Correlation of severity of chronic venous disease with capillary morphology assessed by capillary microscopy
Article Outline
Background
In patients with lipodermatosclerosis caused by chronic venous disease of the lower limb, the skin capillaries show proliferation and convolution. To our knowledge, no previous attempt has been made to correlate the clinical severity of venous disease based on the CEAP classification with the capillary morphology changes. The purpose of this study was to correlate the clinical severity with the capillary morphology changes in patients with chronic venous disease by using capillary microscopy and to explore the significance of atypical capillary morphology in patients with uncomplicated varicose veins.
Method
Patients attending the vascular clinic for management of chronic venous disease were examined clinically and by duplex ultrasonography scans, and they were assigned to the appropriate CEAP clinical stage (C1, n = 15; C2, n = 20; C3, n = 15; C4a, n = 15; C4b, n = 15; C5, n = 15). Also studied were 10 control subjects with no arterial or venous disease of the lower limb. In part 1 of the study, a capillary microscope was used to obtain digital images of the skin microcirculation in the gaiter region. The capillary density and capillary convolutions were counted in a 2.4 mm2 region of skin. In part 2 of the study, a further 33 C2 and C3 patients were studied to provide more detailed information on a small subgroup with unusual capillary morphology.
Results
In part 1 of the study, the capillary count was similar in controls and patients in CEAP stages C2 to C4a, but decreased in patients of groups C4b and C5 (median count: C2, 15; C4b, 4; and C5, 7). The median number of the capillary convolutions was 1 per capillary in patients of groups C1 to C3 and controls; C4a had 4 convolutions, C4b had 7, and C5 had ≥10. In part 2 of the study, an expanded group of 66 C2 and C3 patients was analyzed, of whom 14 had a mean capillary loop count of more than two. Biologic markers of inflammation were investigated for their correlation with capillary convolution, but no relationship was found.
Conclusions
Among patients with venous disease, increased capillary convolution is strongly associated with the more severe stages (lipodermatosclerosis and healed ulceration). The significance of atypically increased capillary convolutions in a small subgroup of C2/C3 patients remains unknown.
The Edinburgh Vein Study found that varicose veins are present in 40% of men and 32% of women, with skin changes attributable to venous disease in 9% of men and 7% of women.1 The center of the disease process in these cases is the subpapillary capillary plexus in the skin.2 The microcirculation of the skin has been studied by histologic and capillary microscopy during the last 20 years.3, 4, 5, 6, 7 Although capillary microscopy has been used to assess the association between the severity of venous disease assessed by Widmer’s classification and nail fold changes,8 to our knowledge, quantification of capillary morphologic changes in the skin in the gaiter region of the leg has not been reported. The aim of this study was to quantify changes in the subpapillary capillaries by using capillary microscopy and relate this to the clinical severity of venous disease categorized according to the CEAP classification.
It has been suggested that cutaneous microangiopathy precedes the development of trophic skin changes caused by chronic venous disease and that the clinical degree of skin change and the severity of microangiopathy are closely related.3, 7 It would therefore be beneficial to detect the group of patients at risk of developing skin changes and ulceration at the earliest possible stage. It would then be possible to prioritize treatment to prevent further complications.
At present, there is no measure to indicate which of the patients with uncomplicated varicose veins will develop skin changes that might lead to ulceration. In a previous histologic study, a small number of patients with uncomplicated varicose veins showed proliferation of subpapillary capillaries in a similar manner to those patients with more severe venous disease.9 We hypothesized that these patients among those with uncomplicated varicose veins (C2/C3) are at risk of developing clinical skin changes. The aim of second part of the study was to detect these patients and assess whether they could be distinguished from other patients of the same CEAP clinical stage by testing for biologic markers of inflammation that have previously been reported to be elevated in patients with the more severe stages of venous disease10, 11, 12 and to symptoms of venous disease.
Methods
Permission to conduct the study was obtained from UCL Medical School Committee for Medical Ethics. Patients with chronic venous disease were recruited from the vascular clinic at the Middlesex Hospital, and the controls were healthy volunteers recruited from the staff and the general public. A medical history was taken and a clinical examination was performed. Patients who gave informed written consent were studied further and were assigned to the appropriate CEAP13 clinical stage by a surgeon experienced in the management of venous disease (Table I).
