the hallmark of autoimmune diseases is the pathologic activity of the immune system of an organism directed against its own cells and tissues .
the disease is a direct consequence of tissue and/or organ damage as mediated by autoreactive components of the immune system , that is , autoreactive t - lymphocytes and/or autoantibodies . for diagnostic purposes ,
autoantibodies are the most important analytes . within the systemic autoimmune rheumatic diseases ( sard ) , anti - nuclear antibodies ( ana ) , directed against various cellular components , and associated autoantibodies , such as antibodies reactive with dsdna and extractable nuclear antigens ( ena ) , are fundamental for diagnosis [ 13 ] .
traditionally , ana are detected by indirect immunofluorescence ( iif ) performed on human epithelial cells ( hep-2 ) .
this technique requires a multistage process consistent with visual determination of the staining pattern , serial titrations of positive sera , followed by a second test in which autoantigen specificity is determined [ 2 , 4 ] .
recently , the american college of rheumatology ( acr ) stated that ana detection by iif is still considered the gold standard .
this was primarily based on the high sensitivity of the iif assay and the inclusion of ana detection by iif assay in diagnostic criteria of systemic lupus erythematosus ( sle ) and autoimmune hepatitis ( aih ) [ 68 ] .
in addition , ana can also be considered as a screening test for samples that require further testing for autoantigen specificity , that is , dsdna and ena [ 2 , 9 ] .
the specificity of ana detection by iif , however , is relatively poor , especially when low titres are used for screening . indeed , at a 1  :  40 serum dilution ,
2530% of healthy individuals may test positive for ana and this increases even further upon ageing [ 1 , 10 ] .
overall , it is recommended that the serum dilution that gives a specificity of 95% in healthy individuals should be used as cut - off .
moreover , the clinical significance rises with increasing titres [ 11 , 12 ] , as well as with the identification of the responsible autoantigen [ 1 , 9 ] .
obviously , a positive ana test must always be interpreted cautiously and only within the clinical context of the patient . in a clinical setting where the pretest probability of sard is generally low , as in primary care , the added value of a positive ana test is lower as compared to secondary and tertiary care situations where pretest probabilities of sard are often higher . in the current study
, we determined the prevalence of ana in primary ( general practices ) , secondary ( regional hospital ) , and tertiary care ( university hospital ) . besides data on ana prevalence , also ana titres and anti - ena and anti - dsdna antibodies were included in our analyses .
we hypothesize that ana prevalence , ana titre , and anti - ena / dsdna reactivity increase from primary to tertiary care as these situations are expected to be also associated with an increasing pretest probability of sard .
in the present study , three different patient populations from the southern part of the netherlands were evaluated and compared with each other .
these three populations consisted of patients who were tested for ana between november 2011 and august 2012 in suspicion of an autoimmune disease .
all ana requests were considered to involve the diagnostic workup since none of the patients had requests for ana ( and/or anti - ena / dsdna ) at least 4 years prior to the study period . in the first patient population
( n = 1453 ) ana were requested by general practitioners ( primary care ) . the second population ( n = 1621 ) had an ana request in a regional hospital ( secondary care ) , while the third population ( n = 1168 ) had an ana request in a university hospital ( tertiary care ) . testing for ana in the first and second cohorts
was performed in the atrium mc ( heerlen , the netherlands ) , while the ana tests in the third cohort were performed in the maastricht university medical centre ( mumc , maastricht , the netherlands ) .
furthermore , in both regional and university hospitals , the origin , that is , hospital department , of ana requests was documented .
ana detection by iif was performed on hep-2000 cells according to the instructions provided by the manufacturer ( immuno concepts , sacramento , ca ) .
hep-2000 cells are transfected with the gene for ssa-60 , which makes these cells more sensitive for ssa - antibody detection [ 14 , 15 ] .
fitc - conjugated goat anti - human igg antibody was used for detection of ana .
five staining patterns were considered ana positive : homogenous , atypical speckled , speckled , centromere , and nucleolar . in case of mixed - patterns ,
slides were evaluated with a fluorescent microscope ( axioskop , carl zeiss microscopy gmbh , jena , germany ) with led light source .
all slides were evaluated by two independent observers ; in case of a difference in opinion , a third observer was decisive .
the presence of anti - ena antibodies was screened by a commercially available line immunoassay ( ana 3 profile euroline , euroimmun , lbeck , germany ) .
