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Trace Element Analysis in Hair
   
Citation:  Bass, Dean A.  (2001).  Trace Element Analysis In Hair: Factors Determining Accuracy, Precision, And Reliability.  Altern Med Rev. 6(5):472-481

Abstract:  Trace element analysis in biological samples has improved significantly over the last 40 years. Improvements in instrumentation such as inductively coupled plasma-mass spectrometry and microwave digestion have resulted in improved precision, accuracy, reliability, and detection limits. The analysis of human scalp hair has benefited significantly from these improvements. A recent article in the Journal of the American Medical Association found significant inter-laboratory variation amongst several laboratories performing trace metal hair testing. It concluded that standardization was necessary to improve inter-laboratory comparability, and an accompanying commentary described the characteristics of a laboratory that should be used in performing hair analysis. The objective of this study is to demonstrate that good laboratory practices will generate precise, accurate, and reliable results. A method for establishing reference ranges and specific data on an analytical method will also be presented. The use of prescribed clinical quality control, including method validation, proficiency testing, split sampling, and good laboratory practices clearly demonstrates that measuring trace elements in hair can be analytically valid. (Altern Med Rev 2001;6(5):472-481)

Introduction

Scalp hair has been used as early as 1929 to assess human systemic levels of elements. (1) Hair is widely accepted for assessing toxic element exposures and measured by most clinical laboratories capable of making trace element measurements. Using hair to assess essential elements is more controversial, yet researchers have found many correlations of essential elements to diseases, metabolic disorders, environmental exposures, and nutritional status. (2-14) Compared to other types of clinical specimens, hair has different uses and even advantages over blood or urine. While urine and blood tend to show current or recent body status, hair represents a longer time frame, potentially years. Elements also occur in hair at higher levels, allowing for more sensitive and, because of the higher levels, more analytically accurate results. Hair is easier and safer to collect, ship, and store than blood or urine and the analysis is less expensive. This makes hair an excellent choice in certain situations and as a screening tool. Other challenges that must be addressed when measuring trace elements in hair include external contamination, lack of standardization, and analytical accuracy.

Making accurate and precise measurement in trace metal analysis of hair samples is important to the validity and usefulness of the test. Literature reviews well characterize analytical considerations and limitations of trace metal analysis in a variety of clinical samples. (15-19) Biological trace element analysis has improved significantly over the last 40 years. Improvements in instrumentation have resulted in improved precision, accuracy, reliability, and detection limits. The analysis of human scalp hair has benefited significantly from these improvements.

A recent article by Seidel et al (20) found significant inter-laboratory variation and concluded that standardization was necessary. An accompanying commentary by Steindel and Howanitz (21) stated, "A sample for hair analysis is best sent first to a laboratory that can validate its certification or accreditation for performing the test and that reports test characteristics, such as the hair-washing procedure, digestion techniques, recovery rates for the elements, internal quality control performance over time, and the minimum detection limits for each element." Puchyr et al reported such specific test characteristics for 36 elements in hair. (22) Rodushkin and Axelsson also reported on 71 elements in hair and nails using high-resolution inductively coupled plasma-mass spectrometry (ICP-MS). (23)

The objective of this paper is to demonstrate that good laboratory practices and validated methodology will generate precise, accurate, and reliable results. Variations in hair cleaning procedures, analytical methodology, and types of quality control performance assessments will be reviewed. A method for establishing reference ranges and data on analytical variability and accuracy will also be presented. The data presented in this paper follow the guidelines of the Clinical Laboratory Improvement Act (CLIA) and the National Committee for Clinical Laboratory Standards (NCCLS) for test method validation and quality assurance.

Pre-analytical Variables

One issue often raised with trace metal analysis of hair is the potential for contamination from external sources. Shampoo has been shown by LeBlanc, Dumas, and Lefebvre (24) not to have a significant impact on hair element levels for most elements. Unpublished results in the authors' laboratory confirm these findings. However, several hair preparation products contain metals that will significantly impact the levels of metals in hair. Most notably is selenium in shampoos that contain selenium sulfide, and lead in hair dyes that contain lead acetate.

