Intra-individual variability of fibrinogen levels

J Clin Epidemiol Vol. 50, No. 4, pp. 393-399, Copyright 0 1997 Elsevier Science Inc.

0895s4356/97/$17.00 PII SO895~4356(97)00044-9

1997

ELSEVIER

Intra-Individual

Variability

D. De Buap.er,‘*G. ‘DEPARTMENT

OF PUBLIC

HEALTH,

UNIVERWT

of Fibrinogen

Levels

De Bucket,’ L. Braeckmun,’ and G. Baele* OF GENT, HOSPITAL,

BELGIUM,

GENT,

AND

‘COAGULATION

LABORATORY,

UNIVERSTY

BELGIUM

ABSTRACT.

Elevated fibrinogen concentrations are recognized as playing an important role in the pathogenesis of atherosclerosis. In the framework of a risk factor survey in 342 middle-aged working men, screened twice over a period of five months, plasma fibrinogen levels were found to be fairly unstable as large discrepancies between both measurements were observed. Due to a substantial proportion of within-person variability, the reliability coefficient was only R = 0.56. Repeatability was highest in higher educated and physically more active men. Our data suggest that the impact of elevated fibrinogen levels on the development of ischemic heart disease and stroke, is likely to be under-estimated. J CLIN EPIDEMIOL 50;4:393-399, 1997. 0 1997 Elsevier Science Inc.

KEY WORDS.

Fibrinogen,

reproducibility,

regression

INTRODUCTION During the last decades several longitudinal epidemiological studies [l-5] have identified an elevated plasma fibrinogen level as a major risk factor for developing ischemic heart disease (IHD) and stroke. This relation is found to be independent of other major cardiovascular risk factors and may be stronger than the association between serum cholesterol and IHD. Some authors [6-91 even suggest that impaired fibrinolysis might be an important link between lifestylerelated risk factors and the subsequent incidence of IHD. For example, Meade et al. [lo] conclude that a substantial part of the relation between smoking and IHD may be mediated through the fibrinogen concentration. Obviously one of the key issues in evaluating these associations is how precisely an individual is characterized through a single fibrinogen measurement. If the fibrinogen level is to be included in an individual patient’s cardiovascular risk profile, it ought to be reproducible over time within certain limits, not merely over a short period but also if measured months apart. In situations where the within-person variability of a biological measure is relatively high compared to its between-person variability, it is well recognized that the association of the result based upon a single measurement with subsequent events may well be underestimated, a phenomenon known as “regression dilution bias” [11,12]. We used replicate data from a risk factor survey in a cohort of middle-aged working men to meet the following objectives: *Address reprint requests to: Dirk De Bacquer, Department of Public Health, University Hospital, De Pintelaan 185-Block A, B-9000 Gent, Belgium. Accepted for publication on 6 January 1997.

dilution

1. to evaluate the intra-individual variability of plasma fibrinogen values by estimating the range of differences to be expected over a 5month period 2. to identify subgroups according to other risk factors in which this intra-individual variability is more pronounced The potential role of individual spontaneous variability in the interpretation of results from clinical and epidemiological studies is discussed.

METHODS Study Population As part of a nutritional intervention study in male employ ees aged 35 to 59 years, four Belgian companies volunteered to participate in a longitudinal study at the worksite. Two of these factories were allocated as experimental groups while the other two served as control companies not being subjected to any form of intervention. The latter two control companies consisted of the subjects from which data were abstracted for the present paper. Out of the 409 eligible employees from these two factories, 342 (participation rate 84%) had fibrinogen measured twice with a time interval of about five months. All first blood samples were drawn in the period January-March 1993 for the first company and October-November 1993 for the second, while second blood samples were gathered in the periods June-July 1993 and February 1994, respectively. Blood

Samples

Venous blood samples were drawn by clean venipuncture from the non-fasting subjects in sitting position. Specimens

