Determination of 25-hydroxyvitamin D 25(OH)D represents a unique challenge, considering its lipophilic na - ture. Considering the widespread prevalence of vitamin D deficiency, which leads to ...increasing number of requests for 25(OH)D determination, immunoassay measurements adjusted to automated analyzers are being developed. Because of the variability among assays, it is often difficult to monitor vitamin D status and supplementation. The aim of this study was to compare the results of two immunoassays with high performance liquid chromatography with ultraviolet detection (HPLC-UV). Also, the aim was to estimate vitamin D status, since up to date the prevalence of vitamin D deficiency in Serbia was not examined. We have evaluated analytical characteristics of two automated immunoassays for 25(OH)D determination, from Roche (CobasR e601) and Abbott (Architect). For comparison studies we used HPLC analysis of 25-(OH)-Vitamin D3/D2 from Chromsystems (Waters isocratic system). In order to estimate vitamin D status in general population, we have searched the database of the laboratory information system and analyzed the data from 533 patients whose 25(OH)D was determined together with intact parathyroid hormone (iPTH). For imprecision assessment, four serum pools were prepared with following 25(OH)D concentrations: 35 nmol/L, ∼50 nmol/L, ∼75 nmol/L and ∼125 nmol/L. Obtai ned CVs for Roche method were 1.5-2.8% for within-run and 4.0-6.7% for between-run imprecision. For Abbott method, CVs were 0.7-4.4% for within- run and 3.8-7.2% for between-run imprecision. Inaccuracy was analyzed with commercial control sera. Obtained deviations from target value were 2.1% for Roche assay and 1.3-1.5% for Abbott method, and were not statistically significant (P>0.05). Comparison of Roche and HPLC-UV methods using Passing-Bablok regression analysis gave the following equation for the regression line y=0.937x+9.518 (r=0.739; n=97) and the regression line equation from the comparison of Abbott and HPLC-UV methods was y=0.745x+10.343 (r=0.793; n=97). Mean difference and SD for Bland-Altman plot were -4.5 nmol/L and 21.75 nmo/L, respectively for Roche method and 6.4 nmol/L and 18.8 nmol/L, respectively for Abbott. Statistical analysis (Chi-square test) of frequency distribution among different vitamin D status categories (<25 nmol/L severe deficiency, 25-50 nmol/L deficiency, 50-75 nmol/L insufficiency and >75 nmol/L sufficiency) showed that the frequency distribution obtained with Abbott method was significantly different from the distribution of the HPLC results, in contrast to Roche results frequency distribution which did not differ significantly. Also, statistical analysis of the agreement between the three methods for each vitamin D status category showed that results of both Roche and Abbott methods were significantly higher than HPLC in the two deficiency categories (P=0.005 for Roche, P=0.0407 for Abbott), and in the sufficiency category Abbott method significantly underestimated concentration of 25(OH)D compared to HPLC results (P<0.0001). Median population values of 25(OH)D and iPTH were 41.8 nmol/L and 76.6 ng/L, respectively. ANOVA analyses showed significant (P<0.05) decrease in iPTH and Ca2+ concentrations across the 25(OH)D concentration categories. Stepwise multiple linear regression analysis indicated independent correlation of iPTH with 25(OH)D concentration (b=-0.290, P=0.0008). Also, one-way ANOVA with Student-Newman-Keuls test demonstrated that 25(OH)D concentrations measured in summer and autumn were significantly (P<0.001) higher compared to those determined in winter and spring. Despite acceptable imprecision and inaccuracy of both examined methods, results obtained with them did not correlate well with HPLC-UV (r<0.9), which was used as a reference. However, methods showed satisfactory ability to classify patients into vitamin D status categories, which is important for diagnosis of vitamin D deficiency and therapy follow-up. About two thirds (68.5%) of the examined po pulation had vitamin D deficiency (25(OH)D<50 nmol/L) and only 8% had sufficient 25(OH)D concentration (>75 nmol/L).