Hair collection protocols (25) recommend clean hair that has not been dyed, permed, bleached, or straightened for three months and that only the newest hair growth be sampled. This procedure is designed to prevent major sources of contamination, which include the environment (water, grease, oil, dust, dirt, air) and hair treatments (dyes, perms, and other chemical treatments). However, these protocols are not always adhered to or contamination occurs in spite of these protocols.

Cleaning procedures should be employed by laboratories to remove exogenous contaminants. An ideal washing procedure would remove only external contaminants and leave endogenous elements intact. A variety of cleaning procedures are used and their efficacy may vary based on the nature of the contaminant and the element.

Most researchers who study trace metals in hair report the method they use for cleaning hair prior to analysis. Numerous authors have conducted studies of hair washing procedures. (26-32) Other authors have used radioactive labeling, (33) scanning electron microscopy, (34) and other methods (35-43) to assess the efficacy of removal of exogenous and endogenous elements from hair using various cleaning procedures. A recent study that assessed lead and mercury in the hair of President Andrew Jackson (44) used Triton X100, water and ethanol, and sonication, and showed that endogenous lead was not appreciably removed by this washing procedure.

Studies by the International Atomic Energy Agency (IAEA) examined different washing procedures as well, and found that even in cases where endogenous elements were removed it was not to the extent that would render the sample unusable. They concluded that while there are many variables associated with the washing procedure, including incomplete removal of exogenous contamination or partial removal of endogenous elements, representative measurements could be made if standardized washing procedures were employed. (45,46)

A standardized cutting and washing procedure has been described in detail elsewhere. (22) The hair specimen is cut and washed using a modified method developed by the IAEA. (44) The hair is first cut into approximately 0.125-inch (0.3-cm) pieces and mixed to allow a representative sub-sampling of the hair specimen. After cutting, each sample is washed four times with a 1:200 V/V dilution of Triton X-100. The samples are then rinsed with acetone and allowed to drain. This is followed by three rinses with de-ionized water (18-MW) and two rinses with acetone. The samples are then dried in an oven at 75 [+ or -] 5 [degrees] C.

The accuracy and reproducibility of this procedure has been documented in other studies (45,46) and in studies looking at blind split samples, results from identical twins, and repeat testing from the same individual. The expected reproducibility from such procedures is presented later in this paper. As indicated in the literature cited in this section, variation from this methodology may result in different measured element levels.

Trace Metal Analysis (Digestion and Analysis)

Analytical methodology choices can also introduce biases and variability, which can result from volatilization losses during digestion, contamination during digestion or analysis, or from interferences during analysis. Microwave digestion has been recommended as the sample preparation method most suitable for standardization. In addition, ICP-MS is the best technique for ultra-trace, multi-element analysis. (47,48) To assure accurate and precise measurements, it is important to characterize and validate the method used. The most important parameters in method validation are precision, accuracy, and detection limits. Table 1 shows the precision, accuracy, and detection limits for a method using temperature-controlled microwave digestion, followed by analysis using ICP-MS (Elan 5000, Perkin Elmer, Danbury, CT). As shown in this table, spike recovery values were generally between 90-110 percent, which is within expected ICPMS performance. Table 1 also shows good agreement for all elements when compared to a certified reference material (CRM). Only chromium, nickel, and arsenic showed significant deviation from the certified values. However, spike recoveries (Table 1) and testing another CRM (Table 2), indicate more accurate measures are being made.

The digestion method utilizes 0.2 g of washed hair samples accurately weighed into 50-mL disposable polypropylene centrifuge tubes. Concentrated trace-metal-grade nitric acid (3 mL) is added and the tubes are capped and placed in a microwave oven (MDS 2000 and MDS 2100; CEM Corporation, Matthews, NC). The digestion sequences are controlled by temperature through use of a fiber optic temperature probe placed in one of the samples. The process causes a refluxing of the sample, with no visible loss of liquid volume. The resulting digestate is a clear liquid with a yellow tint. After digestion an internal standard mixture is added, and the samples are diluted to a final volume of 50 mL. Recovery of all elements, including volatile elements such as mercury, was verified by CRMs and spike recoveries as shown in Tables 1 and 2.

