Epicutaneous Patch Testing Research Articles

Value Proposition of Epicutaneous Patch Testing: A Win-Win for the Patient and the Medical System

Value Proposition of Epicutaneous Patch Testing: A Win-Win for the Patient and the Medical System

A cross-sectional review of contact allergens in popular self-tanning products

Background: In recent years, self-tanners have become a well-liked alternative to sun tanning and tanning bed usage, as strikingly similar results can be achieved without the harmful side effects of ultraviolet exposure. Objective: The aim of this study is to investigate the presence and prevalence of potential allergens in the most popular self-tanning products. Methods: Five major retailers in the United States were evaluated, from which 17 different brands and 44 unique self-tanning products were analyzed. The ingredients in each self-tanning product were compared with 80 and 36 notable contact allergens taken from the North American Contact Dermatitis Group and Food and Drug Administration–approved T.R.U.E (Thin-Layer Rapid Use Epicutaneous Patch Test), respectively. Results: We found that contact allergens are frequently present in self-tanning products; allergens especially common are propylene glycol, linalool, polysorbate, d-limonene, benzyl alcohol, tocopherol (vitamin E), fragrances, and other scented botanicals. On average, each self-tanner we analyzed contained 11.86 allergens. Limitations: The limitation is that commercial names could not be eliminated from the analysis, introducing potential bias. Conclusion: While self-tanning products are a safer alternative to tanning bed use or sunbathing, consumers and clinicians alike must be aware that they may cause an allergic reaction of the skin for some users.

Patch test in the diagnosis of food allergy

Food allergens are capable of producing adverse reactions through multiple mechanisms of an allergic or non-allergic nature, and through different routes of exposure; generally by ingestion or contact, as in protein contact dermatitis or contact urticaria, including inhalation. Food allergy reactions, in turn, can be mediated by immediate hypersensitivity mechanisms, delayed hypersensitivity or mixed immediate-delayed mechanisms. The reference diagnostic method in food allergy is the double-blind placebo-controlled food challenge test (DBPCFC), but skin and serological tests are important in the clinical context. The diagnosis of immediate food allergy depends on well-standardized allergological tests, such as the skin prick test (SPT) or specific IgE dosing, which are ideally tested by food challenge testing. However, the diagnosis of delayed mechanism food allergy and mixed allergies, which combine both immune mechanisms, is more complex. Delayed hypersensitivity reactions are evaluated with the epicutaneous patch test, or patch testing, for the diagnosis of contact dermatitis. The atopy patch test is initially used for the investigation of inflammatory reactions, which may be linked to food allergens in patients with atopic dermatitis. It was later applied in other diseases, whose pathogenesis is mainly mediated by a mechanism of delayed hypersensitivity to protein allergens: eosinophilic esophagitis, enterocolitis induced by food proteins, protein contact dermatitis, contact urticaria, among other disorders.

Contact Allergy in Atopic Patients

Summary Atopic dermatitis (AD) is a chronic inflammatory disease based on genetic and immune alterations and is part of the atopic symptom complex, including allergic rhinitis, allergic conjunctivitis, and bronchial asthma. A disturbed barrier function facilitates antigen penetration through the skin, with the subsequent development of allergic contact dermatitis (ACD). The gold standard for diagnosing ACD is epicutaneous (patch) testing, also applied to objectify contact sensitization in AD. This study aimed to determine the frequency of contact allergy (CA) among individuals with atopic history and the allergens that caused ACD in those cases. We studied 453 individuals tested in the period 2009-2022. Of these, a subpopulation of 189 individuals with atopic diathesis was identified. A retrospective analysis was used. Using clinical and allergological methods, we divided the tested patients according to sex, age, professional occupation, and areas of the body affected by dermatitis and identified the most common contact allergens that cause positive reactions and ACD. In conclusion, our results highlight the possibility of developing ACD in people with atopic diathesis. As far as we know, our study is the first one in Bulgaria to examine the frequency of contact sensitization in AD patients.

