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Clinical Chemistry 54: 1883-1891, 2008. First published August 29, 2008; 10.1373/clinchem.2008.108761
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(Clinical Chemistry. 2008;54:1883-1891.)
© 2008 American Association for Clinical Chemistry, Inc.

General Clinical Chemistry

Effects of Delayed Sample Processing and Freezing on Serum Concentrations of Selected Nutritional Indicators

Bakary S. Drammeh1, Rosemary L. Schleicher1, Christine M. Pfeiffer1,a, Ram B. Jain1, Mindy Zhang1 and Phuong Hong Nguyen2

1 National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA; 2 Emory University, Atlanta, GA.

aAddress correspondence to this author at: Centers for Disease Control and Prevention, 4770 Buford Hwy., MS F55, Atlanta, GA 30341. Fax 770-488-4139; e-mail CPfeiffer{at}cdc.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Environmental conditions during sample processing, shipping, and storage are often suboptimal, particularly in less developed countries. We used samples from US volunteers to investigate the effects of delayed whole blood (WB) processing and delayed freezing of serum on selected nutritional indicators.

Methods: WB tubes (n = 35) were either stored at 32 °C for up to 3 days before serum separation or centrifuged within 2 h of collection; serum samples were stored at 11 °C for up to 14 days to simulate delayed shipping. We assessed analyte stability by comparing results with data from optimally prepared/stored serum samples (<2 h on the clot, frozen at –70 °C) and by using clinical-acceptability criteria based on combined analytical imprecision and intraindividual biologic variability.

Results: Clinically acceptable changes in concentration varied from 3%–15%. Delayed WB processing did not unacceptably affect concentrations of carotenoids and vitamins B12, D, and E; however, we obtained clinically unacceptable changes for ferritin (+9%), soluble transferrin receptor (sTfR) (+5%), and folate (–30%) after 1 day, and for vitamin A (–10%) after 3 days. Delayed freezing of serum did not affect concentrations of ferritin, sTfR, carotenoids, and vitamins A, B12, and E; however, we obtained clinically unacceptable changes for vitamins C (–20%) and D (+7%) after 7 days and for folate after 14 days (–22%).

Conclusions: Despite substantial delays in WB processing or in the freezing of serum samples, most nutritional indicators showed remarkable stability. This information is important for both the design of field studies and the use of residual samples subjected to suboptimal preanalytical factors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Micronutrient deficiencies are a widespread public health problem in developing countries and, to a lesser extent, in the industrialized world. Globally, over 2 billion people are at risk for vitamin A, iodine, and/or iron deficiency (1). Other micronutrient deficiencies that have received less attention but are also of public health concern include deficiencies for zinc, riboflavin, folate, vitamin B12, calcium, vitamin D, and selenium (2). To assess the prevalence of micronutrient deficiencies before and/or after an intervention aimed at improving nutritional status, clinicians frequently collect blood during nutrition surveys or clinical trials.

Unfavorable environmental conditions, such as increased temperatures, a weak infrastructure, and a shortage of adequately trained staff in many developing countries, make it difficult to follow proper procedures for sample processing, shipping, and storage. Irregular access to cold packs, dry ice, centrifuges, refrigerators, and freezers and unstable or unavailable electricity, particularly in remote locations, pose challenges to maintaining a proper cold chain and ensuring timely processing. Although such situations are rare in industrialized countries, inadvertent delays in sample processing or shipping and exposure of samples to increased temperatures can occur from time to time. Inherent in all of these scenarios is the question of whether the analysis of samples exposed to unfavorable preanalytical conditions will produce valid results.

To date, numerous reports have provided information about the stability of nutritional indicators, but only a few have studied analytes and conditions that are relevant for our investigation. Most reported studies have been limited in scope to single or few analytes (3)(4)(5)(6)(7)(8) or to particular panels, such as antioxidant (pro)vitamins (9)(10)(11)(12). Only a few studies have evaluated the effects of preanalytical factors on a broader list of nutritional indicators (13)(14)(15), and all but one of these studies (14) have had small sample sizes (12 individuals or fewer). Of the reports that have investigated delayed processing of whole blood (WB)1 (3)(5)(9)(10)(13)(14)(15), the most extreme delays were up to 1 day for WB stored at 32 °C (13) and up to 7 days for WB stored at room temperature (9). Some reports have evaluated the question of delays in the shipping and/or freezing of serum (4)(5)(6)(7)(8)(11)(12). With the exception of the article by Zhang et al. (13), most reports lack a clinical interpretation of any statistically significant changes, making it difficult to evaluate the relevance of the findings.

