Discussion and Conclusions

1. Performance Characteristics of Six Intact Parathyroid Hormone Assays Sonia L. La’ulu1, William L. Roberts2 1ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, 2Department of Pathology, University of Utah, Salt Lake City, UT American Association for Clinical Chemistry Annual Meeting, Chicago, Illinois. July 19-23, 2009 Abstract (revised) OBJECTIVE: The aim of this study was to evaluate the performance characteristics of six intact parathyroid hormone (PTH) assays: Beckman Coulter Access, Abbott ARCHITECT i2000SR, Siemens ADVIA Centaur, Roche Modular E170, Siemens IMMULITE 2000, and DiaSorin LIAISON. METHODS: Imprecision studies were performed using three concentrations of commercially available quality control materials. Two runs of duplicate testing were conducted per day, for 5 days, with a minimum of 2 hours between runs. For method comparison, 203 serum and 193 EDTA plasma samples were tested by all methods and analyzed using the E170 as the comparison method. To determine the reference interval, serum samples were drawn from 130 subjects in the summer (August) and winter (February) and a frozen aliquot was tested by all methods. A study comparing sample types and their stabilities was conducted where 15 individuals had 5 types of BD Vacutainer tubes drawn: red top, SST, PST, green top, and EDTA. An aliquot from each tube was tested after being subjected to one of the following conditions: tested immediately, frozen immediately and tested after thawing, and after 24 hours and 48 hours refrigerated. RESULTS: For imprecision, total CV’s ranged from 1.6 to 10.9%, 1.5 to 8.1%, and 1.1 to 5.5% for levels 1, 2, and 3, respectively. The E170 always had the lowest within laboratory CV and the LIAISON had the highest. Method comparison results by Passing-Bablok regression had slopes of 0.98 – 1.83 and correlation coefficients of 0.92 – 1.00. The lower limit of the reference interval ranged from 10.8 to 21.8 and the upper limit was 56.7 to 123.4. The results of the stability study are summarized (Table 4). CONCLUSIONS: In summary, imprecision was acceptable for all methods with total CV’s ≤10.9%. All methods correlated well to the E170 (r = 0.92 to 1.00). The IMMULITE exhibited the highest positive bias. The lower limit of the reference interval is not influenced by vitamin D status while the upper reference limit is affected. The types of collection tubes and sample storage conditions are more important for some methods than others. Table 2. Method comparison Table 4. Sample stability and tube type Table 1. Imprecision Table 3. Reference interval Introduction Numerous studies have demonstrated the lack of comparability among PTH assays. In addition, many note that the differences observed may potentially influence clinical decision making and prove to be problematic for subsequent diagnosis and treatment. This variability can be influenced by a variety of conditions such as the assay used, the population evaluated, vitamin D status, and numerous pre-analytical conditions. In the present study, we set out to evaluate performance characteristics that potentially play a role in inter-method variability of PTH assays namely, imprecision, comparison of methods including two sample types, reference intervals with consideration of vitamin D status, sample storage conditions, and sample collection tubes. Figure 1. Method comparison by Passing-Bablok regression for plasma and serum samples • Materials and Methods • Testing was performed with six PTH assays: Beckman Coulter Access, Abbott ARCHITECT i2000SR, Siemens ADVIA Centaur, Roche Modular E170, Siemens IMMULITE 2000, and DiaSorin LIAISON. Testing by all methods was performed according to manufacturers’ instructions. • Imprecision studies were performed using three concentrations of commercially available quality control materials. Two runs of duplicate testing were conducted per day, for 5 days, with a minimum of 2 hours between runs. • Method comparison was evaluated by testing 203 serum and 193 EDTA plasma samples by all methods. Results were analyzed by Passing-Bablok regression with the E170 arbitrarily chosen as the comparison method. • To determine the reference interval, serum samples were drawn from 130 subjects in the summer and winter seasons and a frozen aliquot