Handbook of Direct Immunofluorescence A Pattern-Based Approach to Skin and Mucosal Biopsies
Handbook of Direct Immunofluorescence A Pattern-Based Approach to Skin and Mucosal BiopsiesDouglas R Fullen MD Professor of Pathology and Dermatology, University of Michigan, Ann Arbor, MI, USA May P Chan MD Associate Professor of Pathology and Dermatology, University of Michigan, Ann Arbor, MI, USA Aleodor A Andea MD Professor of Pathology and Dermatology, University of Michigan, Ann Arbor, MI, USA David P Arps MD Dermatopathologist, Consolidated Pathology Consultants, Libertyville, IL, USA
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Handbook of Direct Immunofluorescence: A Pattern-Based Approach to Skin and Mucosal Biopsies is a guide for practicing general pathologists, dermatopathologists, and oral and ophthalmic pathologists who interpret biopsies from patients with inflammatory and blistering disorders requiring direct immunofluorescence examination. The book will also serve as a resource for clinicians who care for patients with these disorders and trainees who are studying in these fields.
This book consists of an introductory section that describes immunofluorescence techniques and protocols, followed by 29 chapters covering the range of conditions which the pathologist or clinician is likely to encounter using direct immunofluorescence examination. Each chapter succinctly describes the clinical presentation, pathogenesis, histopathology, direct immunofluorescence and, when appropriate, indirect immunofluorescence and enzyme-linked immunosorbent assays for the specific disorders. Multiple images show the histopathologic and direct immunofluorescence findings of the entities discussed in each chapter. An additional strength of this book is the inclusion of nonspecific direct immunofluorescence findings that are commonly observed in practice. A limited number of essential references are included in each chapter.
We believe this book will aid in understanding the technical aspects and problems that affect specimen procurement, transportation and processing for direct immunofluorescence examination. The pattern-based approach to direct immunofluorescence will serve as a logical framework for learning about these disorders, for reaching diagnoses, for understanding when additional serologic studies may be helpful, and in recognizing nonspecific findings on biopsy specimens submitted for this purpose.
Douglas R Fullen
May P Chan
Aleodor A Andea
David P Arps
Introduction: immunofluorescence techniques and protocols
To Angela, Peter, Aileen and my mentors, Dr J T Headington,
Dr J A Reed and Dr N S McNutt, for their support and encouragement
To Alice, Danny and all of my mentors
To my son, Andrew
To Joyce, Jennifer, Nicholas, Monisha, Nikolai, David, Gideon and Ladd
Immunofluorescence is a laboratory technique used to document the presence of auto-antibodies either in tissues or circulating in blood. It provides useful information for the diagnosis and treatment of certain skin disorders. All immunofluorescence tests are based on the detection of fluorescence-linked antibodies against immunoglobulins, complement or proteins such as fibrinogen.
Fluorescent techniques use fluorochromes: when stimulated with light of a specific wavelength, these substances emit light with a longer wavelength. The most commonly used fluorochrome in dermatologic immunofluorescence is fluorescein isothiocyanate (FITC). Less commonly, tetramethylrhodamine isothiocyanate (TRITC) is employed. The fluorochrome is linked to the antibody via a thiocarbamide linkage which does not destroy its immunoreactivity.1
Biopsy and tissue transport
Choosing an appropriate skin biopsy site is an important component for optimal results using direct immunofluorescence (DIF) and the selection depends on the suspected clinical diagnosis. In the absence of a favored clinical diagnosis perilesional skin should be sampled.2 However, depending on the clinical differential diagnosis the biopsy protocol can be modified as follows:
For autoimmune blistering diseases, the optimal biopsy is from the perilesional area.2,3 In connective tissue diseases, the biopsy should be taken from active lesions in evolution if chronic cutaneous lupus is suspected, or from both lesional and nonlesional sun-protected skin if systemic lupus erythematosus is considered (see Chapter 20). In cases where vasculitis is suspected, the biopsy should be collected from recent lesions, preferably under 24 hours of evolution.3 Since often a lesional biopsy for histologic diagnosis is collected at the same time with the DIF biopsy, one can consider performing a biopsy at the edge of the lesion and bisecting the tissue. This practice is controversial, with some authors opposing it due to the risk of inadvertently detaching the epidermis; however, one study seems to suggest that bisection of the tissue does not result in a higher rate of inadequate specimens.4
For lesions of the oral mucosa, it is recommended that two biopsies be taken, one from lesional tissue and one from adjacent normal mucosa. A punch biopsy is usually performed; however, incisional biopsy is the technique of choice in case of hard palate and gingival lesions because punch at these sites often provides unsatisfactory tissue for DIF.5–7
If the test is not performed immediately and the specimen needs to be transported to another location (which is more often the case), the biopsies should be placed in Michel (or Zeus) transport medium which contains ammonium sulfate, N-ethyl maleimide and magnesium sulfate in citrate buffer8 and allows the specimens to maintain immunoreactivity for about 2 weeks at room temperature (up to 6 months in some cases).7,9 Alternatively, the specimen can be placed on saline-soaked gauze if it is submitted directly to the laboratory to be processed the same day or snap-frozen immediately if the test is performed on site.
Biopsies for DIF should not be placed in formalin. Tissues accidentally immersed in formalin for <10 min may still yield positive results for pemphigoid and dermatitis herpetiformis (but not for pemphigus). Longer exposure to formalin compromises the DIF diagnosis for all entities.10
For indirect immunofluorescence (IIF), blood is collected without anticoagulant. Serum is separated from the clotted blood. If it is not tested immediately it should be stored at –20°C.
