcium is available with 3.8%
sodium citrate (0.129M) than with 3.13%
(0.105M) or 3.2% (0.109M). Adcock and
colleagues examined the effect of underfilling tubes containing 0.129M citrate
compared with underfilling 0.105M citrate tubes.23 They found that the prolongation of the APTT is more pronounced
with underfilling in 0.129M citrate tubes
than in 0.105M tubes; similar results
were found with PT. In this sense, the
lower citrate concentration is more “forgiving” with respect to underfilling (or
overfilling) the tube; 0.105M citrate is
also less sensitive to variability in the
hematocrit. For these reasons, the
NCCLS recommends the use of 3.13% or
3.2% sodium citrate tubes,18 and in recent
years there has been a significant shift
away from 3.8% citrate to the lower concentrations.
It is important to quickly invert the
sodium citrate tube after it is filled in
order to ensure adequate mixing and prevent clotting in the tube. However, an excessive number of inversions, or vigorous
shaking of the tube, can lead to platelet
activation and artificial shortening of clotting times, particularly in heparinized
samples. Thus, it is optimal to invert the
tube gently 3 to 4 times.
Specimen Transportation and
Handling
Coagulation proteins, particularly
factors V and VIII, undergo in vitro
degradation that increases with time. This
loss of cofactor activity is accelerated at
increased temperatures, and it results in
artifactual prolongation of PT and APTT
in most patient populations when there
are delays between sample acquisition
and analysis. While elevated temperatures
enhance factor V and VIII degradation,
refrigeration can result in cold-induced
activation of the intrinsic coagulation system, subsequent factor VII activation, and
artifactual PT shortening if unsiliconized
glass citrate tubes are used.24
The NCCLS recommends that specimens for PT assays uncentrifuged or centrifuged with plasma remaining on top of
the cells in an unopened tube should be
tested within 24 hours from the time of
specimen collection.18 For APTT and
other coagulation assays on
nonheparinized patients, uncentrifuged
samples or samples centrifuged with
laboratorymedicine> january 2003> number 1> volume 34
©
plasma remaining on top of the cells
should be tested within 4 hours of specimen acquisition.18 However, in the latter
case, if agitation of the specimens is
likely after centrifugation (such as remote
transportation of the sample), then the
plasma should be removed within 1 hour
of collection and tested within 4 hours.18
Adcock and colleagues reported that in
patients undergoing unfractionated heparin therapy, citrate samples demonstrate
a clinically significant shortening of the
APTT when stored uncentrifuged at room
temperature.25 This shortening was attributable to release of platelet factor 4.
Therefore, for APTT specimens suspected
to contain unfractionated heparin, centrifugation should be performed within 1
hour of collection, and testing should be
performed within 4 hours from time of
collection.18 If testing is not completed
within these time periods, plasma should
be removed from the cells and frozen at
–20ºC for up to 2 weeks. If the samples
are kept at –70ºC, they may be stored for
up to 6 months. A frost-free freezer
should not be used, and frozen plasma
samples should be rapidly thawed at 37ºC
while gently mixing, and tested immediately (the sample may be held for a maximum of 2 hours at 4ºC before testing).18
Platelets also rapidly lose their reactivity
after storage, so platelet aggregation studies should typically be initiated within an
hour after sample acquisition and completed within 2 hours.
Traumatic specimen handling, such
as in some older pneumatic tube systems,
can lead to platelet activation and shortening of clotting times. Centrifugation
conditions are critical for coagulation
testing. The NCCLS H21-A3 guideline
recommends that centrifugation be performed at room temperature at a speed
and time that consistently produces
platelet-poor plasma with a platelet count
under 10,000/µl—eg, 1,500 g x 15 minutes or longer.18 Higher platelet counts
can neutralize anti-phospholipid antibodies, resulting in false-negative lupus anticoagulant assays. Just as in the blood
bank and other sections of the laboratory,
unsuitable coagulation samples should be
rejected for analysis; otherwise,
erroneous results may be reported. When
YLF1_Jan03.qxd
12/4/02
10:47 AM
Page 53
testing is performed on aliquots derived
from a primary citrate tube, errors in
pipetting and sample misidentification
can occur.
Coagulation Tubes
Citrate tubes need to be compatible
with the holders, centrifuges, and coagulation analyzers present in the laboratory.
It is also desirable that the tubes protect
health care workers from injury from
bloodborne pathogen exposure due to
blood splatter and tube breakage. Safety
closures and plastic tubes offer such protection; however, with the latter it is important to establish that the coagulation
test results are not compromised.
