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Blood Lead Measurement F
or most people, the words "lead poisoning" conjure up Time magazine cover photographs of inner-city toddlers sucking on paint chips, surrounded by neglect and decay. But in the past five years, numerous studies have found subtle but widespread physiological, neurological, and intelligence-deficit damage a t blood lead concentrations as low as 10 pg/dL-a level previously considered normal-in asymptomatic children from all areas of the United States and all economic groups. About 74% of American homes built before 1980 still contain lead paint. Although lead-based paint, contaminated soil, and lead dust are still considered the major sources of childhood lead exposure, drinking water is also a problem. I t is estimated that among young children, 10-20% of total lead exposure comes from drinking water. The Environmental Protection Agency (EPA) estimates that from 1976 to 1980,10.7% of US. children under six years old had blood lead concentrations > 25 pg/dL, and 91% had concentrations > 10 pg/dL. By 1990, only 1%had concentrations > 25 pg/dL, but 15% still had concentrations > 10 pg/dL. Pnolornlcmprapn d lead acetate cadnesy M cnael W Daadson. Florida Slate Univenily
On the basis of these and other studies, the Centers for Disease Control and Prevention (CDC) lowered its acceptable limit for pediatric blood lead concentrations in 1991 from 25 pg/dL, which had been the cutoff since 1985, to 10 pggldL, and recommended that all children under age six be screened for blood lead at the new lower levels. The CDC also recommended that a "direct" blood lead assay be used rather than calculating blood lead concentration from measurements of lead's physiological effects on red blood cells (an "indi-
rect" assay). Only a direct measurement of lead in blood is accurate for lead concentrations below 10 pg/dL. These recommendations were incorporated into the fmst stages of a Department of Health and Human Services ( H H S ) 2 0 - y e a r p l a n for eliminating childhood lead poisoning. The new standard posed a technical challenge to manufacturers and state health departments alike. The available direct blood lead measurement techniques that were sensitive enough to pick up the lower concentrations accurately were neither por-
table nor cheap enough to use in the mass screenings mandated by HHS, especially given most state health departments' budgets. From 1985 to 1991 the most widely used tests to detect lead poisoning were the free erythrocyte protoporphyrin (EP) and, later, zinc protoporphyrin (ZPP) assays, two indirect functional tests for predicting the physiological effects of lead poisoning. Lead interferes with a t least one of the heme synthesis steps leading to incorporation of iron into the hemoglobin functional group, thereby leaving free protoporphyrin to chelate cytosolic zinc. Free protoporphyrins fluoresce deep red, whereas heme with bound iron is not fluorescent. In EP, protoporphyrin M is extracted from blood with acetic acid and ethyl acetate, then backextracted into HC1 before determining its concentration with a conventional fluorometer. When the hematofluorometer, a portable front-face fluorometer, was developed, it was used for ZPP, which measures the ratio of free or zinc protoporphyrin fluorescence to the light absorbance of functional heme. From this ratio the hematofluorometer estimates the concentration of protoporphyrin by electronically converting the results to predicted con-
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centrations. EP and ZPP give indirect indications of long-term lead exposure, but both methods-although suitable for predicting very high lead concentrations-are insensitive to values below 20 or 25 pg/dL, because little change occurs in heme synthesis in response to lower concentrations of blood lead. HHS still recommends that state health departments facing budget constraints continue to use EP/ZPP tests if they cannot yet afford to replace them with direct lead methods. Even though EP/ZPP does not predict lead poisoning a t the levels of concern in the new guidelines, the CDC and other health agencies generally prefer t h a t states do some screening rather t h a n none, said Daniel Paschal, a chemist in the Environmental Health Laboratory Sciences Division of the CDC National Center for Environmental Health. The main budgetary concern for most state health departments is the price hike for switching to direct blood lead measurement. The lower blood lead limit also means a great increase in the number of children expected to meet the new “action levels” that require repeat testing and eventual treatment. ZPP tests, which currently cost from $1to $5 per test, were developed as a mass screening tool, whereas the more sensitive, direct-lead assay methods such as anodic stripping voltammetry (ASV) and atomic absorption spectrometry ( U S )cost anywhere from $10 to $70 per test and are generally difficult to transport, set up, and calibrate in nonlaboratory settings such as elementary schools. Graphite furnace atomic absorption spectrometry (GFAAS) equipment requires