ULTRASOUND N November 2009 N Volume 17 N Number 4

The Detection of Early Atherosclerosis in Healthy Male Relatives of Men with Peripheral Arterial Disease: a Feasibility Study
Stephen Wolstenhulme1, Stefano Ricci2, Robert M West3, J Anthony Evans4 & D Julian A Scott5 1 Ultrasound Department, Leeds NHS Teaching Hospitals, UK, 2Electronics and Telecommunications Department, University of Florence, Italy, 3Division of Biostatistics, Centre for Epidemiology and Biostatistics, LIGHT, University of Leeds, UK, 4Division of Medical Physics, LIGHT, University of Leeds, UK and 5Division of Cardiovascular and Diabetes Research, LIGHT, University of Leeds, UK

Aim: A feasibility study to attempt to determine if conventional ultrasound and multi-gate ultrasound could differentiate between men with peripheral arterial disease (PAD), healthy first-degree male relatives (FDRs) of men with PAD, and age/sex matched controls in identifying early atherosclerosis. Methods: three groups were studied using conventional ultrasound: first, four men with premature PAD ((65 years); second, four healthy first-degree relatives; third, seven male controls with no known family history or evidence of premature cardiovascular disease. Primary outcome measures were plaque and intima-media thickness of the common carotid artery, flow mediated vasodilatation of the brachial artery, and lower extremity arterial plaque. Multigate Doppler ultrasound blood velocity profile measurements were obtained for three controls, but this series of measurements was discontinued due to technical difficulties. Statistics: mean (standard deviation) and analysis of variance (ANOVA) (p,0.05) Results: Conventional ultrasound demonstrated lower extremity arterial lesions in all four of the patients with PAD. One of their first-degree relatives was found to have a non-significant lesion. No significant carotid artery lesions were found in any of the patients, but males with PAD had significantly greater carotid artery intima-media thickness than a combined group of first-degree relatives and controls (0.750.06 mm, versus 0.560.07 mm; p(0.01). The mean (standard deviation) flow mediated vasodilation per cent change of the brachial artery was for males with PAD, 3.494.15; relatives, 6.172.53; and control subjects, 3.536.63. Results from the multigate system were inconclusive. Conclusion: Carotid artery intima-media thickness seemed able to differentiate between patients with PAD and the other two groups. This measurement could be used in future case-control family PAD studies and possibly eventually as a screening tool. Flow mediated dilatation per cent change showed a wide variation in measurements and was not able to differentiate between the three groups. For researchers considering future studies, it is worth noting that recruitment figures for this pilot study were much lower than quoted in previous reports. Keywords: Atherosclerosis, Duplex Ultrasound, Multigate Doppler, Peripheral Arterial Disease

Introduction
Cardiovascular disease (CVD) is a common disease, which affects multiple sites: including cerebrovascular disease, coronary artery disease (CAD) and peripheral arterial disease (PAD). It is known that family history is independently associated with CAD.1,2 It has only recently been shown, based on atherothrombotic risk factor clustering, that a prethrombotic state exists in asymptomatic rst-degree male relatives (FDRs) of subjects with PAD.3,4 These subjects are thought to be at high risk of developing premature CVD. Epidemiological studies have identied several risk factors that are associated with all CVD sites: smoking, obesity, insulin resistance, hypertension, type II diabetes mellitus and hyperlipidemia. Peripheral arterial disease commonly affects the femoral artery. Leng et al.5 examined the common femoral artery using duplex ultrasound and reported a prevalence of arterial plaques in 64% of male subjects under 60 years of age. In 60% of cases the plaques were asymptomatic and only 4% had both plaque and intermittent claudication. The German Epidemiological Trial on Ankle Brachial Index study reported a
Correspondence: Stephen Wolstenhulme, MHSc, Ultrasound Department, Leeds NHS Teaching Hospitals, Beckett Street, Leeds, LS9 7TF, UK. swolstenhulme@leeds.ac.uk Ultrasound 2009;17(4):220226 British Medical Ultrasound Society 2009