The tube’s stopper plays a key role in
maintaining vacuum and liquid retention
in the tube. It must be readily penetrated
by the non-patient end of the needle
within evacuated tube holders and remain
seated in the tube, and it must not leach
calcium, magnesium, and zinc into the
citrate additive. This requires vigilant
quality control since rubber preparations
must be specially formulated to prevent
divalent cation leaching.26 The draw volume of the tube must be adequate to support the plasma volume necessary for
testing without leading to unnecessary
blood loss for the patient. The tube’s
sodium citrate concentration (eg, 3.2%
versus 3.8%) affects coagulation results
through its effect on ionized calcium
availability, with relatively longer clotting
times observed with 3.8% citrate.27
While early coagulation tubes were
manufactured with untreated glass, current coagulation assays have been standardized using full-draw sodium citrate
tubes made of siliconized glass.
Siliconization was introduced because of
the increased activation of the contact
portion of the intrinsic coagulation cascade observed with unsiliconized glass,
leading under certain conditions to shortened clotting times.24 Certain plastic formulations show similar properties.
Clinical correlation studies in which outcomes such as hemorrhage and thrombosis are linked to PT and APTT values
have used full draw siliconized glass
tubes. This is critical because the bloodsurface interaction with tubes made of
other materials, such as unsiliconized
glass or various plastics (such as polyethylene terephthalate or polypropylene),
may differ significantly from that
observed with siliconized glass.28 Furthermore, the effect of the inner surface
of coagulation tubes on test results is difficult to predict because the degree of
hydrophobicity of surfaces has opposite
effects on coagulation activation and
platelet activation.29 Because of these
complexities, the bias in coagulation test
results exhibited by different plastic
tubes, relative to siliconized glass tubes,
is unpredictable and may differ for different assays.30-34 The magnitude, and even
the direction, of the bias may differ according to the instrument and reagent
used.
Unlike with clinical chemistry and
other laboratory disciplines, since clotbased coagulation tests are reported in
seconds [or International Normalized
Ratio (INR) units based upon seconds],
there are no primary standards to use for
calibration and validation of these assays.
Instead, clinical validation of the PT and
APTT is ultimately based upon the association of particular clotting time values
with important clinical outcomes such as
hemorrhage and thrombosis in randomized, controlled clinical trials. For example, Azar and colleagues studied 3,404
myocardial infarction patients receiving
oral anticoagulant therapy.35 They found
that as the PT increased, the incidence of
thromboembolic events declined while
the incidence of hemorrhage rose. The
clinically optimized INR range is defined
as that which minimizes the combined
incidence of adverse events (ie, thromboembolic + hemorrhagic). Similar studies in venous thrombosis and pulmonary
embolism patients established the range
of 2.0 to 3.0 as the optimal INR range for
anticoagulation of such patients, while for
patients with mechanical heart valves and
others at particularly high risk of thrombosis, the recommended INR therapeutic
range is 2.5 to 3.5.36 These studies (as
well as trials of heparin therapy
monitored with the APTT) have all been
conducted with full-draw sodium citrate
tubes made of siliconized glass. If a laboratory uses any other type of coagulation
©
laboratorymedicine> january 2003> number 1> volume 34
tube (such as partial draw and/or plastic
tubes), it is critical to demonstrate clinical
equivalence of the PT or APTT results
obtained with the new tube to those obtained with the clinically-defined “gold
standard” full draw siliconized glass
tubes. Otherwise the optimal range of
INR or APTT results observed with the
new tube may not be the same as found
in standard recommendations in the literature derived from trials using the “gold
standard” tubes, and patients could be at
risk of over- or under-anticoagulation.
The manufacturer is responsible for
demonstration of equivalent clinical performance of newly introduced tubes.
However, under CLIA ’88 and CAP requirements, the laboratory is responsible
to obtain such documentation from the
manufacturer.
One should also be careful that the
documentation provided by the vendor
reflects patient populations actually at
risk for hemorrhagic or thrombotic complications, particularly heparin patients.
This is critical because the typical studies
performed in normal subjects rarely detect analytical bias that occurs in anticoagulated patients. Recently, plastic
coagulation tubes with a novel design
permitting both a full draw configuration
and lower blood draw volumes than previous full draw glass tubes (ie, 2.7 and
1.8 mL) have been introduced. In clinical
trials with >1,000 patients, these tubes
demonstrated equivalence to the
siliconized glass “gold standard” tube in
normal subjects, warfarin and heparin
patients, and patients with a variety of
coagulopathies, across the spectrum of
instruments and reagents present in most
clinical laboratories. Some of these comparative data are presented as an example
of the kinds of clinical trials necessary to
compare the clinical performance of new
citrate tubes with that of full draw siliconized glass tubes [T3].