was tested by all methods. Samples were also tested for calcium, creatinine, and 25-hydroxyvitamin D (vitamin D). Those with an eGFR ≥60 and normocalcemic were used to calculate the reference interval. Reference interval results were further analyzed based on vitamin D results using cutoffs of 10, 20, and 30 ng/mL. • A study comparing sample types and their stabilities was conducted where 15 individuals had 5 types of BD Vacutainer tubes drawn: red top, SST, PST, green top, and EDTA. An aliquot from each tube was tested after being subjected to one of the following conditions: tested immediately (fresh), frozen immediately and tested after thawing (frozen), and after 24 hours and 48 hours refrigerated. • Results from samples that were frozen or refrigerated were compared to fresh samples using paired t-test and percent recoveries. Also, to evaluate possible differences among sample types, all collection tubes were compared to red tops for fresh samples. *Greater than 12.5% desirable analytical bias than compared to red top. †Greater than 12.5% desirable analytical bias than compared to samples tested immediately (fresh). • Discussion and Conclusions • Imprecision was acceptable for all methods with total CVs of 10.9% or less (Table 1). The E170 always had the lowest total CV and the LIAISON had the highest. • All methods correlated well to the E170 (Figure 1, Table 2). The IMMULITE exhibited the highest positive bias for both sample types, with the largest bias seen for EDTA plasma samples. • No statistically significant difference was observed for serum samples drawn in the summer vs. winter (p≥0.418). Therefore, both seasons were combined and used to calculate the reference interval (Table 3). • The lower limit of the reference interval is not affected by vitamin D status for any method. However, the upper limit does seem to be influenced by the vitamin D cutoff used, except for the LIAISON. In most cases, the upper limit for vitamin D >30 ng/mL does not fall within the confidence limits of the calculated reference interval for all normal subjects as well as those with vitamin D >10 ng/mL. • While it remains controversial regarding the threshold used to define vitamin D insufficiency, some have reported using a vitamin D of 20 ng/mL. When analyzing our reference interval data at this cutoff, the upper reference limit for the majority of the methods, overlapped the 90% confidence intervals for the other cutoffs we looked at. • Analysis of sample stability showed many statistically significant differences compared to fresh analysis. However, when using a previously defined desirable analytical bias of 12.5% to evaluate if the differences where clinically significant, only 6 were ≥12.5% (Table 4). • No clinically significant difference was observed with any method for storage at 24 hours refrigerated vs. tested immediately. • Evaluation of changes among the different collection tubes revealed 4 which had a significant difference compared red top: PST, green top, and EDTA for IMMULITE and EDTA for the E170. • The types of collection tubes and sample storage conditions are more important for some methods than others. References 1. Joly D, Drueke TB, Alberti C, Houillier P, Lawson-Body E, Martin KJ, et al. Variation in serum and plasma PTH levels in second-generation assays in hemodialysis patients: a cross-sectional study. Am J Kidney Dis 2008;51:987-95. 2. Lips P. Vitamin D deficiency and secondary hyperparathyroidism in the elderly: consequences for bone loss and fractures and therapeutic implications. Endocr Rev 2001;22:477-501. 3. Malabanan A, Veronikis I, Holick M. Redefining vitamin D insufficiency. Lancet 1998;351:805-6. 4. Souberbielle JC, Fayol V, Sault C, Lawson-Body E, Kahan A, Cormier C. Assay-specific decision limits for two new automated parathyroid hormone and 25-hydroxyvitamin D assays. 5. Viljoen A, Singh DK, Twomey PJ, Farrington K. Analytical quality goals for parathyroid hormone based on biological variation. Clin Chem Lab Med 2008;46:1438-42. Acknowledgements Support for this study was provided by Abbott Diagnostics and the ARUP Institute for Clinical and Experimental Pathology. We gratefully acknowledge Abbott Diagnostics, Beckman Coulter, and Roche Diagnostics for providing instrumentation to perform testing using their methods.