The main objective of the current study was to evaluate the stability of commonly measured nutritional biomarkers [representatives of fat- and water-soluble (pro)vitamins and iron-status indicators] under previously unstudied conditions that simulate extreme conditions encountered in a hot environment or one with a poor infrastructure. To mimic delays in processing or shipping, we focused on 2 preanalytical conditions: a delay in processing of WB stored at 32 °C for up to 3 days and a delay in freezing of serum samples stored at 11 °C for up to 14 days. We used acceptability criteria based on combined analytical imprecision and intraindividual biologic variation (13) to evaluate whether changes in concentrations due to unfavorable sample treatment were clinically acceptable.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
participants
Thirty-five volunteers (15 men and 20 women, all ≥18 years of age) from the CDC staff provided written consent to participate in this study. There were no exclusion criteria for participation in the study. The study was approved by the CDC Institutional Review Board.

blood collection and processing
Venous blood was collected from each participant into seven 7-mL and five 10-mL red-topped tubes (BD Medical Systems). To study the effects of delayed WB processing, we incubated two 7-mL tubes per each treatment in a 32 °C incubator (Precision; Labcare America) for 1, 2, and 3 days. We randomly assigned the 7-mL tubes to each of the 3 treatment conditions to eliminate any effects of the blood-drawing sequence. At the end of each treatment period, we centrifuged the tubes at 1800g for 15 min (Allegra 6 Centrifuge; Beckman Coulter) and transferred the serum sample from each treatment condition into 12 cryovials (Nalgene; Fisher Scientific) to allow a separate analysis for each assay and to provide some spare vials for reanalysis. We recorded the color of the sample following incubation to document the degree of hemolysis and stored the serum samples at –70 °C until analysis.

To study the effects of delayed freezing of serum samples, we allowed all 10-mL tubes to clot for 30–90 min at room temperature and then centrifuged the samples at 1800g for 15 min. We combined the serum samples from the 5 tubes from each participant into a 50-mL tube, mixed the tube, and transferred the serum aliquots (0.3–0.6 mL) into 12 cryovials for each of 5 treatment conditions: optimally prepared/stored serum (serum frozen immediately at –70 °C) and serum stored at 11 °C in a refrigerator for 2, 7, 10, and 14 days. To prepare samples for vitamin C measurement, we stabilized 100-µL serum samples from a subset of 12 participants with 400 µL of 60 g/L metaphosphoric acid and stored the samples at 11 °C. All sample processing was conducted under gold fluorescent lights.

serum analyses
Samples were analyzed at the CDC Nutritional Biomarkers Laboratory. To eliminate run-to-run imprecision, we assayed all serum samples from an individual in the same run. Table 1 specifies the methods used to analyze each nutritional indicator. We analyzed 3 levels of QC samples [2 for soluble transferrin receptor (sTfR)] at the beginning and end of each assay. We measured all samples (approximately 300 samples/assay) in only a single replicate, with the exception of 25-hydroxyvitamin D (25OHD), for which the assay manufacturer requires duplicate analysis. Our laboratory participated regularly in external quality-assurance programs and verified the methods with standard reference materials when available.


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  		Table 1. Methods characteristics and baseline concentrations of nutritional indicators in optimally prepared and stored serum samples.