Cutaneous adverse drug reactions

Cutaneous adverse drug reactions (ADRs) represent a heterogeneous field including various clinical patterns without specific features suggesting drug causality. Maculopapular exanthema and urticaria are the most common types of cutaneous ADR. Serious cutaneous ADRs, which may cause permanent sequelae or have fatal outcome, may represent 2% of all cutaneous ADR and must be quickly identified to guide their management. These serious reactions include bullous manifestations (epidermal necrolysis i.e. Stevens-Johnson syndrome and toxic epidermal necrolysis), drug reaction with eosinophilia and systemic symptoms (DRESS) and acute generalized exanthematous pustulosis (AGEP). Some risk factors for developing cutaneous ADRs have been identified, including immunosuppression, autoimmunity or genetic variants. All drugs can cause cutaneous ADRs, the most commonly implicated being antibiotics (especially aminopenicillins and sulfonamides), anticonvulsants, allopurinol, antineoplastic drugs, non-steroidal anti-inflammatory drugs and iodinated contrast media. Pathophysiology is related to immediate or delayed "idiosyncratic" immunologic mechanisms, i.e., usually not related to dose, and pharmacologic/toxic mechanisms, commonly dose-dependent and/or time-dependent. If an immuno-allergic mechanism is suspected, allergological explorations (including epicutaneous patch testing and/or intradermal test) are often possible to clarify drug causality, however these have a variable sensitivity according to the drug and to the ADR type. No in vivo or in vitro test can consistently confirm the drug causality. To determine the origin of a rash, a logical approach based on clinical characteristics, chronologic factors and elimination of differential diagnosis (especially infectious etiologies) is required, completed with a literature search. Reporting to pharmacovigilance system is therefore essential both to analyze drug causality at individual level, and to contribute to knowledge of the drug at population level, especially for serious cutaneous ADRs or in cases involving newly marketed drugs.

Detecting Skin Reactions in Epicutaneous Patch Testing with Deep Learning: An Evaluation of Pre-Processing and Modality Performance.

Epicutaneous patch testing is a well-established diagnostic method for identifying substances that may cause Allergic Contact Dermatitis (ACD), a common skin condition caused by exposure to environmental allergens. While the patch test remains the gold standard for identifying allergens, it is prone to observer bias and consumes valuable human resources. Deep learning models can be employed to address this challenge. In this study, we collected a dataset of 1579 multi-modal skin images from 200 patients using the Antera 3D® camera. We then investigated the feasibility of using a deep learning classifier for automating the identification of the allergens causing ACD. We propose a deep learning approach that utilizes a context-retaining pre-processing technique to improve the accuracy of the classifier. In addition, we find promise in the combination of the color image and false-color map of hemoglobin concentration to improve diagnostic accuracy. Our results showed that this approach can potentially achieve more than 86% recall and 94% specificity in identifying skin reactions, and contribute to faster and more accurate diagnosis while reducing clinician workload.

Dermatological adverse effects of hair dye use: A narrative review.

Hair dyeing is a popular practice dating back to ancient Egyptian times. Initially, hair dye use was restricted to concealing grey and white hairs of the elderly population. However, in recent times, its use is common among the younger generation as a fashion statement. Hair dye contact dermatitis is a common dermatological condition encountered by dermatologists. It is a delayed type of hypersensitivity reaction that commonly affects the scalp and the vicinity of hair line and neck. Para-phenylenediamine (PPD), a synthetic aromatic amine is the most common allergen specifically implicated in hair dye contact dermatitis. Para-phenylenediamine was announced as the allergen of the year in 2006 by the American Contact Dermatitis Society. Contact allergy to para-phenylenediamine can occur in 0.1-2.3% of the general population. Epicutaneous patch testing is the gold standard test for the diagnosis of hair dye contact dermatitis. However, para-phenylenediamine carries a risk of cross-sensitivity and co-sensitization to other allergens. Apart from contact dermatitis, hair dye use is also associated with various other cutaneous adverse effects such as pigmentary changes, hair loss, skin malignancies and autoimmune disorders. Due to the various adverse effects associated with hair dye use, it is prudent to look for safer alternatives to allergenic hair dyes. In this article, we review the epidemiology, cutaneous and systemic adverse effects associated with hair dye use, patch testing, preventive strategies to minimize the risk of hair dye contact dermatitis, and treatment aspects.

What should be considered during epicutaneous patch testing?

