Vitamin D Assay May Give Misleading Results

NEW YORK (Reuters Health) - In certain circumstances one widely used test for vitamin D intoxication, the Diasorin radioimmunoassay, may not be entirely reliable, according to two case studies by U.S. and Irish investigators.

"Our study," Dr. Michael A. Levine told Reuters Health by email, "highlights the continuing challenge that we face when using current assay technologies to measure vitamin D metabolites." The patients involved "developed vitamin D toxicity from inadvertent overdosage using standard over-the-counter preparations of vitamin D."

In a June 22 online paper in the Journal of Clinical Endocrinology & Metabolism, Dr. Levine, of the University of Pennsylvania, Philadelphia, and colleagues note that vitamin D intoxication is characterized by elevated serum 25-hydroxyvitamin D (25(OH)D) and suppressed serum 1,25-dihydroxvitamin D (1,25(OH)2D).

The team used both the Diasorin radioimmunaossay test (RIA) and liquid chromatography and tandem mass spectrometry (LC-MS/MS) to evaluate samples from two retrospectively identified patients with hypercalcemia. One was a 15-year-old male with a two-week history of postprandial vomiting, abdominal pain and polyuria. The other, a 17-year old female, had a history of weight loss.

Both had elevated serum 1,25(OH)2D by RIA, but normal serum 1,25(OH)2D concentrations by LC-MS/MS. To help explain these surprising findings the team conducted further in vitro experiments on serum samples from a random set of inpatients and outpatients.

The team noted that concentrations of 25(OH)D2 or 25(OH)D3 increased as expected based on the amount of vitamin D metabolite added to pooled serum samples or artificial serum matrix in all experiments.

The addition of 100 ng/mL of 25(OH)D3 to pooled patient serum resulted in a median increase of 114% in measured 1,25(OH)D2 via RIA and a 21% increase via LC-MS/MS. At 700 ng/mL, the increase was 349% with RIA and 117% with LC-MS/MS.

Thus, wrote the researchers, "We recommend measurement of serum 24,25(OH)2D and use of LC-MS/MS, which appears less susceptible to this interference, to reassess serum levels of 1,25(OH)2D when the clinical scenario is confusing."

Summing up, Dr. Levine said, "Assessment of plasma levels of the most active vitamin D metabolite, 1,25(OH)2D, using a common laboratory immunoassay pointed away from nutritional vitamin D intoxication and suggested other more worrisome diagnoses. Repeating the testing with a mass spectrometer assay confirmed the clinical diagnosis of vitamin D intoxication."

He concluded, "Clinicians must remember that laboratory tests are not 100% reliable, and they must continue to rely upon their clinical judgment when confronted with test results that do not make sense."

Diasorin did not respond to a request for comment.

The authors reported no financial disclosures or competing interests.

NEW YORK (Reuters Health) - In certain circumstances one widely used test for vitamin D intoxication, the Diasorin radioimmunoassay, may not be entirely reliable, according to two case studies by U.S. and Irish investigators.

"Our study," Dr. Michael A. Levine told Reuters Health by email, "highlights the continuing challenge that we face when using current assay technologies to measure vitamin D metabolites." The patients involved "developed vitamin D toxicity from inadvertent overdosage using standard over-the-counter preparations of vitamin D."

In a June 22 online paper in the Journal of Clinical Endocrinology & Metabolism, Dr. Levine, of the University of Pennsylvania, Philadelphia, and colleagues note that vitamin D intoxication is characterized by elevated serum 25-hydroxyvitamin D (25(OH)D) and suppressed serum 1,25-dihydroxvitamin D (1,25(OH)2D).

The team used both the Diasorin radioimmunaossay test (RIA) and liquid chromatography and tandem mass spectrometry (LC-MS/MS) to evaluate samples from two retrospectively identified patients with hypercalcemia. One was a 15-year-old male with a two-week history of postprandial vomiting, abdominal pain and polyuria. The other, a 17-year old female, had a history of weight loss.

Both had elevated serum 1,25(OH)2D by RIA, but normal serum 1,25(OH)2D concentrations by LC-MS/MS. To help explain these surprising findings the team conducted further in vitro experiments on serum samples from a random set of inpatients and outpatients.

