University of Manchester Rheumatic Diseases Centre, Hope Hospital, Salford M6 8HD and
1 Department of Mathematics and Statistics, Lancaster University, Lancaster LA1 4YF, UK
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Abstract |
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Methods. Using the technique of nailfold video capillaroscopy, capillary density and dimensions were averaged from all visible capillaries in a 3 mm length of the nailfold from right and left ring fingers of each subject. Twenty healthy control subjects, 15 patients with PRP, 13 patients with DSSc and 21 patients with LSSc were examined. Intra-observer and inter-observer variability were calculated in 18 and 23 patients, respectively.
Results. There were significant trends for capillary density to fall and for all dimensions to rise across the four groups (P < 0.0001 for density and all dimensions, order healthy controls, PRP, DSSc and LSSc). Intra- and inter-observer reproducibility studies showed that although there was good correlation between and within observers, the limits of agreement were between ±2550% indicating lack of reproducibility.
Conclusions. Microcirculatory abnormalities can be quantified using the technique of video capillaroscopy and were most marked in patients with LSSc.
KEY WORDS: Nailfold video capillaroscopy, Capillary dimensions, Raynaud's phenomenon, Systemic sclerosis.
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Introduction |
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Patients and methods |
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Video capillaroscopy
Patients and controls were asked to refrain from smoking and caffeine-containing drinks for 4 h prior to examination. Each patient/control was acclimatized for 20 min at a room temperature of 23°C prior to video capillaroscopy. The ring finger of each hand was examined.
Capillaroscopy equipment.
The capillaroscopy equipment includes a dissecting microscope with two interchangeable lenses giving magnifications of x200 and x600. A Flexilux 300 long-life fibreoptic light source and filter (Scholly Fibreoptic GmbH, Germany; model D-7819 denzlingen) provides cold illumination. The video camera used with the microscope is the Moritex Europa Ltd, UK camera, model MS-500. This allows the image of the nailfold capillaries to be captured and transferred in an analogue format to a Hitachi M930E video cassette recorder. The capillaries are projected from the video cassette recorder on to a 14 inch television screen which is connected to a personal computer. The software used is Capiflow software as developed by the Karolinska Institute for Microelectronics and Diagnostika HB (Stockholm, Sweden). This software enables the image on the television screen to be analysed.
Measurement of capillary dimensions.
The dimensions of nailfold capillaries from both right and left ring fingers were measured. A length of 3 mm of the distal capillary row was defined by finding an arbitrary start capillary and then measuring 3 mm on the screen by moving the calibration square a distance of 1 mm at a time and reproducing the capillaries by hand on a grid which showed how many capillaries were to be measured. This was done at x200 magnification.
A magnification of x600 was then used to find the start capillary and to measure the capillary dimensions. For this part of the procedure the snapshot mode of the Capiflow software was used, giving a picture on the video screen of a single capillary. Capillary density was determined by counting the total number of capillaries in the 3 mm length, and a mean result taken from right and left hands. The dimensions measured in each capillary were as follows (Fig. 1): the arterial diameter of the capillary (a), the venous diameter of the capillary (v), the width of the capillary at its apex, or apical loop diameter (l) and the total capillary width at its widest point or total loop diameter (t).
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Measurement of inter- and intra-observer variability.
All measurements were originally performed by a vascular flow technician (TM). After this initial analysis the stored videotapes from 18 subjects (spread across all four control/patient groups) were reanalysed to allow calculation of intra-observer variability. To calculate inter-observer variability, a clinician (MB) reanalysed 23 (spread across all groups) of the stored videotapes. For each reanalysis, the observer attempted to find the same start capillary from the sketch of capillary morphology drawn when the capillaries were first measured. However, sometimes it was difficult to be certain that the same start capillary had been identified on each occasion.
Statistical analysis
Demographic data were compared across the groups using analysis of variance (age), the chi-squared test (sex, smoking) or KruskalWallis non-parametric analysis of variance. Density and dimensions were compared across groups using analysis of covariance, adjusted for age, sex and smoking status. Three pre-planned comparisons were carried out between the four groups (PRP vs controls, PRP vs SSc, LSSc vs DSSc): these were based on the pooled variance from the analysis of covariance. All dimensions were log transformed prior to analysis to achieve normality and the results are reported as geometric mean with coefficient of variation (CV).
Intra- and inter-observer reproducibility were assessed using Pearson's correlation, and bias with limits of agreement [5].
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Results |
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These dimensions are smaller than those previously published in abstract form [6, 7]. This is because of a previously unrecognized fault in the computing of the nailfold dimensions, suggesting a magnification of x450 instead of x600. This fault also affected the analysis of the results in our previously published paper [3], but because the fault was consistent the analysis of comparisons between groups is not affected, only the absolute measurements.
Reproducibilityintra- and inter-observer
This is shown in Table 3. Repeated measurements by the same observer (TM2) and a different observer (MB) were highly correlated with the original measurements (TM1), but the limits of agreement were between ±2550%, indicating lack of reproducibility. The actual measurements showed small, but often statistically significant, bias both within and between observers.
