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Methods & Techniques 2
Methods & Techniques
2
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NON-‐INVASIVE TOOLS TO INVESTIGATE MICROVASCULAR STRUCTURE AND
FUNCTION
Because of their accessibility, skin and nailfold capillaries are often used to investigate the
microcirculation. We choose two non-‐invasive techniques to study microcirculatory
structure and function respectively: nailfold video capillary microscopy, and iontophoresis
combined with laser Doppler flowmetry.
SKIN MICROCIRCULATION
Nailfold capillaroscopy and iontopheresis should be considered against the background of
the anatomy and physiology of the skin microcirculation. The microvascular bed of the skin
consists of nutritive capillaries, a subpapillary plexus and deeper arteriovenous anastomoses
(figure 1).
Epidermis
Capillary
Plexus
Anastomoses
Figure 1. Skin anatomy
The nutritive capillaries are the most superficial ones (10-‐50 μm from the skin surface). In
most areas of the fingers, the nutritional capillary loops run perpendicular to the skin surface
and only the apex of the capillary loops can be visualised (figure 2).
In the nailfold area however, the capillary loops run more parallel with the skin surface, and
the distal row capillaries can be visualised in their full length (figure 3).
Figure 2. In most areas of the finger, only the apex of capillaries can be visualised as dots
Figure 3. In the nailfold, capillaries can be visualised as loops
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NON-‐INVASIVE TOOLS TO INVESTIGATE MICROVASCULAR STRUCTURE AND
FUNCTION
Because of their accessibility, skin and nailfold capillaries are often used to investigate the
microcirculation. We choose two non-‐invasive techniques to study microcirculatory
structure and function respectively: nailfold video capillary microscopy, and iontophoresis
combined with laser Doppler flowmetry.
SKIN MICROCIRCULATION
Nailfold capillaroscopy and iontopheresis should be considered against the background of
the anatomy and physiology of the skin microcirculation. The microvascular bed of the skin
consists of nutritive capillaries, a subpapillary plexus and deeper arteriovenous anastomoses
(figure 1).
Epidermis
Capillary
Plexus
Anastomoses
Figure 1. Skin anatomy
The nutritive capillaries are the most superficial ones (10-‐50 μm from the skin surface). In
most areas of the fingers, the nutritional capillary loops run perpendicular to the skin surface
and only the apex of the capillary loops can be visualised (figure 2).
In the nailfold area however, the capillary loops run more parallel with the skin surface, and
the distal row capillaries can be visualised in their full length (figure 3).
Figure 2. In most areas of the finger, only the apex of capillaries can be visualised as dots
Figure 3. In the nailfold, capillaries can be visualised as loops
20
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In the subpapillary vascular bed (approximately 0.05-‐2.0 mm from the skin surface) the
dominating vessels are venules and, to a smaller extent, arterioles. The deeper
arteriovenous anastomoses have a thermoregulatory function. They are especially numerous
in the fingertips, ears and nose, whereas the skin of the dorsal finger and forearm are
thought to lack arteriovenous anastomoses.1 Under conditions of normal environmental
temperature (20-‐25 ºC), the majority (>90%) of total skin blood flow passes through the
arteriovenous anastomoses. Skin microcirculatory blood flow and pressure are determined
by a balance of central and local mechanisms. In addition, microvascular perfusion is
influenced by the rheological properties of blood. Central control is achieved by sympathetic
adrenergic vasoconstrictor nerves, through the action of norepinephrin on alpha-‐receptors,
which control blood flow through the arteriovenous anastomoses. Capillaries themselves
have no nerve supply. Local autoregulatory mechanisms are usually direct responses of
vascular smooth muscle to local metabolites, changes in transmural pressure or shear stress.
These local autoregulatory mechanisms may in part be endothelium-‐dependent.1,2
NAILFOLD CAPILLAROSCOPY
The technique of nailfold capillaroscopy as a tool to study structural microcirculatory
changes in the nailfold has been widely used in the assessment of patients with Raynaud's
Phenomenon (RP) and SSc. The nomenclature of structural capillary abnormalities in the
literature has not been consistent.3 Hildegard Maricq has worked extensively to refine the
assessment of the skin capillaries using microscopic and photographic equipment.4,5 She
distinguished the following characteristics of the nailfold capillary bed, although exact
quantification of the capillary dimensions could not be made:
1. Scleroderma capillary pattern. Based on overall estimation of the
microvascular abnormalities. Presence of enlarged capillaries of the
scleroderma (SD)-‐ type: increased diameter (> 25 µm) of all three portions of
the capillary loop (arterial, apical, and venular), often associated with
avascular areas: none, slight (0.4-‐2 mm2), moderate (2-‐4 mm2) extensive (>4
mm2), and a combination of other morphological features such as capillary
haemorrhages, disorganisation of the capillary bed, edema and discoloration
of the cuticle.