prevalence of PAD in 17.1% of men (65 and 70 years).6 The disease is progressive and approximately 50 000 patients are admitted to UK hospitals with chronic limb ischaemia;7 of these 22 000 patients required peripheral vascular surgery.8 Furthermore, these patients have a high mortality from underlying cardiovascular disease.9 For these reasons the identication of high risk prethrombotic rst-degree relatives, without overt disease, is an important challenge. Lifestyle changes and drug therapies could have a profound effect on the reduction in morbidity and premature mortality from CVD. A screening tool is needed to identify rst-degree relatives of men with PAD who have early atherosclerosis (sub-clinical disease). Several studies have used ultrasound to measure dimensions and velocities in subjects with a family history of CAD,1012 and arterial plaques in subjects with a family history of PAD,13,14 to detect early atherosclerosis. Flow mediated brachial artery dilatation (FMD) has been used as a biomarker in subjects with familial hypercholesterolemia,15 adults at risk of early atherosclerosis16 and subjects with a family history of CAD.17 One limitation of these ultrasound studies is the unknown sensitivity/specicity of ultrasound measurements for the detection of early atherosclerosis. This is due to the lack of a gold standard; since no longitudinal study has yet determined whether young asymptomatic subjects with early atherosclerosis detected by ultrasound will go on to develop PAD. Unfortunately, such a study would take decades.
DOI: 10.1179/174227109X12500830049915

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Detection of early atherosclerosis in healthy male relatives of men with PAD

Figure 1. Flowchart to demonstrate the recruitment and enrolment of men with PAD and healthy rst-degree male relatives.

Previously Tortoli et al. developed a multigate Doppler ultrasound system (MGUS) which can be used to display the velocity prole within each vessel.18 MGUS extends analysis of a single ultrasound sample volume located at a specic depth to analysis of 128 sample volumes located along the direction of the beam.19 This technique allows investigation of blood velocity proles in real time.19 In previous work, this system was successfully applied to assessment of the mechanics and haemodynamics of the carotid artery.1820 Tortoli et al.21 have also used a novel dual-beam method, coupled to multigate Doppler, to estimate wall shear rate and wall distension. Multigate Doppler ultrasound studies have demonstrated early atherosclerosis by assessing dimensions, compliance, distensibility and endothelial function,22,23 but to the best of our knowledge have not been applied to family case-control studies investigating PAD. In this study, MGUS was implemented using a customised circuit board connected to a modied commercial ultrasound system. The primary aim of our feasibility study was to explore whether ultrasound methods could be used as an early marker for PAD. Theoretically this could be achieved by testing whether ultrasound can differentiate between men with PAD (group 1), high-risk healthy rst-degree male relatives of men with PAD (group 2), and age/sex matched controls (group 3). However, the need for a feasibility study was initially identied since there were insufcient data upon which to undertake reliable sample size calculations. The original aim of our study was to enrol 10 subjects per group to provide an estimate of the size of the effect together with standard deviations for the outcomes. The intention was to then use these outcomes to power a full-sized trial. The study also sought to evaluate which measurements would be most useful for identifying the onset of PAD, including the potential use of MGUS.

Patients and Methods

This case-control family, feasibility study was carried out at the Leeds Teaching Hospitals NHS Trust, UK. All subjects were recruited from the general population within the geographical location of West Yorkshire. Ethical approval for the study was obtained from the Leeds (East) Research Ethics Committee. Figure 1 demonstrates the recruitment and enrolment of patients. Although we approached 67 patients with conrmed PAD, only four pairs of patients and rst-degree relatives agreed to take part. Table 1 shows the reasons why males with PAD and their relatives declined to participate in the study. Table 2 shows the clinical characteristics of the four male white patients with PAD. These four male patients and their rst-degree relatives were compared with seven control subjects matched for age, sex and race, recruited at random from the Leeds Teaching Hospital NHS Trust staff. The controls had no known history of CVD, diabetes or hypertension, although one was taking statins. None of the controls had any family history of CVD, which was dened as up to 65 years of age for this study. All subjects gave informed consent, fasted for 10 h overnight and were asked to refrain from smoking, taking vasoactive medication, and exercising for the same period. A detailed medical history was taken, including current drug history, nicotine smoking, and consumption of alcohol. Relatives and control subjects were established to be free from both intermittent claudication and angina according to the Edinburgh Claudication Questionnaire.24 Blood pressure was determined with an automated cuff to the nearest 2 mmHg. Body mass index (BMI) was calculated as weight 221