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Discussion |
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While we have confirmed our previous finding that capillary dimensions are larger in patients with PRP compared with healthy controls, this was only a trend that was not statistically significant and differences between the two groups were small. Nonetheless these findings lend some further support to our previous suggestion that PRP is not an entirely benign vasospastic phenomenon, but may be associated with subtle microcirculatory changes. In the present study we averaged dimensions across all measurable capillaries in two 3 mm lengths of nailfoldone from the left and one from the right ring finger. This would seem to be a more objective method of evaluation than in our previous study, in which we chose the largest capillary of the distal row.
The technique of video capillaroscopy facilitates the quantitation of the nailfold capillary abnormalities which have been long recognized in patients with connective tissue disease [1]. Because images are recorded on video, images can be stored for analysis and reanalysis (as in this study). So far only a small number of studies have applied this technique in patients with primary and secondary Raynaud's. Kabasakal et al. used the technique to define a set of abnormal findings including enlarged capillaries, giant capillaries, haemorrhage, and avascularity and compared frequencies of these abnormal findings (and also capillary density) between patients with connective tissue disease and healthy controls [11], but did not compare capillary dimensions between groups. Patients with SSc demonstrated a reduced capillary density compared with patients with systemic lupus erythematosus, undifferentiated connective tissue disease or healthy controls, and patients with SSc had the highest frequency of severe avascularity, giant capillaries and haemorrhage [11]. Studer et al. used the technique of quantitative television microscopy (capillary microscopy connected to a television camera) to measure a number of parameters in patients with localized scleroderma, SSc, cutaneous and systemic lupus erythematosus, and healthy control subjects: the visibility of the venous plexus and the number of capillary haemorrhages were measured in addition to capillary dimensions and density [12]. Dimensions were greatest in patients with systemic connective tissue disease, especially in those with SSc [12].
There is also the potential to extend the technique of video capillaroscopy to examine red blood cell velocity in response to a dynamic challenge such as cooling [1315] and to examine capillary permeability [16, 17]. The study by Studer et al. included measurement of the stop-flow duration [12].
A larger number of studies quantified nailfold capillary abnormalities using primarily the widefield technique without continuous recording. Different methods of quantifying capillary abnormalities have included the following: the number of capillaries in the distal row measured over 5 mm [18]; an index derived from capillary apex width and maximum limb width [19]; afferent and efferent luminal diameters [20]; the mean diameter of the capillary loops, averaged across the whole nailfold, and capillary density [21]; mean capillary surface area [22]; computer-based analysis of capillary density and median capillary loop area [8]. In a study of 800 healthy individuals and using a widefield technique, Andrade et al. proposed a method of objectively assessing nailfold capillaries which included assessment of devascularization, morphology of the distal loops, endothelial damage (microhaemorrhage) and plexus visibility [23].
Although the technique of video microscopy clearly has the potential for allowing us to quantify microcirculatory abnormalities in patients with Raynaud's phenomenon and connective tissue disease, the current method of quantification has its limitations. Calculating mean dimensions using our current software is extremely laborious and time consuming. Some capillaries are difficult to visualize, and there is therefore a subjective element in deciding which capillaries to include in the analysis, which capillaries belong to the distal row (some capillary loops fall short of others) and, especially for tortuous capillaries, at which point to measure dimensions. To gain insight into this subjective element of the analysis, we studied both intra-observer and inter-observer variability. The results from this analysis showed that the usefulness of video microscopy is somewhat limited by its lack of reproducibility as evidenced by the substantial intra- and inter-observer variability, even though both observers had considerable experience in the technique. Although the between-group differences seen in this study are much larger than could be accounted for by measurement error alone, our present technique of quantifying abnormalities could not be used to measure change within an individual over time or in response to treatment, as small differences would not be reliably detectable. In the future more sophisticated image analysis programmes [24] may partially obviate the above problems and improve upon intra- and inter-observer variability. At present, measurement of capillary dimensions by nailfold video microscopy is purely a research tool, as opposed to widefield microscopy which is useful in clinical practice.
As a final point, the high number of smokers among the three patient groups is of interest. We have previously reported that patients with Raynaud's phenomenon who smoked cigarettes had lower plasma concentrations of ascorbic acid than did non-smokers, and suggested that cigarette smoking may be a risk factor [25]. There is therefore a need for studies addressing the specific issue as to whether cigarette smoking is an independent risk factor for developing severe Raynaud's phenomenon.
In conclusion, video microscopy allows capillary abnormalities to be quantified, and in this study has demonstrated how these abnormalities in patients with SSc are most pronounced in the LSSc subtype of disease. Further studies examining capillary density and dimensions in different patient groups prospectively, using improved image analysis programmes, are now required to elucidate whether video capillaroscopy might be a useful tool in the assessment of disease progression.
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Notes |
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References |
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