**Videocapillaroscopy in Connective Tissue Diseases**

**Videocapillaroscopy in Connective Tissue Diseases**

DOI: 10.5772/intechopen.69520

### Simone Parisi and Maria Chiara Ditto Simone Parisi and Maria Chiara Ditto Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69520

### **Abstract**

Videocapillaroscopy is a noninvasive, quick, and easy examination method to indicate if there is clinical suspicion of microangiopathy. It provides the rheumatologist indispens‐ able information on the microcirculation state. Recently with the development of the new classification criteria of systemic sclerosis (ACR 2013), capillaroscopy has become even more important. It is currently the only instrumental test whose result is pathognomonic for diagnosis of systemic sclerosis. During videocapillaroscopy, the following param‐ eters are evaluated: density, structure, hemosiderin deposition, bloodstream, presence of megacapillaries, presence of subpapillary venous plexus, and edema. It can distin‐ guish several patterns, especially scleroderma pattern, as follows: (1) "Early" pattern: few enlarged/giant capillaries, few capillary hemorrhages, relatively well‐preserved capillary distribution, no evident loss of capillaries; (2) "Active" pattern: frequent giant capillaries, frequent capillary hemorrhages, moderate loss of capillaries, mild disorganization of the capillary architecture, absent or mild ramified capillaries; (3) "Late" pattern: irregular enlargement of the capillaries, few or absent giant capillaries and hemorrhages, severe loss of capillaries with extensive avascular areas, disorganization of the normal capil‐ lary array, ramified/bushy capillaries. Although capillaroscopic examination is easy to perform, it is essential that the operator has been properly trained on the instrument's function and on correct method of image acquisition to avoid misinterpretation.

**Keywords:** nailfold capillaroscopy, Raynaud's phenomenon, scleroderma pattern, prognostic score

### **1. Introduction**

Capillaroscopy is a noninvasive, fast, and easy imaging technique to evaluate the assessment of the microcirculation. It is indicated in patients with suspect of microangiopatia [1].

It gives precise information about capillaries' conditions and related diseases to rheumatologist.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Recently, after the development of new systemic sclerosis classifications criteria [2], the role of capillaroscopy became more important. In fact a score of two out of nine is assigned in the case of significant capillaroscopic abnormalities to diagnose this disease (**Table 1**).

In the past, the study of capillaries was performed by instruments that could zoom, take pic‐ tures, or film the blood microcirculation, such as the ophthalmoscope and the dermatoscope, the stereo microscope, and tools for macrophotography.

The modern capillaroscopy, equipped with optical probes, is today commonly used in rheu‐ matological and dermatological practice, and it is able to zoom (magnificat) capillaries in order of 200×, obtaining much qualitative measurements that could be reproducible.

The instrument consists of an optical probe fitted with an adjustable magnification and illu‐ mination ring for focusing and a personal computer (with high resolution color screen) with software for data processing. Several new models interface with iOS or Android system, and they are more portable (**Figure 1**).

Although capillaroscopic examination is easy to perform, it is essential that the operator has been properly trained about the instrument's functioning and correct method of image acqui‐ sition to avoid misinterpretation [3].


**Table 1.** Systemic sclerosis classification criteria 2013.

**Figure 1.** Example of Wi‐fi capillaroscope.

Recently, after the development of new systemic sclerosis classifications criteria [2], the role of capillaroscopy became more important. In fact a score of two out of nine is assigned in the

In the past, the study of capillaries was performed by instruments that could zoom, take pic‐ tures, or film the blood microcirculation, such as the ophthalmoscope and the dermatoscope,

The modern capillaroscopy, equipped with optical probes, is today commonly used in rheu‐ matological and dermatological practice, and it is able to zoom (magnificat) capillaries in

The instrument consists of an optical probe fitted with an adjustable magnification and illu‐ mination ring for focusing and a personal computer (with high resolution color screen) with software for data processing. Several new models interface with iOS or Android system, and

Although capillaroscopic examination is easy to perform, it is essential that the operator has been properly trained about the instrument's functioning and correct method of image acqui‐

case of significant capillaroscopic abnormalities to diagnose this disease (**Table 1**).

order of 200×, obtaining much qualitative measurements that could be reproducible.

the stereo microscope, and tools for macrophotography.

they are more portable (**Figure 1**).

114 Systemic Sclerosis

sition to avoid misinterpretation [3].

**Table 1.** Systemic sclerosis classification criteria 2013.

## **2. The capillaroscopic technique**

Capillaroscopy should be always performed in conditions of constant temperature. By con‐ vention, it is considered that the patient needs an acclimatization of at least 15 min at the temperature of 21–24°C before performing the examination.

When booking the videocapillaroscopy, it is necessary to give to the patient a few sugges‐ tions, especially to avoid the manicure in 7–10 days prior to the examination and application of nail polish.

At the time of the examination, the patient should be sat with his hands gently resting on the table on the palm with the fingers slightly apart.

Before starting the exam, it is necessary to apply a drop of oil (usually cedar oil) on the nail fold of each district that should be examined. At this level, the capillaries shall run parallel to the skin plane and, therefore, in normal subjects, are visible an afferent branch, a loop and an efferent branch (capillary hairpin).

Usually, they are investigated four districts in each hand leaving the first finger that usually has a bad view of the vascular widespread and nonspecific alterations. Furthermore, several studies have shown that, even in the presence of Raynaud phenomenon exclusively localized at the foot, capillaroscopy of the hands provides the same information that would be obtained by investigating the lower limbs and, therefore, for convenience, the examination, even in these cases, is usually performed exclusively in the hand level.

Modern software let you select on the monitor the investigation from time to time in the dis‐ trict, so you can compare any changes in respect of each finger. Also, they include measuring systems for dimensional analysis [4].

Although there are some parameters to indicate a normal/healthy capillaroscopic, it is impor‐ tant to consider that there is great variability in the capillary structure both interindividual and intraindividual. This variability depends on many factors such as employment, racial, and environmental.

In particular, in patients underwent to repeated microtrauma for professional reasons, such as typists, jackhammer users, pianists, etc., it is not uncommon to observe widespread phe‐ nomena of microhemorrhages and neoangiogenesis. As well as, patients who smoke have often shortened and tortuous capillaries. Moreover, in patients with dark or very dark skin color (e.g., for racial factors), it is often difficult to correctly visualize the capillaries and almost impossible to see the subpapillary venous plexus. Modern capillaroscopies with editable light intensity solve this problem in part. It is also important that the capillaroscopic be set from time to time for each patient calibrating on the "contrast", "range," and "saturation" functions to obtain an image that is as clear as possible [5].

### **3. The capillaroscopic parameters in normal/healthy conditions**

A good capillaroscopic examination is achieved by positioning the probe plumb to the district under consideration and to obtain the correct visualize of the dermal papilla roughly between the middle and the upper third of the monitor (photo 2—correct assess‐ ment of the image). The area of interest is in fact just the dermal papilla and the capil‐ laries residing there, or should reside within it, although the outside alterations are also important [6–8].

During capillaroscopy, the following parameters are evaluated:


**Density:** In a healthy subject, the number of capillaries per millimeter is equal to 11 ± 2 while the number of capillaries for dermal papilla is equal to 2 ± 1 (picture 3: number of capillaries normal, equal to 12 per mm). The conditions under which this number is reduced may be described simply as "density reduction capillary" up to extreme situations of complete absence capillary describable as "areas avascular" or, in the case of a specific papilla, "vacuous papilla" (**Figure 2**: normal density; **Figure 3**: (a) and (b) area avascular papillary or "papillae vacuous.")

**Structure:** The capillaries of the healthy subject, in most cases, have an aspect so‐called "hair‐ pin shape" or "U‐shaped" and are well aligned, in an orderly manner, the one beside the other with a "comb arrangement" (**Figure 4**: distribution capillary ordered to comb with

**Figure 2.** Example of normal pattern.

Although there are some parameters to indicate a normal/healthy capillaroscopic, it is impor‐ tant to consider that there is great variability in the capillary structure both interindividual and intraindividual. This variability depends on many factors such as employment, racial,

In particular, in patients underwent to repeated microtrauma for professional reasons, such as typists, jackhammer users, pianists, etc., it is not uncommon to observe widespread phe‐ nomena of microhemorrhages and neoangiogenesis. As well as, patients who smoke have often shortened and tortuous capillaries. Moreover, in patients with dark or very dark skin color (e.g., for racial factors), it is often difficult to correctly visualize the capillaries and almost impossible to see the subpapillary venous plexus. Modern capillaroscopies with editable light intensity solve this problem in part. It is also important that the capillaroscopic be set from time to time for each patient calibrating on the "contrast", "range," and "saturation" functions

A good capillaroscopic examination is achieved by positioning the probe plumb to the district under consideration and to obtain the correct visualize of the dermal papilla roughly between the middle and the upper third of the monitor (photo 2—correct assess‐ ment of the image). The area of interest is in fact just the dermal papilla and the capil‐ laries residing there, or should reside within it, although the outside alterations are also

**Density:** In a healthy subject, the number of capillaries per millimeter is equal to 11 ± 2 while the number of capillaries for dermal papilla is equal to 2 ± 1 (picture 3: number of capillaries normal, equal to 12 per mm). The conditions under which this number is reduced may be described simply as "density reduction capillary" up to extreme situations of complete absence capillary describable as "areas avascular" or, in the case of a specific papilla, "vacuous papilla" (**Figure 2**: normal density; **Figure 3**: (a) and (b) area avascular papillary or "papillae vacuous.") **Structure:** The capillaries of the healthy subject, in most cases, have an aspect so‐called "hair‐ pin shape" or "U‐shaped" and are well aligned, in an orderly manner, the one beside the other with a "comb arrangement" (**Figure 4**: distribution capillary ordered to comb with

**3. The capillaroscopic parameters in normal/healthy conditions**

During capillaroscopy, the following parameters are evaluated:

and environmental.

116 Systemic Sclerosis

important [6–8].

• Microhemorrhages

• Edema (soft focus effect)

• Subpapillary venous plexus

• Bloodstream

• Density • Structure

to obtain an image that is as clear as possible [5].

**Figure 3.** (a) and (b) Black arrows indicate papillae vacuous.

aspect hairpin), all similar in form and with some dimensional variability (length approxi‐ mately between 200 and 500 μm).

You can observe the modest structural disorganization even in healthy individuals. Instead, the complete subversion of it is attributable to pathological conditions.

They should not be present ramifications that often indicate a poor vascularization with angiogenesis to provide the blood supply of avascular areas.

Tortuosities are often found, for the most in apical zone, and the capillary has a distorted aspect. It manifested as single or multiple cross/overs and/or patterns described as "trefoil," "antler," "glomerular loop," and "treble clef." These anomalies can be isolated or diffuse (**Figure 5**: tortuosities).

The apical tortuosities, by themselves, are not a pathologic finding. They are frequently found in heavy smokers, in patients underwent to repeated microtrauma, in patients with psoriasis and in various other conditions.

Otherwise, the tortuosity can be contextualized within a framework frankly altered or a real scleroderma pattern.

**Figure 4.** Normal structure: hairpin shape.

**Figure 5.** Tortuosity in apical zone.

The capillary branches (afferent and efferent loop) of the healthy subject normally have a diameter between 8 and 20 μm depending on the capillary portion considered. In fact, usu‐ ally, the loop efferent, due to venous stasis, is slightly larger than that afferent.

They are defined "enlarged" capillaries with a diameter between 30 and 50 μm measured at the level of the two branches and the loop, "mega" capillaries with diameter >50 μm at the level of the two branches and the loop, and "giant" capillaries with diameters >100 μm wide and the two branches of the loop (**Figure 6**: megacapillaries and giant capillaries).

Morphostructural minor anomalies (e.g., tortuosity) are found in approximately 10–20% of healthy subjects.

Among irregularly enlarged capillaries, loop size can vary considerably in different segments, with normal portions alternating with extremely enlarged areas, sometimes giving a "micro‐ aneurysmatic" or "rosary‐like" appearance (**Figure 7**).