argon cylinders and an autosampler for any reasonably large number of samples (100 per day). However, these methods are now being adapted to meet growing public health demands. Part of the CDC plan in 1991 was to seek out companies to develop lead-testing technology for assays that were accurate enough to meet the guideline specifications of < 10 pg1dL but were cheap enough to use in large - scale state public health screening programs. ESA of Bedford, MA, is one of the companies the CDC approached last year. In order to meet the new demands on instrumentation, the company is developing a more sensitive version of its ASV method, which has been in use for 20 years. In ASV, the lead in a sample is first made available for electrolysis by dilution with a decomplexing re266 A
agent, then reduced a t a controlled potential for a fiied length of time, which causes it to deposit a t the cathode surface. The working electrode is usually a mercury film on a wax-impregnated graphite electrode. Once the lead is deposited, it is anodically dissolved or “stripped” by oxidation during a n anodic scan. The amount of lead in the sample is calculated by integrating the current. ASV has the drawback that the reduction potential of Cu is near that of Pb, but the two can be resolved by running the scans under analytical conditions that favor their separation. ASV is a likely candidate for mass screenings, said Paschal, because portable versions are available and can be taken to schools, community centers, and other mass screening sites for operation. He said that ESA has completely modified the electronics of one of its current models and is close to achieving the 10ygldL detection level in a portable ASV lead analyzer. Paschal said that another electrochemical method also looks promising. Potentiometric stripping analysis (PSA) works almost the same way as ASV but instead of removing the amalgam by shifting the potential toward the positive, PSA holds the potential constant and uses chemical oxidation with mercuric ion to strip the deposited lead from the amalgam - coated electrode. This technique is in the prototype stage a t Radiometer, based in Copenhagen. Paschal estimated the real cost per test by PSA would be $2 or $3, which is comparable to the cost per test by ZPP. AAS is less likely to find a niche as a mass screening tool for blood lead, said Paschal, because the equipment is cumbersome and not very portable, is harder to set up and calibrate on relocation than are ASV and ZPP test methods, and requires an argon or acetylene source. Many central laboratories are reluctant to acquire AAS technology because the initial equipment costs are “a multiple” of those for the other methods, Paschal said. However, the lower operational costs make up for the initial investment over time because there is very little reagent use, and AAS is both sensitive and accurate for low-level blood lead determination. Direct blood lead measurement may be a new requirement of the CDC guidelines, but Patrick J. Parsons of the New York State Department of Health Wadsworth Center for Laboratories and Research noted that it is not really new. The earliest
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approaches were manual spectrophotometric and colorimetric methods such as extraction and complexation of lead with diphenylthiocarbazone, which required large sample volumes of several milliliters. AAS methods for blood lead determination were developed in the 1960s and required chelation and extraction of lead into methylisobutylketone (MIBK) for aspiration into an air-acetylene flame. Precision was generally poor, however, with standard deviations o f f 9 or 10 a t 40 pg1dL. Delves cup AAS (DCAAS), with its revolutionary microsampling technology and high accuracy, replaced MIBK-extraction AAS as a practical screening method for lead testing in the early 19708, and Parsons’ lab has performed DCAAS blood lead assays for years. With DCAAS, a 10-pL sample of whole blood is dried and oxidized with hydrogen peroxide in a small nickel crucible, then placed in a loop by the operator and manually inserted into the air-acetylene flame of an AA spectrometer for vaporization. The vapor is collected in a nickel or ceramic absorption tube, where a light beam passes through it and a detector identifies the smoke and analyte peaks. Although DCAAS can determine blood lead concentrations e 10 pgl dL, the method has several disadvantages for large-scale use. It requires determinations in triplicate for good precision, and aqueous standards cannot be used because lead vaporizes differently from water than it does from blood in the flame technique. Therefore blood with known lead content is used as a standard; NIST provides blood pooled from lead-dosed cows as a Standard Reference Material. Because sample insertion is done manually, Parsons said, operation requires the full-time attention of a laboratory technician for the length of the run. The method never took off commercially because it could not be automated. When EP/ZPP was introduced as a cheap, portable, and contaminationfree functional method for predicting lead poisoning, most commercial sources for DCAAS stopped producing the nickel and ceramic accessories necessary for operation. Parsons said his laboratory now has to purchase cut sheet nickel forms from one company and send them to another company to be stamped into the proper shapes. GFAAS, using readily available graphite tube platforms, is more practical, can be automated, and can achieve better precision at low concentrations because samples