Since platelets are prone to activation
during phlebotomy, tube inversion, and
centrifugation, and with the attendant risk
of interference with coagulation assays,
coagulation tubes have been developed in
which metabolic inhibitors have been
added to sodium citrate for platelet stabilization. Mody and colleagues studied the
53
YLF1_Jan03.qxd
12/4/02
10:47 AM
Page 54
Warfarin Donor Prothrombin Time
Instrument: ELECTRA 1400C Coagulation Analyzer (Instrumentation Laboratory, Lexington, MA)
Reagent
Hemoliance Brain Thromboplastin (n=30)
Draw volume
Tube type
Mean + SD
4.5 mL
Siliconized glass
17.43+3.19
2.7 mL
Plus Plastic
17.43+3.18
T3
Dade Innovin (n=30)
1.8 mL
Plus Plastic
17.41+3.00
4.5 mL
Siliconized glass
19.76+7.17
2.7 mL
Plus Plastic
19.46+7.17
1.8 mL
Plus Plastic
19.52+7.13
None of the differences was statistically significant.
Unfractionated Heparin Donor Activated Partial Thromboplastin Time
Instrument: ELECTRA 1400C Coagulation Analyzer (Instrumentation Laboratory, Lexington, MA)
Reagent
Hemoliance ThrombosilI (n=58)
Draw volume
Tube type
Mean + SD
4.5 mL
Siliconized glass
50.89+13.80
2.7 mL
Plus Plastic
49.30+12.83
Dade ACTIN FSL (n=59)
1.8 mL
Plus Plastic
48.86+12.85
4.5 mL
Siliconized glass
46.19+7.17
1.8 mL
Plus Plastic
45.67+13.83
None of the differences was statistically significant.
Anticoagulated warfarin and unfractionated heparin donor samples were collected, transported, processed, and tested with the designated PT and APTT reagents and coagulation
analyzer according to NCCLS Guideline H21-A3.18 Samples were collected in BD Vacutainer siliconized glass 4.5 ml 0.105 M sodium citrate tubes, and 2.7 and 1.8 mL Plus Plastic
0.109 M sodium citrate tubes, and test results with each of the Plus Plastic tubes were statistically compared for equivalence with the “gold standard” siliconized glass tubes.42
effect of one such additive, CTAD (containing sodium citrate, theophylline,
adenosine, and dipyridamole) on platelet
activation over time.37 They reported that
for both uncentrifuged and centrifuged
tubes, CTAD significantly inhibited
platelet activation. This resulted in a significant reduction in preanalytical variability
in APTT results in heparinized patients.
54
Greatest Susceptibility to
Preanalytical Variability
A number of coagulation assays that
show high susceptibility to preanalytical
variability are predominantly research
tools. These include highly sensitive immunoassays for thrombin generation (the
F1+2 prothrombin activation peptide and
thrombin-antithrombin complex) and
peptides indicative of thrombin activity
(fibrinopeptide A), as well as markers of
fibrinolysis (plasminogen activator
inhibitor-1, tissue plasminogen activator,
and plasmin-antiplasmin complex). The
time-dependence of platelet aggregation
studies was noted before, and flow cytometric analysis of platelets is also highly
susceptible to preanalytical variability, as
are assays for cytokines such as
interleukin-1 and tumor necrosis factor.
The coagulation assays in which the
greatest clinical risk is associated with
preanalytical variability are the following:
PT monitoring of warfarin therapy, APTT
monitoring of unfractionated heparin
therapy, platelet count verification in
pseudothrombocytopenia, and lupus anticoagulant testing.
Warfarin Monitoring
Introduction of the INR system significantly improved management of warfarin patient by removing a substantial
proportion of the variability associated
with thromboplastin sensitivity. However,
the coagulation studies by which the
manufacturers and contracted reference
laboratories make International Sensitivity Index (ISI) assignments to a thromboplastin show the same sensitivity to
preanalytical variables such as sodium
citrate concentration, coagulation tubes,
time between sample acquisition and
analysis, temperature, etc.31-34,38 For example, it has been reported that ISI assignments are approximately 10% lower
when determined with samples collected
in 0.129M sodium citrate than with
0.109M citrate.38 Thus, if a laboratory is
relying upon the manufacturer’s ISI assignment for INR determinations, it
laboratorymedicine> january 2003> number 1> volume 34
should verify that its preanalytical practices are consistent with those used by the
manufacturer. As noted, use of
unsiliconized glass is potentially
hazardous for warfarin monitoring, since
cold-induced PT shortening can lead to
inappropriate increases in patients’ warfarin dosages.24 Fortunately, warfarin
monitoring using the PT is overall significantly less susceptible to preanalytical
variability than heparin monitoring with
the APTT, since the former is insensitive
to platelet activation.