after the first incubation with diluted serum , a second incubation was performed with goat anti - human igg linked to alkaline - phosphatase . finally , a third incubation took place with bromochloroindolyl phosphate and nitro blue tetrazolium chloride ( bcip / nbt ) as substrate to detect anti - ena antibodies .
although the ana 3 profile euroline kit enables detection of 15 different antigens , only eight were evaluated in the current study : ro52 , ss - a / ro60 , ss - b / la , nrnp / sm , sm , scl-70 ( topoisomerase 1 ) , jo-1 , and cenp - b .
reading of the results was automated and the colour intensities of the reactions were evaluated by the eurolinescan program ( euroimmun ) to enable semiquantitative determination , that is , equivocal , 1 + , 2 + , and 3 + .
results were considered positive for sm , scl-70 , and jo-1 if the intensity was at least equivocal , while for ss - a / ro60 , ss - b / la , nrnp / sm , and cenp - b an intensity of at least 1 + was required . finally , the cutoff for ro52 was 2 + .
positive lia results , as defined above , were confirmed with a commercially available feia ( elia , immunodiagnostics , thermo fisher scientific , freiburg , germany ) .
this method uses highly purified ( smd ) or recombinant ( ss - a / ro60 , ro52 , ss - b / la , cenp - b , scl-70 , jo-1 , rnp70 , and u1rnp ) human antigens that are coated on irradiated polystyrene cups . the assay was performed according to the manufacturer 's instructions .
the sera were diluted 1  :  50 with dilution buffer . after binding of anti - ena antibodies , the cups were washed and subsequently incubated with mouse anti - human igg ( heavy chain specific ) conjugated to  -galactosidase . in case of antibody association , binding was detected fluorometrically using 4-methylumbellifery--d - galactoside ( 0.01% ) as substrate .
for all antigens , values above 10  u / ml were considered positive . in primary and secondary care , anti - dsdna antibodies
were detected with a commercially available feia ( elia , immunodiagnostics , thermo fisher scientific ) .
this method uses a circular plasmid dsdna , purified from escherichia coli , as antigen .
the assay was performed according to the manufacturer 's instructions and as described for the anti - ena antibodies .
the sera were diluted 1  :  10 in dilution buffer and values above 15  u / ml were considered positive . in tertiary care ,
anti - dsdna antibodies were detected by the crithidia luciliae immunofluorescence test ( clift ; immuno concepts ) .
fitc - conjugated goat anti - human igg antibody was used for detection of anti - dsdna antibodies .
slides were evaluated with a fluorescent microscope ( axioskop , carl zeiss microscopy gmbh ) with led light source .
all slides were evaluated by two independent observers ; in case of a difference in opinion , a third observer was decisive .
all samples were tested first by ana iif . if the result was negative , no further testing was performed , unless specifically requested .
however , the results of these additional tests were not included in the present study .
if the ana iif was positive , irrespective of the staining pattern , titration was performed ( 1  :  320 and 1  :  1280 ) .
if a homogenous ana pattern was detected , testing for anti - dsdna antibodies was performed by feia ( primary and secondary care ) or clift ( tertiary care ) .
additionally , in case of a homogenous , ( atypical ) speckled , or centromere pattern , typing for anti - ena antibodies was performed by lia .
because of the high correlation between the atypical speckled ana pattern and ss - a / ro60 antibodies , these antibodies were considered positive if the atypical speckled ana pattern was observed in combination with either a positive lia or a positive feia .
similarly , anti - cenp - b antibodies were considered positive when a centromere ana pattern was observed in combination with a positive anti - cenp - b result in at least one of both anti - ena antibody test systems .
since the lia does not enable specific distinction of anti - rnp antibodies , positivity for the nrnp / sm complex was followed by testing for antibodies against rnp70 and u - rnp by feia .
chicago , il ) or graphpad prism version 6 ( graphpad software inc . , la jolla , ca ) .
the kolmogorov - smirnov analysis was performed to determine whether the age distributions of the three study populations were normally distributed .
furthermore , the chi square test , with yates ' correction if appropriate , was performed when comparing proportions of groups . in the case of small samples ,
of the three patient populations included , 90 ( 6.2% ) , 175 ( 10.8% ) , and 187 ( 16.0% ) patients from primary ( n = 1453 ) , secondary ( n = 1621 ) , and tertiary care ( n = 1168 ) , respectively , were tested positive for ana and were therefore eligible for the current study .