2. ‘active’ or ‘slow’ capillary pattern, based on the size of the nailfold terminal
row capillary loops: normal, definitely enlarged (total width 91-‐150 µm), or
extremely enlarged (>150 µm), and on the extent of the avascular areas.
‘Active’ is defined as moderate to extensive avascular areas without capillary
telangiectases (clusters of dilated capillaries) or extremely enlarged loops.
‘Slow’ is defined by extremely enlarged capillaries with no or minimal
avascularity with or without capillary telangiectases.
The ‘active’ and ‘slow’ patterns were defined on the basis of an observed correlation
between the activity of the microvascular lesions and the clinical progression of the disease
in small series of cases.5
In 2000, Cutolo et al, classified microvascular changes in SSc patients into 3 distinct
patterns6:
1. early pattern: few enlarged/giant capillaries, few capillary hemorrhages, no
evident loss of capillaries.
2. active pattern: frequent giant capillaries, frequent capillary hemorrhages, mild
disorganisation of the capillary network.
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In the subpapillary vascular bed (approximately 0.05-‐2.0 mm from the skin surface) the
dominating vessels are venules and, to a smaller extent, arterioles. The deeper
arteriovenous anastomoses have a thermoregulatory function. They are especially numerous
in the fingertips, ears and nose, whereas the skin of the dorsal finger and forearm are
thought to lack arteriovenous anastomoses.1 Under conditions of normal environmental
temperature (20-‐25 ºC), the majority (>90%) of total skin blood flow passes through the
arteriovenous anastomoses. Skin microcirculatory blood flow and pressure are determined
by a balance of central and local mechanisms. In addition, microvascular perfusion is
influenced by the rheological properties of blood. Central control is achieved by sympathetic
adrenergic vasoconstrictor nerves, through the action of norepinephrin on alpha-‐receptors,
which control blood flow through the arteriovenous anastomoses. Capillaries themselves
have no nerve supply. Local autoregulatory mechanisms are usually direct responses of
vascular smooth muscle to local metabolites, changes in transmural pressure or shear stress.
These local autoregulatory mechanisms may in part be endothelium-‐dependent.1,2
NAILFOLD CAPILLAROSCOPY
The technique of nailfold capillaroscopy as a tool to study structural microcirculatory
changes in the nailfold has been widely used in the assessment of patients with Raynaud's
Phenomenon (RP) and SSc. The nomenclature of structural capillary abnormalities in the
literature has not been consistent.3 Hildegard Maricq has worked extensively to refine the
assessment of the skin capillaries using microscopic and photographic equipment.4,5 She
distinguished the following characteristics of the nailfold capillary bed, although exact
quantification of the capillary dimensions could not be made:
1. Scleroderma capillary pattern. Based on overall estimation of the
microvascular abnormalities. Presence of enlarged capillaries of the
scleroderma (SD)-‐ type: increased diameter (> 25 µm) of all three portions of
the capillary loop (arterial, apical, and venular), often associated with
avascular areas: none, slight (0.4-‐2 mm2), moderate (2-‐4 mm2) extensive (>4
mm2), and a combination of other morphological features such as capillary
haemorrhages, disorganisation of the capillary bed, edema and discoloration
of the cuticle.
2. ‘active’ or ‘slow’ capillary pattern, based on the size of the nailfold terminal
row capillary loops: normal, definitely enlarged (total width 91-‐150 µm), or
extremely enlarged (>150 µm), and on the extent of the avascular areas.
‘Active’ is defined as moderate to extensive avascular areas without capillary
telangiectases (clusters of dilated capillaries) or extremely enlarged loops.
‘Slow’ is defined by extremely enlarged capillaries with no or minimal
avascularity with or without capillary telangiectases.
The ‘active’ and ‘slow’ patterns were defined on the basis of an observed correlation
between the activity of the microvascular lesions and the clinical progression of the disease
in small series of cases.5
In 2000, Cutolo et al, classified microvascular changes in SSc patients into 3 distinct
patterns6:
1. early pattern: few enlarged/giant capillaries, few capillary hemorrhages, no
evident loss of capillaries.