Unfractionated Heparin
Monitoring
Intravenous heparin therapy has become standard practice for patients with
acute coronary syndrome, in addition to
its established role in the treatment of
deep venous thrombosis and pulmonary
embolism. However, this life-saving therapy is also associated with life-threatening risks of hemorrhage. Hirsh and
Fuster reviewed a series of published
studies of intravenous heparin therapy,39
and reported that while heparin is effective in preventing fatal pulmonary embolism and reducing recurrent
thrombosis, major bleeding occurred in
from 1.9% to 6.5% of patients (with a
©
YLF1_Jan03.qxd
12/4/02
10:47 AM
Page 55
combined rate in the reviewed studies of
3.8%). A significant number of these
life-threatening hemorrhagic events are
likely attributable to problems in monitoring this therapy with the APTT, and of
these errors, a substantial proportion may
be preanalytical in origin.
APTT reagent systems show significant variability in their sensitivity to heparin. However, despite multiple
attempts, no standardization system comparable to the INR has been devised.
While many laboratories continue to use
the traditional therapeutic range for heparin of 1.5 to 2.5 times the midpoint of
the population reference range, the College of American Pathologists Conference on Laboratory Monitoring of
Anticoagulant Therapy made recommendations for defining a local therapeutic
range that takes into consideration the
varying sensitivity of APTT reagent systems.40 This involves studying a series of
patients on a stable heparin dose (without
accompanying oral anticoagulants) by
measuring both the APTT using the local
preanalytical and reagent/instrument systems and heparin activity using a chromogenic anti-factor Xa assay. Values for
APTT associated with heparin levels between 0.3 and 0.7 U/mL define the therapeutic range. However, preanalytical
variability complicates this system, as the
APTT is much more sensitive to the
sample quality than the PT. This difference in sensitivity is because the former
assay is significantly affected by platelet
activation and secretion, and it reflects
many more biochemical reactions
encompassing both the intrinsic and
common coagulation pathways. This renders the APTT significantly more sensitive to sodium citrate concentrations as
well as to sample handling (eg, tube inversion), specimen transport time and
conditions, and centrifugation. In addition to these preanalytical factors, APTT
results in the presence of heparin are
highly sensitive to properties of coagulation tubes, such as their configuration
(eg, full-draw versus partial-draw) and
composition (ie, siliconized glass versus
unsiliconized glass versus plastic).
In response to increasing concerns
about iatrogenic blood loss, sodium citrate
tubes were designed to draw 2.7 mL or
1.8 mL blood into the same 13 x 75 mm
configuration as traditional full-draw 4.5
mL tubes. This was achieved by creating
a reduced vacuum in the tube. This design is referred to as a partial-draw tube.
In the first several years after this product’s release, it performed equivalently to
the standard full-draw “gold standard”
sodium citrate tubes with the coagulation
analyzers and reagents in clinical use.
However, as APTT reagents were
developed with greater sensitivity to heparin, lupus anticoagulants, and factor
deficiencies, and as coagulation instrumentation became more robust, it
became apparent that partial-draw tubes
were associated with artifactual shortening of the APTT in heparinized patients
with certain instrument/reagent combinations. Siegel and colleagues studied a
series of heparinized patients by comparing APTT results on samples collected in
full-draw and partial-draw sodium citrate
tubes.41 They reported that there was a
significant negative bias with the partialdraw tubes in heparinized patients that
could result in excessive anticoagulation
in these patients. No significant differences were observed with results within
the reference range. Similar bias in heparin monitoring has been observed in
plastic partial-draw citrate tubes.
This negative bias in APTT results
in partial-draw tubes has been shown to
be due to platelet activation during the
slower filling of these tubes. Shear stress
is known to activate platelets. In partialdraw tubes, platelets are exposed for a
longer period of time to altered shear
forces related to the tubes’ increased
headspace. This activates the platelets,
leading to release of platelet factor 4
from the alpha granules, which in turn
neutralizes the heparin in the sample and
artificially shortens the APTT.25
Thus, if a laboratory is planning to
use coagulation tubes other than the fulldraw siliconized glass tubes that were used
in the clinical trials which formed the basis
for the recommended heparin therapeutic
range, it is critical for the manufacturer
and/or laboratory to verify equivalent clinical performance of the new tubes to those
of these “gold standard” tubes.