Direct immunofluorescence is used to identify the presence of autoantibodies, complement and fibrinogen bound to tissue antigens. It is a one-step procedure in which fluorescent-labelled antibodies against immunoglobulins, complement and fibrinogen are incubated with the patient's skin biopsy. The usual protocol consists of the following steps:
- Optimal cutting temperature (OCT) embedding compound is distributed onto a cold chuck which is placed in the cryostat and allowed to freeze. The skin biopsy is rinsed in phosphate buffered saline (PBS) and then placed onto the chuck. Proper orientation of the specimen with the epidermis embedded perpendicular to the cutting plane is critical. Tissue is covered with OCT and snap-frozen in liquid nitrogen or hexane bath.
- Specimens are either cut the same day or can be stored at –80°C for later sectioning. The block is faced until full epidermis is present, and then 7 slides (or more depending if additional complement factors are performed) are cut at 4–5 μm thickness. Multiple (usually 4) profiles should be placed on one slide to maximize the chance that at least one will have intact epidermis.
- Slides are dried, washed with PBS to remove the remaining OCT compound and dried again. One slide is stained with hematoxylin and eosin (H&E) and reviewed to check the orientation and ensure the presence of the basement membrane zone.
- Five slides are incubated with FITC-labelled primary antibodies including anti-IgG, anti-IgA, anti-IgM, anti-C3 and anti-fibrinogen.
Positive controls for IgG, IgA, IgM, and C3 are run with each DIF batch. The laboratory needs to identify recent tissue samples positive for each antibody that can be used as positive controls. Slides are cut and stored at –80°C. Control slides are stained in an identical fashion to the patients' slides. They are replaced as needed and as good control tissue becomes available. One slide per patient will also be run with the primary antibody step omitted (only reaction buffer) to serve as a negative reagent control.
Controls are routinely reviewed by a laboratory director and any problems are discussed with the technical staff. Staining is repeated for any isolated failure; antibody incubation times may need to be readjusted if problem persists.
Indirect immunofluorescence (IIF) is used to detect the presence of circulating autoantibodies. IIF is a two-step procedure.
In the first step the patient's serum is incubated with frozen section slides from an appropriate substrate (such as human epidermis, monkey esophagus or rat bladder). If autoantibodies are present in the serum they will bind to the substrate.
In the second step a FITC-labelled antibody of defined type (usually anti-IgG or anti-IgA) is incubated with the tissue. The usual protocol consists of the following steps:
- Serial dilutions of the patient's serum in PBS are incubated with the substrate in a moist chamber for about 30 min.
- Slides are rinsed with PBS to remove any unbound antibodies.
- A second FITC-labelled anti-IgG or anti-IgA antibody is incubated with the substrate.
- Following a rinse with PBS the slides are cover slipped using a mounting media suitable for fluorescence microscopy and examined on a fluorescence microscope.
The initial serum dilution is usually 1:10 or 1:80. If it is positive, higher dilutions are used to determine the serum titer.2 Control sera from known positive and negative patients are run along with the samples.
This technique is used in autoimmune subepidermal blistering diseases to map the location of the antigen with respect to the lamina lucida. It can be employed for both DIF and IIF; however, it is usually used for the latter. The protocol entails incubation of the patient's perilesional skin (for DIF) or normal human skin (for IIF) with 1M NaCl solution for 48–72 hours. This step induces an artificial split through lamina lucida. The regular protocols for DIF or IIF are employed next and the biopsy is examined under a fluorescent microscope.
- Mohan KH, Pai S, Rao R, Sripathi H, Prabhu S. Techniques of immunofluorescence and their significance. Indian J Dermatol Venereol Leprol 2008;74:415–419.
- Huilgol SC, Bhogal BS, Black MM. Immunofluorescence of the immunobullous disorders part one: Methodology. Indian J Dermatol Venereol Leprol 1995;61:187–195.
- Aoki V, Sousa JX, Jr., Fukumori LM, et al. Direct and indirect immunofluorescence. An Bras Dermatol 2010;85:490–500.
- Loh E, Armstrong AW, Fung MA. Pre-bisection of a single skin biopsy does not produce technically inadequate specimens for direct immunofluorescence: a review of 3450 specimens. J Cutan Pathol 2014;41:890–892.
- Sano SM, Quarracino MC, Aguas SC, et al. Sensitivity of direct immunofluorescence in oral diseases. Study of 125 cases. Med Oral Patol Oral Cir Bucal 2008;13:E287–291.
- Siegel MA. Intraoral biopsy technique for direct immunofluorescence studies. Oral Surg Oral Med Oral Pathol 1991;72:681–684.
- Tapia JL, Neiders ME, Suresh L. Indications and procedures for direct immunofluorescence biopsies of the oral mucosa. Quintessence Int 2015;46:247–253.
- Michel B, Milner Y, David K. Preservation of tissue-fixed immunoglobulins in skin biopsies of patients with lupus erythematosus and bullous diseases – preliminary report. J Invest Dermatol 1972;59:449–452.
- Vaughan Jones SA, Salas J, McGrath JA, et al. A retrospective analysis of tissue-fixed immunoreactants from skin biopsies maintained in Michel's medium. Dermatology 1994;189:131–132.
- Arbesman J, Grover R, Helm TN, Beutner EH. Can direct immunofluorescence testing still be accurate if performed on biopsy specimens after brief inadvertent immersion in formalin? J Am Acad Dermatol 2011;65:106–111.