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Table 1. Reasons why men with PAD (proband) and male firstdegree relatives did not meet eligibility criteria/declined the invite of recruitment into the study.
Reason No living FDR Proband refused to join the study FDRs live outside West Yorkshire and could not travel to join study No reply to communication FDRs refused to join the study/Estranged Proband deceased Brother leg artery disease, son lives abroad Brother .65, son lives abroad Son diabetic In another study Proband too busy/lack of funds Total FDR5First Degree Relative. Number 27 9 6 8 4 3 1 1 1 1 2 63

Scanning Procedure
Ultrasound examinations were carried out using an Acuson 128 (Acuson, UK) tted with an electrocardiogram (ECG) amplier and broadband 5 and 7 MHz linear array transducers. To test the feasibility of using MGUS, the Acuson system was connected to a MGUS board supplied through a research agreement with Professor Tortoli of the University of Florence. All subjects reclined on an examination bed for 15 min before the study. Scans were carried out in a quiet and temperature-controlled (2224uC) room to stabilise the patient and avoid vasodilatation or constriction of the arteries. An experienced Sonographer (SW), performed all of the conventional and MG ultrasound examinations. The screen was masked, blinding the operator to the measurements to avoid measurement bias. All conventional and MGUS images were recorded on a PC for later analysis. Conventional Ultrasound The operator examined 15 subjects using conventional US (four patients with PAD, four relatives and seven controls). The primary end points for conventional ultrasound examinations were: carotid artery intima-media thickness (IMT), carotid artery plaque, and carotid artery peak systolic velocities (PSV), FMD of the brachial artery, lower limb arterial plaque, PSV ratio of the lower limb arteries. Carotid Artery Assessment Intima-media thickness measurements of the common carotid artery (CCA) were performed according to the method described by Thrush.9 The operator subjectively placed manual callipers to the far wall of the CCA to measure the IMT. The right and left CCA were examined for each subject. The mean IMT was calculated from four measurements. The right and left carotid arteries were assessed using the method described by Thrush.9 Plaque disease was dened using the method described by Valentine et al.14 Haemodynamic signicance of each internal carotid artery plaque was determined using the International Consensus PSV criteria.27 Flow Mediated Dilatation Flow mediated dilatation measurements were obtained at the brachial artery of the right arm, and at the site of the antecubital fossa.16,28 The patients arm and the transducer were held in position using a stereotactic clamp. The diameter of the brachial artery was measured from the anterior to the posterior interface between the media and adventitia. The mean diameter was calculated using an automated walltracking device (Brachial Analyser v.5, Medical Imaging Applications, Iowa, USA) synchronized with the R-wave peaks on the ECG. We rst recorded the baseline diameter over a 5 s period. We then inated the cuff around the lower arm to a pressure of 250 mmHg. After 5 min, we deated the cuff and started image recording. Images were analysed off-line by SW and used to determine the baseline diameter and maximum diameter measured in millimetres. FMD was computed as the percent increase in diameter: FMD5{(maximum2baseline)/ baseline}6100%. Lower Limb Assessment Lower limb vessel wall assessment and haemodynamic measurements were performed according to the method described by Thrush.9 Both right and left iliac, common femoral, supercial femoral and popliteal arteries were assessed. Arterial plaque was scored utilising the technique used by Valentine et al.14 A signicant stenosis was