This method, described in greater detail elsewhere, was found to have the best reliability, as other methods may have a potential loss of analytes, contamination of sample, or incomplete digestion. (22) Because ICP-MS has better sensitivity than other techniques for most elements, if other analysis techniques are used poorer detection limits and poorer reproducibility at lower concentrations would be expected. (49)

Standardization is an important aspect of any clinical test. It has been identified as a problem by Seidel et al, (20) and Ryabukhin (45,46) states that standardization of the hair washing step is critical to obtaining reproducible results. Standardization of sample washing protocols between laboratories should certainly allow better comparability among laboratories using quality analytical methodology. While this would be the ideal, variations in philosophy, the expense of re-engineering laboratory protocols, and laboratory politics may make the setting of standards not acceptable to all laboratories. However, if such a step were accepted by regulatory agencies, as suggested by Steindel and Howantiz, (21) inter-laboratory comparability would be improved.

Quality Assessment

The data shown in Table 1 are typical of the type of information collected in an initial method development and method validation. Once the analytical parameters are determined from the method development, and the method is determined suitable for making routine measurements, controls must be put in place to assure continued quality and to better monitor potential sources of errors. Controls used include pre- and post-digestion in-house controls, and CRMs. In addition, split sampling, proficiency testing, and external audits are important parts of assuring quality, both internally and externally. All of the quality tools described here are part of the method validation and quality control steps prescribed by CLIA and NCCLS. (50)

Certified reference materials are often the best tools for assessing accuracy. Several certified reference materials are available for hair. These include two from China (GBW 09101, 30 elements; and GBW 07601, 60 elements), two from the IAEA (IAEA-085, IAEA-086, both 9 elements), and one from Japan. In addition to using CRMs, in-house controls may be used to assess accuracy and precision. A control comprised of digested hair allows monitoring of between-run variations in the instrument, while a control comprised of finely cut homogenized hair allows monitoring of run-to-run variation in the digestion procedure. Together these controls provide a good means of assessing accuracy and precision. Because in-house control values are not certified, some effort must be made to "certify" these values. This can be done by spiking the control with a known quantity of analyte and measuring the recovery, and by measuring the control in conjunction with a CRM. In addition, the control or other samples can be sent to other quality laboratories and results can be compared.

Split sampling is useful in assessing precision and accuracy. Sending the same sample to the same laboratory at different times is useful in assessing reproducibility. Results of such blind split sampling are shown in Table 3. For assessing accuracy, part of a sample is analyzed in-house and part is sent to another laboratory. Results of samples tested in the authors' laboratory and in another laboratory are summarized in Table 4. This particular study was conducted by a university research laboratory and was submitted blind to our laboratory.

An important aspect of split testing is sample homogenization. When trying to homogenize samples for inter-laboratory comparisons, or preparing CRMs, organizations often powder the hair sample to assure reproducible sub-sampling. This has been shown to provide a more homogenous sample. However, the sample is different than a normal hair specimen and may have different characteristics.

While appropriately identified by Seidel et al (20) as an added variable to the results, we have found the homogenization procedure they described to be sufficient to demonstrate acceptable precision and accuracy for measuring trace metal in hair. While variations in results occasionally occur, split sampling results generally confirm acceptable analytical precision and accuracy; i.e., representative results of an individual's relative trace element levels are attained.

Typical results are shown in Tables 3 and 4. Variations shown may be the results of variations in the hair sample. When variations occur with between-laboratory split sampling, other measures of accuracy are assessed to confirm accuracy or quantify potential biases. As shown in these tables, precise and accurate measurement of hair can be made.

Some researchers requiring accurate and reliable trace element testing in hair also develop their own reference material. One such user (51) obtained a large sample of hair, homogenized it, and had it tested at numerous laboratories. This approach produced results similar to that described by Seidel et al. (20) The researcher concluded some laboratories were of better quality and the poorer quality laboratories were eliminated from consideration. The accuracy of the better laboratories was confirmed with continued testing.