The team noted that concentrations of 25(OH)D2 or 25(OH)D3 increased as expected based on the amount of vitamin D metabolite added to pooled serum samples or artificial serum matrix in all experiments.

The addition of 100 ng/mL of 25(OH)D3 to pooled patient serum resulted in a median increase of 114% in measured 1,25(OH)D2 via RIA and a 21% increase via LC-MS/MS. At 700 ng/mL, the increase was 349% with RIA and 117% with LC-MS/MS.

Thus, wrote the researchers, "We recommend measurement of serum 24,25(OH)2D and use of LC-MS/MS, which appears less susceptible to this interference, to reassess serum levels of 1,25(OH)2D when the clinical scenario is confusing."

Summing up, Dr. Levine said, "Assessment of plasma levels of the most active vitamin D metabolite, 1,25(OH)2D, using a common laboratory immunoassay pointed away from nutritional vitamin D intoxication and suggested other more worrisome diagnoses. Repeating the testing with a mass spectrometer assay confirmed the clinical diagnosis of vitamin D intoxication."

He concluded, "Clinicians must remember that laboratory tests are not 100% reliable, and they must continue to rely upon their clinical judgment when confronted with test results that do not make sense."

Diasorin did not respond to a request for comment.

The authors reported no financial disclosures or competing interests.

NEW YORK (Reuters Health) - In certain circumstances one widely used test for vitamin D intoxication, the Diasorin radioimmunoassay, may not be entirely reliable, according to two case studies by U.S. and Irish investigators.

"Our study," Dr. Michael A. Levine told Reuters Health by email, "highlights the continuing challenge that we face when using current assay technologies to measure vitamin D metabolites." The patients involved "developed vitamin D toxicity from inadvertent overdosage using standard over-the-counter preparations of vitamin D."

In a June 22 online paper in the Journal of Clinical Endocrinology & Metabolism, Dr. Levine, of the University of Pennsylvania, Philadelphia, and colleagues note that vitamin D intoxication is characterized by elevated serum 25-hydroxyvitamin D (25(OH)D) and suppressed serum 1,25-dihydroxvitamin D (1,25(OH)2D).

The team used both the Diasorin radioimmunaossay test (RIA) and liquid chromatography and tandem mass spectrometry (LC-MS/MS) to evaluate samples from two retrospectively identified patients with hypercalcemia. One was a 15-year-old male with a two-week history of postprandial vomiting, abdominal pain and polyuria. The other, a 17-year old female, had a history of weight loss.

Both had elevated serum 1,25(OH)2D by RIA, but normal serum 1,25(OH)2D concentrations by LC-MS/MS. To help explain these surprising findings the team conducted further in vitro experiments on serum samples from a random set of inpatients and outpatients.

The team noted that concentrations of 25(OH)D2 or 25(OH)D3 increased as expected based on the amount of vitamin D metabolite added to pooled serum samples or artificial serum matrix in all experiments.

The addition of 100 ng/mL of 25(OH)D3 to pooled patient serum resulted in a median increase of 114% in measured 1,25(OH)D2 via RIA and a 21% increase via LC-MS/MS. At 700 ng/mL, the increase was 349% with RIA and 117% with LC-MS/MS.

Thus, wrote the researchers, "We recommend measurement of serum 24,25(OH)2D and use of LC-MS/MS, which appears less susceptible to this interference, to reassess serum levels of 1,25(OH)2D when the clinical scenario is confusing."

Summing up, Dr. Levine said, "Assessment of plasma levels of the most active vitamin D metabolite, 1,25(OH)2D, using a common laboratory immunoassay pointed away from nutritional vitamin D intoxication and suggested other more worrisome diagnoses. Repeating the testing with a mass spectrometer assay confirmed the clinical diagnosis of vitamin D intoxication."

He concluded, "Clinicians must remember that laboratory tests are not 100% reliable, and they must continue to rely upon their clinical judgment when confronted with test results that do not make sense."

Diasorin did not respond to a request for comment.

The authors reported no financial disclosures or competing interests.