Introduction

With a global prevalence over 1 billion, zinc deficiency ranks as one of the top nutrition challenges worldwide, especially in low and middle-income countries [1–3]. Inadequate zinc intake can result in increased susceptibility to infectious disease, impaired child growth, and other serious morbidities [4–6]. To address zinc deficiency, several international agencies monitor and, when necessary, implement nutrition intervention programs to improve zinc nutrition in populations of concern [6–8]. Zinc status is usually determined by sampling the circulating zinc levels, yet there are important technical challenges that should be considered when attempting to measure zinc within blood [6, 8, 9]. First, zinc is a common environmental and industrial contaminant, so all supplies that could have contact with the blood should be certified for use with trace metal analysis, or at least tested beforehand for background zinc levels [4, 9–12]. This includes phlebotomy supplies, laboratory plasticware, and reagents used in the preparation of the plasma or serum samples within the laboratory. Second, the blood samples must be handled carefully and processed quickly in order to minimize hemolysis, thought to cause the majority of errors in the clinical lab [13]. The concentration of zinc in the erythrocytes is more than 10 times greater than the concentration of zinc in plasma or serum levels, so even a small degree of hemolysis can significantly increase the zinc levels in a blood sample [14–16]. The risk of zinc contamination from internal or external sources is further complicated by the fact that circulating zinc levels are tightly maintained within a narrow homeostatic range under physiological conditions [6, 17, 18]. Thus, even small differences in zinc measurements can have important effects on determination of zinc status for a population, or evaluation of a nutrition intervention program. The choices that researchers make for supplies and procedure, i.e. pre-analytic variables, are crucial to success for measuring zinc [19, 20]. Many of these variables are discussed on the International Zinc Nutrition Consultative Group (IZiNCG) website (https://www.izincg.org).

Pre-analytic variables that affect the way blood is collected include the site of blood draw, use of plasma or serum matrix, and type of blood collection tube (BCT). For blood draw site, the conventional approach for assessing zinc uses venous blood obtained by standard phlebotomy [8]. Phlebotomy is a well-established procedure that can produce substantial blood volume from the veins, but requires trained technicians and specific sterile supplies, leading to expense and logistics challenges for larger studies. The most common alternative to phlebotomy is the use of a fingerstick, which is a simple procedure requiring low-cost supplies and minimal training, but typically yields just a few drops of blood. Historically, this low volume of blood prevented the widespread use of fingerstick sampling for zinc analysis, but technological improvements have reduced the amount of blood volume needed for measurement so that fingerstick samples are now more feasible for zinc assessment [12, 21]. However, fingerstick sampling provides blood from the capillary circulation, which is not the same as venous blood. At present, there are few reports comparing capillary to venous samples for zinc assessment. For blood matrix, the choices are almost always plasma or serum. Plasma includes all constituents of the blood except cells, but plasma also requires anticoagulant additives that could potentially interact with zinc [22]. Serum is similar to plasma but with the clotting factors removed so that anticoagulation is not necessary, but serum requires time for clotting and results in loss of plasma proteins which may affect zinc homeostasis. While there have been several studies comparing zinc levels in plasma and serum, there is no consensus as to which matrix type is best for mineral assessment, with some favoring plasma [23, 24] and others favoring serum [25, 26]. A third format variable relates to manufacturer of the selected BCT(s). Becton, Dickinson and Company (BD) has a major market share for BCTs in the US, whereas Sarstedt AG & Co (Sarstedt) is more common in some European countries. To our knowledge, comparisons of different BCT manufacturers for zinc assessment has not received attention previously.

Pre-analytic variables that affect the way blood is prepared for analysis include processing time and holding temperature for collected samples. It is considered best practice for the whole blood samples to be processed immediately to plasma or serum for the best chance of high-quality data [9]. However, immediate processing is not always possible, particularly in studies in which the laboratory is a substantial distance from the phlebotomy site or when many participants are managed at the same time. Some reports have investigated the impact of processing time on zinc values, but there is no consensus as to which timepoint(s) should be used as a limit for zinc assessment [27–31]. Similarly, it is considered best practice to store whole blood samples in a refrigerated area prior for later processing [9]. Again, this may not always be possible, particularly in field studies where access to a cold chain is limited. Some reports have investigated the impact of holding temperature on zinc values, but there is no consensus as to what temperature protocols should be in place for zinc assessment. Moreover, delays in processing and storage at higher temperatures are likely to co-occur in larger studies and in areas with limited infrastructure, so interactions between the two variables should also be examined.

