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E-IJD® - ORIGINAL ARTICLE
Year : 2017  |  Volume : 62  |  Issue : 2  |  Page : 226
Clinicopathological significance among patients with in vivo epidermal nuclear staining by direct immunofluorescence study


Department of Dermatology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

Date of Web Publication9-Mar-2017

Correspondence Address:
Papapit Tuchinda
Department of Dermatology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok
Thailand
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0019-5154.201749

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   Abstract 

Context: In vivo epidermal nuclear staining (ENS) can be found in patients with autoimmune connective tissue diseases (CNTDs) and other diseases. Aims: The aim of this study was to reveal the underlying diseases and direct immunofluorescence (DIF) characters of patients with in vivo ENS and association of in vivo ENS with circulating autoantibodies. Settings and Design: A retrospective analysis was conducted involving skin biopsy specimens submitted for DIF study at the Dermatoimmunology Laboratory at Siriraj Hospital between 2002 and 2012. Subjects and Methods: The findings of DIF study, clinical manifestations, and diagnosis of patients who had positive ENS were investigated. Statistical Analysis Used: The SPSS software version 18.0. Descriptive statistics were used to report demographic data, clinical characteristics, and laboratory investigation results. Moreover, Chi-squared test or Fisher's exact test were used to compare the categorical variables. Results: One hundred and thirty-eight out of 3735 submitted specimens (3.7%) showed positive ENS. The most common diagnosis was CNTD (79%) followed by vasculitis (10.1%). Lupus erythematosus was the most common diagnosis among CNTD (78%). No association between levels of serum antinuclear antibodies (ANA) titer and intensity of ENS (P = 0.660). However, we found that patients with positive in vivo ANA had lower prevalence of systemic involvement. Conclusions: Although lupus erythematosus was the most common diagnosis among patients with in vivo ENS, the presence of ENS does not indicate any specific diagnosis. However, patients with ENS tend to have less systemic involvement.


Keywords: Autoantibodies, epidermal nuclear staining, in vivo antinuclear antibody, in vivo epidermal nuclear staining autoantibodies


How to cite this article:
Sirikudta W, Tuchinda P, Chularojanamontri L, Kulthanan K, Pinkaew S. Clinicopathological significance among patients with in vivo epidermal nuclear staining by direct immunofluorescence study. Indian J Dermatol 2017;62:226

How to cite this URL:
Sirikudta W, Tuchinda P, Chularojanamontri L, Kulthanan K, Pinkaew S. Clinicopathological significance among patients with in vivo epidermal nuclear staining by direct immunofluorescence study. Indian J Dermatol [serial online] 2017 [cited 2017 May 26];62:226. Available from: http://www.e-ijd.org/text.asp?2017/62/2/226/201749

What was known?
In vivo epidermal nuclear staining can be found in about half of patients with connective tissue diseases.



   Introduction Top


Direct immunofluorescence (DIF) study of the skin biopsy specimen is the useful method to diagnose autoimmune vesiculobullous diseases, connective tissue diseases (CNTDs), and vasculitis. The presence of in vivo epidermal nuclear staining (ENS) represents the antinuclear antibody (ANA) deposition in the nucleus of keratinocytes which is usually found in 55% of patients with CNTD.[1],[2] Most of the previous publications about in vivo ENS were conducted among Caucasians and the number of patients included in studies is limited.[3],[4],[5],[6],[7]

Thus, this study aimed to reveal the characters of in vivo ENS among Thai patients and to find the association of in vivo ENS with circulating autoantibodies and correlated clinical manifestations.


   Subjects and Methods Top


Ethical approval was granted by Siriraj Institute Review Board, Siriraj Hospital, Mahidol University, Bangkok, Thailand. We retrospectively reviewed the data records of skin biopsy specimens submitted for DIF study at Dermatoimmunology Laboratory at Siriraj Hospital between January 2002 and December 2012. Those with immunoglobulin (Ig) and/or complement deposition at epidermal nucleus were included in this study. Clinical and immunological profiles of patients were reviewed. Definite diagnosis was based on clinical, histopathological, and immunofluorescent findings. If CNTD was suspected, the work up of such patients was done such as anti-Smith antibody, anti-ribonucleoprotein antibody, anti-double stranded DNA antibody, anticardiolipin antibody, antinuclear cytoplasmic antibody, anti-Ro antibody, anti-La antibody, lupus anticoagulant, and anti-Scl70 antibody. Patients without final definite diagnosis were excluded from the study.

