March 2010: Volume 36, Number 3
Meeting the Analytical Demands of Identifying Exposed Children
Kathleen L. Caldwell, PhD, and Robert L. Jones, PhD
A naturally occurring, malleable, dense, blue-gray metal, lead has been used in commercial products such as fossil fuels, house paint, batteries, ammunition, solder, pipes, devices to shield X-rays, and craft-making products. But because of health concerns, government agencies imposed regulations that either eliminated or dramatically reduced the amount of lead added to gasoline, paints, food cans, ceramic products, caulking, and pipe solder.
Although lead reduction represents one of the best-known public health success stories, the problem is not fully resolved. Lead is particularly harmful to the developing brain and nervous system of fetuses and young children, and lead-based paint remains the most common source of lead exposure for children in the U. S. Older housing, especially pre-1978, is more likely to have leaded paint, putting millions of children who live in these homes at risk for lead exposures. Because a higher proportion of people in lower socio-economic classes tend to live in older, substandard housing, socio-economic status is a common predictor of possible lead exposure.
Less common sources of exposure also still exist, including lead-glazed ceramic pottery, plumbing systems with lead-soldered joints or lead pipes, lead-containing folk remedies, and lead-contaminated dust in indoor firing ranges. Recently, another seemingly innocent source was discovered that put children at risk of lead poisoning: inexpensive toys and trinkets. In fact, some cases of severe lead poisoning occurred in which the child swallowed and retained the toy. Children may also be exposed to lead that is brought into the home on the clothing of adults whose work or recreational activities involve lead.
Here we describe the advantages and disadvantages of the blood lead analysis methods, as well as why public health efforts to identify children who have been exposed to lead are so critical.
The Public Health Problem
Lead affects almost every organ system in the body, but it is especially harmful to the central nervous system. At high levels, lead results in seizures, coma, and even death. Even at low exposure levels, lead has been documented to cause learning disabilities and behavioral problems. What constitutes a safe level of exposure to lead is still a point of debate among public health experts, but no safe blood lead level in young children has been identified.
Not only are the lives of children and their families greatly affected by lead exposure, but society also pays a high price to care for these children. In fact, even though asthma affects many more children in this country, the total economic costs of asthma pale in comparison to those of lead exposure. As recently as 2002, researchers studying the contribution of environmental pollutants to the incidence, prevalence, mortality, and costs of pediatric disease in American children estimated the cost of asthma at $2 billion annually, while that of lead exposure was $34 billion. The negative effects of even small amounts of lead exposure on a child's developmental status and educational achievement have significant effects throughout the child’s entire life, leading to the much higher economic burden on society.
Elevated Blood Lead in Children
Blood lead levels, defined as ≥10 µg/dL, have steadily declined in children 1–5 years old in the U.S.
Source: Jones RL, Homa DM, Meyer PA, Brody DJ, et. al. Trends in blood lead levels and blood lead testing among U.S. children aged 1 to 5 years, 1988–2004. Pediatrics 2009;123(3):376–85.
Blood: The Preferred Matrix
Even at blood lead levels that can result in neurologic damage, there may not be obvious physical symptoms, so excessive blood lead can go unrecognized and undetected. Laboratory testing of blood is the only reliable way to identify lead-exposed individuals. The concentration of lead in whole blood has gained wide acceptance as the best available measurement of cumulative exposure, because blood levels reflect both recent intake and an equilibration with stored lead in other tissues, particularly the skeleton.
Although a number of alternate matrices have been proposed and discussed, including saliva, hair, and nails, their use in measuring lead exposure remains problematic for a number of reasons. Any matrix used as a tool for biomonitoring must have certified or standard reference materials of the analyte that is being tested; however, these alternative matrices lack such materials. The alternative matrices also lack reliable reference ranges or correlations to the analyte/matrix combination for human populations. Laboratories analyzing these matrices should have matrix-matched internal quality control material and matrix-matched calibration curves so that matrix effects will not bias the analytical results. Furthermore, alternate matrices may have a high potential for pre-collection environmental contamination that cannot be effectively eliminated. Such contamination can contribute to a high analytical bias and therefore produce a false-positive clinical result. Finally, there are no proficiency testing programs established for lead testing in saliva, hair and nails. For these reasons, this article will address only the measurement of lead in whole blood.
Public Health Guidance
In 1985, the Centers for Disease Control and Prevention (CDC) established ≥25 µg/dL as an elevated blood lead level in children and recommended the erythrocyte protoporphyrin assay (EPP) as a screening test for lead exposure. But in 1991, the agency lowered the level of concern for blood lead in children under age 6 years to 10 µg/dL. At this new lower level, the EPP test was no longer sensitive. Consequently, in its 1991 statement CDC also changed its recommendation concerning the best test for detecting lead exposure to direct measurement of lead in whole blood rather than the extraction method used in the EPP test.
