**2.1 Assessment of clinical scoring systems**

In the past, assessing the severity of AD, at levels of either overall or individual parameters, was mostly dependant on clinical scoring systems, such as the Scoring Atopic Dermatitis index (SCORAD)( European Task Force on Atopic Dermatitis, 1993), Atopic Dermatitis Area and Severity Index score(ADASIS) (Bahmer, 1992), Eczema Area and Severity Index (EASI) (Hanifin, 2001), are the most widespread used methods, to name a few(Charman &Williams, 2000).All of these tools, based on the entirely subjective score, have considerable inter-observer variability owing to the observers bias.

ADASIS was based on the lesion areas by point counting. On special body diagrams, the areas were color coded according to the severity of inflammation (green for mild, blue for moderate, and red for severe dermatitis).The result was evaluated by applying a transparent grid and fractionated lesion areas were weighted and multiplied by subjective pruritus intensity score. Haniffin et al suggested EASI index to assess the disease extent and percents of lesion area in four body regions (head and neck, lower limbs, upper limbs and trunk), borrowed from the psoriasis area and severity index (PASI) (Hanifin, 2001). The study by Rullo et al informed that EASI is more suitable for detection of subtle alterations of the severity of AD during drug effect studies, or follow-up of progression for research purposes (Rullo, 2008).

Charman and Williams evaluated 13 AD clinical scoring systems and confirmed that the simple and rapid SCORAD index was the most widely used one and its validity, reliability, sensitivity, and acceptability have been widely confirmed(Charman &Williams, 2000).The

During the past decades about 10 diagnostic criteria for AD have been set up(Brenninkmeijer et al., 2008),while the Hanifin and Rajka diagnostic criteria(in 1980) and Williams diagnostic criteria(in 1994) are the two most widely used methods in the world(Roguedas et al., 2004).The H-R criteria is such a detailed one (4 major and 23 minor criteria) that it is time consuming and not easy to operate, and thus mainly suitable for investigative studies and impractical for epidemiologic studies. What's more, some of the minor features described in the criteria, such as anterior neck folds and the Dennie-Morgan infra-orbital fold, has been challenged for their diagnostic significance in a study on Swiss AD (Mevorah et al., 1988). The concise Williams criteria (1 mandatory and 5 major criteria), are suitable for clinical and epidemiological studies. Williams criteria has been extensively validated(Brenninkmeijer et al., 2008). Due to its inherent nature of easy operation, this diagnostic criterion could be recommended in future international studies. In 1995, International Study of Asthma and Allergies in Childhood (ISAAC) set up ISAAC questionnaire for prevalence survey of childhood AD(Asher et al., 1995).Taken together, an ideal diagnostic criteria need to be well-validated ,easy to apply and suitable for

The methods of measuring disease severity of AD include the evaluation of lesion manifestations in combination with disease extent and intensity, subjective symptoms, quality of life (QOL), and the assessment of skin barrier function by bioengineering methods

In the past, assessing the severity of AD, at levels of either overall or individual parameters, was mostly dependant on clinical scoring systems, such as the Scoring Atopic Dermatitis index (SCORAD)( European Task Force on Atopic Dermatitis, 1993), Atopic Dermatitis Area and Severity Index score(ADASIS) (Bahmer, 1992), Eczema Area and Severity Index (EASI) (Hanifin, 2001), are the most widespread used methods, to name a few(Charman &Williams, 2000).All of these tools, based on the entirely subjective score, have considerable

ADASIS was based on the lesion areas by point counting. On special body diagrams, the areas were color coded according to the severity of inflammation (green for mild, blue for moderate, and red for severe dermatitis).The result was evaluated by applying a transparent grid and fractionated lesion areas were weighted and multiplied by subjective pruritus intensity score. Haniffin et al suggested EASI index to assess the disease extent and percents of lesion area in four body regions (head and neck, lower limbs, upper limbs and trunk), borrowed from the psoriasis area and severity index (PASI) (Hanifin, 2001). The study by Rullo et al informed that EASI is more suitable for detection of subtle alterations of the severity of AD during drug effect studies, or follow-up of progression

Charman and Williams evaluated 13 AD clinical scoring systems and confirmed that the simple and rapid SCORAD index was the most widely used one and its validity, reliability, sensitivity, and acceptability have been widely confirmed(Charman &Williams, 2000).The

**1.2 Diagnostic criteria of AD** 

interventional multicenter trials.

and laboratory parameters.