2. active pattern: frequent giant capillaries, frequent capillary hemorrhages, mild
disorganisation of the capillary network.
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3. late pattern: irregular enlargement of the capillaries, few or absent giant
capillaries, hemorrhages, and extensive avascular areas.
These patterns were found to correlate significantly with disease duration, and it was
hypothesised that these patterns characterise the evolution of SSc associated
microangiopathy, and even predict future organ complications.6,7 It should be noted
however, that the scleroderma capillary pattern is not enitrely specific for SSc: the same
pattern can be observed in other CTD.
Video capillary microscopy is a further development which allows quantification of the
nailfold abnormalities: using high magnification (figure 4) combined with a video camera and
digitising system, dimensions of individual capillaries can be measured.
Recently, newly developed software to analyze digitised images makes it possible to create a
panoramic mosaic of the nailfold (figure 5).8
Figure 4. Nailfold capillaroscopy
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Applying this technique to a control group and a group of patients with Primary RP and SSc,
a clear distinction between the SSc group versus the control and PRP group, became clear.
The most powerful discriminator between groups was the number of loops per mm in the
terminal row of the nailfold, and the mean total width of the capillaries.8 The technique of
digitised panoramic mosaic images was used in this thesis.
THE ROLE OF NAILFOLD CAPILLAROSCOPY IN CLINICAL PRACTICE
Nowadays, nailfold capillaroscopy is used for the differentiation between primary and
secondary (CTD associated) RP and the early diagnosis of SSc. In patients with RP, a normal
nailfold capillary pattern combined with negative or low antinuclear antibodies (ANA <160,
or <++) has a negative predictive value of CTD development of 98% (prior likelihood of CTD :
13%).9 For patients with RP, the positive predictive value of scleroderma specific antibodies
or abnormal capillaroscopy for a scleroderma spectrum disorder within 5 years (SSc, MCTD,
poly/dermatomyositis) is 47% in a group with an prior likelihood of 13%.10 If both specific
antibodies and nailfold abnormalities are present, the positive predictive value increases to
Figure 5. With the use of software to create digitised panoramic mosaic images, it is possible to combine the high magnification of nailfold videocapillaroscopy with the ability to view the whole nailfold (as a mosaic) and make quantitative measurements (courtesy of A.L. Herrick)
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3. late pattern: irregular enlargement of the capillaries, few or absent giant
capillaries, hemorrhages, and extensive avascular areas.
These patterns were found to correlate significantly with disease duration, and it was
hypothesised that these patterns characterise the evolution of SSc associated
microangiopathy, and even predict future organ complications.6,7 It should be noted
however, that the scleroderma capillary pattern is not enitrely specific for SSc: the same
pattern can be observed in other CTD.
Video capillary microscopy is a further development which allows quantification of the
nailfold abnormalities: using high magnification (figure 4) combined with a video camera and
digitising system, dimensions of individual capillaries can be measured.
Recently, newly developed software to analyze digitised images makes it possible to create a
panoramic mosaic of the nailfold (figure 5).8
Figure 4. Nailfold capillaroscopy
CHAP
TER
2 M
etho
ds &
Tec
hniq
ues
Applying this technique to a control group and a group of patients with Primary RP and SSc,
a clear distinction between the SSc group versus the control and PRP group, became clear.
The most powerful discriminator between groups was the number of loops per mm in the
terminal row of the nailfold, and the mean total width of the capillaries.8 The technique of
digitised panoramic mosaic images was used in this thesis.