AbstractIndications for epicutaneous patch testing include the detection or exclusion of allergic contact dermatitis of the skin, transitional mucosa or oral mucosa, suspected delayed-type immune reactions to ingredients of implants, and certain drug reactions (maculopapular exanthema, symmetrical drug-related intertriginous and flexural exanthema [SDRIFE], acute generalized exanthematous pustulosis [AGEP], and fixed drug eruption). When available, allergen preparations that have been pharmaceutically tested and that are approved or marketable as medicinal products should be used. Existing diagnostic gaps can be closed by testing the patient’s own materials in a suitable preparation. Interferences of ultraviolet (UV) light exposure or drugs with patch test reactions have to be considered. In addition to the reading after 48 h and 72 h, a further reading between day 7 (168 h) and day 10 (240 h) is useful, since about 15% of the positive test reactions remain undetected without this late reading. All positive patch test reactions considered allergic must be evaluated with regard to their clinical relevance based on existing exposures.

Screening for Allergic Contact Dermatitis Based on Pattern of Patch Test Results: A Retrospective Study

Abstract Background: Contact allergies are associated with morbidity worldwide. Purpose: To investigate the patterns of sensitization to contact allergens in a local population. Methods: A retrospective study investigated patch test reactivity among patients with a clinical diagnosis of contact dermatitis referred to the dermatology clinic at King Fahad Armed Forces Hospital, Jeddah, between January 2021 and January 2023. Pregnant women, immunosuppressed patients, and those receiving immunosuppressive therapy were excluded. Only 92 of 101 referred patients underwent patch testing after fulfilling requirements. The patch test was performed using a Thin-layer Rapid-Use Epicutaneous patch test containing 35 contact allergenic elements and one control (total = 36). Results: Among 92 patients, 35 (38%) were males, and 57 (62%) were females. Of these, 62 (67.4%) patients had a positive patch test and reacted to one or more allergens. The most common sensitizing allergens were p-phenylenediamine (PPD), gold sodium thiosulfate, and disperse blue 106, with equal positive reactions in 16 (25.8%) patients, followed by nickel sulfate in 13 (21%), p-tert-butylphenol formaldehyde resin in eight (13%), fragrance mix in 7 (10.8%), and epoxy resin in six (9.2%) patients. No significant sex differences were found among patients with contact dermatitis where notable patch test allergen reactivity was observed. Conclusion: Patch test reactivity to PPD, gold sodium thiosulfate, and disperse blue 106 was high. The pattern of contact allergy observed here highlights the need for large-scale investigations to identify local allergens for contact allergy and formulate policies for avoiding exposure.

THE "FLARE-UP PHENOMENON": NEW ONSET OF DISTANT DERMATITIS DURING PATCH TESTING

<h3>Introduction</h3> Allergic contact dermatitis is common, affecting ∼20% of adults, and is often evaluated via diagnostic patch testing.[1] In rare cases, patch testing may result in worsening of pre-existing dermatitis, but development of de novo lesions is unusual.[2] <h3>Case Description</h3> In 2018, a 46-year-old woman experienced severe, bilateral eyelid swelling with intense itching and skin cracking, along with an eczematous rash on her chest and hands. Inpatient workup revealed no infectious/rheumatologic cause, and patient was diagnosed with contact dermatitis from an unknown trigger. After a period of remission, in 2021 patient again developed bilateral eyelid rash without involvement of the hands or chest. Following treatment with topical steroids, eyelid rash resolved. Epicutaneous patch testing was performed (T.R.U.E. TEST). 36h after patch test placement, patient experienced, rapid intensification of eczematous lesions around the eyelids, as well as new onset of rash on the dorsa of her hands, which had been unaffected prior to patch testing (Figure 1). 72h read was positive to colophony and fragrance mix (+1). Systemic and topical corticosteroids were prescribed for presumed flare-up phenomenon, with subsequent resolution of periorbital and hand rash. <h3>Discussion</h3> While generally well-tolerated, diagnostic patch testing has been associated with the "flare-up" phenomenon, worsening pre-existing eczema—and, in this case, leading to the development of de novo lesions.[3] Flare-up is more common in polysensitized patients, as was seen in our case.[2] The mechanism of the "flare-up phenomenon" is poorly understood, but is believed to involve reactivation of memory T cells at previous sites of disease.[3]

No Clinical or Radiographic Differences Between Cemented Cobalt-Chromium and Titanium-Niobium Nitride Mobile-Bearing Unicompartmental Knee Arthroplasty.