gender ( f / m ) and age ( median and range ) distribution were as follows : 78/12 and 57.2 ( 1595 ) for primary care , 129/46 and 57.0 ( 1793 ) for secondary care , and 130/57 and 57.3 ( 384 ) for tertiary care .
the gender distribution differed significantly ( p = 0.009 ) due to a strong female preponderance in primary care .
the age distribution differed significantly ( p = 0.005 ) due to the fact that in tertiary care age distribution was skewed negatively .
evaluation of the origin , that is , hospital departments , of the ana requests in secondary and tertiary care revealed four departments , that is , rheumatology , dermatology , internal medicine , and neurology , which requested the majority of the ana screening tests ( figure 1(a ) ) , that is , 86% and 68% , respectively .
the prevalence of ana in the 3 distinct clinical settings is depicted in figure 2(a ) .
the higher relative ana prevalence in tertiary care ( 16.0% ) versus secondary care ( 10.8% ) was apparent in all 4 clinical disciplines that requested the majority of ana screening test ( figure 1(a ) ) ; this also holds for the other clinical disciplines ( data only shown as pooled results )
. typically , in secondary care 39.5% of overall ana requests came from the rheumatology department , while in tertiary care this was 15.4% . within the positive ana cohorts , patients from primary care had relatively low titres
out of 90 positive ana tests 63 sera ( 70% ) revealed an ana titre of 1  :  80 . in secondary and tertiary care , a titre of 1  : 
consequently , higher titres were observed more frequently in secondary and tertiary care than in primary care .
the distribution in titres between secondary and tertiary care was not different . at first glance
, there is no apparent difference in the distribution of ana patterns between the three health care levels ( figure 2(c ) ) .
also , comparison of the distribution of ana patterns with a titre of 1  :  1280 , considered to have the highest positive likelihood ratio , showed no significant differences ( data not shown ) .
the prevalence of anti - ena and anti - dsdna antibodies , as defined by the algorithm described above , is presented in figure 3(a ) . in the primary care , 19 ( 21.1% )
patients with positive ana ( n = 90 ) were tested positive for anti - ena and/or anti - dsdna antibodies , that is , 1.3% of the total cohort . in the secondary and tertiary care ,
68 of 175 ( 38.9% ) and 40 of 187 ( 21.4% ) ana positive patients revealed anti - ena and/or anti - dsdna reactivity , respectively .
this is 4.2% and 3.4% of the total secondary and tertiary care cohorts , respectively ( p = 0.367 ) .
significantly more positive anti - ena and/or anti - dsdna results were found in the total secondary and tertiary care cohorts than in the primary care cohort ( p < 0.0001 and p = 0.0006 , resp . ) . in secondary care ,
the prevalence of anti - ena and/or anti - dsdna reactivity within the positive ana samples was the highest in the requests from rheumatology ( figure 1(b ) ) .
in tertiary care , however , departments of rheumatology and neurology had , on average , reduced prevalence of anti - ena and/or anti - dsdna reactivity as compared to the departments of dermatology and internal medicine ( figure 1(b ) ) .
since the relevance of anti - ena and anti - dsdna antibodies is considered to increase when combined reactivity is observed , we analysed the prevalence of anti - ena and/or anti - dsdna antibodies in relation to combined reactivity ( figure 3(b ) ) . in the primary care setting
, single positivity appeared to be more abundant than in secondary and tertiary care , but this difference was statistically not significant .
no differences were observed between secondary and tertiary care settings either . with respect to the antigens recognized by the specific antibodies ,
no apparent differences were observed in the prevalence of antibodies reactive to rnp , ssa60 , ro52 , ssb , cenp - b , and dsdna ( figure 3(c ) ) .
anti - scl70 antibodies were only detected in samples of patients from secondary and tertiary care .
however , the absolute number , one in each cohort , was low in both of these settings .
in the present study on the analyses of ana prevalence , ana titre and anti - ena specificity in the primary , secondary , and tertiary care , our results indicate that ( i ) ana prevalence significantly increases from primary to tertiary care , ( ii ) low titres ( 1  :  80 ) are more frequently observed in the primary care , and ( iii ) anti - ena and anti - dsdna specificities are significantly more prevalent in the secondary care than in the primary care .
