Immunofluorescence testing occupies an important place in the diagnosis and evaluation of many diseases. Most testing occurs either with affected tissue samples (direct immunofluorescence) or with serum of affected individuals, looking for circulating antibodies (indirect immunofluorescence).
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STORAGE AND TRANSPORTATION MEDIA CHARACTERIZATION MICHEL'S MEDIUM
The use of Michel's transport medium for immunofluorescence and immunoelectron microscopy in autoimmune bullous diseases.
Vaughn Jones SA, Palmer I, Bhogal BS, Eady RA, Black MM.
Department of Immunofluorescence, St. John's Institute of Dermatology, St. Thomas Hospital, London, UK.
J Cutan Pathol 1995 Aug;22(4):365-70 Abstract quote
We report the use of Michel's solution, already a well established transport medium, in the combined use of direct immunofluorescence (IMF) and pre-embedding immunoelectron microscopy in 3 subepidermal bullous diseases--bullous pemphigoid (BP), epidermolysis bullosa acquisita (EBA) and dermal binding linear IgA disease (LABD).
Our studies demonstrated that electron microscopy of normal skin maintained in Michel's medium for up to 28 days showed remarkable preservation of all components of the basement membrane zone, including the ultrastructure of the basal keratinocytes, dermoepidermal junction and papillary dermis. However, epidermal cell cytolysis occurred after just 48 hours. Immunoelectron microscopy using a gold probe has enabled us to localise the immunoreactants in bullous pemphigoid, epidermolysis bullosa acquisita and dermal-binding linear IgA bullous dermatoses.
Our findings are comparable to and as equally reliable as those on immunoelectron microscopy of fresh skin biopsies with no loss of antigen deposition, and demonstrate an effective new use of a well established transport medium.
STORAGE OF SLIDES
The reliability of long-term storage of direct immunofluorescent staining slides at room temperature.
Dikicioglu E, Meteoglu I, Okyay P, Culhaci N, Kacar F.
Department of Pathology, and Department of Public Health, Medical School of Adnan Menderes University, Aydin, Turkey.
J Cutan Pathol. 2003 Aug;30(7):430-6. Abstract quote
BACKGROUND: Long-term preservation of immunofluorescence is important for re-examinations. We investigated whether the storage of direct immunofluorescent (DIF)-positive slides at room temperature was reliable in daily practice.
METHODS: One hundred and twenty-five DIF-positive slides from the skin of 52 patients were evaluated. Sections were examined for the presence of immunoglobulin A (IgA), IgG, IgM, C3, and fibrinogen using fluorescein isothiocyanate-conjugated antisera and mounted with a ready-to-use permanent mounting medium containing an antifading reagent and sodium azide (DAKO, Glostrup, Denmark, S3023). The slides were stored at room temperature for 16-24 months. Changes in diagnostic pattern, fluorescence intensity, and the form and location of accumulation of immunoreactants and technical deformation were investigated.
RESULTS: Over the entire observation period, 49.6% of the slides faded away; the median length of survival was 16 months. Before 12 months, the survival rate of slides was 92.0%, whereas after 20 months it was 28.0%. In the early faded slides, which faded away before 16 months following the first examination, C3 and IgA were the most frequently observed immunoreactants. The technical deformations did not prevent the diagnosis.
CONCLUSIONS: The preservation of fluorescence in DIF-positive slides using mounting media with an antifading reagent is possible for 2 years at room temperature. However, in daily practice, storage for longer than 11 months prevents a reliable diagnosis.
SITE OF THE BIOPSY CHARACTERIZATION Skin 3 mm biopsy with both the edge of a fresh lesion and some adjacent normal skin May take a biopsy of normal skin and lesional skin Mucosa Perilesional with apparently normal mucosa with intact epithelium Pemphigus or pemphigoid (Skin) Edge of lesion, second biopsy 3 mm from lesion Pemphigus or pemphigoid (Mouth) 1st biopsy 3mm from lesion, 2nd at edge Purpura and vasculitis Biopsy 10mm from lesion Stasis dermatitis and livedo vasculitis Biopsy from edge of lesions Dermatitis herpetiformis Biopsy normal skin about 3 mm. from the lesion Porphyria and pseudoporphyria Biopsy from edge of a fresh lesion with edge of normal skin Screen Biopsy edge of fresh, intact skin and include scales if present
Epidermolysis bullosa: variability of expression of cicatricial pemphigoid, bullous pemphigoid, and epidermolysis bullosa acquisita antigens in clinically uninvolved skin.
