Microvascular Research 80 (2010) 552–555

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Microvascular Researchj o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y m v r e

Short Communication

Dynamic ﬂuorescence imaging of indocyanine green for reliable and sensitive
diagnosis of peripheral vascular insufﬁciency
Yujung Kang a,b, Jungsul Lee a,b, Kihwan Kwon c,⁎, Chulhee Choi a,b,⁎
abc

Department of Bio and Brain Engineering, KAIST, Korea
KI for the BioCentury, KAIST, Korea
Department of Cardiology, School of Medicine, Ewha Woman's University, Korea

a r t i c l ei n f oArticle history:
Received 28 May 2010
Accepted 8 July 2010
Available online 15 July 2010
Keywords:
Peripheral vascular disease
Perfusion
Arterial stenosis
Optical imaging
Indocyanine greena b s t r a c t
Quantitative measurement of functional tissue perfusion is essential for early diagnosis and proper
treatment of peripheral arterial occlusive disease (PAOD). We have previously demonstrated that dynamic
imaging of near-infrared ﬂuorophore indocyanine green (ICG) can be a noninvasive and sensitive tool tomeasure tissue perfusion. In the present study, we investigated the clinical efﬁcacy of ICG perfusion
imaging method for the diagnosis of PAOD. Total nineteen PAOD patients and age-matched controls(n = 10) were evaluated for lower extremity tissue perfusion using ICG perfusion imaging. The perfusion
rates of the lower extremities with severe PAOD (n = 25 legs, 16.6 ± 8.3%/min) were signiﬁcantly lower
than those of normal controls (38.1 ± 17.3%/min, p b 0.001). In cases of mild PAOD, the perfusion rates(n = 11 legs, 18.3 ± 10.3%/min) were also signiﬁcantly lower compared to the control; while theconventional ankle-brachial index (ABI) test failed to detect mild functional impairment. These results
collectively indicate that ICG perfusion imaging can be a very effective tool for diagnosis of PAOD with asuperior efﬁcacy in comparison to conventional ABI test.
© 2010 Elsevier Inc. All rights reserved.

Introduction
Peripheral vascular disease (PVD) has been deﬁned as lower
extremity arterial atherosclerosis because most PVD occurs in themajor arterial segments of aortoiliac, femoropopliteal, and tibioperoneal arteries. This syndrome encompasses a wide range of diseases
from asymptomatic arterial narrowing to critical limb ischemia andgangrene (Khan and Shearman, 2005). Hyperglycemia, dyslipidemia,
and hypertension are well-known potentially modiﬁable risk factors
for the development of PVD (Adler et al., 2002). Although PVD isprevalent in the primary care setting, it has also been unrecognizedand undertreated. However, based on an association with fatal
cardiovascular diseases, high-risk patients have been recommended
to receive a periodic medical checkup for PVD in the primary caresetting (Stein et al., 2006).
Diagnostic methods presently used include the ankle brachialindex (ABI), plethysmography, and angiography by X-ray orcomputed tomography (CT), all of which have limitations. The ABI
is highly sensitive and speciﬁc for diagnosing PVD in patients withsevere arterial stenosis, but the utility of the ABI in patients with lesssevere stenosis and calciﬁed vessels is questionable (Stein et al.,⁎ Corresponding authors. Kwon is to be contacted at Department of Cardiology, Ewha
Woman's University, Seoul 158-710, Korea. Fax: + 82 2 2650 2640. Choi, Department of
Bio and Brain Engineering, KAIST, Daejeon 305-751, Korea. Fax: +82 42 350 4380.
E-mail addresses: kankadin@ewha.ac.kr (K. Kwon), cchoi@kaist.ac.kr (C. Choi).
0026-2862/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.mvr.2010.07.004

2006). Plethysmography, applying the strain-gauge technique,detects pulsatile pressure changes in a nearby artery; therefore,the arterial pressure measured by plethysmography is not a reliable
index of arterial ﬂow in cases of abnormal vascular impedance. X-ray
and CT angiography can only visualize the vessel lumen, and not thewall, and cannot assess the end-organ effects of peripheral artery
occlusive disease (PAOD) (Kramer, 2007). Because of the procedure
invasiveness, arteriography is usually reserved for patients withoccult symptoms sufﬁcient to make them surgical candidates and is
not usually required for an early PAOD diagnosis. Diagnostic
modalities that allow study of progression and regression of PVD
are clearly needed. The ability to assess functional tissue perfusion
will be ideal for the periodic checkup, early diagnosis, and adequate
treatment of PVD.
Near-infrared (NIR) ﬂuorescence optical imaging has been studied
as a noninvasive and non-ionizing diagnostic modality for in vivo
imaging of the vasculature and estimating functional perfusion.
Optical imaging as a screening method has several advantages: noor minimal invasiveness, low cost, safety, ease of use, and bedsideassessment (Cheng et al., 2005; Frangioni, 2003; Keller et al., 2001).
We previously developed an ICG perfusion imaging method thatemploys time-series planar NIR ﬂuorescence imaging and analyzes
modeling of spatiotemporal dynamics to assess perfusion in the lower
extremities (Kang et al., 2009a, 2009b, 2010). In this study, weperformed a pilot clinical study to validate the efﬁcacy of this methodon the diagnosis of mild as well as severe PAOD.

