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Effects of anticoagulants and contemporary blood collection containers on aluminum, copper, and zinc results.

Laboratory analysis is used to evaluate both deficiency and excess of trace elements (1). The intravascular concentrations of many of these elements are maintained within narrow limits. For this reason, preanalytical loss of analyte and spuriously high values attributable to contamination are concerns. Blood collection, processing, and storage before analysis are critical for accurate trace element analysis. Decreased concentrations of analytes may result from adsorption onto collection container surfaces or from the use of anticoagulants that complex metals (2, 3). Sources of contamination include patient clothing and skin; blood collection materials, including needles, anticoagulants, stoppers, serum separators, and glass containers; and particulate matter in laboratory air.

Aluminum, copper, and zinc are three metallic elements commonly monitored by the clinical laboratory. Although aluminum may have a physiological role in the action of a few enzymes, such as succinic dehydrogenase and porphobilinogen synthase, it is typically monitored to evaluate toxicity in patients subjected to hemodialysis for renal failure (3, 4). These patients may be exposed to high aluminum concentrations in their treatment regimen but lack an efficient physiological means to remove this element. Aluminum accumulation may lead to dialysis encephalopathy and osteomalacia. Toxicity is known to occur at concentrations >100 /,g/L, although symptoms may occur at 60 /,g/L or lower in dialysis patients (3,5). Because aluminum is ubiquitous and pervasive, contamination is a serious concern for the analytical laboratory. Falsely high concentrations measured in contaminated specimens may affect clinical decisions.

Copper is an essential trace element necessary for the function of several enzymes involved in electron transport, free radical defense, and other biological oxidation-reduction reactions (1). Copper has a role in iron metabolism and is an important indicator of Wilson disease and Menkes kinky hair syndrome. Copper concentrations vary with age, gender, and ethnic group.

Zinc is an essential trace metal that is second in abundance physiologically only to iron (1). Zinc is a necessary component of the active sites of many enzymes and contributes to the structural stability of numerous metallo-enzymes and other proteins. More than 75% of whole blood zinc is present in erythrocytes. Hemolysis can falsely increase zinc concentration and should be avoided in specimens used to measure zinc. Copper and zinc usually are measured to evaluate deficiency. Higher concentrations, however, may be associated with toxicity, and laboratory methods to determine increased concentrations are necessary.

We evaluated the effects of common blood collection tubes on the measured concentrations of these three trace elements in serum and plasma. The sampling protocol was devised to minimize contamination during specimen collection and sample processing. This study was approved by the University of Washington Human Subjects Review Committee. Specimens (n = 164) were collected from 23 fasting subjects by venipuncture using sterile butterfly needles. The first milliliter of drawn blood was discarded. Seven different commercial evacuated glass tubes were cooled to 4 [degrees]C before filling. The order of filling and tube characteristics are shown in Table 1. Filling order was based on NCCLS guidelines devised to minimize cross-contamination from tube additives (6). Royal-blue top tubes are acid-washed and have been tested for trace element background concentration. Plasma tubes were centrifuged cold within 10 min of collection. Serum tubes were placed in a 22 [degrees]C water bath for 30 min, and then centrifuged at room temperature. Specimens were transferred immediately into two acid-washed plastic tubes, capped, and stored at -70 [degrees]C before analysis. One aliquot was used for the determination of the aluminum concentration, and the second aliquot was used for analysis of copper and zinc by inductively coupled plasma mass spectrometry. This sensitive technique allows simultaneous multielement measurement and the use of an internal standard to monitor background signal, instrument drift, and matrix effects (7). Aluminum was analyzed separately to minimize contamination of the specimen. This is consistent with our usual laboratory protocol, which was adopted to decrease potential aluminum contamination from particulate matter and other sources in the laboratory that cannot be entirely controlled. Ions generated by the argon plasma were separated by their mass-to-charge (m/z) ratios and measured by mass spectrometry. We monitored masses 27, 65, and 64 for aluminum, copper, and zinc, respectively. Quantification was accomplished by comparison with an internal standard. Beryllium (isotope mass, 9) was used as an internal standard in the measurement of aluminum. Yttrium (isotope mass, 89) was used as an internal standard in the measurement of copper and zinc.