J Invest Dermatol 1985 Jul;85(1):47-9 Abstract quote
Indirect immunofluorescence was performed on skin from 13 patients with epidermolysis bullosa (EB) (simplex, 6; junctional, 2; dystrophic, 5) to compare the expression of 3 basement membrane components, bullous pemphigoid (BP) antigen, cicatricial pemphigoid (CP) antigen, and epidermolysis bullosa acquisita (EBA) antigen, in clinically uninvolved tissue.
In addition, expression of laminin, type IV collagen, and KF-1 antigen was also evaluated. Whereas laminin, type IV collagen, and KF-1 antigen were each detectable in EB skin in a manner identical to that previously reported, marked variability was noted in the expression of BP, CP, and EBA antigens. However, no correlation was noted comparing lack of expression of any one of these latter antigens with either of the remaining two. Of these 3 antigens, BP antigen was the least often detectable, particularly in skin from patients with EB simplex.
The lack of detectable BP antigen in EB simplex skin appeared to correlate with more extensive disease involvement and/or younger patient age. These findings may, therefore, limit the usefulness of BP serum in immunofluorescence mapping. Additionally, the disparity in expression of basement membrane antigens defined by BP and CP sera suggests that BP antigen and CP antigen are distinct entities.
DIRECT IMMUNO-FLUORESCENCE CHARACTERIZATION Salt Split Skin (1M)
Incubate normal skin with 1M NaCl
Separates the epidermis from dermis
Epidermal half Upper lamina lucida and hemidesmosomes BP antigen Dermal half Laminin 5 Lamina densa, anchoring fibrils Limitations
Specimens with multiple, subepidermal or subepithelial vesicles or parts of bullae
Split induced by traction cannot be distinguished from pre-existing vesicles or bullae
J Cutan Pathol. 2006 Mar;33(3):220-6. Abstract quote
BACKGROUND: Artificial separation of oral mucosa into epithelium and connective tissue represents the first step in investigating both the basement membrane zone and its two components (epithelium and connective tissue) separately. It is a useful tool in analysing the possible role of hepatitis C virus in the pathogenesis of oral lichen planus (OLP). The aim of the present study was to test different methods to separate the epithelium from the connective tissue of oral mucosa affected by OLP as a preliminary step for further immunohistochemical or molecular investigations.
PATIENTS AND METHODS: Fourteen tissue biopsies of the oral mucosa of clinically and histopathologically confirmed OLP lesions underwent epithelial connective separation (ECS) using six different reagents.
RESULTS AND CONCLUSIONS: Among the separation methods tested, CaCl2 1 M at 37 degrees C for 30 min, NaCl 1 M at 4 degrees C for 26 h and elastase at 4 degrees C for 22 h allowed good ECS without structural alteration or tissue loss. The present study provides useful information regarding techniques to separate the epithelium from the connective tissue as a preliminary step to investigate oral mucosa affected by OLP from an immunohistochemical or molecular viewpoint.
Direct immunofluorescence microscopy of 1 mol/L sodium chloride-treated patient skin
Domloge-Hultsch N, Bisalbutra P, Gammon WR, Yancey KB.
Department of Dermatology, Uniformed Services University of the Health Sciences (USUHS), Bethesda, MD 20814-4799.
J Am Acad Dermatol 1991 Jun;24(6 Pt 1):946-51 Abstract quote
Patients with bullous pemphigoid and those with epidermolysis bullosa acquisita often demonstrate virtually identical clinical, histologic, and immunopathologic features. Although some patients can be distinguished by their pattern of circulating IgG anti-basement membrane zone antibody binding to 1 mol/L sodium chloride-split human skin, approximately 20% and 50% of bullous pemphigoid and epidermolysis bullosa acquisita patients, respectively, do not possess such antibodies.
Hence this study sought to determine whether these patients can be distinguished by mapping the distribution of basement membrane zone immunoreactants in patient skin split in vitro by 1 mol/L sodium chloride. All sodium chloride-treated samples from patients with bullous pemphigoid (n = 8), epidermolysis bullosa acquisita (n = 4), or other bullous skin diseases (n = 6) contained a lamina lucida cleavage plane bounded by bullous pemphigoid antigen and laminin; moreover, treatment of patient samples was performed without loss of tissue substrate or in situ immunoreactants. Deposits of IgG were found on the epidermal side of sodium chloride-treated skin from 13 of 14 bullous pemphigoid samples; IgG deposits in bullous pemphigoid samples were exclusively epidermal in eight, epidermal and dermal in five, and solely dermal in one.