THE ROLE OF NAILFOLD CAPILLAROSCOPY IN CLINICAL PRACTICE
Nowadays, nailfold capillaroscopy is used for the differentiation between primary and
secondary (CTD associated) RP and the early diagnosis of SSc. In patients with RP, a normal
nailfold capillary pattern combined with negative or low antinuclear antibodies (ANA <160,
or <++) has a negative predictive value of CTD development of 98% (prior likelihood of CTD :
13%).9 For patients with RP, the positive predictive value of scleroderma specific antibodies
or abnormal capillaroscopy for a scleroderma spectrum disorder within 5 years (SSc, MCTD,
poly/dermatomyositis) is 47% in a group with an prior likelihood of 13%.10 If both specific
antibodies and nailfold abnormalities are present, the positive predictive value increases to
Figure 5. With the use of software to create digitised panoramic mosaic images, it is possible to combine the high magnification of nailfold videocapillaroscopy with the ability to view the whole nailfold (as a mosaic) and make quantitative measurements (courtesy of A.L. Herrick)
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78%.10 SSc is a CTD characterised by auto-‐immunity, fibrosis and vasculopathy with
substantial morbidity and mortality. The diagnosis of SSc is based on the criteria of the
American College of Rheumatology (ACR). However, by the time SSc is diagnosed, many
(organ) complications may already be present.11,12 New criteria have been proposed for the
early identification of SSc, based on a combination of RP, specific scleroderma antibodies,
and nailfold capillaroscopic abnormalities.13 SSc is diagnosed according American College of
Rheumatology (ACR) preliminary classification for defenite SSc.14 However, the ACR
classification criteria for SSc were not developed for diagnostic purposes, but rather with the
intent to “establish a standard for definite disease in order to permit comparison of groups
of patients from different centres”.14 One study evaluated a group of 259 ‘definite’ SSc
patients, who were diagnosed by expert clinicians. The investigators wanted to evaluate the
sensitivity of the ACR criteria and to determine whether addition of nailfold capillaroscopy
could increase their sensitivity in this group. The study showed an increase in the sensitivity
of the ACR criteria for SSc from 34% to 89% when nailfold capillaroscopy was added.12 As
expected, most patients with limited SSc (scleroderma skin changes below the elbows) were
excluded by the ACR criteria and identified by detection of characteristic nailfold capillary
changes by nailfold capillaroscopy. The same procedure was followed in another cohort of
101 patients, showing an increase from 67% to 99% in the sensitivity of SSc diagnosis when
nailfold capillaroscopy was added to the ACR criteria.11 Whether the early diagnosis of
patients with SSc improves prognosis in terms of morbidity (development of organ
complications) and mortality remains to be seen.
IONTOPHORESIS AND LASER DOPPLER FLOWMETRY
Iontophoresis is a non-‐invasive method of drug application that allows the local transfer of
charged substances across the skin by use of a small electric current. The principle is based
on the fact that, when an electrical voltage difference is applied to a solution, solute ions will
migrate towards an electrode of opposite charge. Thus, positively charged drug ions can be
introduced through the skin under a positively charged electrode (anodal iontophoresis) and
vice versa (cathodal iontophoresis)(figure 6).
To investigate endothelial function, skin micovascular responses to iontophoresis of
acethylcholine, an endothelium-‐dependent vasodilator and sodium nitroprusside, an
endothelium independent vasodilatator can be studied using a laser Doppler flowmetry.
Laser Doppler flowmetry is a noninvasive method to measure skin perfusion. A laser beam
penetrates the skin and a fraction of the light is backscattered by moving blood particles,
undergoing a frequency shift according to the Doppler principle. From the frequency shift,
tissue perfusion can be derived in arbitrary units. After refraining from eating, smoking and
beverages for at least 4 h and acclimatisation for 20 minutes at 23 oC, iontophoresis
combined with laser Doppler flowmetry was performed. Acetylcholine (1%) was delivered
using anodal current, and sodium nitroprusside (0.01%) was delivered with a cathodal
current. Laser Doppler flux was measured on the middle phalanx of the left and right third
finger with the Periflux 4000 system (Perimed) and expressed as arbitrary perfusion units.
Figure 6. Iontopheresis of the skin
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78%.10 SSc is a CTD characterised by auto-‐immunity, fibrosis and vasculopathy with
substantial morbidity and mortality. The diagnosis of SSc is based on the criteria of the
American College of Rheumatology (ACR). However, by the time SSc is diagnosed, many
(organ) complications may already be present.11,12 New criteria have been proposed for the
early identification of SSc, based on a combination of RP, specific scleroderma antibodies,
and nailfold capillaroscopic abnormalities.13 SSc is diagnosed according American College of
Rheumatology (ACR) preliminary classification for defenite SSc.14 However, the ACR
classification criteria for SSc were not developed for diagnostic purposes, but rather with the
intent to “establish a standard for definite disease in order to permit comparison of groups
of patients from different centres”.14 One study evaluated a group of 259 ‘definite’ SSc
patients, who were diagnosed by expert clinicians. The investigators wanted to evaluate the
sensitivity of the ACR criteria and to determine whether addition of nailfold capillaroscopy
could increase their sensitivity in this group. The study showed an increase in the sensitivity
of the ACR criteria for SSc from 34% to 89% when nailfold capillaroscopy was added.12 As
expected, most patients with limited SSc (scleroderma skin changes below the elbows) were
excluded by the ACR criteria and identified by detection of characteristic nailfold capillary
changes by nailfold capillaroscopy. The same procedure was followed in another cohort of
101 patients, showing an increase from 67% to 99% in the sensitivity of SSc diagnosis when
nailfold capillaroscopy was added to the ACR criteria.11 Whether the early diagnosis of
patients with SSc improves prognosis in terms of morbidity (development of organ
complications) and mortality remains to be seen.
