COAGULATION AND TRANSFUSION MEDICINE
Original Article
Prolonged Prothrombin Time and Activated Partial
Thromboplastin Time Due to Underfilled Specimen Tubes
With 109 mmol/L (3.2%) Citrate Anticoagulant
JOHANNA RENEKE, PhD, MD,1 JOAN ETZELL, MD,2 SUSAN LESLIE, MD,2 VALERIE L. NG, PhD, MD,1
AND EUGENE L. GOTTFRIED, MD1
Underfilling of specimen tubes containing 129 mmol/L (3.8%)
buffered citrate prolongs prothrombin time (PT) and activated
partial thromboplastin time (APTT) values. We studied this phe-
nomenon by using 109 mmol/L (3.2%) buffered citrate as the anti-
coagulant, anticipating some increase in tolerance to underfilling.
Venous blood drawn from 12 healthy subjects and 30 patients
receiving long-term oral warfarin therapy was mixed with 109
mmol/L buffered citrate solution in proportions equivalent to fill-
ing the collection tubes from 52% to 100% of capacity. Accurate PT
values were obtained from normal specimens if the tubes were
filled to 65% or more of capacity. Accurate PT results in the thera-
peutic range were obtained only with filling to 80% or more of
The results of prothrombin time (FT) and activated par-
tial thromboplastin time (APTT) tests are known to
become prolonged if there is too high a concentration of
citrate, as may occur if the specimen collection tube is
insufficiently filled, and the proportion of whole blood
to anticoagulant solution is less than 9:1.1-2 Guidelines
based on this observation have been established in most
clinical laboratories for the rrtiriimum acceptable volume
of blood in specimen tubes submitted for coagulation
tests. The National Committee for Clinical Laboratory
Standards recommends a final citrate concentration of
10.9 to 12.9 mmol/L in the specimen, corresponding to
complete filling of a tube containing one of the two com-
mon types of buffered citrate anticoagulant.3
At present, two different concentrations of buffered
citrate solution are in widespread use for coagulation
From the Department of Laboratory Medicine, University of
California San Francisco School of Medicine; 1Clinical Laboratories, San
Francisco General Hospital and 'Clinical Laboratories, Veterans Affairs
Medical Center, San Francisco, California.
Manuscript received June 23, 1997; revision accepted August
22,1997.
Address reprint requests to Dr Gottfried: Clinical Laboratories,
San Francisco General Hospital, 1001 Potrero Ave, San Francisco,
CA 94110.
754
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capacity (using a "moderately sensitive" thromboplastin reagent,
International Sensitivity Index [ISI] = 2.06) or 90% or more of
capacity (using a "highly sensitive" thromboplastin reagent, ISI =
1.01). In contrast, APTT was much less tolerant to underfilling,
with prolonged values observed in most specimens filled to less
than 90% of capacity. No false l o w v a l u e s were observed.
Specimen tubes should be filled to at least 90% of capacity to
avoid falsely elevated PT or APTT results, but values within the
reference range may be acceptable even from underfilled tubes.
(Key words: Prothrombin time; PT; Activated partial thromboplas-
tin time; APTT; International Sensitivity Index; ISI; Underfill; 109
mmol/L citrate; 3.2% citrate) Am J Clin Pathol 1998;109:754-757.
testing throughout the world. In the United States,
about 70% of clinical laboratories use an anticoagulant
solution with a concentration of 129 mmol/L (3.8%),
whereas in Europe, more laboratories prefer a concen-
tration of 109 mmol/L (3.2%).4'5 Previous studies, how-
ever, have demonstrated that the citrate concentration
may have a clinically important effect on PT, interna-
tional normalized ratio (INR), and APTT results. 5 - 7
This observation has led to increasing support for the
adoption of a single standard concentration of buffered
citrate solution for these tests. The 109 mmol/L (3.2%)
concentration is favored, because it is the one generally
used by manufacturers of thromboplastin reagents to
determine the International Sensitivity Index (ISI) used
in the calculation of the INR.
Previous studies d e m o n s t r a t i n g the effects of
underfilling (or decreasing the relative proportion of
blood to anticoagulant) on PT and APTT values used
the higher concentration anticoagulant, viz, 129
mmol/L (3.8%) buffered citrate solution. 1 ' 2 In prepa-
ration for a change to 109 mmol/L (3.2%) buffered cit-
rate anticoagulant solution in our specimen tubes, and
in the expectation of some increase in tolerance to
underfilling with the lower concentration of citrate,
we performed the current study using 109 m m o l / L
(3.2%) buffered citrate solution as the anticoagulant.
 RENEKE ET AL
Prolonged PT and APTT Due
MATERIALS AND METHODS
Study Locale and Patient Population
This study was conducted simultaneously at two
medical centers, San Francisco General Hospital (Lab
A) and the San Francisco Veterans Administration
Medical Center (Lab B). Subjects included 12 healthy
volunteers and 30 outpatients (16 at Lab A and 14 at
Lab B) receiving long-term oral anticoagulant therapy
with warfarin. Specimens collected from healthy vol-
unteers were tested in both laboratories. The speci-
mens d r a w n from outpatients receiving long-term
oral anticoagulant therapy were tested only in the cor-
responding on-site laboratory.
Specimen Collection
All specimens were obtained in accordance with
guidelines specified by the University of California,
San Francisco C o m m i t t e e on H u m a n Research.
Specimens were collected by venipuncture into evacu-
ated specimen tubes (Sherwood Medical, St Louis,
Mo; lot 019476) designed to draw 4.5 mL of venous
blood into a tube containing 0.5 mL of 109 mmol/L
(3.2%) buffered citrate anticoagulant solution (10.9
mmol/L final citrate concentration). Aliquots of these
specimens were transferred to tubes containing addi-
tional 109 mmol/L (3.2%) buffered citrate solution in
amounts sufficient to produce final whole blood cit-
rate concentrations between 12.1 m m o l / L and 20.7
m m o l / L , corresponding to the concentrations that
would be produced in tubes filled to between 90%
and 52% of the nominal capacity, respectively. All
specimens were centrifuged (2500g, 10 minutes, room
temperature) and assayed within 2 hours.
PT and APTT Determinations
PT and APTT were determined in duplicate in
Lab A w i t h an a u t o m a t e d c o a g u l o m e t e r (MLA
1000C, Medical Laboratory Automation,
Pleasantville, NY) and Ortho Brain Thromboplastin
(Ortho Diagnostics Systems, Raritan, NJ; lot OBT280,
ISI = 2.06) a n d O r t h o T h r o m b o s i l ( O r t h o ; lot
1TH240), respectively, and in Lab B with an auto-
mated coagulometer (MLA 1400, Medical Laboratory
Automation) and Ortho RecombiPlasTin (Ortho; lot
RTF157, ISI=1.01) and Ortho Synthasil (lot SSL104),
respectively. A PT or APTT result obtained at a final
citrate concentration greater than 10.9 m m o l / L was
c o n s i d e r e d significantly p r o l o n g e d if the v a l u e
Vol. 1 « • N o . 6
755
Underfilled Specimen Tubes
exceeded the interassay imprecision for a single
specimen by at least threefold.
RESULTS
Subjects included 12 healthy volunteers (6 men and 6
women; age, 23 to 45 years; hemoglobin range, 12.1-15.1
g/dL [121-151 g/L]) and 30 adult outpatients, 16 at Lab
A and 14 at Lab B, receiving long-term oral anticoagu-
lant therapy with warfarin (age range, unavailable;
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hemoglobin range, 10.8-15.3 g/dL [108-153 g/L]). No
polycythemic patients were included in the study.
Prothrombin Time
Specimens obtained from healthy subjects showed
little change in the PT values of tubes filled to at least
65% of capacity, whether a "moderately sensitive"
thromboplastin reagent (Fig 1, A) or a "highly sensi-
tive" reagent (Fig 1, B) was used. Some of the speci-
mens collected from patients receiving long-term oral
anticoagulant therapy, especially those with higher
initial PT values, responded to increasing citrate con-
centration by prolongation of the test result. This
effect became a p p a r e n t at citrate concentrations
equivalent to 70% or less of the tube filled at Lab A
(using a " m o d e r a t e l y sensitive" t h r o m b o p l a s t i n
reagent) and 80% or less at Lab B (using a "highly
sensitive" thromboplastin reagent).
Activated Partial Thromboplastin Time
All APTT values became prolonged with increas-
ing citrate c o n c e n t r a t i o n s (Fig 2). The o b s e r v e d
response was steeper in the specimens with higher
initial APTT values and was more prominent with the
reagent-instrument combination used in Lab B (see
Fig 2, B) than that used in Lab A (see Fig 2, A).
DISCUSSION
Previous studies have demonstrated the effects of a
"short d r a w " on coagulation tests performed on
blood collected into tubes containing 129 m m o l / L
(3.8%) buffered citrate. 1,2 We conducted the current
study to determine the relative tolerance to underfill-
ing in collection tubes containing a lower concentra-
tion of buffered citrate, viz, 109 mmol/L (3.2%).
The lower citrate concentration (109 mmol/L [3.2%])
yielded stable PT results at concentrations equivalent
to those previously reported with 129 mmol/L (3.8%)
buffered citrate. 1,2 PT determined on specimens from
756 COAGULATION AND TRANSFUSION MEDICINE
Original Article