In some cases, we lacked unthawed spare vials for reanalysis after a QC failure. To demonstrate that reanalysis after a limited number of freeze/thaw cycles is not a problem, we have assembled in-house data on freeze/thaw stability of nutritional indicators (see Table 1Up in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol54/issue11). All reported changes were within acceptable clinical limits according to the same criteria used to judge the effects of time and temperature variation.

statistical analysis
Observations outside the Tukey inner fences were considered potential outliers. Because of the limited size of the study and the fact that we identified only a few outliers [5% of the data, on average, for each analyte–treatment combination (n = 105; 4 vitamin C combinations were excluded because of small sample sizes)], we did not remove any observations from the statistical analysis, with the exception of folate and vitamin B12 because of extremely high concentrations in 1 participant (248 nmol/L folate and 2803 pmol/L vitamin B12). Eliminating these samples left data from 34 participants in the analysis for these analytes. The data analyzed for ferritin (n = 34) and cis-β-carotene (n = 33) were also from fewer than 35 participants because of a laboratory error during the processing of samples from 1 and 2 individuals, respectively.

For descriptive statistics, we calculated the arithmetic mean and the SD for each analyte under the optimal conditions and, when available, under the conditions of delayed WB processing (32 °C) and delayed freezing of serum (11 °C). We computed mean differences between the data obtained at delayed time points and data for optimally prepared/stored serum samples and calculated their 95% confidence limits. We also expressed the mean differences as percent changes. We used the 2-tailed paired t-test (SAS software; SAS Institute) to test whether differences between the delayed time points and the optimal conditions in analyte concentrations were significantly different from zero. Differences were considered statistically significant at P values of <0.05. Because the data were generally not normally distributed, we verified that results obtained with alternative analyses (log transformation and nonparametric testing) produced similar conclusions (see Text 1 in the online Data Supplement). We chose to present the results for parametric testing of nontransformed data.

We evaluated whether changes in concentrations due to unfavorable sample treatment were clinically acceptable by comparing the percent difference between the delayed treatment and the optimum treatment to the clinically acceptable percent difference derived from a combination of analytical variation (CVA) and intraindividual biologic variation (CVI) (13). A combined CV (CVC) was calculated as: CVC = (CVI2 + CVA2)1/2. We then calculated 95% confidence limits (upper and lower clinically acceptable limits) around the mean result for the optimum treatment (x) by using twice the formula for the clinically acceptable process limit (13): 0.975 x CVC x x/N1/2, where N is the number of blood donors used for each test. The clinically acceptable percent change was calculated as the difference between the upper limit and the optimum and between the lower limit and the optimum, with the mean difference expressed as a percentage of the optimum. For example, if the mean for the optimum treatment was 100 nmol/L and the clinically acceptable limits were calculated as 90 and 110 nmol/L, then the clinically acceptable percent change would be ±10%.

For most nutritional analytes (folate, vitamins A, B12, and E, trans-β-carotene, β-cryptoxanthin, lutein/zeaxanthin, and trans-lycopene), we used biologic variability information from the Third National Health and Nutrition Examination Survey (NHANES) for thousands of persons in the United States (16). For other analytes (ferritin, vitamin C, and {alpha}-carotene), we used information from a comprehensive database (www.westgard.com/guest17.htm) (17), or from studies of individual analytes [sTfR (18), {gamma}-tocopherol(19), and 25OHD (personal communication, L. Ovesen, 2007)]. Because we found no information on intraindividual biologic variability for retinyl palmitate and cis-β-carotene, we could not calculate the maximum clinically acceptable changes for these analytes.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Table 1Up summarizes characteristics of the methods, such as analytical imprecision and acceptability based on biologic variability, as proposed by Fraser et al. (20). The mean interassay CVs derived from analyses of multilevel QC samples over at least 20 days before this study were ≤5% for vitamin A, vitamin E, and sTfR; ≤10% for folate, ferritin, vitamin B12, vitamin C, 25OHD, and most carotenoids ({alpha}-carotene, trans-β-carotene, β-cryptoxanthin, and trans-lycopene); 11% for lutein/zeaxanthin; 13.7% for retinyl palmitate; and 21.3% for cis-β-carotene. On the basis of objective performance criteria with intraindividual variation (20), most methods displayed desirable precision (CVA ≤ 0.50 CVI), and only 1 method (25OHD) did not meet the minimum-precision requirements (CVA ≤ 0.75 CVI). The 25OHD method, however, had the strictest precision requirements because of the small degree of intraindividual variation (personal communication, L. Ovesen, 2007).

Table 1Up also summarizes baseline characteristics of the nutritional indicators, such as the mean concentrations for the 35 participants and the range of concentrations in optimally prepared/stored serum samples. Comparisons of each participant’s analyte concentrations with the traditionally used cutoff concentrations revealed that most participants had an adequate nutritional status. Small percentages of participants had low ferritin (5.9%), increased sTfR (11.4%), low 25OHD (11.4%), and low vitamin C (16.7%) concentrations.

Compared with optimally prepared/stored serum samples, serum samples that had been prepared from WB that had undergone delayed processing (Table 2 ) or serum samples that had undergone delayed freezing (Table 3 ) typically had small but statistically significant differences for most indicators. WB stored at 32 °C for up to 3 days before processing showed significantly increased values for ferritin (9%), sTfR (5%), vitamin E (3%), most carotenoids (2%–25%, except for lutein/zeaxanthin), and vitamin B12 (3%). A significant decrease (30%) was seen for folate after just 1 day, and significant decreases were also apparent for retinol (2%) and lutein/zeaxanthin (5%) after 2 days and 3 days, respectively. We observed no significant changes for 25OHD and retinyl palmitate, even after 3 days.


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  		Table 2. Mean differences (95% confidence limits) in concentrations of nutritional indicators for serum samples prepared from WB stored at 32 °C for up to 3 days.


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  		Table 3. Mean differences (95% confidence limits) in concentrations of nutritional indicators for serum samples stored at 11 °C for up to 14 days.

Table 3Up shows that serum samples stored at 11 °C for up to 14 days before freezing showed a significant decrease for vitamin C (5%) after 2 days; significant increases for 25OHD (7%), vitamin E (1%), and some carotenoids (1%–2% for {alpha}-carotene, trans-β-carotene, and trans-lycopene) after 7 days; a significant decrease for folate (5%) after 7 days; a significant increase for cis-β-carotene (1%) after 10 days; and significant changes for retinol (+3%) and retinyl palmitate (–4%) after 14 days. We observed no significant changes for ferritin, sTfR, and 2 carotenoids (β-cryptoxanthin and lutein/zeaxanthin), even after 14 days.

To assess whether these statistically significant changes were of clinical relevance, we used a combination of analytical and biologic variation in calculating the maximum allowable change (Table 4 ). Because of analyte-specific differences in analytical and biologic variation, the acceptability criteria for differences between the delayed treatments and the optimum treatment varied from 3% to 15%. Delayed WB processing did not affect the concentrations of vitamin B12, 25OHD, vitamin E, and carotenoids unacceptably; however, we did observe clinically unacceptable changes for ferritin (+9%), sTfR (+5%), and folate (–30%) after 1 day and for vitamin A (–10%) after 3 days. Delayed freezing of serum samples did not unacceptably affect the concentrations of ferritin, sTfR, carotenoids, and vitamins A, B12, and E; however, we did obtain clinically unacceptable changes for vitamin C (–20%) and 25OHD (+7%) after 7 days and for folate after 14 days (–22%).


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  		Table 4. Percent changes in concentrations of nutritional indicators in serum samples prepared after delayed processing or freezing, and assessment of clinical acceptability of changes based on a combination of assay imprecision and intraindividual biologic variation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Preanalytical factors are known to affect the concentrations of nutritional biomarkers. We therefore designed 2 experiments to assess the extent to which delays in WB processing or the freezing of serum may cause unacceptable changes in the concentrations of nutritional indicators, compared with optimally prepared/stored samples. Our approach differs from many others in that we have examined the stabilities of a comprehensive list of nutritional indicators under the same preanalytical conditions and have used protracted times and increased temperatures. We delayed WB processing for up to 3 days (32 °C) to mimic field conditions in a hot climate and kept serum samples unfrozen (11 °C) for up to 2 weeks to mimic delays in shipping and/or freezing. Extended delays were important to simulate field studies of nutrition in developing countries, where environmental conditions often cannot be carefully controlled and sample-collection protocols are frequently fraught with uncertainties. Our findings are also applicable to studies with well-designed sample-collection protocols in which accidental procedural violations occur and samples cannot be replaced.

We found that prolonged contact of serum with the clot at 32 °C was clinically acceptable for vitamin B12, 25OHD, vitamin E, and carotenoids. We saw small unacceptable changes for vitamin A (10% decrease after 3 days), ferritin (9% increase after 1 day), and sTfR (5% increase after 1 day) and large unacceptable changes for folate (32% decrease after 1 day). Maintenance of serum at 11 °C for up to 2 weeks was clinically acceptable for all analytes except vitamin C (20% decrease after 7 days), 25OHD (7% increase after 7 days), and folate (24% decrease after 14 days). Thus, many nutritional indicators are stable despite considerable deviation from standard blood-processing and storage protocols that recommend separating serum from blood cells within 20 min to 2 h and storing serum samples immediately at –20 °C to –80 °C.

Our findings regarding analyte stability after delayed WB processing are generally in agreement with those in the literature; however, none of the previous studies tested conditions as extreme (combination of temperature and length of time) as in ours. Zhang et al. studied the effects of the serum–clot contact time (up to 1 day) at 32 °C on several nutritional indicators (13). Similar to our study, these investigators found clinically acceptable stabilities for vitamins A, B12, and E for this period. They also found acceptable stabilities for ferritin (9% increase) and folate (15% decrease), whereas the changes we obtained for these analytes (9% increase for ferritin and 32% decrease for folate) were unacceptable. This difference in acceptability stems mainly from the difference in the number of study participants: the acceptability limits in the Zhang et al. report were generally based on data from 4 individuals and were therefore wider, whereas the limits in our study were based primarily on data from 35 individuals and were consequently tighter. Zhang et al. accepted changes of up to 15% for ferritin and up to 23% for folate, whereas we accepted changes only up to 6% and 12%, respectively.

Not surprisingly, other studies that stored WB under milder conditions, such as room temperature, found smaller changes in the concentrations of the nutritional indicators vitamin A: [<3% change after 4 days (14), ≤1% change per day for up to 7 days (9), no change after 1 day (3)], 25OHD [no change after 3 days (5)], vitamin E [no change after 3 days (10)], carotenoids [≤1% change per day for up to 7 days (9)], folate [up to 20% decrease after 4 days (14), 17% decrease after 3 days (15)], and ferritin [7% increase after 3 days (15), ≤3% change after 4 days (14)]. The increased ferritin concentrations obtained not only with prolonged serum–clot contact at 32 °C in our study and in the study of Zhang et al. (13) but also at room temperature in the study of Chu and McLeod (15) could be explained by hemolysis and ferritin leakage into the serum (21). Indeed, we observed that the degree of hemolysis increased with storage time at 32 °C.

A few older studies reported results that are contrary to our findings. When WB was stored at room temperature for 3 days, Hankinson et al. found that the vitamin A concentrations decreased 5% per day (10), and Chu and McLeod found a 27% increase in vitamin B12 (15). The reason for the increase in vitamin B12, which we also observed when we used an alternative test method (the Bio-Rad Laboratories Quantaphase II radio protein-binding assay; data not shown), is unclear. We considered the possibility that the swelling of cells had caused a subsequent increase in the serum concentration; however, because we did not observe this effect with the Abbott AxSYM® method (see footnote a in Table 1Up ), a more likely possibility was that the Bio-Rad assay detected a method-specific artifact that was produced during the prolonged contact of the serum with the clot at 32 °C. This finding shows that method-specific differences in the response to unconventional preanalytical variables have to be considered and that the findings obtained with one method cannot necessarily be applied to another.

To simulate delayed freezing, we stored serum samples at 11 °C for up to 2 weeks. Generally, our findings on analyte stability are in agreement with findings from previous studies. Folate concentrations were reported to be stable at 4 °C for 7 days (6)(8) but were no longer stable in refrigerated samples after 14 days (6). Similarly, our data showed clinically acceptable changes in folate concentrations only up to 10 days of refrigerated storage. Good stability was reported for ferritin stored at 4 °C for 14 days (7). Lastly, Margolis et al. reported a degree of stability for vitamin C that was similar to that in our study: Storage at 4 °C for 1 day produced a 7% change in concentration, whereas 2 weeks of storage produced 30%–80% reductions in vitamin C concentrations (4). Some reports in the literature present conflicting results on analyte stabilities. According to Craft et al., the concentrations of carotenoids and vitamins A and E in samples stored at room temperature in the dark for 1 day were not significantly different from those of samples stored frozen (12); however, Su et al. reported that these fat-soluble (pro)vitamins showed small but significant changes when they were stored at 4 °C for only 3 days (11). Kubasik et al. reported good stability for vitamin B12 at 4 °C for 14 days (6), but Komaromy-Hiller et al. reported variation in vitamin B12 concentrations of up to 28% after refrigerated and frozen storage, possibly because of poor assay precision (8).

Except for the study by Zhang et al. (13), none of the previous published studies assessed the clinical acceptability of changes in concentrations of nutritional indicators. Most often, the statistical significance of the bias introduced by the preanalytical variation was used to draw conclusions about acceptability. Because of the repeated-measures design of most such experiments, small differences can be statistically significant but not clinically important. This was the case in our study. Fewer treatment conditions were clinically unacceptable (Table 4Up ) than statistically significant (Tables 2Up and 3Up ) in comparison with optimally prepared/stored serum samples.

Because changes in analyte concentrations can be explained by analytical and biologic variations, an approach that combines method imprecision with intraindividual variation seems most appropriate for assessing the acceptability of treatment-induced changes; however, because the acceptability limits largely depend on the number of individuals from whom the data are derived, only adequate sample sizes can ensure reasonable acceptability limits. As mentioned earlier, the acceptability limits used by Zhang et al. (13) appear too wide because of the small number of individuals in their study. Depending on the purpose, clinically unacceptable changes may still be valid. For example, they might allow ranking of individuals in large-scale epidemiologic studies.

In addition to the use of objective criteria to judge clinical acceptability and an adequate sample size, our study had other strengths. The concentrations of the measured analytes were sufficiently high to ensure precise measurements. We minimized analytical variation by analyzing all of the samples from an individual in a single run. Lastly, the majority of the data on biologic variation used for this report were either from NHANES (16), a large national study representative of the US population, or from a large database (www.westgard.gom/guest17.htm) that subjected data from several studies to a scoring system to derive reliable information on biologic variation (17). Generally, the study populations in the reports used for our analysis (16)(17)(18)(19) were similar to our study population, i.e., healthy adults with a balanced sex ratio (40%–50% male). NHANES also included children, however, and 1 report (25OHD) was based exclusively on postmenopausal women (personal communication, L. Ovesen, 2007). As a result, the biologic variation in reference populations in some cases could be different from that of our study population.

This study simulated some practical laboratory and field conditions applicable to hot climates and areas with weak infrastructure. We conclude that most of the nutritional indicators studied have remarkable stability when exposed to extreme temperatures and prolonged storage times. As expected, folate and vitamin C emerged as the least stable analytes. Furthermore, to obtain accurate concentrations of iron-status indicators (ferritin and TfR), investigators should let serum sit on the clot for <1 day. It is not clear whether these findings are also valid for malnourished individuals. Studies with nutritionally deficient populations should be conducted to verify these findings.


   Acknowledgments
 
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, acquisition of data or analysis, and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors’ Disclosures of Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: We thank Drs. Joanne V. Mei and Michael E. Rybak (CDC, Atlanta, GA) for their valuable input to this study. We also thank staff members of the CDC Nutritional Biomarkers Laboratory for analyzing the samples for this study.


   Footnotes
 
The findings and conclusions in this report are those of the authors and do not necessarily represent the official views or positions of the US federal government, including the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry, the National Institutes of Health, the Food and Drug Administration, and the Department of Health and Human Services.

1 Nonstandard abbreviations: WB, whole blood; sTfR, soluble transferrin receptor; 25OHD, 25-hydroxyvitamin D; NHANES, National Health and Nutrition Examination Survey.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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