Evaluation of salt split technique of immunofluorescence in bullous pemphigoid

Introduction

Bullous pemphigoid is an autoimmune subepidermal blistering disorder characterized by outoantibodies to the basement membrane zone (BMZ). These are detected as a linear band of fluorescence, along the basement membrane zone, of C3 in 100%, IgG in 90-95% and less commonly IgM, IgA on direct immunofluorescence (DIF). [1] Circulating autoantibodies are detected in around 60-80% of cases on indirect immunofluorescence (IIF). [1] Recent studies suggest that skin which has been split through the BMZ using 1 mol/L sodium chloride to create an artificial blister is a more sensitive substrate than intact skin for this purpose. [2],[3] The present study was undertaken with the aim of evaluating this recently introduced modification and comparing it with the routine method of immunofluorescence in bullous pemphigoid.

Materials and Methods

Thirty - two cases which were clinically and histologically bullous pemphigoid were included in the study. Serum samples were available in 25 cases. The samples for both routine and salt split method were collected and processed in the following manner: [Figure - 1]

Direct immunofluorescence

A perilesional skin biopsy with 5mm punch was collected in Michel medium (transport medium) and stored at room temperature. Biopsy specimens were rinsed in Phosphate Buffered Saline (PBS) at pH 7.2 for 30 minutes. Snap freezing with embedding medium (Jung tissue freezing medium, Leica Instruments, Germany) was carried out in the cryostat at-20° C and 3-4 micron section obtained. A minimum of 3 sections were washed with PBS for 10 minutes thrice and air dried. A drop of specific regent-Fluorescein isothiocyanate (FITC) labeled antihuman IgG, IgM, IgA, complement C3 and fibrin were used. These slides were then incubated at 37°. C for 30 minutes in a moist chamber and then again rinsed with PBS (3 washing of 10 minutes each) to remove the conjugate. The slides were air dried and mounted with buffered glycerol (pH 8). Reading was done under the fluorescence microscope (Nikon model HB 1010 AF).

Indirect immunofluorescence:

Serum samples were available in 25 cases. These were stored at 4° C prior to testing. Substrate used was normal human skin tissue, obtained from the plastic surgery department. The skin was transported in saline, rinsed briefly in PBS. [Figure - 2]

For routine method the tissue was directly mounted in embedding medium, snap frozen and 4 micron sections taken. Initial screening dilutions of sera in titres of 1:10 and 1:100 were made with PBS. Few drops of the dilutions of sera were poured on labeled slides and incubated at 37° C for 30 minutes in a moist chamber. Second incubation of the slides with FITC tagged anti human IgG was done for 30 minutes. The slides were washed, air dried and mounted with buffered glycerol and results read under the fluorescence microscope. Higher dilution was tested if sera was positive on 1 : 100. The fluorescence titre was determined as the highest dilution of serum giving visible fluorescence under the microscope. [Figure - 3]

Skin separation method

For salt split, the method of obtaining tissue samples, transportation and processing remains the same for both DIF and IIF Tissue samples, if stored in Michel medium, were rinsed well with PBS for 30 minutes. Incubation in 10-15ml1 mol/ L sodium chloride at 40 C for 72 hours was done. The epidermis was then separated from the dermis with use of fine forceps to tease it gently. The specimens were processed in the same manner as described above to obtain 4 micron sections. DIF and IIF staining was carried out as above. The fluorescence was graded as follows:

1+

When seen only in high power (40 X)

2+

When seen with low and high power (20X & 40X)

3+

When seen even on scan-ner view (1 OX) [Figure - 4]

Results

Direct immunof-luorescence showed 100% positivity with both methods. C3 alone or in combination was found in 90. 60% cases on routine method. IgG deposits alone were seen in 2 cases. With salt the most common pat-tern was combined roof and floor in 50% cases. Roof alone was noted in 40. 6% and floor in 9. 4%. With salt split method an increase in the intensity of fluorescence was noted [Table - 1]. On routine method it was additionally noted with IgG in 7 cases. With salt split DIF, the strength of fluores-cence increased or remained the same but never decreased. Additional immunoreactant deposits were seen in 5 cases using split technique (15. 6%). They were IgG in 3, IgM in 2 and IgA in a single case [Table - 2].

On IIF by routine method positivity was 36%. This positivity was increased to 68% by salt split technique (p< 0. 05, chi squre test). There was increase in the titres in 2 cases from 1:80 to 1:100 and 1:20 to 1:40. The pattern of fluorescence on DIF and IIF with salt split method remained consistent.

Discussion

Salt split technique of immunofluores-cence has recently been described in evaluation of subepidermal blistering disorders. [2],[1] We under-took this study to evaluate this technique in our set up for bullous pemphigoid. Routine DIF positivity of 100% persisted with salt split method. This fig-ure is similar to that reported by Beutner et al. [1] On routine DIF, intensity of fluorescence increased but never decreased. Maximum intensity was ad-ditionally noted with IgG on split tissue testing. Enhanced intensity of fluorescence as observed has been shown in [Table - 1]. This shows that incu-bation of tissue in salt solution does not result in reduction of fluorescence in terms of both positiv-ity and intensity. With salt split method the pattern of linear roof and floor fluorescence was noted in maximum cases. Floor pattern was noted in 3 cases. Studies have shown that roof, roof and floor occur in bullous pemphigoid frequently. [3],[4] Floor pattern has rarely been reported.[4],[5],[6] This could be explained by heterogeneity of bullous pemphig-oid antigen and steric modification of the antigen due to such treatment, resulting in floor pattern. [7] This makes differentiation of bullous pemphigoid from epidermolysis bullosa acquisita (EBA) diffi-cult. However these can be differentiated by elec-tron microscopy, immunoelectron microscopy or response to therapy. [7] One study carried out re-cently has described the use of toad skin to differ-entiate between bullous pemphigoid and EBA when floor pattern is present. [7] In our study all 3 patients with dermal pattern of fluorescence were females with mild, moderate and severe disease respec-tively. No specific correlation, clinical and histo-pathological could be found. Additional immun-oreactant deposition was found in a total of 5 cases comprising IgG, IgM, IgA. No references regard-ing such a finding could be found. We think that this could be due to splitting resulting in exposing or making the BMS deposits more available for detection thereby increasing the sensitivity of the test.

On IIF with salt spilt the positivity increased to 68% from 36% from on routine method. Various studies have shown increased sensitivity of salt split skin as a substrate for IIF[8],[9] In addition, 2 cases showed increase in the titre of circulating autoantibodies when using split skin as substrate have been reported. [8] It has been hypothesised that sodium chloride therapy of substrate exposes or chemically modifies the BMZ antigens so that they react avidly with autoantibodies in the patients′ sera. [8] Pattern on salt split IIF was consistent with that noted on salt split DIF. In cases of mixed pattern, fluorescence was more intense or equal in intensity to dermal fluorescence but never less.

Salt split technique in immunofluo-rescence is an additional tool to study and further elucidate immunopathology of bullous pemphigoid. Salt split skin as a substrate for IIF shows higher sensitivity in detecting circulating antibodies. The procedure is cheap, easy to carry out and does not create any extra burden on the immunofluorescence lab and should be employed as a routine in immunofluorescence study of patients with bullous pemphigoid.