in kilograms divided by height-squared in metres (kg m22). Waist-hip ratio was calculated as minimal abdominal girth (to the nearest 0.5 cm) divided by maximal protrusion of the hips (in centimetres) at the level of the symphysis pubis. A 12-lead electrocardiograph was performed to exclude myocardial ischemia or previous infarction. Resting ankle brachial pressure index (ABPI) was measured in both legs with a hand-held 8 MHz Doppler probe (Huntleigh, Cardiff, UK). An ABPI of less than 0.9 is 95% sensitive in detecting angiogram positive disease and ABPI 0.9 or greater is almost 100% specic in detecting healthy subjects.25 Free-owing venous blood (50 ml) from an antecubital vein with a 19-gauge needle was drawn. Blood was transferred to a room temperature tube for serum lipid prole and glucose assay. All subjects underwent a 75 g oral glucose tolerance test, and serum glucose concentration was determined at 2 h. Glycaemic state was classied in accordance with World Health Organization criteria.26

Laboratory Tests
Sample tubes at room temperature were centrifuged at 3000 rev min21 (Rotanta 460R, Hettich) for 10 min. Serum glucose was measured with the glucose oxidase (Trinder) method (analyser Bayer ADVIA 1650/2400 (Siemens)), total cholesterol, high-density lipoprotiein cholesterol and triglyceride levels with a Bayer ADVIA 1650/2400 analyser, and lowdensity lipoprotein cholesterol with the Friedwald equation. All assays were performed only once. Table 2. Clinical characteristics of men with intermittent claudication.
Men with intermittent claudication Age, year Age at diagnosis, year Claudication distance Ankle-brachial pressure index Peripheral vascular intervention Angioplasty or stenting Surgical endarterectomy or bypass{ Both radiological and surgical intervention Coexistent CVD1 Coexistent diabetes 60 (5565)* 59 (5260)* 100 (50200)* 1.25 (0.27){ 3 of 4 (75%) 1 of 4 (25%) 3 of 4 (75%) 1 of 4 (75%) 2 of 4 (50%) 1 of 4 (25%)

*Non-parametric data expressed as median (range). {Parametric data expressed as mean (standard deviation, SD). {Bypass (femoropopliteal) or femoral endarterectomy. 1Coronary artery disease.

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Table 3. Clinical and biochemical characteristics of study subjects according to the presence or absence of a family history of premature PAD.
Probands (n54) Age, year BMI,* kg m22 Waist-hip ratio Systolic blood pressure, mmHg Diastolic blood pressure, mmHg ABPI* Heart rate, bpm Current smoker, % Previous smoker, % Antihypertensive drugs Lipid lowering therapy Cholesterol, mmol L21 LDL,* mmol L21 HDL,* mmol L21 Triglyceride, mmol L21 0 h glucose level, mmol L21 2 h glucose level, mmol L21 IGR or diabetes,* % 60.5 (5564) 29.5 (2.24) 0.99 (0.12) 140 (7.43) 79 (5.4) 1.25 (0.27) 56 (10.66) 1 of 4 (25%) 3 of 3 (100%) 3 of 4 (75%) 3 of 4 (75%) 4.4 (1.34) 2.58 (1.39) 1.15 (0.13) 1.53 (0.9) 5.53 (0.85) 11.15 (6.7) 3 of 4 (75%) Relatives (n54) 38 (2352) 27.9 (5.43) 0.87 (0.07) 129 (10.25) 78 (4.17) 1.4 (0.16) 60 (1.83) 0 of 4 (0%) 2 of 4 (50%) 0 of 4 (0%) 1 of 4 (25%) 5.1 (1.58) 3.03 (1.44) 1.38 (0.13) 1.6 (0.84) 5.1 (0.27) 5.98 (1.86) 0 of 4 (0%) Controls (n57) 43 (3562) 26.8 (3.86) 0.88 (0.08) 117 (6.06) 79 (5.46) 1.57 (0.33) 56 (6.16) 2 of 7 (28.6%) 0 of 5 (0%) 0 of 4 (0%) 1 of 4 (25%) 4.9 (0.68) 3.1 (0.53) 1.21 (0.23) 1.27 (0.62) 4.97 (0.27) 5.43 (1.95) 1 of 7 (15%) p 0.03{ 0.49 0.19 ,0.01 0.19 0.14 0.56 1 1 1 1 0.68 0.73 0.24 0.76 0.22 0.08 1

