Indian Journal of Dermatology
: 2013  |  Volume : 58  |  Issue : 1  |  Page : 22--25

Direct immunofluorescence in cutaneous vasculitis: Experience from a referral hospital in India

BN Nandeesh, Rajalakshmi Tirumalae 
 Department of Pathology, St. John's Medical College, Bangalore, India

Correspondence Address:
B N Nandeesh
Assistant Professor, Department of Pathology, St. John«SQ»s Medical College, Bangalore - 560 034


Background : Cutaneous vasculitis is commonly recognized and biopsied, owing to ease of access. Most biopsies are also subjected to direct immunofluorescence (DIF), though the rates of positivity vary. This is an attempt to assess the utility of DIF and glean data that will help optimize the test. Objective : To assess the diagnostic utility of DIF in cutaneous vasculitis. Materials and Methods : All cases of suspected cutaneous vasculitis submitted for DIF between 2004 and 2010 were included. Clinical data, histopathologic diagnosis, DIF findings and additional tests such as anti nuclear antibody (ANA), anti neutrophil cytoplasmic antibody (ANCA) (where done) were noted. Results: There were 198 patients in the study group, with a female predominance. Purpura was the commonest clinical presentation. Extracutaneous involvement was noted in 29% of patients«SQ» i.e., joint pain, abdominal pain and hematuria. Leukocytoclastic vasculitis was the commonest histologic diagnosis. DIF showed an overall positivity of 39% (n = 77) with C3 in 26% (n = 52) and IgA in 23% (n = 46) cases. Forty one cases of suspected Henoch Schonlein Purpura (HSP) showed IgA positivity. The timing of biopsy ranged from <3 days to six months, with 38% being done within seven days. DIF was positive in 86% of biopsies performed within seven days of onset of lesions. Sixty percent of patients with extracutaneous manifestations showed deposits. Vascular deposits were also noted in dermatitis herpetiformis, dematomyositis and prurigo. Conclusion : DIF positivity is strongly influenced by the timing of the biopsy and the presence of extracutaneous features. Its clinical value is greatest in patients with HSP, being contributory in 90% of cases. Vascular deposits may be seen in non-vasculitic conditions and need clinicopathologic correlation.

How to cite this article:
Nandeesh B N, Tirumalae R. Direct immunofluorescence in cutaneous vasculitis: Experience from a referral hospital in India.Indian J Dermatol 2013;58:22-25

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Nandeesh B N, Tirumalae R. Direct immunofluorescence in cutaneous vasculitis: Experience from a referral hospital in India. Indian J Dermatol [serial online] 2013 [cited 2022 Jun 25 ];58:22-25
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Vasculitides represent a group of disorders characterized by inflammation of the blood vessel wall. [1] Cutaneous vasculitis (CV) comprises a wide spectrum of diseases that involve predominantly the blood vessels and surrounding tissues of the skin. The vasculitis may be idiopathic or with an identifiable cause such as drugs, infection or connective tissue disease. Cutaneous involvement in vasculitis may be primary or reflect a potentially fatal systemic disease. Cutaneous vasculitis is commonly recognized and biopsied, owing to ease of access. Most biopsies are also subjected to direct immunofluorescence (DIF), though the rates of positivity vary. Histopathologic examination is essential for confirmation and classification of vasculitis. [2] DIF, though deemed sensitive, has variable yield and is influenced by several factors. The positivity rates are highest in early disease, with most studies using a 48-hour cutoff period. [3],[4]

However, in practice, biopsies from much older lesions are sent for DIF, especially in our setting. This study attempts to understand the relationship of the positive yield of DIF in patients with vasculitis with the timing of the sample and various clinical parameters. The data gleaned from this exercise will help optimize the test.

 Materials and Methods

The study included skin biopsies (of the lesional tissue) from patients of clinically suspected cutaneous vasculitis and biopsies that were sent for histopathology and direct immunofluorescence. The retrospective study extended over a period of six years between 2004 and 2010. Clinical details were noted, particularly the presence of extracutaneous features and time period between onset of symptoms and biopsy. The biopsies were transported in Michel's transport media. All biopsies were frozen in a cryostat and sectioned at 4 μm. Sections were incubated with fluorescein isothiocyanate (FITC) conjugated, Fc-specific F (ab) 2 antisera directed against IgG (1:30 diluted), IgA (1:30 diluted), IgM (1:30 diluted), and complement C3 (1:30) (Dako), respectively. A specimen was considered to be positive if granular deposits of one or more immunoreactants were found in the walls of one or more vessels. A quantitative scoring of the intensity was also assessed. The DIF findings from the skin biopsy were compared with duration of illness/timing of biopsy and with clinical manifestations. Wherever possible the most recent crop of the lesion was biopsied and healed lesions were preferably avoided. Statistical analyses were performed using SPSS for Windows version 16.0.


