Underestimation of Serum 25-Hydroxyvitamin D by the Nichols Advantage Assay in Patients Receiving Vitamin D Replacement Therapy

Abstract

washed, the Vitros Signal Reagent was added to the well, and the luminescence was measured (ALOKA luminometer). The IgG fraction of serum samples from the patient and from five control individuals was purified with a MAbTrap TM Kit (Amersham Biosciences), and the IgG concentration was adjusted to 4.0 g/L. The purified IgG (0.08 mL) was incubated at 4°C for 24 h with 0.08 mL of PBS alone or with T 2 , T 3 , or T 4 (Sigma; at 4570, 42, and 768 nmol/L, respectively) dissolved in PBS. Each sample was mixed vigorously with 1.2 mL of polyethylene glycol (PEG; 125 g/L), centrifuged at 2800g for 30 min, aspirated, and washed with 1.2 mL of PEG (125 g/L). After the precipitates were dissolved in 0.001 mol/L hydrochloric acid (0.04 mL) and neutralized by equal amounts of 0.001 mol/L sodium hydroxide, T 2 and T 3 were measured by the FT 3 assay and T 4 by the FT 4 assay. The concentrations of T 2 were expressed as T 3 concentrations. Each sample was analyzed in duplicate. The ability of the purified IgG to bind T 2 , T 3 , or T 4 was defined as the difference between the FT 3 or FT 4 assay result and the respective blank value and is reported as the ␦T 2 , ␦T 3 , or ␦T 4 value.The ratios of FT 3 and FT 4 concentrations in PEG-treated samples (6 ) to those in untreated samples were significantly lower for the patient than for 37 other patients (Table 1 in the online Data Supplement); therefore, immunoglobulins in the patient's serum interfered with both the FT 3 and FT 4 assays. FT 3 and FT 4 values in the mixtures of serum with sheep IgG, bovine globulin, and gelatin did not differ significantly from those in the mixtures of serum and PBS only, suggesting that heterophilic antibodies and anti-gelatin antibodies did not cause the high FT 3 and FT 4 values.When we examined the patient's IgG binding with Vitros FT3II and FT 4 assay wells, the luminescence generated by the patient's serum was higher than that of the 5 control individuals (Table 1 in the online Data Supplement). This suggested that the patient's IgG bound to T 2 -and T 3 -gelatin.The FT 3 and FT 4 concentrations in purified IgG and in treated samples of purified IgG (6 ) were below the lower detection limits of the Elecsys assays, suggesting an absence of T 3 and T 4 contamination in the IgG fractions. The patient's ␦T 2 and ␦T 3 values were higher than those of the 5 control individuals, but the ␦T 4 value was within 2 SD of the values for the 5 controls (Table 1 in the online Data Supplement). This finding implies that the patient's IgG interacted with T 2 and T 3 but not with T 4 . The crossreactivity of the anti-T 3 antibody with T 2 in the Vitros FT3II assay was very low, whereas the patient's ␦T 2 value was evidently higher than that of 5 control individuals. We conclude that T 2 was bound to the patient's IgG.Because anti-gelatin antibodies in the patient's serum were not recognized, we suggest that the interfering substance were antibodies to T 2 and T 3 . As the interfering antibodies did not interfere with the Elecsys FT 3 assay, the interfering antib...

Vitamin D Status in Children and Young Adults With Inflammatory Bowel Disease

“…Assay-related underrepresentation of serum 25OHD concentrations in subjects taking vitamin D supplements was considered, and is possible, in view of the recently reported Nichols' Advantage assay's inadequacy in measuring exogenous sources of vitamin D 2 . 41,42 Nevertheless, our screened subjects were not receiving therapeutic doses of vitamin D 2 (>800 IU of vitamin D 2 ) when their serum 25OHD concentration was measured. Two of the 3 studies in pediatric subjects with IBD to report vitamin D status used a serum 25OHD concentration <15 ng/mL as the cutoff value for vitamin D deficiency and could have, as a result, underestimated the prevalence of this condition.…”

“…Sixty of the above patients qualified and enrolled in the trial, and the abovementioned laboratory values were obtained during enrollment. The remaining 70 did not enroll (either did not qualify [42] or declined enrollment [28]), and the same laboratory values were obtained during a regular clinic visit, as part of a quality improvement nursing initiative in our IBD center. All of the patients had their vitamin D status evaluated, regardless of their BMD status.…”

“…40 Recent studies have found this assay to be inadequate in measuring exogenous sources of vitamin D 2 . 41,42 To further investigate whether possible assay-related underrepresentation of serum 25OHD concentrations contributed (especially in patients who reported taking vitamin D supplements) to the lack of a relationship between serum PTH and 25OHD concentration, we performed the analysis separately for participants who reported taking vitamin D supplements and those who did not, and we still did not find a significant relationship (Spearman's ρ (r) = 0.13, P = .57 in those not taking vitamin D supplements; r = −0.12, P = .26 in those taking such supplements). To account for effects of puberty, we evaluated the relationship between serum PTH and 25OHD concentrations separately in patients ≤18 years and those >18 years, and we still did not find it to be significant (Spearman's ρ (r) = −0.05, P = .64 in patients ≤18 years; r = −0.43, P = .11 in patients >18 years).…”

HPLC Method for 25-Hydroxyvitamin D Measurement: Comparison with Contemporary Assays

“…Likewise, a serum product Some VDBP-based assays forgo the traditional liquidliquid or solid-phase sample clean-up for convenience and to minimize the need for manual intervention. Unfortunately, reports of inaccuracy have brought into question the reliability of the VDBP-based assays (26,27 ).…”

25- Hydroxyvitamin D: Explosion in Clinical Interest and Laboratory Requests

“…This is particularly critical as vitamin D supplements that contain only vitamin D2 may not be detected and therefore, follow-up of vitamin D deficient patients who are on D2 replacement or prophylactic therapy will be challenging, leading to inappropriate supplementation, inaccurate monitoring or possible misdiagnosis. 18,19,20 Despite being reported by many workers, this problem may be underestimated by the manufacturers or physicians alike. 21,22 Vitamin D2 is less physiologically active than vitamin D3, and may be less commonly available in supplementations.…”