Skin Biopsy for Diagnosing Discoid Lupus Erythematosus

Skin lesions are common and important features in many rheumatic diseases, and biopsy with pathological analysis plays a critical role in diagnosis. This article provides a detailed overview of the standardized skin biopsy procedure and, using discoid lupus erythematosus as an example, highlights the essential role of dermatopathology in diagnosing rheumatic diseases.

Discoid lupus erythematosus (DLE) is the most common variant of cutaneous lupus erythematosus (CLE). It typically affects the scalp, face, auricle, and lips. When lesions extend beyond these areas to involve the trunk and limbs, the condition is termed disseminated DLE (DDLE). The pathognomonic skin lesions present as well-demarcated discoid erythematous plaques with firmly adherent scales or crusts, central atrophy, characteristic scarring, and pigmentary changes, potentially leading to irreversible scarring alopecia. Histopathological and immunopathological examination of the skin lesions is instrumental in diagnosing DLE. Histologically, DLE is characterized by hyperkeratosis, follicular plugging, focal epidermal thinning, vacuolar alteration of the dermo-epidermal interface, thickening of the epidermal basement membrane, and a superficial and deep perivascular and periadnexal lymphocytic infiltrate, often in conjunction with interstitial mucin. Direct immunofluorescence (DIF) may reveal deposits of immunoglobulins and complement at the dermo-epidermal junction (DEJ). The following article outlines the protocols for DLE lesion assessment, skin biopsy procedures, histopathological and immunopathological analysis, and management options, with the aim of providing a comprehensive, standardized, and replicable diagnostic and therapeutic process to assist clinical physicians in practice.

Skin lesions are common and crucial manifestations, often serving as early indicators in many rheumatic diseases. These lesions play an essential role in diagnosis and disease monitoring and are frequently included in the classification criteria for conditions such as systemic lupus erythematosus, dermatomyositis, scleroderma, adult-onset Still's disease, and Behçet's disease¹. A thorough examination of the skin can assist in the early diagnosis, effective treatment, and monitoring of these conditions, helping to prevent severe complications.

Skin biopsy is one of the most commonly performed diagnostic procedures in rheumatic diseases with cutaneous manifestations. As a simple and cost-effective diagnostic modality, skin biopsy allows for the visualization of morphological features that are not visible to the naked eye. Histopathology provides a detailed examination of a small portion of the lesion, viewed perpendicular to the skin surface. This process is crucial for both clinicians and pathologists in diagnosing connective tissue diseases (CTD).

The formalin-fixed, paraffin-embedded technique is widely established and offers several advantages, such as the ability to archive samples long-term and the potential for immunohistochemical processing. Hematoxylin and eosin (HE) stain is the most commonly used stain for histopathology due to its capacity to highlight both normal and abnormal cellular and tissue components. The hematoxylin component binds to acidic structures, such as DNA and RNA, coloring them blue. In the skin, this stains cell nuclei, rough endoplasmic reticulum, and ribosomes². Eosin stains eosinophilic structures, typically composed of intracellular or extracellular protein (e.g., cytoplasm and most connective tissue fibers), in varying shades of red. As a result, all relevant tissue structures in the specimen are effectively stained³.

Immunofluorescence (IF) is a critical diagnostic tool in evaluating patients with suspected autoimmune, vasculopathic, and cutaneous vesicobullous diseases, as it helps identify antigen-bound antibodies in tissues or body fluids. There are two main types of IF assays: direct IF (DIF) and indirect IF (IIF). The more commonly used DIF assay employs fluorochrome-labeled antibodies to detect the deposition of immunoreactants in patient skin. A panel of antibodies targeting immunoglobulin (Ig) G, IgA, IgM, and complement C3 has been utilized⁴.