are injected into the furnace rather than manually inserted into a flame. By 1985, most federal guidelines had abandoned direct blood lead testing for screening purposes in favor of EPIZPP. But last year, direct blood lead testing was restored when the CDC again dropped the lowest acceptable blood concentration. Although the Delves cup method cannot match the precision needed for efficient mass screenings, comparative studies (conducted by Parsons and Walter Slavin, former chief atomic spectroscopist with Perkin Elmer and currently a consultant with Bonaire Technologies, Ridgefield, CT) indicate that a variation on GFAAS has better precision than either DCAAS or conventional GFAAS. As Parsons put it, "It's ironic that we're now back to using direct blood lead determination methods like the ones we started with 20 years ago." Parsons' laboratory has automated GFAAS to handle larger workloads by using an autosampler, with a n adaptation of conventional GFAAS equipment that reduces the analysis t i m e per sample. Conventional GFAAS hah been p d m e h on an AA spectrometer with transverse nally heated graphite furnace. Parsons said that his laboratory has recently developed a rapid &d more sensitive method using a spectrometer with longitudinal Zeeman bdckground correction and a transversely heated graphite tubelplatform that more closely realizes stabilized temperature platform fnmace (STPF) atomization conditions. The transverse heating produces uniform temperature along the length of the tube for a nearly isothermal system, thereby allowing lower atomization temperatures, cutting down on the time needed per sample, and improving precision. With these modifications, said Parsons, a single blood lead determination takes 90 s, including cooldown and an autosampler wash cycle, instead of the 2-3 min per sample needed for conventional GFAAS. STPF conditions in the graphite furnace eliminate many of the chemical and matrix interferences that affect DCAAS a n d c o n v e n t i o n a l GFAAS, Parsons said, so matrixmatched standards are unnecessary. Sample preparation is "dilute and shoot": Samples of EDTA-anticoagulated fmgerstick or venous blood are diluted 10-fold in a chemical modifier (''matrix modifier") solution of ammonium dihydrogen phosphate, Triton X-100, and nitric acid; mixed
by pipetting up and down directly in the autosampler cups to lyse the cells; and injected into the furnace in 12-pL aliquots. Samples are dried at moderate heat (175 and 260 "C), pyrolyzed at 900 "C, and atomized at 1900 "C. The system can run 90-100 samples per day with duplicate injections, and the computer-controlled system automatically runs bilevel quality-control checks every 11 samples. Within-run precision (CV) at blood lead concentrations of 10 )rg/dL is typically < 5% and falls to < 1%for samples with lead concentrations above 20 pg/dL. Parsons said the operational capacity of the method is limited only by the lifetime of the graphite tube, which lasts for a day's worth of samples, standards, blanks, and calibrators. However, overall success depends on obtaining clean fmgerstick samples fmm children, and on comprehensive quality control programs that include the use of external proficiency-testing programs. Fingerstick sampling is the most convenient sample collection method for child screenings. There are some consiherations i n collecting lingerstick blood samples i n general;
squeezing or 'milking" the finger, for instance, leads to dilution errors because it also produces interstitial fluids that mix with the blood. For blood lead determination, fmgerstick sampling also presents the problem of contamination by lead on the skin. Careful scrubbing gets rid of most of it, b u t to prevent contamination more rigorously, the New York State Health Department uses a silicone spray to seal off any residual contamination on the child's fmger before lancing. The silicone spray also helps the blood bead up for easier collection. Venous blood is usually taken for diagnostic (confirmatory) tests because it is less likely to be contaminated during collection, and results are more representative of the patient's general blood lead concentration than are results from fmgerstick samples. Sample containers and analytical equipment, of course, have always had to be essentially free of lead contaminants, but the new blood lead concentration requirements may mean that fingerstick samples and sample containers have to meet more exacting standards tobe deemed uncontaminated. Deborah Noble
&her ...the first labomtory chemical rnan&cturerhAmericambringyo1 the reassuranceof IS09002 Intemtional Quallycertification fbr testingand mandacturing.
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