Clinical Chemistry 55:9 1737–1741 (2009


Clinical Case Study

A Case of Renal Osteodystrophy with Unexpected Serum Intact Parathyroid Hormone Concentrations
Danni L. Meany,1 Suzanne M. Jan de Beur,2 Mary Jo Bill,1 and Lori J. Sokoll1*

CASE A 64-year-old female with long-standing end-stage renal disease (ESRD),3 status post 2 failed renal transplants, was evaluated for management of renal osteodystrophy with particular concern for adynamic bone disease (ABD). ABD was suspected because of low normal serum intact parathyroid hormone (PTH) concentrations (range 2.5–54 ng/L, reference range 10 – 65 ng/L), intermittently increased serum calcium concentrations (range 88 –107 mg/L, reference range 84 –105 mg/L), and severe osteoporosis. However, her mildly increased serum alkaline phosphatase activities (range 149 –196 U/L, reference range 30 –120 U/L) were inconsistent with the low bone turnover observed in ABD. This discrepant clinical profile prompted investigation into the PTH assay used at our institution. Simultaneous samples were analyzed for intact PTH on our Roche Elecsys 2010 immunoassay analyzer and at a reference laboratory (Quest Diagnostics) on the Siemens Immulite 2000 immunoassay analyzer. Discrepant values of 48 and 786 ng/L were obtained, respectively. DISCUSSION Parathyroid hormone functions to maintain serum calcium concentrations within a tight physiologic range. Patients with chronic renal failure develop secondary hyperparathyroidism owing to decreased renal production of 1,25-dihydroxyvitamin D and hyperphosphatemia, both of which result in hypocalcemia. These derangements in mineral metabolism stimulate PTH production to raise serum calcium and promote phosphorus excretion. Increased serum PTH leads to excessive bone resorption through stimulation of os-

Department of Pathology; and 2 Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD. * Address correspondence to this author at: Department of Pathology, Johns Hopkins Medical Institutions, 600 N. Wolfe St., Meyer B-125, Baltimore, MD 21287. Fax 410-614-7609; e-mail Received December 5, 2008; accepted February 18, 2009. DOI: 10.1373/clinchem.2008.121921 3 Nonstandard abbreviations: ESRD, end-stage renal disease; ABD, adynamic bone disease; PTH, parathyroid hormone; ROD, renal osteodystrophy; HAAA, human antianimal antibody.


teoblasts and osteoclasts. The combination of secondary hyperparathyroidism and mineralization defects (osteomalacia) represents the most common form of renal osteodystrophy (ROD). In contrast, a subtype of ROD known as adynamic bone disease can be observed in the setting of prolonged peritoneal or hemodialysis, oversuppression of PTH with calcitriol or calciumbased phosphate binders, or the use of bisphosphonates for osteoporosis treatment (1 ). ABD is characterized by marked suppression of bone remodeling that leads to fracture. Common biochemical hallmarks of ABD include hypercalcemia, low or inappropriately normal PTH concentrations, and reduced markers of bone turnover (e.g., alkaline phosphatase) (2 ). In this case, the increased intact PTH result from the reference laboratory (786 ng/L) was more consistent with the patient’s clinical picture, suggesting that the 48 ng/L value obtained in our clinical laboratory was falsely low. The magnitude of the difference between the 2 results suggested an assay interference as opposed to method differences or specificities (3 ), and therefore the following studies were performed. Dilution studies confirmed the presence of a negative interference, since PTH concentrations were higher in the diluted samples than in the undiluted sample (undiluted, 48 ng/L; diluted 1:20, 567 ng/L). Treatment with heterophilic blocking reagent (Scantibodies Laboratory) had no effect (intact PTH posttreatment 68 ng/L), suggesting that heterophile antibodies or human antianimal antibodies (HAAAs) were not likely to be the cause of the interference. Further review of the patient’s medical history revealed that she was ingesting 10 mg biotin per day for restless leg syndrome and had been doing so for at least the past 2 years. In humans, biotin is a coenzyme for 4 important carboxylases in fatty acid synthesis, branched-chain amino acid catabolism, and gluconeogenesis (4, 5 ). Although not a widely used regimen, high doses of biotin (10 mg/day) have been reported to improve symptoms of encephalopathy and peripheral neuropathy in patients with renal failure and undergoing chronic hemodialysis (6 ). In addition, it has been used for patients with diabetic peripheral neuropathy (7, 8 ). Biotin is recognized as a potential interferent in PTH and other assays on the Elecsys platform, and it is recommended in the product insert that samples from

