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Effects of Tourniquet Technique, Order of Draw, and
Sample Storage on Plasma Fibrinogen, Robert S. Rosenson,* Beth A. Staffileno, and Christine C. Tangney1 (Preventive Cardiol. Center, Lipoprotein and Hemorheol. Res.
Facility, Depts. of Med., Pathol., and 1Clin. Nutr., Rush–
Presbyterian–St. Luke’s Med. Center, 1653 W. Congress
Pkwy., Chicago, IL 60612; *author for correspondence: fax
312-226-7013, email [email protected])
The use of plasma fibrinogen concentrations to predict
clinical ischemic events in subjects with and without prior
evidence of atherosclerosis [1–14] has led to recommendations to incorporate fibrinogen into the cardiovascular
risk profile [15]. Such use of plasma fibrinogen requires
careful attention to potential sources of variability, as only
small (5%) absolute differences in fibrinogen distinguish
subjects who develop cardiovascular disease from those
subjects who do not [16].
Variability in fibrinogen results apart from intraindividual differences is difficult to quantify, because the
few groups who have examined these issues used
different assays for fibrinogen determination. Moreover, the functional assay for fibrinogen is highly
influenced by technician expertise and manipulation
[17]. Previously, we examined within-run analytical
CV, within-person, within-day biological variation, and
a 6-week within-person biological variation with the
Clauss method for fibrinogen determination [16].
We describe here selected effects of sample collection
and storage on fibrinogen results and we propose guidelines for sample collection and storage.
Blood was collected in evacuated tubes (5-mL capacity)
containing 0.5 mL of 0.129 mol/L (3.8%) buffered citrate
solution (Becton Dickinson). Fibrinogen concentrations
were determined by thrombin clottable time with the
Clauss method (Dade Data-Fi, Baxter Diagnostics) [18]
with bovine thrombin (100 kU/L) and 2.8 3 1022 mol/L
sodium barbital in 0.125 mol/L sodium chloride. Duplicate plasma samples were diluted 1:10 (by vol) with an
imidazole buffer (Organon Teknika) and measured at 20and 40-s intervals at 37 °C in a fibrometer (Model BBL,
Becton Dickinson).
All samples were acquired from healthy, nonsmoking
volunteers who were seated $5 min before phlebotomy
[19]. Volunteers were excluded if they had current ill-
nesses, regular and recent use of medications during the
preceding 3 months, or an acute-phase illness in the
preceding 2 months [20].
The impact of tourniquet pressure and duration was
evaluated in a cross-over study design on 38 subjects. The
tourniquet pressure was standardized with a sphygmomanometer and a C-clamp that was applied to the tubing
to maintain the specified pressure. Mean arterial pressure
(MAP) was calculated from three blood pressure recordings obtained at 1-min intervals from seated volunteers.
Low pressure was defined as 40 mmHg tourniquet pressure. High pressure was calculated as the MAP 1 10
mmHg (MAP 1 10) for each subject. Fibrinogen samples
were procured at three intervals of equal duration: 0 –1
min, 2–3 min, and 4 –5 min. For each interval, three
replicate samples were acquired at each interval, and the
mean value is reported.
On the basis of the findings of the tourniquet pressure
and duration study, 14 consecutive blood samples were
acquired from each of 38 subjects at low pressure within
1 min. All but the first acquired sample (which was
discarded) were centrifuged to obtain plasma specimens.
The first four plasma samples were delivered to the
Coagulation Laboratory for analysis within 10 min. The
remaining nine plasma samples were refrigerated at 4 °C.
Three sets of plasma samples were delivered to the
Coagulation Laboratory at 6 h, 24 h, and 48 h.
All statistical analyses were performed with SAS statistical package (ver. 6.10) [21], except for the Friedman test
performed with SPSS for Windows [22]. Results are
expressed as mean 6 SD. The first-stick phenomenon was
evaluated with a two-tailed paired t-test. The Friedman
test was used to examine the influence of the duration of
the tourniquet application on fibrinogen concentrations at
low and high tourniquet pressures, and the influence of
storage time on fibrinogen concentrations. The fibrinogen
concentrations at each tourniquet pressure were compared at each time interval by using the paired-samples
sign test. A 0.05 significance level was used for statistical
tests. A critical difference or relative change value in
fibrinogen measurement was defined as 5% on the basis
of prospective population studies [16]. The percentage of
subjects with a difference in fibrinogen values $5% was
determined at low tourniquet pressure that was applied
for 0 –1 min, 2–3 min, and 4 –5 min.
The first-stick phenomenon was evaluated in 115 subjects by comparing the fibrinogen concentration from the
first-acquired sample (2.58 6 0.47 g/L) with the average
fibrinogen concentration of the next four samples (2.57 6
0.47 g/L). No statistically significant difference was observed (P 5 0.73).
The application of low tourniquet pressure was associated with a progressive increase in the concentration of
plasma fibrinogen (P ,0.00005) across the three sample
acquisition time periods (Table 1). The fibrinogen concentrations were significantly higher for samples acquired
during intervals at 2–3 min (2.68 6 0.70 g/L, P ,0.00005)
and 4 –5 min (2.70 6 0.71 g/L, P 5 0.0005) compared with
those acquired during the first minute (2.62 6 0.63 g/L).
