the detection of anti - dsdna autoantibodies represents a hallmark in the diagnosis of systemic lupus erythematosus ( sle )   and these antibodies are part of the classification criteria for sle [ 2 , 3 ] .
additionally , anti - dsdna antibodies measured by certain assays have been reported to correlate with disease activity and lupus nephritis . despite the fact that several assay systems have been developed during the last decades
, controversies persist on which assay is the best test to measure anti - dsdna antibodies [ 4 , 5 ] .
although the farr assay has been considered the  gold standard  for the detection of anti - dsdna antibodies in many laboratories [ 4 , 6 , 7 ] , radioimmunoassays in general have recently been falling into disuse in routine practice based on several factors , including the radioactive nature of the assay .
therefore , it is important to evaluate new fully automated technologies which have recently become available as viable alternatives to farr assay .
the objective of the present study was to compare quanta flash dsdna , a chemiluminescent immunoassay ( cia ) on the bio - flash , a rapid - response chemiluminescent analyzer , to three other anti - dsdna antibody assays and to crithidia luciliae indirect immunofluorescence test ( clift ) .
in the first part of the study , 161 samples , 61 from patients suffering from sle and 100 controls , including hashimoto 's thyroiditis ( n = 40 ) , rheumatoid arthritis ( n = 40 ) , and blood donors ( n = 20 ) from apparently healthy individuals , were tested for anti - dsdna igg by quanta flash dsdna cia ( inova diagnostics , san diego , ca , usa ) , quanta lite dsdna sc elisa ( inova diagnostics inc . ) , bioplex 2200 mfi ( bio - rad , usa ) , immulisa dsdna elisa ( immco diagnostics , buffalo , ny , usa ) , and nova lite clift ( inova diagnostics ) .
a second cohort of 69 sle patients was tested by quanta flash dsdna cia and nova lite clift to expand the first part of the study for these methods .
the diagnoses were established as described in a previous paper before or , if applicable , according to the standard disease criteria .
this study meets and is in compliance with all ethical standards in medicine , and informed consent was obtained from all patients according to the declaration of helsinki .
assay is a novel cia that is used on the bio - flash instrument ( biokit s.a . , barcelona , spain ) ,
fitted with a luminometer , as well as the hardware and liquid handling accessories necessary to fully automate the assay .
the principle of the bio - flash system has recently been described [ 9 , 10 ] .
the quanta flash assay for this study was developed using synthetic dsdna ( see table 1 ) coupled to the surface of paramagnetic beads .
. a patient serum sample is prediluted with the bio - flash sample buffer in a small disposable plastic cuvette .
small amounts of the diluted patient serum , the beads , and the assay buffer are all combined into a second cuvette , mixed , and then incubated for 9.5 minutes at 37c .
the magnetized beads are sedimented using a strong magnet in the washing station and washed several times followed by addition of isoluminol conjugated anti - human igg and again incubated for 9.5 minutes at 37c .
the isoluminol conjugate is oxidized when sodium hydroxide solution and peroxide solutions ( triggers ) are added to the cuvette , and the flash of light produced from this reaction is measured as relative light units ( rlus ) by the bio - flash optical system .
the rlus are proportional to the amount of isoluminol conjugate that is bound to the human igg , which is in turn proportional to the amount of autoantibodies bound to the antigen on the beads .
bioplex 2200 ( bio - rad , hercules , ca ) system is an automated analyzer that uses multiplex bead technology ( luminex , austin , tx , usa ) to simultaneously detect antibodies to several antigens in a single tube . the bioplex 2200 ana screen is intended for the qualitative screening of ana , the quantitative detection of antibody to dsdna , and the semiquantitative detection of ten separate antibodies ( chromatin , ribosomal p , ss - a , ss - b , sm , smrnp , rnp , scl-70 , jo-1 , and centromere b ) [ 11 , 12 ] in human serum and/or edta or heparinized plasma .
both the immulisa dsdna ( immco diagnostics ) and quanta lite dsdna sc ( inova diagnostics inc . )
are enzyme linked immunosorbent assays ( elisa ) for the quantitative or semiquantitative detection of igg specific for dsdna in human serum as an aid in the diagnosis of sle in conjunction with other laboratory and clinical findings .