The methods for blood collection and processing can influence the quality of zinc measurements, but it is difficult to isolate the impact that specific pre-analytic variables have on this assessment. Logistical issues, including supply chain choices or procedural steps, are often co-variables that make it challenging to determine which parameters are most consequential for zinc measurements. We sought a more straightforward design that focused on the impact of specific pre-analytic variables on zinc measurement while minimizing the complications from most other technical parameters. Our goal was to determine how blood draw site, blood matrix, tube type, processing time, and holding temperature impact the assessment of circulating zinc levels.

Methods

Study overview

This study on zinc assessment was conducted in parallel with a previously reported study on hemoglobin assessment, with much of the methodology being the same for both reports [32]. The Children’s Hospital Oakland Research Institute (CHORI) Institutional Review Board approved this study (2019–055), and recruitment was conducted over 7 months. Participants completed a demographic questionnaire, underwent anthropometric measurement, and provided sequential blood draws by fingerstick (capillary blood) and then conventional phlebotomy (venous blood). All blood samples were collected at our clinical site and transported to our laboratory under the custody of the study coordinator. Most variables associated with the blood draws and processing were minimized, including the use of the same clinical site, phlebotomist, blood draw procedures, and sample handling procedures (Table 1). Although there is no established convention for the description of pre-analytical variables in this type of study, we followed the Checklist for Reporting Stability Studies (CRESS) developed by the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) to allow for allow greater comparability with other studies on assessment of zinc [33].

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Table 1. Clinical and processing variables controlled in this study.

https://doi.org/10.1371/journal.pone.0286073.t001

Clinical procedures

This study was conducted entirely on the campus of CHORI, now part of the University of California San Francisco. All clinical visits were scheduled within the same 2-hour window of the day (10:00AM–12:00PM), as time of day is known to have a strong influence on circulating zinc levels [6, 34]. The clinical procedures began with each participant reviewing and signing the consent form and completing a demographic questionnaire. Height and weight were measured using a Harpenden 602VR stadiometer and Scaletronix ST scale, respectively; these values were used to calculate body mass index (BMI) as mass (kg)/height (m)². The participants then provided non-fasting blood samples beginning with fingerstick(s) then immediately followed by phlebotomy [32]. The timing for the blood draws and approximate blood volumes in the BCTs were recorded by the study coordinator. After blood collection, participants were provided juice and snack food while they were observed for recovery. Participants received a gift card after completing the study protocol.

A set of 26 BCTs were prepared and pre-labeled for each participant (Fig 1), including 8 BD plasma tubes (6 ml-size Vacutainer containing dipotassium ethylenediaminetetraacetic acid (K₂EDTA), product number 368381, lot number 8099632), 8 BD serum tubes (6 ml-size Vacutainer containing clot activator additive, product number 368380, lot number 8099631), 1 Sarstedt plasma tube (7.5 ml-size Monovette containing K₂EDTA, product number 01.1605.100, lot number 8031811), 1 Sarstedt serum tube (7.5 ml-size Monovette containing clot activator additive, product number 01.1601.100, lot number 8033711), 4 BD capillary tubes (0.5 ml-size Microtainers containing K₂EDTA, product number 365974, lot number 8311522), and 4 Sarstedt capillary tubes (0.5 ml-size Microvette containing tripotassium ethylenediaminetetraacetic acid (K₃EDTA), product number 20.1341.102, lot number 9482211). These BCTs were selected for the goal of comparing zinc levels in blood samples for each participant. For venous blood samples, BD Vacutainer products 368381 and 368380 were used because they are certified for trace metal analysis, rated at a limit of 0.04 mg/L zinc according to manufacturer’s instructions. Sarstedt also had venous BCTs designed for trace metal analysis, but only with lithium heparin as the anticoagulant. Since we wished to directly compare BCTs with the same additives, the Sarstedt Monovette products 01.1605.100 and 01.1601.100 were chosen instead even though they were not certified for trace metal analysis. We tested samples from the same lot number of the Sarstedt BCTs and found them to be below detection for zinc content (<0.005 mg/L). For capillary blood samples, there are currently no BD products certified for zinc analysis, but the BD Microtainer product 365974 is certified for measuring lead according to manufacturer product information, which may indicate low levels of environmental contaminants. Thus we used the BD 365974 product for capillary blood collection but tested samples from the same lot number and found them to be below detection for zinc content. Similarly, there are currently no Sarstedt products certified for zinc analysis, but the Sarstedt Microvette product 20.1341.102 uses a similar anticoagulant and is also certified for measuring lead according to manufacturer product information. Thus we used the Sarstedt 20.1341.102 product for capillary blood collection but tested samples from the same lot number and found them to be below detection for zinc content.