Specimens with high concentrations of biotin may prevent the binding of the sandwich complex to the streptavidin-coated microparticles. An aliquot of the specimen was sent to Cambridge Biomedical Research Group for determination of the biotin concentration using a bioassay.8 g/L. 1 illustrate that biotin did inhibit the measured recovery of PTH. and no decrease at all in a specimen with an increased PTH concentration. a concentration similar to the concentration in the patient’s specimen. The first approach was carried out by adding various amounts of free biotin (0. we hypothesized that biotin was in fact the cause of the falsely low result. 80. The biotin concentration in the specimen was 4. caused a 3% decrease in PTH concentration compared with an untreated serum specimen with a normal PTH concentration. 40. Per the manufacturer. The discordant results from these 2 investigations may be due to differences between the free biotin we spiked in and biotinylated molecules such as enzymes and metabolites endogenously present in this patient’s specimens. streptavidin microparticles (magnetically extracted from 250 L of reagent M from the intact PTH immunoassay) for 1 h at room temperature with shaking. Two different approaches were subsequently used to examine the interfering role of biotin: (1) studying the effect of added biotin on the Roche assay and (2) removing the effect of biotin (10 ). catabolism of biotin leads to formation of many metabolites such as bisnorbiotin and biocytin. the Immulite 2000 intact PTH assay does not use biotin–streptavidin interaction. 20. respectively). after which streptavidin-coated microparticles are added to magnetically separate out the sandwich complex via biotin and streptavidin interaction. In contrast.8 g/L. 40. 20. Despite the fact that the sample in question was collected 8 h after biotin administration. 10. In this approach. 160 g/L) (Sigma-Aldrich) into sera with normal and increased intact PTH concentrations (33 and 487 ng/L. The solid diamond and solid squares indicate specimens with normal and increased concentrations of intact PTH (33 and 487 ng/L. 50 L of the patient’s specimen was incubated with the 1738 Clinical Chemistry 55:9 (2009) Fig. intact PTH results are purportedly unaffected (recovery within 10% of the target) when biotin concentrations are 50 g/L (9 ). 5. The recovery curves shown in Fig. In the Elecsys intact PTH assay. approximately 10-fold higher than the reference range upper limit (200 –500 ng/L). 10. as recommended by the manufacturer. biotin is a coenzyme for 4 carboxylases. biotin is covalently attached to these carboxylases via an amide linkage to a specific lysine residue (4 ). Effect of biotin on serum intact PTH concentrations determined using the Roche Elecsys 2010 analyzer. Percent recovery was calculated as the ratio of PTH concentration after the addition of biotin at various concentrations (5. Because the microparticles were isolated out of solution. When this plasma sample was analyzed by the Roche Elecsys intact PTH assay (suitable for both serum and EDTA plasma). As described above. in this case using streptavidin-coated microparticles. the plasma (EDTA) specimen sent to the reference laboratory was obtained and used for subsequent investigational experiments. 160 g/L) to the PTH concentration without biotin addition. Only when biotin concentrations were increased to 160 g/L did PTH results decrease 50%. this treatment did not dilute the patient’s specimen. respectively). In its physiologically active form. In addition. 80. undiluted and diluted results from this plasma sample were similar to our previous results. The PTH recovery was 1000% compared to recoveries of approximately 80% in 3 control specimens from patients not taking large doses of biotin. the first approach by addition of free biotin into . We measured the biotin concentration in the patient’s specimen using a microbial growth assay that underestimates biotinylated molecules (11 ). Surprisingly. a biotinylated anti-PTH monoclonal antibody and a ruthenium-labeled antiPTH monoclonal antibody form a sandwich complex with PTH. addition of 5 g/L of biotin. thus giving falsely low signals. In this experiment.Clinical Case Study patients receiving high biotin doses of 5 mg/day be collected at least 8 h after biotin administration (9 ). the second approach using streptavidin microparticles clearly identified biotin as the interference. suggesting that the actual concentration of biotinylated molecules present may be substantially higher than 4. Due to insufficient volume of our clinical laboratory specimen (serum). Hence. In contrast. 1. the intact PTH concentration in the patient’s specimen increased from 32 ng/L pretreatment to 419 ng/L posttreatment.