*BMI, Body mass index; ABPI, ankle-brachial pressure index; LDL, low-density lipoprotein; HDL, high-density lipoprotein; IGR, impaired glucose regulation. {Nonparametric data expressed as median (range). 1 Categorical data expressed as a proportion (%) IGR 2 new diagnosed probands and 1 new diagnosed control; 1 known proband diabetic.

determined using the PSV ratio and waveform shape. The ratio is calculated by dividing PSV in the stenosis by PSV in normal artery just proximal to the stenosis. A PSV ratio greater than 2 was used to dene a signicant stenosis.9 Multigate Doppler Owing to reasons highlighted in the discussion section only three control subjects were examined using MGUS. After conventional US scanning, SW and SR activated the MGUS board, which processed in real time the data extracted from the ultrasound system, to produce the blood velocity prole of the investigated vessel.20 The primary end points for MGUS were blood velocity proles of the carotid, brachial, femoral and popliteal artery. Repeatability All subjects were assessed using conventional ultrasound (carotid artery assessment, FMD and lower limb assessment) repeated at an interval of one to three weeks. The operator was blind to previous ultrasound examination measurements to prevent measurement bias.

Results
Clinical and Biochemical Characteristics The clinical and biochemical characteristics of the probands are shown in Tables 2 and 3. As shown in Table 3, there was no signicant difference between the relatives of patients with PAD and controls for age; anthropometric characteristics; diastolic blood pressure; serum cholesterol, HDL; low-density lipoprotein; triglycerides glycaemic state and smoking. However, the FDRs had one atherogenic risk factor, a signicant difference in systolic blood pressure. The results in Table 3 also showed the four rst-degree relatives recruited to this study to have a signicantly higher intake of alcohol than the other two groups. However, this is probably an artefact of the small number of patients recruited. Ultrasound Characteristics Conventional Ultrasound Duplex ultrasound scans showed lower extremity lesions in all of the patients with PAD and in one of their rst-degree relatives (a previous smoker). The distribution of leg artery lesions for the three groups of subjects is shown in Table 4. All probands and the relative with lesions were affected in more than one artery. The lower extremity plaque lesions in the relative had luminal encroachment of less than 50%. Carotid lesions were detected in all probands; none produced a signicant stenosis (Table 4). None of the rst-degree relatives or controls had carotid artery plaque disease (Table 4). The characteristics of the brachial and carotid arteries in subjects with PAD, rst-degree relatives of patients with PAD and control subjects are shown in Table 5. There was no signicant difference in the mean (SD) combined (right and left) carotid IMT measurements for relatives compared to controls (0.53 [0.09] and 0.59 [0.08]; p5NS). The IMT measurements for the relatives and controls were therefore combined. Carotid IMT was signicantly higher in the probands than in the combined rst-degree relatives and controls (0.75 [0.08] versus 0.56 [0.07]; p(0.01) All probands underwent FMD; one FMD in the rst-degree relatives and two FMDs in the control subjects were excluded due to poor image quality. The results for the subjects in all 223

Statistical Analysis
Parametric data are presented as a mean and associated standard deviation. Nonparametric data are presented as a median together with the range. Categorical data are expressed as a proportion (%). An analysis of variance (ANOVA) test was used to compare differences in mean values of various ultrasound parameters for each of the three groups. ANOVA was chosen instead of a multiple t-test, because with multiple testing the analysis becomes cognitively difcult, while ANOVA organises and directs the analysis, allowing easier interpretation of results. The Bland and Altman29 method was used to assess the intraobserver repeatability coefcient (2 SD) of the ultrasound measurements. Data were stored in a Microsoft Excel le. Statistical analysis carried out by SW and RW using STATA 10 (Statacorp LP, USA). A value of p,0.05 was considered statistically signicant.