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Fig 1. Schematic of sample processing.

Blood was collected in up to 26 blood collection tubes (BCT) for each participant. Comparison between blood draw location was conducted with venous (taller tubes) and capillary (shorter tubes) blood. Comparison between blood format was conducted with plasma (lavender cap) and serum (red cap) tubes. Comparison between BCT manufacturer was conducted with Becton, Dickinson and Company (BD) and Sarstedt AG & Co (Sarstedt) tubes. From the initial group of BCTs, 6 BD venous plasma and 6 BD venous serum BCT were placed at 3 different holding temperatures (4°C, 20°C, and 37°C) for 2 different delayed processing times (4 hours or 24 hours), as indicated. All samples were measured for zinc content using inductively coupled plasma optical emission spectrometry (ICP-OES). Tube illustrations are not to scale.

https://doi.org/10.1371/journal.pone.0286073.g001

Capillary blood collection.

To begin the blood collection protocol, each participant was asked to stand to maximize the effects of gravity on blood release from the fingerstick while leaning against a bench for comfort. The participants’ hands were first warmed in a waterbath for 10–20 minutes, dried, and then disinfected using alcohol wipes. Capillary blood was then collected by fingerstick using a high flow lancet (BD product number 366594, lot number V3V51E9) while the participant remained standing. A fingerstick was first applied to the non-dominant hand thumb and then middle (3^rd) or ring (4^th) finger if blood volume from the thumb was insufficient. Explanation for the use of the thumb for capillary blood sampling can be found in a previous report [32]. The phlebotomist gently pressed on the base of the hand to start blood flow but avoided directly squeezing or milking the thumb or finger [35]. The first drop of capillary blood was discarded, then the remaining drops were pooled into a BD Microtainer plasma BCT followed by a Sarstedt Microvette plasma BCT as volume allowed. Capillary blood volume target was 0.5 ml or greater but was stopped short of target volume if the pooled blood from both thumb and finger failed to reach 0.5ml or if the participant requested to end the procedure. If more than 0.5ml was collected, multiple capillary BCTs would be used up to 2ml total volume. Using this protocol, 1–2 ml of capillary blood was collected from most participants [32]. Note that a larger volume of blood was collected in order to have enough sample for multiple analytic tests in addition to zinc content; if only zinc analysis is needed, then less blood volume is needed so traditional fingerstick methods should suffice. Once the capillary blood was collected in all BCTs, tubes were inverted 4–6 times according to manufacturer’s instructions and placed on ice while venous blood collection was completed.

Venous blood collection.

After capillary collection, each participant was moved to a blood collection chair and seated position for phlebotomy [35]. Venous blood was then collected by venipuncture in the arm of non-dominant hand, typically at the antecubital vein using a Sarstedt Safety Multifly Needle 21Gx3/4”-size with Multi-Adaptor (product number 85.1638.200, lot number 9050111), as this blood draw system accommodated both BD and Sarstedt BCT types. Venous blood was collected in the following order: Sarstedt Monovette serum BCT, Sarstedt Monovette plasma BCT, BD Vacutainer serum BCTs, and then BD Vacutainer plasma BCTs. Between the BD and Sarstedt BCTs, a discard BCT with no additive was connected to push through a few drops of blood to prevent any contamination when moving to the different BCT manufacturers. Once the venous blood was collected in all BCTs, the tubes were inverted 8–10 times without shaking according to manufacturer’s instructions and held on ice until all samples were taken to the laboratory. Blood samples were transported directly from the clinical site to the laboratory within 10–20 minutes on average and 60 minutes at maximum.