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553

Lower extremity CT angiography

Table 1
Demographics of patients enrolled in the study.
Arterial stenosis

Less than 20%
n = 10 (20 legs)

20–50%
n = 8 (11 legs)More than 50%
n = 16 (25 legs)Age, years
MedianGender, male/female
Risk factors
HTN (mean duration)
DM (mean duration)
Smoking
Dyslipidemia

59.6 ± 8.2⁎
61
2/8

66.5 ± 8.7⁎
68
5/3

68.7 ± 9.9⁎
71
12/3

2 (4)
––
1

2 (3.5)–––5 (5)3 (10)
1–*F2,31 = 3.154, p = 0.057 by ANOVA test.

Materials and methods
Subjects
All protocol, survey, and consent forms were approved by theInstitutional Review Board of Ewha Woman's University Mokdong
Hospital. Written informed consent was obtained from all subjects.
Twenty-nine subjects were recruited for the analysis of perfusion ratesof the lower extremities to test the efﬁcacy of ICG perfusion imaging forthe diagnosis of PAOD. PAOD was deﬁned on lower extremities by CTangiography as a diameter reduction of N50% in the major peripheralarteries, and leg showing diameter reduction less than 20% was deﬁned
as a normal; leg with arterial stenosis between 20% and 50% of in themajor peripheral arteries, as a mild PAOD. Twenty-ﬁve of the PAOD legs
were recruited from nine patients with bilateral stenosis and sevenpatients with unilateral stenosis (arterial stenosis more than 50% in onlyone leg), and 11 mild-PAOD legs were collected from 3 subjects withbilateral and 5 patients with unilateral mild stenosis. Finally, twenty ofnormal legs were examined from 10 age-matched subjects (Table 1).
Lower extremity CT angiography, ABI, and the ICG perfusion imaging
test were performed in all subjects.

Lower extremity CT angiography, ABI, and the ICG perfusion imaging
test were performed in all subjects. CT angiography was performed witha 16-MDCT scanner (SOMATOM Sensation; Siemens Medical Solutions,Erlangen, Germany), and volume data sets were acquired with
12 × 0.75 mm collimation, a gantry rotation time of 420 ms, a table
feed of 2.8 mm per 360° rotation, and an effective tube current-time
product of 400 mAs at a tube voltage of 120 kV. An 18-gauge catheter
was placed into the antecubital vein, and 120 ml of nonionic contrast
material (Iohexol, Omnipaque 300, Nycomed; Amersham, Princeton,
UK) was injected at 4 ml/s. CT scans were obtained from above the iliac
bifurcation to the feet. Postprocessing for CT angiography resulted inimages rotated over 360° for the aortoiliac, femoropopliteal, and tibial
arteries using commercially available cardiac reconstruction software(Syngo, version A50A; Siemens Medical Solutions). Two doctors withextensive experience interpreting CT angiograms evaluated all images
for arterial stenosis and assessed the degree of stenosis.
Ankle brachial index (ABI)
After remaining supine for 5–10 min, blood pressures wererecorded in both brachial arteries and in the dorsalis pedis andposterior tibial arteries (VasoGuard P84; SciMed Ltd., Bristol, UK). TheABI was calculated for each leg by dividing the highest ankle systolicpressure by the highest brachial systolic pressure. Generally, an
ABI b 0.9 was interpreted as legs with PAOD.
ICG perfusion imaging
We recently developed a novel optical imaging-based technology fortranslating ICG time-series images into a quantitative perfusion rate (%/min), which was deﬁned as the fraction of blood exchanged in thevascular volume per minute (Kang et al., 2009a, b, 2010). Fig. 1 illustrates

Fig. 1. ICG perfusion imaging system. (A and B) Diagram of the imaging system. (B) Outside view of the system. (C) Capturing an ICG serial imaging for perfusion analysis.