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Table 4. Distribution of arterial lesions and degree of luminal narrowing detected with duplex ultrasound in probands, firstdegree relatives of men with premature PAD and subjects without such a history.
Artery Carotid, % ,50 5069 7099 Total Iliac, % ,50 .50 Total Femoral, % ,50 .50 Total Popliteal, % ,50 .50 Total Probands (n58) Relatives (n58) Controls (n514)

8 0 0 8 5 0 5 1 2 3 3 0 3

0 0 0 0 1 0 1 1 0 1 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0

groups are displayed in Table 5. Variations in the FMD % change and the FMD absolute change were large. Multigate Ultrasound To briey assess the feasibility of using MGUS, blood velocity proles of the carotid, brachial, femoral and popliteal artery were obtained in a small selection of three control subjects. These were assessed subjectively, but the image quality was poor and no meaningful results were obtained. The use of MGUS in the study was therefore discontinued. Repeatability One rst-degree relative and one control subject were unable to attend for their repeat ultrasound examination. In total, 13 cases was available for analysis. Given there were no signicant carotid or leg artery stenosis in rst-degree relatives or controls and a large variation in the FMD per cent change and FMD absolute change existed, only the carotid artery IMT measurement was analysed to assess repeatability. For the three groups combined the repeatability coefcient (2 SD) was 0.22 mm.

Discussion
This feasibility study highlights a number of challenges in assessing the potential role of ultrasound in early assessment

of PAD. To the best of our knowledge, this study is the rst to attempt to use conventional and MGUS in assessing symptomatic male patients with PAD (probands), who we assume to be the gold standard (the end point of disease), asymptomatic rst-degree relatives and control subjects. Information gained from this pilot was intended to inform the design of future larger studies aiming to determine if ultrasound measurements showed a signicant difference between probands, their relatives, and controls. Case-control studies are prone to bias and a tight method of selection was used to minimise this. The presence of lower limb atherosclerosis in all patients was determined both clinically and by duplex-ultrasound to identify an unequivocal family history of PAD. To further reduce bias, we simultaneously recruited well-characterised and comparable close male relatives and control subjects from the general population of Leeds. A major limitation in the study is the small sample size. Of the 67 patients we approached only 4 had a suitable male relative. This is signicantly less than 31 asymptomatic siblings of the 100 families recruited by Valentine et al.14 and 78% of all patients with an asymptomatic FDR in studies by Parry et al.3,4 The reasons for poor recruitment are listed in Table 5. The low level of recruitment suggests that future case-control PAD family research may require a multicentre study to recruit sufcient relatives. All rst-degree relatives and controls had clinical CVD excluded using validated and reproducible means. Valentine et al.14 used duplex ultrasound to assess for occult arterial lesions in asymptomatic relatives of male and female patients with PAD. They concluded that family history and smoking were independent risk factors for the development of PAD. The current feasibility study showed all male patients to have nonsignicant carotid artery disease and both signicant and nonsignicant leg artery disease (Table 2), suggesting plaque is probably present in all patients with PAD. One relative had non-signicant (,50%) leg artery lesions (Table 2). This is at variance with the results of Valentine et al.14 who showed 40% of asymptomatic rst-degree relatives had raised carotid and leg artery atherosclerotic plaques. This suggests that raised arterial lesions are not a useful marker of atherosclerotic disease in assessing asymptomatic relatives. Carotid IMT has been used as either a single measurement or rate of change of measurement and has been shown to be a surrogate marker for CAD.11,12 In our small sample size no statistical difference was observed between the carotid IMT of relatives and control subjects (0.53 [0.09] versus 0.59 [0.08]; p5NS). However, carotid IMT was signicantly higher in the probands than in a combined group of the relatives and controls (0.75 [0.08] versus 0.56 [0.07]; p(0.01). These results are similar to the family history CAD population,1012 and as such, we conclude that carotid IMT may be an appropriate early atherosclerotic marker in future PAD casecontrol family studies.

