Name: a reproducible computerized method for quantitation of capillary density using nailfold capillaroscopy
Uploaded: 2015-10-27T15:00:00
Description: Watch this Scientific Journal Video about A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy at JoVE.com

This project was supported by Grant Numbers HL96593 from NIH and D56HP20783 from HRSA/ HHS. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH or HRSA / HHS.

Note: The acquisition process for obtaining capillary images has previously been published and is accomplished using a still digital camera with a corresponding image acquisition and analysis computer program. 1121 This lab utilizes still images for analysis, not videos, simplifying image acquisition for analysis. The following describes the new technique for quantitating the capillaries from the images.
1. Image Enhancement Process
<ol>
	<li>Obtain digital imag...

<ol>
	<li>Gronenschild, E. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semi-automatic+assessment+of+skin+capillary+density:+proof+of+principle+and+validation.">Semi-automatic assessment of skin capillary density: proof of principle and validation.</a> Microvasc Res. 90, 192-198 (2013).</li><li>Allen, J., Howell, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+imaging:+techniques+and+opportunities+for+clinical+physiological+measurements.">Microvascular imaging: techniques and opportunities for clinical physiological measurements.</a> Physiol Meas. 35, R91-R141 (2014).</li><li>Boettcher, M., Gloe, T., de Wit, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semiautomatic+quantification+of+angiogenesis.">Semiautomatic quantification of angiogenesis.</a> J Surg Res. 162, 132-139 (2010).</li><li>Wild, R., Ramakrishnan, S., Sedgewick, J., Griffioen, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+angiogenesis+and+tumor+vessel+architecture+by+computer-assisted+digital+image+analysis:+effects+of+VEGF-toxin+conjugate+on+tumor+microvessel+density.">Quantitative assessment of angiogenesis and tumor vessel architecture by computer-assisted digital image analysis: effects of VEGF-toxin conjugate on tumor microvessel density.</a> Microvasc Res. 59, 368-376 (2000).</li><li>Tresadern, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simulating+nailfold+capillaroscopy+sequences+to+evaluate+algorithms+for+blood+flow+estimation.">Simulating nailfold capillaroscopy sequences to evaluate algorithms for blood flow estimation.</a> Conf Proc IEEE Eng Med Biol Soc. , 2636-2639 (2013).</li><li>Anderson, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computerized+nailfold+video+capillaroscopy--a+new+tool+for+assessment+of+Raynaud's+phenomenon.">Computerized nailfold video capillaroscopy--a new tool for assessment of Raynaud's phenomenon.</a> J Rheumatol. 32, 841-848 (2005).</li><li>Neubauer-Geryk, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+reactivity+of+skin+microcirculation+in+response+to+L-arginine+in+later-onset+type+1+diabetes.">Decreased reactivity of skin microcirculation in response to L-arginine in later-onset type 1 diabetes.</a> Diabetes Care. 36, 950-956 (2013).</li><li>Pazos-Moura, C. C., Moura, E. G., Bouskela, E., Torres-Filho, I. P., Breitenbach, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nailfold+capillaroscopy+in+diabetes+mellitus:+morphological+abnormalities+and+relationship+with+microangiopathy.">Nailfold capillaroscopy in diabetes mellitus: morphological abnormalities and relationship with microangiopathy.</a> Braz J Med Biol Res. 20, 777-780 (1987).</li><li>Antonios, T. F., Singer, D. R., Markandu, N. D., Mortimer, P. S., MacGregor, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+skin+capillary+rarefaction+in+essential+hypertension.">Structural skin capillary rarefaction in essential hypertension.</a> Hypertension. 33, 998-1001 (1999).</li><li>Kaiser, S. E., Sanjuliani, A. F., Estato, V., Gomes, M. B., Tibirica, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antihypertensive+treatment+improves+microvascular+rarefaction+and+reactivity+in+low-risk+hypertensive+individuals.">Antihypertensive treatment improves microvascular rarefaction and reactivity in low-risk hypertensive individuals.</a> Microcirculation. 20, 703-716 (2013).</li><li>Cheng, C., Diamond, J. J., Falkner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+capillary+rarefaction+in+mild+blood+pressure+elevation.">Functional capillary rarefaction in mild blood pressure elevation.</a> Clinical and Translational Science. 1, 75-79 (2008).</li><li>Debbabi, H., Bonnin, P., Levy, B. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+blood+pressure+control+with+perindopril/indapamide+on+the+microcirculation+in+hypertensive+patients.">Effects of blood pressure control with perindopril/indapamide on the microcirculation in hypertensive patients.</a> Am J Hypertens. 23, 1136-1143 (2010).</li><li>He, F. J., Marciniak, M., Markandu, N. D., Antonios, T. F., MacGregor, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+modest+salt+reduction+on+skin+capillary+rarefaction+in+white,+black,+and+Asian+individuals+with+mild+hypertension.">Effect of modest salt reduction on skin capillary rarefaction in white, black, and Asian individuals with mild hypertension.</a> Hypertension. 56, 253-259 (2010).</li><li>Murray, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+measurement+location+on+reliability+of+quantitative+nailfold+videocapillaroscopy+in+patients+with+SSc.">The influence of measurement location on reliability of quantitative nailfold videocapillaroscopy in patients with SSc.</a> Rheumatology (Oxford). 51, 1323-1330 (2012).</li><li>Ingegnoli, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+of+different+capillaroscopic+measures+for+identifying+nailfold+microvascular+alterations.">Feasibility of different capillaroscopic measures for identifying nailfold microvascular alterations.</a> Semin Arthritis Rheum. 38, 289-295 (2009).</li><li>Debbabi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+skin+capillary+density+in+treated+essential+hypertensive+patients.">Increased skin capillary density in treated essential hypertensive patients.</a> Am J Hypertens. 19, 477-483 (2006).</li><li>Serne, E. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+skin+capillary+recruitment+in+essential+hypertension+is+caused+by+both+functional+and+structural+capillary+rarefaction.">Impaired skin capillary recruitment in essential hypertension is caused by both functional and structural capillary rarefaction.</a> Hypertension. 38, 238-242 (2001).</li><li>Shore, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillaroscopy+and+the+measurement+of+capillary+pressure.">Capillaroscopy and the measurement of capillary pressure.</a> Br J Clin Pharmacol. 50, 501-513 (2000).</li><li>Rieder, M. J., O'Drobinak, D. M., Greene, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computerized+method+for+determination+of+microvascular+density.">A computerized method for determination of microvascular density.</a> Microvasc Res. 49, 180-189 (1995).</li><li>Vermeulen, P. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+angiogenesis+in+solid+human+tumours:+an+international+consensus+on+the+methodology+and+criteria+of+evaluation.">Quantification of angiogenesis in solid human tumours: an international consensus on the methodology and criteria of evaluation.</a> Eur J Cancer. 32A, 2474-2484 (1996).</li><li>Cheng, C., Daskalakis, C., Falkner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+Assessment+of+Microvascular+and+Endothelial+Function.">Non-invasive Assessment of Microvascular and Endothelial Function.</a> Journal of Visualized Experiments. , (2012).</li><li>Antonios, T. 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Nailfold capillaroscopy shows promise as a clinically useful tool in the future for various oncology, cardiovascular, and rheumatologic disease applications. The image acquisition process is fairly consistent among researchers, yet there are currently multiple methods for image processing and analysis. Methods currently include computerized and manual capillary counts. Manual counts are problematic as they are time-consuming, and subject to user subjectivity and fatigue. Current computer based methods require a high leve...

