Blood Lead Measurement Takes the Direct Approach
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or most people, the words "lead poisoning" conjure up Time mag azine cover photographs of in ner-city toddlers sucking on paint chips, surrounded by neglect and de cay. But in the past five years, nu merous studies have found subtle but widespread physiological, neurologi cal, and intelligence-deficit damage at blood lead concentrations as low as 10 μg/dL—a level previously con sidered n o r m a l — i n asymptomatic children from all areas of the United States and all economic groups. About 74% of A m e r i c a n 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. It is esti mated t h a t among young children, 10-20% of total lead exposure comes from drinking water. The Environ mental Protection Agency (EPA) esti mates that from 1976 to 1980, 10.7% of U.S. children under six years old had blood lead concentrations > 25 μg/dL, and 91% had concentrations > 10 μg/dL. By 1990, only 1% had concentrations > 25 μg/dL, but 15% still had concentrations > 10 μg/dL. Photomicrograph of lead acetate courtesy Michael W. Davidson, Florida State University.
On the basis of these and other studies, the Centers for Disease Con trol and Prevention (CDC) lowered its acceptable limit for p e d i a t r i c blood lead concentrations in 1991 from 25 μg/dL, which had been the cutoff since 1985, to 10 μg/dL, and recommended t h a t all children under age six be screened for blood lead at the new lower levels. The CDC also recommended t h a t a "direct" blood lead assay be used rather than calcu lating blood lead concentration from measurements of lead's physiological effects on red blood cells (an "indi
FOCUS rect" assay). Only a direct measure ment of lead in blood is accurate for lead concentrations below 10 μg/dL. These recommendations were incor porated into the first stages of a De partment of Health and Human Ser v i c e s ( H H S ) 2 0 - y e a r p l a n for eliminating childhood lead poisoning. The new standard posed a techni cal challenge to manufacturers and state health departments alike. The available direct blood lead measure ment techniques that were sensitive enough to pick up the lower concen trations 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 protopor phyrin (EP) and, later, zinc protopor phyrin (ZPP) assays, two indirect functional tests for predicting the physiological effects of lead poison ing. Lead interferes with at least one of the heme synthesis steps leading to incorporation of iron into the he moglobin functional group, thereby leaving free protoporphyrin to che late cytosolic zinc. Free protoporphy r i n s fluoresce deep red, w h e r e a s heme with bound iron is not fluores cent. In EP, protoporphyrin IX is ex tracted from blood with acetic acid and ethyl acetate, then backextracted into HC1 before determin ing its concentration with a conven tional 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 hematofluorome ter estimates the concentration of protoporphyrin by electronically con verting the results to predicted con-
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FOCUS centrations. EP and ZPP give indi rect indications of long-term lead exposure, b u t both m e t h o d s — a l though suitable for predicting very high lead concentrations—are insen sitive to values below 20 or 25 μg/dL, because little change occurs in heme synthesis in response to lower con centrations of blood lead. HHS still recommends t h a t state health d e p a r t m e n t s facing budget constraints continue to use EP/ZPP tests if they cannot yet afford to re place them with direct lead methods. Even though EP/ZPP does not pre dict lead poisoning at the levels of concern in the new guidelines, the CDC and other health agencies gen erally prefer t h a t s t a t e s do some screening r a t h e r t h a n none, said Daniel Paschal, a chemist in the En vironmental Health Laboratory Sci ences 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 lev els" t h a t require repeat testing and eventual treatment. ZPP tests, which currently cost from $1 to $5 per test, were developed as a mass screening tool, whereas the more sensitive, di rect-lead assay methods such as an odic stripping voltammetry (ASV) and atomic absorption spectrometry (AAS) cost anywhere from $10 to $70 per test and are generally difficult to transport, set up, and calibrate in nonlaboratory settings such as ele mentary 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 meth ods are now being adapted to meet growing public health demands. Part of the CDC plan in 1991 was to seek out companies to develop l e a d - t e s t i n g technology for assays that were accurate enough to meet the guideline specifications of < 10 μg/dL but were cheap enough to use in large-scale s t a t e public h e a l t h screening programs. ESA of Bedford, MA, is one of the companies the CDC approached last year. In order to meet the new demands on instru mentation, the company is develop ing 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 re
agent, then reduced at a controlled potential for a fixed length of time, which causes it to deposit at the cathode surface. The working elec trode is usually a mercury film on a wax-impregnated graphite electrode. Once the lead is deposited, it is anodically dissolved or "stripped" by oxi dation during an anodic scan. The amount of lead in the sample is cal culated by integrating the current. ASV has the drawback that the re duction potential of Cu is near that of Pb, but the two can be resolved by running the scans under analytical conditions t h a t favor their separa tion. 