Technical Abstract: Much of the information presented in this article is taken from the Quality Control manual of the Mineral Analysis Laboratory and references for this information may be obtained on request. Contact Sandy Gallagher at sgallagh@gfhnrc.ars.usda.gov for more information. The primary analytical problem encountered in trace mineral analysis is EXTERNAL CONTAMINATION. Many trace elements are present in the laboratory in microgram and even nanogram amounts, hence, contamination may contribute substantially to an analytical value for a trace mineral unless extraordinary measures are taken. A laboratory contemplating trace mineral analysis must be prepared to take precautions to the point of fanaticism; through all sampling, preparation, and analytical procedures to ensure that contamination is minimized. Common sources of trace mineral contamination include rubber stoppers, wood applicators, paper products, metal surfaces, dust, skin, dandruff, and hair. Plastic, borosilicate glass (do not use for boron or silicon) and stainless steel equipment (do not use for chromium) are best suited for trace mineral analysis. Phlebotomy Only plastic disposable syringes with stainless steel needles (siliconized needles are needed for chromium and nickel draws) should be used for blood collection. Evacuated blood collection tubes specified for trace mineral analysis are also suitable if the tops are removed after collection and the sample is not allowed to come in contact with the stopper. Leaching of metals from the stoppers of these and other types of tubes may contaminate the specimen. Other materials involved in sample collection or processing, such as clotting aids or gel serum separators, should be avoided. The amount of trace mineral contamination should be checked in all anticoagulants, tubes, syringes and reagents that are used. When new lot numbers are used, tubes and equipment can be checked for contamination by allowing the equipment to come into contact with a dilute acid solution for one hour and analyzing this solution. This ensures that all equipment is as low in trace minerals as is possible. Common laboratory practices that can contribute to contamination include the use of wooden applicator sticks to ring blood clots of tube walls, the wiping of pipette tips with paper tissues, and the mixing or storing of samples in rubber stoppered tubes. Skin contact with any surface that will be exposed to samples or solution must be avoided. Tubes must be covered at all times. Sample Many things affect the integrity of the sample when reporting a value for trace minerals such as hemolysis, icteric, lipemic, loss of element in the tube, heterogeneity, mixing, contamination during processing, fasting, hemostatsis, posture, exercise, and use of supplements. Physiological conditions should be standardized to minimize these possible sources of sampling variation. Distribution of water depends on posture: standing up leads to a loss of plasma water, and to an apparent increase of approximately 10% in the concentration of nondiffusible blood constituents such as proteins or cells. Similar changes in water distribution take place locally, for example, when a tourniquet is used for blood specimen collection. Most trace minerals are transported in the blood bound to proteins or cells. Changes in posture and application of a tourniquet will therefore change apparent concentrations. Exercise, even of short duration, causes changes in blood serum constituents, possibly caused by leakage of intracellular components. Continuous physical training may cause hemodilution; which may in turn falsely reduce values. Changes caused by physical strain may even be seen in urinary excretion of trace minerals. Evaporation, adsorption on container walls, and precipitation may be sources of variability in biological samples. Adsorption of mineral cations onto surfaces of different types of glass or plastics is a well recognized problem. Polypropylene tubes are suitable for storage of serum and plasma for most trace minerals. Use of micro centrifuge tubes cuts down on the surface area of sample and in turn reduces evaporation for samples that are stored for long periods of time. It is important to avoid hemolysis of blood samples, because erythrocytes contain substantial concentrations of elements such as potassium, iron, zinc, and manganese which may falsely elevate values. Freeze/thaw cycles can increase the concentration of some of the trace minerals because of evaporation and possible contamination from multiple pipetting of the sample. Samples kept in the freezer for long periods of time evaporate by freeze drying. Specific recommendations for centrifuging, aliquoting and storing may differ depending on which mineral is being analyzed. The following is general information about the collection of samples for trace minerals. Specific trace minerals maybe require special handling. Serum Blood is drawn by using a trace metal free syringe and the sample is allowed to clot. Syringe designs such as the Sarstedt® syringe eliminate the need to transfer the blood to another tube. The plunger can be removed and the syringe becomes a tube. Once the blood is clotted, the tube is centrifuged at 3000 RPM¿s for 10 minutes at 4°C. The serum is removed by using a trace mineral free plastic transfer pipette and is then stored in a trace mineral-free container. Plasma We routinely use 4.5 % Disodium EDTA solution (30 ul/ml of blood) when analyzing plasma for zinc, copper, and phosphorus. The correct amount of blood is drawn into the syringe and gently mixed and transferred to a trace metal free tube. The tube is capped and centrifuged at 3000 RPM¿s for 10 minutes at 4°C. The plasma is transferred to a trace mineral clean tube. Care must be taken NOT to transfer RBC¿s or the buffy coat. If the plasma is contaminated with either, the sample should be centrifuged again. Erythrocytes (RBC) A 4.5% Disodium EDTA solution (30 ul/ml of blood) is the anticoagulant of choice. Approximately 3 ml of packed cells are required for analysis in our laboratory. The sample is drawn by using a trace metal free syringe containing the measured amount of Disodium EDTA solution. The syringe of blood is gently mixed and then transferred to a trace mineral free tube. The tube is capped and centrifuged for 10 minutes at 3000 RPM at 4°C. The plasma and buffy coat are carefully removed with a plastic transfer pipette. Some trapped plasma (usually 5-8%) is unavoidable and must be removed by washing the red cells 3 times with 0.85% saline (if sodium is the analyte of interest, another wash solution must be used). Saline is added to the packed cells, mixed well, and centrifuged at 3000 RPM at 4°C for 10 minutes. The supernatant is discarded along with any visible buffy coat. The procedure is repeated a total of 3 times. Leukocytes (WBC): The anticoagulant of choice is 4.5% Disodium EDTA 30ul/ml of blood). Approximately 15 ml of whole blood is needed to isolate enough WBC¿s for trace mineral analysis. Cell separation procedures such as Percoll® separation can be used to isolate mononuclear, PMN¿s, total leukocytes and/or platelets. Urine, Fecal, Diet Care must be taken to ensure that all equipment and procedures are as free from trace mineral contamination as possible. Plastic collection containers, stainless steel blenders (should not be used for chromium) and all supplies should be checked for trace mineral contamination. Depending on the mineral of interest, processing (wet or lypholized), digestion methods, and analysis may vary. A 24 hour urine collection is the specimen of choice and should be acidified to preserve the sample and maintain mineral solubility. Individual fecal samples or a homogenous 24 hour sample may be collected in trace mineral-free containers. Procedures can be requested for the collection, processing and analysis of these types of samples. Hair Lack of s