DIF was done according to the standard method described previously.[8] Skin biopsy specimens were fixed with acetone at 2°C–8°C for 10 min and air-dried. These specimens were then washed twice with phosphate buffer saline pH 7.2 for 10 min. Fluorescein isothiocyanate-conjugated rabbit antihuman IgG, IgA, IgM, C3, and fibrinogen (Dako Patt, Copenhagen, Denmark) (catalog number: F 0202, F 0204, F 0203, F 0201, and F 0111, respectively) were added. Dilution factor for IgG was 1:80. The dilution factor for other Ig, complement and fibrinogen was 1:40. Then, specimens were incubated in a moist chamber at room temperature for 30 min, and excess antibodies were washed off with phosphate buffer saline for 10 min for two times and mounted by mounting medium. The specimens were analyzed under an immunofluorescence microscopy. Interpretation included the presence and intensity of staining at structure of skin (intercellular space, basement membrane zone (BMZ), superficial and deep blood vessels, appendages, colloid bodies, and epidermal nucleus). The intensity was graded in three levels (1+, 2+ and 3+). The presence of any Ig and/or complement at epidermal nucleus meant positive in vivo ENS.

Statistical analysis was done using the SPSS software version 18.0. Descriptive statistics were used to report demographic data, clinical characteristics, and laboratory investigation results. Moreover, Chi-squared test or Fisher's exact test were used to compare the categorical variables.


   Results Top


The total number of 3735 specimens were submitted for DIF at Dermatoimmunology Laboratory at Siriraj Hospital during January 2002 and December 2012. One hundred and thirty-eight specimens which showed positive ENS were included in this study [Figure 1]. The majority of patients with in vivo ENS were women (90.6%) and age of onset of disease ranged from 12 to 82 years. Mean age of onset were 42.5 years. CNTD was the most prevalent diagnosis (79.0%) followed by vasculitis (10.1%). Among patients with CNTD, systemic lupus erythematosus (SLE) was the most common diagnosis followed by cutaneous lupus erythematosus (CLE) [Table 1]. Other definite diagnoses included eczema, lichen planus, exfoliative dermatitis, psoriasis, vitiligo, erythema nodosum, sweet's syndrome, subcutaneous panniculitis-like T-cell lymphoma, sarcoidosis, granuloma annulare, erythema multiforme, linear morphea, follicular mucinosis, and alopecia areata.
Figure 1: Immunoglobulin G deposited in the nucleus of keratinocytes (epidermal nuclear staining) (DIF, ×40)

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Table 1: Demographic data of patients with in vivo epidermal nuclear staining (n=138)

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Of 126 patients, serum ANA was detected in almost all patients (124 patients, 98.4%) with titers varying from 1:40 to 1:2,560. Most patients had high serum ANA titer (1:640 or higher). Speckled pattern was the most frequent ANA pattern in serum (82.8%). Homogenous, rim, and nucleolar pattern were detected in 32.8%, 13.1%, and 12.3% of tested patients, respectively. Intensity of ENS among patients with high serum ANA titer was 1+ in 91.7%, 2+ in 7.3%, and 3+ in 1%, respectively. All patients with serum ANA titer lower than 1:640 had 1+ intensity ENS. There was no statistically significant association between serum ANA titer and intensity of ENS (P = 0.660) [Table 2]. Among 124 patients with positive serum ANA, 63.7% had either SLE or CLE, 16.1% were diagnosed with other CNTD than SLE/CLE, and 10.5% had vasculitis. Two patients who had negative serum ANA were female aged 27 and 38 years old, respectively. They were diagnosed with localized CLE without any systemic involvement. Other serum autoantibodies found in patients with in vivo ENS are shown in [Table 3].
Table 2: Serum antinuclear antibodies titer in patients with in vivo epidermal nuclear staining (n=126)

