Preanalytical Factors in the Measurement of Intact Parathyroid Hormone with the DPC IMMULITE Assay

Intact parathyroid hormone (iPTH) is commonly measured with two-site immunometric assays. The DPC IMMULITE 2000 assay (Diagnostics Products Corporation) measures iPTH by a chemiluminescence reaction with a monoclonal murine capture antibody and a polyclonal caprine signal antibody conjugated to alkaline phosphatase. Accurate and precise iPTH measurements are needed for the correct triage of individuals with hyper- or hypocalcemia and for the evaluation of PTH function in bone and mineral disorders (1)(2). In end-stage renal failure, iPTH measurements are commonly used to determine medical management (administration of vitamin D) and surgical therapeutic options (subtotal parathyroidectomy for extreme hyperparathyroidism) (3)(4). Collection of whole blood into EDTA-containing sample tubes offers increased iPTH stability at room temperature (5) compared to serum samples (6) and is recommended by the manufacturer of the DPC IMMULITE 2000 iPTH assay. However, insufficient sample volume can lead to increased final EDTA concentration in the sample-reagent mixture, causing chelation of metallic cations, and this can affect the activity of the alkaline phosphatase label used in the chemiluminescent reaction. We determined the magnitude of effect of limited sample volume in EDTA-containing tubes on results of the DPC IMMULITE iPTH assay. We evaluated randomized samples from 20 patients with end-stage renal failure: 12 with increased iPTH, 5 with iPTH within the reference interval, and 3 with suppressed iPTH, along with 5 patients with healthy renal function. We collected ∼15 mL of whole blood from each patient, before the commencement of dialysis in end-stage renal failure, into preweighed sample tubes; one EDTA tube was completely filled (reference sample) and five other EDTA tubes were underfilled by approximately 20%, 33%, 50%, 67%, and 80%, as estimated visually and confirmed by weighing after sample collection. PTH was determined by the DPC Intact PTH assay with the IMMULITE 2000 automated immunoassay analyzer. Subsequently, PTH was remeasured after the addition of 120 mmol/L magnesium sulfate to the DPC IMMULITE assay reagent in 10 of the patients (60 individual samples). When EDTA tubes were <50% filled with whole blood, the reported iPTH value was <75% of the target value (where the iPTH was >10 pmol/L) or, in absolute terms, >2.5 pmol/L (where the iPTH was <10 pmol/L) less than the target value (Fig. 1A). Both of the above criteria are used by the Royal Australasian College of Pathologists Quality Assurance Program to determine allowable limits of performance for this assay. The effect was demonstrable with both the old and the new (reformulated) DPC IMMULITE iPTH assays. Furthermore, the effect was present regardless of renal function or whether the target iPTH value was suppressed, within the reference interval, or increased. Statistical analysis (repeated-measures ANOVA and Dunnett’s post hoc test) of results, expressed as a percentage of the target value, indicated that all sample iPTH values were significantly different from the target value when the EDTA sample tube was ≤50% filled with blood (P <0.001). Sample size and magnesium-supplemented reagent effects on accuracy of iPTH measurements. (A), effect of sample size on iPTH measurement. Mean ± 1 SD for each sample size was expressed relative to the target value, obtained from completely filled EDTA samples. Sample iPTH values from EDTA-containing tubes filled with ≤50% blood are statistically different from the target iPTH values (P <0.001; repeated measures from ANOVA and Dunnett’s post hoc test). •, patient samples (n = 12) studied with increased iPTH; ▴, samples (n = 13) studied with suppressed or iPTH within the reference interval. (B), recovery with magnesium-supplemented reagent and diluted EDTA whole blood patient samples (n= 10). Mean ± 1 SD was expressed relative to the target value obtained from completely filled EDTA samples. When assayed with magnesium-supplemented reagent, only samples that were collected into EDTA-containing tubes and in which there was >80% underfilling have iPTH values that are statistically different from the target iPTH value (P <0.0001; repeated measures from ANOVA and Dunnett’s post hoc test). Using five samples from patients with target iPTH values within the reference intervals and five samples from patients with increased target iPTH values, we performed repeat assays with the magnesium-supplemented DPC reagent. All samples, except those <33% full, conformed to the criteria used by the Royal Australasian College of Pathologists Quality Assurance Program for allowable limits of performance with values ≥75% of the target value (Fig. 1B). We conclude that filling EDTA-sample tubes to ≤50% affects iPTH measurements by the DPC IMMULITE iPTH assay. Incomplete sample filling increases the final EDTA concentration in the sample and consequently affects the alkaline phosphatase secondary enzyme used in the DPC IMMULITE chemiluminescent assay. The addition of supplementary magnesium to the DPC IMMULITE iPTH assay reagent appears to be a simple solution to this problem and can recover most iPTH values. These findings are not only important for the DPC IMMULITE iPTH assay, but potentially applicable to other chemiluminescence assays that use conjugated alkaline phosphatase. We thank the medical and nursing staff of the renal unit at Royal Perth Hospital, especially Dr. A. Irish and J. Gobener for access to patient samples.