Table 5. Characteristics of brachial and carotid arteries in men with PAD, first-degree relatives of men with PAD and asymptomatic subjects without such a history.
Variable Functional measure of brachial artery Baseline diameter, mm Maximum diameter during reactive hyperaemia, mm FMD per cent change FMD absolute change, mm Structural measures of CCA IMT, mm Right artery Left artery Combined arteries FMD, Flow Mediated Dilatation; IMT, Intima Media Thickness. Controls (n57) 4.61 (0.47) 5.08 (0.47) 9.47 (8.1) 0.4 (0.29) 0.6 (0.08) 0.57 (0.08) 0.59 (0.08) FDRs (n53) 4.67 4.96 6.17 0.28 (0.97) (1.03) (4.38) (0.23) Probands (n54) 5.18 5.31 3.49 0.13 (1.06) (0.82) (8.29) (0.42) p 0.61 0.68 0.95 0.91 ,0.01 ,0.01 ,0.01

0.58 (0.05) 0.48 (0.1) 0.53 (0.09)

0.78 (0.08) 0.73 (0.05) 0.75 (0.08)

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and was not able to differentiate between the three groups. Owing to technical difculties, the usefulness of MGUS could not be determined. We found that it was not possible to combine multigate Doppler with modern ultrasound systems and recommend that manufacturers work closely with research teams to develop systems to realise the potential of this innovative technique. We felt that the low level of recruitment in this study, compared to previous reports, was worth highlighting to future researchers. We suggest that future case-control family PAD research be conducted in a multicentre study to recruit sufcient rst-degree relatives to obtain signicant results.

Acknowledgements
This research was funded by a Leeds Teaching Hospitals Charitable Foundation Project Support Award, Ref 9/R21/ 2003. The authors would like to thank Professor Piero Tortoli, University of Florence for the research agreement to supply the multi-gate ultrasound board; Mrs Anne Johnson and Penny Rice, Research Nurses, LIGHT, Leeds, for taking the bloods; and Mrs Marion Lindsay, Clinical Physiologist, General Infirmary, Leeds, for doing the ECGs. We also extend our thanks to the participants for taking part in this study.

Figure 2. Multi-gate Doppler blood velocity prole of the common carotid artery.

Flow mediated brachial artery dilatation has been used to measure impaired release of nitric oxide, a physiological feature of endothelial injury. The decrease in FMD and/or progression of IMT is associated with classical risk factors, including smoking and hyperlipidaemia15,16 and family history of CAD.17 Table 5 shows a large variation in the FMD per cent change and the FMD absolute change for the subjects in all groups, which is at odds with studies that have shown that FMD per cent change may be used to detect endothelial dysfunction in offspring of patients with hyperlipidaemia15 and family history of CAD.16,17 The problems encountered in this study may have been due to the operators limited experience with the technically challenging FMD technique and inability of the Brachial analyser to detect the anterior and posterior intimal layers because of arm movement, despite the use of the stereotactic clamp, and poor image resolution. These pitfalls highlight that standardised and reproducible protocols, training and ongoing quality improvement are necessary to obtain valid and reproducible data.28 Use of the MGUS system was also more limited than we would have hoped. We found that it was not possible to access the raw radio-frequency data signal from a modern real-time ultrasound system and therefore, in this study, the MGUS board could only be used in conjunction with an Acuson 128 through a non-optimised connection. This made it necessary to remove the side panel of the ultrasound system, which was felt to be a health and safety hazard for clinical use. Moreover, the suboptimal connection resulted in a limitation of the quality of the MG data recorded. Due to the poor quality of the blood velocity prole (Fig. 2) it was not possible to compare the proles even in controls and use of the MGUS was discontinued.19

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Conclusion
Carotid artery IMT was able to differentiate between men with PAD and the combined rst-degree relatives and control subject groups. This measurement seemed the most promising for use in future case-control family PAD studies and possibly eventually as a screening tool. Flow mediated dilatation per cent change has a wide variation in measures

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