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

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Underfilling of specimen tubes containing 129 mmol/L (3.8%) capacity (using a "moderately sensitive" thromboplastin reagent,
buffered citrate prolongs prothrombin time (PT) and activated International Sensitivity Index [ISI] = 2.06) or 90% or more of
partial thromboplastin time (APTT) values. We studied this phe- capacity (using a "highly sensitive" thromboplastin reagent, ISI =
nomenon by using 109 mmol/L (3.2%) buffered citrate as the anti- 1.01). In contrast, APTT was much less tolerant to underfilling,
coagulant, anticipating some increase in tolerance to underfilling. with prolonged values observed in most specimens filled to less
Venous blood drawn from 12 healthy subjects and 30 patients than 90% of capacity. No false l o w v a l u e s were observed.
receiving long-term oral warfarin therapy was mixed with 109 Specimen tubes should be filled to at least 90% of capacity to
mmol/L buffered citrate solution in proportions equivalent to fill- avoid falsely elevated PT or APTT results, but values within the
ing the collection tubes from 52% to 100% of capacity. Accurate PT reference range may be acceptable even from underfilled tubes.
values were obtained from normal specimens if the tubes were (Key words: Prothrombin time; PT; Activated partial thromboplas-
filled to 65% or more of capacity. Accurate PT results in the thera- tin time; APTT; International Sensitivity Index; ISI; Underfill; 109
peutic range were obtained only with filling to 80% or more of mmol/L citrate; 3.2% citrate) Am J Clin Pathol 1998;109:754-757.

The results of prothrombin time (FT) and activated par- testing throughout the world. In the United States,
tial thromboplastin time (APTT) tests are known to about 70% of clinical laboratories use an anticoagulant
become prolonged if there is too high a concentration of solution with a concentration of 129 mmol/L (3.8%),
citrate, as may occur if the specimen collection tube is whereas in Europe, more laboratories prefer a concen-
insufficiently filled, and the proportion of whole blood tration of 109 mmol/L (3.2%).4'5 Previous studies, how-
to anticoagulant solution is less than 9:1.1-2 Guidelines ever, have demonstrated that the citrate concentration
based on this observation have been established in most may have a clinically important effect on PT, interna-
clinical laboratories for the rrtiriimum acceptable volume tional normalized ratio (INR), and APTT results. 5 - 7
of blood in specimen tubes submitted for coagulation This observation has led to increasing support for the
tests. The National Committee for Clinical Laboratory adoption of a single standard concentration of buffered
Standards recommends a final citrate concentration of citrate solution for these tests. The 109 mmol/L (3.2%)
10.9 to 12.9 mmol/L in the specimen, corresponding to concentration is favored, because it is the one generally
complete filling of a tube containing one of the two com- used by manufacturers of thromboplastin reagents to
mon types of buffered citrate anticoagulant.3 determine the International Sensitivity Index (ISI) used
At present, two different concentrations of buffered in the calculation of the INR.
citrate solution are in widespread use for coagulation 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
From the Department of Laboratory Medicine, University of the higher concentration anticoagulant, viz, 129
California San Francisco School of Medicine; 1Clinical Laboratories, San mmol/L (3.8%) buffered citrate solution. 1 ' 2 In prepa-
Francisco General Hospital and 'Clinical Laboratories, Veterans Affairs ration for a change to 109 mmol/L (3.2%) buffered cit-
Medical Center, San Francisco, California.
rate anticoagulant solution in our specimen tubes, and
Manuscript received June 23, 1997; revision accepted August in the expectation of some increase in tolerance to
22,1997. underfilling with the lower concentration of citrate,
Address reprint requests to Dr Gottfried: Clinical Laboratories,
San Francisco General Hospital, 1001 Potrero Ave, San Francisco,
we performed the current study using 109 m m o l / L
CA 94110. (3.2%) buffered citrate solution as the anticoagulant.

754
 RENEKE ET AL 755
Prolonged PT and APTT Due Underfilled Specimen Tubes

MATERIALS AND METHODS exceeded the interassay imprecision for a single

specimen by at least threefold.
Study Locale and Patient Population
RESULTS
This study was conducted simultaneously at two
medical centers, San Francisco General Hospital (Lab Subjects included 12 healthy volunteers (6 men and 6
A) and the San Francisco Veterans Administration women; age, 23 to 45 years; hemoglobin range, 12.1-15.1
Medical Center (Lab B). Subjects included 12 healthy g/dL [121-151 g/L]) and 30 adult outpatients, 16 at Lab
volunteers and 30 outpatients (16 at Lab A and 14 at A and 14 at Lab B, receiving long-term oral anticoagu-
Lab B) receiving long-term oral anticoagulant therapy lant therapy with warfarin (age range, unavailable;