Proficiency testing is often used to allow laboratories to assess and improve performance. It also serves as an independent assessment of accuracy. Unfortunately, few proficiency testing programs exist for hair. The National Public Health Institute of Quebec (INSPQ) Toxicology Center (Laboratory) conducts trace metal proficiency programs for blood and urine and, on a rotating basis, other clinical matrices such as hair (studies with hair conducted in 1997, 1999, and 2001). The study is an ICP-MS inter-laboratory comparison for hair and found significantly different results than did Seidel et al. (20) There were some key differences, however; for example INSPQ asked for volunteer laboratories to participate, they looked at more laboratories, they used a homogenized hair sample, and all the laboratories used ICP-MS. The results showed much lower inter-laboratory variability, and support the thesis that trace elements can be accurately measured in hair.

External assessment is often used to evaluate the quality of laboratories, often in the form of audits for purposes of certification. Audits typically look at all aspects of quality control described in this paper, with the intent of assessing whether a laboratory can make an accurate measurement of the clinical specimen of interest. Although certification is not available for many types of testing, including hair, as laboratories follow proper laboratory procedures and quality control programs, such certifications should be attainable when they become available.

Reference Ranges and Interpretation

Once a measurement is made, what does it mean? Is 5 mcg/g mercury in hair high or low? The determination of reference ranges is an important part of providing this interpretation. Guidelines for determining reference ranges are available from NCCLS. (52) Druyan et al described a detailed method for determining references ranges in hair using results from a large patient population. (14) Because of potential bias in using a general patient population, a smaller, physician-defined "Healthy American Population Study" was used to verify the ranges established with Druyan's patient population. Druyan's work further compared these ranges to literature values and ranges established by other laboratories.

For non-essential or potentially toxic elements with a single-tailed distribution, the reference limit is that seen at the 95th percentile. The designation of an expected range (as opposed to reference limit) is taken to be 68 percent of the reference population. For physiologically essential analytes, this range covers the 16th through 84th percentiles. (53) Individuals outside this limit may or may not have a problem that warrants attention. Clinicians making diagnoses and designing treatment plans based on test results should interpret the data in terms of a patient's history, medical condition, physical examination, diet, lifestyle, and environment, as well as other laboratory data.

Conclusion

Published research demonstrates that testing of trace metals in hair can be done with accuracy and precision. The work by Seidel correctly identified the need for standardization. The interest and use of hair testing they identified should call for more proficiency testing programs and better CRMs. The lack of inter-laboratory comparability is not supportive of the conclusion that hair testing is not valid, especially when it was inferred that some of the laboratories compared were unscrupulous and others had method-related differences. Like the findings of Walsh, Seidel could have drawn the conclusion that some laboratories do not perform quality work.

If hair can be reliably measured and is useful in screening for trace element status, why is its use so controversial? Why did Seidel come to the conclusion that hair testing is invalid when their data does not support it? The authors believe a general distrust of the utilization of hair for analyzing the presence of trace metals exists among some individuals who may feel some laboratories or doctors misuse or over-interpret the results of hair element analysis. The key to obtaining reliable, accurate, and precise hair element analysis, as with any other laboratory test, is to ensure that the laboratory of choice is appropriately licensed and employs standardized and documented procedures such as those described in this article.
 
 

Table 1. Analytical Characteristics
Element Within Run  Between Run Measured Value Certified Value SR (%) MDL
[micro] g/g
Li  11 16

91 0.003
Be



101 0.01
B 8 24

97 0.1
Na 3.0 3.8 268 266 90 1
Mg 4.2 6.2 94 105 95 7
Al 8.6 20 9.2 13.3 91 1
P 13 20 207 (184) 104 30
S

42300 (46900) 94 2000
K 12 15 5.3 (11.8)
3
Ca 5 7.1 1022 1090 90 90
Cr 13 15 2.88 4.77 107 0.04
Ti 10 17


.02
V 7.3 8.1 0.062 (0.069+) 97 0.005
Mn 4.3 6.3 2.5 2.94 99 0.05
Fe 9.7 11


2
Co 7.2 8.4 0.095 0.135 92 0.002
Ni 9.7 12 2.11 3.17 96 0.02
Cu 4.1 5.7 22 23 92 0.3
Zn 3.3 6.2 192 189 91 6
Ge 14 18


0.003
As 7.1 8.6 0.78 0.59 116 0.02
Se 7.2 8.5 0.61 0.58 120 0.8
Rb 8.9 9.0


0.003
Sr 6.3 7.2 0.22 4.19 98 0.07
Zr 4.1 5.6

104 0.03
Mo 6.2 7.8 0.39 (0.58) 105 0.006
Ag 8.0 11 0.30 (0.35)
0.02
Cd 9.4 15 0.091 0.095 100 0.02
Sb 6.6 11


