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EDTA is essential to recover lead from dried blood spots on filter paper
Clinica Chimica Acta 350 (2004) 143 – 150 www.elsevier.com/locate/clinchim
EDTA is essential to recover lead from dried blood spots on filter paper Maria T. Di Martino, Andzelika Michniewicz, Maria Martucci, Giuseppe Parlato* Department of Experimental and Clinical Medicine bG.SalvatoreQ, Chair of Chemistry, Clinical Chemistry Unit and Regional Centre for Neonatal Screenings, University Magna Gr& cia of Catanzaro, Via T. Campanella, 115-I-88100 Catanzaro, Italy Received 12 February 2004; received in revised form 10 July 2004; accepted 15 July 2004
Abstract Background: Residual dried blood spots (DBSs) on filter paper from neonatal screening have been proposed as samples for population survey of lead contamination. We have investigated the EDTA effect on lead release in the eluting solution. Methods: Furnace atomic absorption spectrophotometry has been used for lead measurements. Standard, blank and sample solutions contained 2% m/v NH4H2PO4, 0.5% v/v Triton X-100 and 0.2% v/v HNO3 as matrix modifier solution (MMS) with or without EDTA. A calibration curve was established from aqueous standard solutions. Paper discs from DBS and blank, punched near the DBS, were eluted in MM solution and, where required, EDTA at different concentrations. Specimens were leftover DBSs with different storage times, matched samples from 20 adult patients consisting of liquid whole blood (LWB) containing 5 mmol/L EDTA, DBSs eluted in MM solution with 5 mmol/L EDTA or without EDTA. Results: Optimal lead recovery from DBS required 5 mM EDTA in the eluting solution. Mean lead levels of LWB and DBSs eluted with EDTA were similar and higher than DBSs without EDTA ( Pb0.001). Without EDTA, the median value of lead optical density was lower for 6-month-old DBSs than for blanks ( Pb0.001). Conclusions: Residual DBSs can be used for population survey, but 5 mmol/L EDTA in the extracting solution is required to fully recover lead. D 2004 Elsevier B.V. All rights reserved. Keywords: Dried blood; Filter paper; Lead; Neonatal screening; EDTA
1. Introduction Abbreviations: EDTA, ethylen-diamine tetra-acetic acid; NA2EDTA, ethylen-diamine tetra-acetic acid, disodium salt; K3EDTA, ethylen-diamine tetra-acetic acid tripotassium salt; GFAAS, graphite furnace atomic absorption spectrophotometry; DBS, dried blood spot; MMS, matrix modifier solution. * Corresponding author. Tel.: +39 961772090; fax: +39 961773672. E-mail address: [email protected] (G. Parlato). 0009-8981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2004.07.019
Measurements of lead levels in whole blood samples of large population could be used to detect environmental contamination. The graphite furnace atomic absorption spectrometry (GFAAS) is the most common technique to determine blood lead levels, due to high sensitivity and specificity combined to a
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2. Materials and methods
trophotometer (mod. GBC 903), equipped with deuterium lamp for background correction, equivalent to Zeeman correction [14]. The furnace was protected from the oxidation on the outside and cleaned on the inside with external and internal argon flow, which was stopped during the atomization step. A working calibration curve was prepared with aqueous standard solutions at lead concentration of 5, 10, 15, 20 Ag/L. Each standard, blank and sample solution contained 2% m/v NH4H2PO4, 0.5 v/v Triton X-100 and 0.2% v/ v HNO3 as matrix modifier solution (MMS), with or without Na2EDTA at concentrations, depending on experimental conditions. Paper discs 6.3 mm in diameter were punched from dried blood spot (DBS) on filter paper, grade 2992, from Schleicher and Schqell. Lead was extracted in 5 ml capped lead-free plastic tubes from the discs by over-night elution under gentle agitation with 150 AL of solution containing MMS and, where required, Na2EDTA salt at the indicated concentrations. A paper disc, punched near the DBS and eluted like the paper disc with blood sample, was used as blank. Twenty-microliter aliquots of 1/10 diluted blood in MMS or standard solution or eluted sample from DBS or blank discs were deposited into graphite furnace tubes. For deposition, a 1–20 AL variable Eppendorf Research pipette equipped with metal-free tips was used. Samples and blanks were measured in duplicate. When difference between duplicates was higher, then 10% further measurement was performed. The lead optical density was calculated by subtraction of the means of the duplicates. To measure lead concentration in DBS, prepared with venous blood of hospital patients, the blood volume absorbed onto paper disc (Vpd) was determined by the following procedure: 1 mL of blood with 44% hematocrits, corresponding to adult mean value was gently mixed with 50 AL of 125I labelled thyrotropin aqueous solution (2105 cpm); the gamma radioactivity measured in 50 AL of this blood (R50) and in 6.3 mm paper disc punched from DBS prepared by deposing blood on filter paper and dried at room temperature (R6.3); Vpd was calculated as follows: 50 AL (R6.3/R50).
