direct immunoflourescence ( dif ) has become an indispensable diagnostic tool in the diagnosis of immunobullous lesions of the skin and has been widely used to supplement the clinical and histologic features of various vesiculobullous disorders .
the utility of this technique is limited by cost , site , and time of the biopsy , technical and tissue processing factors , history of treatment and the nature of the disease .
this study was undertaken to set up the immunofluorescence facility in our department and to evaluate the practical utility of dif in immunobullous dermatoses at our institution as a university granted research project .
the objectives of this study was to evaluate the sensitivity of dif in immunobullous dermatoses and to study the pattern of dif .
the study also aimed to correlate dif with clinical and histologic findings and to analyze discrepancies .
a cross - sectional study was undertaken at the department of pathology and dermatology of a tertiary care hospital in western india from august 2010 to september 2012 .
one hundred patients , of any age and sex , with strong clinical suspicion of an immunobullous lesion were included in the study .
patients with a known infective etiology of the bullous lesions were excluded . after taking written informed consent of the patients ,
two biopsies were performed in each case ; one from the fully developed vesiculobullous lesion ( lesional biopsy ) and the other from the perilesional area within 2 cm diameter of the lesion ( perilesional biopsy ) .
histopathological examination ( hpe ) finding were used as a further aid in the diagnosis whereas the perilesional biopsy , placed in normal saline / phosphate buffered saline was immediately taken to the laboratory for dif .
the tissue was embedded in the cryostat embedding medium , frozen , and cut at 5 m thickness at 22c .
sections were taken on poly l lysine coated glass slides , fixed with ether - alcohol mixture , and air dried for 10 min .
later the slides were rinsed in phosphate buffered saline at ph 7.3 for 15 min .
the sections were treated with fluorescein isothiocyanate labeled and optimally diluted ( 1:40 ) antisera , i.e. , igg , iga , igm , and c3 respectively .
the slides were incubated in wet chamber for 1 h in dark room , washed with phosphate buffered saline pbs and mounted with glycerine jelly .
dif was reported based on nature and site of immune deposits , semi quantitative grading of strength of fluorescence and pattern of immune complex deposits .
statistical methods used to analyze this study include , percentages , ratio , sensitivity , specificity , and positive and negative predictive values .
statistical methods used to analyze this study include , percentages , ratio , sensitivity , specificity , and positive and negative predictive values .
ages of our patients ranged from 16 years ( a girl diagnosed as pemphigus vulgaris ) to 87 years ( a male diagnosed as bullous pemphigoid [ bp ] ) with a mean age of 57 years . the maximum ( 33% ) cases belonged to the age group of 41 - 50 years , pemphigus group being maximum ( 27% ) .
fluid filled lesions ( 95% ) were the most common clinical finding followed by erosion and crusting ( 73% ) . itching ( 63% ) was the most common symptom followed by pain ( 57% ) and burning ( 55% ) .
subcorneal blister was seen in 12% , subepidermal in 28% , and suprabasal in 60% of the cases .
histopathological diagnosis of various immunobullous lesions [ figures 1a , 1b , 3a 
 4a , 5a , 6a , 7a , 7b ] along with corresponding dif findings are depicted in table 1 .
( h and e , 100 ) , ( b ) blister contains eosinophils and few neutrophils .
h and e , 400 ) direct immunoflourescence of pemphigus vulgaris ( a ) lacelike intercellular space deposition of igg , ( b ) lacelike ics deposition of c3 pemphigus foliaceus ( a ) subcorneal blister with dyskeratotic acantholytic granular cells .
secondary cleft formation seen in the mid - level of epidermis ( h and e , 100 ) , ( b ) direct immunofluorescence shows intercellular space deposition of igg in upper dermis bullous pemphigoid ( a ) subepidermal blister formation with an inflammatory infiltrate in the bullous cavity .
( h and e , 100 ) , ( b ) direct immunoflourescence shows linear deposition of igg in the basement membrane zone linear iga bullous dermatosis ( a ) neutrophils along the dermo - epidermal junction ( h and e , 400 ) , ( b ) direct immunoflourescence showing linear deposits of iga along the dermoepidermal junction dermatitis herpetiformis ( a ) subepidermal bulla ( h and e , 400 ) , ( b ) direct immunoflourescence showing granular deposits of iga along the dermal papillae pemphigus erythematosus ( a ) subcorneal bulla containing acantholytic cells .
( h and e , 100 ) , ( b ) subcorneal bulla contains neutrophils .