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% Tube Fill % Tube Fill

FIG 1. Prothrombin time (PT) values obtained by adding buffered citrate solution, 109 mmol/L, in amounts corresponding to the concentra-
tions that would be present in tubes filled to the indicated percentage of the rated capacity. Specimens obtained from healthy subjects are
represented by solid circles, and specimens obtained from patients receiving oral anticoagulant therapy are represented by open squares. A,
Tests performed in the San Francisco (Calif) General Hospital with a "moderately sensitive" thromboplastin reagent (International
Sensitivity Index [ISI] = 2.06). B, Tests performed in the San Francisco Veterans Administration Medical Center with a "highly sensitive"
thromboplastin reagent (ISI = 1.01).

1a.=
<

% Tube Fill % Tube Fill

FIG 2. Activated partial thromboplastin time (APTT) values obtained by adding buffered citrate solution, 109 mmol/L, in amounts cor-
responding to the concentrations that would be present in tubes filled to the indicated percentage of the rated capacity. Specimens
obtained from healthy subjects are represented by solid circles, and specimens obtained from patients receiving oral anticoagulant ther-
apy are represented by open squares. A, Tests performed in the San Francisco (Calif) General Hospital with a "moderately sensitive"
partial thromboplastin reagent. B, Tests performed in the San Francisco Veterans Administration Medical Center with a "highly sensi-
tive" partial thromboplastin reagent.

AJCP • June 1998

 RENEKE ET AL
Prolonged PT and APTT Due to Underfilled Specimen Tubes
healthy subjects with a moderately or highly sensitive
thromboplastin reagent were relatively unchanged,
even at citrate concentrations corresponding to grossly
underfilled tubes (ie, 65% of capacity). For the patients
whose PT values were initially prolonged, results
obtained w i t h the " m o d e r a t e l y sensitive" or the
"highly sensitive" thromboplastin reagent remained
relatively unchanged at citrate concentrations corre-
sponding to filling the specimen tubes to 70% or more
or 80% or more of capacity, respectively.
The APTT, in contrast, remained relatively intolerant
to underfilling even with the reduced citrate concentra-
tion of 109 mmol/L. Substantial APTT prolongation was
observed if tubes were filled to less than 90% capacity.
Thus, a prolonged APTT result derived from even a
minimally underfilled specimen tube is unreliable.
It should be noted that the specimens with initially
prolonged PT and APTT values were all derived from
p a t i e n t s receiving oral a n t i c o a g u l a n t t h e r a p y .
Consequently, there are no data to indicate whether
specific factor deficiencies would show a different
response to underfilling.
The extent of prolongation of a PT or APTT result
was dependent on the initial value, the degree of
underfilling, and differences in the reagent-instrument
combination used for the determination. Many of the
specimens with normal initial values showed minimal
prolongation, and some of the PT and APTT results
remained within the reference range. No falsely short-
ened values were observed. In contrast to a prolonged
test result, therefore, a result that falls within the refer-
ence range—even if derived from an underfilled speci-
men that would otherwise have been rejected—may
be regarded as acceptably close to the true value.
Vol. 109 • No. 6
757
In setting a policy for the m i n i m u m acceptable
specimen volume in standard tubes containing citrate
anticoagulant for routine coagulation assays, clinical
laboratories must weigh the need for reliable results
against the inconvenience of a rejected specimen to
patients and health care providers. Our study and
previous studies suggest that underfilled tubes col-
lected exclusively for PT may be acceptable, whereas
the fill requirements for APTT (and perhaps other
coagulation tests) are far more stringent.
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Acknowledgments: We gratefully acknowledge the technical assistance
of Gerry Amato, Michele Cloutier, Ambee Miranda, and John Johnson.
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