This study aimed to compare the clinical and radiographic outcomes of patients with positive patch tests undergoing a medial mobile-bearing titanium-niobium nitride (TiNbN) unicompartmental knee arthroplasty (UKA) to patients undergoing standard UKA (cobalt-chromium [CoCr] implants). Two successive groups of patients, amounting to a total of 246 individuals, who received Oxford (Zimmer-Biomet, Warsaw, Indiana, USA) UKA were included. The first group was composed of a series of 203 consecutive standard CoCr UKAs (Standard Group), while the second group comprised 43 consecutive hypoallergenic TiNbN UKAs (HA group). The patients of the second group had a positive epicutaneous patch test result for metals. Each patient was evaluated using the Oxford Knee Score (OKS) and Knee Society Score (KSS) a day prior to the surgery (T 0) and at two consecutive follow-ups, namely T 1 (minimum follow-up of 12months) and T 2 (minimum follow-up of 34months). Radiographic measurements were performed at the final follow-up (T 2). No statistical differences were noted between the two groups regarding demographic data (p > 0.05). No clinical or radiographic differences were found between the HA and standard groups at any follow-up (p > 0.05). A statistically significant improvement was found at any follow-up for both OKS and KSS (p < 0.05). No clinical or radiographic differences between the hypoallergenic and standard cobalt-chromium groups at any follow-up were found, with a clinically significant improvement being experienced by both groups during the entire follow-up. Level II-comparative prospective study. The online version contains supplementary material available at 10.1007/s43465-021-00486-3.

Evaluating the inclusiveness of common allergens in expanded series patch tests for children in the pediatric allergic contact dermatitis registry

Evaluating the inclusiveness of common allergens in expanded series patch tests for children in the pediatric allergic contact dermatitis registry

Automated detection of skin reactions in epicutaneous patch testing using machine learning

Patch testing is the diagnostic gold standard to identify causes of allergic contact dermatitis (ACD); however, the paucity of specialized patch testing clinics along with the need for multiple clinic visits can be burdensome for patients and healthcare systems. Automated classification of allergic reactions from photographs offers the opportunity to reduce patient, physician, and clinic time and improve access to care.

Epicutaneous Patch Testing in Type IV Allergy Diagnostics: State of the Art and Best Practice Recommendations.

This chapter summarises all relevant aspects of patch testing, closely following recommendations outlined in a recent European, and a German S3 guideline on diagnostic patch testing with contact allergens and medicinal products (drugs). Patch testing is indicated in patients suspected of suffering, or having been suffering, from delayed-type hypersensitivity leading to allergic contact dermatitis or other skin and mucosal diseases. Sections of this chapter include detailed indications, reasons for possibly postponing the test, considerations on choosing haptens (contact allergens) to test, various aspects of the application of patch test allergen preparations (storage, dosing) and of testing with individual materials provided by the patients. Special aspects of patch testing in cutaneous adverse drug reactions, children, or occupational contact dermatitis are outlined. Supplemental test methods, notably the repeated open application test, are briefly described. Finally, the final evaluation in terms of assessment of clinical relevance of reactions and patient counselling are outlined.

Dermatite de Contato Alérgica aos (Meta)Acrilatos- estudo retrospetivo de sete anos num Hospital Público Português

Introduction Acrylates are salts or esters of the acrylic acid, widely used as part of cosmetic or dental products, surgical equipment, medical devices, household items, building materials and printing inks. Allergic contact dermatitis (ACD) caused by (metha)acrylates could be described in occupational settings, mainly in beauticians using acrylic manicure and, specifically, the photo-bonded long-lasting nail polish. The use of gloves, fingertips made of Polyethylene/ Ethylvinyl-alcohol, mask, safety glasses and an appropriate uniform are recommended in these cases. Materials and methods We present a retrospective study of ACD to acrylates diagnosed at an Hospital in Braga (Portugal), between January 2012 and March 2018. In all cases the epicutaneous patch tests used included the Portuguese Contact Dermatitis Research Group baseline series and an extended series of (meth)acrylates. Results A total of 39 female patients had a sensitization to (metha)acrylates, aged between 21-66 years, with personal history of atopic eczema and allergic rhinitis in 23 and 26% of cases, respectively. The most frequent presentation was hand eczema (87%). Dorsal and palms were affected in an equal percentage of cases (38%) and nail dystrophy in 8% of patients. Ectopic lesions on the face were detected in 33% of cases, mostly with the involvement of the eyelids. Occupational related exposure due to (metha)acrylates was found in 69% of cases, mostly of them beauticians/ artificial nail technicians. The most common sensitizers among the 39 allergic individuals were 2-hydroxyethyl methacrylate (HEMA; positive in 97% of cases) and ethyleneglycol dimethacrylate (positive in 69% of cases). Conclusion In our population, we highlight the high number of cases of ACD from (meth)acrylates in relation to artificial nails. Concerning to the health and safety of workers, it certainly deserves better preventive measures at the occupational level and specific regulation from the authorities concerned with the safety of consumers. HEMA was the most frequent positive allergen (above 90% of tests) and, in agreement with previous studies, can be considered a good screening allergen.