IONTOPHORESIS AND LASER DOPPLER FLOWMETRY
Iontophoresis is a non-‐invasive method of drug application that allows the local transfer of
charged substances across the skin by use of a small electric current. The principle is based
on the fact that, when an electrical voltage difference is applied to a solution, solute ions will
migrate towards an electrode of opposite charge. Thus, positively charged drug ions can be
introduced through the skin under a positively charged electrode (anodal iontophoresis) and
vice versa (cathodal iontophoresis)(figure 6).
To investigate endothelial function, skin micovascular responses to iontophoresis of
acethylcholine, an endothelium-‐dependent vasodilator and sodium nitroprusside, an
endothelium independent vasodilatator can be studied using a laser Doppler flowmetry.
Laser Doppler flowmetry is a noninvasive method to measure skin perfusion. A laser beam
penetrates the skin and a fraction of the light is backscattered by moving blood particles,
undergoing a frequency shift according to the Doppler principle. From the frequency shift,
tissue perfusion can be derived in arbitrary units. After refraining from eating, smoking and
beverages for at least 4 h and acclimatisation for 20 minutes at 23 oC, iontophoresis
combined with laser Doppler flowmetry was performed. Acetylcholine (1%) was delivered
using anodal current, and sodium nitroprusside (0.01%) was delivered with a cathodal
current. Laser Doppler flux was measured on the middle phalanx of the left and right third
finger with the Periflux 4000 system (Perimed) and expressed as arbitrary perfusion units.
Figure 6. Iontopheresis of the skin
26
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Day-‐to-‐day reproducibility was assessed previously in our institute and was 15.9%±8.4% for
acetylcholine and 13.9%±9.0% for nitroprusside, as determined in 5 subjects on 2
occasions.15
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REFERENCE LIST 1. Coffman JD. Effects of endothelium-‐derived nitric oxide on skin and digital blood flow
in humans. Am. J. Physiol 1994; 267: H2087-‐H2090.
2. Morris SJ, Shore AC, Tooke JE. Responses of the skin microcirculation to acetylcholine and sodium nitroprusside in patients with NIDDM. Diabetologia 1995; 38: 1337-‐1344.
3. Jones BF, Oral M, Morris CW, Ring EF. A proposed taxonomy for nailfold capillaries based on their morphology. IEEE Trans. Med. Imaging 2001; 20: 333-‐341.
4. Maricq HR, LeRoy EC. Patterns of finger capillary abnormalities in connective tissue disease by "wide-‐field" microscopy. Arthritis Rheum. 1973; 16: 619-‐628.
5. Maricq HR, Valter I. A working classification of scleroderma spectrum disorders: a proposal and the results of testing on a sample of patients. Clin. Exp. Rheumatol. 2004; 22: S5-‐13.
6. Cutolo M, Sulli A, Pizzorni C, Accardo S. Nailfold videocapillaroscopy assessment of microvascular damage in systemic sclerosis. J. Rheumatol. 2000; 27: 155-‐160.
7. Cutolo M, Pizzorni C, Tuccio M, Burroni A, Craviotto C, Basso M et al. Nailfold videocapillaroscopic patterns and serum autoantibodies in systemic sclerosis. Rheumatology. (Oxford) 2004; 43: 719-‐726.
8. Anderson ME, Allen PD, Moore T, Hillier V, Taylor CJ, Herrick AL. Computerized nailfold video capillaroscopy-‐-‐a new tool for assessment of Raynaud's phenomenon. J. Rheumatol. 2005; 32: 841-‐848.
9. Spencer-‐Green G. Outcomes in primary Raynaud phenomenon: a meta-‐analysis of the frequency, rates, and predictors of transition to secondary diseases. Arch. Intern. Med. 1998; 158: 595-‐600.