( h and e , 100 ) histopathological diagnosis and dif findings of immunobullous lesions on dif examination , intercellular space ( ics ) deposition of igg was present in 51 out of 58 cases of pemphigus vulgaris resembling fishnet pattern [ figure 2a ] .
all the cases of pemphigus foliaceus showed ics deposition of igg in upper dermis [ figure 3b ] .
majority ( 18/25 ) of bp showed linear igg and c3 deposition in the basement membrane zone ( bmz ) [ figure 4b ] .
single patient of linear iga disease showed linear deposition of iga along the bmz [ figure 5b ] .
both cases of dermatitis herpetiformis ( dh ) had granular iga deposited along the bmz [ figure 6b ] .
single case of pemphigus erythematosus showed granular deposition of igg at ics and also showed igm deposition along the bmz [ figures 8a and b ] .
direct immunoflourescence of pemphigus erythematosus ( a ) deposition of igg in intercellular space ( arrow ) , ( b ) granular deposition of igm at dermoepidermal junction ( arrow ) as regards the 11 dif negative cases , seven were of pemphigus vulgaris ( three had no epidermis ) and four were in remission being on long - term cyclophaphamide pulse and steroid therapy .
negative immunofluorescence may be viewed as the state of immunological remission in pemphigus because most patients with negative immunofluorescence did not have relapse after discontinuation of the treatment .
a total of 4 cases with specific histopathological findings of bp had negative dif due to lack of epidermis in one and technical / sampling errors in the three cases .
dif was diagnostic in all the pemphigus variants including the pemphigus foliaceus ( 12% ) and the pemphigus vegetans ( 1% ) .
dif also helped to confirm the diagnosis of dh , linear iga dermatosis and pemphigus erythematosus in these solitary cases respectively .
the autoimmune bullous lesions result from an immune response against adhesion molecules of epidermis or bmz .
there is clinical overlap among various groups of bullous diseases for example : linear iga dermatosis may mimic bp or dh .
hpe reveals the site of formation of bulla , the presence of infiltrate and its composition .
a differential diagnosis is generated on the basis of combination of findings in the biopsy specimen .
subepidermal bullous diseases have overlapping hpe findings and hence their diagnosis on hpe findings alone is difficult . at present ,
diagnosis of autoimmune bullous disorders is based on immunological and molecular findings rather than clinical and hpe diagnosis alone .
the ideal site for the biopsy specimen depends on the type of disorder . for bullous diseases
, dif is performed using the perilesional skin that is normal appearing skin immediately adjacent to a lesion .
the immune deposits are partially or completely degraded in inflamed or blistered skin and dif may be negative .
the differential diagnosis of a dif test depends on primary site of immune deposition , the class of immunoglobulin or other type of immune deposits , number of immune deposits and if multiple , the identity of the most intense deposits and deposition in other sites besides the main site . with these parameters ,
the most frequent disorder in our study was pemphigus vulgaris and the finding is in accordance with other regional and international studies .
male to female ratio was 1:1.7 , which is comparable to the findings of shamim et al .
dif findings in pemphigus vulgaris showed igg positivity in ics in fishnet pattern in 51% .
dif is positive in 90 - 100% of patients with active disease if an appropriate biopsy specimen has been obtained .
occasionally , the fluorescence may be limited to or most intense at the level of the epidermis that is involved with blister formation that is lower epidermal layer for pemphigus vulgaris and superficial epidermal layers in pemphigus foliaceus .
this variation in the intensity of fluorescence at various layers of the epidermis may be caused by differences in the relative amounts of the target desmosomal proteins for each of the two diseases , namely desmoglein 1 for pemphigus foliaceus , and desmoglein 3 for pemphigus vulgaris .
immunofluorescence is required to differentiate staphylococcal scalded skin syndrome ( ssss ) from pemphigus foliaceus , since a small number of acantholytic cells are seen in case of the former ssss .
the lesions of pemphigus foliaceus can become impetiginized , which produces pustules as in impetigo and subcorneal pustular dermatosis .
pemphigus vulgaris and vegetans have similar dif findings and hence need to be differentiated by clinical and hpe characteristics .
the combination of ics and bmz deposition of igg is seen in pemphigus erythematosus , in which immunopathologies of pemphigus foliaceus and lupus erythematosus co - exist .
we had one such case , where the patient had clinical features of systemic lupus erythematosus , her rheumatoid factor and anti - nuclear antibody tests were positive and dif revealed findings of pemphigus and lupus erythematosus .
thus , dif helped to confirm the diagnosis of senear - usher syndrome , a rare variant of pemphigus foliaceus .
bp affects the elderly during their fifth to seventh decade of life , with an average age of onset being 65 years .
nearly , 48% of the patients were in the age group of 61 - 70 years .
the reported sex ratios in bp have varied from 2.4:1 to 1:5.75 in different studies due to variation in the sample size . in our present study ,
herpes gestationis , linear iga dermatosis and epidermolysis bullosa acquisita show subepidermal bulla with neutrophil rich infiltrate , which can be confused with bp . however , dif helps to differentiate these conditions from bp . in our present study ,
only two cases of dh were seen both being males aged 37 years and 38 years .