Table I. Clinical classification of lower limb chronic venous disease13
| C0 | No visible or palpable signs of venous disease |
| C1 | Telangiectases or reticular veins |
| C2 | Varicose veins; distinguished from reticular veins by a diameter of 3 mm or more |
| C3 | Edema |
| C4 | Changes in skin and subcutaneous tissue secondary to chronic venous deficiency |
 C4a | Pigmentation or eczema |
| C4b | Lipodermatosclerosis or atrophie blanche |
| C5 | Healed venous ulcer |
| C6 | Active venous ulcer |
All patients underwent a venous duplex examination to establish venous reflux and to localize it anatomically. The venous segments evaluated were the femoral, popliteal, and deep veins of the calf (posterior tibial, peroneal, gastrocnemius). Reflux in any of these regions was classified as deep vein incompetence. The great saphenous vein was evaluated in the thigh and calf and the small saphenous vein in the calf. Reflux in any of these segments was considered to be superficial venous reflux (SSV).
Because we planned to use blood tests to assess systemic inflammatory markers, we excluded those patients with diseases likely to lead to elevation of biochemical markers of inflammation not related to venous disease. Exclusion criteria were patients treated for venous disease ≤3 months, diabetes mellitus, any concomitant active disease, patients treated with vasoactive drugs, deep venous thrombosis ≤12 months, superficial venous thrombosis ≤3 weeks, significant peripheral vascular diseases (ankle-brachial pressure index <0.8), and infection or inflammation in the legs other than that attributable to venous disease. Patients with leg ulcers were also excluded.
In all, 192 patients were screened for this research, of whom126 patients and 10 healthy volunteers drawn from the staff of the department and patients with unrelated disease were studied. Although this work was conducted before the most recent revision of the CEAP classification was published, we subdivided patients of CEAP clinical stage C4 into C4a with hemosiderosis and venous eczema, and C4b with lipodermatosclerosis. Our previous work had suggested that this division most accurately reflected the clinical severity of venous disease.
We divided this work into two studies with different aims. In the first part of the study, we analyzed the data from groups of 15 to 20 patients taken from each of the CEAP clinical stages C1 to C5. We also investigated healthy control subjects who we considered to belong to CEAP clinical stage C0. While conducting the first part of the investigation, we found a small group of patients with mild venous disease (CEAP clinical stages C2 and C3) who appeared to have atypically increased capillary convolution. We had observed similar findings in an earlier histologic study.9
In the second part of the study, we recruited a larger group of patients with mild venous disease to study this phenomenon in more detail. In all, a further 28 patients in stage C2 and three in stage C3 were included. Including those patients already studied in part 1 of the investigation, we had 66 patients with mild venous disease, 48 patients with varicose veins (C2), and 18 patients with varicose veins and edema (C3).
Part 1 procedure
The main part of this study was to investigate the capillary circulation of the skin using a capillary microscope. To minimize venous pressure in the leg, subjects lay supine in an environmental chamber at 22°C. Patients and controls acclimatized for 10 to 20 minutes before the start of the experimental protocol. A small amount of liquid paraffin was wiped over the examination area to minimize specular reflection from the skin surface. In each patient and volunteer, the supramalleolar skin 5 cm proximal to the medial malleolus or the region most severely affected by lipodermatosclerosis in patients with venous disease was investigated. At least four representative areas of capillaries were studied in each limb.
We used a CAM1 Capillary Anemometer (KK Technology, Devon, UK) capillary microscope with a high-resolution (752 × 582 pixel) monochrome charge-coupled-device video camera. Four ultra-bright light-emitting diodes epi-illuminated the skin with a peak wavelength of 525 nm (green). The microscope was attached to a heavy support to allow it to be positioned on the skin in the gaiter region. The microcirculation was viewed through a 10-mm diameter acrylic disc placed in contact with the skin that provided mechanical stability to the microscope system. The image was magnified ×6.3 to give an overall magnification of about 1.23 μm/pixel. The area of each microscope field was 0.6 mm2.
Images were recorded at the time of the investigation and stored on the computer disk drive as JPEG files. These were analyzed at a later time to speed the investigation and ensure that the author responsible for image analysis was unaware of the clinical status of the volunteer from which they came.