Discoid lupus erythematosus (DLE) is the most prevalent form of cutaneous lupus erythematosus (CLE), typically affecting areas such as the scalp, face, auricle, and lips. When lesions extend beyond these regions to involve the trunk and limbs, the condition is referred to as disseminated DLE (DDLE)⁵. Characteristic skin lesions appear as well-demarcated discoid erythematous plaques, often accompanied by firmly adherent scales or crusts, central atrophy, characteristic scarring, and pigmentary changes, which may result in irreversible scarring alopecia if occurring on the scalp. Although many patients remain asymptomatic, some may experience mild itching^6,7. DLE lesions are typically marked by epidermal atrophy, follicular plugging, basement membrane thickening, and lymphocytic infiltration. DIF demonstrates the deposition of immunoglobulin and complements along the dermo-epidermal junction, present in both lesional and non-lesional skin, known as the lupus band test^8,9. The diagnosis of DLE is primarily based on clinical presentation, along with histopathological and immunopathological findings. When histopathological and immunopathological information is necessary, a skin biopsy becomes essential.

The choice of biopsy type and location significantly impacts the histological assessment. Understanding the appropriate timing, location, and technique of biopsy is crucial to obtaining accurate pathological results. Several factors must be considered to ensure a precise diagnosis, highlighting the importance of addressing potential challenges in the biopsy process to enhance both the quality of the biopsy and the accuracy of the diagnosis. This article provides a detailed overview of the standardized skin biopsy procedure, using DLE as an example, and highlights key considerations to ensure accurate and effective diagnosis.

This protocol was approved by the Ethics Committee of Shanghai Dermatology Hospital, and written informed consent was obtained from the participants for the publication of clinical images and data. A skin biopsy typically refers to the traditional excision of skin lesions using a fusiform technique with a surgical scalpel. Using excisional biopsy as an example, the following protocol provides a detailed description of the surgical procedure for performing a skin biopsy (Figure 1).

Participants were eligible for inclusion if presenting with distinct skin lesions requiring biopsy for definitive diagnosis, demonstrating normal coagulation function, and showing no signs of acute infection or purulent lesions at the intended biopsy site. Exclusion criteria included a known tendency for keloid or hypertrophic scar formation, long-term use of immunosuppressants, or other conditions that could impair wound healing. Pregnant or lactating women were excluded due to potential risks. Patients unable to cooperate due to mental illness or other factors were also excluded from the study. All participants provided written informed consent and voluntarily agreed to undergo the skin biopsy procedure. The details of the reagents and the equipment used are listed in the Table of Materials.

1. Patient preparation

1. Verify the patient's identity, including name, medical record number, and the biopsy site.
2. Conduct a thorough review of the patient's medical history, including any bleeding tendencies, hypertension, diabetes, heart disease, and current use of anticoagulants or antiplatelet medications.
3. Perform preoperative evaluations, including routine blood tests, coagulation function assessments, and screening for infectious disease indicators^10,11.
4. Clearly explain the purpose, method, potential complications, and precautions associated with the skin biopsy to both the patient and their family.
5. Ensure that the patient has signed the informed consent form.

2. Equipment and materials preparation

1. Prepare a sterile biopsy kit containing all necessary instruments, including a scalpel, forceps, scissors, and other required tools. Ensure that all instruments are properly sterilized.
2. Prepare antiseptic solution (e.g., alcohol, iodophor, or chlorhexidine), local anesthetic (e.g., lidocaine), sterile injection syringe, sterile needles, sterile sutures, sterile gloves, sterile gauze, and dressing materials. Arrange all necessary instruments and medications within easy reach to maximize efficiency.

3. Preparation of the biopsy site

1. Select the biopsy site carefully, especially when multiple lesions are present. Choose a non-treated, non-traumatized, fully developed lesion that displays distinct characteristic features. Ensure that the biopsy is deep enough to capture the full thickness of the lesion, including the epidermis, dermis (especially the deep dermis), and a small portion of adjacent normal tissue to serve as a control.
2. Mark the planned excision path prior to the procedure to ensure precision. Shave any hair around the surgical site if necessary.
3. Take photographs of both the lesions and the biopsy site for future reference in medical decision-making and treatment planning.
4. Position the patient properly, ensuring that the biopsy site is well exposed and aligned parallel to the floor.