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Y. Kang et al. / Microvascular Research 80 (2010) 552–555

an ICG ﬂuorescence imaging system manufactured by Vieworks Corporation (Seongnam, Gyeonggi-do, Korea). After remaining supine for 5–
10 min, 120 serial images (748×518 pixels) of both feet were taken at 5-sintervals for 600 s after an intravenous bolus injection of ICG (0.16 mg/kg)for time-series analysis of ICG ﬂuorescence. The perfusion rate wascalculated using nonlinear regression and differential evolution methods
as described previously (Kang et al., 2010).
Statistical analysis
Data are expressed as the mean± SD. Statistical signiﬁcance wasassessed by Student's two-tailed t-tests for analysis of the PAOD andnormal control. Correlation between the perfusion rate and the ABI wasanalyzed with an ANOVA and Bonferroni post hoc test on the groups of
mild-PAOD, PAOD and normal control. The difference between samples
was considered statistically signiﬁcant is the p value was less than 0.05.
Results and discussion
The perfusion rates of normal legs were signiﬁcantly higher(38.1 ± 17.3%/min) compared to those of the PAOD legs (16.6 ±8.3%/min; Fig. 2A). Setting the value of 24.4%/min perfusion rate

as a cutoff, this optical approach predicted the presence of PAOD
with a sensitivity of 0.92 and a speciﬁcity of 0.90 with an area
under the ROC curve of 0.895 (95% conﬁdence interval, 0.79 to1.0) for the angiographically documented PAOD (Fig. 2B). Becauseour novel optical method detects functional perfusion impairment
rather than structural abnormalities, this method might be useful
not only for conﬁrming obvious occlusive cases but also for
detecting mild impairment, especially in the early stages ofatherosclerotic changes. To test this idea, the perfusion rates oflower extremities from 8 subjects with asymptomatic PAOD wereevaluated and compared with those of normal and severe PAODlegs. As expected, the perfusion rates of mild PAOD cases weresigniﬁcantly lower compared to those of normal control (18.3 ±10.3%/min, F2,53 = 18.0, p b 0.001 by ANOVA test ) (Fig. 2C leftpanel). Although the ABIs of mild-PAOD, PAOD and normal groups
were signiﬁcant differences (1.2 ± 0.1, 1.0 ± 0.2, 0.6 ± 0.2, respectively; F2,53 = 57.7, p b 0.001 by ANOVA test), there was no
signiﬁcant difference in the ABIs between normal and mildPAOD legs (p = 0.14 by Bonferroni post hoc test, Fig. 2C rightpanel).
Consistent with a previous report (Lijmer et al., 1996), the ABI
could detect PAOD legs as well as ICG perfusion imaging method.

Fig. 2. The performance of the perfusion rate to discriminate PAOD legs from normal legs. (A) Perfusion rates from the PAOD and Normal legs. Each dot represents one leg. The graydashed line indicates a cutoff value for diagnosis of PAOD. p Value was evaluated by Student's t-test. (B) ROC curve of the perfusion rate to diagnose PAOD legs. The ideal criterion wasdeﬁned as the white point on the ROC curve. (C) Perfusion rates and ABIs of the mild-PAOD, PAOD and normal groups. *p b 0.001 by Bonferroni post hoc test.

Y. Kang et al. / Microvascular Research 80 (2010) 552–555

On the contrary, this conventional method failed to differentiate
mild cases of PAOD from a normal group; while the ABI has been
widely applied to detect the functional decline in peripheral
vasculature due to its sensitivity and noninvasiveness (McDermottet al., 2009). Despite these advantages, the ABI shows poor
discriminative power in distinguishing mild and moderate degreesof stenosis (McDermott et al., 2005), as well as in providing
diagnostic information, particularly in patients with diabetes andlimb claudication (Lindner et al., 2008). Although large population
studies have demonstrated a signiﬁcant correlation between theABI and clinical symptoms, normal ABIs are commonly encountered in patients with symptomatic PAD (Wang et al., 2005).
Considering the high sensitivity of ICG perfusion imaging fordetection of mild functional impairments, we suggest a combined
use of the ABI and perfusion imaging for a complementary
diagnosis of various stages of perfusion impairment.
Although the small number of patients had been enrolled in thecurrent study, our pilot clinical trial clearly demonstrated that thisnovel dynamic optical imaging combined with modeling can provide
highly reliable and quantitative estimates of peripheral tissue
perfusion sensitive enough for diagnosis of mild cases of PAOD.
This observation on efﬁcacy of ICG perfusion imaging test prompts
us to pursue a large-scale study to unveil the role of various risk
factors such as metabolic abnormalities on peripheral vascularinsufﬁciency.
Acknowledgments
We thank Dr. D. Kim (Department of Cardiology, School ofMedicine, Ewha Woman's University) for help with the clinical trial.
This work was supported by the Korea Healthcare Technology R&D555

Project (A091258), Ministry for Health, Welfare and Family Affairs ofthe Republic of Korea.
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