In contrast, IgG was found exclusively on the dermal side of sodium chloride-treated samples from patients with epidermolysis bullosa acquisita. Although IgG mapping distinguished bullous pemphigoid and epidermolysis bullosa acquisita patients in 94% of these samples, the distribution of C3 in sodium chloride-treated patient skin was more variable and less predictive diagnostically.
Floor-pattern salt-split skin cannot distinguish bullous pemphigoid from epidermolysis bullosa acquisita. Use of toad skin.
Pang BK, Lee YS, Ratnam KV.
National Skin Centre, Singapore.
Arch Dermatol 1993 Jun;129(6):744-6 Abstract quote
BACKGROUND AND DESIGN--Patients with epidermolysis bullosa acquisita (EBA) and bullous pemphigoid (BP) can present with similar clinical features. These antigens have different phylogenetic origins. It was thought that sodium chloride split-skin immunofluorescence could reliably distinguish between BP and EBA. However, it has now been recognized that both diseases can present with a floor pattern fluorescence on salt-split skin. A simple method is required to distinguish these diseases.
RESULTS--Serum specimens from two patients presenting with pruritic blisters showing a floor pattern immunofluorescence on salt-split skin were found to have positive fluorescence on toad skin that has the BP and not the EBA antigen. The diagnosis of BP in these patients was confirmed by a prompt response to treatment with systemic steroids. Electron microscopy showed a split at the lamina lucida.
CONCLUSIONS--Bullous pemphigoid can present with a floor pattern immunofluorescence on salt-split skin. The use of the skin from invertebrates like the toad may help in distinguishing BP from EBA.
Optimal conditions of 1 M NaCl splitting technique to demonstrate basement membrane zone antigens in bullous pemphigoid, epidermolysis bullosa acquisita and linear IgA bullous dermatoses.
Jenkins RE, Rodenas J, Bhogal BS, Black MM.
St. John's Institute of Dermatology, St. Thomas' Hospital, London, UK.
Dermatology 1994;189 Suppl 1:133-4 Abstract quote
We compare a series of conditions for inducing lamina lucida separation through normal skin specimens using 1 M NaCl for the detection of basement membrane zone auto-antibodies using indirect immunofluorescence. Skin was split at room temperature and at 4 degrees C with and without phenylmethylsulphonyl fluoride.
Our results demonstrate that for the detection of bullous pemphigoid and epidermal-binding linear IgA bullous dermatoses antibodies skin should be split at 4 degrees C with enzyme inhibitors. For epidermolysis bullosa acquisita and dermal-binding antibodies skin can be split at room temperature.
Clinical, histopathologic, and therapeutic aspects of subepidermal autoimmune bullous diseases with IgG on the floor of salt-split skin.
Barnadas MA, Gonzalez MJ, Planaguma M, Romani J, Curell R, de Moragas JM, Alomar A.
Department of Dermatology and Pathology, Hospital de la Sta. Creu i St Pau, C/St Antoni M Claret, 167, 08025 Barcelona, Spain.
Int J Dermatol 2001 Apr;40(4):268-72 Abstract quote
BACKGROUND: About 12% of patients with subepidermal autoimmune bullous disease and immunoglobulin G (IgG) at the dermal-epidermal junction present diseases other than bullous pemphigoid.
MATERIALS AND METHODS: We report the clinical, histopathologic, and therapeutic aspects of eight cases of subepidermal bullous disorder with IgG on the floor of salt-split skin.
RESULTS: A predominant neutrophilic infiltrate was detected in six of the eight patients. In one patient, the inflammatory infiltrate was neutrophilic and eosinophilic in the same proportion. A good response to dapsone alone or combined with prednisone was observed in six patients.
CONCLUSIONS: The salt-split skin direct immunofluorescence test is useful for its diagnostic and therapeutic implications.
A practical technique for differentiation of subepidermal bullous diseases: localization of in vivo-bound IgG by laser scanning confocal microscopy.
Wozniak K, Kazama T, Kowalewski C.
Departments of Dermatology, Medical University of Warsaw, Warsaw, Poland.