©
laboratorymedicine> january 2003> number 1> volume 34
Platelet Counts in Citrate Tubes
A small proportion of inpatients (1%
to 2%) and outpatients (0.1% to 0.2%)
demonstrate platelet clumping in the
presence of EDTA due to EDTA-dependent antibodies reactive with platelet antigens. Most cases of clumped platelets are
due to phlebotomy technique or failure to
adequately mix the specimen. Standard
protocols designed to obtain reliable
platelet counts in such patients include
preparing blood smears from a fingerstick specimen and measuring the
platelet count in a sodium citrate tube.
However, preanalytical variability,
such as excessive inversion or shaking of
the tube, and/or use of tubes with a partial draw configuration, can result in erroneously low platelet counts in these
patients. F1 shows the effect of platelet
activation occurring in partial-draw citrate tubes on the platelet count in 30
healthy donors. The platelet counts with
EDTA tubes and full-draw sodium citrate
tubes were equivalent. However, platelet
activation associated with 2.7 mL and 1.8
mL partial-draw tubes led to platelet aggregation and resulted in artificially reduced platelet counts. Consequently, use
of partial draw citrate tubes to obtain accurate platelet counts in patients with
EDTA-associated pseudothrombocytopenia would be associated with the risk of
clinically significant preanalytical error.
The hypothesis that platelet activation is
responsible for this phenomenon is confirmed by use of the CTAD metabolic
platelet inhibitor cocktail. Platelet counts
were equivalent in full-draw 4.5 mL
CTAD tubes and 2.7 mL and 1.8 mL partial draw CTAD tubes [F1].
Lupus Anticoagulant Testing
Sensitive and accurate detection of
the presence of the lupus anticoagulant
is critical for the evaluation of patients
with thrombotic disorders and recurrent spontaneous abortion. Assays to
detect the lupus anticoagulant are very
sensitive to the presence of platelets,
since false negative results can occur if
the antiphospholipid antibody activity
present in the patient’s plasma is neutralized by platelet phosphoplipids.
Thus, compliance with the NCCLS
55
YLF1_Jan03.qxd
12/4/02
10:47 AM
Page 56
3. Emery JD, Leifer DW, Moura GL, et al. Whole
blood platelet aggregation predicts in vitro and in
vivo primary hemostatic function in the elderly.
Arterioscl Thromb Vasc Biol. 1995;15:748-753.
4. Sagripanti A, Carpi A. Natural anticoagulants,
aging, and thromboembolism. Exp Gerontol.
1998;33:891-896.
5. Lawrence JB, Leifer DW, Moura GL, et al. Sex
differences in platelet adherence to
subendothelium: Relationship to platelet function
tests and hematologic variables. Am J Med Sci.
1995;309:201-207.
6. Campbell NR, Hull RD, Brant R, et al. Different
effects of heparin in males and females. Clin
Invest Med. 1998;21:71-78.
[F1] Phlebotomy was performed on 30 healthy donors; samples were collected, transported, and
processed according to NCCLS Guideline H21-A321; and platelet counts were measured on a
Coulter STKS hematology analyzer. Samples were collected in the following BD Vacutainer tubes:
• 4.5 mL glass K3EDTA tubes
• Siliconized glass 0.105 M sodium citrate tubes: 4.5 mL full-draw, and 2.7 and 1.8 mL partialdraw tubes
• Siliconized glass CTAD tubes (containing 0.109 M sodium citrate, 15 mM theophylline, 3.7 mM
adenosine, and 0.198 mM dipyidamole): 4.5 mL full draw, and 2.7 and 1.8 mL partial draw tubes
The platelet counts reported by the hematology analyzer with citrate and CTAD tubes were
multiplied by 1.1 (to compensate for the dilution arising from the liquid citrate or CTAD additive) to
give the values to be compared with the platelet counts obtained with K3EDTA tubes; the bars in
the figure represent the mean of these platelet counts for the 30 healthy donor samples collected
with each tubes type. The 2.7 and 1.8 mL partial draw citrate tubes gave statistically significantly
lower mean platelet counts than the K3EDTA or 4.5 mL full draw citrate tubes. No significant
differences were observed in the platelet counts obtained in full draw or partial draw CTAD tubes;
these values were also statistically equivalent to the platelet counts obtained with the K3EDTA and
4.5 mL full draw citrate tubes.
*:P<0.001.
centrifugation guidelines18 is critical for
lupus anticoagulant testing.
56
Implications for Clinical
Laboratories
The information described in this
article suggests that of all possible
approaches to improve the quality of coagulation test results standardizing preanalytical practices probably offers the
greatest payoff because preanalytical variability is the greatest contributor to laboratory errors.1 Laboratories must ensure
that all their preanalytical practices (including phlebotomy, specimen identification, transportation and handling, and
citrate tubes) minimize bias.
