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Analysis of vitamin D status at two academic medical centers and a national reference laboratory: result patterns vary by age, gender, season, and patient location
© Genzen et al.; licensee BioMed Central Ltd. 2013
Received: 13 June 2013
Accepted: 28 October 2013
Published: 5 November 2013
Testing for 25-hydroxyvitamin D [25(OH)D] has increased dramatically in recent years. The present report compares overall utilization and results for 25(OH)D orders at two academic medical centers - one in New York and one in Iowa – in order to characterize the vitamin D status of our inpatient and outpatient populations. Results are also compared to those from a national reference laboratory to determine whether patterns at these two institutions reflect those observed nationally.
Retrospective data queries of 25(OH)D orders and results were conducted using the laboratory information systems at Weill Cornell Medical College / New York Presbyterian Hospital (WCMC), University of Iowa Hospitals and Clinics (UIHC), and ARUP Laboratories (ARUP). Chart review was conducted for cases with very high or low serum 25(OH)D levels in the WCMC and UIHC datasets.
The majority of tests were ordered on females and outpatients. Average serum 25(OH)D levels were higher in female versus male patients across most ages in the WCMC, UIHC, and ARUP datasets. As expected, average serum 25(OH)D levels were higher in outpatients than inpatients. Serum 25(OH)D levels showed seasonal periodicity, with average levels higher in summer than winter and correlating to regional UV index. Area plots demonstrated a peak of increased 25(OH)D insufficiency / deficiency in adolescent females, although overall worse 25(OH)D status was found in male versus female patients in the WCMC, UIHC, and ARUP datasets. Surprisingly, improved 25(OH)D status was observed in patients starting near age 50. Finally, chart review of WCMC and UIHC datasets revealed over-supplementation (especially of ≥ 50,000 IU weekly doses) in the rare cases of very high 25(OH)D levels. General nutritional deficiency and/or severe illness was found in most cases of severe 25(OH)D deficiency.
25(OH)D status of patients seen by healthcare providers varies according to age, gender, season, and patient location. Improved 25(OH)D status was observed later in life, a finding that may reflect the previously described increased use of vitamin D-containing supplements in such populations. Severe vitamin D deficiency is much more common than vitamin D toxicity.
Vitamin D is a fat-soluble vitamin important for calcium absorption and bone health. Vitamin D also plays an important role in a variety of other functions, including muscle strength, cellular proliferation, differentiation, and immunomodulation to name a few (see  for review), and deficiency may be associated with a variety of clinical conditions and disease states . Vitamin D2 (ergocalciferol) is obtained exclusively from dietary sources, whereas vitamin D3 (cholecalciferol) is obtained from dietary sources as well as the conversion of endogenous 7-dehydrocholesterol by UV-B exposure to the skin. Vitamin D2 and vitamin D3 are hydroxylated to 25-hydroxyvitamin D2 (25(OH)D2) and 25-hydroxyvitamin D3 (25(OH)D3) respectively [collectively referred to as 25(OH)D; calcidiol] in the liver. 25(OH)D is then hydroxylated to the biologically active hormone 1,25-dihydroxyvitamin D (1,25(OH)2D; calcitriol) in the kidney and to some extent in peripheral tissues. 25(OH)D, while an inactive precursor, is the best measurement of vitamin D nutritional status (versus 1,25(OH)2D or vitamin D itself) due to its longer half-life, less day to day variation, and ease of measurement in the clinically relevant concentration range .
Growing interest in vitamin D has led to a dramatic increase in 25(OH)D testing in recent years . This surge in 25(OH)D orders has been a challenge for clinical laboratories deciding whether to handle this testing in-house or as send-outs to commercial reference laboratories. Multiple vendors now offer assays for 25(OH)D testing, which include immunochemical, chromatographic, and mass spectrometric platforms [5, 6]. Numerous studies, however, have demonstrated variability of 25(OH)D results across assays [7–10]. Unfortunately, variability between assays can impact studies that examine the prevalence of vitamin D deficiency in populations, as well as clinical decisions for an individual patient [11, 12].