2.1. Lead measurement
2.2. Blood specimens
The measurement of lead was carried out at 283.3 nm by using electrothermal atomic absorption spec-
Blood specimens on filter paper were: leftover DBSs with different storage times from the neonatal
good level of automation [1]. Normally the specimen for measurements consists of venous or capillary blood collected in tubes containing EDTA as anticoagulant. Whole blood absorbed onto filter paper and subsequently dried (DBS), remaining after newborn screening analytical tests for metabolic and endocrine diseases has been proposed as an easy source of biological specimen for such environmental investigation [2]. The validity of DBS specimen, recognized for population surveys of lead contamination, is largely discussed for children screening of lead exposure [3–5]. However, recently a filter paper method to measure blood lead levels in a large population of young children has been successfully evaluated [6]. DBSs obtained from blood specimens, used in the proficiency testing program to evaluate the suitability of filter paper for blood collecting [5] and the recently proposed screening method [6], differ from DBSs specimen for neonatal screening in that the latter are obtained from blood collected directly from the heel and deposited on filter paper in the absence of EDTA; in addition, they are stored at room temperature for several months before being eluted in aqueous solution to extract lead, which is maximally localized in red cells [7]. The storage at room temperature decreases the release of biological material and heavy metals from the filter paper [8]. According to studies that compare the filter blood lead technique to either graphite furnace or ICP-MS, low lead recovery from filter paper has been reported [9]. EDTA as a chelating agent is effective to mobilize lead, to eliminate this excess in case of exposure [10–13] and, as a solubilizer agent, promotes the solubility of soil-contaminating lead [13]. Here we show that adding EDTA to the DBS eluting solution is essential for the optimal recovery of lead during the extraction step from blood absorbed and dried on filter paper. In addition, we have found that the efficiency of the recovery increases with the storage time of DBS.
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screening tests; DBSs matched with venous blood of 20 hospital patients on the occasion of blood drawing for routine diagnostic blood test; residual 2 month old DBSs obtained with whole blood from two phenylketonuric children during the routine monitoring for dietary control. DBSs for neonatal screening were obtained by depositing two to three blood drops on filter paper from heel puncture at 3–5 days after birth. DBSs from informed hospital patients were prepared by depositing on filter paper through the needle two to three drops from residual blood in plastic tube, after blood collection in vacutainer tube (Becton Dickinson) containing 7.5% (m/v) K3EDTA; liquid blood remaining after the routine tests was used to control the lead recovery from paper discs. 2.3. Analytical reagents Triton X-100 (BDH), Na2EDTA, NH4H2PO4, HNO3, 65% w/v, (Farmitalia C. Erba) were analytical grade reagents. Standard solutions were prepared from a lead atomic absorption solution, 1.010 mg/ mL (Aldrich). Before use, plastic tubes were tested for lead release under the same conditions of sample treatments. Eppendorf Clear Tips, metal-free (SigmaAldrich) with a 2–20 AL variable Eppendorf Research pipette were used. Glass-ware, first treated
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with sulpho-chromic mixture, was extensively rinsed with deionized water and finally with double glassdistilled water, which was also used to prepare all solutions. To control accuracy of lead measurement in venous liquid blood from hospital patients, control blood Seronorm, Trace elements, Whole Blood, Nycomed, was tested. The same control blood before each series, after 10 samples and at the end, was used to control the precision of the analytical procedure at level of the instrumental measurement, when the optical density of eluted samples from DBS was measured. 2.4. Statistical analysis Statistical significance of differences between lead levels in matched blood sample groups was evaluated with All Pairwaise Multiple Comparison Procedure (Tukey test). To verify if the differences in the median values among EDTA-treated DBSs and -untreated were statistically significant, Friedman Repeated Measures One-Way Analysis of the Variance on Ranks was performed. The mean or median value were considered when the normality test with One-Way Analysis of the Variance passed or failed, respectively. For statistical treatment, Sigma Stat program, SPSS, 1997, was used.
Fig. 1. Effect of EDTA concentration on the lead optical density at 283.3 nm. Samples were prepared by elution using matrix modifier solution in the presence of EDTA at increasing concentrations. Samples were from 2-month-old dried blood spots remaining after ordinary diet control test for 2 PKU children. Full squares and open circles represent optical density of samples eluted from 6 mm paper discs punched from dried blood spot of both children.
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3. Results Fig. 1 shows the lead optical density at 283.3 nm of blood samples from two residual DBSs stored for 2 months eluted in the presence of Na2EDTA at increasing concentrations up to about 10 mmol/L. For both series, the optical density from a value of nearly zero in the absence of Na2EDTA increases to maximum value (0.046 and 0.026, respectively) at 5 mmol/L Na2EDTA and remains almost constant between 0.015 and 0.030 in the range 7–10 mmol/L, where smaller differences of values for two groups are measured. Results shown in Fig. 2 confirm that 5 mmol/L Na2EDTA was optimal for lead extraction. Lead levels in three matched groups of blood samples from 20 hospital patients were measured: group 1 consisted of blood samples collected in vacutainer tube with K3EDTA as anticoagulant and diluted 1:10 in MM solution plus 4.6 mmol/L Na2EDTA, in order to reach for EDTA a 5 mmol/L concentration; groups 2 and 3 consisted of DBSs prepared by dropping blood, residual in the plastic tube after the venous drawn, on filter paper and eluted in MM solution with addition or in absence of 5 mmol/L Na2EDTA, respectively. The lead extraction from DBSs and measurements were performed one week after sample collection. The storage was for DBSs at room temperature, for whole blood at 4 8C. Groups 1 and 2 showed similar blood lead mean values, which were
Table 1 Mean lead concentration in liquid whole blood plus 5 mmol/L EDTA and in samples eluted from dried blood spots with or without 5 mmol/L EDTA and statistical significance of differences Samples (no. 20)
Mean lead concentration (Ag/mL)
Statistical significance of differences at Pb0.001
1 DBS+EDTA 2 DBS EDTA 3 WB+EDTA
6.58 3.62 6.36
P 12b0.001 P 13=0.975 P 23b0.001
Specimens for sample preparation were whole blood (WB) collected as liquid in vacutainer tube containing 7.5% (m/v) K3EDTA and as dried spot on paper filter from the same patient; for whole dried blood on paper filter (DBS), blood remaining in plastic tube was used to prepare blood spot without EDTA. When EDTA was present in the eluting solution, the concentration was adjusted with the required addition of Na2EDTA.
higher than group 3 mean value and with statistically significant difference, as shown in Table 1. The recovery of blood lead from filter paper in absence of EDTA was dependent on storage time of paper DBSs. The lead optical density at 283.3 nm of 20 blood samples (not shown), prepared by eluting in MMS without EDTA 6.3 mm discs from 2 week-old and 3-month-old DBSs, respectively, were not different from their respective blanks, consisting of paper disc punched as close as possible to blood spot and treated like the blood disc. Blood samples from more recent DBSs showed values in same case a little higher or similar to blanks or lower than the blanks,
Fig. 2. Comparison of lead levels in three matched whole blood samples from 20 patients: liquid whole blood plus 5 mmol/L EDTA (full squares), whole blood collected and dried on filter paper and eluted from 6 mm paper discs in the presence of 5 mmol/L EDTA (open circles) or in the absence of EDTA (stars).
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Fig. 3. Effect of time storage on lead recovery by extraction from 6-month-old dried blood spots on filter paper without addition of EDTA to eluting solution. Open circles represent the lead optical density of samples eluted from blanks, i.e. 6.3-mm paper discs punched as close as possible as to dried blood spot, full squares of samples eluted from 6.3 mm paper discs punched from dried blood spots.
for older samples the optical density was similar or lower than blanks. In Figs. 3 and 4, we show the optical density of samples eluted from 6-month-old DBSs and their respective blanks with or without the addition of EDTA to the extracting solution. The median values for blanks were about the same in both conditions, for DBSs higher values than blanks were measured, when elution was carried out in presence of EDTA and the differences between the median values were statistically significant ( Pb0.001), as indicated in Table 2. When EDTA was absent in the eluting
solution, the blanks showed optical density higher than DBS samples ( Pb0.001).
4. Discussion The use of DBS on filter paper for lead blood analysis with GFAAS has been the object of many discussions [2,5,15–17]. The main criticisms concern poor recovery, high scattering of results, and uncertainty about the volume of absorbed blood.
Fig. 4. Effect of 5 mmol/L EDTA in the eluting solution on lead recovery from 6-month-old dried blood spots on filter paper. Open circles represents the lead optical density of samples eluted from blanks, i.e. 6.3 mm paper discs punched as close as possible to dried blood spot, full squares of samples eluted from 6.3 mm paper discs punched from dried blood spots.
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Table 2 Optical density median value at lead 283.3-nm wavelength of samples prepared by elution of 6-month-old dried blood spots and respective blanks in the presence or in the absence of 5 mmol/L EDTA and statistical significance of differences Samples (no. 40)
Optical density at 283.3 nm (median value)
Statistical significance of differences at Pb0.001
1 2 3 4
0.064 0.044 0.033 0.050
P 12b0.001 P 34b0.001 P 24=0.136
DBSs+EDTA BLANKS+EDTA DBSs EDTA BLANKS EDTA
From dried blood spots (DBSs), 6.3 mm paper discs were punched and eluted with matrix modifier solution (see Material and methods) with or without 5 mmol/L EDTA. Blanks were 6.3 mm paper discs punched as close as possible to whole spots and eluted under the same conditions as for dried whole blood discs.
To evaluate the performance of methods using DBS on filter paper to determine lead blood, a proficiency testing program has been carried out [5]. The results of this program do not eliminate the doubts about the validity of filter paper collected blood samples to screen lead levels in children. Two reasons support this conclusion: (1) unacceptable results according to the criteria of Clinical Laboratory Improvement Amendments of 1988 (CLIA ’88) were obtained from three of six laboratories participating to the program [5]; (2) the blood used for the proficiency testing program was collected in tubes containing EDTA as anticoagulant, then spotted onto filter paper and dried at room temperature, in contrast, blood as dried spots for routine screening is collected without EDTA and it retains intact properties of clotting. Five Laboratories participating in the proficiency testing program have extracted lead from DBS, the sixth laboratory used a Delves cup flame atomic absorption with a preashing step. This last laboratory along with other two extracting lead from DBS with a HNO3-Triton X-100 solution disagreed with CLIA ’88 criteria because of substantial negative bias in the slope of their regressions. The bias was probably due to factors not depending on the extraction procedure. Among the Laboratories respecting the CLIA ’88 criteria, two extracted blood spots with (NH4)H2PO4-Triton X 100 solution, the remaining one added also HNO3 to the extracting solution. Although we extracted lead from DBS by reproducing identical conditions of this last Laboratory, at lead wavelength absorption of 283.3 nm, the optical
density of the sample solution obtained by eluting lead from 3- (not shown) and 6-month-old (Fig. 3) DBSs was lower than the blank solution and near the background. When we extracted lead in the presence of 5 mmol/L Na2EDTA (Fig. 4), optical density of DBS was significantly higher ( Pb0.001) than the blanks (Table 2). This result allows us to measure lead concentration in DBS sample to carry out epidemiological investigations. In the absence of EDTA in the eluting solution, the difference between blanks and blood samples decreased significantly as the storage time increased; the difference between blanks and sample has a low statistical significance ( P=0.038) after 2 weeks of storage and no significance ( P=0.522) after 3 months; after 6 months, the blanks show optical density significantly higher ( Pb0.001) than the samples. Stanton et al. [5] suggested, as a possible cause for negative bias, a negative effect of EDTA on the efficiency of extraction with HNO3 in the blood samples, in the absence of (NH4)H2PO4. According to our results, the negative bias was probably due to the inability of EDTA to bind lead fixed on DBS, at low pH. The addition of 5 mmol/L EDTA in the extracting solution containing 0.2% v/v HNO3 and 2% m/v (NH4)H2PO4 increases the lead recovery (Fig. 1). The presence of EDTA at 5 mmol/L (Fig. 2, Table 1) enabled total recovery: the lead level was substantially the same for samples eluted from DBSs in presence of EDTA and liquid whole blood samples; differences between the mean values were not statistically significant ( P=0.975). At lead level higher than 10 Ag/dL (Fig. 2), values were comparable for whole blood and untreated EDTA DBSs; these results were in agreement with conclusion of Srivuthana et al. [18] and Shen et al. [6], who found that the filter method for blood analysis is convenient to examine children with elevated blood lead levels. Our results offer suggestions useful to the lead blood screening proposed by Shen et al. [6]. These authors obtained good correlation between lead levels in matched samples of venous liquid blood collected in vacutainer tube containing EDTA and dried blood absorbed on filter paper from finger. Furthermore, they tested lead stability on filter paper after 4 weeks, and they concluded that blood collection on filter paper represents an optimal solution to send blood sample to specialized labo-
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ratory for lead analysis from remote regions of China. In this regard, we feel that the conditions for lead screening should be different from those described by Shen et al. In fact, blood on filter paper was dried for 10 min, then transported in hermetically closed plastic bag to the laboratory for lead analysis, the extracting step was started in the presence of 5% HNO3. As the arrival time from remote region of China could be longer and consequently also the contact of blood with filter paper, the Authors carried out test to verify lead stability, i.e. lead recovery, after 1, 2, 3 and 4 weeks from blood spotting on filter paper. About 50% of older samples show lower lead levels compared to lead levels in 1-week-old blood spot. The recovery was more efficient for sample with high lead level, according to our results indicating similar value for whole blood, EDTA-treated and -untreated DBS (Fig. 2). In the test performed by Shen et al. on 30 venous blood samples spotted on filter paper, 24 samples showed lead concentration N15 Ag/dl. According to our results, EDTA effect is stronger when lead concentration is less than 10 Ag/dl (Fig. 2), that is considered cut-off value for normal children [19]. EDTA at 5 mmol/L concentration gave optimal results in lead extraction from old DBSs as indicated in Fig. 1, showing the dose response of EDTA on lead extraction. The rapid decrease of the absorbance at 5 mmol/L could be due to the accumulation of carbon residues in the graphite tube at the level of atomization. This condition was suggested by Nakajima et al. [20], who observed irreproducible absorbance due to accumulation of carbon residues, at high EDTA concentrations, when lead, eluted with increasing EDTA concentrations from a lead selective resin, was injected in the graphite furnace. We observed that, at 283.3 nm, the absorbance of a solution containing lead in MMS, at concentration similar to most concentrated samples described in Fig. 1, was almost constant, ranging from 0.062 to 0.055, with 11.2% decrease, at EDTA concentrations between 0 and 5 mmol/L. On the other hand, a 50% decrease in the absorbance was observed at higher EDTA concentrations between 5 and 10 mmol/L, thus suggesting that the decrease in the absorbance, at EDTA concentration higher than 5 mmol/L, does not depend on the extraction efficiency but on lead
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atomization. The almost overlapping absorbances for EDTA concentrations above 6 mmol/L, measured for both samples (Fig. 2), suggest that EDTA does not specifically affect lead mesurement. The role of EDTA in lead recovery from blood absorbed on filter paper is based on the well-known chelant properties of EDTA, which forms a very stable complex with lead, log K stability=18 [21], in aqueous solution at pH as low as 3. Because of these chelant properties, EDTA is used as therapeutic agent in case of lead intoxication [11–13], or as solubilizing agent to eliminate soil pollution [14] or as analytical reagent in complexometry [22]. In our study, EDTA contributed to solubilize lead blocked on filter paper through protein matrix, as better as the storage time increases. The results of our study confirm that residual DBSs, stored after laboratory tests for neonatal screening of endocrine and metabolic diseases, could be useful tool for epidemiological survey of lead pollution. However, the lead must be extracted from DBS in the presence of optimal amounts of EDTA. Further investigations are needed to verify whether DBSs can be used for diagnostic results of lead intoxication.
Acknowledgements This work was supported by grant of Regione Calabria. We thank Prof. Nicola Perrotti for reviewing the English language of this text.
References [1] Bannon DI, Murashchik C, Zapf CR, Farfel MR, Chisolm Jr JJ. Graphite furnace absorption spectroscopic measurement of blood lead in matrix-matched standards. Clin Chem 1994; 40:1730 – 4. [2] Morrow JC, Hannon WH, Henderson LO, Paschal D. Reliability of lead measurement of blood collected on filter paper and their potential application to epidemiological survey. In: Farriaux JP, Dhondt JL, editors. New horizons in neonatal screening. Amsterdam7 Elsevier; 1994. p. 289 – 92. [3] Yee HY, Holtrop TG. An improved capillary blood filter paper graphite furnace atomic absorption spectrometric method for lead screening. J Anal Toxicol 1997;21:1 – 7. [4] Holtrop TG, Yee HY, Simpson PM, Kauffmann RE. A community outreach lead screening program using capillary
150
[5]
[6]
[7]
[8]
[9] [10]
[11]
[12]
[13]
M.T. Di Martino et al. / Clinica Chimica Acta 350 (2004) 143–150 blood collected on filter paper. Arch Pediatr Adolesc Med 1998;152:455 – 8. Stanton NV, Maney JM, Jones R. Evaluation of filter paper blood lead methods: results of a pilot proficiency testing program. Clin Chem 1999;45:2229 – 35. Shen X-M, Zhang Y-W, Wu S-H, Yan C-H, Ying J-M, Li R-Q. Applicability of a filter paper method to measure blood lead levels in large populations of Chinese children. Clin Chim Acta 2003;328:99 – 104. Ewers U, Turfeld M, Jermann E. Lead. In: Herber RFM, Stoeppler M, editors. Trace element analysis in biological specimens. Amsterdam7 Elsevier; 1994. p. 371 – 84. Makowski GS, Davis ET, Hopfer SM. The effect of storage of Guthrie cards. Implication for deoxyribonucleic acid amplification. Ann Clin Lab Sci 1996;26:458 – 63. Moyer TP, Nixon DE, Ash OK. Filter paper-collected blood lead testing in children. Clin Chem 2000;46:1026 – 8. Markowitz ME, Bijur PE, Ruff H. Effects of calcium Disodium Versenate (CaNa2EDTA) chelation in moderate childhood lead. Pediatrics 1993;92:265 – 71. Guzman DD, Velez LI, Shepherd JG, Goto CS. Calcium EDTA and DMSA chelation of a neonate with congenital lead intoxication. J Toxicol Clin Toxicol 2000;38:548 – 9. Brownie CF, Aronson AL. Comparative effects of Ca ethylenediaminetetraacetic acid (EDTA), ZnEDTA and ZnCaEDTA in mobilizing lead. Toxicol Appl Pharmacol 1984;75:167 – 72. Kedziorek MAM, Dupuy A, Bourg ACM, Compere F. Leaching of Cd and Pb from a polluted soil during the percolation of EDTA: laboratory column experiments mod-
[14]
[15] [16] [17] [18]
[19]
[20]
[21]
[22]
elled with a non-equilibrium solubization step. Environ Sci Technol 1998;32:1609 – 14. Qiao H, Parsons PJ, Slavin W. Transferability of blood lead determination by furnace atomic absorption spectrophotometry and continuum background correction. Clin Chem 1995;41: 1451 – 4. Moyer TP, Nixon DN, Ash OK. Filter paper lead testing. Clin Chem 1999;45:2055 – 6. Vereby K. Filter paper-collected blood lead testing in children. Clin Chem 2000;46:1024 – 8. Stanton N, Maney J, Jones R. More on filter paper lead testing. Clin Chem 2000;46:1028 – 9. Srivuthana K, Yee HY, Bhambhani K, Elton RM, Simpson PM, Kauffmann RE. A new filter paper method to measure capillary blood lead level in children. Arch Pediatr Adolesc Med 1996;150:498 – 502. Centers for disease Control and Prevention. Preventing lead poisoning in young children. Atlanta, GA7 US Dept. of Health and Human Services, Public Health Service, CDC; 1991. Nakajima J, Hirano Y, Oguna K. Determination of lead in seawater by flow-injection on-line preconcentration electrothermal atomic absorption spectrometry after coprecipitation with iron (III) hydroxide. Anal Sci 2003;19:585 – 8. Ringbom A, Vanninen E. Theory of photoelectric complex formation titrations using metal indicators. Anal Chim Acta 1954;11:153 – 67. Skoog DA, West DM. Fundamentals of analytical chemistry. New York7 Holt, Rinehart and Winston; 1965.
EDTA is essential to recover lead from dried blood spots on filter paper Maria T. Di Martino, Andzelika Michniewicz, Maria Martucci, Giuseppe Parlato* Department of Experimental and Clinical Medicine bG.SalvatoreQ, Chair of Chemistry, Clinical Chemistry Unit and Regional Centre for Neonatal Screenings, University Magna Gr& cia of Catanzaro, Via T. Campanella, 115-I-88100 Catanzaro, Italy Received 12 February 2004; received in revised form 10 July 2004; accepted 15 July 2004
Abstract Background: Residual dried blood spots (DBSs) on filter paper from neonatal screening have been proposed as samples for population survey of lead contamination. We have investigated the EDTA effect on lead release in the eluting solution. Methods: Furnace atomic absorption spectrophotometry has been used for lead measurements. Standard, blank and sample solutions contained 2% m/v NH4H2PO4, 0.5% v/v Triton X-100 and 0.2% v/v HNO3 as matrix modifier solution (MMS) with or without EDTA. A calibration curve was established from aqueous standard solutions. Paper discs from DBS and blank, punched near the DBS, were eluted in MM solution and, where required, EDTA at different concentrations. Specimens were leftover DBSs with different storage times, matched samples from 20 adult patients consisting of liquid whole blood (LWB) containing 5 mmol/L EDTA, DBSs eluted in MM solution with 5 mmol/L EDTA or without EDTA. Results: Optimal lead recovery from DBS required 5 mM EDTA in the eluting solution. Mean lead levels of LWB and DBSs eluted with EDTA were similar and higher than DBSs without EDTA ( Pb0.001). Without EDTA, the median value of lead optical density was lower for 6-month-old DBSs than for blanks ( Pb0.001). Conclusions: Residual DBSs can be used for population survey, but 5 mmol/L EDTA in the extracting solution is required to fully recover lead. D 2004 Elsevier B.V. All rights reserved. Keywords: Dried blood; Filter paper; Lead; Neonatal screening; EDTA
1. Introduction Abbreviations: EDTA, ethylen-diamine tetra-acetic acid; NA2EDTA, ethylen-diamine tetra-acetic acid, disodium salt; K3EDTA, ethylen-diamine tetra-acetic acid tripotassium salt; GFAAS, graphite furnace atomic absorption spectrophotometry; DBS, dried blood spot; MMS, matrix modifier solution. * Corresponding author. Tel.: +39 961772090; fax: +39 961773672. E-mail address: [email protected] (G. Parlato). 0009-8981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2004.07.019
Measurements of lead levels in whole blood samples of large population could be used to detect environmental contamination. The graphite furnace atomic absorption spectrometry (GFAAS) is the most common technique to determine blood lead levels, due to high sensitivity and specificity combined to a
144
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2. Materials and methods
trophotometer (mod. GBC 903), equipped with deuterium lamp for background correction, equivalent to Zeeman correction [14]. The furnace was protected from the oxidation on the outside and cleaned on the inside with external and internal argon flow, which was stopped during the atomization step. A working calibration curve was prepared with aqueous standard solutions at lead concentration of 5, 10, 15, 20 Ag/L. Each standard, blank and sample solution contained 2% m/v NH4H2PO4, 0.5 v/v Triton X-100 and 0.2% v/ v HNO3 as matrix modifier solution (MMS), with or without Na2EDTA at concentrations, depending on experimental conditions. Paper discs 6.3 mm in diameter were punched from dried blood spot (DBS) on filter paper, grade 2992, from Schleicher and Schqell. Lead was extracted in 5 ml capped lead-free plastic tubes from the discs by over-night elution under gentle agitation with 150 AL of solution containing MMS and, where required, Na2EDTA salt at the indicated concentrations. A paper disc, punched near the DBS and eluted like the paper disc with blood sample, was used as blank. Twenty-microliter aliquots of 1/10 diluted blood in MMS or standard solution or eluted sample from DBS or blank discs were deposited into graphite furnace tubes. For deposition, a 1–20 AL variable Eppendorf Research pipette equipped with metal-free tips was used. Samples and blanks were measured in duplicate. When difference between duplicates was higher, then 10% further measurement was performed. The lead optical density was calculated by subtraction of the means of the duplicates. To measure lead concentration in DBS, prepared with venous blood of hospital patients, the blood volume absorbed onto paper disc (Vpd) was determined by the following procedure: 1 mL of blood with 44% hematocrits, corresponding to adult mean value was gently mixed with 50 AL of 125I labelled thyrotropin aqueous solution (2105 cpm); the gamma radioactivity measured in 50 AL of this blood (R50) and in 6.3 mm paper disc punched from DBS prepared by deposing blood on filter paper and dried at room temperature (R6.3); Vpd was calculated as follows: 50 AL (R6.3/R50).
2.1. Lead measurement
2.2. Blood specimens
The measurement of lead was carried out at 283.3 nm by using electrothermal atomic absorption spec-
Blood specimens on filter paper were: leftover DBSs with different storage times from the neonatal
good level of automation [1]. Normally the specimen for measurements consists of venous or capillary blood collected in tubes containing EDTA as anticoagulant. Whole blood absorbed onto filter paper and subsequently dried (DBS), remaining after newborn screening analytical tests for metabolic and endocrine diseases has been proposed as an easy source of biological specimen for such environmental investigation [2]. The validity of DBS specimen, recognized for population surveys of lead contamination, is largely discussed for children screening of lead exposure [3–5]. However, recently a filter paper method to measure blood lead levels in a large population of young children has been successfully evaluated [6]. DBSs obtained from blood specimens, used in the proficiency testing program to evaluate the suitability of filter paper for blood collecting [5] and the recently proposed screening method [6], differ from DBSs specimen for neonatal screening in that the latter are obtained from blood collected directly from the heel and deposited on filter paper in the absence of EDTA; in addition, they are stored at room temperature for several months before being eluted in aqueous solution to extract lead, which is maximally localized in red cells [7]. The storage at room temperature decreases the release of biological material and heavy metals from the filter paper [8]. According to studies that compare the filter blood lead technique to either graphite furnace or ICP-MS, low lead recovery from filter paper has been reported [9]. EDTA as a chelating agent is effective to mobilize lead, to eliminate this excess in case of exposure [10–13] and, as a solubilizer agent, promotes the solubility of soil-contaminating lead [13]. Here we show that adding EDTA to the DBS eluting solution is essential for the optimal recovery of lead during the extraction step from blood absorbed and dried on filter paper. In addition, we have found that the efficiency of the recovery increases with the storage time of DBS.
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screening tests; DBSs matched with venous blood of 20 hospital patients on the occasion of blood drawing for routine diagnostic blood test; residual 2 month old DBSs obtained with whole blood from two phenylketonuric children during the routine monitoring for dietary control. DBSs for neonatal screening were obtained by depositing two to three blood drops on filter paper from heel puncture at 3–5 days after birth. DBSs from informed hospital patients were prepared by depositing on filter paper through the needle two to three drops from residual blood in plastic tube, after blood collection in vacutainer tube (Becton Dickinson) containing 7.5% (m/v) K3EDTA; liquid blood remaining after the routine tests was used to control the lead recovery from paper discs. 2.3. Analytical reagents Triton X-100 (BDH), Na2EDTA, NH4H2PO4, HNO3, 65% w/v, (Farmitalia C. Erba) were analytical grade reagents. Standard solutions were prepared from a lead atomic absorption solution, 1.010 mg/ mL (Aldrich). Before use, plastic tubes were tested for lead release under the same conditions of sample treatments. Eppendorf Clear Tips, metal-free (SigmaAldrich) with a 2–20 AL variable Eppendorf Research pipette were used. Glass-ware, first treated
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with sulpho-chromic mixture, was extensively rinsed with deionized water and finally with double glassdistilled water, which was also used to prepare all solutions. To control accuracy of lead measurement in venous liquid blood from hospital patients, control blood Seronorm, Trace elements, Whole Blood, Nycomed, was tested. The same control blood before each series, after 10 samples and at the end, was used to control the precision of the analytical procedure at level of the instrumental measurement, when the optical density of eluted samples from DBS was measured. 2.4. Statistical analysis Statistical significance of differences between lead levels in matched blood sample groups was evaluated with All Pairwaise Multiple Comparison Procedure (Tukey test). To verify if the differences in the median values among EDTA-treated DBSs and -untreated were statistically significant, Friedman Repeated Measures One-Way Analysis of the Variance on Ranks was performed. The mean or median value were considered when the normality test with One-Way Analysis of the Variance passed or failed, respectively. For statistical treatment, Sigma Stat program, SPSS, 1997, was used.