0.02
I 9.0 12


0.7
Ba 9.1 9.6 5.54 5.41 96 0.1
Au 7.7 11


0.004
Hg 8.5 10


0.07
Pb 4.5 4.9 7.77 7.72 94 0.2
Bi 6.5 8.0

95 0.005
U 6.8 6.32

90 0.003

SR = spike recovery 
MDL = method detection limit
RSD = relative standard deviation

Certified value is for human hair control CRM GBW 09101, Shanghai
Institute of Nuclear Research Academia Sinica, P.O. Box 8204,
Shanghai 201849, China.
 

Table 2.  Accuracy for China Hair CRM GBW 07601

Element Certified Value ([micro]g/g) Measured Value ([micro]g/g)
Chromium 0.37 0.37
Nickel 0.83 0.73
Arsenic 0.28 0.27

Table 3. Repeated Testing Results of an Individual Sample Submitted at Different Times
(in [micro]g/g)

Element Report 1 Report 2 Report 3 % RSD
Al 20 21 23 7.1
Sb 0.064 0.069 0.046 20
As <0.01 0.013 <0.01
Be <0.002 <0.002 <0.002
Bi 0.286 0.270 0.262 4.5
Cd 0.379 0.373 0.399 3.5
Pb 6.2 6.3 6.7 4.1
Hb 1.06 1.01 1.00 3.1
Ni 3.39 3.32 3.66 5.2
Pt <0.001 <0.001 <0.001
Ag 0.18 0.20 0.13 21
Ti <0.001 <0.001 0.002
Th <0.001 <0.001 <0.001
Sn 1.6 1.4 1.6 7.7
U 0.099 0.091 0.105 7.2
Ca 2216 2137 2465 7.5
Mg 464 8.6 487 33
Na 1195 969 1324 15
K 2 5 2 58
Cu 95 90 100 5.3
Zn 276 266 281 2.8
Fe 19 21 18 7.9
Mn 2.23 2.10 3.36 27
Cr 0.89 0.77 0.70 10
Co 4.48 4.37 4.69 3.6
V 0.17 0.18 0.17 3.3
Mo 0.064 0.068 0.089 18
B 1.8 1.74 1.47 11
I 0.7 0.6 0.7 11
Li 0.207 0.185 0.221 8.9
P 167 171 160 3.4
Se 1.39 1.42 1.19 9.1
Sr 4.79 4.66 1.90 2.5
S 38500 39800 40800 2.8
Ba 29.4 26.4 28.2 5.5
Ge 0.017 0.017 0.021 13
Rb 0.028 0.026 0.026 4.2
Ti <0.1 <0.1 <0.1
Zr 0.175 0.275 0.197 36

          RSD = relative standard deviation
 

Table 4.  Results of Split Samples from Two Laboratories
(in [micro]g/g)


Element
Lab #1
Lab #2
Lead 6.6 6.85
Mercury 0.56 0.51
Cadmium  0.335 0.33
Silver .031 0.42
Barium 8.04 8.62
Arsenic 0.03 <0.04
Antimony 0.095 0.041
Aluminum 30 32.5
Bismuth 0.057 0.012
Nickel 7.60 7.68
Lithium <0.007 <0.03
Sodium 52 83
Potassium <2.0 <0.05
Phosphorum 182 160
Boron 014 0.3
Calcium 938 878
Magnesium 82 68
Vanadium 0.107 0.050
Chromium 0.17 <0.30
Iron 38 36.1
Manganese 2.34 2.21
Copper 27 28.9
Zinc 492 481
Molybdenum 0.041 0.035
Strontium 6.97 6.31
Selenium 1.18 0.7
Germanium 0.12 <0.001
Cobalt 0.178 0.17

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