We assessed both the capillary density (number of capillaries per unit area) and the complexity of the capillary morphology. All clearly identifiable capillary loops in a field were considered to be part of the total capillary count for that area. These were considered to be visible capillaries. Because not all parts of every capillary were in focus or within the image, not all of the visible capillaries could be evaluated for their morphology. Only those capillaries that were clearly visible were considered to be evaluable. The total number of visible capillaries was counted in a 2.4-mm2 region, where one field is 0.6 mm2. Within each capillary, the number of convolutions was also counted. Capillary density was expressed as the number of capillaries per mm2 area of skin. Morphology was assessed by counting the number of convolutions or loops per capillary. We counted a loop as a 180° reversal of direction. The number of convolutions for each evaluable capillary loop were counted, and when more complex capillaries contained more than 10 loops, they were recorded as >10. An average number of convolutions per capillary were calculated for each patient based on all evaluable capillary loops. We had established that this method of quantification was reliable and reproducible in a previous study.14
Part 2 procedure
In part 2, we identified a group of patients with CEAP C2 and C3 stage venous disease, which we defined as those with an average of more than two convolutions per capillary. We considered that these were the atypical group. Examples of capillary morphology changes in various grades of CEAP clinical group are illustrated in Fig 1.
Fig 1.
A, Simple loop capillaries (arrow) commonly seen in control subjects and in patients of CEAP clinical stages C1, C2, and C3. B, This photograph shows early capillary convolutions with variable number of loops (arrow) usually seen in C4a patients. C, Photograph shows convoluted tortuous capillaries seen in CEAP stages C4b and C5. D, Typical glomerulus-shaped capillary seen in lipodermatosclerosis and healed ulceration (C4b and C5). Here, the multiple proliferation of single capillary has formed a “glomerulus.”
Patients assessed their symptoms of venous disease by using a 10-cm-long visual analogue scale. The scale was marked 0 to 10, severe symptoms were graded 10, and an absence of symptoms was marked as 0. We inquired about pain, cramps, heaviness, paraesthesia, and edema. The distance along the scale at which the patient placed a mark was recorded in centimeters.
Hematologic tests were performed to assess systemic inflammatory markers. A Butterfly 23-gauge canula (Venisystems, Abbott, Ireland) was placed in the distal long saphenous vein or dorsal foot vein of the most affected leg of the C2 and C3 patients. Five milliliters of blood was collected into a citrated tube and 8 mL of blood was collected into two Vacutainer tubes containing ethylene diamine tetraacetic acid (Becton Dickinson Vacutainer Systems Europe, BP No 37-38241, Meylan Cedex, France). Plasma from the samples was separated by centrifugation at 20,000 rpm for 10 minutes at 4°C within 1 hour of collection and promptly frozen at –84°C before analysis of enzyme-linked immunoabsorbent assay (ELISA).
Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were assayed by using the commercially available kit manufactured by Diaclone (Stamford, Conn). E-selectin, L-selectin, vascular endothelial growth factor (VEGF) and the cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured by commercially available ELISA kits (R & D Systems Europe, Abingdon UK) once all the samples were collected. Von Willebrand factor was measured with the kit manufactured by Shield Diagnostics, Aberdeen, UK. The separate citrated samples were analyzed for leucocyte surface CD11b and L-selectin expression by whole blood assay using fluorescent-labelled monoclonal antibodies in a flow cytometer.
Statistical analysis
Descriptors used to represent the data are the median and interquartile ranges for the capillary analysis and biologic markers. Differences between medians were calculated by using the Wilcoxon method.
Results
Table II summarizes the basic information concerning the patients included in both parts of this study. The average age of patients was similar in each of the CEAP clinical stages, including that of the patients included in part 2 of the study. The sex distribution was not equal in all groups, however. In CEAP stages C1 to C3, the ratio of female to male patients was 3.5:1, whereas in the C4a to C5 groups, it was 0.5:1. Because patients were recruited directly from an outpatient clinic, this distribution probably reflects the referral pattern to our hospital. We suspect that more women than men with less severe venous disease are referred because this is partly a cosmetic problem as well as resulting in symptoms. In the more severe stages, men predominate, although there is no suggestion from epidemiologic studies that this should be the case. Table II also shows the duplex ultrasound findings, which have been summarized in a simple way showing those with superficial venous incompetence and those with combined deep and superficial venous incompetence.