Lead Analytical Methods
Today, labs primarily assess lead exposure with whole blood lead measurements. Although a number of human tissues and fluids also reflect lead exposure, most of the published information related to human exposure and health effects is based upon the concentration of lead in whole blood. In blood, lead binds to erythrocytes and then is distributed initially to soft tissues. Because lead has a relatively long half-life of 1–2 months, the fraction of absorbed lead not promptly excreted can become incorporated into bone and even teeth. Consequently, lead concentration in whole blood serves as a measure of recent exposure, whereas bone lead is an indicator of long-term exposure.
In cases of very high exposure, in vivo measurements may be necessary to reflect the exposure accurately. Lead concentration in vivo may be determined by non-invasive, in vivo X-ray fluorescence (XRF); however, XRF is still an emerging technique available only in research settings. Several research teams are working to understand the localization of lead in bone, its mode of chemical binding, and its chemical and physiologic distribution.
Typically, labs collect venous blood samples for lead assessment; however, finger-stick capillary samples may prove to be equally useful. For the latter, the sample collection process must be performed carefully to avoid external contamination. Three common analytical techniques are often used to measure lead in whole blood. These include anodic stripping voltammetry (ASV), atomic absorption spectroscopy (AAS), and inductively coupled plasma mass spectrometry (ICP-MS).
Zinc protoporphyrin (ZPP) measurements were also used for assessing lead exposure in children prior to 1991; however, the method still has utility today for cases in which blood lead levels are ≥25 µg/dL. In cases of acute exposure, although blood lead levels decrease, according to the 1–2 month half-life, ZPP lead levels remain elevated for the lifetime of the involved red blood cells. Lead levels measured by ZPP do not reflect recent or acute lead exposure, and they do not change quickly when a person’s source of lead exposure is removed. ZPP measurements may aid in detecting a person’s average exposure to lead over the last 3–4 months; however, the method is not sensitive enough for use as a screening test in children.
Although the AAS method, with either a flame or an electrothermal atomization furnace, is outdated technology, it is specific and sensitive and provides reliable laboratory data for lead levels. The method involves the use of graphite furnace atomic absorption spectrometry (GFAAS) and is based on the fact that free atoms will absorb light at frequencies or wavelengths characteristic of the element of interest. Measuring light absorbed at 283.3 nm by ground-state atoms of lead from either an electrodeless discharge lamp (EDL) or a hollow cathode lamp (HCL) source provides useful information: the amount of light absorbed can be linearly correlated to the concentration of analyte. Typically, 100 µL of blood is diluted with a matrix modifier and dropped onto the graphite furnace, which is a small graphite tube. When the graphite furnace is heated, the sample is vaporized and the lead is atomized. Although GFAAS has been shown to be precise and dependable, it can only measure one element at a time.
ICP-MS’s multi-element analysis capability enhances lab productivity, and therefore many public health and private clinical labs use the method. ICP-MS offers a high degree of specificity, sensitivity, and selectivity, as well as the ability to analyze other toxic and essential metals from a small sample. This technique can also determine the isotope ratios of the lead in a set of samples, which is not possible with AAS. Such ratios help determine if a particular source of lead is a possible contributor to poisoning of an individual.
In a routine clinical mode that uses a whole blood sample of <100 µL (either venous or finger-stick capillary), ICP-MS analysis provides an adequate limit of detection (often <0.5 µg/dL) that can be achieved with minimal sample preparation. This multi-element analytical technique is based on quadrupole ICP-MS technology, which couples radio frequency power into a flowing argon stream seeded with electrons, creating the plasma. The predominate species in the plasma are positive argon ions and electrons. Use of a nebulizer inserted within a spray chamber converts diluted whole blood samples into an aerosol, and a portion of the aerosol then is transported first through the spray chamber followed by the central channel of the plasma, where the temperature is 6000–8000° K. This thermal energy atomizes and ionizes the sample, and the ions, along with the argon gas, then enter the mass spectrometer through an interface that separates the ICP from the mass spectrometer. Once inside the mass spectrometer, the ions pass through the ion optics, and the mass analyzing quadrupole before they strike the surface of the detector. Electrical signals resulting from the ions are processed into digital information that is used to indicate the intensity of the ions and subsequently the concentration of the element.
Analytical methods such as GFAAS and ICP-MS work well for routine measurement of blood lead levels <0.5 µg/dL. However, as the cutoff for a blood lead level of concern drops, laboratories wishing to engage in routine biomonitoring must evaluate pre-analytic variables with care, eliminating as much background contamination as possible. The use of “lead-free” blood collection devices, alcohol wipes, laboratory reagents, and sample vials is essential.