Because of the complexity of preanalytical operations within hospitals and
other health care facilities, it is essential
to maintain accountability for implementation of best practices at each step—
from the patient to the coagulation
analyzer—regardless of institutional reporting relationships. Furthermore, preanalytical educational efforts must be
persistent and directed at all personnel
involved, regardless of department.
7. Gill JC, Endres Brooks J, Bauer PJ, et al. The
effect of ABO blood group on the diagnsosis of
von Willebrand’s disease. Blood. 1987;69:16921695.
8. Tofler GH, Brezinski D, Schafer AI, et al.
Concurrent morning increase in platelet
aggregability and the risk of myocardial infarction
and sudden cardiac death. N Engl J Med.
1987;316:1514-1518.
9. Mavri A, Guzic-Salobir B, Salobir-Pajnic B, et al.
Seasonal variation of some metabolic and
haemostatic risk factors in subjects with and
without coronary artery disease. Blood Coagul
Fibrinolysis. 2001;12:359-365.
Finally, the requirements of good
clinical and laboratory practice and our
obligations under CLIA ’88 and CAP
requirements teach us that laboratories
have the right and duty to request documentation from manufacturers about citrate tubes in all relevant patient
populations. Preanalytical variability can
be critical for accurate coagulation results
in patients receiving warfarin or heparin
and those undergoing testing for lupus
anticoagulants, while normal individuals
and certain other patient populations are
significantly less sensitive to preanalytical
practices. Therefore, validation studies in
healthy subjects are not necessarily predictive of results in patients with coagulopathies due to inherited or acquired
conditions or anticoagulant therapy. Laboratories should require manufacturers to
demonstrate clinical performance of their
tubes in these key patient populations.
1. Plebani M, Carraro P. Mistakes in a stat
laboratory: Types and frequency. Clin Chem.
1997;43:1348-1351.
2. Andrew M, Paes B, Johnston M. Development of the
hemostatic system in the neonate and young infant.
Am J Pediatr Hematol Oncol. 1990;12:95-104.
laboratorymedicine> january 2003> number 1> volume 34
©
10. Ceriello A, Bortolotti N, Motz E, et al. Red wine
protects diabetic patients from meal-induced
oxidative stress and thrombosis activation: A
pleasant approach to the prevention of
cardiovascular disease in diabetes. Eur J Clin
Invest. 2001;31:322-328.
11. Miller GJ, Bauer KA, Cooper JA, et al. Activation
of the coagulation pathway in cigarette smokers.
Thromb Haemost. 1998;79:549-553.
12. Zhang QH, Das K, Siddiqui S, et al. Effects of
acute, moderate ethanol consumption on platelet
aggregation in platelet-rich plasma and whole
blood. Alcohol Clin Exp Res. 2000;24:528-534.
13. Walsh BW, Kuller LH, Wild RA, et al. The effects
of raloxifene HCl on serum lipids and coagulation
factors in healthy postmenopausal women. JAMA.
1998;279:1445-1551.
14. Kadir RA, Economides DL, Sabin CA, et al.
Variations in coagulation factors in women:
Effects of age, ethnicity, menstrual cycle and
combined oral contraceptive. Thromb Haemost.
1999;82:1456-1461.
15. Weiss C, Bierhaus A, Kinscherf R, et al. Tissue
factor-dependent pathway is not involved in
exercise-induced formation of thrombin and fibrin.
J Appl Physiol. 2002;92:211-218.
16. Von Kanel R, Mills PJ, Fainman C, et al. Effects
of psychological stress and psychiatric disorders
on blood coagulation and fibrinolysis: A
biobehavioral pathway to coronary artery disease?
Psychosom Med. 2001;63:531-544.
17. Koepke JA, Rodgers JL, Ollivier MJ. Preinstrumental variables in coagulation testing. Am J
Clin Pathol. 1975;64:591-596.
18. NCCLS. Collection, Transport, and Processing of
Blood Specimens for Coagulation Testing and
General Performance of Coagulation Assays;
Approved Guideline—Third Edition. NCCLS
document H21-A3. 1998 NCCLS, Wayne, PA.
YLF1_Jan03.qxd
12/4/02
10:47 AM
Page 57
19. Valeri CR, Cassidy G, Pivacek LE, et al. Anemiainduced increase in the bleeding time:
Implications for treatment of nonsurgical blood
loss. Transfusion. 2001;41:977-983.
32. D’Angelo G, Villa C. Measurement of
prothrombin time in patients on oral anticoagulant
therapy: Effect of two different evacuated tubes.
