Evaluation of the analytical performance of the Beckman Coulter Unicel DXI 800 Access Total 25(OH) Vitamin D immunoassay

Study design

The study was conducted with 148 patient samples with either low, normal or high vitamin D levels, which were sent to Ankara Numune Training and Research Hospital for routine vitamin D testing. Every sample was aliquoted following centrifugation at 2000 g for 15 min and stored at −80°C until the time of analysis. For every sample, vitamin D level was analyzed with both Beckman Coulter Access Total 25(OH) Vitamin D assay on the Unicel DXI 800 analyzer and LC-MS/MS.

Imprecision, interference, limit of blank (LOB), recovery, linearity and carry-over studies were performed for the Beckman Coulter Unicel DXI 800 analyzer.

Analytic method

The Access Total 25(OH) Vitamin D assay is a two-step competitive binding immunoenzymatic assay using sheep monoclonal anti-25(OH) vitamin D antibodies. Initially, 25(OH) vitamin D is removed from the vitamin D-binding protein (DBP) and bound to the immobilized monoclonal anti-25(OH) vitamin D in the solid phase. Subsequently, a 25(OH) vitamin D analog-alkaline phosphatase conjugate is added which competes for binding to the immobilized monoclonal anti-25(OH) vitamin D. After a second incubation, materials bound to the solid phase are held in a magnetic field while unbound materials are washed away. Then, the chemiluminescent substrate Lumi-Phos* 530 is added to the vessel and light generated by the reaction is measured using a luminometer. The light production is inversely proportional to the concentration of 25(OH) vitamin D in the sample. The amount of analyte in the sample is determined from a stored, multi-point calibration curve.

The measurand (analyte) in the Access Total 25(OH) Vitamin D calibrators is traceable to a Joint Committee for Traceability in Laboratory Medicine (JCTLM)-approved isotope dilution mass spectrometry (ID-LC-MS/MS) reference measurement procedure (RMP) developed at Ghent University.

Reference method: LC-MS/MS

An AB Sciex API 3200 triple quadropol mass spectrometer connected to a Shimatzu HPLC (Kyoto, Japan) system was used. In HPLC column (Phenomenex, CA, USA) mobile phase A was water, and mobile phase B consisted of 0.1% formic acid in acetonitrile. For sample preparation step, 200 μL serum (calibrator, control, or patient samples) and 75 μL internal standard were pipetted to a standard microcentrifuge tube. Upon addition of 1000 μL acetonitrile, the mixture was vortexed for 30 s, and incubated at +4°C for 10 min. Then, the mixture was centrifuged at 20,000 g for 5 min. The supernatant was transferred to a clean tube and evaporated to dryness with nitrogen. Then, the samples were reconstituted in 100 μL of 50% acetonitrile and vortexed. Fifty microliters of the sample was injected to the HPLC system. The HPLC system had a flow rate of 0.3 mL/min. The API 3200 system was employed in the electrospray mode. Using a multiple reaction monitor, mass/charge spectra of the samples were determined. The following m/z parameters were obtained: 25(OH)D3: 383.4/211.1; 25(OH)D2: 395.4/209.1; and IS: 407.2/389.4.

Precision, linearity, carry-over, interference, recovery and method comparison studies were carried out in accordance with the Clinical & Laboratory Standards Institute (CLSI) protocol.

Imprecision

Precision studies were performed with two levels of pooled serum samples. For intra-assay precision study, each level of the samples was analyzed in 10 replicates in a single run. The low and high serum pool specimens were measured as 5×2 in a single day for the inter-assay precision study. For between-day precision study, each level of the samples was run in duplicate, with two runs per day, over 5 days (first run in the morning and second run in the afternoon). The intra-assay, inter-assay, between-day and total coefficient of variation (CV%) values were calculated in accordance with the EP 5A protocol. Acceptable CV% values were determined as ≤10% [12, 13].

Limit of blank (LOB) and linearity

For determination of LOB, zero concentration calibrator provided by the manufacturer was run in 10 replicates, and the lowest concentration calibrator other than the zero calibrator was run in three replicates. LOB, limit of detection (LOD) and limit of quantitation (LOQ) were calculated according to the following formulas:

LOB=Average blank+1.65*SD blank.

LOD=LOB+1.65*SD(low concentration sample)

LOQ= Average(low concentration sample)+ bias(low concentration sample)+2SD(low concentration sample)

The linearity study was performed by diluting high patient serum pool with low patient serum pool. For this purpose, high serum pools and low serum pools were mixed in the proportions of 1:3, 1:1 and 3:1; thus, five samples were obtained. Acceptable recovery was determined as ±15% of the target value [12].

Recovery

For recovery analysis, serum samples with low and high concentrations were mixed in the proportions of 1:3, 1:1 and 3:1, or 1:2, 1:1 and 2:1 [14].

