Direct immunofluorescence

Viegas MS, Martins TC, Seco F, et al. An improved and cost-effective methodology for the reduction of autofluorescence in direct immunofluorescence studies on formalin-fixed paraffin-embedded tissues. Ear. I. Histochem. 2007 51 59-66. 

The methods outlined below include protocols for direct and indirect immunofluorescence staining, that can be adapted easily for the cell type of interest as indicated in the relevant notes. The principal approaches to flow cytometric analysis, standardization and calibration are then given, followed by two more detailed protocols illustrating quantitation using direct immunofluorescence, and a competitive binding assay, which demonstrates the application of linear amplification of fluorescence. 

A 27-year-old woman, a pharmacist, had dermatitis on three separate occasions a few hours after she started to take oral deflazacort 6 mg for vesicular hand eczema (185). On each occasion, her symptoms included a widespread macular rash mainly on the inner aspects of her arms and legs and buttocks. She also had severe scaling, fever, nausea, vomiting, malaise, and hypotension. A skin biopsy was consistent with erythema multiforme, and direct immunofluorescence showed granular deposits at the dermoepidermal junction. Patch tests to the commercial formulation of deflazacort 6 mg (1% aqueous solution) and to pure deflazacort (1% aqueous solution) were positive, but there were no cross-reactions to other glucocorticoids. 

The two main methods of immunofluorescent labeling are direct and indirect. Less frequently used is direct immunofluorescence whereby the antibody against the molecule of interest is chemically conjugated to a fluorescent dye. In indirect immunofluorescence, the antibody specific for the molecule of interest (called the primary antibody) is unlabeled, and a second anti-immunoglobulin antibody directed toward the constant portion of the first antibody (called the secondary antibody) is tagged with the fluorescent dye (Figure 1). 

Advantages of direct immunofluorescence include shorter sample staining times and simpler dual and triple labeling procedures. In cases where one has multiple antibodies raised in the same species, for example two mouse monoclonals, a direct labeling may be necessary. 

Disadvantages of direct immunofluorescence include lower signal, generally higher cost, less flexibility and difficulties with the labeling procedure when commercially labeled direct conjugates are unavailable. 

Advantages of indirect immunofluorescence include greater sensitivity than direct immunofluorescence. There is amplification of the signal in indirect immunofluorescence because more than one secondary antibody can attach to each primary (see Figure 1). Commercially produced secondary antibodies are relatively inexpensive, available in an array of colors, and quality controlled. 

Direct immunofluorescence studies are performed on 4mm thick cryostat sections, which are incubated with fluorescein isothiocyanate-labeled goat anti-rat IgG, goat anti-rat C3, goat anti-rat fibrinogen and goat anti-rabbit IgG. (Note these procedures may also be performed on the paraffin sections using standard immunohistochemistry techniques). 

Rocamora A, Matarredona J, Sendagorta E, Ledo A. Sweat gland necrosis in drug-induced coma a light and direct immunofluorescence study. J Dermatol 1986 13(l) 49-53. 

Ruchel R, Zimmermaim F, Boning-Stutzer B, Helmchen U Candidiasis visualised by proteinase-directed immunofluorescence. Virch Arch Pathol Anat 1991 419 199—202. 

Diagnosis of chlamydial ophthalmia neonatorum is estabUshed by conjunctival smears that reveal typical basophihe intracytoplasmic inclusions with Giemsa stain and by traditional specimen culture (Figure 25-21). Direct immunofluorescent, immunoenzyme antibody or NAAT testing can also be helpful in confirming the diagnosis. 

To demonstrate that 3D STORM can resolve the 3D morphology of nanoscopic structures in cells, we imaged clathrin-coated pits using a direct immunofluorescence scheme. The clathrin in the cell was stained with... 

A 67-year-old man presented with an acute bullous eruption 6 weeks after starting bumetanide. He had numerous large tense bullae on erythematous skin, with superficial ulceration on the thighs, arms, and anterior trunk. Pruritus was severe. Routine laboratory tests were normal, except for blood eosinophilia. Biopsy of a blister showed subepidermal bullae associated with dermal infiltrates of neutrophils and eosinophils. Direct immunofluorescence showed continuous linear deposits of C3 and IgG at the basement membrane zone, confirmed by immunoelectron microscopy. Circulating IgG antibasement membrane antibodies were localized in the roof of the blister. Compete clinical heahng and normalization of immunology occurred within 2 months of withdrawal of bumetanide. 

A 75-year-old man with prostatic carcinoma took flutamide for 18 months and developed blisters on the back of the hands and fingers after exposure to the sun (4). The bullae were associated with skin fragility and atrophic scarring. Histopathology and direct immunofluorescence showed ultrastructural features similar to those described in porphyria cutanea tarda. However, porphyrin concentrations in the urine and blood were normal. Flutamide was withdrawn and the lesions healed, without relapse after 11 months. 

Henoch-Schdnlein purpura developed in an 84-year-old Indian woman 10 days after she started to take clarithromycin (250 mg bd) for pneumonia (52). She was otherwise healthy and taking no regular medications. Histology confirmed a leukocytoclastic vasculitis of superficial vessels, with extravasation of erythrocytes, and direct immunofluorescence showed immunoglobulin A in superficial dermal vessels. Treatment with prednisone (1 mg/kg/day) was required. Most of the symptoms and signs resolved within a few days, but renal function remained impaired. 

A 64-year-old woman, who had used penicillamine 500 mg/day for 3 years for rheumatoid arthritis, developed a bullous skin eruption affecting her neck and limbs (299). After treatment with prednisolone (dose not specified) she improved, but relapsed when the dose was reduced below 10 mg. Eleven months after the onset of blistering, penicillamine was discontinued and within 2 months the prednisolone was also stopped, with no recurrence of the eruption during 12 months follow-up. Direct immunofluorescence was positive for immunoglobulin G and complement component C3,... 

A previously healthy 63-year-old man, who had taken quinidine gluconate 972 mg/day for 9 months, developed diffuse edematous erythema on the extensive surfaces of the hands, arms, and face, with marked accentuation over the joints. His nail-fold capillaries were dilated and the shoulder abductors were shghtly weak. His erythrocyte sedimentation rate was shghtly raised (29 mm/hour) and there was a positive ANA titer (1 640) with a speckled pattern. There were no antibodies to Sm, ribonucleoprotein, SSA or SSB antigens, or histones. There was no evidence of inflammatory myopathy on electromyography, and a skin biopsy showed a mild, superficial, perivascular, lymphocytic inflammation with positive direct immunofluorescence for IgG and IgM at the dermoepidermal junction. There was no evidence of malignancy. All these abnormalities resolved rapidly after quinidine withdrawal. 

Linear IgA bullous disease is an autoimmune subepider-mal disorder, characterized by a linear deposition of IgA along the blister base, with a predominantly neutrophilic dermal infiltrate. Most often idiopathic, a subset of linear IgA bullous disease is induced by drugs, and intravenous vancomycin is the best-documented drug that triggers it (71). The diagnosis can be confirmed by direct immunofluorescence (72,73). In some cases the blisters resolve only after withdrawal of therapy and in others glucocorticoid therapy is required. 

Direct immunofluorescence. Rapid simple procedure. Multiple labeling is possible using different filter combinations. Confocal microscopy may be used. Extravagant when more than just a few target antigens are to be studied. 

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