antinuclear antibodies ( ana ) are a marker of several autoimmune diseases including autoimmune thyroid disease ( aitd ) and systemic lupus erythematosus ( sle ) and they can be detected many years before disease onset [ 15 ] . ana positivity can also be found in patients with malignant or infectious diseases as well as in healthy subjects [ 25 ] . the aitd , graves ' disease ( gd ) , and chronic lymphocytic thyroiditis ( clt ) are organ - specific autoimmune disorders that are defined by lymphocytic infiltration of the thyroid   and autoantibodies against thyroid antigens [ i.e. , thyroid peroxidase antibody ( tpoab ) , thyroglobulin antibody ( tgab ) , and anti - tsh - receptor antibody ( trab ) ] . aitd is frequently associated with other organ and non - organ - specific autoimmune disorders [ 810 ] . this association has been also reported in juvenile forms of chronic arthritis and sle [ 11 , 12 ] . ana prevalence up to 45% has been reported in adult patients with aitd [ 13 , 14 ] . to the best of our knowledge , so far , only one study analyzed ana positivity in children with aitd . we investigated the prevalence of serum ana in pediatric patients with aitd and their association with signs and symptoms related to immune - rheumatic diseases . we studied 93 consecutive children ( 86 patients with clt and 7 with gd , 76 females ) referred for aitd to the pediatric endocrinology unit of  sapienza  university in rome . clt was defined on the basis of the presence of thyroid autoantibodies more than two times the upper normal value ( nv ) ( [ tpoab nv < 20  iu / ml ] and/or tgab [ nv < 50  iu / ml ] ) and thyroid ultrasound evaluation showing reduced echogenicity compatible with thyroiditis , regardless of thyroid function . gd was defined by clinical and biochemical hyperthyroidism and by positivity for trab . the enrolled patients underwent a complete physical examination and the clinical and laboratory data were collected in a standardized form , which includes demographics , past medical history with date of diagnosis , comorbidities , and previous and concomitant treatments . all children and parents were interviewed according to a questionnaire seeking signs and symptoms related to rheumatic diseases in children . the questionnaire took into account the following signs and symptoms : joint pain , joint swelling , back pain , morning stiffness , asthenia , raynaud 's phenomenon , xerostomia , xerophthalmia , pleuritis , and pericarditis . children with signs and/or symptoms suggestive of immune - rheumatic diseases were examined by a rheumatologist ( fc ) . patients underwent peripheral blood sample collection and sera were stored at 20c until being assayed . free t3 , free t4 , tsh , tpoab , and tgab serum levels were determined in all patients and trab were determined in presence of hyperthyroidism . ft3 and ft4 were determined by ria and tsh ( upper normal value 3.5  u / ml ) was determined by immunoradiometric assay ( all by byk sangtec diagnostica , dietzbach , germany ) . trab were detected by radioreceptor assay ( radim , angleur , belgium ) and tpoab and tgab by immunoradiometric assay ( icn pharmaceutical inc . ana were detected by indirect immunofluorescence assay ( iifa ) on hep-2 cells ( ana nova lite tm hep-2 , inova diagnostics inc . , san diego , ca 92131 , usa ) . anti - double dna antibodies ( anti - dsdna ) were investigated by crithidia luciliae immunofluorescence test ( clift ) ( a. menarini diagnostics , 50131 florence , italy ) . ana were considered positive at a titer  1  :  80 , anti - dsdna at a titer  1  :  10 . serum antibodies against extractable nuclear antigen ( anti - ena screening and in the case of ena positivity : sm , rnp , ss - a , ss - b , scl-70 , jo-1 ) were determined by elisa ( quanta lite ena 6 , inova diagnostics inc . , san diego , ca 92131 , usa ) . in addition , anti - cyclic citrullinated peptide antibodies ( anti - ccp ) and rheumatoid factor ( rf ) were determined . anti - ccp were determined by fluoroenzyme - immunoassay ( eliaccp ) , using immunocap 100 analyzer ( phadia ab , 75002 uppsala , sweden ) and rf was determined by nephelometry using bn2 automate ( n latex rf kit , siemens healthcare diagnostics products , marburg 35037 , germany ) . statistical calculations were performed using spss for windows version 17 ( spss , chicago , il , usa ) . ana positivity was found in 66/93 children ( 71% ) , anti - ena was found in 4/93 ( 4.3% ) , anti - dsdna antibodies were found in 1/93 ( 1.1% ) , rf was detectable in 