Interference

Interference analysis was performed in accordance with CLSI [12, 15]. Interfering substances (hemolysate, bilirubin and intralipid) at three different concentrations were added to the pooled serum samples and they were run in duplicates. The following formula was used: ([Interferer-added] – [interferer-not added])/interferer-not added*100. More than 10% deviation from the target concentration was accepted as clinically meaningful.

Carry-over

A serum sample with high vitamin D concentration (191.14 ng/mL; HC sample) was aliquoted to 10 portions; and another serum sample with low vitamin D concentration (15.39 ng/mL; LC sample) was aliquoted to 11 portions, in order to obtain 21 separate samples in total. These 21 samples were tested in a single run as in the following sequence: 3 LC, 2 HC, 1 LC, 2 HC, 4 LC, 2 HC, 1 LC, 2 HC, 1 LC, 2 HC and 1 LC sample. The average measurement result of the LC samples tested after an LC sample was subtracted from the average measurement result of the LC samples tested after a HC sample in order to determine the limit of error. The limit of error was accepted in case it was lower than 3 times the standard deviation (SD) of the measurement results of LC samples tested after an LC sample [13].

Method comparison

Totally 148 patient samples with either low, normal or high concentrations of vitamin D (48 samples with vitamin D concentrations of <30 ng/mL; 60 samples with vitamin D concentrations of 30–100 ng/mL; and 40 samples with vitamin D concentrations of >100 ng/mL) were analyzed with the Access Total 25(OH) Vitamin D immunoassay on the Beckman Coulter Unicel DXI 800 analyzer, and then with the gold standard method, LC-MS/MS, in an accredited laboratory. The results were compared statistically.

Statistical analysis

Statistical analysis was performed using SPSS software (version 17.0 SPSS Inc., Chicago, IL, USA) and MedCalc Statistics software (version 12.0 MedCalc software, Mariakerke, Belgium). The concordance of the results was evaluated with Passing-Bablok regression analysis and Bland-Altman plot. The concordance correlation coefficient (CCC) was calculated. CCC>0.99 denotes excellent agreement; CCC between 0.95 and 0.99 denotes substantial agreement, CCC between 0.90 and 0.95 denotes moderate agreement and CCC<0.90 denotes poor agreement.

Repeatability

The intra-assay, inter-assay and total CV% were 3.3%, 5.3%, and 8.3%, respectively, for 31.7 ng/mL concentration, and 2.1%, 3.2%, and 7%, respectively, for 66.8 ng/mL concentration (Table 1).

Limit of blank

The LOB for the Access Total 25(OH) Vitamin D immunoassay was found to be 1.5 ng/mL. The LOB value specified by the manufacturer was <2 ng/mL. The LOD and LOQ values were 2.05 ng/mL and 2.57 ng/mL, respectively.

Linearity

Serum samples obtained by mixing the samples with high and low concentrations of vitamin D in the predefined proportions were analyzed, and the measured 25(OH) vitamin D concentrations were plotted against the expected values to draw the regression line. Accordingly, the linear regression equation was: measured value=expected value*0.979+2.660; correlation coefficient, r=0.998. Maximum bias was calculated as 9.2% for a concentration of 45.4 ng/mL (Table 2 and Figure 1).

Figure 1:

Linearity graph of the Unicel DXI 800 Access Total 25(OH) Vitamin D assay.

Recovery

Serum samples with high and low concentrations of 25(OH) vitamin D were mixed in the proportions of 3:1, 1:1 and 1:3, and 2:1, 1:1 and 1:2. Measured values of 25(OH) vitamin D concentrations in these samples are listed in Table 3 against the expected values. The recovery values obtained varied between 101% and 111%.

Carry-over

The difference between the average of low-level results tested following low-level samples and the average of low-level results tested following high level samples was 0.724. The limit of error, which is 3 times the value of SD of low-level results, was 6.59.

Interference

In interference study, the presence of hemoglobin at 400 mg/dL, bilirubin at 18 mg/dL and intralipid at 428 mg/dL concentrations in the serum resulted in greater than 10% bias in the results (Table 4).

Method comparison

Compared to the LC-MS/MS method, the Access Total 25(OH) Vitamin D immunoassay on the Beckman Coulter Unicel DXI 800 analyzer demonstrated an R-value of 0.957 (intercept: −3.938 (−6.740–[−0.226]), slope: 1.185 [1.124–1.251]) and a mean bias of 9.5%. The CCC between the two methods was 0.916 (Table 5, Figures 2 and 3).

Figure 2:

Passing-Bablok regression analysis of the Unicel DXI 800 Access Total 25(OH) Vitamin D assay compared to LC-MS/MS methods.

Solid line, regression; dashed lines, 95% confidence intervals.

Figure 3:

Bland-Altman %bias of the Unicel DXI 800 Access Total 25(OH) Vitamin D assay compared to LC-MS/MS method.

Solid lines indicate average %bias. Dashed lines demonstrate the 95% limits of agreement (bias ±1.96 SD).

A concordance according to the percentage of patients classified in the same group with both methods, we found this rate between the Access Total 25(OH) Vitamin D immunoassay and LC-MS/MS to be 75%.