There is actually little consensus on reference intervals used to determine what are optimal, sufficient, insufficient, deficient, and/or toxic levels of 25(OH)D . For example, the Institute of Medicine (IOM) has recently defined four categories of 25(OH)D status: risk of deficiency (<30 nmol/L; <12 ng/mL), risk of inadequacy (30–49 nmol/L; 12–19 ng/mL), sufficiency (50–125 nmol/L; 20–50 ng/mL); and above recommended levels (>125 nmol/L; >50 ng/mL) . Data from the National Health and Nutrition Examination Survey (NHANES) was originally analyzed to assess the vitamin D status of the United States population , and has more recently been reviewed with these categories in mind . The Endocrine Society’s 2011 clinical practice guidelines, however, describe vitamin D deficiency as 25(OH)D of < 50 nmol/L (<20 ng/mL) and insufficiency as 52.5-72.5 nmol/L (21–29 ng/mL) . Reference intervals may also vary based on assay package inserts, population-specific studies, and/or reference laboratory .
In the present report, we compare the ordering and result patterns of 25(OH)D testing at two large academic medical centers: one in New York City (WCMC) and one in Iowa City (UIHC). We subsequently compare 25(OH)D results, as well as the distribution of results into reference intervals by age, to those observed at a national reference laboratory (ARUP). Finally, chart review was performed for cases of vitamin D toxicity and deficiency to identify possible patterns in clinical presentation and/or causality.
This report presents data from three separate retrospective reviews of 25(OH)D tests ordered by practitioners in clinical practice. No 25(OH)D testing was ordered specifically for this report. Patients for whom 25(OH)D tests were ordered represent a mixture of healthy and ill individuals, and no screening was done to include and/or exclude patients based on patient medical history in the WCMC, UIHC, and ARUP datasets. Data were not adjusted for survival, population trends, or relative probability of visiting a healthcare provider.
Two different 25(OH)D assays were available for ordering by clinicians in the inpatient and outpatient electronic health systems during the period of time investigated (October 2010 through May 2012). These were 25(OH)D by DiaSorin immunoassay (sent to ARUP Laboratories, Salt Lake City, UT) and 25(OH)D by LC-MS/MS (sent to Quest Diagnostics, Teterboro, NJ). Using a protocol approved by the Weill Cornell Medical College Institutional Review Board (WCMC IRB), the WCMC Laboratory Information System (LIS) (Millennium, Cerner Corporation, North Kansas City, MO) was queried for all vitamin D-related tests sent out over this 20 month interval. Historical ordering data (2008–2010) were reviewed to determine trends in frequency. In accordance with the WCMC IRB protocol, chart review was then conducted to determine causes of vitamin D toxicity (100 ng/mL or greater based on elevated 25(OH)D immunoassay results) or extreme deficiency [based on both 25(OH)D2 and 25(OH) D3 results < 4 ng/mL on 25(OH)D assays by LC-MS/MS].
Using a protocol approved by the University of Iowa Institutional Review Board Biomedical (01) subcommittee (UI IRB), the UIHC electronic health system (EpicCare, Epic Systems Corporation, Verona, WI) was queried for all 25(OH)D tests ordered from January 2000 through October 2012. Several distinct 25(OH)D assays were utilized during this interval of time, although none simultaneously. From January 2000 to mid-July 2005, the Nichols ADVANTAGE® 25-OH Vitamin D immunoassay was performed in-house. From late-July 2005 to January 2012 specimens were sent to ARUP Laboratories (DiaSorin immunoassay). An in-house assay, Abbott Architect 25-OH Vitamin D (Abbott Laboratories, Abbott Park, IL) was used from mid-January 2012 to mid-October 2012. Starting in mid-October 2012, the laboratory moved to the Roche Elecsys Vitamin D assay (Roche Diagnostics, Basel, Switzerland) on the in-house Modular E platform. Since only a partial month of data was available for October 2012, the October 2012 data were excluded from this report. In accordance with the UI IRB protocol, chart review was performed on all patients with 25(OH)D immunoassay results of 100 ng/mL or greater, or less than 5 ng/mL. That chart review was directed at medical history and potential reasons for having elevated or severely deficient 25(OH)D serum concentrations.
Using a protocol approved by the University of Utah Institutional Review Board, a de-identified list of immunoassay results for 25(OH)D (DiaSorin immunoassay) was obtained from the ARUP LIS. Exclusion criteria were any specimens potentially received from WCMC or UIHC to prevent duplication of results.