**Microhemorrhages:** Although the microhemorrhages are frequent elements in the pathologi‐ cal or scleroderma pattern, it is not uncommon to observe them in healthy individuals as a

**Figure 6.** (a) and (b) Megacapillaries and giant capillaries.

**Figure 7.** Capillaries with "rosary‐like" aspect.

The capillary branches (afferent and efferent loop) of the healthy subject normally have a diameter between 8 and 20 μm depending on the capillary portion considered. In fact, usu‐

They are defined "enlarged" capillaries with a diameter between 30 and 50 μm measured at the level of the two branches and the loop, "mega" capillaries with diameter >50 μm at the level of the two branches and the loop, and "giant" capillaries with diameters >100 μm wide

Morphostructural minor anomalies (e.g., tortuosity) are found in approximately 10–20% of

Among irregularly enlarged capillaries, loop size can vary considerably in different segments, with normal portions alternating with extremely enlarged areas, sometimes giving a "micro‐

**Microhemorrhages:** Although the microhemorrhages are frequent elements in the pathologi‐ cal or scleroderma pattern, it is not uncommon to observe them in healthy individuals as a

ally, the loop efferent, due to venous stasis, is slightly larger than that afferent.

and the two branches of the loop (**Figure 6**: megacapillaries and giant capillaries).

aneurysmatic" or "rosary‐like" appearance (**Figure 7**).

healthy subjects.

**Figure 5.** Tortuosity in apical zone.

**Figure 4.** Normal structure: hairpin shape.

118 Systemic Sclerosis

result of trauma. In these cases, bleeding is well‐defined, unique, and usually transient, not necessarily directly related to the underlying capillaries. It is also not uncommon to observe very distant from the papilla or much below it.

The extravasation blood of pathological capillaries assumes a characteristic aspect in the sup‐ ply chain "a strung pearls" (**Figure 8a** and **b**: microhemorrhages to "strung pearls"), or mold on the capillary (**Figure 8c** and **d**: "Napoleonic hat").

It can get an idea more or less realistic age of bleeding based on the analysis of the same color that tends to move from dark red to light yellow before disappearing altogether.

In the case of disease patterns may be encountered persistent deposits hemosiderin standing.

Nailfold microbleeds may also be related to capillary thrombosis which can occur in some pathological conditions and is often misinterpreted as hemorrhages. A key distinguishing feature of capillary thrombosis is the configuration of the dark area, which mirrors that of the capillary loops.

**Figure 8.** (a) and (b) Microhemorrhages to "strung pearls"; (c) and (d) microhemorrhages to "Napoleonic hat."

**Bloodstream:** The capillaroscopy is a dynamic examination and allows to study blood flow, although in an approximate manner when compared to the Doppler technique.

A capillary with continuous blood flow is normal index. It will appear constantly full, and in the case when pressure is exerted by the probe on the same papilla, these will quickly fill the cessation of the stimulus. In the case of slowed blood flow, this will assume a granular appearance in particular in correspondence of the capillary walls and, in the case a pressure is exerted with the capillaroscopic probe the capillary will fill up slowly to cease the stimulus.

In the case where there is capillary thrombosis, typical of giant capillaries or megacapillari, the flow will appear static, and neither the pressure exerted by the probe nor the subsequent stop of the stimulus will show variations in the capillary which will appear always full.

**Edema (soft focus effect):** The flou effect in photography is a special effect that is achieved by reducing the contrast of the image without really being blurred. In capillaroscopy, it is talking about flou effect when, at the subpapillary, capillaries appear poorly visible in an edematous context of vascular congestion.

Occasionally, it is seen in healthy subjects. Alone it does not constitute a significant fault (**Figure 9**: flou effect).

**Subpapillary venous plexus:** The plexus is visible in the case of good skin transparency and is best viewed in the extreme ages of life (**Figure 10(a)** and **(b)**).

The venous plexus vessels have a perpendicular progress to the capillaries and are larger.

**Figure 9.** Black arrow → indicates flou effect.

**Figure 10.** (a) and (b) Good visibility of the subpapillary venous plexus.

In cases of serious and widespread destruction capillaries, such as in the advanced stages of systemic sclerosis, the subpapillary venous plexus may constitute the only identifiable vascular element.

In healthy individuals, it is not always detectable.

### **4. Capillaroscopy in pathologic subjects**

### **4.1. Introduction**

**Bloodstream:** The capillaroscopy is a dynamic examination and allows to study blood flow,

**Figure 8.** (a) and (b) Microhemorrhages to "strung pearls"; (c) and (d) microhemorrhages to "Napoleonic hat."

A capillary with continuous blood flow is normal index. It will appear constantly full, and in the case when pressure is exerted by the probe on the same papilla, these will quickly fill the cessation of the stimulus. In the case of slowed blood flow, this will assume a granular appearance in particular in correspondence of the capillary walls and, in the case a pressure is exerted with the capillaroscopic probe the capillary will fill up slowly to cease the stimulus. In the case where there is capillary thrombosis, typical of giant capillaries or megacapillari, the flow will appear static, and neither the pressure exerted by the probe nor the subsequent stop of the stimulus will show variations in the capillary which will appear always full.

**Edema (soft focus effect):** The flou effect in photography is a special effect that is achieved by reducing the contrast of the image without really being blurred. In capillaroscopy, it is talking about flou effect when, at the subpapillary, capillaries appear poorly visible in an edematous

Occasionally, it is seen in healthy subjects. Alone it does not constitute a significant fault

**Subpapillary venous plexus:** The plexus is visible in the case of good skin transparency and

The venous plexus vessels have a perpendicular progress to the capillaries and are larger.

is best viewed in the extreme ages of life (**Figure 10(a)** and **(b)**).

although in an approximate manner when compared to the Doppler technique.

context of vascular congestion.

(**Figure 9**: flou effect).

120 Systemic Sclerosis

Secondary Raynaud's phenomenon refers to the clinical manifestation in the presence of an underlying systemic disease.

Among the diseases of rheumatologic interest, the more strongly and inseparably linked to Raynaud's phenomenon is the systemic sclerosis, even if this appears also in the presence of other autoimmune diseases such as systemic lupus erythematosus, dermatomyositis, undif‐ ferentiated connective, and mixed connective, in a small percentage of cases, in the presence of rheumatoid, psoriatic, or juvenile idiopathic arthritis.

Capillaroscopy does not constitute, in itself, a diagnostic test. Especially not usefull to diag‐ nostic Raynaud's phenomenon, whose diagnosis is exclusively linked to the clinic. The exami‐ nation provides a specific view on the state of the microcirculation and in particular on its integrity and is always related to the clinical and laboratory data. Then we can determine whether the Raynaud's phenomenon is referable to a damage of the microcirculation [8, 9].

However, capillaroscopy plays a key role in the diagnosis of connective tissue diseases and especially of the scleroderma spectrum disorders, which include, in addition to systemic scle‐ rosis, the dermatomyositis, the undifferentiated, and mixed connective tissue disease [10, 11].

In fact, historically, the typical capillaroscopic alterations for scleroderma spectrum disorders have always played a fundamental role in the diagnosis of systemic sclerosis, being included in all the diagnostic criteria formulated over the years for this condition, including the latest ACR criteria of 2013.

In contrast to what happens in the case of primitive Raynaud's phenomenon, in the presence of secondary Raynaud's phenomenon, the capillaroscopy is able to reveal specific anomalies.

Over the years, we have been implemented many efforts in an attempt to identify specific capilla‐ roscopic paintings for a specific pathology. These efforts have hesitated in identifying a capillaro‐ scopic pattern defined as "scleroderma pattern." More than 95% of patients with overt systemic sclerosis have morphological markers of microvascular disorganization, including giant capillar‐ ies, microhemorrhages, loss of capillaries, avascular areas, and angiogenesis [12–14].

### **4.2. Systemic sclerosis**

Specific capillaroscopic alterations are found in the majority of cases of systemic sclerosis and often several years before diagnosis. The key element that distinguishes the scleroderma pat‐ tern is megacapillare. Another common finding is typical microhemorrhage called "pearls strung" or "Napoleon hat" usually overlying dilated capillaries or megacapillaries. Third distinctive element is represented by avascular areas. In 2000, the group of Genoa, headed by professor Cutolo, has identified three types of scleroderma pattern, referred to as "early," "active," and "late" [15–17].

We analyze below a capillaroscopic examination as a "scleroderma pattern."

**Density:** As is known in a healthy subject, the number of capillaries per millimeter is equal to 11 ± 2 while the number of capillaries for dermal papilla is equal to 2 ± 1. In the case of sclero‐ derma, pattern is common to find a reduction in the number of capillaries. In particular, the reduction of the density can range from focused frameworks (e.g., to a single papilla "vacu‐ ous papilla") to framework of rarefied widespread, until the total disappearance of capillaries with large subpapillary vascular areas. The most dramatic paintings are typical of late stages or framework identified by Cutolo et al. as "late," even if similar findings can be found in patients with recent symptoms onset, especially in progressive cases.

Among the diseases of rheumatologic interest, the more strongly and inseparably linked to Raynaud's phenomenon is the systemic sclerosis, even if this appears also in the presence of other autoimmune diseases such as systemic lupus erythematosus, dermatomyositis, undif‐ ferentiated connective, and mixed connective, in a small percentage of cases, in the presence

Capillaroscopy does not constitute, in itself, a diagnostic test. Especially not usefull to diag‐ nostic Raynaud's phenomenon, whose diagnosis is exclusively linked to the clinic. The exami‐ nation provides a specific view on the state of the microcirculation and in particular on its integrity and is always related to the clinical and laboratory data. Then we can determine whether the Raynaud's phenomenon is referable to a damage of the microcirculation [8, 9].

However, capillaroscopy plays a key role in the diagnosis of connective tissue diseases and especially of the scleroderma spectrum disorders, which include, in addition to systemic scle‐ rosis, the dermatomyositis, the undifferentiated, and mixed connective tissue disease [10, 11].

In fact, historically, the typical capillaroscopic alterations for scleroderma spectrum disorders have always played a fundamental role in the diagnosis of systemic sclerosis, being included in all the diagnostic criteria formulated over the years for this condition, including the latest

In contrast to what happens in the case of primitive Raynaud's phenomenon, in the presence of secondary Raynaud's phenomenon, the capillaroscopy is able to reveal specific anomalies.

Over the years, we have been implemented many efforts in an attempt to identify specific capilla‐ roscopic paintings for a specific pathology. These efforts have hesitated in identifying a capillaro‐ scopic pattern defined as "scleroderma pattern." More than 95% of patients with overt systemic sclerosis have morphological markers of microvascular disorganization, including giant capillar‐

Specific capillaroscopic alterations are found in the majority of cases of systemic sclerosis and often several years before diagnosis. The key element that distinguishes the scleroderma pat‐ tern is megacapillare. Another common finding is typical microhemorrhage called "pearls strung" or "Napoleon hat" usually overlying dilated capillaries or megacapillaries. Third distinctive element is represented by avascular areas. In 2000, the group of Genoa, headed by professor Cutolo, has identified three types of scleroderma pattern, referred to as "early,"

**Density:** As is known in a healthy subject, the number of capillaries per millimeter is equal to 11 ± 2 while the number of capillaries for dermal papilla is equal to 2 ± 1. In the case of sclero‐ derma, pattern is common to find a reduction in the number of capillaries. In particular, the reduction of the density can range from focused frameworks (e.g., to a single papilla "vacu‐ ous papilla") to framework of rarefied widespread, until the total disappearance of capillaries with large subpapillary vascular areas. The most dramatic paintings are typical of late stages

ies, microhemorrhages, loss of capillaries, avascular areas, and angiogenesis [12–14].

We analyze below a capillaroscopic examination as a "scleroderma pattern."

of rheumatoid, psoriatic, or juvenile idiopathic arthritis.

ACR criteria of 2013.

122 Systemic Sclerosis

**4.2. Systemic sclerosis**

"active," and "late" [15–17].

**Structure:** The most significant finding of scleroderma pattern, as said, is the megacapillare or a capillary which measures a greater diameter than 50 μm in correspondence with the two branches of the ascending and descending loops. In the case of diameters greater than 100 μm, it is called the capillary giant. The presence of only one megacapillare within a nor‐ mal framework is not sufficient to define scleroderma pattern although suggests the need a follow up. The presence of two or more megacapillaries leads to a scleroderma pattern even in the absence of microhemorrhages or avascular areas.

Generally, the presence of megacapillari in the context of apparently normal capillaries, with few or absent microhemorrhages, characterizes the earliest stages of the disease and it is iden‐ tified by Cutolo et al. as "early." Instead, in the presence of numerous or ubiquitous megacap‐ illari in the context of rare normal capillaries or ectatic capillaries, in the presence of numerous microhemorrhages in more districts, we will be faced to a capillary framework called "active."