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Table 3: Autoantibodies found in patients with in vivo epidermal nuclear staining

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The most common immunoreactant deposit at ENS was IgG (135/138 patients, 97.8%). Considering the location of immune deposition from DIF, half of patients who showed only ENS had SLE or CLE. If deposit also presented at both ENS and BMZ, the diagnosis of SLE or CLE was more likely (74.0%). The presence of ENS and superficial blood vessel staining was more prevalent in vasculitis and other CNTD than SLE or CLE (P = 0.002) [Table 4]. Deposition at DEJ or superficial blood vessel were not different in patients with SLE and CLE (P = 0.471 and 0.774, respectively).
Table 4: Diagnosis in patients with in vivo epidermal nuclear staining and staining at basement membrane zone and/or superficial blood vessels

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Among patients with SLE, cutaneous symptoms were the most common presentation (87.2%). Cutaneous presentations were discoid rash (54.8%), photosensitivity (45.2%), oral ulcer (42.9%), and malar rash (38.1%). One-third of patients had arthritis (33.3%). Hematologic and renal abnormalities were presented in 22.0% and 19.0%, respectively. Serositis and neurological involvement were infrequent (2.5% and 2.4%, respectively).


   Discussion Top


DIF in patients with CNTD, either from lesions or normal skins, usually reveals epidermal nuclear staining.[2],[4] This phenomenon was previously thought to be in vitro phenomenon resulted from high serum anti-ribonucleoprotein antibody titer.[9] However, later publication demonstrated that ENS was in vivo phenomenon and caused by penetration of antibodies through live mononuclear cells by Fc-receptor-mediated endocytosis.[10] Furthermore, binding of autoantibodies to antigen on epidermal cells leads to posterior internalization and antibodies can diffuse into living cells.[11]

The prevalence of in vivo ENS in CNTD varies among studies. ENS was found in 32%–66% of patients with SLE, 10.7%–100% of patients with mixed connective tissue diseases (MCTD), 33.3%–56% of patients with systemic sclerosis.[2],[5],[12] Wells et al. [4] studied 33 patients with positive in vivo ENS and reported that SLE was the most prevalent diagnosis (69.7%), followed by MCTD (9.1%), Sjogren's syndrome (6.1%), dermatomyositis (6.1%), progressive systemic sclerosis (6.1%), and rheumatoid arthritis (3.0%). In our study, SLE (43.1%) or CLE (34.9%) was also the most prevalent diagnosis [Table 1]. Taken together the results, it may be suggest that other evidence of autoimmune diseases should be sought in patients with positive ENS. Similar to previous studies, we also found that 98.4% of patients with ENS showed positive serum ANA.[3],[4] Thus, further serum ANA testing should be done in these ENS positive patients.

Focusing on intensity of ENS and level of serum ANA titer, Wells et al .[4] found that intensity of ENS could not predict the serum ANA titer. Similarly, almost all patients in our study had ENS with 1+ intensity regardless of serum ANA titer and we did not find the correlation between intensity of ENS and level of serum ANA.

All patients with SLE had positive serum ANA. However, high serum ANA titer did not correlate with any cutaneous manifestation such as malar rash, discoid rash, and photosensitivity. In addition, there were no relationships between high titer of serum ANA and hematologic, renal, neurological involvement, or serositis. Previous report among Thai patients with SLE showed that hematologic abnormalities are the most frequent systemic involvement (76.2%) followed by nephropathy (66.2%), arthritis (53.9%), neuropathy (19.2%), and serositis (19.2%).[13] Compare to our study, all patients with positive in vivo ANA had lower prevalence of systemic involvement, and arthritis is the most common (33.3%). Further studies are needed to clarify whether positive in vivo ENS could predict any systemic involvement in SLE patients.