Odre|ivanje 25-hidroksivitamina D 25(OH)D predstavlja jedinstven izazov, s obzirom da je visoko lipofilno jedinjenje. Visoka prevalencija deficijencije vitamina D uzrok je pove}anja broja zahteva za odre|ivanjem 25(OH)D, zbog ~ega se razvijaju imunohemijske metode prilago| ene auto ma tizovanim sistemima. ^esto je te{ko pratiti status vitamina D i suplementaciju zbog varijabilnosti izme|u testova. Cilj ove studije bio je da se uporede rezultati dve imunohemijske metode sa te~nom hromatografijom visoke efikasnosti sa detekcijom u ultraljubi~astom delu spektra (HPLC-UV). Ta ko|e, cilj je bio i procena statusa vitamina D, po{to do sada nije ispitivana prevalencija deficijencije vitami - na D u Srbiji. Ispitivane su karakteristike dve imunohemijske me tode za odre|ivanje 25(OH)D, proizvo|a~a Roche (analizator CobasR e601) i Abbott (na analizatoru Architect). Me - to de su pore|ene sa rezultatima HPLC analize kori{}enjem 25-(OH)-Vitamin D3/D2 reagenasa firme Chromsystems (Waters izokratski sistem). Da bi se procenio status vitamina D u op{toj populaciji, pretra`ena je baza podataka laborato - rijskog informacionog sistema i analizirani su rezultati 533 pacijenata kojima je odre|en 25(OH)D zajedno sa intaktnim paratiroidnim hormonom (iPTH). Pripremljena su ~etiri se - rumska pool-a sa koncentracijama 25(OH)D ∼ 35 nmol/L, ∼50 nmol/L, ∼75 nmol/L i ∼125 nmol/L za procenu nepreciznosti imunohemijskih odre|ivanja. Dobijeni koeficijenti va - rijacije za Roche metodu su se kretali u opsegu 1,5-2,8% u seriji i 4,0-6,7% izme|u serija. Za Abbott metodu su koeficijenti varijacije iznosili 0,7-4,4% u seriji i 3,8-7,2% izme|u serija. Neta~nost je ispitivana pomo}u komercijalnih kontrolnih uzoraka. Dobijena odstupanja od deklarisane vrednosti su iznosila 2,1% za Roche i 1,3-1,5% za Abbott, i nisu bila statisti~ ki zna~ajna (P>0,05). Pore|enjem Roche i HPLC-UV metoda pomo}u Passing-Bablok regresione analize dobijena je slede}a regresiona jedna~ina y=0,937x+9,518 (r=0,739; n=97), dok regresiona jedna~ina dobijena pore|enjem Ab - bott i HPLC-UV metoda glasi y=0,745x+10,343 (r=0,793; n=97). Srednja vrednost razlika na Bland-Altman dijagramu razlika i standardna devijacija su iznosile -4,5 nmol/L i 21,75 nmo/L, redom, za Roche metodu i 6,4 nmol/L i 18,8 nmol/L, re dom, za Abbott metodu. Statisti~ka analiza (Chi-kvadrat test) distribucije frekvencija me|u razli~itim kategorijama statusa vitamina D (<25 nmol/L te{ka deficijencija, 25-50 nmol/L deficijencija, 50-75 nmol/L insuficijencija i >75 nmol/L preporu~ena koncentracija) je pokazala da je distri - bucija frek ven cija dobijena Abbott metodom zna~ajno raz li - ~ita od distribucije HPLC rezultata, za razliku od ras po dele frekvencija dobijene Roche metodom koja se nije zna~ajno razlikovala. Tako|e, statisti~ka analiza slaganja izme|u ispitivane tri me tode u svakoj od kategorija statusa vitamina D je pokazala da su rezultati i Roche i Abbott metoda zna~ajno ve}i od HPLC-UV u kategorijama deficijencije vitamina D (P=0,005 za Roche; P=0,0407 za Abbott), i u kategoriji sa preporu~enom koncentracijom vitamina D Abbott metoda je zna~ajno potcenjivala koncentraciju 25(OH)D u po re |e - nju sa HPLC rezultatima (P<0,0001). Medijana za 25(OH)D u ispitivanoj po pulaciji bila je 41,8 nmol/L, i 76,6 za iPTH. ANOVA analiza je pokazala zna~ajan pad (P<0,05) koncentracija iPTH i jonizovanog kalcijuma izme|u kategorija koncentracija 25(OH)D. Multiplom linearnom regresionom ana - lizom utvr|ena je ne zavisna korelacija izme|u koncentracija iPTH i 25(OH)D (b =-0,290; P=0,0008). Tako|e, ANOVA za jedan kriterijum klasifikacije sa Student-Newman-Keuls testom je pokazala da su koncentracije 25(OH)D odre|ene u leto i jesen zna~ajno vi{e (P<0,001) u pore|enju sa onima odre|enim u zimu ili prole}e. Uprkos prihvatljivoj nepreciz - nosti i neta~nosti obe ispitivane imunohemijske metode, do - bijeni rezultati nisu u zadovoljavaju}oj korelaciji sa HPLC-UV metodom (r<0,9), koja je kori{}ena kao referentna u ovom slu~aju. Uprkos ovoj ~injenici, metode su pokazale zadovo - ljavaju}u sposobnost klasifikacije pacijenata u kategorije statusa vitamina D, {to je va`no za dijagnozu deficijencije vitami - na D i pra}enje terapije. Oko dve tre}ine (68,5%) ispitivane populacije je imalo deficijenciju vitamina D (25(OH)D<50 nmol/L) i samo 8% je imalo preporu~enu koncentraciju 25(OH)D (>75 nmol/L).
PDF