Table II. Distribution of subjects according to age, sex, and CEAP clinical stage
| Stage (n) | Sex (F:M) | Age⁎ | SVI (n) | DVI/SVI (n) |
|---|---|---|---|---|
| Part 1 | ||||
| C0 (n = 10) | 6:4 | 43(32-56) | ||
| C1 (n = 15) | 13:2 | 56(53-61) | ||
| C2 (n = 20) | 14:6 | 53(47-60) | 16 | 4 |
| C3 (n = 15) | 12:3 | 50(41-59) | 11 | 4 |
| C4a (n = 15) | 5:10 | 52(40-67) | 8 | 7 |
| C4b (n = 15) | 5:10 | 64(55-68) | 9 | 6 |
| C5 (n = 15) | 5:10 | 50(44-65) | 7 | 8 |
| Part 2 (after 28 C2 patients and 2 C3 patients were added) | ||||
| C2 (n = 48) | 35:13 | 52(44-59) | 37 | 11 |
| C3 (n = 18) | 15:3 | 52(42-61) | 14 | 4 |
| Atypical | ||||
| C2 (n = 12) | 7:5 | 56(52-66) | 10 | 2 |
| C3(n = 2) | 1:1 | 47(44-50) | 2 | 0 |
⁎ Years (mean value) and range in parenthesis. |
In patients with mild venous disease (CEAP C2 and C3), superficial venous reflux alone was the most frequent finding. In the more severely affected patients, approximately equal numbers of patients had isolated superficial venous reflux and combined deep and superficial venous reflux. Therefore, deep venous reflux was strongly associated with the more severe stages of venous disease. In view of this uneven distribution of deep vein incompetence, we considered that analysis of our measurements according to whether there was deep or superficial venous reflux would not lead to any useful conclusion. Of all the patients included in our investigation, only three had an established history of previous venous thrombosis. However, deep venous thrombosis ≤1 year was an exclusion criterion for patients in this study. In no patient was there duplex ultrasound evidence of chronic obstruction of the deep veins.
Table II also summarizes the data from the 66 patients included in part 2 of the study. Here, 35 patients from part 1 of the study are combined with a further 31 C2 and C3 patients to investigate the phenomenon of atypical capillary convolution. Among this group, 14 patients had an average of more than two convolutions per capillary.
Fig 2, Fig 3 show the results of the quantitative analysis of the capillary morphologic changes. The capillary density was similar in control patients and in those of CEAP clinical stage C2 to C4a (Fig 2). Capillary density was reduced in C4b and C5 patients, however. This corresponds to previous publications on this subject that have shown reduced capillary density in patients with lipodermatosclerosis and atrophie blanche. Capillary convolution (Fig 3) was identical in the C0 to C3 stages, with only one capillary loop seen on average. Increased capillary convolution was seen in patients of clinical stages C4a to C5, with the most severely affected patients showing the greatest capillary convolution. Patients in the C1 group who had only telangiectases and reticular veins showed no evidence of increased capillary counts or convolution. Proliferation of vessels in these patients was confined to much larger veins than were evaluated in this study.
Fig 2.
This graph shows the number of visible capillaries per mm2 in the six CEAP stages studied. The CEAP stages are shown on the horizontal axis and the capillary density on the vertical axis. The horizontal bar shows the median capillary density.
Fig 3.
This graph shows the number of convolutions per capillary on the vertical axis in six CEAP stages. Horizontal bars represent the median value of each group.
In the second part of the study, the median number of capillary convolutions was one (interquartile range [IRQ] 1 to 1.2) in the 52 patients without capillary convolution. In the 14 patients with capillary convolution it was 3.1 (IQR 2.2 to 4.0), with a median difference of 2 (95% confidence interval, 1.2 to 2.9, Wilcoxon method). The capillary density was identical in the two groups of patients evaluated in part 2 of this investigation.
Table III compares the inflammatory mediators and symptoms in the atypical group with the remainder of the C2/C3 patients. For several of the inflammatory markers, distinct differences are present between the control group and the disease groups (data not shown). These included increased levels of VEGF, IL-6, and soluble(s) L-selectin, sICAM, and sVCAM in the venous disease patients compared with control subjects. We found no difference between the atypical group and the remaining C2/C3 patients, however. Neither did symptoms recorded on the visual analogue scale (VAS) differ between these groups.