nova lite dsdna clift ( inova diagnostics inc . ) is an indirect immunofluorescence ( iif ) assay for the screening and titration based determination of anti - dsdna antibodies in human serum .
this single - cell organism possesses a giant mitochondrion containing a highly condensed mass of circular dsdna .
the assay was performed by a single operator using an led microscope at inova diagnostics according to the direction insert .
clift results were graded from 0 to 4 according to the intensity ( see also direction insert of the kit ) ; 4 is brilliant apple green fluorescence ; 3 is bright apple green fluorescence ; 2 is clearly distinguishable positive fluorescence ; 1 is lowest specific fluorescence that enables the kinetoplast staining to be clearly differentiated from the background fluorescence ; 0 is negative .
precision and linearity of the quanta flash dsdna cia were verified by performing the required testing according to the clinical and laboratory standards institute ( clsi ) guidelines . for the precision study ,
the within - run , between - day , between - run , and total precision were determined by running two aliquots of the precision samples twice a day in random order , with a minimum of 2 hours between each run .
the samples were run on the same instrument for each assay and repeated for at least 20 days , according to clsi guideline ep5-a2 .
linearity testing was performed according to clsi guideline ep6-a ( volume 23 , number 16 ) , which involved diluting several high titer sera in a serial dilution scheme to span the analytical measuring range ( amr ) for each assay .
the data were statistically evaluated using the analyse - it software ( version 1.62 ; analyse - it software , ltd . ,
spearman 's correlation and cohen 's kappa agreement test were carried out to analyze the agreement between portions and p values < 0.05 were considered significant .
receiver operating characteristics ( roc ) analysis was used to analyze the discriminatory ability of different immunoassays .
cluster analysis was used to illustrate the relationship between different assays   and to display the reactivity pattern of the patients .
hierarchical clustering was performed using average linkage clustering where patient correlation was performed uncentered and the reactivities were uncentered . for the assays having an equivocal range ,
all data was analyzed using the applied cut - offs listed in table 1 , where the equivocal results were considered positive .
for the precision testing of quanta flash dsdna cia , seven samples ranging from 27.3 to 402.8  iu / ml were tested . for all samples , the within - run varied between 4.7 and 6.9% , the between - day varied between 2.3 and 4.0% , and
the between - run varied between 0.0 and 4.9% . the total precision for the assay varied between 6.6 and 8.2% .
the linearity study showed linearity of the quanta flash dsdna cia from 7.8 to 683.8  iu / ml with a slope of 1.06 ( 95% confidence interval 1.041.08 ) , a y - intercept of 2.0 ( 6.92.8 ) , and an r of 0.99 .
the overall qualitative agreements varied between 77.0% ( nova lite clift versus quanta lite ) and 89.4% ( immulisa versus nova lite clift ) .
kappa agreements were low ranging from 0.17 to 0.42 ( quanta lite and quanta flash versus bioplex ) .
total percent agreement , kappa values , and spearman 's rho between dsdna methods can be found in table 2 ( spearman correlation graphs between dsdna methods are not shown ) .
the clinical sensitivities at the recommended cut - off were 8.2% ( 95% confidence interval , ci 2.718.1% ) for nova lite clift , 26.2% ( 95% ci 15.839.1% ) for immulisa , 39.3% ( 95% ci 27.252.7% ) for quanta flash , 44.3% ( 95% ci 31.557.6% ) for bioplex , and 54.1% ( 95% ci 40.866.9 ) for quanta lite .
the clinical specificities at the recommended cut - off were 100.0% ( 95% ci 96.4100.0% ) for nova lite clift , 96.0% ( 95% ci 90.198.9% ) for immulisa , 96.0% ( 95% ci 90.198.9% ) for quanta flash , 88.0% ( 95% ci 80.093.6% ) for bioplex , and 91.0% ( 95% ci 83.695.8% ) for quanta lite .