Haematologica 1999;84:656-672.
20. Reneke J, Etzell J, Leslie S, et al. Prolonged
prothrombin time and activated partial
thromboplastin time due to underfilled specimen
tubes with 109 mmol/L (3.2%) citrate
anticoagulant. Am J Clin Pathol. 1998;109:754-757.
33. van den Besselaar AMHP, Bertina RM, van der
Meer, et al. Different sensitivities of various
thromboplastins to two blood collection systems
for monitoring oral anticoagulant therapy.
Thromb Haemost. 1999;82:153-154.
21. NCCLS. Procedures for the Collection of
Diagnostic Blood Specimens by Venipuncture;
Approved Standard – Fourth Edition. NCCLS
document H3-A4. 1998 NCCLS, Wayne, PA.
34. Biron-Adreani C, Mallol C, Seguret F, et al.
Plastic versus siliconized glass tubes: Evaluation
in current laboratory practice. Thromb Haemost.
2000;83:800-801.
22. Adcock DM, Kressin DC, Marlar RA. Are discard
tubes necessary in coagulation studies? Lab Med.
1997;28:530-533.
35. Azar AJ, Cannegieter SC, Deckers JW, et al.
Optimal intensity of oral anticoagulant therapy
after myocardial infarction. J Am Coll Cardiol.
1996;27:1349-1355.
23. Adcock DM, Kressin DC, Marlar RA. Minimum
specimen volume requirements for routine
coagulation testing. Dependence on citrate
concentration. Am J Clin Pathol. 1998;109:595-599.
24. Palmer RN, Gralnick HR. Inhibition of coldpromoted activation of the prothrombin time:
Studies of new siliconized borosilicate collection
tubes in normals and patients receiving warfarin.
Am J Clin Pathol. 1985;83:492-494.
25. Adcock D, Kressin D, Marlar RA. The effect of
time and temperature variables on routine
coagulation tests. Blood Coag Fibrinolysis.
1998;9:463-470.
26. Cummings J. Evacuated tubes for monitoring heparin
treatment. Thromb Haemost. 1981;34:939-940.
27. Adcock D, Kressin D, Marlar R. Effect of 3.2%
vs. 3.8% sodium citrate concentration on routine
coagulation testing. Am J Clin Pathol.
1997;107:105-110.
28. Vogler EA, Graper JC, Harper GR, et al. Contact
activation of the plasma coagulation cascade. I.
Procoagulant surface chemistry and energy.
J Biomed Mater Res. 1995;29:1005-1016.
29. Hunt BJ, Parratt R, Cable M, et al. Activation of
coagulation and platelets is affected by the
hydrophobicity of artificial surfaces. Blood
Coagul Fibrinolysis. 1997;8:223-231.
36. Hirsh J, Hoak J. Management of deep venous
thrombosis and pulmonary embolism: A
statement for healthcare professionals from the
Council on Thrombosis (in consultation with the
Council on Cardiovascular Radiology),
American Heart Association. Circulation.
1996;93:2212-2245.
37. Mody M, Lazarus AH, Semple JW, et al.
Preanalytical requirements for flow cytometric
evaluation of platelet activation: choice of
anticoagulant. Transfus Med. 1999;9:147-154.
38. Duncan EM, Casey CR, Duncan BM, et al.
Effect of concentration of trisodium citrate
anticoagulant on calculation of the international
normalized ratio and the international sensitivity
index of thromboplastin. Thromb Haemost.
1994;72:84-88.
39. Hirsh J, Fuster V. Guide to anticoagulant therapy.
Part 1: Heparin. Circulation. 1994;89:1449-1468.
40. Olson JD, Arkin CF, Brandt JT, et al. College of
American Pathologists Conference XXXI on
laboratory monitoring of anticoagulant therapy:
Laboratory monitoring of unfractionated
heparin therapy. Arch Pathol Lab Med.
1998;122:782-798.
30. Polack B, Barro C, Pernod G, et al. Impact of the
blood collection tube on the activation of
coagulation. Thromb Haemost. 1997;77:217-218.
41. Siegel JE, Bernard DW, Swami VK, et al.
Monitoring heparin therapy: APTT results from
partial- vs full-draw tubes. Am J Clin Pathol.
1998;110:184-187.
31. van den Besselaar AMHP, Meeuwisse J, Wineveen
E, et al. Effect of blood collection tubes on
thromboplastin calibration. Thromb Haemost.
1998;79:1062-1063.
42. Berger RL, Hsu JC. Bioequivalence trials,
intersection-union tests, and equivalence
confidence sets. Statistical Science. 1996;11:282302.