interpretation of the data obtained in the current study is highly dependent on the viewpoint on clinical utility of an ana test result .
this result may help the clinician to identify a patient with sard , but especially in situations with low pretest probabilities of such diseases , the risk of false interpretation of a positive ana is high .
this risk of false positive interpretation will decrease if the positive ana is characterized by high titre and includes ( multi)-reactivity for ena and/or dsdna , since these characteristics are associated with higher positive likelihood ratios [ 11 , 12 , 17 ] .
next , one has to realize that ana testing is performed in the context of multiple diseases , varying from distinct sard to autoimmune liver diseases .
interpretation of a positive ana test may be different for each distinct disease : patients with , for instance , aih or systemic sclerosis often have a positive ana with no ena reactivity , while a positive ana test as such is part of the classification criteria for sle , as well as aih [ 68 ]
. a negative ana test result , on the other hand , may also be very useful to exclude a specific set of diseases and may drive attention to other diseases .
again , this differs for the distinct sard : a negative ana test has a high negative predictive value for sle and systemic sclerosis but is less helpful to exclude sjgren 's syndrome or myositis [ 2 , 3 ] .
obviously , definite interpretation of our dataset is hampered by the lack of clinical data from the patients of the three cohorts .
the assumption of the primary care having a relatively low pretest probability was based on previous studies [ 13 , 18 , 19 ] . for future studies
, we recommend the inclusion of clinical data in order to be able to thoroughly assess pretest probabilities and strengthen our assumptions .
this study , however , also has noteworthy strengths , that is , only data obtained during the diagnostic workup of patients ( not follow - up samples ) were included and the same testing algorithm and reagents ( except for detection of anti - dsdna antibodies ) were used for all three patient cohorts .
as expected , we observed a gradual increase in the prevalence of ana from primary to tertiary care . in our present study ana
was detected by iif in a screening dilution of 1  :  80 . in several studies
it has been reported that 1015% of healthy controls are ana positive in this serum dilution [ 1 , 10 , 20 ] .
obviously , a positive ana test result is not only dependent on the dilution factor but also on the quality of other reagents , that is , fluorescent conjugate , cell substrate , and the fluorescent microscope . for standardization purposes ,
it has , therefore , been recommended to screen for ana with a specificity of 95% .
taking this into account , the mere presence of ana in our primary care cohort ( prevalence 6.2% ) lacks discriminating power to identify patients with sard . on the other hand , due to the high negative predictive value of a negative ana result
furthermore , since both high titre and anti - ena and/or anti - dsdna ( multi-)reactivity may increase the likelihood of identifying a patient with sard [ 11 , 12 , 17 ] , it is apparent in our primary care cohort that 30% of ana positive sera are of medium to high titre ( n = 27 ) , 21% contain anti - ena and/or anti - dsdna reactivity ( n = 19 ) , and 37% of the latter reveal multireactivity ( n = 7 ) . in our secondary and tertiary cohorts , the ana prevalence is also relatively low ( 11 and 16% , resp . ) , but in both clinical settings about half of the ana positive sera are of high titre ( 48 - 49% ) . as expected , the observed anti - ena and/or anti - dsdna reactivity of ana positive sera in the secondary care ( 39% ) is higher than in the primary care .
surprisingly , anti - ena and/or anti - dsdna reactivity in the tertiary care ( 21% ) is lower than in the secondary care and similar to the primary care .
this might be related to the spectrum of diseases investigated in the tertiary versus the secondary centres .
also the higher number of ana requests by tertiary care departments not typically involved in the diagnosis of sard might be the result of academic profiling of the respective departments .
for instance , the cardiology department of the mumc is specialized in inflammatory cardiomyopathies and a possible autoimmune aetiology of these diseases .
cardiology requested 108  ana tests ( 9.2% ) , revealing 13% ana positive results of which 20% was anti - ena and/or anti - dsdna antibody positive .
a second important difference is the presence of a division of clinical immunology within the department of internal medicine of the tertiary care hospital , implying that many patients with sard are evaluated by clinical immunologists instead of rheumatologists .
the latter difference might at least explain the lower relative prevalence of anti - ena and anti - dsdna antibodies in the rheumatology department of the tertiary care hospital .