Lead Testing at the Point of Care
Recognizing the need to add point-of-care (POC) capability to the arsenal of blood lead methodologies, CDC spearheaded development of a first-of-its-kind, portable blood lead instrument that has since been used to indentify lead poisoning globally. In 1997, scientists at CDC, in collaboration with ESA Biosciences, a supplier of medical analytical instruments, developed the first portable blood lead testing device approved for field use in healthcare clinics and in targeted blood lead investigations. The LeadCare instrument uses electrochemistry with a small, screen-printed colloidal gold electrode to measure the amount of lead in whole blood by ASV. At the beginning of a blood lead measurement, the operator mixes 50 μL of fresh, whole blood with a treatment reagent. This process disrupts the red blood cells that contain most of the lead and chemically disassociates the lead from the red blood cell components. “Free” lead is then released in the treatment reagent in the form of divalent lead, which is then available for detection on the sensor electrodes. After the blood treatment reagent mixture is transferred to the sensor and the test is started, an electrical potential is applied by the analyzer, causing the lead to plate onto the test electrode. The analyzer strips the lead from the plate via a voltage shift to a more positive potential. The device measures the resultant current associated with stripping and automatically converts it into a blood lead value that is displayed on the analyzer.
The Food and Drug Administration (FDA) classified the first-generation ASV device as ‘moderately complex’, a classification that limited its use in the U.S. However, design improvements eventually led to the 2007 release of a CLIA-waived device. The second-generation LeadCare II Blood Lead Test System is a fast and simple system that allows healthcare providers to detect blood lead levels in patients. It is now widely available in a variety of patient care settings, such as doctors’ offices and public health clinics.
Given the public health need for lead testing, CDC funded development of the LeadCare II as a CLIA-waived device. This POC device has led to a paradigm shift in blood lead testing. Health practitioners can now perform in-office tests during a single well-child visit and avoid unnecessary delays in the detecting, counseling, and treatment of cases of lead poisoning. Where it has been implemented, the device provides a significant public health benefit, empowering parents and the public health community to help prevent future lead poisoning.
Being able to educate parents about the dangers of lead at the same time they learn of their child's lead levels has been proven to be effective in rectifying and reducing lead exposure. But given that lead poisoning is an environmental health condition, monitoring by public health officials is also crucial. Therefore, it is critically important that labs and other healthcare providers report all blood lead test results to public health officials. Many states already mandate such reporting, and CDC has worked with ESA to develop user friendly software that supports the reporting requirement.
The Goal: Improved Public Health Outcome
In 1991, CDC defined the blood lead level of concern that should prompt public health actions as 10 µg/dL, and subsequently the U.S. Department of Health and Human Services (HHS) established a national goal to eliminate blood lead levels ≥10 µg/dL in children younger than age 6 by 2010. Remarkable progress has been made towards this goal. As a result of the reduced use of lead in commercial products and public health efforts to educate the public about the dangers of lead exposure, data from the National Health and Nutrition Examination Survey (NHANES) indicate the number of children 1–5 years with blood lead levels ≥10 µg/dL has fallen from an estimated 8.6% during 1988–1991 to 1.4% during 1999–2004.
NHANES, a national survey of the health of the non-institutionalized U.S. population, also indicates that blood lead levels have continued to decline since 1976 (Figure 1). NHANES data from survey periods 2007–2008 indicate that approximately 1.2% (95% CI of 0.2–3.9) of 817 children tested who were representative of the U.S. population had blood lead levels ≥10 µg/dL. Of these children, 100% in the 1–5 years age group had detectable lead values, defined as CDC’s 0.25 µg/dL limit of detection. For the NHANES 2007-2008 survey period, the geometric mean for children 1–5 years old was 1.24 µg/dL (95% CI of 1.18–1.29), compared with 1.57 µg/dL (95% CI of 1.5–1.7) for survey years 2003–2008 .
As the blood lead levels for U.S. children steadily fall, laboratorians will need to continue to evaluate analytical methods and instruments that will enable them to provide public health officials with the best means to monitor the population. Although widespread exposure to lead sources such as leaded gasoline and paint have diminished, exposure resulting from toys and other consumer products has become a source of concern. These new exposure risks emphasize the need for laboratorians to provide analytical methods capable of detecting low levels of blood lead and to test blood lead in non-traditional settings, such as doctor’s offices, walk-in clinics, or field study locations. Efforts to identify affected children and eliminate lead poisoning will require continued innovation and persistence from both public health officials and lab professionals.
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Kathleen L. Caldwell, PhD is inorganic laboratory chief in the Inorganic and Radiation Analytical Toxicology Branch in the Division of Laboratory Sciences, National Center for Environmental Health, CDC.
Robert L. Jones, PhD is branch chief in the Inorganic and Radiation Analytical Toxicology Branch in the Division of Laboratory Sciences, National Center for Environmental Health, CDC.
The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.