394

D. De Bacquer et al.

were collected in VacutainerR tubes of 5 ml, containing 0.129 M trisodium citrate (1: 10 vol), for fibrinogen determination. Personnel were instructed to fill the tubes correctly and not to stop drawing before the tube was as full as intended. Incorrectly filled tubes were discarded. No hematocrit was measured to adjust the anticoagulant volume or to correct the fibrinogen results to the in viva level. All overtly hemolyzed or lipemic samples were discarded to avoid interference with the optical density method used in measuring fibrinogen. Blood samples were also drawn in 10 ml VacutaineF tubes for total and HDL cholesterol measurements. All tubes were transferred the same day to the central laboratory, shipped on ice in a cooling box. They were centrifuged there immediately for 20 minutes at 3000 g at room temperature. Serum samples were frozen at minus 70°C until analyzed for total and HDL cholesterol. Plasma fibrinogen determinations were carried out the next day.

Luboratorv

Measurements

All fibrinogen measurements were prothrombin time (PT) derived on the same ACL-200 nephelometric centrifugal analyzer. Light scattering, before, during and after clot formation, was continuously registered. The light scattering increase reached at equilibrium is proportional to the fibrin concentration, and therefore to the total clottable fibrinogen. Calibration of the ACL analyzer was done every run using calibration plasma from Instrumentation Laboratory. Regular (three per month) obligatory external quality control measurements organized by the Belgian government were performed. Over the study period, 10 control plasma samples were distributed to 35 reference laboratories using the same nephelometric method. The measurements performed on these samples in our coagulation laboratory revealed differences of 0 to 6% with the median of the 35 external measurements. Method-specific (nephelometric) coefficients of variation over the study period ranged from 7 to 11% for the 10 control samples. Total serum cholesterol and HDL cholesterol were assayed enzymatically with a Technicon Autoanalyzer. HDL cholesterol was measured on serum after precipitation with manganese chloride and phosphotungstate.

Other

ees of higher educational level were those having finished their second school education. Subjects were initially grouped into four levels of physical activity, based on the reported frequency and intensity of exercise, sporting and other leisure activities. Since the beneficial effect of exercise on fibrinogen is largest in the most active subjects [13], men with the highest energy expenditure level were compared to individuals less physically active. Body mass index (BMI) was calculated as weight (in kilograms) divided by height (in meters) squared. Systolic and diastolic blood pressures (SBP and DBP, respectively) were each measured twice with a random-zero sphygmomanometer after a resting time of 5 min while the subjects were seated. The average of the two blood pressure measurements was used in our analysis. Hypertension was defined according to the WHOguidelines [14] or based upon a positive answer to the question: “Are you presently using medication for high blood pressure?” Individuals with one or both of these conditions were classified as being hypertensive.

Measurements

Data on smoking habits, physical activity, education, and parental history of myocardial infarction were collected by self-administered questionnaires and checked by interview. Average daily alcohol consumption was calculated from questions probing for the usual weekly intake of beer, wine and spirits. Current smokers included cigar, cigarette and pipe smokers. Coding of educational level was based on the highest diploma or professional training received. Employ-

Statistical

Methods

To estimate the size of individual time variation in fibrinogen concentration, the method as proposed by Bland and Altman [15] was applied by constructing 95% confidence intervals for the observed intra-individual changes, under the assumption that no systematic differences existed between the two measurements. To fully meet the constantvariance assumption a natural logarithmic transformation was used. The repeatability coefficient, as adopted by the British Standards Institution [16], was defined as twice the standard deviation (around zero) of the sample differences between both measurements. This coefficient measures the extent to which the replicates agree with each other. Under the assumption that no systematic within-person error is present, repeated measurements on an individual are expected to fluctuate at random about the true value. This error may well represent true biological variation as well as method-dependent error. In our study, such methodrelated variability can be the result of several factors such as blood sampling technique, transport and storage of blood samples, laboratory instrument variability and data entry. Decomposition of the total variability of the pool of fibrinogen measurements in between-person (&) and withinperson (c&r) components, was done using analysis of variance techniques based upon the fitting of a random effects model [ 171. The reliability coefficient R (intra-class correlation) is defined as the proportional contribution of the between-person variation to the total variance:

Obviously this measure is inversely related to the ratio of within-person to between-person variation and varies between 0 and 1. In case of two replicate samples with no