The mean and standard deviation for each analyte are plotted by collection container type in Fig. 1. Two specimens collected in tiger top serum separator tubes produced spuriously high aluminum values and were excluded from further analysis. For all other tube types and analytes, results from 23 specimens are plotted. In general, plasma values were lower than serum concentrations, but these differences were not statistically significant. We recommend a royal-blue top collection tube with no additive or with heparin for trace element testing. Mean values for specimens collected in these tubes were compared with mean values for the other anticoagulant collection tube systems using a paired t-est.


Comparison of aluminum measured in serum collected in a royal-blue top trace metal collection tube with aluminum measured in other collection containers showed a statistically significant difference only for EDTA plasma collected in the lavender top tube. Comparison of aluminum in plasma collected in a royal-blue heparinized tube with aluminum in all other plasma collection containers gave similar results, i.e., only the aluminum concentration found in the EDTA plasma was significantly different. We found no statistically significant differences between serum copper concentrations measured in any collection container and the copper concentration measured in serum collected in a royal-blue top trace metal collection tube. Likewise, no statistically significant differences were seen between plasma copper concentrations compared with copper measured in plasma collected in a royal-blue top tube, although the P value for the EDTA result was 0.09. Copper concentrations measured in EDTA plasma were lower than copper measured in any other collection container. Chelation of copper in the specimen by EDTA should not substantially affect the copper concentration measured by inductively coupled plasma mass spectrometry; it is possible that less copper contamination was present in these tubes. Comparison of zinc measured in serum collected in a royal-blue top trace metal collection tube showed statistically significant differences for serum collected in red top tubes and for plasma collected using lithium heparin and a polymer gel separator as well as for specimens collected in EDTA. Zinc concentrations in EDTA plasma were much higher than zinc measured in other collection systems. This could reflect contamination of the collection container or the EDTA anticoagulant with zinc. Plasma zinc concentrations in all tubes other than EDTA showed no statistically significant differences when compared with the royal-blue heparinized tube.

To evaluate the contribution of an osmotic effect on differences seen between serum and plasma, we measured the albumin concentration in serum collected in royal-blue top serum collection containers and in plasma anticoagulated with heparin (royal-blue top tube) and EDTA (lavender top tube). The mean albumin concentration was decreased by 2.6% in heparinized plasma (44.6 g/L; SD, 2.4 g/L) and by 6.6% in EDTA plasma (42.8 g/L; SD, 2.1 g/L) compared with the mean serum albumin concentration (45.8 g/L; SD, 2.1 g/L). The difference was significant for EDTA specimens (P <0.01), but not significant for heparinized specimens (P = 0.08). The decrease in the aluminum concentration observed for the heparinized royal-blue top tube was equivalent to the decrease in the albumin measured in the same tube type and could be explained by the osmotic redistribution of water between cells and plasma. This observation is consistent with those of several other investigators and has been attributed to a fluid shift from erythrocytes into plasma in response to increased osmolality produced by the addition of anticoagulants such as citrate or EDTA (8). These authors found decreased concentrations of albumin, cholesterol, and total protein in plasma specimens compared with the same analytes measured in serum specimens. Heparinized plasma concentrations of copper and zinc were much lower, reduced by 7% and 16%, respectively.

Metal contamination of EDTA in the collection container was evaluated by drawing a volume of distilled, deionized water into each of three royal-blue top and lavender top tubes and analyzing each sample. The mean concentrations of aluminum, copper, and zinc measured in the royal-blue top serum containers were 0.67 [micro]g/L (SD, 0.59 [micro]g/L), 0.38 [micro]g/L (SD, 0.18 [micro]g/L), and 4.8 [micro]g/L (SD, 0.34 [micro]g/L), respectively. The mean concentrations of aluminum, copper, and zinc measured in the EDTA plasma containers were 13.1 [micro]g/L (SD, 2.61 [micro]g/L), 0.67 [micro]g/L (SD, 0.15 [micro]g/L), and 698.9 [micro]g/L (SD, 442.7 [micro]g/L), respectively. We conclude that some component of the lavender top collection container, most likely the EDTA, contributes to the aluminum and zinc concentrations of specimens collected in these tubes. The extent of zinc contamination varies considerably from tube to tube.