A total of 198 patients were studied. Women outnumbered men in a ratio of approximately 1.6:1 (122 female versus 76 male). The age of the patients ranged from eight to 82 yrs with a mean age of 29.6 years. Seventy percent of the patients were below 40 years. The biopsies were taken between less than 3 days (<72 hrs) to 6 months after the onset of lesions. The commonest site was lower extremity (n = 117, 47%) followed by upper extremity (n = 59). [Figure 1] show the distributions of lesions from which biopsy were taken. Palpable purpura was the main clinical finding seen in all the patients. Sixty six patients in addition showed extracutaneous manifestations [Table 1]. [Table 2] presents IF findings of skin biopsies. DIF showed an overall positivity of 39% (n = 79) with C3 in 26% (n = 52) and IgA in 23% (n = 46) cases. The timing of biopsy ranged from < three days to six months, with 110 (56%) being biopsied within 7 days of onset of lesions (25% being within 72 hrs) [Figure 2]. Histopathology showed leukocytoclastic vasculitis as the commonest diagnosis, observed in 66% of the patients, and lymphocytic vasculitis in 18%. Sixteen percent of the patients showed perivascular inflammation.{Figure 1}{Figure 2}{Table 1}{Table 2}

[Figure 2] shows relation of DIF findings with the timing of biopsy. DIF was positive in 85% of biopsies performed within seven days and 70% (forty six out of sixty six patients) of patients with extracutaneous manifestations showed deposits. Forty three out of 48 suspected cases (90%) of Henoch Schonlein Purpura showed positive staining for IgA (60% of these also showed C3 deposits). Four of the total cases studied, also showed immune deposits in the epidermal basement membrane zone in addition to blood vessels and they had positive antinuclear antibodies. Overall, positive anti-nuclear antibodies were detected in 28% of cases. ANCA was done in fifteen percent of cases, all of which were negative.


Cutaneous vasculitis is an inflammatory disease of dermal blood vessels that presents clinically as palpable purpura located primarily on the lower extremities. [4],[5],[6] It has many causes, although they result in only a few histologic patterns of vascular inflammation. Cutaneous vasculitis is the most commonly encountered vasculitic manifestation in clinical practice and is frequently biopsied. While histopathology is essential for confirmation and sometimes, in identifying the cause, DIF adds credence to the diagnosis. But the DIF result is affected by several factors. Despite knowledge of these limitations, most biopsies are subjected to DIF. It is necessary to evaluate the utility of DIF in such cases and help triage cases where it might be beneficial.

A few earlier studies and our current results shed new light on the question whether DIF results yield contributory results in cutaneous vasculitis. In the present study, a substantial number of skin biopsies showed no immune deposits on DIF, which is routinely done with the belief of confirming vasculitis. Gaffo AL has supported this view. [7] However, very few studies have actually tried to compare the general positivity in the routine biopsies of skin for vasculitis, a few of which are depicted in [Table 3]. [1],[8],[9],[10]{Table 3}

One of the important reasons for a low positive DIF results in the present study is patient selection that is, varying time period of biopsy after the onset of the lesions, reflecting the practical situation in a general dermatology practice. Previous studies indicated in the table had definite patient selection criteria as their objective was not specific to test DIF positivity in vasculitis. Also, the age of the lesions at biopsy in most of these studies varied from one to seven days. The importance of timing is paramount. [11] A biopsy specimen taken too late (i.e., after 48 hrs) may show more of the pathology of repair than of the initial injury and may have negative DIF because immune deposits are degraded rapidly. [12],[13],[14],[15],[16] Nearly fifty percent of the cases in the present study had their biopsy done after 1 week of the onset of lesions. Also most of the patients are evaluated are not very sure of the exact duration of illness. The diagnosis of cutaneous vasculitis may be slightly delayed as patients tended to self-medicate in the initial phases of their disease or the condition may be misdiagnosed by primary level physicians who are unfamiliar with the presentation of cutaneous vasculitis. We observed that most of our patients had a history of drug intake for the lesions, which may have affected the DIF positivity. Also, the present study has a larger sample size. The most frequent deposit in the present study was C3, similar to other documented studies in the literature. The deposition is usually granular or fibrillar. [12],[14]