Intra-Individual

Variability

of Fibrinogen

395

Levels

systematic bias, the reliability coefficient can be interpreted as a correlation between the two series of measurements. The within-person coefficient of variation was calculated as: cvwp

= loo*owP/~(o~P

+ $),

where p is estimated by the sample mean. Although the homoscedasticity assumption (within-person variance constant over the whole range of fibrinogen values) was not fully met in our data, we chose nevertheless to model the untransformed fibrinogen values since variance components and reliability coefficients do not retransform easily. Moreover, in our study the reliability coefficients obtained on the log-transformed fibrinogen values were exactly the same as for the untransformed data. The different variance estimates were obtained using the NESTED-procedure in the SAS software package [18].

RESULTS

At the time of first measurements, the mean age of the 342 participants was 43.7 years (SD = 4.9 yr) and 34.5% of them were current smokers while 38.0% reported themselves as former smokers. The median alcohol intake amounted to 14.2 grams per day and their mean body mass index was 26.5 kg/m* (SD = 3.5 kg/m*). According to the definition described above, 25.2% of these working men were labeled as hypertensive. Average total and HDL cholesterol levels were 226 mg/dl and 43.7 mg/dl, respectively. One-quarter of the group (26.5%) reported a history of myocardial infarction in at least one of their parents. The individuals under study had their second blood sample taken after a median time period of 4.9 months from their first, with a range from 4.3 to 5.3 months. Over this period, lifestyles remained fairly constant as no changes in daily alcohol consumption or quantity of cigarettes smoked was observed. The two plasma fibrinogen distributions at these measurement occasions are characterized in Table 1. Clearly these distributions at time points one and two are comparable regarding measures of location (means = 315

TABLE 1. Distribution ment occasions (n

of fibrinogen = 342)

levels Fibrinogen (mEId

Mean SD SEM Median Interquartile Range

range

at two

measure-

levels

First

Second

Difference

316.6

314.4

72.2 3.90 305 265-355

66.4 3.59 300 270-345

175-620

195-570

2.21 65.2 3.53 0 -30-35 -315-282

mg/dl) and spread (SD = 70 mg/dl). The mean of individual differences in fibrinogen level between both examinations is 2.21 mg/dl with a 95% confidence interval of -4.70 to 9.12 mg/dl. Hence it may be concluded that fibrinogen levels are not significantly different between the two timepoints 5 months apart; there is no systematic bias. When first measurements were taken into account, 5.8% of the individuals had a fibrinogen level exceeding the limit of 450 mg/dl, which has been put forward in clinical practice to characterize risk of IHD, while on the second occasion 5.0% reached this threshold. In Fig. lA, a scatter plot of first versus second fibrinogen values is shown. Obviously data points are clustered around the first diagonal line although rather large discrepancies are observed. To get a better insight in the magnitude of the deviations, these are plotted against the means of the two measurements in Fig. 1B. Since the spread of the differences increased slightly over the range of measurements, data are presented after logarithmic transformation. The mean of the differences on a log-scale is 0.0031 and their standard deviation is 0.1928. Hence 95% of these deviations may be expected to lie in the interval of -0.3749 to +0.3811. Taking the antilog of these limits of agreement reveals that first measured fibrinogen levels may well differ from the second ranging from 31% below to 46% above. The 95% confidence intervals for these limits of agreement are estimated to be 29%-34% for the lower limit and 41%-52% for the upper limit. The repeatability coefficient for the untransformed fibrinogen values, as defined by the British Standard Institution, is 130 mg/dl (= 2 X 65.2 mg/dl). Analysis of the frequency distribution of the absolute discrepancies between both sets of measurements revealed that 32.7% of all subjects had differences larger than 50 mg/dl and 8.5% showed changes of more than 100 mg/dl. (For 72 individuals (2 1.1%) a difference of 70 mg/dl or larger was noted. From Table 1 it can be seen that for these subjects their within-person deviation exceeds the between-person standard deviation of the genuine fibrinogen distribution.) To judge whether any particular subgroups could be differentiated, Table 2 displays the repeatability coefficients for fibrinogen measurements in some strata defined on the basis of some established cardiovascular risk factors. No major differences in these coefficients are found except for the relatively low intra-subject variability in individuals with a higher education and in those reporting being highly physically active. On the other hand, workers with a lowered HDL cholesterol level show a much larger spread in their fibrinogen deviations as can be concluded from the repeatability coefficient of 162 mg/dl. In our data, changes in cardiovascular profile did not seem to be associated with changes in fibrinogen levels. Differences over the 5-month period in body mass index, alcohol consumption, daily number of cigarettes smoked, blood