UV index information is available in the public domain from the Climate Prediction Center, National Weather Service/National Oceanic and Atmospheric Administration website . UV index is defined by the National Weather Service as “forecast of the amount of skin damaging UV radiation expected to reach the earth’s surface at the time when the sun is highest in the sky (solar noon)” . The daily “Issued UV Index” is used throughout this paper and incorporates cloud information (as opposed to “clear sky UV index”). Issued UV index was used from the weather station at the John F. Kennedy International Airport (~ 40.64°N latitude) in New York (near WCMC ~ 40.77°N latitude) and the weather station at the Des Moines International Airport (~ 41.53°N latitude) in Iowa (near UIHC ~41.66°N latitude).
Limit of quantitation and exclusions
Assays for 25(OH)D have a lower limit of quantitation. Results below this are typically reported with a “less than” (<) symbol. For the duration of time evaluated in this study, the following were the lower limits of quantitation evident in results of our queries from 2000 through 2012: Nichols Advantage (< 7 ng/mL), ARUP (< 4 ng/mL or < 7 ng/mL), Abbott (< 4 ng/mL or < 7 ng/mL), Roche (< 5 ng/mL). Analysis of ordering patterns and frequency was not impacted by these “less than” results and includes all available data. Area plot analysis (see below) also includes all “less than” results, as they could be fit within the same interval category (ex. 0–10 ng/mL). For analysis of 25(OH)D immunoassay results by sex, age, and month, however, “less than” results were not included, as an actual value was unknown. For WCMC, 59 results (~0.1% of total results; 40 female, 19 male) were excluded from analysis of 25(OH)D averages due to “less than” results. For UIHC, 378 results (~0.6% of total results; 235 female, 143 male) were excluded due to “less than” results. For ARUP, ~0.09% of total results (64.4% female, 35.6% male) were excluded due to “less than” results. The difference in the relative percent of excluded results from UIHC (versus WCMC or ARUP) is likely due to the much longer period of data available for analysis from UIHC and corresponding improvements in 25(OH)D immunoassay performance and sensitivity over time. An additional 30 “results” (20 female, 10 male) were excluded from analysis at WCMC, as text comments instead of numeric results were retrieved from the de-identified LIS queries.
For the LC-MS/MS total 25(OH)D and fractionated 25(OH)D2 and 25(OH)D3 result analysis from the WCMC dataset, exclusion of values below the lower limit of quantitation (< 4 ng/mL; n = 6,445 for 25(OH)D2 and n = 75 for 25(OH)D3) would lead to a marked over-estimation of average 25(OH)D2 concentrations. For these figures, “less than” results were therefore tabulated as “zeros” for the purpose of graphical display and average 25(OH)D2 and 25(OH)D3 concentration by age. Area plot analysis of LC-MS/MS data includes all “less than” results, as they could be fit within the same interval category.
Finally, it should be noted that repeat testing on the same patient (pseudoreplication) could not be avoided in the WCMC, UIHC, and ARUP datasets, as patient-level exclusion was not possible on our IRB-approved de-identified result queries. A separate analysis of WCMC 25(OH)D ordering, however, revealed the following distribution of 25(OH)D “orders per patient” in that interval of time studied (20 months): 1 order, 69.3%; 2 orders, 19.5%; 3 orders, 6.2%; 4 orders, 2.4%; 5 orders, 1.2%; 6 orders, 0.6%; 7 orders, 0.3%; 8 orders, 0.2%; 9 orders, 0.1%; ≥ 10 orders, 0.2%.
Microsoft Excel 2007 and SigmaPlot 11 (Systat Software, Inc, Chicago, IL) were used to analyze and visualize data by age, sex, order month, and patient location. Patient age was rounded to the nearest year to facilitate binning and comparison. Alternate (quantitative) method comparison between immunoassay and LC-MS/MS (including bias, correlation coefficient, and Deming regression) was conducted in EP Evaluator 9 (Data Innovations, South Burlington, VT) and plotted in SigmaPlot 11. Cross-tabulation in SPSS 18 (PASW; IBM, Armonk, NY) was used to calculate the proportion of results by age that fall into specific reference intervals. These results were used to generate area plots in SigmaPlot, which display stacked, proportionate areas such that the sum at age is equal to 100% (see corresponding legend for a description). Data throughout the manuscript are presented as mean ± standard deviation (SD) unless otherwise indicated. Since results demonstrated a non-normal 25(0H)D distribution of scores, statistical significance was evaluated using the Mann Whitney rank sum test (with alpha set at 0.05 for the threshold of significance).
25(OH)D by immunoassay
25(OH)D orders and immunoassay results by institution, patient location, and sex
Average 25(0H)D Results ± SD (ng/mL)
# Orders females
# Orders males
F vs M
30.5 ± 13.3
28.4 ± 12.7
29.8 ± 13.2
p < 0.001
Hospital Outpatient Clinics
27.2 ± 12.4
24.9 ± 11.3
26.6 ± 12.1
p < 0.001
25.8 ± 19.2
21.9 ± 12.1
24.3 ± 16.9
p < 0.001
30.5 ± 14.6
29.8 ± 14.3
30.2 ± 14.5
p < 0.001
26.1 ± 13.0
32.6 ± 16.3
29.0 ± 14.8
p = 0.031
28.8 ± 17.5
24.8 ± 10.5
27.6 ± 15.8
p = 0.574
27.3 ± 16.0
25.7 ± 18.3
26.7 ± 17.0
p < 0.001
22.6 ± 13.4
24.8 ± 14.0
23.8 ± 13.7
p = 0.119
29.4 ± 13.8
28.1 ± 13.0
29.0 ± 13.6
p < 0.001
25(OH)D immunoassay results by assay platform – UIHC dataset
Average 25(0H)D Results ± SD (ng/mL)
# Orders females
# Orders males
Nichols ADVANTAGE® (in-house)
Jan 2000 - Jul 2005
27.4 ± 16.6
24.9 ± 18.0
26.5 ± 17.2
DiaSorin Immunoassay (send-out)
Jul 2005 -Jan 20012
30.6 ± 14.7
30.0 ± 15.0
30.4 ± 14.8
Abbott Architect (in-house)
Jan 2012 -Oct 2012
29.4 ± 14.2
27.6 ± 12.2
28.8 ± 13.6
Table 1 presents average 25(OH)D results (including all ages) for female and male patients by location. The only locations where average 25(OH)D results were statistically higher in men than women had relatively small population sizes (<200 per group). At WCMC, both outpatient populations (private ambulatory and hospital-owned clinics) showed higher 25(OH)D results than inpatients for both women and men (p < 0.001). Both female and male 25(OH)D levels were significantly lower in patients at the hospital-owned clinics versus private ambulatory clinics (p < 0.001), a factor that may be related to different patient populations served and/or ordering practices at these two categories of clinics. At UIHC, 25(OH)D results were also higher in outpatients than inpatients for both genders (p < 0.001). 25(OH)D results were significantly lower in female ICU inpatients than female non-ICU inpatients (p < 0.001), although this was not true when comparing male results between ICU and non-ICU inpatients (p = 0.648).
A peak of sub-optimal 25(OH)D status is again evident in female adolescents (see Figure 5G ARUP data), although in general 25(OH)D status is lower in male patients in the WCMC, UIHC, and ARUP datasets (as suggested by upper border of magenta interval, Figure 5C-H). A relative stability of 25(OH)D status is again evident between the ages of approximately 20–50 years old in WCMC (Figure 5C,D), UIHC (Figure 5E,F), and ARUP (Figure 5G,H) datasets. An increase in optimal 25(OH)D levels (yellow shading) was observed after approximately age 50 in the WCMC, UIHC, and ARUP datasets.
25(OH)D by LC-MS/MS
The LS-MS/MS dataset also allowed us to plot 25(OH)D2 and 25(OH)D3 by age for female (Figure 6C) and male (Figure 6D) patients. While there is significant scatter in the data due to the relatively small number of specimens for patients at each age (Figure 6E, females; Figure 6F, males; 1 yr bins), the overall trend in 25(OH)D3 results is similar to the 25(OH)D immunoassay results observed in Figure 2A-C, supporting a hypothesis that the increase in optimal 25(OH)D concentrations observed in later adulthood (see Figures 2, 3, and 5) are primarily due to increased concentrations of serum 25(OH)D3. Reference range area plots were also generated for total 25(OH)D by LC-MS/MS at WCMC (Figure 6G, females; Figure 6H, males). While increased scatter is again noted due to the relatively small number of specimens, the overall pattern is relatively consistent with that observed in 25(OH)D immunoassay results (Figure 5).
Chart review – elevated 25(OH)D
Chart review of patients with 25(OH)D ≥ 100 ng/mL
Number of patients
25(OH)D level (ng/ml)
Number of patients
Average age ± SD (yrs)
Number of females
Number of pediatric patientsa
Suspected cause of elevated 25(OH)D
Hypercalcemia at time of 25(OH)D measurement
Over-the-counter vitamin D supplements
43.9 ± 18.3
151 – 200
58.0 ± 19.6
126 – 150
53.1 ± 17.4
100 – 125
49.5 ± 20.0
58.8 ± 21.5
151 – 200
46.0 ± 14.9
126 – 150
58.8 ± 12.1
100 – 125
57.0 ± 16.8
In the UIHC review there were only 3 pediatric patients (17 years or younger) who had 25(OH)D of 126 ng/mL or greater. The pediatric patients were 23 months old (182 ng/mL), 4 years old (292 ng/mL) and 17 years old (146 ng/mL). The elevated 25(OH)D in the first two patients were attributed to high prescription vitamin D doses which were adjusted once the elevated 25(OH)D serum concentration was known.
At WCMC, over the 20 months of results reviewed, only 15 patients (~0.03%) had 25(OH)D levels > 150 ng/mL, with an additional 35 (~0.06%) having a measurement between 100 and 150 ng/mL. Vitamin D supplementation was specifically mentioned in 29 of these 50 cases, with actual and/or estimated doses documented in 24 of these cases. In 10 cases “vitamin D deficiency” was mentioned in prior clinic notes, suggesting that the elevated 25(OH)D result was in response to therapy. Four of the 50 WCMC patients with 25(OH)D levels of 100 ng/mL or greater had hypercalcemia, and each of these four had 25(OH)D levels > 150 ng/mL. As with UIHC, there were no cases of vitamin D-based rodenticide poisoning in the WCMC dataset.
The highest 25(OH)D level at WCMC (409 ng/mL) was in a patient hospitalized for symptoms associated with severe hypercalcemia. This patient subsequently disclosed to clinicians that she had been consuming massive amounts of over-the counter vitamin D and calcium-containing supplements. Only one other patient had a 25(OH)D level of 300 ng/mL or greater. That patient (an elderly male with dementia, chronic renal insufficiency, and recurrent Staghorn calculi) had been diagnosed one year earlier by his clinician with “vitamin D deficiency” due a 25(OH)D level of 24 ng/mL. It was unclear what the prescribed and/or consumed amount of vitamin D supplementation was, nor was it evident whether the history of renal calculi pre-dated the vitamin D supplementation. At WCMC, there was only one pediatric patient with a 25(OH)D value of 100 ng/mL or greater, a 4 month old diagnosed with osteogenesis imperfecta (25(OH)D result of 130 ng/mL) receiving vitamin D supplementation.
In 12 of the 50 WCMC patients identified with 25(OH)D levels of 100 ng/mL or greater, bone disease (osteoporosis, osteopenia, scoliosis, and/or fracture) was documented by the clinicians. Four of the 50 WCMC cases showed vitamin D supplementation in the context of Crohn’s disease, ulcerative colitis, and/or inflammatory bowel disease. Five of the 50 WCMC cases showed vitamin D supplementation in the context of multiple sclerosis.
Chart review – decreased 25(OH)D
Clinical characteristics for patients with extremely low 25(OH)D results a
Number of patients
Decreased production or intake of vitamin D
Skin damage (e.g., burns)
Malabsorption of vitamin D or deficient 25-hydroxylation
Liver failure and/or biliary tract dysfunction
Other disorder with lipid malabsorption
Increased loss of 25(OH)D
Increased catabolism or 1α-hydroxylation of 25(OH)D
Liver-enzyme inducing medications
Other conditions possibly linked to 25(OH)D deficiency
Morbid obesity and/or status post bariatric surgery
Systemic lupus erythematosus without lupus nephritis
Systemic lupus erythematosus with lupus nephritis
Perinatally acquired HIV; now in adolescence or adulthood
For the WCMC dataset, chart review was conducted for cases of “severe deficiency” where 25(OH)D2 and 25(OH)D3 results by LC-MS/MS were both <4 ng/mL. Of 8,439 orders for 25(OH)D by LC-MS/MS, 14 distinct patients (0.17%) met this criteria. One of these 14 patients, however, had a separate 25(OH)D result by immunoassay reported on the same specimen with discordant results of 21 ng/mL. As no clinical history was available for this patient, it was excluded from further analysis due to possible mix-up and/or analytical error. Of the remaining 13 patients (see Table 4), three had a history of prior bariatric surgery (one with complications), one presented for a pre-operative evaluation for bariatric surgery, three had renal failure (two were status post renal transplant while the other had nephrotic syndrome), one had liver failure (pre-transplant evaluation), and three did not have a clear documented cause. It should be noted that in six of the cases, signs of malnourishment and/or poor appetite were documented by the clinicians. Interestingly, two patients had a history of perinatally acquired HIV but were now in late adolescence or adulthood, although it should be emphasized that poor dietary habits were noted for one of these two cases as well. Chart review revealed documentation of subsequent vitamin D supplementation in 8 of the 13 cases reviewed.
A number of studies have examined vitamin D status in large populations. For example, comprehensive analyses of NHANES data have previously been conducted [14, 15, 19]. A few of the findings of NHANES differ, however, from observations described here for the WCMC, UIHC, and ARUP clinical datasets. For example, the NHANES studies indicated that 25(OH)D concentrations are, in general, higher in males than females [15, 19]. A second observation of the published NHANES data is that children tended to have higher 25(OH)D concentrations than adults . While we observed a general pattern of more optimal 25(OH)D status in childhood in all datasets analyzed, an improvement in 25(OH)D status later in adulthood was observed at WCMC, UIHC, and ARUP, demonstrating that 25(OH)D status changes throughout life. For the WCMC dataset, we were able to demonstrate this pattern using both immunoassay and LC-MS/MS 25(OH)D results.
It is likely that consumption of vitamin D fortified foods, as well as UV exposure during childhood [20, 21] support 25(OH)D status during youth. Studies of the Canadian Health Measures Surveys data have been able to demonstrate that vitamin D supplementation contributes to improved 25(OH)D status , as was frequent milk consumption and white racial background among other factors . It should be noted that American female adolescents report consuming less milk than male adolescents , and in general, girls (during childhood and adolescence) have been reported to spend less time outdoors than boys . These factors may contribute to the patterns observed for female adolescents throughout this report.
We suspect that increased use of vitamin D-containing supplements (specifically vitamin D3) during later adulthood contributes to the improved 25(OH)D status observed at those ages . Elevation of 25(OH)D3 levels as measured by LC-MS/MS (Figure 6C,D) supports this hypothesis. Increased use of supplemental vitamin D in women versus men is also likely to contribute to the more optimal 25(OH)D status in women seen in our report .
It should be noted that another large study of approximately 158,000 individuals who had 25(OH)D testing performed at Calgary Laboratory Services in Alberta, Canada also found higher 25(OH)D levels in female than male patients and improved mean 25(OH)D levels later in adulthood (described as a nadir in early adulthood) . A population-based study in São Paulo, Brazil of 636 participants showed similar correlations regarding age, sex, and mean 25(OH)D concentrations . Other population studies, however, have shown higher 25(OH)D values in males than females . Finally, seasonal/UV-B dependent variation in 25(OH)D levels have been well-characterized in numerous prior studies [29–35], as well as a more recent excellent report analyzing 3.44 million U.S. patient samples .
It is tempting to speculate that differences in 25(OH)D levels between females and males could also be due to differences in circulating vitamin D binding protein (DBP; also known as Gc globulin). For example, some previous studies have found higher circulating DBP levels in women versus men [37, 38], including higher levels in pregnant women [9, 38]. Interestingly, two prior studies have not shown a correlation between DBP levels and age, although their population sizes [n = 100 participants, men >45 years old and women >55 year old, ref ; n = 228 participants, age ranges 18–69, ref ] may theoretically preclude the ability to see any subtle trends if present. Furthermore, one of these studies actually demonstrated lower 25(OH)D levels in women versus men . Until clinically-approved DBP assays are commercially available and/or DBP research assays are included in larger population studies, we may not have a complete picture of whether DBP levels vary throughout one’s lifetime. While the clinical relevance of circulating DBP levels to vitamin D status is not fully understood, studies have demonstrated that DBP polymorphisms can affect circulating 25(OH)D levels, as well as response to vitamin D supplementation and sun exposure [39–42]. Of particular relevance to the clinical laboratory, new evidence demonstrates an inverse correlation between the deviation of most vendor 25(OH)D immunoassay kits (versus an LC-MS/MS) and DBP levels, a finding that may explain some of the inaccuracies observed with 25(OH)D immunoassays .
One limitation of the present study is that patient ethnicity was not available in our datasets; therefore, results could not be subdivided by race. It is reasonable to assume that WCMC and UIHC 25(OH)D datasets may not be fully representative of national demographic patterns. U.S. Census data for 2010 reports an 86.1% white ethnicity in zip code 10065 (WCMC) and 88.8% white ethnicity in zip code 52242 (UIHC) . These percentages, however, are likely much higher than the percent of white populations observed at these facilities, which both provide inpatient and outpatient care to patients far beyond a single zip code. No demographic information was available for the ARUP dataset, but it may be more representative of the U.S. population as a whole due to its status as a national reference laboratory. Finally, we were not able to control for any potential gender differences in the relative likelihood of visiting a healthcare provider in our patient populations.
Other limitations of the present report are the variability inherent to including multiple immunoassay platforms in our datasets (see Methods), as well as any potential short- and/or long-term changes in assay performance characteristics that can sometimes be observed in the clinical laboratory setting. While we observed differences in average 25(OH)D results across different assay platforms (see Table 2), it is important to note that ordering practices for 25(OH)D testing have dramatically shifted over time. A change from prior testing primarily for clinically suspected deficiency toward the more recent widespread utilization of such testing in otherwise healthy individuals likely confounds the attempt to utilize our dataset for direct assay comparisons. As our all of the testing, however, was ordered by clinicians and used in the management of patient care, we believe that it is appropriate to include all available data for patient averages and reference interval distributions in the present report, while acknowledging that some degree of bias may be introduced by including multiple assays in such analysis.
In our chart review, most cases of 25(OH)D levels greater than 150 ng/mL (potentially toxic) were due to vitamin D over-supplementation (especially use of 50,000 IU prescription vitamin D2 at weekly or greater intervals, intake of prescribed daily vitamin D that approximated or exceed weekly totals of >50,000 IU, and/or very heavy use of over-the-counter supplements outside of physician recommendations). Frequently, however, vitamin D intake was not well documented. In some cases, the dose recorded in the patient’s chart was far below what would be expected to cause potentially toxic levels. It is unclear whether this represented inaccurate/incomplete documentation or hypersensitivity to vitamin D.
It should be noted that elevated 25(OH)D results were uncommon at both UIHC and WCMC. For the UIHC population, only 27 of 60,237 tests (~0.05%) ordered over an approximately 13 year period had values >150 ng/mL. Of these 27 patients, only three had hypercalcemia and only one was hospitalized in critical condition. At WCMC, 15 of 57,433 orders for 25(OH)D by immunoassay (~0.03%) had values > 150 ng/mL. Of these 15, only four had hypercalcemia and only one was hospitalized (also in critical condition). While it was clear that documentation of over-the-counter supplementation was frequently absent or incomplete in our chart review, it is possible that vitamin D toxicity may only been suspected when symptoms (and/or laboratory findings) consistent with hypercalcemia were observed. In this era of increased attention to (and supplementation of) vitamin D, it would be prudent to keep the possibility of vitamin D toxicity in the differential diagnoses of patients receiving large doses of vitamin D2 or vitamin D3.
In conclusion, the present report describes the order and result patterns for 25(OH)D testing at two large academic medical centers, and shows that these data are reflective of results observed at a national reference laboratory. Future studies on large populations may be necessary to fully understand vitamin D status throughout life.
JRG is Medical Director of the Automated Core Laboratory at ARUP Laboratories and Assistant Professor in the Department of Pathology at the University of Utah. JRG previously served as Assistant Director of the Core Laboratory at Weill Cornell Medical Center/New York Presbyterian Hospital. JTG is a visiting professor at Sacred Heart University and teaches undergraduate and graduate courses including research methods. TCW and ER are pathology residents in the Department of Pathology at the University of Iowa Hospitals and Clinics. MDK is Director of the Clinical Laboratories and Clinical Associate Professor in the Department of Pathology at University of Iowa Hospitals and Clinics.
The authors would like to thank Christina Bjurstrom for providing the pre-October 2010 data on WCMC order frequency. Preliminary data has been presented at annual meetings of the American Association for Clinical Pathology (2012) and the Academy of Clinical Laboratory Physicians and Scientists (2013), and abstracts have been included in the respective conference proceedings [44, 45]. The authors would also like to thank Dr. A. Wayne Meikle and Dr. Joely Straseski for providing comments on a draft of this manuscript.
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