nicolas et al . reported dh most commonly between the age of 25 - 55 years .
inchara reported four cases of dh . but none of the four biopsy proven cases showed immunoglobulin deposits . in absence of characteristic , dif pattern they recommended to use a combination of the clinical and the serological data .
differential diagnosis for deposition of igg and/or c3 at the bmz includes bp , cicatricial pemphigoid , pemphigoid gestationis , epidermolysis bullosa acquisita , and bullous systemic lupus erythematosus .
c3 deposits with significantly higher intensity than igg favor the pemphigoid group of diseases such as bp , cicatricial pemphigoid , and pemphigoid gestationis . in bp and pemphigoid
gestationis , the deposition consists predominantly and occasionally exclusively ( especially in pemphigoid gestationis ) , of c3 .
they are present in continuous , fine , and linear patterns . in bullous systemic lupus erythematosus ,
approximately 60% of cases reveal bmz deposition of igg indistinguishable from that of epidermolysis bullosa acquisita .
differentiation between bullous systemic lupus erythematosus and epidermolysis bullosa acquisita is based on the presence of serological evidence of systemic lupus erythematosus . in these subepidermal bullous lesions , dif / indirect immunoflourescence if on salt split skin yields higher rate of positivity in contrast with conventional dif
. in bp , patterns of fluorescence was in the roof ( 40.60% ) , floor ( 9.4% ) , and combined roof and floor ( 50% ) . on indirect immunoflourescence , positivity was almost doubled with salt split technique ( 68% ) as compared to routine methods ( 36% ) .
our study revealed 90% ppv and 94% sensitivity and the sensitivity was 84% in bp .
false negativity was seen in 7/58 cases of pemphigus vulgaris and 4/25 cases of bp group .
this was mainly due to technical errors like lack of epidermis in the biopsy ( n = 4 ) , exposure to light ( n = 1 ) , improper site of the skin biopsy ( n = 1 ) and longer stay of the biopsy in the normal saline ( n = 1 ) .
total 4 histologically proven cases of pemphigus vulgaris , who were on the treatment revealed the dif negativity .
this finding was against the previous documentation , which states that dif is a very reliable diagnostic test for pemphigus , which becomes positive at an early stage and remains positive for a long period after clinical remission .
hence , in the absence of the characteristic dif pattern , one needs the combination of clinical , histologic , and immunologic data to support the definite diagnosis of different immunobullous disorders .
however , a diagnosis based solely on the clinical or histologic findings may not be accurate .
dif is extremely helpful in confirming a suspected diagnosis and in distinguishing among closely related cases of immunobullous lesions .
our study concludes that the dif is an essential for diagnosing autoimmune blistering diseases with the clinical and the histopathological overlap .
however , larger studies with proper selection of cases and judicious use are mandatory to optimize its utility . 

dif helped in confirmimg the diagnosis of bullous lesions with the clinical and histopathological overlap.in this study , a rare case of senear - usher syndrome could be diagnosed due to dif.sampling errors contributed to fn results . 
 
dif helped in confirmimg the diagnosis of bullous lesions with the clinical and histopathological overlap . in this study , a rare case of senear - usher syndrome could be diagnosed due to dif .
 background : autoimmune blistering diseases are a group of bullous disorders characterized by pathogenic antibodies directed at the target antigens , which are components of the desmosomes or adhesion complex at the dermoepidermal junction . 
 direct immunofluorescence ( dif ) is invaluable in the diagnosis of these lesions.aim:the aim of this study was to evaluate the sensitivity of dif in immunobullous dermatoses and to study the pattern of dif . 
 the study also aims to correlate dif with clinical and histologic findings and to analyze discrepancies.materials and methods : total 100 biopsies received over a period of 2 years in the department of pathology were analyzed . 
 dif , histopathology and clinical data were reviewed.results:out of 100 , 89 cases showed dif patterns concordant with clinical / histologic diagnosis . 
 the sensitivity of dif was 94.44% ( 51/58 ) in the pemphigus and 84% ( 21/25 ) in the bullous pemphigoid ( bp ) group , 100% each in dermatitis herpetiformis ( 2/2 ) and linear iga disease ( 1/1 ) . 
 a total of 11 histologically proven cases of immunobullous lesions were dif negative - four ( three of pemphigus vulgaris and one of bp ) due to having no epidermis , three ( cases of bp ) owing to sampling / technical errors and the remaining four ( cases of pemphigus vulgaris ) due to being on treatment.conclusion:immunofluorescence helps confirm the diagnosis of bullous lesions in which there is clinical and the histopathologic overlap . 
 sampling errors contributed to false negative ( fn ) results . 