396

D. De Bacquer

600

TABLE ments

2. Repeatability coefficients in several subgroups

of fibrinogen

n Age

et al.

measureRepeatability coefficient (mddl)

(yr)

<45 245

214 128

134 124

199 143

134 126

224 118

126 138

268 74

130 130

282 60

137 93

297 45

135 92

250 90

123 135

255 86

133 117

253 89

129 134

73 269

162 120

BMI (kg/m*) ~27 227 .

.

Smoking No

-

.

Yes

‘

Alcohol

intake

(g/day)

<30 230

100

I 300

I 200 First

fibrinogen

I 400

I 600

measurement

Img/dl)

I 600

Educational level Lower Higher Physical activity Lower High Parental history of AMI No

Yes Hypertension No

.

*.

Yes

*

Total

cholesterol

(mg/dl)

~250 2250 HDL

.

- . . ..* . . ..

-

and blood lipid levels showedno appreciablecorrelations with changesin fibrinogen. Five subjects(1.5%) reported quitting smoking between both measurementoccasions. Their changes in fibrinogen level were however comparable to the group of those remaining smokers. Table 3 provides uswith information about the decomposition of the total variance for fibrinogen levels in its between-person and within-person components. A relatively high proportion of the total variability can be attributed to

pressure

.

5.2

B FIGURE (Second differences brinogen

5.4 Average

(mg/dl)

.

.

-11

cholesterol

<35 235

5.6 of two

5.8 log(fibrinogen)

6

6.2

6.4

values

1. (A) Scatter plot of observed fibrinogen versus first measurement), and (B) scatter between measurements versus averages levels (after logarithmic transformation).

levels plot of of fi-

TABLE liability

3. Variance coefficient

components

of fibrinogen

levels

and

re-

Fibrinogen h&-W Sample

mean

Total variance Between-person variance Within-person variance Reliability coefficient R Within-person standard deviation Within-person coefficient of variation

315.5

4808.8 2685.7 2123.0

0.56 46.1 14.4%

Intra-Individual

TABLE

4.

Variability

Agreement

of groups with elevated

(1-W-b

300 350 400 450 500

110 219 278 311 329

397

Levels

Number

Cutpoint hzW

“Number cutpoint;

of Fibrinogen

fibrinogen

level based on different

cutpoints

of subjects in each cell”

(l+w-)

(l-)(2+)

138 51 18 6 3

42 33 19 11 4

(l+w-)

Kappa

52 39 27 14 6

0.45 0.45 0.36 0.29 0.36

95% 0.35-0.54 0.34-0.55 0.22-0.51 0.08-0.49 0.05-0.67

of subjects in each cell: ( 1-) = first fibrinogen measurement lower than cutpoint; (1 +) = first fibrinogen measurement equal to or higher (2-) = second fibrinogen measurement lower than cutpoint; (2+) = second fibrinogen measurement equal to or higher than cutpoint.