**2. Assessment of the disease severity in AD** 

**2.1 Assessment of clinical scoring systems** 

inter-observer variability owing to the observers bias.

for research purposes (Rullo, 2008).

SCORAD index includes objective symptoms (extent and intensity) and subjective criteria (daytime pruritus and sleep loss). The severity assessment of each items were evaluated at its average intensity. Due to that the subjective symptoms represented 20 % of total of interobserver variation, European Task Force on Atopic Dermatitis recommended severity scale based only on the objective criteria of the SCORAD index to define mild, moderate, and severe AD (Kunz, 1997). It remains to be investigated that quite some laboratory parameters obtained from the lesion, serum, and other body fluids, have no significant correlation with SCORAD index. SCORAD index can not reflex the whole aspects of AD.

Most of the severity index has been applied for a given short-term therapeutic intervention, while they are insensitive in reflecting the overall disease activity to the chronic and fluctuating course of AD. Nottingham Eczema Severity Score (NESS) was introduced to be a clinical severity score method that documents AD severity over a long -term period of 12 months(Emerson et al., 2000; Hon et al., 2006).

For evaluating the life quality in AD, Quality of Life Index for Atopic Dermatitis (QoLIAD) is designed for adults with AD based on Quality of Life (QoL)and Children's Dermatology Life Quality Index (CDLQI) for children AD. These instruments could be used to evaluate the impact degree of AD and also measure the therapeutic effect (Whalley et al., 2004; Ben-Gashir et al., 2004).

Taken together, though well recognized and widely applied, the implementation of various scoring systems is complicated and subject to a variety of bias. Noninvasive, objective measurement has been developed and in use in recent year. The objective severity assessment of atopic dermatitis system (OSAAD) (Sugarman et al., 2003), which take advantage of instrumentations to measure water content (stratum corneum hydration, SCH), sebum content and trans-epidermal water loss (TEWL) of the skin, features the recent development of objective evaluation of AD.

OSAAD is a severity evaluation scale in AD including TEWL, SCH using a noninvasive bioengineering methods and estimation of affected body surface area (BSA). The method is suitable for both children and adults with AD compared with classical scoring system (Angelova-Fischer et al., 2005). The OSAAD score have several advantages over the clinical scoring methods. Measurement of TEWL and SCH is reproducible and reliable under given standard environment condition and instrument devices(Angelova-Fischer et al., 2005), able to evaluate quantitatively the severity scale of skin barrier impairment, sensitive to minor fluctuations in the course of AD development, and consequently applicable to assessment of treatment effects. The limitations of this technique are the inter- or intra- variability in AD patients, influenced by anatomic sites, environmental changes, etc. So the standardization of the conditions is very important in multicenter trials .There is no unified standard sites suitable for evaluation. Choi et al showed that antecubital fossa is the most favorable site for TEWL evaluation (Choi et al., 2003), while flexure surface is unsmooth. The '2 cm' site under the antecubital fossa resembles a flexure and is conveniently flat for measurements (Hon et al., 2008).

#### **2.2 Evaluation of skin function and properties**

The skin barrier is primarily in the outer 15μm of the epidermis, the stratum corneum (SC) (Landmann, 1988). Breakdown of skin barrier is the primary event in the development of AD, which may result in atopic march (Lipozencic & Wolf, 2007) . The evaluation of skin barrier and properties is based on several variances: such as TEWL, natural moisturizing factors (NMF), skin sebum content, skin pH and skin hydration (SH). The study