689
Clinical Chemistry 44, No. 3, 1998
Table 1. Influence of tourniquet pressure on fibrinogen concentration.a
Tourniquet duration, min
Tourniquet pressure
Low
High
P valuec
a
b
c
0–1
2–3
4–5
P value for trendb
2.62 6 0.63
2.59 6 0.42
0.87
2.68 6 0.70
2.62 6 0.41
0.13
2.70 6 0.71
2.69 6 0.45
0.62
,0.00005
,0.00005
Fibrinogen concentrations (g/L) are expressed as mean 6 SD.
P value reflects the comparison of fibrinogen values obtained at the three time intervals of tourniquet application by the Friedman test.
P value reflects the comparison of fibrinogen values at a particular time interval between low and high pressures with a paired-samples sign test.
There was no statistically significant difference between
samples obtained at 2–3 min and those acquired at 4 –5
min (P 5 0.23).
Similarly, samples acquired under conditions of high
tourniquet pressure increased across the three sampling
time periods (P ,0.00005). The concentration of fibrinogen was greater for samples acquired at 4 –5 min (2.69 6
0.45 g/L) than those obtained within the first minute after
tourniquet application (2.59 6 0.42 g/L, P ,0.00005) or at
2–3 min (2.62 6 0.41 g/L, P ,0.00005). There was no
statistically significant difference between samples acquired at 2–3 min and those obtained within the first
minute (P 5 0.11).
Mean fibrinogen was not influenced by the tourniquet
pressure at 0 –1 min (P 5 0.87), 2–3 min (P 5 0.13), or 4 –5
min (P 5 0.62) (Table 1).
Fibrinogen remained stable at 4 °C for 48 h (P 5 0.11)
with mean values of 2.53 6 0.35 g/L at baseline, 2.51 6
0.36 g/L at 6 h, 2.54 6 0.40 g/L at 24 h, and 2.57 6 0.45
g/L at 48 h.
The current recommended procedure for acquiring
samples for fibrinogen analysis by the Clauss method
mandates that the first sample be discarded [23–25].
Discarding of the first tube of blood or the “first-stick
phenomenon”, although not evaluated, has been considered standard practice. This recommendation has been
promulgated because tissue thromboplastin could interfere with coagulation assays by activating the intrinsic
pathway. For example, the Atherosclerosis Risk in Communities (ARIC) investigators clearly state that the first 3
mL obtained after venipuncture must be discarded [25].
We demonstrate that the first acquired sample can be
submitted for fibrinogen analysis.
We have found several published reports that investigate the influence of tourniquet pressure or duration on
fibrinogen measurements. The Cardiovascular Health
Study investigators reported tourniquet use for only a
brief duration in nearly all of the 5000 samples, but 1.2%
of phlebotomies required tourniquet application for .2
min [26]. The influence of longer periods of venous
occlusion on fibrinogen concentrations was evaluated in
several studies. In 25 hyperlipidemic patients, venous
occlusion for 10 min resulted in a 3.7% increase in
fibrinogen (3.27 to 3.39 g/L, P 5 0.052) [27]. Larger
increases in fibrinogen after 10 min of tourniquet application were reported for 20 hypertriglyceridemic patients
(4.86 to 6.12 g/L or 26%, P ,0.005) and 20 normolipidemic
patients (3.42 to 3.96 g/L or 16%, P ,0.005) [28]. A 14.5%
increase in fibrinogen concentration was reported after 15
min of tourniquet application (3.24 to 3.71 g/L, P ,0.001)
in 28 healthy subjects [29]. These studies support our
results that the duration of venous occlusion has a significant influence on fibrinogen concentrations.
The stability of fibrinogen throughout storage is important for multicenter studies that involve delays from
shipping and sample processing. We demonstrate that
plasma samples stored at 4 °C remain stable for 48 h. The
ARIC investigators examined the impact of temporary
storage time on fibrinogen concentration assayed by the
Clauss method (1 week at 270 °C vs 30 min before the
assay at 4 °C) [26]. They found no significant difference
in fibrinogen values with such conditions. Similar findings were obtained in plasma samples stored at 0, 1, 4,
and 7 days and assayed fibrinogen by radial immunodiffusion [30].
In this report, we identify tourniquet duration as a
critical determinant of preanalytical fibrinogen variability.
On the basis of a critical fibrinogen variability of 5%, the
application of low tourniquet pressure for 2–3 min resulted in 6 of 38 subjects with differences that exceeded
5%, and this doubled (12 of 38) with 4 –5 min of tourniquet
application.
We conclude that variability in fibrinogen measurements can be minimized by acquiring samples within 1
min after venipuncture and analyzing samples that have
been stored at 4 °C up to 48 h. The magnitude of tourniquet pressure has no influence on fibrinogen concentrations, and the first acquired blood sample can be used for
fibrinogen analysis.
This work was supported by an unrestricted research
grant from Leo Burnett and Co., Chicago, IL. We acknowledge the contributions of Amy E. McCormick for sample
processing, Susan Shott for statistical analyses, and Mary
Lou Briglio for manuscript preparation.
This paper was presented on May 4 – 6, 1996 at the
Third International Conference on “Fibrinogen: A Cardiovascular Risk Factor” in Ulm, Germany.
690
Technical Briefs
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