To our knowledge, this study was conducted in the largest number of patients with in vivo ENS. However, the limitations are that patients with negative in vivo ENS were not included and full panel autoantibodies were not tested in all patients.

Acknowledgement

We are grateful to Assistant Prof. Dr. Chulaluk Komoltri, Department of Clinical Epidemiology for her very kind support.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
Buch AC, Kumar H, Panicker N, Misal S, Sharma Y, Gore CR. A cross-sectional study of direct immunofluorescence in the diagnosis of immunobullous dermatoses. Indian J Dermatol 2014;59:364-8.  Back to cited text no. 1
[PUBMED]  [Full text]  
2.
Rao R, Balachandran C. Epidermal nuclear staining: A distinct reaction pattern in connective tissue diseases. Indian J Dermatol Venereol Leprol 2007;73:120-1.  Back to cited text no. 2
[PUBMED]  [Full text]  
3.
Bukilica MN, Andrejevic SB, Bonaci-Nikolic BM, Nikolic MM. Speckled antinuclear antibodies in keratinocytes – What does it mean? Clin Exp Rheumatol 2002;20:499-504.  Back to cited text no. 3
    
4.
Wells JV, Webb J, Van Deventer M, Fry B, Pollard KM, Raftos J, et al. In vivo anti-nuclear antibodies in epithelial biopsies in SLE and other connective tissue diseases. Clin Exp Immunol 1979;38:424-35.  Back to cited text no. 4
    
5.
Velthuis PJ, Kater L, van der Tweel I, Meyling FG, Derksen RH, Hené RJ, et al. In vivo antinuclear antibody of the skin: Diagnostic significance and association with selective antinuclear antibodies. Ann Rheum Dis 1990;49:163-7.  Back to cited text no. 5
    
6.
Prystowsky SD, Tuffanelli DL. Speckled (particulate) epidermal nuclear IgG deposition in normal skin. Correlation of clinical features and laboratory findings in 46 patients with a subset of connective tissue disease characterized by antibody to extractable nuclear antigen. Arch Dermatol 1978;114:705-10.  Back to cited text no. 6
    
7.
Burrows NP, Bhogal BS, Russell Jones R, Black MM. Clinicopathological significance of cutaneous epidermal nuclear staining by direct immunofluorescence. J Cutan Pathol 1993;20:159-62.  Back to cited text no. 7
    
8.
Beutner EH, Kumar V, Krasny SA, Chorzelski TP. Defined immunofluorescence immunodermatology. In: Beutner EH, Chorzelski TP, Kumar V, editors. Immunopathology of the Skin. 3rd ed. New York: John Wiley and Sons; 1987. p. 3-40.  Back to cited text no. 8
    
9.
Iwatsuki K, Tagami H, Imaizumi S, Ginoza M, Yamada M. The speckled epidermal nuclear immunofluorescence of mixed connective tissue disease seems to develop as an in vitro phenomenon. Br J Dermatol 1982;107:653-7.  Back to cited text no. 9
    
10.
Alarcon-Segovia D, Ruiz-Arguelles A, Fishbein E. Antibody to nuclear ribonucleoprotein penetrates live human mononuclear cells through Fc receptors. Nature 1978;271:67-9.  Back to cited text no. 10
    
11.
Alarcon-Segovia D, Ruiz-Argüelles A, Llorente L. Broken dogma: Penetration of autoantibodies into living cells. Immunol Today 1996;17:163-4.  Back to cited text no. 11
    
12.
Sousa JX Jr., Miyamoto D, Zimbres JM, Costa DV, Aoki V. Clinicopathological evaluation of in vivo epidermal nuclear fluorescence. Clin Exp Dermatol 2009;34:314-8.  Back to cited text no. 12
    
13.
Kasitanon N, Louthrenoo W, Sukitawut W, Vichainun R. Causes of death and prognostic factors in Thai patients with systemic lupus erythematosus. Asian Pac J Allergy Immunol 2002;20:85-91.  Back to cited text no. 13
    

What is new?
Serum antinuclear antibody titer was not correlated with disease severity, but patients with epidermal nuclear staining tend to have less systemic involvement.


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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