Results

Comparison of blood draw site on zinc measurement

A comparison of zinc concentrations in capillary and venous blood samples was conducted from study participants collected in BD BCTs (Table 2 and Fig 2A and 2B). For capillary plasma measurements, one sample did not have enough volume, one sample was found to have excessive hemolysis, and one sample was identified as an outlier, resulting in a total of 57 available plasma samples. For venous plasma measurements, data for 60 plasma samples were available with none found to have insufficient volume, excessive hemolysis, or identified as outliers; however, only venous samples with matching capillary values were compared, resulting in a total of 57 datapoints. Both datasets passed normality testing, so parametric statistics were used. The mean zinc concentration was 0.700 ± 0.094 mg/L for capillary and 0.646 ± 0.084 mg/L for venous, indicating that capillary values were elevated by 0.054 mg/L (8%) in comparison to venous blood values with a sequential blood draw from the same participants. This difference was statistically significant (p<0.0001) using a two-tailed paired t-test. Bland-Altman analysis revealed a similar bias of 0.063 mg/L for capillary zinc values compared to venous zinc values.

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Fig 2. Zinc concentrations differ depending on blood draw site, blood matrix type, and blood collection tube (BCT) manufacturer.

Correlation plots comparing circulating zinc values from (A) capillary plasma and venous plasma (using only BD BCTs), (C) venous plasma and venous serum (using only BD BCTs), and (E) capillary plasma from BD and Sarstedt BCTs are shown. Each circle represents the zinc level (mean ± SD, n = 1–3) for an individual participant, with linear regression and 95% confidence interval indicated by a solid red and dotted red line, respectively. The line of concordance is shown as a solid black line for comparison. Bland-Altman plots of these same data revealed a bias of (B) 0.054 ± 0.047 mg/L for capillary over venous plasma samples, (D) 0.029 ± 0.025 mg/L for serum over plasma venous samples, and (F) 0.036 ± 0.033 mg/L for BD over Sarstedt BCTs for capillary plasma. Each circle represents the zinc level for an individual participant, with average distance and 95% confidence interval indicated by a solid red and dotted red line, respectively.

https://doi.org/10.1371/journal.pone.0286073.g002

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Table 2. Zinc concentrations differ by three pre-analytic variables.

https://doi.org/10.1371/journal.pone.0286073.t002

Comparison of processing time and holding temperature on zinc measurement

The effects of delay time and holding temperature on zinc measurements were tested in venous plasma from study participants collected in BD BCTs (Fig 3A). Whole blood samples were processed immediately or after a delay of 4 or 24 hours while held at 4°C, 20°C, or 37°C for 1 hour. For plasma samples at time 0-hour, data for 60 plasma samples were available with none found to have insufficient volume, excessive hemolysis, or identified as outliers. For plasma samples at time 4-hour at all temperature settings, three samples were found to have insufficient volume, none had excessive hemolysis, and none were identified as outliers, resulting in 57 available plasma samples. For plasma samples at time 24-hour at all temperature settings, four samples were found to have insufficient volume, none had excessive hemolysis, and none were identified as outliers, resulting in 56 available plasma samples. Only samples with matching values at all temperatures were compared resulting in a total of 56 datapoints. Zinc concentrations for the 4-hour and 24-hour timepoints were all greater than the time 0-hour zinc concentration, ranging from 5–12% higher. Two-way ANOVA of this data indicated that both temperature (p = 0.0007) and processing delay (p<0.0001) were a significant variation, but without significant interaction (p = 0.1007).

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Fig 3. Zinc concentrations differ depending on blood processing time and holding temperature.