our study shows that in secondary care the majority of positive ana results ( 92.6% ) , as well as the positive anti - ena and anti - dsdna results ( 95.6% ) , are linked to the departments of rheumatology , dermatology , internal medicine , and neurology .
obviously , patients with the highest pretest probabilities for sard , as associated with the initial clinical presentation , are most likely referred to these clinical departments .
in clinical settings with lower pretest probabilities , ana positive sera are more likely to be of no clinical significance [ 13 , 18 , 19 ] .
it might therefore be an option , in particular in primary care settings , to move away from the traditional ana screening test . in this context , the recent discovery of the dense fine speckled ( dfs70 ) antigen is promising and could offer a possible solution for the identification and exclusion of positive sera with no clinical relevance [ 21 , 22 ] . the typical ana dense fine speckled pattern ( dfs ) , known to be associated with the dfs70 antigen , has been found to be commonly prevalent in healthy individuals with ana positive sera ( 33.1% ) , whereas in sard patients 0.0% of the sera revealed a dfs pattern .
another study revealed that 2 - 3% of sard patients had antibodies directed to the dfs70 antigen . obviously , in sard patients other anti - ena antibodies might be present that hamper correct recognition of the dfs iif pattern
indeed , the identification and correct interpretation of the dfs pattern might prove to be challenging for diagnostic laboratories and would require additional training .
moreover , the majority of patterns recognized do not seem to be compatible with a dfs pattern , but this might be the consequence of only interpreting the strongest pattern in the current study .
since the identification of the dfs pattern might be challenging for routine diagnostic laboratories and inaccurate interpretation can have significant consequences , an immunoadsorption protocol to diminish anti - dfs70 antibody reactivity to hep-2000 cells could be implemented in the current iif assay in order to significantly improve the performance characteristics of the ana iif test [ 21 , 25 ] .
this approach sustains recognition of sard - related autoantibodies in sera with combined reactivities , that is , anti - dfs70 and other anti - ena antibodies .
another alternative testing algorithm could , instead of ana iif , include solid phase assays including multiplex and screening assays for well - defined anti - ena and anti - dsdna reactive antibodies [ 11 , 12 , 26 , 27 ] . both approaches enable to distinguish or reduce the number of positive ana results lacking clinical relevance .
in addition , multiplex anti - ena screening assays are considered to better recognize particular antigens , for example , ssa and jo-1 , as compared to ana iif testing [ 3 , 11 , 12 , 28 ] .
altogether , the results indicate that in the primary care the usage of traditional ana screening tests is more prone to false - interpretation of positive ana results
. rather , an alternative testing algorithm for detection of patients with sard might be more appropriate .
this might either be achieved by immunoadsorption of anti - dfs70 antibodies or direct screening for anti - ena antibodies .
obviously , a patient with severe clinical manifestations typical for sard presenting in a primary care setting should be referred directly , that is , without any laboratory testing , to the rheumatologist or clinical immunologist .
this recommendation may not only apply for general practitioners but may also hold for clinical departments that are less likely to encounter patients suspected of sard .
 for the diagnosis of systemic autoimmune rheumatic diseases ( sard ) , patients are screened for anti - nuclear antibodies ( ana ) . 
 ana , as assessed by indirect immunofluorescence ( iif ) , have a poor specificity . 
 this hampers interpretation of positive results in clinical settings with low pretest probability of sard . 
 we hypothesized that the utility of positive ana iif results increases from primary to tertiary care . 
 we retrospectively determined ana , anti - ena , and anti - dsdna antibody prevalence in patient cohorts from primary ( n = 1453 ) , secondary ( n = 1621 ) , and tertiary ( n = 1168 ) care settings . 
 results reveal that from primary care to tertiary care , ana prevalence increases ( 6.2 , 10.8 , and 16.0% , resp . ) . moreover , in primary care low titres ( 70% versus 51% and 52% in secondary and tertiary care , resp . ) 
 are more frequent and anti - ena / dsdna reactivities are less prevalent ( 21% versus 39% in secondary care ) . 
 typically , in tertiary care the prevalence of anti - ena / dsdna reactivities ( 21% ) is lower than expected . from this descriptive study 
 we conclude that positive ana iif results are more prone to false interpretation in clinical settings with low pretest probabilities for sard , as in primary care . whether alternative approaches , 
 that is , immunoadsorption of anti - dfs70 antibodies or implementation of anti - ena screen assays , perform better , needs to be determined . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion