cilj ove multicentrine studije bio je da se rezultati sedam automatizovanih komercijalnih imunoeseja uporede sa referentnom tehnikom hplc .
je , podeljeno u alikvote , smrznuto i poslato laboratorijama koje su uestvovale u istraivanju .
je pomou referentnog sistema hplc i pomou sedam automatizovanih komercijalnih imunoeseja ( roche cobas e601 , beckman coulter unicel dxi 800 , ortho vitros es , diasorin liaison , siemens advia centaur , abbott architect i system i ids isys ) .
u poreenju sa referentnim metodom , koeficijenti regresije kretali su se izmeu 0,923 i 0,961 ( svuda p<0,001 ) .
nagib za demingovu regresiju kretao se od 0,95 do 1,06 , dok je kriva obuhvatila podruje izmeu 15,2
i razliitih imunoeseja pri koncentraciji od 50 nmol / l 25oh vitamina d variralo je izmeu 0,61 i 0,85 ( svuda p<0,001 ) .
procenat uzoraka ispod ove cut - off vrednosti znaajno se razlikovao samo za jedan imunoesej .
odlina korelacija sa referentnom tehnikom hplc potvruje da se svih sedam automatizovanih imunoeseja mogu sa sigurnou koristiti za rutinsko odreivanje nivoa 25oh - d u klinikim laboratorijama .
znaajno odstupanje izmeu razliitih metoda po svoj prilici se moe uglavnom pripisati nedostatku standardizacije i zahteva dodatne napore kako bi se popravila harmonizacija imunoeseja za 25oh - d .
vitamin d is a fat soluble compound , which exerts a kaleidoscope of biological functions in humans ( 1 ) .
basically , vitamin d2 ( ergocalciferol ) derives from ergosterol , a membrane sterol produced by phytoplankton , invertebrates and fungi in response to ultraviolet ( uv ) irradiation , but it is not constitutively synthesized in plants or vertebrates .
vitamin d3 ( cholecalciferol ) is produced in the skin or can be received through diet from animal sources , principally fish , eggs yolks or liver .
the synthesis of 25-hydroxyvitamin d ( 25oh - d ) occurs by hydroxylation of vitamin d3 ( or d2 ) in the liver , which is then followed by a further hydroxylation in the kidney , to generate 1-,25-dihydroxycholecalciferol ( i.e. , calcitriol or 1,25oh-2d ) . according to this complex metabolic pathway
, the concentration of 25oh - d is currently regarded as the most suitable indicator of vitamin d body stores ( 1 ) .
the 25oh - d deficiency , which is currently defined as a value lower than 50 nmol / l ( i.e. , 20 ng / ml ) by the us institute of medicine ( iom ) ( 2 ) and the clinical practice guideline of the endocrine society ( 3 ) , has recently emerged as a public healthcare issue . beside the well - know function in bone metabolism , 25oh - d deficiency
is increasingly associated with a number of human disorders , including cardiovascular disease ( 4 ) , cancer ( 5 ) , infectious diseases ( 6 ) , and frailty ( 7 ) among others .
the measurement of 25oh - d in serum or plasma has hence become a cornerstone for overall health and well - being , and vitamin d testing volumes continue to grow in clinical laboratories ( 8) . the current techniques for measuring 25oh - d entail liquid chromatography ( lc ) methods coupled with automated uv or mass spectrometric ( ms ) detection , and immunochemistry techniques , which are based on polyclonal or monoclonal antibodies directed against 25oh - d and have been developed for use on a variety of automated clinical chemistry platforms ( 2 , 9 ) .
although isotope dilution lc - ms / ms is considered the candidate reference method for accurate quantification of 25oh - d , high - pressure ( hplc ) techniques with uv detection provide comparable results , allow simultaneous measurement of either 25oh - d2 or 25oh - d3 , and are also more affordable to routine clinical laboratories ( 9 ) .
both lc - ms / ms and hlpc techniques have several drawbacks compared to automated immunoassays , including higher complexity , longer turnaround time and the need for skilled personnel , which make them virtually unavailable to some laboratories , especially the smaller ones or those for which lc equipment is unaffordable .
therefore , automated assays are typically regarded as the best choice for a number of laboratory services , provided that these methods display satisfactory analytical performance and optimal agreement with the reference lc techniques .
some previous articles have been published about the analytical comparison of automated immunoassays with lc methods ( 1014 ) , showing rather heterogeneous results .
therefore , the aim of this multicenter study was to compare the results of 25oh - d obtained with a reference hplc technique , with the results of seven different automated commercial immunoassays .
blood collection ( 13  100 mm x 6.0 ml bd vacutainer plus plastic serum tube ; becton dickinson , franklin lakes , nj , usa ) was centralized at the academic hospital of parma , italy . in brief , 120 consecutive outpatient samples
( 58 males and 72 females ; mean age 5418 years ) referred to the local laboratory medicine service with a specific request for 25oh - d testing were centrifuged , separated and divided in 5 aliquots of 0.6 ml each .
the first aliquot was used for routine measurement of 25oh - d as for the physician s prescription , whereas the remaining 4 aliquots were stored at 70 c for delayed testing .
after one week of storage , all aliquots were transported to the participating centers using certified transport boxes , under controlled conditions of temperature and humidity .
upon arrival to the different laboratories , the samples were kept stored at 70 c until all centers had received the shipment , thus allowing a simultaneous start of measurements . before analysis ,
the analytical characteristics of the 25oh - d immunoassays are synthesized in table i. the reference hplc method used in this study ( chromsystems instruments & chemicals gmbh , grfelfing , germany ) allows simultaneous chromatographic determination of both 25-oh - d3 and 25-oh - d2 on a simple isocratic hplc system with uv detection ( gilson aspec xl , middleton , wi , usa ) .
the method was calibrated using a commercial proprietary standard ( chromsystems instruments & chemicals gmbh ) .
the basic characteristics of this method have been previously described elsewhere ( 15 ) . in brief , interfering components are removed from the samples by protein precipitation and selective solid phase extraction .
the analytes are then quantified with a hplc test system , by inclusion of a stable internal standard .
the results of all measurements were analyzed with deming fit , spearman s correlation , kappa statistic ,  square test with yates correction and bland & altman plots , using analyse - it ( analyse - it software ltd , leeds , uk ) .
the study was based on preexisting samples , so that ethical permission and informed consent were unnecessary , according to our local ethical committee .
the study was , however , performed in accordance with the declaration of helsinki and under the terms of all relevant local legislations .
the values of 25oh - d measured in the 120 out - patient serum samples with the reference hplc were evenly distributed throughout the relevant biological range of concentrations ( median 78.7 nmol / l ; inter - quartile range 53.7115 nmol / l ; range 8.2255 nmol / l ) .
the results of deming fit and spearman s correlation obtained by comparison with the hplc reference method are shown in table ii .
briefly , the regression coefficients were always optimal , ranging from 0.923 to 0.961 ( all p<0.001 ) .
the slope of the deming fit ranged from 0.95 to 1.06 , whereas the intercept was comprised between 15.2 and 9.2 nmol / l .
the bias from the reference hplc technique , calculated from bland & altman plots , is shown in table iii and figure 1 . in general , the mean bias varied from 14.5 to 8.7 nmol / l .
accordingly , the minimum performance goal for bias suggested by the endocrine society ( i.e. 15.8% ) was slightly exceeded by only one immunoassay ( i.e. unicel dxi 17.1% , 95% ci 21.7 to 12.4% ) , but not by the other methods ( i.e. cobas e601 11.9% , 95% ci 17.2 to 6.5% ; advia centaur 11.1% , 95% ci 6.6 to 15.6% ; vitros es 6.7% ; 95% ci 11.9 to 1.5% ; liaison 5.4% , 95% ci 8.8 to 2.0% ; isys 10.7% , 95% ci 7.5 to 14.0% ; architect 7.9% , 95% ci 4.0 to 11.8% ) ( 3 ) .
the agreement ( kappa statistics ) between hplc and the different immunoassays at the 50 nmol / l 25oh - d threshold is also shown in table iii , and varied between 0.61 and 0.85 ( all p<0.001 ) .
the percentage of samples below this cut - off was 22% ( 26/120 ) with hplc , 24% ( 29/120 ; p=0.76 versus hplc ) with cobas e601 , 11% ( 13/120 ; p=0.036 ) with advia centaur , 29% ( 35/120 ; p=0.24 ) with unicel dxi , 28% ( 33/120 ; p=0.37 ) with vitros es , 23% ( 28/120 ; p=0.88 ) with liaison , 14% ( 17/120 ; p=0.18 ) with isys , and 20% ( 24/120 ; p=0.87 ) with architect .
according to recent evidence , the frequency of 25oh - d deficiency ranges from 22 to 26% across ages and genders ( 16 ) .
these figures are mirrored by a constant increase in demand for vitamin d measurement in clinical laboratories ( 8) . although it is rather understandable that 25oh - d should be preferably assessed with lc techniques , either hplc or lc - ms ( 2 , 3 ) , this approach is virtually unsuitable in laboratories where these techniques are unaffordable due to economic or organizational issues . the gradual introduction into the diagnostic market of a variety of automated immunoassays should hence be regarded as a viable alternative for routine 25oh - d assessment , provided that results are accurate and ultimately correlated with a reference method .
various previous studies showed that some commercial methods fail to meet stringent analytical criteria ( 10 , 14 ) , whereas others reported more favorable data for certain assays ( 1113 ) . beside a simple comparison of data throughout the biological range of 25oh - d ,
the most critical issue is indeed the agreement at the current threshold used for defining vitamin d deficiency that is 50 nmol / l ( 2 , 3 ) .
the results of this investigation clearly attest that only one assay ( i.e. advia centaur ) displayed a significant disagreement at the diagnostic threshold of 25oh - d deficiency among the seven that we have tested , wherein the rate of vitamin d deficiency determined with this method appeared nearly half that measured by using hplc ( 11% versus 24% ; p=0.036 ) .
interestingly , we found an overall positive bias for advia centaur as compared with hplc ( + 11% ) , which was more evident in samples with low values of 25oh - d ( i.e. < 50 nmol / l ) ( figure 1 ) .
( 17 ) , wherein samples with very low 25oh - d were shown to have an approximately 50% positive bias compared to a reference lc - ms / ms technique . however , the overall correlation of this assay was excellent both in our study ( r=0.955 ) and in the analytical evaluation of janssen et al .
an opposite trend was found with cobas e601 . in agreement with two previous studies ( 12 , 18 ) ,
this is probably attributable to the fact that , at variance with the reference hplc assay used in this study , the roche immunoassay is traceable to the national institute of standards and technology ( nist ) serum - based standard reference material ( srm ) 972 ( 19 ) .
it is also noteworthy , however , that the overall agreement of this assay was the third highest among all immunoassays and the correlation was excellent ( i.e. r=0.923 ; p<0.001 ) , very similar to the one previously reported by chen et al .
( 12 ) ( r=0.945 ) . as regards the other immunoassays , despite a good agreement at the diagnostic cut - off and an excellent correlation ( r=0.945 ; p<0.001 ) with hplc , the method that has been made recently available on the unicel dxi exhibited the highest ( negative ) bias ( i.e. 14.5
nmol / l ) and a percentage difference that slightly exceeded the minimum performance goal for bias ( i.e. 17.1% versus 15.8% ) . specifically , this bias was mostly attributable to samples with 25oh - d > 75 nmol / l ( figure 1 ) , and hence with analyte concentrations greater than the conventional threshold of moderate 25oh - d deficiency
. this may be attributable to the specific immunoreactivity of the antibodies against the samples tested , but also to the fact that this is a 2-step chemiluminescent method , whereas the other immunoassays are based on direct ( i.e. 1-step ) competitive binding against 25oh - d or vitamin d binding protein ( vdbp ) ( table i ) .
interestingly , the percentage bias calculated on samples with 25oh - d < 75 nmol / l was much lower ( n=56 ; 12.8% ; 95% ci 21.1 to 4.5% ) .