Table III. Association of inflammatory markers and symptoms with the groups of patients studied
| C2/C3 excluding atypical group | Atypical group | Median difference (95% CI)⁎ | |
|---|---|---|---|
| Number | 52 | 14 | |
| Age | 51(41-59) | 54(51-65) | |
| Inflammatory markers† | |||
| Neutrophil CD11b (FU) | 202(152-258) | 137(106-256) | 20(−24to62) |
| Neutrophil CD62L (FU) | 58(64-53) | 56(60-53) | 0(−4to5) |
| Monocytes CD11b (FU) | 79(50-117) | 86(41-101) | −5(−32to25) |
| Monocytes CD62L (FU) | 38(46-30) | 41(42-34) | 2(−8to11) |
| VEGF (pg/mL) | 104(37-106) | 60(48-116) | 12(−11to53) |
| TNF-α (pg/mL) | 1.3(0.6-9.2) | 1.4(.8-1.9) | −0.5(−0.4to0.9) |
| IL-6 (pg/mL) | 2.2(1.3-2.8) | 2.1(1.4-2.9) | −1(−0.7to0.5) |
| sL-selectin (ng/mL) | 1052(796-1202) | 671(612-915) | −106(−274to69) |
| sE-selectin (ng/mL) | 39(28-52) | 46(36-57) | 4(−20to17) |
| sICAM-1 (ng/mL) | 547(394-630) | 585(442-701) | −18(−129to24) |
| sVCAM-1 (ng/mL) | 1270(1039-1584) | 1159(868-1554) | 2.5(−224to250) |
| vWF (IU/mL) | 1.3(1.0-1.6) | 1.4(1.2-1.8) | −0.2(−0.5to0.1) |
| Capillary microscopy | |||
| Capillary count | 12(9-16) | 16(10-22) | −3(−7to0) |
| Symptoms recorded in cm on 10-cm visual analogue scale | |||
| Pain | 3(1-4.9) | 2.8(0-5.4) | −1.5(−2.9to0) |
| Heaviness | 3.5(0-5) | 1.9(.1-4.6) | 0(−1.9to1) |
| Cramps | 2.5(0-4.7) | 3.2(0-7) | −1.95(−3.6to0) |
| Paraesthesia | 0.3(0-2.8) | 0(0-0.2) | 0(0to.5) |
| Edema | 0.7(0-4.5) | 0.8(0-5) | 0(−1.5to0.3) |
⁎ Median difference and 95% CI between “normal C2/C3 excluding atypical” and “atypical group” were calculated by using the Wilcoxon method. Patient supine. All data are median and inter-quartile ranges in parenthesis. |
† CD62L values refer to down regulation. |
Discussion
The aim of this study was to quantify the morphologic changes in the capillaries of the subpapillary plexus of skin capillaries, which appear to be in the most severely affected region of damage in patients with venous disease. We relied on assessment of the capillary microscope images by an experienced observer. This clearly has the possibility that the outcome may be biased by the prejudice of the observer, a problem which we investigated previously.14 We found that there was excellent agreement between observers who made capillary counts and assessments of capillary convolution. To minimize bias, we evaluated the capillaries without the assessor being aware of the patient’s identity. This allowed comparison between patients and assessment of capillary morphology. We excluded images of poor quality that were out of focus or in which no recognizable capillaries could be found owing to the presence of large amounts of hemosiderin in the skin. Some images were lost because of technical problems. In patients without obvious skin changes, we used the supramalleolar skin proximal to the medial malleolus; whereas in patients with skin changes, the affected skin was assessed. Clearly, this may have led to slightly different regions being investigated, but this is a common strategy in studies of this type.15
Native capillaroscopy shows only the capillary loops that are perfused or contain red blood cells. Not all capillaries are open at any time, and the count varies depending on the circumstances of the observations. This can be seen from a range of publications on capillary morphology and density studies in chronic venous disease.16, 17, 18, 19 We concede that capillary counting does not count all capillaries present, but we conducted this study under standardized conditions that were the same for all subjects.
Despite our care in selecting well-defined clinical groups, we found that although all CEAP stages contained patients of similar age, there were far more women in the less severe stages of venous disease (C1 to C3) and more men in the more severe stages (C4 and C5). We acknowledge that this may have led to some influence on the results; however, we have been unable to find substantial differences between the capillary density and convolution in patients of similar age but different gender.