In our study, young adult women appeared to be primarily affected. The lower limbs are preferentially involved, and this is likely to be related to the effect of gravitational vascular stasis. It is important to remember that "non-specific trapping" of immunoglobulins in skin vessels occurs as a result of non-pathologic, intense autofluorescence of the vessels. Vessel wall deposition is not diagnostic of LCV and may be seen in biopsy specimens from the lower legs in patients without vasculitis. [17] If a patient with suspected vasculitis has lesions at sites other than the lower legs, it is preferred that the biopsy specimen be obtained from such lesions. The concomitant presence of immune deposits along the dermo-epidermal junction may suggest lupus erythematosus as an underlying cause. We also observed that a few of the non-vasculitic conditions like Dermatitis herpetiformis, Prurigo nodularis and dermatomyositis showed vascular immune deposits, but they were not included in the study as a clinical suspicion of vasculitis was not considered in them.

A diagnosis of LCV should not be made solely on the presence of positive DIF findings, nor should the diagnosis be excluded with a negative DIF test. The findings of DIF should be interpreted along with clinical, microscopic, and other laboratory findings. [12],[14]

Henoch Schonlein Purpura is described in children who have systemic involvement (gastrointestinal, renal, and articular) in addition to cutaneous lesions. The primary immunoglobulin deposited in HSP in both the skin and the kidney is IgA. [18] The prevalence rate of positive DIF in HSP is variable and likely reflects variation in the duration of the lesion. Ninety percent of our patients suspected to have HSP clinically showed IgA deposits. Several studies have investigated the frequency of IgA deposits in cutaneous blood vessel walls in both lesional and clinically normal skin. [19] A strong association exists between IgA deposits on DIF and HSP with the frequency of perivascular deposits of IgA in lesional skin ranging from 75% to 100%., but a subset of HSP cases always exists in which vascular IgA deposition in the skin appears to be negative. Also the presence of the same may not correlate with disease activity. [20] Previous DIF studies showed positive findings mainly in the early stage of the disease. [15],[21],[22],[23]

Ryan [24] believes that the 'vulnerability status' of the vessel determines the development of vasculitis rather than the mere presence of immune complexes. In his model, noxious substances are likely to localize in blood vessels which have sustained microvascular injury with a consequent change in their structure. He predicts that vessels under 'stress' e.g., in areas of gravitational stasis such as the legs, and wounds and scars, are more likely to be affected by vasculitis. Also the presence of IgA may not be very specific, but will be helpful in providing a diagnosis of HSP. [25] When there are atypical clinical features. [25],[26]

It has been suggested that a paucity of immune complexes suggests the presence of an ANCA-Small Vessel Vasculitis (SVV), although many would argue that, because of degradation, the level of immunoreactants is simply inversely correlated with the age of the lesion. Perhaps the presence of high levels of immunoreactants helps to rule out an ANCA-SVV, although this is controversial. [16] Kawakami [27] has proposed a new algorithm for the evaluation of vasculitis where in the clinical delineation of small and medium sized vasculitis can be recognized and the first step in the evaluation would be testing for ANCA. Unfortunately, in our present setting we encountered ANCA being tested only in ten percent of the patients. Use of DIF only comes later down the workup especially for confirming/ruling out a diagnosis of HSP and patients presenting with extracutaneous manifestations and atypical features.

In conclusion, direct immunofluorescence of lesional skin is a useful ancillary diagnostic tool in the evaluation of cutaneous vasculitis (provided that there is optimal Clinicopathological interpretation), but is negative in a significant number of cases, especially when samples are taken more than seven days after onset of lesions. The value of DIF is greatest in HSP (contributory in 90% of cases). Timing of biopsy and presence of extra-cutaneous manifestations is a reliable guide to selection of patients for DIF. Indiscriminate use of the test should be avoided and the same resources can be directed to other tests that guide treatment.


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