In the late stages of systemic sclerosis are sometimes pathognomonic capillary ramifications, with elongated and bizarre capillaries, or overgrowth of the subpapillary venous plexus, last attempt to make up for the total or almost total disappearance of the subpapillary capillaries. This framework configures the "late" scleroderma pattern.

Other detectable morphostructural abnormalities in scleroderma pattern are ectasia (capillaries in the range from 30 to 50 μm), the tortuosities, identifying, based on the forms as "staghorn," "a clef,""a glomerulus,""ball," etc. and microaneurysms. These alterations are not specific and can also be found in healthy subjects.

Microhemorrhages**:** They constitute an extremely common finding in the scleroderma pattern and, especially in the one called "active". The typical hemorrhages of the scleroderma pattern may look as a mold, overlooking a megacapillare, or a giant capillary, or an aspect to "strung pearls" that are stacked in succession in the subpapillary and extrapapillary, never in deep seat. The microhemorrhages indicated breaking of capillary wall and are a sign of microvas‐ cular damage. There may be bleeding in the absence of megacapillari. The lonely bleeding are not considered sufficient for the identification of scleroderma pattern although, the presence of diffuse bleeding, even in the absence of further alterations, suggests necessarily the follow up.

The microhemorrhages should not be confused with traumatic hemorrhages that have a very heterogeneous presentation, generally overlying or adjacent to completely normal capillaries. Usually, they have a greater extension and can be found in the subpapillary‐, deep‐or extra‐ papillary segment.

**Flow:** The typical flow scleroderma patterns abnormalities are linked to the structural dam‐ age, in particular to megacapillaries. In these cases, the blood flow can appear slow or granu‐ lar or even static. In the case of slow or static flow, a stimulus as the modest pressure exercised by the capillaroscopy probe is sufficient to make empty capillaries, and the capillaries will become fills when pressure is stopped. However, megacapillaries, giant capillaries, and the microaneurismatic capillaries remain completely filled both in the presence and in the absence of pressure stimulus, indicating a wall thickening causing static flow.

**Edema (soft focus effect):** Subpapillary edema is a constant finding in the case of capillaries vasodilatation in particular in the presence of megacapillaries and giant capillaries, so it is a typical finding in the scleroderma pattern. Edema could be mild, moderate, or severe and in these cases, subpapillary nous plexus is hardly visible.

**Subpapillary venous plexus:** It is clearly visible in healthy subjects and the extreme ages of life, can be seen in the case of early scleroderma pattern, is usually barely visible or not visible in the "active" phase, and is the only detectable findings in the late stage, where papillary capillaries are absent. Bizarre capillary ramifications are frequent findings in the late stage, as a last attempt to overcome the vascular deficit.

### **Scleroderma pattern:**


**Figure 11.** (a–c) Few megacapillaries without microhemorrhages.

**Figure 12.** (a) and (b) Diffuse megacapillaries with microhemorrhages.

**Figure 13.** (a) and (b) "Papilla vacua," diffuse or localized loss of capillaries.

### **5. Capillaroscopy in other rheumatic diseases**

As mentioned, all of rheumatic diseases included in the scleroderma spectrum disorders may present a capillaroscopic framework suggesting for scleroderma pattern. However, in some diseases, there are typical capillary feature presentations. For example, in rheumatoid arthri‐ tis, extremely elongated capillaries are the principal findings (**Figure 14**) [18, 19].

In dermatomyositis, the most frequent findings are dilated and giant capillaries with tree‐like appearance (**Figure 15(a)** and **(b)**).

Even in the case of psoriatic arthritis, capillaries appear rather short and stubby (**Figure 16**).

However, these features are not enough specific to identify a defined framework.

### **5.1. Scoring method**

**Edema (soft focus effect):** Subpapillary edema is a constant finding in the case of capillaries vasodilatation in particular in the presence of megacapillaries and giant capillaries, so it is a typical finding in the scleroderma pattern. Edema could be mild, moderate, or severe and in

**Subpapillary venous plexus:** It is clearly visible in healthy subjects and the extreme ages of life, can be seen in the case of early scleroderma pattern, is usually barely visible or not visible in the "active" phase, and is the only detectable findings in the late stage, where papillary capillaries are absent. Bizarre capillary ramifications are frequent findings in the late stage, as

• Early scleroderma pattern (**Figure 11(a–c)**): framework characterized by the presence of dilated capillaries and some megacapillaries. Microhemorrhages are poorly represented or

• Active scleroderma pattern (**Figure 12(a)** and **(b)**: framework characterized by the wide‐ spread presence of megacapillaries and/or giant capillaries. A large number of microhem‐ orrhages. No reduction in capillary density or occasional finding of "papilla vacua" in the

• Late scleroderma pattern (**Figure 13(a)** and **(b)**: framework characterized by the presence of rare lasts megacapillaries, abundant ramifications. Rare or absent microhemorrhages.

Large avascular areas. Exuberance of the subpapillary venous plexus.

these cases, subpapillary nous plexus is hardly visible.

a last attempt to overcome the vascular deficit.

absent. No reduction in capillary density.

presence of angiogenesis phenomena.

**Figure 12.** (a) and (b) Diffuse megacapillaries with microhemorrhages.

**Figure 11.** (a–c) Few megacapillaries without microhemorrhages.

**Scleroderma pattern:**

124 Systemic Sclerosis

In present‐day clinical practice, capillaroscopic surveys are usually analyzed qualitatively in order to show patterns of disease (as previously stated). However, some authors share the idea that a normalization of the capillaroscopic pattern may be positive.

**Figure 14.** Capillaroscopy in rheumatoid arthritis.

**Figure 15.** (a) and (b) Tree pattern in dermatomyositis.

**Figure 16.** Capillaroscopy pattern in psoriatic arthritis.

Different scoring methods have been proposed to prospectively evaluate both the trend and the gravity of the scleroderma microangiopathy.

### **5.2. Semiquantitative method**

One of these methods is the semiquantitative assessment which contemplates the analysis of the following capillaroscopic parameters:


Each parameter is given a score based on the given impairment:


The average score for each parameter comes from the analysis of four conterminous capil‐ laroscopic areas (each area consisting of a 1 mm<sup>2</sup> surface) in the central part of the II, III, IV, and V finger of each hand.

The final score of each parameter is given by adding up the average scores of each finger; then the result is divided by 8. Lastly, the sum of the three scores constitutes the *microangiopathy evolution score* whose value may vary from 0 to 9.

On the basis of the capillaries loss only, such score is easier to determine and has been pro‐ posed as a predictor of digital ulcers. An average score of the capillaries number decrease is obtained by using capillary density as only parameter if a 1 mm2 surface on eight fingers is analyzed. Scores that appear to be higher than 1.67 are shown to be a predictive factor of digital ulcers (Se 70%, Sp 69.77%, with a positive likelihood ratio of 2.32 and a negative likelihood ratio of 0.43) since such ulcers occur within 6–12 months after the capillaroscopic evaluation [20] (**Figure 17**).

### **5.3. Quantitative method**

Different scoring methods have been proposed to prospectively evaluate both the trend and

One of these methods is the semiquantitative assessment which contemplates the analysis of

**2.** Disorganization of the capillary architecture: irregular loops distribution, orientation, and

**3.** Tree‐like capillary network: capillaries with skein‐like or shrub‐like branched loops.

**1.** Loss of capillaries: reduction of the number of capillaries to less than 9 per mm

the gravity of the scleroderma microangiopathy.

**Figure 16.** Capillaroscopy pattern in psoriatic arthritis.

**Figure 15.** (a) and (b) Tree pattern in dermatomyositis.

126 Systemic Sclerosis

the following capillaroscopic parameters:

**5.2. Semiquantitative method**

morphology

This method needs a strict standardization to be reproducible and comparable through time. As of today, the only quantitative score to be validated for both replicabilities and the pre‐ dictive value is the *Capillaroscopy Skin Ulcer Risk Index* (CSURI), which is predictive for the incoming appearance of digital ulcers (which are already present within 3 months of the capil‐ laroscopy) and for the nonfulfillment of their recovery (Se 92.3%, Sp 81.4 %, NPV 97.2%, TPV 84.3% with a PPV higher than 81% in the subgroup of patients with a history of ulcers appeared within a year).

The calculation is carried out by analyzing the whole nailfold area (from the II to the V finger of each hand, saving at least an image for each finger). Of all selected images, the one with the highest number of capillaries and the one with the lowest number of megacapillaries are to be considered; then the following formula has to be used: M×DN2 (with M: numbers of capillaries, D: maximum diameter, N: numbers of megacapillaries). Having at least a visible capillary is a prerequisite (otherwise the formula would be equal to 0; in fact, it is not possible to use it in 5% of patients. In this case, the formula is considered positive in principle). The analyzed area must be 1.57 mm wide. CSURI calculation is based on one image chosen between the saved one (the image with the lowest number of capillaries should be preferred) (**Figures 18** and **19**).

Capillaroscopic parameters are defined in a strict way so that reproducibility and replicability are optimized:

**Figure 17.** Number of fields studied: gold standard (F32) and successive simplifications (F16‐F8‐F4). (A) F32: eight fingers (arrows), four fields of 1 mm per finger, giving a total of 32 fields. (B) F16: eight fingers (arrows), two fields of 1 mm per finger, giving a total of 16 fields. (C) F8: eight fingers (arrows), one filed of 1 mm per finger, giving a total of eight fields. (D) F4: one finger (arrows), four fields of 1 mm in that finger, giving a total of four fields [20].


**Figure 18.** Examples of capillaroscopic finding measurements. (A) 4 capillaries, 3 giant capillaries (1 ramified giant capillaries occupying both dermal papillae). (B) 8 capillaries, 5 giant capillaries (every capillary was counted in the distal row even if it was not on the same level). (C) 13 capillaries, 1 giant capillary (1 ramified giant capillary computed as 2 in the total number count). (D) 12 capillaries, 1 giant capillaries (every capillary was counted in the distal row even if it was not on the same level) [14].

• Number of capillaries: all the capillaries in the first row (the ones closest to the papilla)

fields. (D) F4: one finger (arrows), four fields of 1 mm in that finger, giving a total of four fields [20].

**Figure 17.** Number of fields studied: gold standard (F32) and successive simplifications (F16‐F8‐F4). (A) F32: eight fingers (arrows), four fields of 1 mm per finger, giving a total of 32 fields. (B) F16: eight fingers (arrows), two fields of 1 mm per finger, giving a total of 16 fields. (C) F8: eight fingers (arrows), one filed of 1 mm per finger, giving a total of eight

• Megacapillary: maximum measurable diameter in the first row (microaneurysms should

• Tree‐like morphology: a tree‐like capillary is equivalent to the number of taken papillae or

must be counted even if they are all different depths.

not be included).

128 Systemic Sclerosis

to the number of observable loops.

**Figure 19.** Algorithm for capillaroscopic skin ulcer risk index (CSURI) evaluation. D, Maximum diameter of mega capillary; M, number of megacapillaries (diameter ≥ 50 μm); N, number of capillaries [14].

The CSURI value per patient is the maximum computable if the whole nailfold area is ana‐ lyzed so that the highest score of microangiopathy is defined. PROs: number of false nega‐ tives <3%. CONs: a higher risk of increasing the false positives.[21].

### **5.4. Prognostic index**

The *Prognostic Index for Nailfold Capillaroscopic Examination* (PRINCE: **Table 2**) allows to strat‐ ify the risk of development of a scleroderma spectrum disorder over a period of 5 years in


**Table 2.** Prognostic parameters of PRINCE index.

patients with Raynaud syndrome by analyzing the three main capillaroscopic anomalies (with the survey of a 1 mm area per finger):

• Presence of megacapillaries

The CSURI value per patient is the maximum computable if the whole nailfold area is ana‐ lyzed so that the highest score of microangiopathy is defined. PROs: number of false nega‐

The *Prognostic Index for Nailfold Capillaroscopic Examination* (PRINCE: **Table 2**) allows to strat‐ ify the risk of development of a scleroderma spectrum disorder over a period of 5 years in

tives <3%. CONs: a higher risk of increasing the false positives.[21].

**5.4. Prognostic index**

130 Systemic Sclerosis

**Table 2.** Prognostic parameters of PRINCE index.


The inclusion of antinuclear antibodies allowed to develop an additional predictive model with the following risk categories: high (50+ %), medium (10–50%) and low risk (<10%) (**Figure 20**) [22].

**Figure 20.** Prognostic Index for Nailfold Capillaroscopic Examination (PRINCE). A, D, G, and J: possible combination of giant loops and microhemorrhages (0 = absent, 1 = present). The score (on the *y*‐axis) is obtained as a function of the number of capillaries (represented on the *x*‐axis). B, E, H, and K: data used with the corresponding scores in A, D, G, and J to obtain the incidence and thus deduce the risk of developing Raynaud's phenomenon secondary to a scleroderma spectrum disorder. C, F, I, and L: examples of capillaroscopic patterns [22].

## **6. The role of capillaroscopy in the early diagnosis of systemic sclerosis**

The role of capillaroscopy has been considered over the years more and more attention, espe‐ cially in the early diagnosis of systemic sclerosis. Then, to detect valid predictors of early sys‐ temic sclerosis, the European Scleroderma Trials and Research group (EUSTAR) identified three red flags, thanks to the VEDOSS program (very early diagnosis of systemic sclerosis): Raynaud's phenomenon (RP), antinuclear antibodies (ANA) positivity, and puffy fingers are the main ele‐ ments to suspect systemic sclerosis. In the case of these three flags performing, further tests to confirm the diagnosis, in particular nailfold video‐capillaroscopy and evaluation of specific disease antibodies (anticentromere and antitopoisomerase I), are mandatory. The challenge of VEDOSS program is to identify patients who will develop an established systemic sclerosis.

Very recently, the first results of the VEDOSS project were processed and new EULAR/ACR (American College of Rheumatology) classification criteria have been validated and pub‐ lished (2013), in which the capillaroscopic characteristic changes have been included (requir‐ ing at least two, or better, all four items to be present) (**Figure 21**) [23, 24].

**Figure 21.** A behavioral flow chart for patients in whom the very early diagnosis of systemic sclerosis (SSc) should be considered is proposed. Red flags should trigger the differential diagnosis of SSc and guide the general practitioner to send the patient to the referral center where capillaroscopy and specific autoantibodies are ordered and the diagnosis of very early SSc is made. HRCT, high resolution CT; PFT, pulmonary function tests [23].

### **7. Conclusions**

Capillaroscopy is an easily tolerable, noninvasive, important angiologic examination method. In the case of Raynaud's phenomenon, associated or not with signs or symptoms suggestive for connective tissue disorders or in the presence of autoantibodies (in particu‐ lar, antinuclear antibodies (ANA) and extractable nuclear antigens (ENA)), capillaroscopy is a crucial examination that adds irreplaceable information to formulate a diagnosis. In the meantime, capillaroscopy has achieved a firm status in the early diagnosis of systemic sclerosis (SSc).

### **7.1. Capillaroscopic template**

**6. The role of capillaroscopy in the early diagnosis of systemic sclerosis**

The role of capillaroscopy has been considered over the years more and more attention, espe‐ cially in the early diagnosis of systemic sclerosis. Then, to detect valid predictors of early sys‐ temic sclerosis, the European Scleroderma Trials and Research group (EUSTAR) identified three red flags, thanks to the VEDOSS program (very early diagnosis of systemic sclerosis): Raynaud's phenomenon (RP), antinuclear antibodies (ANA) positivity, and puffy fingers are the main ele‐ ments to suspect systemic sclerosis. In the case of these three flags performing, further tests to confirm the diagnosis, in particular nailfold video‐capillaroscopy and evaluation of specific disease antibodies (anticentromere and antitopoisomerase I), are mandatory. The challenge of VEDOSS program is to identify patients who will develop an established systemic sclerosis.

Very recently, the first results of the VEDOSS project were processed and new EULAR/ACR (American College of Rheumatology) classification criteria have been validated and pub‐ lished (2013), in which the capillaroscopic characteristic changes have been included (requir‐

Capillaroscopy is an easily tolerable, noninvasive, important angiologic examination method. In the case of Raynaud's phenomenon, associated or not with signs or symptoms

**Figure 21.** A behavioral flow chart for patients in whom the very early diagnosis of systemic sclerosis (SSc) should be considered is proposed. Red flags should trigger the differential diagnosis of SSc and guide the general practitioner to send the patient to the referral center where capillaroscopy and specific autoantibodies are ordered and the diagnosis of

very early SSc is made. HRCT, high resolution CT; PFT, pulmonary function tests [23].

ing at least two, or better, all four items to be present) (**Figure 21**) [23, 24].

**7. Conclusions**

132 Systemic Sclerosis

The report should include capillaroscopic terms understandable to nonexperts and should be as standardized as possible, using both qualitative and quantitative parameters that reli‐ able and establishing normal limits (**Figure 22**). In the presence of capillaroscopic, more alterations (megacapillaries, microhemorrhages, neoangiogenesis, density decrease,) a greater degree of detail is required, indicating although these alterations are present on only one or a few fingers.


**Figure 22.** Example of Capillaroscopic Template.

### **Author details**

Simone Parisi\* and Maria Chiara Ditto

\*Address all correspondence to: simone.parisi@hotmail.it

Rheumatology Unit, Azienda Ospedaliera Universitaria Città della Salute e della Scienza di Torino, Turin, Italy

### **References**


[15] Bellando‐Randone S, Guiducci S, Matucci‐Cerinic M. Very early diagnosis of systemic sclerosis. Polskie Archiwum Medycyny Wewnętrznej. 2012;**122**(Suppl 1):18‐23

**References**

134 Systemic Sclerosis

9788862613194

S116‐S117

[1] De Angelis R, Ferri C, Sebastiani M, Manfredi A, Grassi W. La capillaroscopia in reu‐ matologia. Lesioni elementari e metodi di scoring 2012 Mattioli 1885 Editore. ISBN:

[2] Van den Hoogen F, et al. Classification criteria for systemic sclerosis: An American College of Rheumatology/European League against Rheumatism collaborative initia‐

[3] Anderson ME, et al. Computerized nailfold video capillaroscopy—A new tool for assess‐ ment of Raynaud's phenomenon. Journal of Rheumatology. 2005 May;**32**(5):841‐848

[4] Cutolo M. Atlas of Capillaroscopy in Rheumatic Disease. Elsevier 2010 ISBN: 9788821433917

[5] Fahrig C, et al. Capillary microscopy of the nailfold in healthy subjects. International

[6] Batticciotto A, et al. Feet nailfold capillaroscopy is not useful to detect the typical sclero‐ derma pattern. Clinical and Experimental Rheumatology. 2012 Mar‐Apr;**30**(2 Suppl 71):

[7] Cutolo M, Sulli A, Pizzorni C, Accardo S. Nailfold videocapillaroscopy assessment of microvascular damage in systemic sclerosis. J Rheumatol. 2000 Jan;**27**(1):155‐60

[8] Hoerth C, et al. Qualitative and quantitative assessment of nailfold capillaries by capil‐

[9] Kabasakal Y, et al. Quantitative nailfold capillaroscopy findings in a population with connective tissue disease and in normal healthy controls. Annals of the Rheumatic

[10] De Angelis R, et al. A growing need for capillaroscopy in rheumatology. Arthritis &

[11] LeRoy EC, Medsger TA Jr. Criteria for the classification of early systemic sclerosis.

[12] Anderson ME, Allen PD, Moore T, Jayson MI, Herrick AL. Computerized nailfold vid‐ eocapillaroscopy—A new tool for assessment of Raynaud's phenomenon. Journal of

[13] Sulli A, Secchi ME, Pizzorni C, Cutolo M. Scoring the nailfold microvascular changes dur‐ ing the capillaroscopic analysis in systemic sclerosis patients. Annals of the Rheumatic

[14] Sebastiani M, Manfredi A, Vukatana G, Moscatelli S, Riato L, Bocci M, Iudici M, Principato A, Mazzuca S, Del Medico P, De Angelis R, D'Amico R, Vicini R, Colaci M, Ferri C. Predictive role of capillaroscopic skin ulcer risk index in systemic sclerosis: A multicentre validation study. Annals of the Rheumatic Diseases. 2012 Jan;**71**(1):67‐70

tive. Arthritis & Rheumatology. 2013 Nov;**65**(11):2737‐2747

Journal of Microcirculation. 1995 Nov‐Dec;**15**(6):287‐292

laroscopy in healthy volunteers. Vasa. 2012 Jan;**41**(1):19‐26

Diseases. 1996 Aug;**55**(8):507‐512

Rheumatology. 2005;**32**:841‐848

Diseases 2008;**67**:885‐887

Rheumatology. 2009 Mar 15;**61**(3):405‐410

Journal of Rheumatology. 2001 Jul;**28**(7):1573‐1576


**Provisional chapter**

### **Ophthalmological Manifestations and Tear Investigations in Systemic Sclerosis Investigations in Systemic Sclerosis**

**Ophthalmological Manifestations and Tear** 

DOI: 10.5772/intechopen.69909

Aniko Rentka, Krisztina Koroskenyi, Jolan Harsfalvi, Zoltan Szekanecz, Gabriella Szucs, Peter Szodoray and Adam Kemeny-Beke Harsfalvi, Zoltan Szekanecz, Gabriella Szucs, Peter Szodoray and Adam Kemeny-Beke Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Aniko Rentka, Krisztina Koroskenyi, Jolan

http://dx.doi.org/10.5772/intechopen.69909

### **Abstract**

Systemic sclerosis (SSc) is a chronic autoimmune disorder characterized by widespread small vessel vasculopathy, immune dysregulation with production of autoantibodies, and progressive fibrosis. There are only few reports available concerning ophthalmological complications in the course of SSc, although ocular manifestations, e.g., dry eye syndrome (DES), occurs frequently and decreases the quality of life of these patients. Vascular endothelial growth factor (VEGF), the major pro-angiogenic factor, plays a key role in the pathomechanism of SSc. Although elevated levels of VEGF in sera have already been demonstrated, VEGF analysis in tears of patients with SSc has not been performed in previous studies. VEGF in the tears of patients with SSc was found to be decreased by 20%, compared to healthy controls. The reason why the VEGF levels are not elevated in the tears of patients with SSc needs further investigations, as does the sera of the same patients. The cytokine array results revealed a shift in the cytokine profile characterized by the predominance of inflammatory mediators. Our current data depict a group of cytokines and chemokines, which play a significant role in ocular pathology of SSc; furthermore, they might function as excellent candidates for future therapeutic targets in SSc with ocular manifestations.

**Keywords:** systemic sclerosis, dry eye syndrome, tear, vascular endothelial growth factor, cytokine, tear sampling, total protein, enzyme-linked immunosorbent assay, cytokine array, multiplex bead assay

### **1. Introduction**

There are few reports, mainly case reports, available concerning ophthalmological complications in the course of systemic sclerosis (SSc). Overall studies are even fewer involving

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

only a small number of patients, since SSc is a rare disease [1, 2]. Changes in the organ of vision are thought to be the consequences of systemic complications of scleroderma or adverse effects of the immunosuppressive treatment applied. Ocular symptoms may occur at any stage of the disease and may involve numerous ocular tissues. Their course can be clinically latent or very intensive. The most prevalent clinical manifestations of soft tissue fibrosis and inflammation in patients with SSc include increased tonus and telangiectasia of the eyelid skin. The most common lesions reported include periorbital edema, palpebral ectropion, and madarosis [3].

The most frequent ocular manifestation of SSc in our studies was dry eye syndrome (DES).

DES is a major healthcare problem because it affects the patient's quality of life. DES in SSc is believed to be caused by fibrosis-related impairment of lacrimal gland secretion, namely, the reduction of the water portion of the tear film. Furthermore, lipid layer disorder is caused by chronic blepharitis and meibomian gland dysfunction (MGD), while increased evaporation of tears from the ocular surface is the consequence of restricted eyelid mobility and the consecutive reduced blinking [2]. DES was recently redefined as a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance, tear film instability, and last but not least damage to the ocular surface [4]. Increased osmolality of the tear film [5] and inflammation of the ocular surface [6] are the two major characteristic features of this ocular surface disease. The most important laboratory findings [6] are increased levels of several inflammatory cytokines. Accordingly, tear cytokine levels are regarded as potential markers of inflammation in DES.

The ophthalmological manifestations in patients with SSc are frequently underestimated and not or not correctly treated. In order to better understand the ocular features and use this body fluid as a potential tool for monitoring these important biomarkers, we have turned our attention to tear investigations.

Precorneal tears as a biological fluid are very easily accessible with non- or very low-invasive methods at a relatively low cost. Tears not only lubricate the ocular surface carrying secreted molecules from corneal epithelial cells and tissues producing tear components but also represent the whole physiological status of the body. Due to the very limited amount of samples and the relative instability of the components, sample collection is a critical step in tear research and diagnostics.

Although tear analysis is of increasing interest in ophthalmology, no studies have investigated tears of patients with SSc as yet, possibly because of the technical challenge posed by the extremely small sample volumes available [7].

Quantitative determination of tear proteins is of increasing interest in ophthalmology, but a technical problem still remains due to small tear sample volumes available on the one hand and the complexity of their composition on the other [7, 8]. Tear sampling performed either directly or indirectly is definitely a major challenge or has a most significant influence on the precision and reproducibility of the analytical results as seen in the summary below.

### **1.1. Direct sampling methods**

only a small number of patients, since SSc is a rare disease [1, 2]. Changes in the organ of vision are thought to be the consequences of systemic complications of scleroderma or adverse effects of the immunosuppressive treatment applied. Ocular symptoms may occur at any stage of the disease and may involve numerous ocular tissues. Their course can be clinically latent or very intensive. The most prevalent clinical manifestations of soft tissue fibrosis and inflammation in patients with SSc include increased tonus and telangiectasia of the eyelid skin. The most common lesions reported include periorbital edema, palpebral

The most frequent ocular manifestation of SSc in our studies was dry eye syndrome (DES).

DES is a major healthcare problem because it affects the patient's quality of life. DES in SSc is believed to be caused by fibrosis-related impairment of lacrimal gland secretion, namely, the reduction of the water portion of the tear film. Furthermore, lipid layer disorder is caused by chronic blepharitis and meibomian gland dysfunction (MGD), while increased evaporation of tears from the ocular surface is the consequence of restricted eyelid mobility and the consecutive reduced blinking [2]. DES was recently redefined as a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance, tear film instability, and last but not least damage to the ocular surface [4]. Increased osmolality of the tear film [5] and inflammation of the ocular surface [6] are the two major characteristic features of this ocular surface disease. The most important laboratory findings [6] are increased levels of several inflammatory cytokines. Accordingly, tear cytokine levels are regarded as potential

The ophthalmological manifestations in patients with SSc are frequently underestimated and not or not correctly treated. In order to better understand the ocular features and use this body fluid as a potential tool for monitoring these important biomarkers, we have turned our

Precorneal tears as a biological fluid are very easily accessible with non- or very low-invasive methods at a relatively low cost. Tears not only lubricate the ocular surface carrying secreted molecules from corneal epithelial cells and tissues producing tear components but also represent the whole physiological status of the body. Due to the very limited amount of samples and the relative instability of the components, sample collection is a critical step in tear

Although tear analysis is of increasing interest in ophthalmology, no studies have investigated tears of patients with SSc as yet, possibly because of the technical challenge posed by

Quantitative determination of tear proteins is of increasing interest in ophthalmology, but a technical problem still remains due to small tear sample volumes available on the one hand and the complexity of their composition on the other [7, 8]. Tear sampling performed either directly or indirectly is definitely a major challenge or has a most significant influence on the precision and reproducibility of the analytical results as seen in the summary

ectropion, and madarosis [3].

138 Systemic Sclerosis

markers of inflammation in DES.

attention to tear investigations.

research and diagnostics.

below.

the extremely small sample volumes available [7].

Direct sampling methods use microcapillary tubes [9] or micropipettes for sampling. This requires previous stimulation or instillation of different volumes of saline (100–200 μl) into the cul-de-sac and collecting after appropriate mixing. The procedure causes dilution and may not permit collection of samples from specific sites of the ocular surface [10].

Kalsow et al. investigated the tear cytokine response to multipurpose solutions in contact lens [11] wearing. Before tear collection, contact lens was removed, and then NST tears were collected from both eyes from the inferior lateral conjunctival cul-de-sac using a 10-μl flame-polished glass micropipette. The collection, a 5.5-μl tear volume, was immediately transported to a sterile 0.2-ml tube containing 49.5 μl of storage solution to produce a 1:10 tear dilution for immediate storage at −80°C [11].

Guyette et al. compared low-abundance biomarker levels in capillary-collected NST tears and washout (WO) tears of aqueous-deficient and normal patients. 10-microliter polished micropipettes were used to collect tears from the inferior marginal strip, taking special care to minimize ocular surface contact. Tear collection rate was continuously monitored. Individual NST tear samples were collected in 10-min aliquots and immediately transferred to a sterile polymerase chain reaction (PCR) tube. An equal volume of assay buffer was added, and the sample was stored at −86°C. A total of at least 6.5-μl NST tears were collected from each study participant, and each 10-min aliquot was transferred into a separate PCR tube and put in the freezer without delay. Prior to WO tear sample collection, 10-μl sterile physiologic saline solution was added to the lower conjunctiva by a digital pipette. The patient was instructed to gently close the eyes and avoid any eye movements for one minute. Tears were then collected using the same method as for NST samples, but a shorter collection time of 5 min per aliquot was used to make up the 6.5-μl minimum volume required. Tear collection volume and time were continuously monitored to measure tear collection rate [12].

There have been several research projects focusing on dry eye syndrome, and nowadays the emphasis has shifted toward the role of inflammation in the anterior surface of the eye [13]. Since inflammatory mediators originating from various ocular surface sources and the main lacrimal gland do not constitute a totally homogenous mix, the way the tears are collected will influence the resulting biomarker profile. NST tear samples from the inferior marginal strip cover a broader spectrum of the sources, whereas ST samples contain a higher proportion of the lacrimal gland secretion [14]. Explicit protein profile differences between NST and ST tears demonstrate that these two sample types are not equivalent [15, 16]. Although NST tears represent specifically the inflammatory status of the ocular surface, the volume of NST tears is limited, especially in aqueous-deficient dry eye. Even though tear sampling frequently makes use of capillaries as they are less irritating and the resulting sample is an exact representative concentration of molecules, the main limitation of the method is the volume of sample (2–3 μl) to be gained [17].

One way to increase the available tear sample volume is to add fluid (e.g., sterile saline) to the eye prior to sample collection, effectively "washing out" ocular surface molecules [18, 19]. In an experimental dry eye study, Luo et al. collected tears from mice with tear fluid washing [20]. Tear fluid washings were collected by a method previously reported by Song et al. [21]. Briefly, 1.5 μl of phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) was instilled into the conjunctival sac. The tear fluid and buffer were collected with a 10-μl volume glass capillary tube by capillary action from the tear meniscus in the lateral canthus. The 2-μl sample of tear washings was pooled from both eyes of each mouse and was stored at −80°C until zymography and enzyme-linked immunosorbent assay (ELISA) were performed.

Validity of the WO method depends on the extent to which it changes the NST tear biomarker profile. By determining tear sIgA, inducement of reflex tearing is easily detected because tear sIgA levels decrease with reflex tear flow rate [16]. Markoulli et al. found equal tear sIgA-total tear ratios in WO and NST tears, which suggests that WO tear samples do not significantly induce reflex tearing [19]. Guyette's study evaluated WO tear collection as a replacement for microcapillary NST tear collection and applied this to compare biomarker levels between aqueous-deficient (AD) dry eye and non-AD patients [12, 15, 22].

### **1.2. Indirect methods**

An indirect method means that collection of precorneal tear film (PTF) is carried out using absorbing supports such as Schirmer test strips (STS), filter paper disks, cellulose sponges, and polyester rods. STS collection is the most commonly used method among them [23].

Acera et al. analyzed the inflammatory markers in the PTF of patients with ocular surface disease. 10 μl of tear samples was collected by a Weck-Cel sponge [24]. The concentrations of IL-1β, IL-6, and pro-MMP-9 were measured by ELISA, and the MMP-9 activity was evaluated by gelatin zymography.

Inic-Kanada et al. compared ophthalmic sponges and extraction buffers for quantifying cytokine profiles in tears using Luminex technology. They found that Luminex detection of cytokine/chemokine profiles of tears collected with Merocel sponges may be useful in clinical studies, for instance, to assess cytokine profile evaluation in ocular surface diseases [25].

Samples obtained from the Schirmer test procedure have been found to have a higher mucus, lipid, and cellular content than microcapillary (MC) samples [26]. STS also suffers from incomplete, nonuniform elution of proteins from the filter matrix [23]. Although micropipette and STS collection provide different biomarker profiles for a given donor, the correctly applied micropipette method has proved to be more consistent [27]. STS is widely applied as the volume of sample collected with this method is larger than other methods, but it can cause reflexive tearing due to irritation, which increases the volume of the samples, thus aggravating the detection of the investigated tear component(s), e.g., drug levels [9].

In comparative studies, the tears of the same patient are collected using several collection methods to determine the same biomarkers from the different tear samples.

Green-Church et al. collected tears using small volume (1–5 μl) Drummond glass MC tubes with 1.6× slit-lamp magnification. Non-reflex tears were collected from the inferior tear prism without contact with the lower lid until a total of 5 μl had been collected. During a separate visit, tear collection was performed by placing an STS over the lower lid. The lid was canthus. The subject was instructed to close his/her eyes for the 5-min test duration; the wet length was not recorded but was observed to be within normal ranges in all cases. The STS was then placed in 1.6-ml amber Eppendorf tube and stored at 4°C until analysis [27].

Lee et al. used two collection techniques for the comparative analysis of polymerase chain reaction assay for herpes simplex virus 1 detection [28]. Tear samples were collected from the lower fornix using STS for 5 min, a method adopted in a previous study of Satpathy et al. [29]. The other collection method they used was micropipetting tears, after irrigating 100-μl saline in the lower fornix, a method that was described in a previous study of Markoulli et al. [19], who validated the "flush" tear collection technique as a viable alternative to basal and reflex tear collection.

## **2. "Main body of the paper"**

In an experimental dry eye study, Luo et al. collected tears from mice with tear fluid washing [20]. Tear fluid washings were collected by a method previously reported by Song et al. [21]. Briefly, 1.5 μl of phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) was instilled into the conjunctival sac. The tear fluid and buffer were collected with a 10-μl volume glass capillary tube by capillary action from the tear meniscus in the lateral canthus. The 2-μl sample of tear washings was pooled from both eyes of each mouse and was stored at −80°C until zymography and enzyme-linked immunosorbent assay (ELISA) were

Validity of the WO method depends on the extent to which it changes the NST tear biomarker profile. By determining tear sIgA, inducement of reflex tearing is easily detected because tear sIgA levels decrease with reflex tear flow rate [16]. Markoulli et al. found equal tear sIgA-total tear ratios in WO and NST tears, which suggests that WO tear samples do not significantly induce reflex tearing [19]. Guyette's study evaluated WO tear collection as a replacement for microcapillary NST tear collection and applied this to compare biomarker levels between

An indirect method means that collection of precorneal tear film (PTF) is carried out using absorbing supports such as Schirmer test strips (STS), filter paper disks, cellulose sponges, and polyester rods. STS collection is the most commonly used method among

Acera et al. analyzed the inflammatory markers in the PTF of patients with ocular surface disease. 10 μl of tear samples was collected by a Weck-Cel sponge [24]. The concentrations of IL-1β, IL-6, and pro-MMP-9 were measured by ELISA, and the MMP-9 activity was evaluated

Inic-Kanada et al. compared ophthalmic sponges and extraction buffers for quantifying cytokine profiles in tears using Luminex technology. They found that Luminex detection of cytokine/chemokine profiles of tears collected with Merocel sponges may be useful in clinical studies, for instance, to assess cytokine profile evaluation in ocular surface diseases [25].

Samples obtained from the Schirmer test procedure have been found to have a higher mucus, lipid, and cellular content than microcapillary (MC) samples [26]. STS also suffers from incomplete, nonuniform elution of proteins from the filter matrix [23]. Although micropipette and STS collection provide different biomarker profiles for a given donor, the correctly applied micropipette method has proved to be more consistent [27]. STS is widely applied as the volume of sample collected with this method is larger than other methods, but it can cause reflexive tearing due to irritation, which increases the volume of the samples, thus aggravating the detection of the investigated tear component(s), e.g.,

In comparative studies, the tears of the same patient are collected using several collection

methods to determine the same biomarkers from the different tear samples.

aqueous-deficient (AD) dry eye and non-AD patients [12, 15, 22].

performed.

140 Systemic Sclerosis

**1.2. Indirect methods**

by gelatin zymography.

drug levels [9].

them [23].

The aims of our studies were the following:


### **2.1. Patients and healthy controls**

In the first study, 43 patients with SSc (40 female and 3 men) and 27 healthy controls were included. In the second study, we enrolled 9 patients and 12 controls. Mean (SD) age of the patients was 61.85 (48–74) years. SSc was diagnosed based on the corresponding international criteria. Patients were enrolled from the outpatient clinic at the Department of Rheumatology. They went through ophthalmological examination and basal tear sample collection at the Department of Ophthalmology. None of the patients had secondary Sjögren's syndrome. The healthy control groups were composed of age- and gender-matched volunteers with no history of any autoimmune or ocular disorder. Patients did not take immunosuppressive medications at the time of the tear sampling.

Written informed consent was obtained from all patients and controls. Study protocol was approved by the local bioethics committee and followed the tenets of the declaration of Helsinki.

### **2.2. Tear sample collection**

Unstimulated, open-eye tear samples were gently collected from the inferior temporal meniscus of both eyes, using glass capillary tubes (Haematokritkapillare, 75 μL, L 75 mm, Hirschmann Laborgerate, Germany), minimizing irritation of the ocular surface or lid margin as much as possible.

In the course of the first study, samples were collected between 11 a.m. and 16 p.m. by the same physician. Tear-secretion velocity was counted by dividing the volume of collected sample with time of secretion. Volume was calculated from the lengths of the fluid column in the capillary tube, measured with a vernier caliper, and from the known diameter of the tube. Time of tear collection was measured with a stopwatch.

In the course of the second study, tear collection was performed between 9 and 11 a.m.

Tears were transferred into low-binding-capacity Eppendorf tubes by the help of a sterile syringe and a needle, carried on dry ice to the laboratory and stored at −80°C until assessment. The samples were obtained from both eyes of each individual and were pooled due to the small volume available.

### **2.3. Quantification of total protein and VEGF levels in tear samples of patients with SSc**

First, as a point of reference for VEGF, total tear protein concentrations were determined using the microplate method of the bicinchoninic acid (BCA) Protein Assay Kit (Pierce Biotechnology, Rockford, USA) adapted to a 384-well microplate due to the small sample amounts. The kit is a two-component, high-precision, detergent-compatible assay. Total protein concentration determination was based on color intensity measurement proportional to the peptide bound and the protein provided with the reagent set. The reaction absorbs visible light, namely, the wavelength 562 nm.

We used a human VEGF immunoassay kit by Quantikine (R&D Systems, Minneapolis, MN, USA) for the quantitative determination of VEGF in tear fluid. This assay employs the quantitative sandwich enzyme immunoassay technique.

### **2.4. Membrane array and multiplex bead analysis of tear cytokines in SSc**

To remove cells, cellular debris, and contaminant particles, tear samples were centrifuged (10 min, 15,000 rpm, 4°C) prior to use.

Tear samples of controls and patients were used for cytokine profiling. The relative levels of 102 different cytokines were determined by Proteome Profiler Human XL Cytokine Array Kit (R&D Systems) using 50-μl samples according to the manufacturer's instructions. The pixel density in each spot of the array was determined by ImageJ software.

Alternatively, the absolute levels of MCP-1, complement factor D (CFD), IP-10, and C-reactive protein (CRP) were determined from diluted tear samples (CFD, MCP-1, and CRP, 1:10; IP-10, 1:40) by Human Luminex Performance Assays (R&D Systems) according to the manufacturer's instructions. The measurement was run on Bio-Plex 200 Systems (Bio-Rad) workstation.

### **2.5. Results**

history of any autoimmune or ocular disorder. Patients did not take immunosuppressive

Written informed consent was obtained from all patients and controls. Study protocol was approved by the local bioethics committee and followed the tenets of the declaration of

Unstimulated, open-eye tear samples were gently collected from the inferior temporal meniscus of both eyes, using glass capillary tubes (Haematokritkapillare, 75 μL, L 75 mm, Hirschmann Laborgerate, Germany), minimizing irritation of the ocular surface or lid margin

In the course of the first study, samples were collected between 11 a.m. and 16 p.m. by the same physician. Tear-secretion velocity was counted by dividing the volume of collected sample with time of secretion. Volume was calculated from the lengths of the fluid column in the capillary tube, measured with a vernier caliper, and from the known diameter of the tube.

In the course of the second study, tear collection was performed between 9 and 11 a.m.

Tears were transferred into low-binding-capacity Eppendorf tubes by the help of a sterile syringe and a needle, carried on dry ice to the laboratory and stored at −80°C until assessment. The samples were obtained from both eyes of each individual and were pooled due to

**2.3. Quantification of total protein and VEGF levels in tear samples of patients with SSc**

First, as a point of reference for VEGF, total tear protein concentrations were determined using the microplate method of the bicinchoninic acid (BCA) Protein Assay Kit (Pierce Biotechnology, Rockford, USA) adapted to a 384-well microplate due to the small sample amounts. The kit is a two-component, high-precision, detergent-compatible assay. Total protein concentration determination was based on color intensity measurement proportional to the peptide bound and the protein provided with the reagent set. The reaction absorbs visible

We used a human VEGF immunoassay kit by Quantikine (R&D Systems, Minneapolis, MN, USA) for the quantitative determination of VEGF in tear fluid. This assay employs the quan-

To remove cells, cellular debris, and contaminant particles, tear samples were centrifuged (10

**2.4. Membrane array and multiplex bead analysis of tear cytokines in SSc**

medications at the time of the tear sampling.

Time of tear collection was measured with a stopwatch.

Helsinki.

142 Systemic Sclerosis

**2.2. Tear sample collection**

the small volume available.

light, namely, the wavelength 562 nm.

min, 15,000 rpm, 4°C) prior to use.

titative sandwich enzyme immunoassay technique.

as much as possible.

*2.5.1. Vascular endothelial growth factor in tear samples of patients with systemic sclerosis*

The average tear secretion velocity in patients was 4.53 μl/min with a median of 3.8 μl/min (1.5–25.6).

Duration of tear sample collection from patients varied between 20 and 313 s, until 5 μl, the minimally required volume was reached.

The average collected tear fluid volume was 10.4 μl (1.6–31.2) in patients and 15.63 μl (3.68– 34.5) in controls.

In tear samples of patients with SSc, the average total protein level was 6.9 μg/μl (1.8–12.3), and the average concentration of VEGF was 4.9 pg/μl (3.5–8.1) in the case of basal tear secretion.

Control tears contained on average 4.132 μg/μl (0.1–14.1) protein and 6.15 pg/μl (3.84–12.3) VEGF.

### *2.5.2. Membrane array and multiplex bead analysis of tear cytokines in systemic sclerosis*

### *2.5.2.1. Cytokine array results*

Nonstimulated tear cytokine profiles of the control groups and patients with SSc were analyzed by cytokine array detecting 102 different cytokines. Array results revealed a shift in cytokine profile characterized by the predominance of inflammatory mediators. The following 9 out of the 102 analyzed molecules were significantly increased in tears of patients with SSc: complement factor D (CFD), chitinase-3-like protein 1 (CHI3L1), C-reactive protein (CRP), epidermal growth factor (EGF), interferon-γ-inducible protein 10 (IP-10, also called CXCL-10), monocyte chemoattractant protein-1 (MCP-1), monokine induced by gamma interferon (MIG), matrix metallopeptidase 9 (MMP-9), and vitamin D binding protein (VDBP) (**Table 1**).

Integrated density values were normalized to positive control spots and total protein content of the samples. Cytokine array data are representative of four control and four SSc samples.


Mean total protein values did not differ significantly in tears of patients and controls. Mean total protein value was 40.9239 μg/ml in tears of patients with SSc and 42.536 μg/ml in tears of healthy controls (p = 0.863604).

**Table 1.** Normalized densities of cytokines and chemokines in patients with SSc and healthy controls.

### *2.5.2.2. Multiplex cytokine bead assay results*

By using the more sensitive and more specific Luminex bead assay, 4 selected molecules were determined in tears of 9 healthy controls and 12 patients with SSc.

Based on the Luminex bead results, mean CRP levels were 103.44 (3.57–359.02) μg/mg protein in tears of patients with SSc and 7.41 (0.87–18.03) μg/mg protein in tears of healthy controls.

Mean IP-10 levels were 564.78 (252.62–1107.2) μg/mg protein in tears of patients with SSc and 196.118 (101.66–514.37) μg/mg protein in tears of healthy controls.

Mean MCP-1 levels were 2626.83 (457.84–5619.4) μg/mg protein in tears of patients with SSc and 661.27 (397.87–1171.4) μg/mg protein in tears of healthy controls.

Mean CFD levels were 15.27 (5.00–35.28) μg/mg protein in tears of patients with SSc and 23.31 (5.18–106.63) μg/mg protein in tears of healthy controls.

Except for CFD all results were significant at p = 0.01 for CRP, p = 0.001 for IP-10, and p = 0.01 for MCP-1, respectively.

Values represent the mean (±SD) of the 9 control and 12 patient samples, which are the fold change of normalized cytokine levels.

The difference between total protein values of control and SSc tear samples was not significant (p = 0.37263). Mean total protein was 818.46 (779.94–1162.4) μg/ml in tears of patients and 872.46 (771.78–1359.5) μg/ml in tears of controls.

Based on both the cytokine array and the multiplex bead assay results, concentrations of IP-10 showed the most significant difference in tears of patients and controls.

### **2.6. Discussion**

*2.5.2.2. Multiplex cytokine bead assay results*

**Name of the cytokines and** 

**chemokines**

144 Systemic Sclerosis

determined in tears of 9 healthy controls and 12 patients with SSc.

**Normalized density patients with SSc**

CFD 50.35 (23.17–53.76) 22.33 (18.39–24.75) 0.002072 CHI3L1 94.41 (31.9–95.98) 31.06 (20.37–45.85) 0.000000 CRP 25.98 (15.28–53.16) 4.55 (4.35–4.66) 0.018250 EGF 53.42 (34.86–70.23) 34.04 (20.42–47.61) 0.032818 IP-10 123.42 (93.81–152.35) 21.99 (12.12–29.01) 0.000000 MCP-1 19.93 (5.38–42.44) 1.72 (1.44–2.27) 0.044726 MIG 22.85 (5.6–64.14) 3.58 (3.29–3.88) 0.033787 MMP-9 49.10 (4.24–129.04) 12.74 (10.29–17.56) 0.000068 VDBP 31.35 (11.87–64.68) 10.18 (8.3–13.84) 0.019733

196.118 (101.66–514.37) μg/mg protein in tears of healthy controls.

(5.18–106.63) μg/mg protein in tears of healthy controls.

for MCP-1, respectively.

change of normalized cytokine levels.

872.46 (771.78–1359.5) μg/ml in tears of controls.

and 661.27 (397.87–1171.4) μg/mg protein in tears of healthy controls.

showed the most significant difference in tears of patients and controls.

By using the more sensitive and more specific Luminex bead assay, 4 selected molecules were

Mean total protein values did not differ significantly in tears of patients and controls. Mean total protein value was

40.9239 μg/ml in tears of patients with SSc and 42.536 μg/ml in tears of healthy controls (p = 0.863604).

**Table 1.** Normalized densities of cytokines and chemokines in patients with SSc and healthy controls.

**Normalized density healthy controls**

**Significance of the difference (p)**

Based on the Luminex bead results, mean CRP levels were 103.44 (3.57–359.02) μg/mg protein in tears of patients with SSc and 7.41 (0.87–18.03) μg/mg protein in tears of healthy controls. Mean IP-10 levels were 564.78 (252.62–1107.2) μg/mg protein in tears of patients with SSc and

Mean MCP-1 levels were 2626.83 (457.84–5619.4) μg/mg protein in tears of patients with SSc

Mean CFD levels were 15.27 (5.00–35.28) μg/mg protein in tears of patients with SSc and 23.31

Except for CFD all results were significant at p = 0.01 for CRP, p = 0.001 for IP-10, and p = 0.01

Values represent the mean (±SD) of the 9 control and 12 patient samples, which are the fold

The difference between total protein values of control and SSc tear samples was not significant (p = 0.37263). Mean total protein was 818.46 (779.94–1162.4) μg/ml in tears of patients and

Based on both the cytokine array and the multiplex bead assay results, concentrations of IP-10

Although ocular manifestations in systemic autoimmune diseases have significant debilitating effects, tear analysis has been missing from the repertoire of investigations. Since tears represent the local homeostasis of the ocular surface better than serum, this makes tears ideal for assessing ocular pathology in the disease. There are two possible ways for cytokines to appear in the precorneal tear film. Some are locally produced and diffuse into the tear film from the corneal and conjunctival epithelia; others leak into the tear film from the conjunctival blood vessels [30]. Tear investigation is a challenging research field; though sample collection is noninvasive, it has an almost insurmountable limitation, the quantity of the sample obtainable [31].

Tear investigation studies have been performed in different ocular and systemic disorders [30, 32, 33]. Leonardi et al. assessed multiple mediators, such as cytokines, matrix metalloproteases, and angiogenic and growth factors in tears of patients with vernal keratoconjunctivitis. These analyses identified previously unreported factors in tears of patients, including MMP-3 and MMP-10 and multiple proteases, growth factors and cytokines, which may all be instrumental in the pathogenesis of conjunctival inflammation. Different molecules were identified in human tear samples that were involved in the development and maintenance of corneal neovascularization. Concentrations of the pro-angiogenic cytokines such as IL-6, IL-8, VEGF, MCP-1, and Fas ligand were determined in blood and tear samples using flow cytometrybased multiplex assay. These investigations resulted in significantly higher concentrations of pro-angiogenic cytokines in human tears compared to their concentrations in serum; furthermore highest levels were revealed in basal tear samples [30]. These findings lend further support to the importance of our current studies.

After reviewing the literature on direct and indirect tear sampling methods in various ocular and systemic disorders, we have chosen the microcapillary method for tear sampling in patients with SSc, since it is safely applicable for the collection of nonstimulated tears. In order to transfer the tear fluid from the microcapillary tube to the collection tube, we applied a sterile syringe and a needle. This tear sampling method proved to be suitable for our experiments on tear cytokines.

### *2.6.1. VEGF in tear samples of patients with SSc*

VEGF is one of the components of normal tear fluid [34]. Vesaluoma et al. determined VEGF concentrations in healthy tears. The median VEGF concentration was 5 pg/μl (4–11) consistent with our results, as control tears contained an average of 6.15 pg/μl (3.84–12.3) VEGF [35].

They calculated the average tear fluid secretion in healthy controls, which was 8.1 μl/min (0.7–20.8), using the same tear collecting method as we did in our study. Results show that patients with SSc have significantly decreased tear secretion that could be explained by DES, which is a probable sequel of the disease or to the side effects of the therapeutic drugs [36].

Tear-secretion velocity was lower by 67% in patients with SSc than in healthy controls. The difference was significant (p < 0.01). The reason for this sign could be explained by the pathophysiology of the disease, namely, fibrotic processes of the lacrimal gland.

Total protein values in patients with SSc were higher by 42% than in healthy controls. This may indicate that total protein production—or simply protein concentration, since patients with SSc have a decreased tear secretion velocity—is only increased because of the smaller tear volume. VEGF in the tears of patients with SSc decreased by 20%, which can be explained also by the decreased tear secretion of patients [36].

The question why contrary to our expectations VEGF levels are not higher in patients with SSc than in the healthy group needs further investigation.

### *2.6.2. Membrane array and multiplex bead analysis of tear cytokines in SSc*

Based on our cytokine array results, nine cytokines and chemokines had significantly higher levels in tears of patients with SSc. This screening method was performed for the assortment of 102 cytokines, selecting the most relevant ones in the pathogenesis of SSc for further experiments. All molecules which appeared to be significantly higher in tears of patients are molecular players of the immune responses and inflammatory processes, which confirms the presence of ocular surface inflammation in patients with SSc possibly as a consequence of DES [36].

CHI3L1, a protein which takes part in the processes of inflammation and tissue remodeling, has not been previously described in relation to the pathomechanism of SSc. We have found elevated levels of CHI3L1 in patients with SSc. This result correlates well with the fact that inflammation and tissue injury caused by hypoxia and oxidative stress are always present in the course of SSc.

In fact, different pathways may lead to vascular dysfunction processes in SSc, such as direct vascular damage or pro-inflammatory responses. Studies in different diseases have shown functional links between activated complement molecules and these pathways. CFD, a serine protease, also known as adipsin, plays a key role in these processes [37, 38]. CFD is the ratelimiting enzyme in the activation cascade of the alternative pathway, and its level in the blood is quite low. Our cytokine array results showed increased CFD levels, which confirm the role of the complement system in the ocular pathology of SSc.

Levels of EGF were also elevated in tear samples of patients with SSc. EGF is a growth factor that stimulates cell growth, proliferation, and differentiation [39]. Elevation of EGF may be explained by the above processes of vasculopathy. The next molecule, which appeared to be higher in patients' tears, is matrix metallopeptidase-9 (MMP-9). As a protease of the MMP family, it is involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, angiogenesis, bone development, wound healing, cell migration, as well as in pathological processes, such as intracerebral hemorrhage, arthritis, and metastasis [40–42]. In a study of Kim et al., serum MMP-9 concentrations were found to be elevated in patients with SSc correlating well with skin scores [43]. Their results suggest that increased MMP-9 concentrations may be due to their overproduction by dermal fibroblasts and also that the enhanced production of MMP-9 may contribute to fibrogenic remodeling during the progression of skin sclerosis in SSc. Our results of tear cytokine array are parallel with the finding that MMP-9 is increased in the course of SSc.

Tear-secretion velocity was lower by 67% in patients with SSc than in healthy controls. The difference was significant (p < 0.01). The reason for this sign could be explained by the patho-

Total protein values in patients with SSc were higher by 42% than in healthy controls. This may indicate that total protein production—or simply protein concentration, since patients with SSc have a decreased tear secretion velocity—is only increased because of the smaller tear volume. VEGF in the tears of patients with SSc decreased by 20%, which can be explained

The question why contrary to our expectations VEGF levels are not higher in patients with SSc

Based on our cytokine array results, nine cytokines and chemokines had significantly higher levels in tears of patients with SSc. This screening method was performed for the assortment of 102 cytokines, selecting the most relevant ones in the pathogenesis of SSc for further experiments. All molecules which appeared to be significantly higher in tears of patients are molecular players of the immune responses and inflammatory processes, which confirms the presence of ocular surface inflammation in patients with SSc possibly as a consequence of

CHI3L1, a protein which takes part in the processes of inflammation and tissue remodeling, has not been previously described in relation to the pathomechanism of SSc. We have found elevated levels of CHI3L1 in patients with SSc. This result correlates well with the fact that inflammation and tissue injury caused by hypoxia and oxidative stress are always present in

In fact, different pathways may lead to vascular dysfunction processes in SSc, such as direct vascular damage or pro-inflammatory responses. Studies in different diseases have shown functional links between activated complement molecules and these pathways. CFD, a serine protease, also known as adipsin, plays a key role in these processes [37, 38]. CFD is the ratelimiting enzyme in the activation cascade of the alternative pathway, and its level in the blood is quite low. Our cytokine array results showed increased CFD levels, which confirm the role

Levels of EGF were also elevated in tear samples of patients with SSc. EGF is a growth factor that stimulates cell growth, proliferation, and differentiation [39]. Elevation of EGF may be explained by the above processes of vasculopathy. The next molecule, which appeared to be higher in patients' tears, is matrix metallopeptidase-9 (MMP-9). As a protease of the MMP family, it is involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, angiogenesis, bone development, wound healing, cell migration, as well as in pathological processes, such as intracerebral hemorrhage, arthritis, and metastasis [40–42]. In a study of Kim et al., serum MMP-9 concentrations were found to be elevated in patients with SSc correlating well with skin scores [43]. Their results suggest that increased MMP-9 concentrations may be due to their overproduction by dermal

physiology of the disease, namely, fibrotic processes of the lacrimal gland.

also by the decreased tear secretion of patients [36].

than in the healthy group needs further investigation.

of the complement system in the ocular pathology of SSc.

DES [36].

146 Systemic Sclerosis

the course of SSc.

*2.6.2. Membrane array and multiplex bead analysis of tear cytokines in SSc*

In a previous study, expression of antiangiogenic chemokines and their receptors were determined in the sera and skin of patients with SSc [44]. Based on their results, MIG and its receptor are elevated in serum and highly expressed in the skin of patients with SSc. We have also found increased levels of MIG in tear samples of patients, which confirm the fact that dysregulated angiogenesis is an important feature in the pathomechanism of SSc. The next protein that appeared to be higher is VDBP, a member of the albumin gene family. VDBP is a multifunctional protein found in plasma, ascitic and cerebrospinal fluid and on the surface of many cell types. It binds to vitamin D and its plasma metabolites and transports them to target tissues [45]. Others have measured significant quantities of VDBP-actin complexes in the plasma following injury [46]. The presence of tissue injury is likely to be the explanation of our results, namely, the elevated levels of VDBP in the tears of patients with SSc.

Based on our results of multiplex bead assay, the three molecules that showed significant differences in tears of patients and controls were IP-10, MCP-1, and CRP. Previous studies have already demonstrated elevated levels of these markers in the sera of patients with SSc.

General markers of inflammation, such as CRP, are expected to be higher in a disease like SSc. In earlier trials, CRP appeared to be elevated in the sera of patients with SSc and was associated with poor survival. Therefore, it may be a useful indicator of disease activity and severity in SSc [47, 48].

Another inflammatory chemokine, IP-10, also called CXCL-10, has often been investigated in SSc studies [44, 49, 50]. IP-10 has an angiostatic function as it suppresses neovascularization; furthermore, it is involved in immune regulation [51].

Recent reports have shown that the serum and/or the tissue expressions of IP-10 are increased in various bacterial, viral, fungal, and protozoal infections [52] and also in autoimmune diseases like rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, autoimmune thyroid diseases, type 1 diabetes mellitus, Addison's disease, and SSc [50, 53–55]. CXCL10 is secreted by CD4+, CD8+, natural killer and natural killer T cells and is dependent on interferon-γ. CXCL10 can also be secreted by several other cell types, including endothelial cells, fibroblasts, keratinocytes, thyrocytes, preadipocytes, etc. Detecting a high level of CXCL10 in peripheral fluids is therefore a marker of host immune response [48], which correlates well with our results of cytokine bead assay measurements.

Finally, MCP-1, which is a key participant of the fibrotic processes in SSc, also appeared to be higher in patients' tears. MCP-1, which recruits monocytes, memory T cells, and dendritic cells to the sites of inflammation, is produced by either tissue injury or infection [56]. It is known as one of the most pathogenic chemokines during the development of inflammation and fibrosis in SSc [57]. MCP-1 is not only a chemoattractant molecule for monocytes and T cells, but it also induces Th2 cell polarization and stimulates collagen production by fibroblasts [58]. Hasegawa et al. have previously shown that serum MCP-1 levels are elevated when the skin and lung are affected in patients with SSc [59]. It has also been reported that cultured dermal fibroblasts from patients with SSc show augmented expressions of MCP-1 mRNA and protein [60].

Of the last three molecules, IP-10 and MCP-1 are the ones whose molecular characteristics single them out as potential candidates for therapies against the pathological consequences of diseases such as SSc.

### **2.7. Novel findings**


### **2.8. Future plans**

Angiogenesis impairment in SSc has been proved by several researchers. A number of serum investigations have been carried out regarding this phenomenon, but there are only scant data concerning tears of SSc patients.

The issue why the VEGF levels are not higher in SSc patients than in the healthy group needs further investigation. Other biochemical methods, like PCR, would be feasible to confirm array results. Furthermore, a longer-term prospective study in a larger population with extension of the ophthalmological examinations is needed to confirm clinical utility.

Our current data depict a group of inflammatory mediators, which may play a significant role in ocular pathology of SSc. Monitoring these factors in the tears of patients with SSc can be a noninvasive alternative to serum investigation. Additionally, in patients with ocular manifestations, such as DES, tear analysis is far more informative; it provides information of the ocular surface; hence it could help us choose the appropriate treatment, in particular artificial tears or anti-inflammatory eye drops [36]. Further studies are needed to understand the signaling pathways regulating pro-inflammatory cytokines, with the aim of developing new interventions against autoimmune diseases mediated by cytokines and chemokines, as well as inventing novel therapeutic possibilities for the ocular manifestations of SSc. New inflammatory mediators are to be searched that might function as excellent candidates for future therapeutic targets in SSc with ocular manifestations.

### **Acknowledgements**

cultured dermal fibroblasts from patients with SSc show augmented expressions of MCP-1

Of the last three molecules, IP-10 and MCP-1 are the ones whose molecular characteristics single them out as potential candidates for therapies against the pathological consequences

**1.** After reviewing the literature of tear sampling techniques, we labored the adequate tear sampling methods and collected tears with capillary system from SSc patients in order to

**2.** We were the first to demonstrate the presence and concentration of VEGF, an element that plays an important vascular role in the pathogenesis of SSc, with the help of a method that

**3.** By the help of our survey, which is based on a quantitative sandwich immunoassay technique, we were able to verify a 20% reduction in the VEGF concentration in tears of SSc

**4.** We were the first to establish a wide cytokine profile in tears of SSc patients using an array

**5.** Based on our cytokine array results, we revealed that 9 out of the 102 cytokines and chemokines had significantly higher levels in tears of patients with SSc. All of them are molecular players of the immune responses and the inflammatory processes. These findings legitimate the existence of ocular surface inflammations which are quite frequent in patients with SSc. In addition, they are in accordance with former study results regarding

**6.** By using a highly sensitive and specific multiplex bead assay, we were the first to demonstrate increased levels of IP-10, MCP-1, and CRP in tear samples of patients with SSc. Previous studies have already demonstrated elevated levels of these biomarkers in the sera of these patients; therefore tear analysis is to be raised as a potential means in dealing with

Angiogenesis impairment in SSc has been proved by several researchers. A number of serum investigations have been carried out regarding this phenomenon, but there are only scant

The issue why the VEGF levels are not higher in SSc patients than in the healthy group needs further investigation. Other biochemical methods, like PCR, would be feasible to confirm array results. Furthermore, a longer-term prospective study in a larger population with exten-

diagnostic, prognostic, and maybe even therapeutic challenges of SSc.

sion of the ophthalmological examinations is needed to confirm clinical utility.

mRNA and protein [60].

148 Systemic Sclerosis

of diseases such as SSc.

investigate VEGF molecule and cytokines.

patients compared to healthy controls.

the pathomechanism of SSc.

data concerning tears of SSc patients.

**2.8. Future plans**

that monitors 102 cytokines simultaneously.

is based on a quantitative sandwich immunoassay technique.

**2.7. Novel findings**

Parts of this chapter are reproduced from authors' recent work mentioned in the "Reference" section and properly cited in the body of the chapter (Refs. [34, 36]). Permission from authors/ publishers for the usage of the content of their articles has been obtained.

### **Nomenclatures**



### **Author details**

Aniko Rentka<sup>1</sup> , Krisztina Koroskenyi2 , Jolan Harsfalvi<sup>3</sup> , Zoltan Szekanecz<sup>4</sup> , Gabriella Szucs<sup>4</sup> , Peter Szodoray5 and Adam Kemeny-Beke<sup>1</sup> \*

\*Address all correspondence to: kemenyba@med.unideb.hu

1 Department of Ophthalmology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary

2 Department of Biochemistry and Molecular Biology, Signaling and Apoptosis Research Group, Hungarian Academy of Sciences, Research Center of Molecular Medicine, University of Debrecen, Debrecen, Hungary

3 Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary

4 Department of Rheumatology, Institute of Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary

5 Institute of Immunology, Rikshospitalet, Oslo University Hospital, Oslo, Norway

### **References**


[3] Albert D, Jakobiec, FA. Principles and Practice of Ophthalmology. 5th ed. Philadelphia: W.B.Saunders Company; 2000

PCR Polymerase chain reaction

sIgA Secretory immunoglobulin A

TGF-β Transforming growth factor-beta TNF-α Tumor necrosis factor-alpha

VEGF Vascular endothelial growth factor

, Krisztina Koroskenyi2

and Adam Kemeny-Beke<sup>1</sup>

\*Address all correspondence to: kemenyba@med.unideb.hu

, Jolan Harsfalvi<sup>3</sup>

1 Department of Ophthalmology, Faculty of Medicine, University of Debrecen, Debrecen,

2 Department of Biochemistry and Molecular Biology, Signaling and Apoptosis Research Group, Hungarian Academy of Sciences, Research Center of Molecular Medicine, University

3 Department of Biophysics and Radiation Biology, Semmelweis University, Budapest,

4 Department of Rheumatology, Institute of Medicine, Faculty of Medicine, University of

[1] Gomes Bde A, Santhiago MR, Magalhaes P, Kara-Junior N, Azevedo MN, Moraes HV, Jr. Ocular findings in patients with systemic sclerosis. Clinics (São Paulo, Brazil).

[2] Waszczykowska A, Gos R, Waszczykowska E, Dziankowska-Bartkowiak B, Jurowski P. Prevalence of ocular manifestations in systemic sclerosis patients. Archives of Medical

5 Institute of Immunology, Rikshospitalet, Oslo University Hospital, Oslo, Norway

\*

, Zoltan Szekanecz<sup>4</sup>

, Gabriella Szucs<sup>4</sup>

,

VDBP Vitamin D binding protein

RNP Ribonucleoprotein

SSc Systemic sclerosis ST Stimulated tear STS Schirmer test strip

TRIM Tripartite motif

of Debrecen, Debrecen, Hungary

Debrecen, Debrecen, Hungary

2011;**66**(3):379-385

Science. 2013;**9**(6):1107-1113

WO Washout

**Author details**

Aniko Rentka<sup>1</sup>

150 Systemic Sclerosis

Peter Szodoray5

Hungary

Hungary

**References**

PTF Precorneal tear filmRNARibonucleic acid


[30] Zakaria N, Van Grasdorff S, Wouters K, Rozema J, Koppen C, Lion E, et al. Human tears reveal insights into corneal neovascularization. PLoS One. 2012;**7**(5):e36451

[18] Bjerrum KB, Prause JU. Collection and concentration of tear proteins studied by SDS gel electrophoresis. Presentation of a new method with special reference to dry eye patients. Graefes Archive for Clinical and Experimental Ophthalmology.

[19] Markoulli M, Papas E, Petznick A, Holden B. Validation of the flush method as an alternative to basal or reflex tear collection. Current Eye Research. 2011;**36**(3):198-207

[20] Luo L, Li DQ, Doshi A, Farley W, Corrales RM, Pflugfelder SC. Experimental dry eye stimulates production of inflammatory cytokines and MMP-9 and activates MAPK signaling pathways on the ocular surface. Investigative Ophthalmology & Visual Science.

[21] Song XJ, Li DQ, Farley W, Luo LH, Heuckeroth RO, Milbrandt J, et al. Neurturin-deficient mice develop dry eye and keratoconjunctivitis sicca. Investigative Ophthalmology &

[22] Senchyna M, Wax MB. Quantitative assessment of tear production: A review of methods and utility in dry eye drug discovery. Journal of Ocular Biology, Diseases, and

[23] VanDerMeid KR, Su SP, Krenzer KL, Ward KW, Zhang JZ. A method to extract cytokines and matrix metalloproteinases from Schirmer strips and analyze using Luminex.

[24] Acera A, Rocha G, Vecino E, Lema I, Duran JA. Inflammatory markers in the tears of patients with ocular surface disease. Ophthalmic Research. 2008;**40**(6):315-321

[25] Inic-Kanada A, Nussbaumer A, Montanaro J, Belij S, Schlacher S, Stein E, et al. Comparison of ophthalmic sponges and extraction buffers for quantifying cytokine profiles in tears

[26] Choy CK, Cho P, Chung WY, Benzie IF. Water-soluble antioxidants in human tears: Effect of the collection method. Investigative Ophthalmology & Visual Science.

[27] Green-Church KB, Nichols KK, Kleinholz NM, Zhang L, Nichols JJ. Investigation of the human tear film proteome using multiple proteomic approaches. Molecular Vision.

[28] Lee SY, Kim MJ, Kim MK, Wee WR. Comparative analysis of polymerase chain reaction assay for herpes simplex virus 1 detection in tear. Korean Journal of Ophthalmology.

[29] Satpathy G, Mishra AK, Tandon R, Sharma MK, Sharma A, Nayak N, et al. Evaluation of tear samples for Herpes Simplex Virus 1 (HSV) detection in suspected cases of viral keratitis using PCR assay and conventional laboratory diagnostic tools. The British Journal

using Luminex technology. Molecular Vision. 2012;**18**:2717-2725

1994;**232**(7):402-405

152 Systemic Sclerosis

2004;**45**(12):4293-4301

Informatics. 2008;**1**(1):1-6

2001;**42**(13):3130-3134

2008;**14**:456-470

2013;**27**(5):316-321

of Ophthalmology. 2011;**95**(3):415-418

Visual Science. 2003;**44**(10):4223-4229

Molecular Vision. 2011;**17**:1056-1063


[57] Distler JH, Akhmetshina A, Schett G, Distler O. Monocyte chemoattractant proteins in the pathogenesis of systemic sclerosis. Rheumatology (Oxford). 2009;**48**(2):98-103

[44] Rabquer BJ, Tsou PS, Hou Y, Thirunavukkarasu E, Haines GK, 3rd, Impens AJ, et al. Dysregulated expression of MIG/CXCL9, IP-10/CXCL10 and CXCL16 and their recep-

[45] Chun RF. New perspectives on the vitamin D binding protein. Cell Biochemistry and

[46] Ge L, Trujillo G, Miller EJ, Kew RR. Circulating complexes of the vitamin D binding protein with G-actin induce lung inflammation by targeting endothelial cells.

[47] Muangchan C, Harding S, Khimdas S, Bonner A, Baron M, Pope J. Association of C-reactive protein with high disease activity in systemic sclerosis: results from the Canadian Scleroderma Research Group. Arthritis Care and Research (Hoboken).

[48] Liu X, Mayes MD, Pedroza C, Draeger HT, Gonzalez EB, Harper BE, et al. Does C-reactive protein predict the long-term progression of interstitial lung disease and survival in patients with early systemic sclerosis? Arthritis Care and Research (Hoboken).

[49] Hasegawa M, Fujimoto M, Matsushita T, Hamaguchi Y, Takehara K, Sato S. Serum chemokine and cytokine levels as indicators of disease activity in patients with systemic

[50] Lee EY, Lee ZH, Song YW. CXCL10 and autoimmune diseases. Autoimmunity Reviews.

[51] Neville LF, Mathiak G, Bagasra O. The immunobiology of interferon-gamma inducible protein 10 kD (IP-10): A novel, pleiotropic member of the C-X-C chemokine superfamily.

[52] Liu M, Guo S, Hibbert JM, Jain V, Singh N, Wilson NO, et al. CXCL10/IP-10 in infectious diseases pathogenesis and potential therapeutic implications. Cytokine & Growth Factor

[53] Narumi S, Takeuchi T, Kobayashi Y, Konishi K. Serum levels of ifn-inducible PROTEIN-10 relating to the activity of systemic lupus erythematosus. Cytokine. 2000;**12**(10):1561-1565

[54] Hanaoka R, Kasama T, Muramatsu M, Yajima N, Shiozawa F, Miwa Y, et al. A novel mechanism for the regulation of IFN-gamma inducible protein-10 expression in rheu-

[55] Fujii H, Shimada Y, Hasegawa M, Takehara K, Sato S. Serum levels of a Th1 chemoattractant IP-10 and Th2 chemoattractants, TARC and MDC, are elevated in patients with

[56] Yoshimura T, Yuhki N, Moore SK, Appella E, Lerman MI, Leonard EJ. Human monocyte chemoattractant protein-1 (MCP-1). Full-length cDNA cloning, expression in mitogenstimulated blood mononuclear leukocytes, and sequence similarity to mouse compe-

matoid arthritis. Arthritis Research & Therapy. 2003;**5**(2):R74-R81

tence gene JE. FEBS Letters. 1989;**244**(2):487-493

systemic sclerosis. Journal of Dermatological Science. 2004;**35**(1):43-51

tors in systemic sclerosis. Arthritis Research & Therapy. 2011;**13**(1):R18

Function. 2012;**30**(6):445-456

154 Systemic Sclerosis

2012;**64**(9):1405-1414

2013;**65**(8):1375-1380

2009;**8**(5):379-383

Reviews. 2011;**22**(3):121-130

Immunobiology. 2014;**219**(3):198-207

sclerosis. Clinical Rheumatology. 2011;**30**(2):231-237

Cytokine & Growth Factor Reviews. 1997;**8**(3):207-219


### **Chapter 7**

**Provisional chapter**