Although there were significant differences between serum and plasma values, with the exception of samples anticoagulated with EDTA, measured aluminum, copper, and zinc values fell within expected reference intervals for all tube types. Results of this study indicate that the best choice for trace metal testing is a royal-blue top tube designed for trace element analysis. Lavender top EDTA tubes should not be used, but other blood collection tubes may be adequate for routine testing. Specimens collected in glass collection containers must be processed and transferred to plastic containers within 1 h of collection to minimize alteration of aluminum concentration (2,3). Plastic vacuum collection containers are available and provide a reasonable alternative if specimen processing will be delayed. Mean concentrations for serum aluminum, copper, and zinc can be expected to be slightly higher than mean plasma concentrations. Plasma specimens may offer an advantage for zinc determinations to minimize release of the high concentration of zinc in erythrocytes and platelets (1).

We thank Robert Costa, Tyler Mendenhall, Delfin G. Landicho, and Teresa Whisler for valuable technical assistance. This work was supported in part by a grant to the University of Washington Clinical Nutrition Research Unit (P30: DK35816) from the National Institutes of Health, and by the ARUP Institute for Clinical and Experimental Pathology, LLC.


(1.) Milne DB. Trace elements. In: Burtis CA, Ashwood ER, eds. Tietz textbook of clinical chemistry, 3rd ed. Philadelphia: WB Saunders, 1999:1029-55.

(2.) Subramanian KS. Storage and preservation of blood and urine for trace element analysis: a review. Biol Trace Elem Res 1995;49:187-210.

(3.) Cornelis R, Heinzow B, Herber RFM, Christensen JM, Poulsen OM, Sabbioni E. Sample collection guidelines for trace elements in blood and urine. J Trace Elem Med Biol 1996;10:103-27.

(4.) Winter M. WebElementS[TM], the periodic table on the WWW. (accessed May 2000).

(5.) Baselt RC. Disposition of toxic drugs and chemicals in man, 5th ed. Foster City, CA: Chemical Toxicology Institute, 2000:919 pp.

(6.) Wiseman JD, Bessman JD, Calam RR, Dawson JB, Fleming DO, Hill BM, et al. Procedures for the collection of diagnostic blood specimens by venipuncture; approved standard, 4th ed. (H3-A4). Wayne, PA: NCCLS, 1998;18:11.

(7.) Ash KO, Komaromy-Hiller G. Analysis of clinical specimens using inductively coupled plasma mass spectrometry. In: Wong SHY, Sunshine I, eds. Handbook of analytical therapeutic drug monitoring and toxicology. Boca Raton, FL: CRC Press, Inc., 1997:107-25.

(8.) Ungaro B, Corso G, Dello Russo A. Cholesterol concentration in serumplasma pairs differs because of water shift from blood cells. Clin Chem 1985;31:1096-7.

Elizabeth L. Frank [1], Martin Patrick Hughes [2], Daniel D. Bankson [3], and William L. Roberts [1] ([1] Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT 84132; [2] Department of Laboratory Medicine, University of Washington, Seattle, WA 98195; [3] Veterans Affairs Puget Sound Health Care System, Seattle, WA 98108; * address correspondence to this author at: c/o ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108; fax 801-584-5207, e-mail
Table 1. Characteristics of specimen collection containers by tube
top color.

 Order of Interior
Tube top color filling Additive Separator coating

Royal-blue 1 None None None
Red 2 None None Silicone
Tiger stripe 3 Clot activator Yes Silicone
Royal-blue 4 Sodium heparin None None
Green tiger
 stripe 5 Lithium heparin Yes None
Lavender 6 Potassium EDTA None None
Green 7 Lithium heparin None None
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No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2001 Gale, Cengage Learning. All rights reserved.

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Title Annotation:Technical Briefs
Author:Frank, Elizabeth L.; Hughes, Martin Patrick; Bankson, Daniel D.; Roberts, William L.
Publication:Clinical Chemistry
Date:Jun 1, 2001
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