Advances in Assessing the Severity of Atopic Dermatitis 173

interpret and operate, particularly in patients sensitive to several kinds of food or potential to anaphylactic Shock. Skin prick test (SPT) is designed for diagnosing food immediate hypersensitivity(Isolauri & Turjanmaa, 1996).The APT reflect delayed-phase allergic reactions, even with a late onset of symptoms (more than 2 hours after food ingestion). APT may provide further diagnostic information in addition to the SPT and serum IgE values used with standardized allergen concentration and vehicle (Niggemann et al., 2005). APT has higher sensitivity than SPT test for food allergy in young children with AD, and can be used to supplement the SPT in diagnosing food allergy in AD children(Stromberg, 2002) and those with late reactions were more easily to have positive APT tests to the relevant foods (Saarinen et al., 2001).The combination of APT, SPT and serum-specific IgE reduce the need for oral food challenges in children with AD (Roehr et al., 2001) and significantly enhance the accuracy in the diagnosis of food allergy when the total IgE is normal or SPT is negative(Boissieu&Dupont,2003). APT test may also help among children with AD to prevent unnecessary restrictive diets (Niggemann et al., 2000).While in a study of allergy to cow's milk and hen's egg in 3 year- old children, no hypersensitivity to cow's milk or hen's egg was predicted by APT alone. The real value of the APT in food allergy in children need

The sensitivity and specificity of the SPT of food allergen was 58% and 70%, while the sensitivity and specificity of the APT for late-phase clinical reactions of food allergen was 76% and 95% respectively (Niggemann et al., 2000). The negative predictive value is over 95%, while the positive predictive value is less than 40% of SPT (Sampson, 2003).So, a positive skin test cannot completely confirm a clinical food hypersensitivity, while a negative result can virtually rules out some kind of IgE-mediated food allergy. It should be noted that SPT may remain positive for many years after the loss of clinical food allergic reactivity. Patients should be re-challenged at intervals to determine whether their food allergy persists (egg, every 2–3 years; milk, soy, wheat, peanut, nuts, fish, and shellfish,

Seeking biomarkers in serum or other body fluid to reflect the severity of AD is an interesting topic. Though the fact that serum IgE level is elevated in more than half of AD has been well established, its correlation with disease severity is disputed, so was the interpretation for peripheral blood eosinophil count, which could be affected by many other factors except for hypersensitivity, such as parasite, drugs, hormone. There were reports that urine eosinophil protein X (Jenerowicz et al., 2006) and leukotriene E4 (Hon et al., 2004) may be used as biomarkers to reflect the severity of AD. Recently, Urinary aquaporin-2 was found to positively correlate with skin dryness of infant AD (Di et al., 2010). The clinical manifestations and distribution pattern of AD may vary at different age-related stages. AD is most prevalent in infants. An objective, easy and non-invasive test for evaluation of infant AD and corresponding management is much expected by

IgE and specific IgE (ELISA or Radioallergosorbent test/RAST) can be used without the need of preparing lesion-free skin and waiting-up of antihistamines drug withdrawal, it is more practical than SPT for the screening of food allergies in most office settings due to its quick and easy- to operate merit. Serum IgE levels are increased in about 80% of adult AD

to be further studied(Osterballe et al., 2004).

every 1–2 years) (Sampson, 2001).

both healthcare workers and parents.

**2.3.2.1 Serum total IgE, specific IgE and IgE autoantibodies** 

**2.3.2 Biomarkers in serum or other body fluid** 

environment (controlled temperature and humidity level) of bioengineering research should be standardized.

From the skin surface, SH is relatively difficult to quantify objectively, instead, SCH level is measured .TEWL is a method to reflex SH, and also correlated well with objective SCORAD score (Gupta et al., 2008).There are two main systems which have been used to measure TEWL by comparing water flux rate following titrated water application: open chamber and closed chamber. The main drawback of open-chamber systems is the disturbance of air movements. The closed-chamber systems conquers this limitation by its closed-chamber, but a major problem with it is that it can not be used to perform continuous measurement, as there is a need of cleaning water vapor of the chamber after a site reading. Elkeeb et al introduced a new closed-chamber systems with a condenser to remove water vapor from chamber and enabling continuous measurement and confirmed significantly correlated with conventional closed and open-chamber systems(Elkeeb et al., 2010) .Later agreed standards of TEWL calibration method is needed to improve the comparability of TEWL measurement devices supplied by different manufacturers.