Arch Dermatol. 2003 Aug;139(8):1007-11. Abstract quote
OBJECTIVE: To develop a practical technique to distinguish autoimmune subepidermal bullous diseases.
DESIGN: A prospective study.
SETTING: Academic referral center-the Department of Dermatology, Medical University of Warsaw.Patients Forty-two patients fulfilling clinical, immunological, and/or immunoelectron microscopic criteria for bullous pemphigoid (n = 31), mucous membrane pemphigoid (n = 6), or epidermolysis bullosa acquisita (n = 5), diagnosed as having disease and treated from January 1, 1997, to December 31, 2002.
MAIN OUTCOME MEASURES: We applied laser scanning confocal microscopy to determine the localization of in vivo-bound IgG at the basement membrane zone in biopsy specimens taken from patients' skin to compare the localization of basement membrane zone markers: antibody against beta4 integrin, antibody against laminin 5, and antibody against type IV collagen. In vivo-bound IgG was visualized by labeling with fluorescein isothiocyanate-conjugated anti-human IgG antibody, whereas basement membrane zone markers were labeled with anti-mouse Cy5-conjugated antibodies.
RESULTS: In patients with bullous pemphigoid, in vivo-bound IgG was localized on the epidermal side of laminin 5 and co-localized with beta4 integrin. In patients with mucous membrane pemphigoid, IgG was in vivo bound to the dermal-epidermal junction between localization of laminin 5 and type IV collagen. In patients with epidermolysis bullosa acquisita, in vivo-bound IgG was present on the dermal side of type IV collagen.
CONCLUSIONS: Laser scanning confocal microscopy allows precise localization of in vivo-bound IgG in patients' skin and, thus, it is a rapid method for the differentiation of mucous membrane pemphigoid from bullous pemphigoid and epidermolysis bullosa acquisita. This tool is suitable for the routine diagnosis of individual patients and for retrospective studies. This method is of special value in those patients in whom circulating autoantibodies are not detectable.
FISH (FLUORESCENCE IN SITU HYBRIDIZATION) CHARACTERIZATION
- Immunofluorescent and FISH analysis of skin biopsies.
Gellrich S, Ventura R, Jones M, Tan SY, Mason DY.
Department of Dermatology, Venereology and Allergy, Medical Faculty, Humboldt-University, Berlin, Germany.
Am J Dermatopathol. 2004 Jun;26(3):242-7. Abstract quote
Cutaneous biopsies are traditionally studied for the expression of cellular markers by immunoenzymatic techniques. However, immunofluorescent analysis is a valuable, and largely overlooked, ancillary technique that can resolve questions arising from conventional immunostaining, since it allows pairs of antigens to be simultaneously visualized.
Furthermore, a novel technique, based on a combination of immunoperoxidase and immunofluorescent staining, allows three markers to be demonstrated together. Fluorescent microscopy also allows skin biopsies from lymphoma cases to be analyzed for chromosomal abnormalities by the fluorescent in situ hybridization (FISH) technique, which is now applicable to routine biopsy samples.
In this review, we describe the technical aspects of immunofluorescent and FISH analysis of routine cutaneous biopsy samples.
LUPUS BAND TEST (LBT) CHARACTERIZATION
Adv in Dermatology 1999;441
Broadly, it is the deposition of Ig at the DEJ in lesional and nonlesional skin
IgM most frequent deposit
IgA least frequent
Granular pattern most frequent
Sharp linear band not accepted
As a general rule, sun-exposed lesional skin should be used to substantiate an initial diagnosis of LE so as to avoid the problem of false negative results due to decreased sensitivity in sun-protected skin
Probably results from UV light denaturing keratinocyte DNA, which diffuses across the basement membrane and becomes trapped due to the natural avidity of native DNA for type IV collagen
The DNA then binds with ANAs creating the granular deposits
J Invest Dermatol 1975;65:154
In spite of the pattern, antibodies directed againts basement membrane component do not play a significant role in generating a positive LBT
- Immunoglobulin M predominance in cutaneous lupus erythematosus.
Kontos AP, Jirsari M, Jacobsen G, Fivenson DP.
Department of Dermatology, Henry Ford Health System, Detroit, MI, USA.
J Cutan Pathol. 2005 May;32(5):352-5. Abstract quote
Background: Direct immunofluorescence (DIF) is a valuable tool in the diagnosis of cutaneous lupus erythematosus (LE). Our goal was to characterize the most frequent immune reactants in the skin biopsies of cutaneous LE and identify the most common immunofluorescence staining patterns.
Methods: DIF results of immunoglobulin G (IgG), IgA, IgM, C3, and fibrinogen from 199 patients between 1989 and 1998 were retrospectively analyzed. Confirmatory clinical and serological diagnosis of LE subtype was available for 95 patients. Intensity of staining was ranked from 0 to 4+ but only included as significant if >/=2+. Laboratory values were gathered and analyzed for all patients who had distinct granular immune deposition for Ig and/or complement.
Results: The most commonly detected individual Ig was IgM in 149 (75%) specimens. IgM and C3 combination was the most common pair expressed with 98 (49%) specimens. The most common triplet was IgM, C3, and fibrinogen in 63 (32%) specimens. The most common quadruplet was the combination of IgG, IgM, C3, and fibrinogen in 42 (21%) specimens. All the five immunoreactants were detected in only 25 (13%) specimens. Systemic LE patients had a higher percent of abnormal laboratory values compared to discoid LE (DLE) and subacute LE (SCLE) patients (p = 0.02). Fibrinogen staining was found to be significantly higher in DLE patients and lowest in SCLE patients (p = 0.05).
Conclusions: This study demonstrates a marked predominance of IgM +/- C3 in cutaneous LE. When used in conjunction with other data, DIF is an extremely powerful tool in the routine evaluation of the LE patient. Our report emphasizes the importance of IgM expression in the diagnosis of LE by DIF and how positive staining with multiple conjugates can raise its sensitivity.
In sun-exposed skin IgM should be continuous deposition over at least 50% of the width of the biopsy with moderate intensity
25% of normal sun-exposed show weak interrupted linear and granular IgM and C1q
20% of normal skin from healthy adults
In sun-protected skin Interrupted band of IgM of at least moderate intensity is positive
If IgA is also present, high specificity
Lupus Band Test-Disease States SLE
Best specificity is to take biopsy of normal skin of sun-exposed forearm (Positive in 67%) Normal unexposed skin will be positive only in severe cases (35-40%)
70% of patients with active nephritis with LBT on normal skin
C1q deposits-higher incidence of renal disease
Predictive value is increased with the following other positive markers:
Look for granules or closely spaced fibrils, only infrequently is it homogeneous in nature
J Invest Dermatol 1980;74:407
Relationship of LBT to disease severity in SLE:
IgG in non-lesional sun protected skin is 3x more common in patients with serious renal disease and 70% have active nephritis-negative LBT does not exclude renal disease
Pure IgM deposition in clinically normal skin have anti-nDNA antibodies restricted to the IgM class and tend to have a more benign course
Positive LBT on buttock skin correlates with active systemic disease
DLE Biopsy of untreated skin lesion in exposed area that has been present for at least 3 months C5b-9
(Terminal complement, membrane attack complex)
Hum Pathol 1996;26:14
J Cutan Pathol 1997;24:543
Usually negative or very weak
May represent activation of complement pathway within the BMZ
Formation of membrane pores may allow circulating Ab access to nucleus and cytoplasm
C5b-9 Disease States SLE Intense granular at DEJ 80%
Distinguish from anti-Ro associated SLES
SCLE DEJ in 66%
Granular nuclear and cytoplasmic epidermal staining
DLE DEJ in 60% MCTD- Granular nuclear and cytoplasmic decoration of keratinocytes 100%
Identical to anti-Ro associated SLE
Dermatomyositis DEJ 90%
Dermal papillary capillaries
Anti-Ro associated SLE, Dermatomyositis, and MCTD Endothelial decoration for C5b-9
Endothelial cell necrosis and denudement Reduction in vascular plexus
Granular and cytoplasmic decoration within keratinocytes for C5b-9
Differentiate by LBT and clinical
The immunofluorescent profile of dermatomyositis: a comparative study with lupus erythematosus.
Magro CM, Crowson AN.
Department of Pathology, Beth Israel Deaconess Medical Centre, Harvard Medical School, Cambridge, MA, USA.
J Cutan Pathol 1997 Oct;24(9):543-52 Abstract quote
We have demonstrated a role for microvascular injury mediated by the membrane attack complex of complement (C5b-9) in the genesis of cutaneous lesions of dermatomyositis (DM) (1).