11:294 –329. Cronan JE Jr.44:511– 4. acquisition of data. Inter-method variability in PTH measurement: implication for the care of CKD patients. Consultant or Advisory Role: S. Boutten A. 2. 4. Kellogg MD. 8. V 3 English. Known specific assay interferences should also be considered for investigation. Head KA. N Engl J Med 2000. J Nutr 1999. Papastephanidis C. Human biotinidase isn’t just for recycling biotin. Molecular biology of biotin attachment to proteins. review and interpretation of data.70:345–50. we found that the biotinylated molecules present in specimens from patients with renal impairment who take high doses of biotin may interfere with immunoassays that use biotin streptavidin mechanisms in their assay designs. Authors’ Disclosures of Potential Conflicts of Interest: Upon manuscript submission.73:1345–54. using the streptavidin microparticle treatment. To further confirm the interfering role of biotin. POINTS TO REMEMBER 1. Jan de Beur. all authors completed the Disclosures of Potential Conflict of Interest form. 6. Agroyannis B.Clinical Case Study the PTH sera did not appear to mimic the interference of endogenously biotinylated molecules present. Altern Med Rev 2006. 9. 7. 11. and accurate measurement of serum parathyroid hormone in patients with ESRD is critical for the proper management of the aberrant bone and mineral metabolism characteristic of renal failure. although the biotin concentration was still increased at 1. Koutsicos D. Marx SJ. Biotin in the management of uremic neurologic disorders. as indicated by similar PTH concentrations before and after streptavidin microparticle treatment (data not shown). Baltimore. in the investigation of this case. heterophilic blocking reagent treatment. Law TC. 158 ng/L. Nephron 1984. Discrepant or unexpected immunoassay results can be investigated in various ways. Role of Sponsor: The funding organizations played no role in the design of study. 10. Hymes J. Rifai N. To summarize.M. High doses of biotin have been used to treat patients with uremic neuropathy and diabetic peripheral neuropathy. Kirin Pharma. the delay in initiating appropriate therapy for severe secondary hyperparathyroidism in ESRD has important clinical consequences that can include tertiary hyperparathyroidism and progressive hyperparathyroid bone disease. Clin Chem 2008. and (c) final approval of the published article. Biomed Pharmacother 1990. Honoraria: None declared. These patients may have biotinylated molecules in their blood specimens that interfere with immunoassays that use streptavidin– biotin interactions. Huang S. Adynamic bone in patients with chronic kidney disease. et al. Kidney Int 2008. Mock DM. Andress DL. Roche Elecsys® Intact PTH assay package insert. Advanced analysis of biotin metabolites in body fluids allows a more accurate measurement of biotin bioavailability and metabolism in humans. Interestingly. Zempleni J. 3.36:183– 6. References 1. 3. Chapman-Smith A. Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design. 2. Wolf B. Yatzidis H.129:485S–9S. Agroyannis B. Carlier MC. A girl with goiter and inappropriate thyroid-stimulating hormone secretion.54:1241– 4. choice of enrolled patients. eliminated the interfering biotin. and measurement with alternate methods. (b) drafting or revising the article for intellectual content. Quest Diagnostics.129:494S–7S. Francos-Plemenos M. 2008-08. was consistent with the reference laboratory PTH concentration measured on the Immulite 2000 (223 ng/L) using the same specimen. LawsonBody E. As in the case of this patient. MD. Acknowledgments: The authors gratefully acknowledge the assistance of Roger Frye. or analysis and interpretation of data. treating the specimen with streptavidin microparticles. Delatola Z. serum intact PTH concentrations were measured in both laboratories after the patient stopped taking biotin for 2 weeks. An in vitro interference study using exogenous biotin failed to mimic the interference. Hyperparathyroid and hypoparathyroid disorders. 5. Biotin for diabetic peripheral neuropathy. Kidney Int 2006. J Nutr 1999. Clinical Chemistry 55:9 (2009) 1739 . this increase did not result in assay interference on the Elecsys 2010 analyzer. J Nutr 1999. Expert Testimony: None declared. Stock Ownership: None declared. Potential conflicts of interest: Employment or Leadership: None declared.129:477S– 84S. PTH plays a critical role in the regulation of serum calcium concentrations.343:1863–75. Souberbielle JC.3 g/L. Peripheral neuropathy: pathogenic mechanisms and alternative therapies. which may be due to the discrepancy between exogenous biotin and endogenous biotinylated molecules. Tzanatos-Exarchou H. whereas the second approach. Research Funding: None declared. The Elecsys 2010 result. Chevenne D. including dilution studies. Coumaros G. or preparation or approval of manuscript. Koutsikos D.