when the sensitivities were compared at a specificity of 94.0% , the values ranged between 8.2% ( 95% ci 2.718.1% ) for clift and 52.5% ( 95% ci 39.365.4% ) for quanta flash dsdna cia .
sensitivities and specificities as well as positive and negative likelihoods ratios ( lr ) and area under the curve ( auc ) values for all assays are shown in table 3 . the odds ratio ( lr+/lr ) for dsdna methods varied between 5.9 ( bioplex ) and 15.6 ( quanta flash ) .
comparative roc curve analysis among sle patients ( n = 61 ) and disease controls ( n = 100 ) for five anti - dsdna antibody assays showed significantly different auc values . quanta flash and
quanta lite had the highest auc values which were significantly higher ( p < 0.05 ) than the auc values of the other assays ( see figure 1 ) . to illustrate the reactivity of various assays in relation to the diagnosis
the cluster analysis shows that the majority of sle patients have multiple positive results ( figure 2 ) .
the dendrogram shows the quanta lite dsdna sc clusters closest to the diagnosis of sle followed by quanta flash and bioplex 2200 .
consequently , we used the clift results ( positive versus negative ) as the reference and analyzed the utility of the other assays to match the sensitivity and specificity of the clift .
all clift positive samples were also positive by all four methods , except for one sample that was missed by the immulisa .
roc analysis ( roc curve not shown ) of the four anti - dsdna antibody assays compared to clift resulted in auc values of 0.93 ( 95% ci 0.851.00 ) for bioplex , 0.94 ( 95% ci 0.881.00 ) for immulisa , 0.95 ( 95% ci 0.910.99 ) for quanta lite , and 0.97 ( 95% ci 0.940.99 ) for quanta flash .
a cut - off of 106.7  iu / ml for quanta flash dsdna would result in an optimal agreement to clift , with a positive agreement of 100.0% ( 95% ci 47.8100.0% ) , negative agreement of 95.5% ( 95% ci 91.098.2% ) , total agreement of 95.7% ( 95% ci 91.298.2% ) , and kappa = 0.57 ( 95% ci 0.290.85 ) . to further validate the good agreement between quanta flash dsdna and clift ,
additional sle patients ( n = 69 , inova diagnostics serum bank ) tested by both methods were added to the study population .
roc analysis of the clinical performance for nova lite clift and quanta flash dsdna among the new study population ( sle = 130 , controls = 100 ) can be found in figure 3 .
roc analysis for the agreement of quanta flash dsdna results to nova lite clift results can be found in figure 4 .
anti - dsdna antibodies are a hallmark in the diagnosis of sle and are part of the classification criteria . during the last decades
, several novel technologies have been developed for anti - dsdna antibody detection including the conventional elisa and , more recently , line immunoassays ( lia ) , cia , and multiplex assays [ 11 , 12 ] .
as elisas are only moderately fast with assay times between 1.5 and 3 hours , the focus has lately shifted towards a decrease in assay time and ease of use . despite significant technological advances ,
controversies persist on the method of choice for the detection of these antibodies [ 4 , 14 ] .
the bioplex 2200 dsdna assay has been compared to other assays ( farr and farrzyme ) in a recent study . additionally , several studies used the bioplex 2200 dsdna assay as the only method for the detection of anti - dsdna antibodies [ 16 , 17 ] .
the sensitivity in our cohort ( 44.3% ) was very similar to the data ( 40% ) presented by op de beck et al .  
the clift assay is an iif test based on the hemoflagellate crithidia luciliae as the substrate .
various commercial elisa tests are available through numerous companies . although based on the same technology , poor agreement can be observed , as seen in previous studies [ 14 , 15 , 19 , 20 ] .
similar to the sensitivity observed for bioplex 2200 dsdna in this study , the sensitivity in sle found for the two elisas was lower than previous studies [ 21 , 22 ] , indicating that the sle sample population for this study could be the reason .
lastly , the farr assay has been considered as the  gold standard  for the detection of anti - dsdna antibodies [ 4 , 6 , 7 ]
. however , not in all studies , the farr assay was superior to other methods for the detection of anti - dsdna antibodies and the discrimination of sle and controls .