intra-individual variability (44%). This is reflected by the quite low value of the reliability coefficient, R, which gives the proportional variation due to between-person variability. As this proportion is not even 60%, it may be concluded that the reproducibility of our fibrinogen determinations is rather weak. The within-person coefficient of variation for the fibrinogen measurements as estimated from our replicate sample was 14.4%. Performing variance component decomposition in the different subgroups as defined in Table 2, revealed that most reliability coefficients were comparable in size (0.50 to 0.60). Only the subgroups that were characterized by a high physical activity or a higher educational level had notable values for R of more than 0.70 (0.72 and 0.73, respectively). In contrast, the worst retest reliability (R = 0.44) was found in those subjects having a HDL cholesterol level lower than 35 mg/dl at their first examination. As mentioned earlier, 5.8% of first fibrinogen measurements exceeded the limit of 450 mg/dl, while on the second occasion 5.0% of all subjects had a value higher than this cut-off. Finally, in Table 4 we see how subgroups, defined on the basis of several cutpoints for plasma fibrinogen, relate to each other. As all values of the kappa-statistic are below 0.50, there might well be only poor agreement if individuals are characterized on the basis of a threshold limit for fibrinogen defining risk. DISCUSSION Replicate measurements were available in 342 middle-aged working men with a time lag of about five months after exclusion of incorrectly taken samples, overtly hemolyzed or grossly lipemic samples, or availability of only a single sample. The large range of individual changes found between both series of measurements and the relatively low value for the reliability coefficient, R, reveal that plasma fibrinogen as a hemostatic parameter is only poorly reproducible. Interestingly the retest reproducibility is better for higher educated and physically more active men, but weaker for persons having a lowered HDL cholesterol. The accuracy of a fibrinogen measurement may be improved by adapting a standardized methodology for blood

CI

than

sampling with respect to the sample volume in proportion to the fixed volume of citrate anticoagulant. Small changes in the total blood volume sampled in the Vacutainera tubes due to some loss of the vacuum or to incomplete filling, may be responsible for some variability in the fibrinogen values. This kind of preanalytical error is not easy to avoid in hemostasis testing. Blood drawing with a syringe technique, although allowing better control of the sample volume, was not considered at the start of our study as the use of a Vacutainera system is common practice in our country. If the maximum amount of blood is drawn into the tube and it has a relatively low hematocrit, the measured fibrinogen will approach the in viwo level. If individual hematocrit measurements can be made, it would be appropriate to adjust the measured fibrinogen level to the in viva level. In this study hematocrit measurements were not available. The PT-derived fibrinogen measurement with the ACL apparatus was chosen as it was easily available, rapid, and the most economical method. In a comparison of six different methods, the von Clauss and the PT-derived methods were considered as reliable although an adequate calibration procedure was indispensable [19]. As mentioned in the method section, calibration was done every run and external quality control measurements were regularly performed. The latter revealed differences of 0 to 6% with the median of 35 participating laboratories. A few other studies have dealt with the intra-individual variability of hemostatic factors. In some of them withinperson variation was further decomposed in method-related within-person variation and individual biological variation. In the NPHS study (Northwick Park Heart Study), Meade and North [20] reported in their substudy of 32 subjects with an average of 10 fibrinogen measurements over a five-year period, that the percentage of variance attributable to laboratory error (gravimetric method) was estimated to be 5% (CV = 4%), much lower than for any other hemostatic variable, but comparable to that for serum cholesterol. Similar findings were obtained in a study of 39 individuals that were measured on three occasions within the framework of the ARIC study (Atherosclerosis Risk in Communities). Chambless et al. [21] estimated the proportional methodrelated variance as 14% of the total variance (CV = 7%).