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with warfarin. Specimens collected from healthy vol- hemoglobin range, 10.8-15.3 g/dL [108-153 g/L]). No
unteers were tested in both laboratories. The speci- polycythemic patients were included in the study.
mens d r a w n from outpatients receiving long-term
oral anticoagulant therapy were tested only in the cor- Prothrombin Time
responding on-site laboratory.
Specimens obtained from healthy subjects showed
Specimen Collection little change in the PT values of tubes filled to at least
65% of capacity, whether a "moderately sensitive"
All specimens were obtained in accordance with thromboplastin reagent (Fig 1, A) or a "highly sensi-
guidelines specified by the University of California, tive" reagent (Fig 1, B) was used. Some of the speci-
San Francisco C o m m i t t e e on H u m a n Research. mens collected from patients receiving long-term oral
Specimens were collected by venipuncture into evacu- anticoagulant therapy, especially those with higher
ated specimen tubes (Sherwood Medical, St Louis, initial PT values, responded to increasing citrate con-
Mo; lot 019476) designed to draw 4.5 mL of venous centration by prolongation of the test result. This
blood into a tube containing 0.5 mL of 109 mmol/L effect became a p p a r e n t at citrate concentrations
(3.2%) buffered citrate anticoagulant solution (10.9 equivalent to 70% or less of the tube filled at Lab A
mmol/L final citrate concentration). Aliquots of these (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
specimens were transferred to tubes containing addi- reagent) and 80% or less at Lab B (using a "highly
tional 109 mmol/L (3.2%) buffered citrate solution in sensitive" thromboplastin reagent).
amounts sufficient to produce final whole blood cit-
rate concentrations between 12.1 m m o l / L and 20.7 Activated Partial Thromboplastin Time
m m o l / L , corresponding to the concentrations that
would be produced in tubes filled to between 90% All APTT values became prolonged with increas-
and 52% of the nominal capacity, respectively. All 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
specimens were centrifuged (2500g, 10 minutes, room response was steeper in the specimens with higher
temperature) and assayed within 2 hours. initial APTT values and was more prominent with the
reagent-instrument combination used in Lab B (see
PT and APTT Determinations Fig 2, B) than that used in Lab A (see Fig 2, A).

PT and APTT were determined in duplicate in DISCUSSION

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, Previous studies have demonstrated the effects of a
Pleasantville, NY) and Ortho Brain Thromboplastin "short d r a w " on coagulation tests performed on
(Ortho Diagnostics Systems, Raritan, NJ; lot OBT280, blood collected into tubes containing 129 m m o l / L
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 (3.8%) buffered citrate. 1,2 We conducted the current
1TH240), respectively, and in Lab B with an auto- study to determine the relative tolerance to underfill-
mated coagulometer (MLA 1400, Medical Laboratory ing in collection tubes containing a lower concentra-
Automation) and Ortho RecombiPlasTin (Ortho; lot tion of buffered citrate, viz, 109 mmol/L (3.2%).
RTF157, ISI=1.01) and Ortho Synthasil (lot SSL104), The lower citrate concentration (109 mmol/L [3.2%])
respectively. A PT or APTT result obtained at a final yielded stable PT results at concentrations equivalent
citrate concentration greater than 10.9 m m o l / L was to those previously reported with 129 mmol/L (3.8%)
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 buffered citrate. 1,2 PT determined on specimens from

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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 757
Prolonged PT and APTT Due to Underfilled Specimen Tubes

healthy subjects with a moderately or highly sensitive In setting a policy for the m i n i m u m acceptable
thromboplastin reagent were relatively unchanged, specimen volume in standard tubes containing citrate
even at citrate concentrations corresponding to grossly anticoagulant for routine coagulation assays, clinical
underfilled tubes (ie, 65% of capacity). For the patients laboratories must weigh the need for reliable results
whose PT values were initially prolonged, results against the inconvenience of a rejected specimen to
obtained w i t h the " m o d e r a t e l y sensitive" or the patients and health care providers. Our study and
"highly sensitive" thromboplastin reagent remained previous studies suggest that underfilled tubes col-
relatively unchanged at citrate concentrations corre- lected exclusively for PT may be acceptable, whereas
sponding to filling the specimen tubes to 70% or more the fill requirements for APTT (and perhaps other
or 80% or more of capacity, respectively. coagulation tests) are far more stringent.
The APTT, in contrast, remained relatively intolerant

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to underfilling even with the reduced citrate concentra- Acknowledgments: We gratefully acknowledge the technical assistance
of Gerry Amato, Michele Cloutier, Ambee Miranda, and John Johnson.
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. REFERENCES
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