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 electron ics of one of its current models and is close to achieving the 1 0 ^ g / d L de tection level in a portable ASV lead analyzer. Paschal said that another electro chemical method also looks promis ing. Potentiometric stripping analy sis (PSA) works almost the same way as ASV but instead of removing the amalgam by shifting the potential to ward the positive, PSA holds the po tential constant and uses chemical oxidation with mercuric ion to strip the deposited lead from the amal gam-coated electrode. This technique is in the prototype stage at Radiome ter, based in Copenhagen. Paschal estimated the real cost per test by PSA would be $2 or $3, which is com parable 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 porta ble, 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 invest ment 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 m e a s u r e m e n t may be a new requirement of the CDC guidelines, but Patrick J. Par sons of the New York State Depart ment 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 de veloped in the 1960s and required chelation and extraction of lead into methylisobutylketone (MIBK) for as piration into an air-acetylene flame. Precision was generally poor, how ever, with standard deviations of ± 9 or 10 at 40 μg/dL. Delves cup AAS (DCAAS), with its revolutionary microsampling tech nology and high accuracy, replaced MIBK-extraction AAS as a practical screening method for lead testing in the early 1970s, and Parsons' lab has performed DCAAS blood lead assays for y e a r s . With DCAAS, a 1 0 ^ L 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 vaporiza tion. 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 < 10 μg/ dL, the method has several disad vantages for large-scale use. It re quires determinations in triplicate for good precision, and aqueous stan dards cannot be used because lead vaporizes differently from water than it does from blood in the flame tech nique. Therefore blood with known lead content is used as a standard; NIST provides blood pooled from lead-dosed cows as a Standard Ref erence Material. Because sample in sertion 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 produc ing the nickel and ceramic accesso ries necessary for operation. Parsons said his laboratory now has to pur chase cut sheet nickel forms from one company and send them to another company to be s t a m p e d into t h e 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 EP/ZPP. But last year, direct blood lead testing was restored when the CDC again dropped the lowest acceptable blood c o n c e n t r a t i o n . Although the Delves cup method cannot match the precision needed for efficient mass screenings, comparative studies (conducted by Parsons a n d W a l t e r S l a v i n , former chief atomic spectroscopist with P e r k i n Elmer and currently a consultant with Bonaire Technologies, Ridgefield, CT) indicate that a variation on GFAAS has better precision t h a n either DCAAS or conventional GFAAS. As Parsons put it, "It's ironic t h a t 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 an adaptation of conventional GFAAS equipment that reduces the analysis time per sample. Conventional GFAAS Yiaà \>een performed on an AA spectrometer w i t h t r a n s v e r s e Zeeman correction and a longitudinally heated graphite furnace. Parsons said t h a t his laboratory has recently developed a rapid and more sensitive method using a spectrometer with longitudinal Zeeman background correction and a transversely heated graphite tube/platform t h a t more closely realizes stabilized temperature platform furnace (STPF) atomization conditions. The transverse heating produces uniform temperature along the length of the tube for a nearly isothermal system, thereby allowing lower atomization temperat u r e s , c u t t i n g down on t h e t i m e needed per sample, and improving precision. With these modifications, said Parsons, a single blood lead det e r m i n a t i o n t a k e s 90 s, including cooldown and an autosampler wash cycle, instead of the 2 - 3 min per s a m p l e n e e d e d for c o n v e n t i o n a l GFAAS. STPF conditions in the graphite furnace eliminate many of the chemical and matrix interferences that affect D C A A S a n d c o n v e n t i o n a l GFAAS, P a r s o n s said, so m a t r i x matched standards are unnecessary. Sample preparation is "dilute and shoot": Samples of EDTA-anticoagulated fingerstick 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 t h e a u t o s a m p l e r cups to lyse the cells; and injected into the furnace in 12 -μϋ, 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 injec tions, and the computer-controlled system automatically r u n s bilevel quality-control checks every 11 sam ples. Within-run precision (CV) at blood lead concentrations of 10 μg/dL is typically < 5% and falls to < 1% for samples with lead concentrations above 20 μg/dL. Parsons said the operational ca pacity of the method is limited only by the lifetime of the graphite tube, which lasts for a day's worth of sam ples, standards, blanks, and calibra tors. However, overall success de pends on obtaining clean fingerstick samples from children, and on com prehensive quality control programs that include the use of external pro ficiency-testing programs. Fingerstick sampling is the most convenient sample collection method for child screenings. There are some considerations in coïiect'ing fingerstick blood s a m p l e s in g e n e r a l ;
squeezing or "milking" the finger, for instance, leads to dilution errors because it also produces i n t e r s t i t i a l fluids that mix with the blood. For blood lead determination, fingerstick sampling also presents the problem of contamination by lead on the skin. Careful scrubbing gets rid of most of it, b u t to p r e v e n t c o n t a m i n a t i o n more rigorously, the New York State Health Department uses a silicone spray to seal off any residual contamination on the child's finger 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 t h a n are results from fingerstick 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 t h a t fingerstick samples and sample containers have to meet more exacting standards to b e deemed uncontaminated. Deborah Noble
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