The effect of a low-nickel diet and nickel sensitization on gastroesophageal reflux disease: A pilot study.

Gastroesophageal reflux disease (GERD) is a common medical condition, frequently refractory to medical therapy. Nickel is a leading cause of allergic contact dermatitis. Although nickel is widely found in foods, the effect of nickel on GERD is unknown. This pilot study sought to evaluate the effect of a low-nickel diet on GERD and determine if epicutaneous patch testing to nickel could predict responsiveness to a low-nickel diet. This prospective, single-site pilot study recruited 20 refractory GERD patients as determined by GERD Health-Related Quality of Life (GERD-HRQL) scores. All patients had epicutaneous patch testing for nickel and were then instructed to follow a low-nickel diet for 8weeks regardless of patch test results. GERD-HRQL was recorded at baseline and following 8weeks of a low-nickel diet. Demographic and clinical data associated with GERD and nickel allergy were recorded. A Wilcoxon signed-rank test and nonparametric analysis of longitudinal data were run to determine statistical significance in pre-and post- GERD-HRQL scores in nickel patch test-positive and negative groups. Nearly all (19/20 [95%]) participants reported reduced GERD symptoms after 8weeks on a low-nickel diet. Mean total GERD-HRQL, regurgitation, and heartburn scores declined (27.05 ± 16.04, 11.45 ± 6.46, 10.85 ± 8.29). Participants with positive vs. negative patch testing to nickel responded equivalently to a low-nickel diet. A low-nickel diet improves GERD symptoms, but responsiveness to a low-nickel diet does not correlate with epicutaneous patch testing to nickel. ClinicalTrials.gov number: NCT03720756.

A discrete subset of epigenetically primed human NK cells mediates antigen-specific immune responses.

Adaptive features of natural killer (NK) cells have been reported in various species with different underlying mechanisms. It is unclear, however, which NK cell populations are capable of mounting antigen-specific recall responses and how such functions are regulated at the molecular level. Here, we identify and characterize a discrete population of CD49a+CD16- NK cells in the human liver that displays increased epigenetic potential to elicit memory responses and has the functional properties to exert antigen-specific immunity in the skin as an effector site. Integrated chromatin-based epigenetic and transcriptomic profiling revealed unique characteristics of hepatic CD49a+CD16- NK cells when compared with conventional CD49a-CD16+ NK cells, thereby defining active genomic regions and molecules underpinning distinct NK cell reactivity. In contrast to conventional NK cells, our results suggest that adaptive CD49a+CD16- NK cells are able to bypass the KIR receptor-ligand system upon antigen-specific stimulation. Furthermore, these cells were highly migratory toward chemokine gradients expressed in epicutaneous patch test lesions as an effector site of adaptive immune responses in the skin. These results define pathways operative in human antigen-specific adaptive NK cells and provide a roadmap for harnessing this NK cell subset for specific therapeutic or prophylactic vaccine strategies.