Microvascular imaging is a rapidly growing field with many potential clinical applications.2 For example, oncologists are using microvessel imaging to determine the extent of tumor angiogenesis, yielding valuable information concerning the state of the tumor and insight into possible treatment options. 34 However, nailfold capillaroscopy is perhaps the most cost efficient and widely applicable form of microvascular imaging. Researchers are using video nailfold capillaroscopy to study blood flow rates and investigate capillary morphology.5 6 Both video and still-picture nailfold capillaroscopy are adjuncts to care for diagnosing and treating Raynaud&#8217;s phenomenon and various connective tissue diseases such as systemic sclerosis.2
Nailfold capillaroscopy has various potential cardiovascular applications as well. Current research using nailfold capillaroscopy suggests that diabetes mellitus Type 1 and Type 2 patients exhibit a high prevalence of abnormal capillary morphology, yet have unchanged capillary densities compared to non-diabetic individuals.7-8 Capillaroscopy has also been studied experimentally in hypertension. Structural capillary rarefaction leading to a reduced capillary density has been demonstrated in hypertensive individuals compared to non-hypertensive individuals.9-10 In contrast to these older hypertensive patients (mean age 40 and above) who exhibit structural rarefaction, more recent research has demonstrated that younger hypertensive patients (mean age under 40 years old) have functional rarefaction without structural rarefaction.11 This suggests that functional rarefaction occurs before and may progress over time to structural rarefaction.
Interestingly, hypertensive patients treated with specific antihypertensive drugs such as Perindopril/Indapamide displayed normal capillary density and endothelial function after treatment, while those treated with ACE (angiotensin-converting-enzyme) inhibitors or diuretics maintained a low capillary density despite comparable blood pressure control.12 This suggests that some antihypertensive medications may normalize capillary density by reversing the capillary rarefaction caused by hypertension. In addition, other researchers have shown that a reduction in salt intake leads to reversal of both functional and structural capillary rarefaction in hypertensive individuals.13
Despite the various potential clinical applications of this technology, there is little standardization in technique for quantitating capillary density images.2 To date, researchers have found that capillary density results are reproducible from both an intra-observer and inter-observer perspective only if the exact same area is being counted each time. 1,14 15 Of note, previous researchers have largely performed capillary counts manually using the naked eye, 9 16 17 18 which is a slow and subjective process.
Standardized, computer based algorithms for quantitation of capillary images theoretically provide more efficient and reproducible data analysis with less subjectivity, facilitating clinical applications of capillaroscopy. Some researchers have indeed used computer-based programs to quantitate the data from nailfold capillaroscopic pictures. 1,6 19 20 However, only one publication to date describes reliability of a complex software program available for quantitating capillaroscopy data,1 and this program is complicated as previously noted above by the requirement to count the exact same visual field. Here, we present a simpler, reliable protocol for quantitating capillaries using a standardized algorithm which allows for the use of multiple visual fields. The use of multiple visual fields not only simplifies the procedure, but also permits the assessment of normal biological variation in capillary count.
The aim of this study is to describe a reproducible and efficient computer based algorithm which standardizes the capillary quantitation process. While these methods are not fully automated they require very little user input, and provide rapid and reliable quantitation of the pictures.