57
©
laboratorymedicine> january 2003> number 1> volume 34
CE_ExamJan03.qxd
12/4/02
9:44 AM
Page 69
Continuing Education Update Exams
Earn 1 CMLE credit hour for each exam (maximum of 4 exams per issue). After processing your examination, ASCP will mail you a certificate of participation and
the answer key. CE Update is approved to meet licensure requirements for California, Florida, and other states.
To complete an exam, fill in the answer section found on the back of this page.
Answers must be received by: March 30, 2003
Hematology 0301 Exam
1. Which of the following preanalytical factors would be expected to
artifactually shorten the prothrombin time/INR in warfarin patients?
a. Ethanol intake
b. Delay between sample acquisition and testing
c. Anemia
d. Underfilling the coagulation tube
e. Use of 3.8% citrate instead of 3.2% citrate
2. The following practice would be expected to increase preanalytical
error in the coagulation laboratory:
a. conversion to evacuated blood collection systems from a needle and
syringe technique
b. inversion of sodium citrate tubes vigorously 8 to 10 times for
APTT monitoring of unfractionated heparin therapy
c. centrifuging coagulation specimens to maintain a platelet count below
10,000/microliter
d. requiring patients to rest comfortably for 30 minutes prior to
phlebotomy
e. use of CTAD tubes for unfractionated heparin monitoring
3. Partial-draw sodium citrate tubes can result in an increased
hemorrhagic risk through which of the following mechanisms:
a. artifactual elevation of the platelet count
b. heparin neutralization by protein S activation
c. inactivation of factor VII
d. degradation of factors V and VIII
e. platelet activation
4. Extended delays between phlebotomy and analysis could be
expected to lead to which of the following:
a. shortening of the prothrombin time/INR in warfarin patients
b. enhanced activity of factor VIII
c. APTT shortening in patients receiving unfractionated heparin therapy
d. elevated platelet counts
e. elevated blood-to-additive ratio
5. Cigarette smoking is associated with which of the following effects:
a. thrombin generation
b. depressed plasma fibrinogen levels
c. platelet inhibition
d. reduced hematocrit
e. hemodilution
69
©
laboratorymedicine> january 2003> number 1> volume 34
CE_ExamJan03.qxd
12/4/02
9:44 AM
Page 70
Fax
Mail
Questions?
Because this exam form will be read by a computer,
please print legibly to ensure accuracy. Remove
carefully from magazine and, not using a cover
sheet, fax this side of the form (or photocopy) with
your credit card information authorizing a $15
processing charge for each exam completed to:
The mailed exam form must be accompanied by
a $15 payment for each exam completed to
cover additional handling, payable by credit card
or check and forwarded to:
If you have questions regarding these exams
please call ASCP Customer Service toll free:
(In Illinois 312.738.1336 x1260)
ASCP
Department 77-3462
Chicago, IL 60678-3462
312.850.8817
Identification Information
800.621.4142
(*Required)
*First Name
*Last Name
*Address (example: 1234 West Main Street)
Address (example: Apt# or P.O. Box Number)
*City
*State
*Social Security Number
*Zip
*Country
*Date Completed (mm-dd-yy)
*Telephone Number
(
)
Payment Information
Credit Card
쏔 Visa
Credit Card Number
쏔 Master Card
Expiration Date
쏔 AMEX
Signature (Required on all submissions)
Your Check Number (Required if submitting check)
Make checks payable to ASCP
Answer Section
(Please select the best answer by filling in the box.)
Hematology 0301
A B C D E
1
2
3
4
5
쏔
쏔
쏔
쏔
쏔
쏔
쏔
쏔
쏔
쏔
쏔
쏔
쏔
쏔
쏔
쏔
쏔
쏔
쏔
쏔
쏔
쏔
쏔
쏔
쏔
Survey
Please let us know your opinion of each CE update article you have read in this issue. If you have read more than one, please fill out the survey for each article
read. 1 = Deficient/5 = Excellent. Thank you for your input.
Hematology
0301
1. The article met the objectives stated.
2. The article provided useful technical data or original ideas.
3. The information provided in the article was new and timely.
4. Technical points were explained clearly and were easy to comprehend.
5. The text was organized logically.
6. Illustrations, charts, and tables helped explain text and added to the article's value.
쏔쏔쏔쏔쏔
쏔쏔쏔쏔쏔
쏔쏔쏔쏔쏔
쏔쏔쏔쏔쏔
쏔쏔쏔쏔쏔
쏔쏔쏔쏔쏔
1 2 3 4 5
Comments: (Attach additional pages if necessary.)
laboratorymedicine> january 2003> number 1> volume 34
©