4. Biopsy procedure

1. Disinfect the biopsy area
   1. Thoroughly disinfect the biopsy area with an appropriate antiseptic solution. Cover a diameter of at least 15 cm. Cleanse in a circular motion, starting from the surgical incision site and moving outward. Allow the antiseptic to dry completely.
      NOTE: Use a non-iodine antiseptic for patients with known allergies.
2. Apply a sterile surgical drape
   1. After disinfection, apply a sterile surgical drape to isolate the surgical field.
3. Administer local anesthesia
   1. Use a 1%-2% lidocaine solution for local anesthesia. Inject subcutaneously at the biopsy site with a fine needle (following institutionally approved protocols). Wait 2-3 min for full effect. Gently palpate the site to confirm the absence of pain.
      NOTE: For children or anxious patients, consider a topical anesthetic in addition to or in place of injectable anesthetic.
4. Perform the incision and tissue excision
   1. Use a sterile scalpel to make a vertical, shuttle-shaped incision extending to the deep dermis or subcutaneous fat. Use biopsy forceps to gently stabilize or lift the tissue.
   2. Excise a representative portion of the lesion, including a small margin of surrounding normal tissue, for comprehensive histopathological evaluation. Aim for a tissue size of 3-5 mm.
   3. Apply steady pressure with sterile gauze for 2-3 min or until the bleeding stops completely.
   4. Use absorbable sutures or electrocautery for larger vessels as needed¹².
      NOTE: Prepare hemostatic tools in advance for high-risk patients.
5. Suture the excision site to promote healing and reduce infection risk. For facial or neck wounds, use 6-0 monofilament non-absorbable sutures. For trunk or limb wounds, use 5-0 non-absorbable sutures. Insert the needle vertically, maintaining a distance between the entry point and wound edge equal to the wound depth.
6. Place a sterile dressing over the biopsy site and secure it with adhesive tape.
7. Advise the patient to keep the wound dry for 24-48 h. Provide detailed post-procedure care instructions, including guidelines for dressing changes and activity restrictions.
   NOTE: Schedule follow-up appointments for high-risk patients to monitor wound healing.

5. Specimen handling

1. Divide the excised tissue into two portions using a sterile scalpel or scissors before placing the specimen in the appropriate transport medium. Label each container clearly with the patient's identification information and the biopsy site. Complete the pathology request form in full.
2. Place one half of the tissue in a container containing a fixative (e.g., 10% formalin solution), and send it to the pathology laboratory for hematoxylin and eosin (HE) staining.
3. Transport the other half either on gauze soaked in ice-cold physiological saline for immediate processing or place it in Michel's medium for delayed processing. Prepare the specimen for direct immunofluorescence (DIF) analysis¹³ as soon as possible.
   NOTE: Among these options, quick freezing is the most commonly used method. Do not store skin biopsy specimens for more than 24 h in saline-soaked gauze or more than one month in Michel's medium at 4-8 °C¹⁴.

Histologically, DLE is characterized by hyperkeratosis, follicular plugging, flattening of the epidermal ridges, focal epidermal thinning, vacuolar alteration of the dermo-epidermal interface, thickening of the epidermal basement membrane, and a superficial and deep perivascular and periadnexal lymphocytic infiltrate, often in conjunction with interstitial mucin^5,15(Figure 2). DIF may reveal deposition of immunoglobulins and complement at the DEJ in lesional skin, with a positivity rate of approximately 80% observed in sun-exposed areas of seventy-one patients with DLE. In contrast, DIF findings in non-lesional skin are generally negative¹⁶. Although DIF is a valuable diagnostic tool, correlating its findings with clinical presentation and histopathological results is essential to achieve the most accurate and reliable diagnosis¹⁷. Emerging studies suggest that features such as mucin deposition in the dermis and epidermis, as well as the intensity of inflammatory infiltrates, may offer insights into disease activity and prognosis. However, further validation of these findings is still needed⁸.

Figure 1: Schematic illustration of the protocol. This diagram outlines the sequence of steps followed during the procedure. Please click here to view a larger version of this figure.

Figure 2: Histopathological features of the skin biopsy (H&E stain). Representative image showing hyperkeratosis, follicular plugging, flattening of the epidermal ridges, focal epidermal thinning, thickening of the epidermal basement membrane, and a superficial and deep perivascular and periadnexal lymphocytic infiltrate. Scale bar: 600 µm. Please click here to view a larger version of this figure.

Skin biopsy is essential for confirming diagnoses and distinguishing between different conditions, as it enables the examination of histological features that provide crucial diagnostic evidence for early detection and treatment of various conditions. An accurate diagnosis empowers physicians to formulate effective treatment plans, thereby preventing inappropriate therapies that might arise from misdiagnosis. Due to its importance, there are many critical steps, potential modifications, and limitations in the biopsy protocol that warrant attention.