Authors’ Disclosures of Potential Conflicts of Interest: Upon manuscript submission. Scantibodies Laboratory. 2009. In this patient’s CKD-MBD.1373/clinchem. however. 9366 Abraham Way. Fax 314-286-2894. Potential conflicts of interest: Employment or Leadership: T. Bone biopsy studies have demonstrated that iPTH is nondiagnostic for ABD when concentrations are 10. Proper diagnosis of ABD is important because administration of potent vitamin D analogs to a patient with ABD (as would be appropriate for a patient with high bone turnover) must be avoided. difficulty in accurately diagnosing chronic kidney disease–mineral bone disorder (CKD-MBD). Cantor.2009.131409 Reference 1. A bone biopsy would have been indicated in the patient presented. and the various assays for iPTH used to estimate bone remodeling have numerous problems in addition to the novel biotin interference discovered in this case. Geng Z.cantor@scantibodies. DOI: 10.6 pmol/L. e-mail tom. 2009. The gold standard for diagnosing ABD is the invasive bone biopsy. Improved assessment of bone turnover by the PTH-(1– 84)/large C-PTH fragments ratio in ESRD patients. was Mawad H. 2009.Clinical Case Study Commentary Tom Cantor ABD is a serious complication of ESRD that leads to soft tissue/vascular calcification and ultimately to myocardial infarction. MO. The cardiovascular risk for the presented 64-year-old woman.2009. Received June 26. Santee. or preparation or approval of manuscript. MO. Role of Sponsor: The funding organizations played no role in the design of study. present a novel cause of a current major problem in clinical medicine. but this is not the standard practice in 2009. The Roche Elecsys iPTH assay was calibrated to the former Nichols Allegro iPTH IRMA (the assay on which treatment guidelines are based). Received June 10. The finding of the adynamic bone disorder would have been associated with attempts to increase bone remodeling by correcting the hypercalcemia and in- 1740 Clinical Chemistry 55:9 (2009) .131417 ter 2 failed kidney transplants and had severe osteoporosis.60:1460 – 8. Cantor TL. Stock Ownership: None declared. Multiple studies have defined the weakness of the standard practice employed here. St. who was on dialysis af- Washington University. Research Funding: None declared. a term coined by the Kidney Disease: Improving Global Outcomes foundation to replace ROD. The current practice is that iPTH assays are used to relate to past bone biopsy studies and to predict the state of bone remodeling. Malluche HH. Commentary Keith Hruska Meany et al. accepted July 2. 2009. The basis for the change in terminology stems from the realization that the skeletal and mineral disorders complicating kidney disease are critical in the pathogenesis of the excess cardiovascular mortality risk associated with CKD. Therefore. Campus Box 8208. 63110. However. which is in the process of being replaced. the case study patient can be reliably concluded to have ABD. Honoraria: None declared. whereas the Siemens DPC iPTH assay generates values that are on average 50% higher than the Roche assay. St. all authors completed the Disclosures of Potential Conflict of Interest form. Monier-Faugere MC. diagnosis of the skeletal remodeling disorder was going to guide critical therapy. (b) drafting or revising the article for intellectual content. Fax 619-258-9366.1373/clinchem. CA. Gao P. Address correspondence to the author at: 5109 McDonnell Pediatric Research Building. Expert Testimony: None declared. and the DPC iPTH should not be used to guide therapy. review and interpretation of data. if iPTH is 10. DOI: 10. and (c) final approval of the published article. Consultant or Advisory Role: None declared. acquisition of data. bonespecific alkaline phosphatase can be used as a marker of ABD. Louis. Scantibodies Laboratory. The case presentation portrays the weakness in the current standard practice. Friedler RM. Kidney Int 2001. Louis. Address correspondence to the author at: Scantibodies Laboratory. CA. there is a 85% probability of ABD (1 ). Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design. e-mail hruska_k@wustl. choice of enrolled patients.6 pmol/L. Measurements of serum calcium and alkaline phosphatase are not reliable for ABD. the leading cause of death in ESRD patients. accepted July 2. or analysis and interpretation of data.

acquisition of data. all authors completed the Disclosures of Potential Conflict of Interest form. Expert Testimony: None declared. (b) drafting or revising the article for intellectual content. Research Funding: K. or analysis and interpretation of data. or preparation or approval of manuscript. choice of enrolled patients. Potential conflicts of interest: Employment or Leadership: None declared. This finding diametrically changes the treatment strategy to controlling the effects of CKD on the cardiovascular system by decreasing the serum phosphorus and on the PTH concentration by using a calcimimetic agent. and Stryker. Honoraria: None declared. Shire. and (c) final approval of the published article. Clinical Chemistry 55:9 (2009) 1741 . The increased PTH concentration results from the reference laboratory and the high alkaline phosphatase concentration confirmed that the patient actually had high-turnover CKD-MBD contributing to her osteoporosis. because the presence of hypercalcemia contraindicates increasing the dose of vitamin D analogs. Role of Sponsor: The funding organizations played no role in the design of study. Fresenius. and Genzyme. Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design.Clinical Case Study creasing the actions of PTH. Genzyme. Hruska. Hruska. Shire. Stock Ownership: None declared. Authors’ Disclosures of Potential Conflicts of Interest: Upon manuscript submission. review and interpretation of data. Consultant or Advisory Role: K.

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