Capillary density in the skin of patients with venous disease has been investigated previously by a small number of authors. It has previously been observed that capillary density is reduced in those patients with the more severe stages of venous disease, resulting in atrophie blanche and lipodermatosclerosis.2, 3 Our findings confirm these observations. We have shown that capillary density is broadly similar in CEAP stages C0 to C4a but decreases in patients of stages C4b and C5. Considerable overlap in the range of capillary density was found between the different CEAP stages, and therefore, reduced capillary density is not necessarily a reliable predictor of the severity of the clinical disease.
In contrast, capillary convolution was greatly increased in the more severe CEAP clinical stages (C4a to C5) and might be regarded as a strong indicator of disease severity. The mean number of convolutions in mild stages of venous disease (C1 to C3) is one per capillary loop, with very little variation from this within capillaries from the same patient and between capillaries from different patients. Increased convolution was observed in patients of stage C4a, a group where reduced capillary density has not been reported. Therefore, the finding of increased capillary convolution may be a more sensitive indicator than capillary density of progression of microvascular injury in patients with venous disease.
We recognize that assessment of capillary convolution is a complex measure to use and is unlikely to be of assistance in clinical practice. However, it may be useful in research projects where the objective assessment of skin changes is required in different patient groups and in response to treatment. Capillary microscopy might avoid the need to take skin biopsy specimens in some investigations.
In part 2 of our study, we detected a small group of patients in CEAP stages C2 and C3 that exhibited abnormally increased convolution of their cutaneous capillaries in the leg. Our definition of these skin changes was an average number of convolutions greater than two per capillary loop. Most patients had three to five convolutions per evaluable capillary, but several had six to 10 convolutions per capillary. The absolute numbers of patients identified with these abnormalities in this study is small, only 14 patients of 66 C2 and C3 patients studied, or about 21%.
Varicose veins are present in 25% to 30% of the population, but only 2% to 5% have skin changes or ulceration, a proportion of 10% to 20%. We suspected that these patients are prone to develop clinical skin changes and investigated whether any of the inflammatory mediators that have previously been reported to be elevated in venous disease differed between these patients and those without capillary convolution. We were unable to find any difference between these groups during this investigation and acknowledge that longitudinal studies would be required to answer this question properly with groups of patients followed up over many years.
When we commenced this investigation, the most recent revision of the CEAP classification of venous disease was still under discussion. Based on our earlier work, we recognized that there were substantial differences between patients with mild skin changes (eczema and hemosiderosis) and those with atrophie blanche and lipodermatosclerosis, both of which were classified as CEAP C4 under the original system. We therefore divided these patients into those with mild skin changes (C4a) and those with more severe skin changes (C4b). The data reported here show reduced capillary density in patients of C4b but not C4a groups. In addition, the capillary convolution was greater in C4b patients compared with C4a. We welcome the international adoption of the revised CEAP classification in which the differences between mild and more severe skin changes are recognized.
The data we have presented show in which CEAP stages the skin capillary changes are most severe. Clearly many inflammatory processes are at work in the skin of patients with lipodermatosclerosis and healed ulcers. The considerable proliferation of cells and increased production of collagen in these regions20 is an attempt at healing. It could simply be the case that vascular proliferation is part of this same process, arising in response to the development of lipodermatosclerosis. However, we have also observed increased capillary convolution in a small proportion of patients with apparently uncomplicated varicose veins and have not found any patient with lipodermatosclerosis who does not have capillary proliferation.
Conclusions
We have confirmed that reduced capillary density and increased capillary convolution are features of the skin changes that occur in chronic venous disease. We consider that capillary convolution may be the more reliable indicator of skin microangiopathy in venous disease and that it might be useful as an indicator of disease progression in clinical trials. We have found that about one fifth of patients with apparently uncomplicated venous disease (C2 and C3) also show increased capillary convolution. Our present study was unable to shed further light on this observation. We suggest that this group may be more susceptible to developing severe skin changes of venous disease than most patients.
Author contributions
References
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Competition of interest: none.
PII: S0741-5214(05)02054-9
doi:10.1016/j.jvs.2005.10.077
© 2006 The Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.