398

This method-variability does not only cover laboratory handling and assay (nephelometric), but also encompasses field center processing, shipping and data transfer. Using data from six healthy men obtained repeatedly throughout a period of one month, Blombsck et al. [22] report that methodvariation for fibrinogen, measured with a polymerization test, is among the lowest compared to other hemostatic components (CV = 3.8%). Thompson et al. [23] report on the sources of variability in coagulation factor assays in a study of 14 volunteers giving blood samples approximately bimonthly for three years. These samples taken on each occasion were analyzed using a gravimetric method in a single analytical batch. The authors estimated the proportional method error variance for fibrinogen measurements to be 4% of the total within-person plus method error variance. In their report, the contribution of between-batch differences to the total variation was found to be less than 1% for plasma fibrinogen determination. In general, the results described above suggest that the relative impact of methodvariation is rather low, proving that blood sampling methods and laboratory analysis techniques are now well standardized. To compare our results on retest reliability of fibrinogen measurements with those obtained from others, two issues have to be taken into account. First, the number of measurement occasions and the length of follow-up period for these other studies differ from ours, ranging from 1 month (ARIC study) to 5 years (NPHS study). Obviously in longer studies, changes in lifestyle-related correlates of fibrinogen are more likely to occur. In our 5-month study period, changes in some of these associated factors such as smoking behavior, body weight and alcohol consumption, were however unrelated to changes in fibrinogen level, as well as were changes in blood pressure and blood lipid levels. The explanation that potential dietary alterations might play an important role in fibrinogen variability is speculative as plasma fibrinogen is not appreciably influenced by dietary changes over a short period. Fluctuations of individual fibrinogen levels can neither be explained by seasonal effects, as a study [24] of over 12,000 subjects did not observe any relevant change in mean fibrinogen levels during different seasons. In a study of healthy Japanese adults [25], plasma fibrinogen was not found to have a diurnal variation. Time of sampling influenced fibrinolytic variables but not plasma fibrinogen. In evaluating long-term changes in hemostatic factors, aging is most likely to result in systematic changes only. However, since fibrinogen is produced in the liver as an acute phase reacting protein, the explanation that transient elevations in fibrinogen levels may therefore represent an acute state of inflammation or infection might be worth considering. As mental stress has been shown [8] to affect fibrinogen levels, the presence of an acute stressor in or outside the working environment could also be partly responsible for short-term increases in fibrinogen levels. Comparing reliability coefficients between studies will

D. De Bacquer et al.

also be confounded through the characteristics of the sample of subjects they are based on, since these coefficients reflect the relative contribution of between-person variability. In a very homogeneous sample with little variation between individuals regarding for example age and smoking behavior, reliability coefficients are more likely to be low. In their study of 17 healthy, young, non-obese male students over a l-year period, Marckmann et al. [26] report the interpersonal contribution to the total variation to be merely 33%. Similarly, in 219 patients suffering from angina pectoris with repeated measurements made 2.5 years apart, reproducibility for fibrinogen levels was also found [27] to be low (R = 0.45). Our results (R = 0.56) agree well with those derived from the population-based sample in the NPHS study [20], in which relative between-person variability over 5 years was estimated at 54%. Using data from the ARIC study, Chambless et al. 1211 concluded fibrinogen to be intermediate in repeatability (R = 0.72), but regarding the small sample size (n = 39), the estimate of the between-person component of variance may be imprecise. In an epidemiological study designed to study how differences between individuals may be related to a certain endpoint (disease), the effect of large within-person variability obviously may bias the association or prediction. Only values for R close to 1 allow for the observed association to be fairly unaffected. The statement that a reliability coefficient of 0.56 is low may seem rather arbitrary. However, these coefficients were much higher for other analytes that were measured repeatedly in our study. For example, the reliability coefficients for total and HDL cholesterol determinations were both R = 0.74. Even blood pressure readings proved much more reproducible than plasma fibrinogen measurements in our study (R = 0.76 for systolic BP). In the statistical literature it has been well recognized that the effect of high intra-individual variability may lead to rather substantial underestimation of the exposure-outcome relationship. In their authoritative paper on the influence of within-person measurement error on logistic regression relative risk estimates, Rosner et al. [28] report that if no systematic bias exists, this “regression dilution bias” or “attenuation” may be corrected for by dividing the original estimate /?for the log(odds) by the reliability coefficient R. Using the results from our study means that the true relation between fibrinogen levels and, for example, IHD may well be underestimated at 79% (l/R = 1.79), a quite substantial increase of clinical significance. This correction however does not affect the statistical significance since the standard error of p is multiplied with the same constant factor. Besides its quantitative impact in epidemiological context, low personal reproducibility does potentially also play an important role in clinical practice. Classifying a patient as being at risk for disease if exceeding a normal range based on a single fibrinogen measurement may be inappropriate. As can be concluded from Table 4, the concordance of such

Intra-Individual

Variability

of Fibrinogen

399

Levels

defined some months apart is low. Of the 20 subjects having a fibrinogen level higher than 450 mg/dl at the first measurement, only six of them had this elevation

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