no previous studies have investigated the performance of this assay , to the best of our knowledge , and thereby comparison with results of other analytical investigations is impossible .
along with cobas e601 , this method is also calibrated against the nist - srm 972 , so that these results are not unexpected .
it has been recently shown that the values of 25oh - d are typically low - biased in methods traceable to nist - srm 972 , especially when measuring samples containing prevalently 25oh - d3 ( 20 ) such as those obtained from our study population of unselected outpatients ( supplementation with 25oh - d2 is not prescribed in our area ) .
the results of other assays were globally satisfactory , with excellent correlations and significant agreement at the cut - off of 25oh - d deficiency ( table ii and iii ) .
a negative bias was also observed for the abbott architect , in agreement with that previously reported by jovii et al . ( 18 ) , who also used an hplc technique as the gold standard . in conclusion ,
the excellent correlations with the reference hplc technique found in this study attest that all seven automated immunoassays may be reliably used for routine assessment of 25oh - d in clinical laboratories .
the differences observed in this study are at least in part attributable to the different test design .
more specifically , the roche cobas e601 electrochemiluminescent immunoassay is a competitive protein binding method which uses a specific protein that binds to vdbp , whereas the other methods are based on the competitive binding of antibodies to 25oh - d ( table i ) .
it is also noteworthy that direct methods , in which 25oh - d and vdbp are not completely separated , may also display heterogeneous immunoreactivity compared to 2-step immunoassays ( 10 ) .
regardless of these differences , only one method slightly exceeded the minimum performance goal for bias as compared with hplc , and another one displayed significant disagreement at the cut - off of 25oh - d deficiency .
in both cases , however , the correlation with the reference method was excellent , thus emphasizing the current issue of poor standardization of vitamin d testing ( table i ) .
this hypothesis is also supported by the rather heterogeneous value of bias ( from 14.5 to 8.5
although this should be regarded as a potential confounding factor when assessing 25oh - d according to the conventional recommendations to maintain the concentration of this vitamin above a certain threshold ( 2 , 3 ) , the objective of harmonization , however , may be achieved with relatively modest efforts . due to the high reliability of individual results against the reference technique ( table ii and iii ) , it is reasonable to hypothesize that extension of traceability to common standards ( e.g. nist - srm 2972 ) across different methods and platforms should be effective to consistently reduce the bias and improve comparability among the various automated 25oh - d immunoassays available on the market .
 summarybackgroundthe measurement of 25oh vitamin d continues to grow in clinical laboratories . 
 the aim of this multi - center study was to compare the results of seven automated commercial immunoassays with a reference hplc technique.methodsone hundred and twenty consecutive outpatient serum samples were centrifuged , divided in aliquots , frozen and shipped to the participating laboratories . 
 25oh vitamin d was measured with a reference hplc system and with seven automated commercial immunoassays ( roche cobas e601 , beckman coulter unicel dxi 800 , ortho vitros es , diasorin liaison , siemens advia centaur , abbott architect i system and ids isys).resultscompared to the reference method , the regression coefficients ranged from 0.923 to 0.961 ( all p<0.001 ) . 
 the slope of deming fit ranged from 0.95 to 1.06 , whereas the intercept was comprised between 15.2 and 9.2 nmol / l . 
 the bias from the reference hplc technique varied from 14.5 to 8.7 
 nmol / l . the minimum performance goal for bias 
 was slightly exceeded by only one immunoassay . 
 the agreement between hplc and the different immunoassays at 50 nmol / l 25oh vitamin d varied between 0.61 and 0.85 ( all p<0.001 ) . 
 the percentage of samples below this cut - off was significantly different with only one immunoassay.conclusionsthe excellent correlation with the reference hplc technique attests that all seven automated immunoassays may be reliably used for routine assessment of 25oh - d in clinical laboratories . 
 the significant bias among the different methods seems mostly attributable to the lack of standardization and calls for additional efforts for improving harmonization of 25oh - d immunoassays . 
Uvod
Metode
Rezultati
Zakljuak
Introduction
Materials and Methods
Results
Discussion