NMF is the most important factor for maintaining the skin proper moisture levels in SC. NMF largely comes from the acid degradation products of filaggrin, including pyrrolidone carboxylic acid (PCA) and urocanic acid (UCA), which also contribute to skin hydration acid mantle in outer SC (O'Regan et al., 2009) .While lowered expression of FLG in AD lesion was found universally (Howell et al., 2009), the loss-of-function mutations of FLG were observed in only less one-third of the general AD population (Marenholz et al., 2006; Palmer et al., 2006; Zhang et al., 2011; Ma et al., 2010). Reduced NMF levels were independently associated with FLG genotype and is a general feature of AD (Kezic et al., 2010).High-Performance Liquid Chromatograph (HPLC) is the common method used to evaluate NMF (Robinson et al., 2010a; Robinson et al., 2010b ).

The skin surface sebum content, hydration and pH can be check by several commercialized instruments with respective probes on the surface of skin (Choi et al., 2003; Firooz et al., 2007; Man et al., 2009). They are easy to operate, but time consuming, difficult to use in infant AD patients. The temperature and humidity levels during their application also should be standardized.

#### **2.3 Laboratory tests**

Laboratory tests may also be applied for AD.

#### **2.3.1 Skin tests (atopy patch test/ APT, skin prick test /SPT)**

APT was traditionally used to assess sensitization to inhalant. Allergens/aeroallergen as a skin test, was used to diagnose AD (Ring et al., 1997).An eczematous reaction based on T cell–specific delayed-type allergic response to the allergens on the healthy skin of the patient's back or forearm is read after 48 and 72 hours. There is no golden standard for aeroallergen provocation of AD. Although the kinds of allergens are different in patients of different environment, race, and regions, the European APT model with standardization of allergen concentration and vehicle may be an useful diagnostic tool to AD patients. However, the clinical relevance of positive APT reactions depends on standardized provocation and avoidance testing verification (Lipozencic & Wolf, 2010).

Although the double-blind, placebo-controlled oral food challenges (DBPCFC) is the golden standard of diagnosis in food allergy(Sampson, 2003).The test is not always suitable to

environment (controlled temperature and humidity level) of bioengineering research

From the skin surface, SH is relatively difficult to quantify objectively, instead, SCH level is measured .TEWL is a method to reflex SH, and also correlated well with objective SCORAD score (Gupta et al., 2008).There are two main systems which have been used to measure TEWL by comparing water flux rate following titrated water application: open chamber and closed chamber. The main drawback of open-chamber systems is the disturbance of air movements. The closed-chamber systems conquers this limitation by its closed-chamber, but a major problem with it is that it can not be used to perform continuous measurement, as there is a need of cleaning water vapor of the chamber after a site reading. Elkeeb et al introduced a new closed-chamber systems with a condenser to remove water vapor from chamber and enabling continuous measurement and confirmed significantly correlated with conventional closed and open-chamber systems(Elkeeb et al., 2010) .Later agreed standards of TEWL calibration method is needed to improve the comparability of TEWL measurement

NMF is the most important factor for maintaining the skin proper moisture levels in SC. NMF largely comes from the acid degradation products of filaggrin, including pyrrolidone carboxylic acid (PCA) and urocanic acid (UCA), which also contribute to skin hydration acid mantle in outer SC (O'Regan et al., 2009) .While lowered expression of FLG in AD lesion was found universally (Howell et al., 2009), the loss-of-function mutations of FLG were observed in only less one-third of the general AD population (Marenholz et al., 2006; Palmer et al., 2006; Zhang et al., 2011; Ma et al., 2010). Reduced NMF levels were independently associated with FLG genotype and is a general feature of AD (Kezic et al., 2010).High-Performance Liquid Chromatograph (HPLC) is the common method used to evaluate NMF

The skin surface sebum content, hydration and pH can be check by several commercialized instruments with respective probes on the surface of skin (Choi et al., 2003; Firooz et al., 2007; Man et al., 2009). They are easy to operate, but time consuming, difficult to use in infant AD patients. The temperature and humidity levels during their application also

APT was traditionally used to assess sensitization to inhalant. Allergens/aeroallergen as a skin test, was used to diagnose AD (Ring et al., 1997).An eczematous reaction based on T cell–specific delayed-type allergic response to the allergens on the healthy skin of the patient's back or forearm is read after 48 and 72 hours. There is no golden standard for aeroallergen provocation of AD. Although the kinds of allergens are different in patients of different environment, race, and regions, the European APT model with standardization of allergen concentration and vehicle may be an useful diagnostic tool to AD patients. However, the clinical relevance of positive APT reactions depends on standardized

Although the double-blind, placebo-controlled oral food challenges (DBPCFC) is the golden standard of diagnosis in food allergy(Sampson, 2003).The test is not always suitable to

should be standardized.

devices supplied by different manufacturers.

(Robinson et al., 2010a; Robinson et al., 2010b ).

Laboratory tests may also be applied for AD.

**2.3.1 Skin tests (atopy patch test/ APT, skin prick test /SPT)** 

provocation and avoidance testing verification (Lipozencic & Wolf, 2010).

should be standardized.

**2.3 Laboratory tests** 

interpret and operate, particularly in patients sensitive to several kinds of food or potential to anaphylactic Shock. Skin prick test (SPT) is designed for diagnosing food immediate hypersensitivity(Isolauri & Turjanmaa, 1996).The APT reflect delayed-phase allergic reactions, even with a late onset of symptoms (more than 2 hours after food ingestion). APT may provide further diagnostic information in addition to the SPT and serum IgE values used with standardized allergen concentration and vehicle (Niggemann et al., 2005). APT has higher sensitivity than SPT test for food allergy in young children with AD, and can be used to supplement the SPT in diagnosing food allergy in AD children(Stromberg, 2002) and those with late reactions were more easily to have positive APT tests to the relevant foods (Saarinen et al., 2001).The combination of APT, SPT and serum-specific IgE reduce the need for oral food challenges in children with AD (Roehr et al., 2001) and significantly enhance the accuracy in the diagnosis of food allergy when the total IgE is normal or SPT is negative(Boissieu&Dupont,2003). APT test may also help among children with AD to prevent unnecessary restrictive diets (Niggemann et al., 2000).While in a study of allergy to cow's milk and hen's egg in 3 year- old children, no hypersensitivity to cow's milk or hen's egg was predicted by APT alone. The real value of the APT in food allergy in children need to be further studied(Osterballe et al., 2004).

The sensitivity and specificity of the SPT of food allergen was 58% and 70%, while the sensitivity and specificity of the APT for late-phase clinical reactions of food allergen was 76% and 95% respectively (Niggemann et al., 2000). The negative predictive value is over 95%, while the positive predictive value is less than 40% of SPT (Sampson, 2003).So, a positive skin test cannot completely confirm a clinical food hypersensitivity, while a negative result can virtually rules out some kind of IgE-mediated food allergy. It should be noted that SPT may remain positive for many years after the loss of clinical food allergic reactivity. Patients should be re-challenged at intervals to determine whether their food allergy persists (egg, every 2–3 years; milk, soy, wheat, peanut, nuts, fish, and shellfish, every 1–2 years) (Sampson, 2001).

#### **2.3.2 Biomarkers in serum or other body fluid**

Seeking biomarkers in serum or other body fluid to reflect the severity of AD is an interesting topic. Though the fact that serum IgE level is elevated in more than half of AD has been well established, its correlation with disease severity is disputed, so was the interpretation for peripheral blood eosinophil count, which could be affected by many other factors except for hypersensitivity, such as parasite, drugs, hormone. There were reports that urine eosinophil protein X (Jenerowicz et al., 2006) and leukotriene E4 (Hon et al., 2004) may be used as biomarkers to reflect the severity of AD. Recently, Urinary aquaporin-2 was found to positively correlate with skin dryness of infant AD (Di et al., 2010). The clinical manifestations and distribution pattern of AD may vary at different age-related stages. AD is most prevalent in infants. An objective, easy and non-invasive test for evaluation of infant AD and corresponding management is much expected by both healthcare workers and parents.