Symptoms of titanium and nickel allergic sensitization in orthodontic treatment

AimThe study aimed to evaluate to which extent self-reported symptomatology, age, and sex are predictors of titanium and nickel allergic sensitization in patients in treatment with fixed orthodontic appliances.MethodsThe study analyzed 228 subjects aged 11–45 years (median 18, interquartile range 16–22); 68% of them were females, and 52% were adolescents. The allergic sensitization testing included epicutaneous patch test to titanium, titanium dioxide, titanium oxalate, titanium nitride, and nickel sulfate. The questionnaire on symptoms potentially linked to titanium and nickel sensitization was used.ResultsPrevalence of the allergic sensitization to titanium in patients undergoing orthodontic treatment was 4% (2% only to titanium without nickel) while to nickel 14% (12% nickel without titanium). Hypersensitivity to both metals at the same time was present in 2% of subjects. Sensitization to nickel was more common in females than in males (17 vs. 8%) and much more common in adults than in adolescents with small effect size (20 vs. 8%; p = 0.013). Sensitization to titanium was more common in females than in males (6 vs. 1%) with no difference in age. Multiple logistic regression analysis revealed that adult age increases the odds for being sensitized to nickel for 2.4 × (95% CI 1.1–5.6; p = 0.044) while watery eyes for 3.7 × (95% CI 1.2–11.1; p = 0.022). None of the symptoms were significant predictors of titanium sensitization.ConclusionAllergic sensitization to titanium and nickel are not very frequent in orthodontic patients, and self-reported symptomatology is a weak predictor of those sensitizations.

Adapting epicutaneous patch testing protocols to assess immediate-type skin reactions.

During the development of cosmetic formulations, in vitro and in vivo methods are essential tools used to reliably assess the skin irritation potential of a product or ingredient. Epicutaneous patch testing (single and/or multiple application protocols) has long been used as an initial in vivo method to screen for possible skin irritation properties of a substance or formulation. To confirm the mildness and dermatological and/or consumer acceptance of a product, use tests are often subsequently conducted. A study was therefore initiated to see how well patch test results correlate with use tests with respect to irritation elicited by skincare (leave-on) products. A number of different cosmetic formulations were assessed in both tests. Although the patch test results did not indicate substantial irritation potentials, immediate-type reactions (stinging and redness) were observed in some volunteers which disappeared within approx. 1h. Although transient, these reactions suggested that consumer acceptance would probably be low and the studies were discontinued. Immediate-type reactions are rare but have been described for some substances used in cosmetics. These unexpected results were nevertheless intriguing and prompted the start of a journey to see if patch test protocols could be modified to assess these reactions. An occlusive short-term patch test protocol with an application period of 20min was developed. Successful identification of the spontaneous reactions became possible. Furthermore, there was a correlation between the intensity of reactions observed in the short-term patch test and those observed in the controlled in-use studies. Short-term patch testing using the developed protocol can therefore reliably be used as a screening method, for example in the development and optimization of cosmetic formulations containing ingredients that could cause spontaneous reactions, for instance of non-immunological contact urticaria type. The lessons learned from this studies indicate that simple modifications of existing test protocols can lead to important insights into skin reactions. These modifications can then be used to create further building blocks in the development and optimization of test strategies for cosmetic formulations which offer reliable study designs for possible reactions product developers may encounter.

Optoacoustic mesoscopy shows potential to increase accuracy of allergy patch testing

Differentiation between irritant and allergic skin reactions in epicutaneous patch testing is based largely on subjective clinical criteria, with the risk of high intraobserver and interobserver variability. Novel dermatological imaging using optoacoustic mesoscopy allows quantitative three-dimensional assessment of microvascular biomarkers. We investigated the potential of optoacoustic imaging to improve the precision of patch test evaluation. Sixty-nine test reactions and 48 healthy skin sections in 52 patients with suspected type IV allergy were examined using raster-scan optoacoustic mesoscopy. We identified biomarkers from the optoacoustic images. Allergic reactions were associated with higher fragmentation of skin vasculature than irritant reactions (19.5 ± 9.7 vs 14.3 ± 3.7 fragments/100 pixels2 ; P < .05), as well as lower ratio of low- to high-frequency acoustic signals (1.6 ± 0.5 vs 2.0 ± 0.6, P < .05). Allergic reactions graded "++" showed higher vessel fragmentation than reactions graded "+" (25.4 ± 13.2 vs 17.1 ± 6.5 fragments/100 pixels2 ; P < .05). A linear model combining the biomarkers fragmentation and frequency ratio could differentiate allergic from irritant test reactions with an area under the receiving operator characteristic curve of 0.80 (95% confidence interval 0.64-0.91), reaching a sensitivity of 81% and specificity of 63%. Optoacoustic mesoscopy shows potential to help in differentiating between allergic and irritant test reactions based on novel biomarkers that may reflect vasodilation, vessel tortuosity, and edema.