The purpose of this study is to revisit the immunofluorescent (IF) profile of DM, to further investigate the role of C5b-9 in the pathogenesis of cutaneous lesions, and to see if any features of the IF profile reliably distinguish DM from LE. Lesional skin biopsies from 24 patients with clinical findings characteristic of DM were received in formalin and in Michel's transport medium. Conventional light microscopy, and IF studies with antibodies monospecific for IgG, IgA, IgM, C3, fibrin and C5b-9 were performed. The control group comprised biopsies from 31 patients with well-documented LE.
A positive lupus band test (LBT) correlated highly with a diagnosis of LE, with a sensitivity of 64.5% and a specificity of 95.6% (p=0.001). The LBT was most sensitive in the setting of DLE and SLE and was least sensitive in the setting of SCLE.
The finding of vascular C5b-9 deposition correlated with a diagnosis of DM versus LE (p=0.001) although the false positive rate was 21.4%. The false negative rate was reduced when vascular C5b-9 was seen in the absence of antibodies to Ro, La, or RNP. While a negative LBT correlated with a diagnosis of DM (p=0.001), the specificity was only 64.5%. However, when it was seen in concert with C5b-9 along the DEJ, specificity was increased to 80.6% (p=0.001).
The presence of C5b-9 in vessels and along the DEJ in concert with a negative LBT was predictive of DM (p=0.001) with a specificity of 93.5%, sensitivity of 78.3%, a false positive rate of 10% and a false negative rate of 14.7%.
The combination of a negative LBT, vascular C5b-9 deposition and negative serology for Ro, La, and RNP was a predictor of DM versus LE with a sensitivity of 90.5%, a specificity of 96.8%, a false positive rate of 5% and a false negative rate of 6.2% (p=0.001).
The IF profile of DM in lesional skin comprises a negative LBT, deposition of C5b-9 within vessels and along the DEJ, and variable keratinocyte decoration for IgG and C5b-9. The most statistically powerful predictor of DM is the combination of a negative LBT with vascular C5b-9 deposition and negative serology for antibodies to Ro, La, Sm, and RNP. Demonstration of a negative LBT in all but 1 case of DM suggests that the DEJ is not a primary site for antigen-antibody interaction.
We postulate that the aforementioned IF findings reflect humorally mediated injury of endothelium and keratinocytes, effected by C5b-9.
DUST-LIKE PARTICLES Antibodies to Ro (SS-A) may have dust-like particles caused by IgG
Usually cytoplasmic and nuclear within the epidermal basal cells
Present within 8/12 (67%) patients with Sjogren's syndrome.
Present in 50% of SCLE patients, usually with a finely granular pattern
In vivo antinuclear antibody of the skin: diagnostic significance and association with selective antinuclear antibodies.
Velthuis PJ, Kater L, van der Tweel I, Meyling FG, Derksen RH, Hene RJ, van Geutselaar JA, Baart de la Faille H.
Department of Dermatology, Utrecht University Hospital, The Netherlands.
Ann Rheum Dis 1990 Mar;49(3):163-7 Abstract quote
Immunofluorescence microscopy of the skin has disclosed antibodies bound to epidermal cell nuclei in several connective tissue disorders.
To establish the diagnostic potential of this phenomenon the results of immunofluorescence microscopy of biopsy specimens from 1651 subjects with various diseases and from 315 patients with systemic connective tissue disorders and related diseases were reviewed. It was found that the predictive value of the phenomenon for the presence of a systemic connective tissue disorder was, in general, 88%. Except for the homogeneous and thready patterns, which seldom appear, but are specific for SLE, in vivo antinuclear antibody (ANA) does not discriminate better between the various disorders than do serum antibodies.
The presence of in vivo ANA in the skin was related to serum antibodies against non-histone nucleoproteins, but not to anti-dsDNA antibodies. Combined with the finding that antibodies against non-histone nucleoproteins can bind on the surface of human keratinocytes, this suggests that ANA of the skin occurs in vivo.
DIFFERENTIAL DIAGNOSIS CHARACTERIZATION Disorders Excluded with Negative DIF IgA pemphigus
IgA vasculitis/Henoch-Schonlein Purpura
Disorders with Negative/Nonspecific IF Subcorneal pustulosis
Bullous insect bite
Bullous drug eruption
Lichen plano pilaris
Drug induced lichenoid photodermatitis
Non-IgA associated vasculitis
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