additionally , in many countries , laboratories are switching from the farr assay to fully automated solutions , which is largely due to the radioactive nature of the farr assay [ 4 , 5 ] .
therefore , considering that newer fully automated technologies are available , it is important to evaluate these methods as viable alternatives as the farr assay becomes less popular .
some studies also investigated the impact of the antigen source on the performance of anti - dsdna antibody assays .
since this study compares not only different technologies but also methods with different antigen sources , it is unclear if antigen source is a contributor to the variability in the results .
this is the first study on quanta flash dsdna , a novel cia for detection of anti - dsdna antibodies . like other assays on the bio - flash [ 9 , 23 , 24 ] , the quanta flash dsdna cia delivers quantitative results with high precision and linearity in as little as 30 minutes .
the quanta flash dsdna cia showed lower sensitivity compared to the quanta lite dsdna elisa ( 39.3% versus 54.1% ) but had a higher specificity ( 96.0% versus 91.0% ) and a higher odds ratio compared to the other methods . by setting the specificity to the clinically relevant level of 94.0% for all dsdna methods , quanta flash dsdna cia achieved the highest sensitivity ( 52.5% ) .
since the clift assay is often used as a confirmatory test for the detection of anti - dsdna antibodies , we aimed to analyze the possibility to use higher cut - off values for quanta flash dsdna to increase the agreement to clift . using a cut - off of 106.7  iu / ml ,
the extended study between quanta flash dsdna cia and nova lite clift with additional sle samples resulted in good agreement between methods around the manufacturer 's cut - off for quanta flash dsdna .
figure 4 displays the agreement of quanta flash dsdna to clift at various cut - offs , which demonstrates the potential to match the samples positive by clift .
this finding is promising that , using the quanta flash dsdna , confirmation testing might become unnecessary .
a limitation of our study is the number of sle patients and controls , but the strength of the study is the number of technologies analyzed .
however , despite the small cohort of patients , we found significant differences between the assays and confirmed the lack of standardization [ 19 , 20 ] .
another shortcoming is the exclusion of the farr assay from our study due to the lack of access to the technology . at our facility ,
assays with radioactive materials can not be performed and the sample volumes were too low to send out samples for testing . on the other hand , we included five methods for the detection of anti - dsdna antibodies .
further studies with larger cohorts are needed to contribute to the understanding of how anti - dsdna antibody assays perform and correlate .
significant variations among anti - dsdna antibody assays were observed confirming the lack of standardization .
quanta flash dsdna cia provides an optimal combination of sensitivity and specificity for the diagnosis of sle .
future studies with larger sample populations are needed to confirm the clinical performance and agreement to clift .
 
 introduction . 
 the objective of the present study was to compare quanta flash dsdna , a chemiluminescent immunoassay ( cia ) on the bio - flash , a rapid - response chemiluminescent analyzer , to three other anti - dsdna antibody assays and to crithidia luciliae indirect immunofluorescence test ( clift ) . 
 methods . in the first part of the study , 
 161 samples , 61 from patients suffering from systemic lupus erythematosus ( sle ) and 100 from a disease control group , were tested by quanta flash dsdna cia , quanta lite dsdna sc elisa , bioplex 2200 multiplex flow immunoassay ( mfi ) , immulisa dsdna elisa , and nova lite clift . 
 a second cohort of 69  sle patients was then tested by quanta flash dsdna and clift to expand the study . 
 results . 
 the overall qualitative agreements varied between 77.0% ( nova lite clift versus quanta lite ) and 89.4% ( immulisa versus nova lite clift ) . the clinical sensitivities for the anti - dsdna antibody tests varied from 8.2% ( nova lite clift ) to 54.1% ( quanta lite ) , while the clinical specificities varied from 88.0% ( bioplex 2200 ) to 100.0% ( nova lite clift ) . 
 good correlation was found between quanta flash dsdna and nova lite clift . 
 conclusion . 
 significant variations among dsdna methods were observed . 
 quanta flash dsdna provides a good combination of sensitivity and specificity for the diagnosis of sle and good agreement to clift . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion