Anodic stripping voltammetry compared with graphite furnace atomic absorption spectrophotometry for blood lead analysis.

According to a recent CDC report, blood lead concentrations in 1 000 000 US children are at values associated with irreversible damage to health (1 ). The effects of chronic lead poisoning on the developing nervous systems have been well documented (2 ), with children in inner city neighborhoods, where older housing stocks have deteriorating lead paint, most vulnerable (3 ). Accurate screening of children for lead exposure is, therefore, of paramount importance. The current biomarker for assessment of lead exposure is venous blood lead, commonly measured by anodic stripping voltammetry (ASV) or graphite furnace atomic absorption spectrometry (GFAA). Although both of these techniques have been used in our laboratory for 15 years, with ASV being our instrument of choice for clinical blood lead analysis, there is surprisingly little published information on ASV as a clinical tool for blood lead analysis. Here we present comparative data on ASV and GFAA analyses of blood lead in our clinic, with a novel reagent for calibration of ASV. For ASV, we used the ESA 3010B Trace Metals Analyzer (Environmental Science Associates) with a mercury-coated graphite electrode, a Ag/ AgCl reference electrode, and a platinum counter electrode. For GFAA (4 ), we used a Zeeman/5100 PC atomic absorption spectrophotometer with HGA-600 graphite furnace and AS-60 autosampler (Perkin-Elmer). For analysis by ASV, instead of the manufacturer’s reagent we used a novel reagent developed at our laboratory that is based entirely on chloride salts and HCl. HCl is suitable for electroplating of metals. Importantly, hemoglobin, the predominant protein found in erythrocytes, is soluble in hydrochloric acid but not in nitric or perchloric acids. In this reagent, Pb ions are found within the pH range 1.3–1.4, above which there is slow conversion to PbOCl, whereas at pH values less than this range, H2 is driven off and recorded as a Pb signal, leading to false positives. We included nickel in the reagent to complex excess EDTA in the anticoagulant, as it has a higher affinity for EDTA than Pb ions have. Additionally, EDTA was preferred to heparin because, in our experience, heparinized samples stored in the refrigerator tend to form gels and give inconsistent analysis (5 ). This reagent is prepared by dissolving 52.6 g of KCl in 1500 mL of deionized water. To remove any trace quantities of lead, this solution was passed through a cationexchange column. The following chemicals were added in the filtrate: 76 mg of HgCl2 (Spex Industries), 320 mg of NiCl2 (Johnson Matthey), 0.2 mL of 2-octanol (Sigma), and 0.4 mL of Triton X-100 (Sigma). The solution was thoroughly mixed and diluted to a total volume of 2 L with deionized water. The pH was adjusted to 1.3–1.4 with 16.7 mL of 6 mol/L HCl [G. Frederick Smith (GFS Chemicals, Powell, OH)]. The manufacturers’ tubes were used for sample analysis. Venous blood samples were collected from children who were referred to the lead clinic at the Kennedy Krieger Institute. Collections were in accordance with the Institute’s protocol on patients and were performed with stainless steel butterflies, polypropylene tubing, and 3-mL Vacutainers containing potassium EDTA as anticoagulant. Acceptable tubes were at least three-fourths filled to avoid interference because of excess EDTA. We combined 100 mL of well-mixed blood with 2.900 mL of reagent and analyzed samples after a few minutes. Several samples can be prepared in advance. Analysis was carried out as recommended by the manufacturer. Calibration of the ASV was carried out with calibrators made from bovine blood, which was filtered and homogenized by sonification. PbNO3 was added to the approximate concentration required and thoroughly mixed. The values of these calibrators were then established by comparisons with human blood that had been assayed by thermal ionization mass spectrometry as described previously (4 ). The calibrators had assigned values of 0.284 mmol/L (59 mg/L), 1.736 mmol/L (360 mg/L), and 3.376 mmol/L (700 mg/L). The quality-control material had values of 0.385 mmol/L (80 mg/L) and 1.736 mmol/L (360 mg/L). These were analyzed after calibration and at least after every 10 samples. Our requirement for process control was a measured value within 0.097 mmol/L (620 mg/L) of the certified value for each high and low qualitycontrol sample for both instruments. We first compared the performance of ASV and GFAA in the Wisconsin State Laboratory of Hygiene Proficiency Testing Program (CDC). From the fitted regression lines (summarized in Table 1), both linear models fit the data and there was no systematic error associated with a plot of the residuals (data not shown). There was no bias associated with either method, although the proportional error, represented by the slope, was slightly greater for GFAA than for ASV. In both cases, the model accounted for .96% of the variance (regression coefficient, R). As expected, these two methods performed well in the measurement of blood lead from single-blind proficiency programs over a representative range of blood lead concentrations. Results of a previous study (4 ) comparing an earlier model, the 3010A, with GFAA were as follows: ASV, y 5 0.984x 1 0.476 (R 5 0.982); GFAA, y 5 0.977x 1 0.292 (R50.996). ASV and GFAA are compared in Fig. 1 for the analysis of human blood samples. The model shows that 98% of