Timecourse of mean circulating zinc values from blood samples held at 4°C (blue circles), 20°C (green circles), or 37°C (red circles) for up to 24 hours in (A) venous plasma and (B) venous serum (using BD only BCTs) are shown. Each circle represents the average zinc level (mean ± SD, n = 55–58) normalized to the ‘time 0’ value for each group of time and temperature condition fit to a point-to-point function. Statistical significance compared to ‘time 0’ of each group is indicated by asterisk of same group color.

https://doi.org/10.1371/journal.pone.0286073.g003

The effects of delay time and holding temperature on zinc measurements were also tested in venous serum from study participants collected in BD BCTs (Fig 3B). Whole blood samples were processed immediately or after a delay of 4 or 24 hours while held at 4°C, 20°C, or 37°C for 1 hour. After 1 hour, the samples from 20°C and 37°C were moved to 4°C for the remainder of their incubation period. For serum samples at time 0-hour, one sample did not have enough volume, one sample was found to have excessive hemolysis, and none were identified as outliers, resulting in 58 available plasma samples. For serum samples at time 4-hour at all temperature settings, no samples were found to have insufficient volume, none had excessive hemolysis, and none were identified as outliers. For serum samples at time 24-hour at all temperature settings, no samples were found to have insufficient volume, none had excessive hemolysis, and none were identified as outliers. Only samples with matching values at all temperatures were compared, resulting in a total of 58 datapoints. Zinc concentrations for the 4-hour and 24-hour timepoints were all greater than the time 0-hour zinc concentration, ranging from 0.5–7% higher. Two-way ANOVA of this data indicated that both temperature (p = 0.0061) and processing delay (p<0.0001) were a significant variation, but without significant interaction (p = 0.1967).

Impact of pre-analytical variables on zinc status assessment

Differences in blood draw site, blood format, and BCT type had significant effects on the average zinc values measured in this study, so the effect of those variables on the overall assessment of zinc status was evaluated in this cohort of healthy adults. Although our study was not specifically designed to investigate zinc deficiency, several participants had circulating zinc values below the conventional cut-off of 0.65 mg/L for morning, non-fasting plasma as defined by IZiNCG [4]. Depending on the choice of draw site, format, or BCT, the number of participants who fell below the threshold for zinc deficiency varied substantially (Table 3). No conclusions can be made from a single study, but these results illustrate that zinc assessment might be influenced by key pre-analytical variables, especially when the average zinc levels are close to the threshold value for zinc deficiency.

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Table 3. The number of participants at risk of zinc deficiency differs with blood draw location, blood matrix, and BCT type.

https://doi.org/10.1371/journal.pone.0286073.t003

Discussion

After two decades of measuring zinc levels in plasma and serum samples for numerous programs, our laboratory has received a variety of inquiries about best practices in zinc assessment, most often about blood draw procedures, tube types, or processing issues. While some of these topics are addressed within the literature on zinc assessment, the available reports are often limited in scope and sometimes conflicting in results. One reason for the lack of consensus may be due to uncontrolled technical parameters that make it difficult to isolate specific pre-analytical variables. The potential effects are magnified due to the tight homeostatic control on circulating zinc levels, such that even small differences in zinc measurements can impact zinc assessment protocols [6, 17, 18]. The impetus for this study was to focus on specific pre-analytical variables that may impact zinc measurement to support best practice guidelines for future assessment of zinc status.

Blood samples from a diverse group of healthy adults were collected and processed under a single chain of custody and highly controlled conditions to test 5 key pre-analytical variables. The mean zinc concentration for venous plasma collected in BD BCTs was 0.647 mg/L, which is near the recommended cutoff of 0.65 mg/L for adequate zinc concentration in adults using morning, non-fasting blood samples [9]. Based on this cutoff, a sizeable number of participants in this study would be categorized as at risk for zinc deficiency despite identifying as healthy. We did not attempt to examine the reasons for the mild zinc deficiency because the primary purpose of this study was only to compare the zinc levels within the same participants and blood draw. In fact, it was helpful to have both zinc sufficient and zinc deficient individuals, as it added to our evaluation of how pre-analytical variables could affect zinc values over a range of circulating zinc concentrations.