10. Koenig M, Joyal F, Fritzler MJ, Roussin A, Abrahamowicz M, Boire G et al. Autoantibodies and microvascular damage are independent predictive factors for the progression of Raynaud's phenomenon to systemic sclerosis: A twenty-‐year prospective study of 586 patients, with validation of proposed criteria for early systemic sclerosis. Arthritis Rheum. 2008; 58: 3902-‐3912.
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Day-‐to-‐day reproducibility was assessed previously in our institute and was 15.9%±8.4% for
acetylcholine and 13.9%±9.0% for nitroprusside, as determined in 5 subjects on 2
occasions.15
CHAP
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2 M
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ds &
Tec
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REFERENCE LIST 1. Coffman JD. Effects of endothelium-‐derived nitric oxide on skin and digital blood flow
in humans. Am. J. Physiol 1994; 267: H2087-‐H2090.
2. Morris SJ, Shore AC, Tooke JE. Responses of the skin microcirculation to acetylcholine and sodium nitroprusside in patients with NIDDM. Diabetologia 1995; 38: 1337-‐1344.
3. Jones BF, Oral M, Morris CW, Ring EF. A proposed taxonomy for nailfold capillaries based on their morphology. IEEE Trans. Med. Imaging 2001; 20: 333-‐341.
4. Maricq HR, LeRoy EC. Patterns of finger capillary abnormalities in connective tissue disease by "wide-‐field" microscopy. Arthritis Rheum. 1973; 16: 619-‐628.
5. Maricq HR, Valter I. A working classification of scleroderma spectrum disorders: a proposal and the results of testing on a sample of patients. Clin. Exp. Rheumatol. 2004; 22: S5-‐13.
6. Cutolo M, Sulli A, Pizzorni C, Accardo S. Nailfold videocapillaroscopy assessment of microvascular damage in systemic sclerosis. J. Rheumatol. 2000; 27: 155-‐160.
7. Cutolo M, Pizzorni C, Tuccio M, Burroni A, Craviotto C, Basso M et al. Nailfold videocapillaroscopic patterns and serum autoantibodies in systemic sclerosis. Rheumatology. (Oxford) 2004; 43: 719-‐726.
8. Anderson ME, Allen PD, Moore T, Hillier V, Taylor CJ, Herrick AL. Computerized nailfold video capillaroscopy-‐-‐a new tool for assessment of Raynaud's phenomenon. J. Rheumatol. 2005; 32: 841-‐848.
9. Spencer-‐Green G. Outcomes in primary Raynaud phenomenon: a meta-‐analysis of the frequency, rates, and predictors of transition to secondary diseases. Arch. Intern. Med. 1998; 158: 595-‐600.
10. Koenig M, Joyal F, Fritzler MJ, Roussin A, Abrahamowicz M, Boire G et al. Autoantibodies and microvascular damage are independent predictive factors for the progression of Raynaud's phenomenon to systemic sclerosis: A twenty-‐year prospective study of 586 patients, with validation of proposed criteria for early systemic sclerosis. Arthritis Rheum. 2008; 58: 3902-‐3912.
28
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11. Hudson M, Taillefer S, Steele R, Dunne J, Johnson SR, Jones N et al. Improving the sensitivity of the American College of Rheumatology classification criteria for systemic sclerosis. Clin. Exp. Rheumatol. 2007; 25: 754-‐757.
12. Lonzetti LS, Joyal F, Raynauld JP, Roussin A, Goulet JR, Rich E et al. Updating the American College of Rheumatology preliminary classification criteria for systemic sclerosis: addition of severe nailfold capillaroscopy abnormalities markedly increases the sensitivity for limited scleroderma. Arthritis Rheum. 2001; 44: 735-‐736.
13. LeRoy EC, Medsger TA, Jr. Criteria for the classification of early systemic sclerosis. J. Rheumatol. 2001; 28: 1573-‐1576.
14. Preliminary criteria for the classification of systemic sclerosis (scleroderma). Subcommittee for scleroderma criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Arthritis Rheum. 1980; 23: 581-‐590.
15. Serne EH, Gans RO, ter Maaten JC, Tangelder GJ, Donker AJ, Stehouwer CD. Impaired skin capillary recruitment in essential hypertension is caused by both functional and structural capillary rarefaction. Hypertension 2001; 38: 238-‐242.