3/93 ( 3.2% ) , and anti - ccp were found in none . ena - specific autoantibodies were determined in 3 out of 4 anti - ena - positive patients , 1 had anti - rnp positive , 1 had anti - jo-1 , and 1 was negative for specific autoantibodies . the ana pattern was homogeneous in 61/66 ( 92.4% ) , coarse / fine speckled in 4/66 ( 6% ) , and nucleolar in 2/66 ( 3% ) . the iifa intensity was + + + in 23/66 ( 34.8% ) , + + in 24/66 ( 36.3% ) , and + in 19/66 ( 28.7% ) cases . among the 93 children with aitd , 20 ( 21% ) had at least one other autoimmune disease : 9 with celiac disease ( cd ) , 2 with autoimmune gastritis ( ag ) , 4 with type 1 diabetes mellitus ( type 1 dm ) , 2 with alopecia , 2 with vitiligo , and 1 with autoimmune thrombocytopenia . we found that 3/7 ( 43% ) of children with gd presented with one other autoimmune disease ( 1 cd , 1 type dm , and 1 ag ) , versus 17/86 ( 20% ) with clt ( 8 cd , 1 ag , 3 type 1 dm , 2 alopecia , 2 vitiligo , and 1 autoimmune thrombocytopenia ) . the difference in the frequency of autoimmune disease associated with clt versus gd was not significant . clinical and laboratory features of ana - positive and ana - negative patients are compared in table 1(b ) . no significant differences were found between the ana - positive and ana - negative groups with respect to age , sex , l - thyroxine treatment , tsh , tpoab , and tgab levels , or presence of other autoimmune diseases in them and in their parents . associated autoimmune diseases in ana - positive and ana - negative children and their parents are reported in table 2 . hereby ana - positive children did not differ for the frequency of additional autoimmune disease , but there was a higher percentage of parental autoimmunity in ana - negative patients , however , without significance . no significant differences were found between ana - positive and ana - negative children with aitd . aitd is a common autoimmune disease and it is frequently associated with other organ and non - organ - specific autoimmune disorders [ 810 ] . a variable ana prevalence up to 45% has been reported in aitd adult patients [ 13 , 14 ] . ana can also be detected in different autoimmune disorders ( i.e. , sle , sjogren 's syndrome , progressive systemic sclerosis , mixed connective - tissue disease , juvenile idiopathic arthritis , primary autoimmune cholangitis , and autoimmune hepatitis ) as well as in infections ( 2,4 ) . in particular , ana can be detected in over ninety percent of patients with sle , a multifactorial autoimmune disease , involving genetic and environmental factors , characterized by a wide range of autoantibodies and clinical manifestations [ 4 , 1625 ] . ana can be also found in healthy people . a recent cross - sectional analysis of 4754 individuals older than 12 years showed a prevalence of ana of 13.8% . a similar prevalence of 12.6% was reported in healthy children , with higher titers found between 5 and 10 years of age . to our knowledge , the authors , using a cut - off of 1  :  40 for ana iifa on hep-2 cells , demonstrated an incidence of ana positivity significantly higher in patients with untreated gd ( 71% ) than in clt ( 33% ) . in addition , ana positivity was identified as a predictive factor for poor response to antithyroid drugs in gd . in our study , clt represented 92% of total enrolled patients versus 36% in the previous study . since we analyzed gd regardless of treatment and we included only 7 children affected with gd , we were not able to correlate gd activity with ana positivity . in clt patients , we did not find any difference in l - thyroxine treatment between ana - positive and ana - negative children . in contrast , a correlation between increased ana levels and reduced thyroid volumes was reported in adult patients affected by vitiligo and aitd . to detect serum ana , we used the same method of inamo and harada , iifa on hep-2 cells , that is considered the most reliable method to search ana [ 2 , 29 ] . using a higher cut - off value of 1  :  80 , we found detectable ana in 71% of children with aitd and the iifa homogeneous pattern was detected in 92% of cases . titers and pattern of ana have been proposed to be a critical parameter for discriminating ana , a homogeneous pattern being suggestive of rheumatic diseases . a nuclear fine speckled pattern was reported in ana - positive healthy children in 77% of cases . we did not find any significant difference in ana - positive and ana - negative group with respect to age , sex , lt4 treatment , tsh , tpoab and tgab levels , and prevalence of other autoimmune diseases in the children or in the parents . no differences were detected investigating signs and symptoms of systemic diseases between ana - positive and ana - negative group . altogether , joint pain was referred by 19% of patients , asthenia was referred by 19% , and raynaud 's phenomenon was present in 4.3% , as well as xerostomia and xerophtalmia . because of the fact that hypothyroidism itself can cause symptoms as asthenia , we confirmed symptoms when children were euthyroid . torok and arkachaisri   in children and adolescents referred to a rheumatological center for ana positivity and investigated for aitd , found arthralgias and fatigue more frequently in ana - positive / antithyroid antibodies - negative subjects . the prevalence of positive antithyroid antibodies in this study was 30% versus 3 - 4% in general pediatric population , and hypothyroidism was found in 14% . it may suggest that to screen for aitd may be worthwhile in apparently healthy children with ana positivity . the clinical and biological meaning of ana is still debated [ 29 , 33 , 34 ] . it has been speculated that a defect in the mechanisms involved in the engulfment of dead cell with inappropriate clearance of self - nucleic acids can cause autoimmune diseases . a deficiency of clearance of apoptotic cells is considered one of the causes of sle and may underlie autoimmunity itself [ 3537 ] . activation of the innate and acquired immune response , which can be induced by infection , inflammation , or tissue injury , may impact on the development of autoimmunity in the thyroid [ 38 , 39 ] . we can speculate that such a high prevalence of ana in juvenile aitd may be read as a link to the involvement of self nucleic acids in the development of aitd . an important point of our study is the observation of parental autoimmunity in up to 30% with only slightly lower percentage ( 20% ) in ana - positive children . whether this reflects ( epi-)genetic heritability or a shared familial environment triggering autoimmunity in parents and children needs to be investigated in prospective family studies . in conclusion , although we analyzed a limited number of cases , our study demonstrated ana positivity in over 70% of children with aitd . ana positivity at the time of the study was not related to overt immune - rheumatic diseases . in our opinion , the finding of ana positivity in children and adolescents affected by aitd needs a careful reevaluation : it can be interpreted as a manifestation of  activated autoimmunity  without clinical relevance at the time of the study . however , because the positivity of ana and other non - organ - specific autoantibodies can occur even many years before the onset of systemic autoimmune diseases [ 5 , 40 , 41 ] , we think that prospective studies are warranted , especially in subjects positive for anti - dsdna and anti - ena .
background . 
 antinuclear antibodies ( ana ) are a hallmark of many autoimmune diseases and can be detected many years before disease onset . 
 autoimmune thyroid diseases ( aitd ) are frequently associated with other organ- and non - organ - specific autoimmune disorders . 
 objectives . to assess the prevalence of ana in pediatric patients with aitd and their clinical correlations . methods . 
 ninety - three consecutive pediatric patients with aitd were enrolled ( 86 children with chronic lymphocytic thyroiditis and 7 with graves ' disease ) . 
 ana , anti - double dna ( anti - dsdna ) antibodies , anti - extractable nuclear antigen ( anti - ena ) , anti - cyclic citrullinated peptide antibodies ( anti - ccp ) , and rheumatoid factor ( rf ) was obtained . 
 signs and symptoms potentially related to rheumatic diseases in children were investigated by a questionnaire . 
 results . 
 ana positivity was found in 66/93 children ( 71% ) , anti - ena in 4/93 ( 4.3% ) , anti - dsdna in 1/93 ( 1.1% ) , rf in 3/93 ( 3.2% ) , and anti - ccp in none . 
 no significant differences were found between the ana - positive and ana - negative groups with respect to age , sex , l - thyroxine treatment , or prevalence of other autoimmune diseases . 
 overall , parental autoimmunity was found in 23% 
 . conclusions . 
 ana positivity was demonstrated in 71% of children with aitd . 
 ana positivity was not related to overt immune - rheumatic diseases . 
 however , because the positivity of ana can occur even many years before the onset of systemic autoimmune diseases , prospective studies are warranted .