The first pre-analytical variable tested was different blood draw sites, specifically the differences in zinc concentration between capillary and venous blood. It is known that capillary and venous blood have small differences in composition, but the blood matrices are considered similar enough that they have the same reference ranges for zinc for both plasma and serum [40, 41]. Comparative studies on nutrient levels in capillary and venous blood have mainly addressed macronutrients like glucose and protein, both of which are 3–10% lower in the venous compared to capillary circulation. A few old reports have addressed mineral micronutrient levels from different blood draw sites, including calcium, potassium, and sodium, but found some differences between capillary versus venous circulation [42–44]. We have found no modern reports that follow up on these studies, which might benefit from the use of more precise methods and sensitive technology. Moreover, zinc was not reported in any of these previous studies, so the impact of blood draw site on zinc assessment is unresolved. In this study, we found that capillary blood plasma had an 8% higher mean zinc concentration than venous blood plasma from the same donors in this cohort. This difference is small but shows that different blood draw sites can influence values derived from zinc assessment. The use of capillary plasma in this study instead of venous plasma resulted in a substantially lower number of participants who would be considered at risk of zinc deficiency (28% compared to 53%).

The second pre-analytical variable tested was different blood matrices, specifically plasma and serum formats. Several studies have examined how zinc levels vary between the two matrices but found no difference in zinc content between plasma and serum samples [11, 26, 45–47]. However, other reports did show modest differences in zinc content between plasma and serum samples, with zinc generally higher in serum compared to plasma samples taken from the same individuals [23, 27, 48], though at least one report found higher zinc in plasma samples [29]. English and Hambidge suggested that the differences in the zinc content of the matrices may be due to the required processing delay to generate serum from whole blood, which could allow more time for zinc to diffuse from cellular sources [27]. Later, more comprehensive analyses of the blood matrix revealed that many nutrients and metabolites were in fact at higher concentration in the serum compared to plasma [25, 49]. These authors proposed that the influence of a volume displacement effect caused by the loss of coagulation proteins led to an increase in the fractional distribution of other metabolites. However, zinc was not measured in those studies, and follow up work to confirm the impact of volume displacement on zinc concentration have yet to be reported. Thus the potential difference in zinc content between plasma or serum remains unresolved, so individual laboratories tend to choose the matrix from their own historical preference, constraints from other co-analytes, or logistical aspects of study design. In fact, much of the commonly cited literature for zinc assessment treat plasma and serum zinc concentrations as equivalent [6, 8, 9]. In this study, we found that venous blood serum had a 5% greater mean zinc concentration than venous blood plasma from the same donors in this cohort. This difference is small but shows that different blood matrices can influence values derived from zinc assessment. The use of (venous) serum in this study instead of (venous) plasma resulted in a somewhat lower number of participants who would be considered at risk of zinc deficiency (47% compared to 53%).

The third pre-analytical variable tested was different BCT manufacturers, specifically testing similar BCT types from BD and Sarstedt. We are unaware of any previous study reporting zinc values from different manufacturers, but this should be an important issue since the market share of these two companies differs internationally. The closest matched BCT types had slightly different anticoagulant additives, with BD using K₂EDTA and Sarstedt using K₃EDTA. Our goal was to compare manufacturers with similar BCT types, and these products were the closest between the two companies. Publications comparing BCTs with K₂EDTA and K₃EDTA for several analytical targets showed minimal differences between the anticoagulants, though zinc was not specifically measured [50, 51]. In this study, the mean zinc concentration for venous blood plasma or serum collected in BD BCTs was not different between the two manufacturers. However, the mean zinc concentration for capillary blood plasma collected in BD BCTs was 6% higher than capillary blood plasma collected in Sarstedt BCTs from the same donors. One explanation for this could related to differences in the manufacturing process of BD and Sarstedt capillary BCTs. Neither BD nor Sarstedt capillary BCTs were certified for use with zinc assessment, but a random sampling of empty tubes from the study lot numbers did not indicate any significant contamination of zinc from either manufacturer. It is important to note that only a single lot number from each manufacturer was used in this study, so it is not possible to state that one manufacturer was superior to another. However, the implication is that even the type of BCTs used can affect zinc assessment, so testing before use and avoiding tube type mixing is highly recommended. The use of capillary plasma from Sarstedt BCTs in this study instead of BD BCTs resulted in a substantially higher number of participants who would be considered at risk of zinc deficiency (50% compared to 28%).