Performing a skin biopsy involves selecting the appropriate biopsy type and site, preparing and positioning the patient, and handling the specimen properly. Various types of skin biopsies include fusiform, punch, shave, and excisional biopsies. Fusiform biopsies are recommended for larger lesions or when deeper tissue sampling is required, as in cases of DLE. Punch biopsies are preferred for small lesions because they provide a full-thickness skin sample with minimal invasiveness, while shave biopsies are suited for epidermal and superficial dermal lesions. Excisional biopsies involve removing a skin lesion in its entirety and are typically used for lesions requiring complete excision¹. Choosing the proper site is essential to obtain a representative sample of the pathology. Ideal biopsy sites should be from new, untreated, untraumatized, and fully developed lesions that clearly display characteristic disease features. For example, erythematous areas are often more diagnostic than older, scarred areas. The biopsy should also be deep enough to capture the lesion's full thickness^1,13. Position the patient with the biopsy site accessible and, if possible, parallel to the floor for easier access, ensuring comfort, as the position may need to be held for several minutes. All necessary instruments and medications should be prepped and arranged nearby to facilitate a smooth procedure. Proper handling of the biopsy specimen is crucial for accurate histopathological analysis. Samples should be immediately placed in an appropriate fixative, typically formalin, for routine histology and subsequently stained with HE. For cases requiring DIF, the sample should be transported in saline-soaked gauze or Michel's medium to preserve immunoreactants in the tissue¹⁸.

Modifications to the biopsy technique may be required based on lesion characteristics or patient considerations. If initial biopsy results are inconclusive, repeat biopsies from alternative sites can improve diagnostic accuracy. Additionally, the biopsy type can also be adjusted based on the lesion type. For instance, in cases of scarring alopecia caused by DLE, a punch biopsy from a well-established area of involvement may be more appropriate¹⁸.

Despite the valuable information provided by skin biopsies, certain limitations should be considered. The diagnostic process may be subjective. Additionally, terms like "not excluded" and "consider in conjunction with clinical findings" in pathology reports may not offer definitive diagnoses, though they can offer valuable clues for further exploration. False-negative results may also occur, particularly when samples are taken from poorly selected sites or late-stage lesions. Although minimally invasive, skin biopsies are not without risk. Potential complications include discomfort, pain, bleeding, scarring, or infection, so patients should be well-informed and adequately prepared. If concern about this invasive test arises, dermoscopy can be considered as an alternative. Dermoscopy serves as a non-invasive tool that can aid in the preliminary diagnosis of DLE. Statistical analysis has shown a strong correlation between dermoscopic findings and histopathological results¹⁹.

Skin biopsies offer significant advantages over alternative diagnostic methods due to their ability to directly assess tissue pathology. They provide histopathological clarity, helping to differentiate among lupus subtypes, and when combined with serologic tests, enhance diagnostic specificity. Advances in diagnostic methods are expanding the applications of skin biopsy, particularly in rheumatology. Future protocols may incorporate biomarker analysis from skin biopsy samples, allowing for improved disease monitoring and insights into personalized therapy²⁰. Advances in molecular biology may also enable gene expression profiling directly from skin biopsy samples, identifying genes related to the clinical heterogeneity in SLE and other phenotype-related loci, thus paving the way for stratified treatment recommendations and precision medicine²¹.

Skin biopsy is an essential diagnostic tool for rheumatic diseases with cutaneous manifestations, serving as a cornerstone for clarifying disease characteristics, confirming diagnoses, and guiding treatment strategies. Despite certain limitations, its value in disease diagnosis is undeniable. By focusing on precise protocol execution, selecting appropriate modifications, understanding limitations, and integrating new technological advancements, the diagnostic utility of skin biopsy can be maximized, providing critical information that improves patient outcomes.

There are no conflicts of interest to disclose.

We warmly thank the following individuals for their assistance and contributions to the video production process: Lili Ma, Ke Xue, Jiaping Qi, and Ruilian Lin from the Department of Rheumatology, Shanghai Skin Disease Hospital, Tongji University School of Medicine.