<table>
	<tbody>
		<tr>
			<td class="xl65">Image-Pro Premier</td>
			<td class="xl65">Media Cybernetics, Inc</td>
			<td align="right" class="xl63">9.1</td>
			<td class="xl63">Image processing software</td>
		</tr>
	</tbody>
</table>

a reproducible computerized method for quantitation of capillary density using nailfold capillaroscopy

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy to learn methodology for directly visualizing the microcirculation. The capillaroscopy technique can provide insight into a patient&#8217;s microvascular health, leading to a variety of potentially valuable dermatologic, ophthalmologic, rheumatologic and cardiovascular clinical applications. In addition, tumor growth may be dependent on angiogenesis, which can be quantitated by measuring microvessel density within the tumor. However, there is currently little to no standardization of techniques, and only one publication to date reports the reliability of a currently available, complex computer based algorithms for quantitating capillaroscopy data.1 This paper describes a new, simpler, reliable, standardized capillary counting algorithm for quantitating nailfold capillaroscopy data. A simple, reproducible computerized capillaroscopy algorithm such as this would facilitate more widespread use of the technique among researchers and clinicians. Many researchers currently analyze capillaroscopy images by hand, promoting user fatigue and subjectivity of the results. This paper describes a novel, easy-to-use automated image processing algorithm in addition to a reproducible, semi-automated counting algorithm. This algorithm enables analysis of images in minutes while reducing subjectivity; only a minimal amount of training time (in our experience, less than 1 hr) is needed to learn the technique.

The goal of this image processing procedure is to differentiate the capillaries from the background image so they can be accurately quantified. Both incomplete image processing and excessive image processing are detrimental to the program's ability to quantify the capillaries. As seen in Figure 3, incomplete image processing makes the capillaries difficult to distinguish from the background. It is critical that the user be able to readily distinguish the border of a capillary since the counting method de...

Watch this Scientific Journal Video about A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy at JoVE.com

A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy-to-learn methodology for directly visualizing capillaries in the microcirculation. However, only one publication to date describes the reliability of a complex software program available for quantitating capillaroscopy data. Here, we present a simple, reliable protocol for quantitating capillaries using a standardized algorithm.

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy to learn methodology for directly visualizing the microcirculation. The ...

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