The fourth and fifth pre-analytical variables were time until processing and holding temperature for blood samples, respectively. These parameters are often examined together given their interrelationship. Several studies have described changes in nutrient levels as a function of processing time and temperature [52–56], but few of these included zinc as an analyte. One of the first reports on zinc was by English and Hambidge who found that both plasma and serum zinc increased by 5% when whole blood processing was delayed by 2 hours, though the samples were kept at ‘room temperature’ [27]. Later studies corroborated these findings by showing significant increases in plasma zinc when samples were held at ‘room temperature’ for 2–4 hours [29–31]. An early comparison of both processing time and temperature by Tamura and colleagues found that plasma and serum zinc were significantly increased between 1–5 hours when whole blood samples were kept at 23°C, but replicate samples kept at 7°C were not significantly increased until 24 hours [28]. An important caveat is that all previously cited studies used heparin as the anticoagulant for plasma samples unlike the current study that used EDTA, though at least one report showed no difference between EDTA and heparin for determining zinc levels in plasma [57]. However, we are not aware of any reports that directly compare zinc from serum samples with plasma samples using EDTA. We also could not find reports in which zinc was measured from blood kept at warmer temperatures, like what might occur in an equatorial country without immediate access to a cold chain. Studies looking at other metabolites did show significant changes when plasma was kept at 37°C for even 1 hour [20]. In this study, the mean zinc concentration for venous blood plasma and serum ranged from 5–12% when processing was delayed 4 or 24 hours after collection. Additionally, the mean zinc concentration for venous blood plasma and serum ranged from 0.5–7% when incubated at 20°C or 37°C after collection. These results are consistent with previous work showing that both time until processing and holding temperature can influence zinc assessment.

A strength of this study is that important pre-analytical variables were evaluated under a single chain of custody with tight control over most of the other environmental and technical parameters that could add to zinc measurement variance. These results help fill in literature gaps on the impact of blood draw site, tube types, and holding procedures during zinc assessment. The highly controlled structure of this study is also a limitation, as the control obtained with the clinical laboratory does not reflect the practical challenges of conducting a large cohort or field assessment of zinc status. However, it seems important to understand the baseline variability within these pre-analytic variables to properly calculate how study factors will affect the dispersion of zinc measurements. Another limitation of this study is that the plasma and serum samples held for 4 or 24 hours for later processing were frozen and analyzed on a different day from the time 0-hour samples due to logistical reasons. The ICP was calibrated with the same standards and validated with the same quality control samples on each run day, but we cannot rule out that the freeze/thaw process or instrument performance differences could have influenced zinc measurements. An additional limitation is that not all BCTs or other clinical supplies used in this study were available as trace metal-certified products. In particular, the lack of commercially available BCTs for capillary blood that are validated for zinc measurement was an unavoidable confounder in this study. We sampled all BCTs and other supply lots for zinc contamination before use, but convenience sampling cannot guarantee every consumable was zinc free. The difficulties in matching the preferred BCT to the study design illustrates the need for more trace metal-certified options for clinical assessments of circulating zinc.

This study attempted to quantify the importance of specific pre-analytical variables on the measurement of circulating zinc concentrations, which is key to determining the public health burden of zinc deficiency in target populations. Blood draw site, blood matrix, blood collection tube type, blood processing delay, and blood holding temperature all had small but significant impacts on the assessment of circulating zinc levels. Yet statistical significance is not the same as clinical significance. Unfortunately, it is not easy to determine the clinical significance of circulating zinc levels since zinc deficiency results in nonspecific shifts in metabolism without unique clinical sign or metabolic changes [6]. Therefore, it is up to each research team to determine if the bias revealed in this study will have an impact in their experimental design. If a research team is trying to reduce all extraneous variability in zinc assessment, then caution is warranted when comparing studies that use different procedural methods to measure zinc concentration in plasma or serum. Future studies should endeavor to control these pre-analytical variables to return the highest quality data for zinc assessment.

Supporting information

Acknowledgments

The authors thank Nyla Sepulveda for expert phlebotomy service during this study. The authors also thank Bonny Alvarenga for laboratory assistance, Dr. Ellen Fung for providing clinical space, Maria Cruz for assistance with supplies, and Sarstedt, Inc. for donating blood collection tubes. Additionally, the authors thank Dr. Christine McDonald, Dr. Mari Manger, and the IZiNCG Steering Committee for helpful feedback.