#### **2.3.2.1 Serum total IgE, specific IgE and IgE autoantibodies**

IgE and specific IgE (ELISA or Radioallergosorbent test/RAST) can be used without the need of preparing lesion-free skin and waiting-up of antihistamines drug withdrawal, it is more practical than SPT for the screening of food allergies in most office settings due to its quick and easy- to operate merit. Serum IgE levels are increased in about 80% of adult AD

Advances in Assessing the Severity of Atopic Dermatitis 175

by Hayashida et al indicated that FoxP3+Treg subsets was similar to that of normal controls in peripheral blood of the acute phase AD and the decreased number of circulating Th17 cells is negatively correlated with CCL17, IgE and eosinophil levels in AD patients (Hayashida et al., 2011). IL-17 has been identified in acute AD lesions (Toda

A complex network of cytokines and chemokines are involved in atopic inflammation, at both the starting and maintainance stages of the inflammation. Scratching injury induce the production of proinflammatory cytokines (such as IL-1, TNF-α, GM-CSF), which in turn induce CCL27 and CCL17 development. Subsequently, CCL27 and CCL17 recruit skinhoming memory T cells into the skin. Within the skin, T cells are activated and release effector cytokines (eg, IL-4, IL-5, IL-13 in acute phase lesion, or IFN-r in the chronic lesion of AD ).These effector cytokines will sustain and amplify the production of chemokines within atopic skin. In this complex procedure, many chemokines, (such as CCL1, CCL2, CCL11, CCL13, CCL18, CCL20, CCL21, and CCL26) interact with various immune effector cells through their receptors(D.Y.Leung et al., 2004; Homey et al., 2006). AD is a disease with the participation of whole body immune system, and that the immunologic abnormality is not limited to the skin. Generally, as is widely accepted that AD has been considered the paradigm of a Th2–mediated disease, characterized by increase Th2 and decrease Th1 and cytokines level, elevated serum IgE levels, eosinophilia in peripheral blood. Recent researched showed that the severity of AD has also been found to be associated with the levels of various other cytokines and chemokines in the lesion and serum, in addition to the conventional serum parameters, such as total serum IgE (tIgE), eosinophilic cationic protein (ECP), increased T helper type 2 (Th2)-skewed cytokine patterns(IL4,13) and decreased IFNγ. Some of them in the serum of AD are correlated with disease severity, and thus may be new biomarkers to reflect AD disease severity. Clinical trials on interleukins and chemokines in the serum of patients with AD were summarized, in table1 and table 2.

Many interleukins participate in AD and it is a consensus that increased IL4, 13 levels are seen in the serum of AD. Recently, many of other interleukins have been shown to play roles in the pathogenesis of AD, and increased serum levels have been detected (in table1). IL-10 is a powerful Th2 cytokines produced by LC in the lesion of AD and exerts its function through inhibition of the secretion of Th1 cytokines. While, the level of IL-10 in the serum may be not a significant marker (Shin et al., 2005). IL-22, a member of the IL-10 family and known to be preferentially produced by Th17 cells, is increased and has significant correlation with CCL17 levels in the serum of AD patients (Hayashida et al., 2011). On the contrary, IL-12 is prominently expressed in the chronic lesion of AD, it is a powerful inducer of Th0 to Th1 conversion and subsequent IFN-γ secretion from Th1.The level of IL-12 in the serum of AD is controversially reported. Piancatelli et al confirmed the serum IL-12 levels increased in paediatric AD (Piancatelli et al., 2008), while the study by Aral, M et al showed that there was no statistically significant difference between children AD and controls in respect of serum levels of IL-12(Aral et al., 2006). IL-16 is a natural ligand of CD4 molecules. Besides its chemotactic properties to CD4-expressing cells, IL-16 amplifies inflammatory processes and possesses immunoregulatory functions (Mathy et al., 2000; Nagy et al., 2011). IL-16 was found to be increased in the serum of AD, both in child and adult patients (Nagy

et al., 2003). The exact role of Treg and Th17 cell in AD is still unclear.

**2.3.2.4 Cytokines and chemokine in the serum** 

**Interleukin/IL** 

(C.A.Akdis et al., 2006). The remaining 20%–30% of patients exhibiting normal serum IgE levels and lack allergen-specific sensitization, are classified as intrinsic AD (IAD) (Novak &Bieber, 2003; Boguniewicz et al., 2006). 28% of AD serum contains IgE autoantibodies which target keratinocytes in AD patients (Altrichter et al., 2008).

For the total IgE and specific IgE, similar to skin tests, a negative result is fairly reliable in ruling out an IgE-mediated reaction. For a positive result, although the lesions significantly regress, there maybe no parallel decrease of serum IgE level. Further large-scale investigations of serum IgE levels between patients with extrinsic atopic dermatitis and intrinsic atopic dermatitis are needed (Ott et al., 2009).