Fig. 1. Effect of EDTA concentration on the lead optical density at 283.3 nm. Samples were prepared by elution using matrix modifier solution in the presence of EDTA at increasing concentrations. Samples were from 2-month-old dried blood spots remaining after ordinary diet control test for 2 PKU children. Full squares and open circles represent optical density of samples eluted from 6 mm paper discs punched from dried blood spot of both children.
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3. Results Fig. 1 shows the lead optical density at 283.3 nm of blood samples from two residual DBSs stored for 2 months eluted in the presence of Na2EDTA at increasing concentrations up to about 10 mmol/L. For both series, the optical density from a value of nearly zero in the absence of Na2EDTA increases to maximum value (0.046 and 0.026, respectively) at 5 mmol/L Na2EDTA and remains almost constant between 0.015 and 0.030 in the range 7–10 mmol/L, where smaller differences of values for two groups are measured. Results shown in Fig. 2 confirm that 5 mmol/L Na2EDTA was optimal for lead extraction. Lead levels in three matched groups of blood samples from 20 hospital patients were measured: group 1 consisted of blood samples collected in vacutainer tube with K3EDTA as anticoagulant and diluted 1:10 in MM solution plus 4.6 mmol/L Na2EDTA, in order to reach for EDTA a 5 mmol/L concentration; groups 2 and 3 consisted of DBSs prepared by dropping blood, residual in the plastic tube after the venous drawn, on filter paper and eluted in MM solution with addition or in absence of 5 mmol/L Na2EDTA, respectively. The lead extraction from DBSs and measurements were performed one week after sample collection. The storage was for DBSs at room temperature, for whole blood at 4 8C. Groups 1 and 2 showed similar blood lead mean values, which were
Table 1 Mean lead concentration in liquid whole blood plus 5 mmol/L EDTA and in samples eluted from dried blood spots with or without 5 mmol/L EDTA and statistical significance of differences Samples (no. 20)
Mean lead concentration (Ag/mL)
Statistical significance of differences at Pb0.001
1 DBS+EDTA 2 DBS EDTA 3 WB+EDTA
6.58 3.62 6.36
P 12b0.001 P 13=0.975 P 23b0.001
Specimens for sample preparation were whole blood (WB) collected as liquid in vacutainer tube containing 7.5% (m/v) K3EDTA and as dried spot on paper filter from the same patient; for whole dried blood on paper filter (DBS), blood remaining in plastic tube was used to prepare blood spot without EDTA. When EDTA was present in the eluting solution, the concentration was adjusted with the required addition of Na2EDTA.
higher than group 3 mean value and with statistically significant difference, as shown in Table 1. The recovery of blood lead from filter paper in absence of EDTA was dependent on storage time of paper DBSs. The lead optical density at 283.3 nm of 20 blood samples (not shown), prepared by eluting in MMS without EDTA 6.3 mm discs from 2 week-old and 3-month-old DBSs, respectively, were not different from their respective blanks, consisting of paper disc punched as close as possible to blood spot and treated like the blood disc. Blood samples from more recent DBSs showed values in same case a little higher or similar to blanks or lower than the blanks,
Fig. 2. Comparison of lead levels in three matched whole blood samples from 20 patients: liquid whole blood plus 5 mmol/L EDTA (full squares), whole blood collected and dried on filter paper and eluted from 6 mm paper discs in the presence of 5 mmol/L EDTA (open circles) or in the absence of EDTA (stars).
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Fig. 3. Effect of time storage on lead recovery by extraction from 6-month-old dried blood spots on filter paper without addition of EDTA to eluting solution. Open circles represent the lead optical density of samples eluted from blanks, i.e. 6.3-mm paper discs punched as close as possible as to dried blood spot, full squares of samples eluted from 6.3 mm paper discs punched from dried blood spots.
for older samples the optical density was similar or lower than blanks. In Figs. 3 and 4, we show the optical density of samples eluted from 6-month-old DBSs and their respective blanks with or without the addition of EDTA to the extracting solution. The median values for blanks were about the same in both conditions, for DBSs higher values than blanks were measured, when elution was carried out in presence of EDTA and the differences between the median values were statistically significant ( Pb0.001), as indicated in Table 2. When EDTA was absent in the eluting
solution, the blanks showed optical density higher than DBS samples ( Pb0.001).
4. Discussion The use of DBS on filter paper for lead blood analysis with GFAAS has been the object of many discussions [2,5,15–17]. The main criticisms concern poor recovery, high scattering of results, and uncertainty about the volume of absorbed blood.
Fig. 4. Effect of 5 mmol/L EDTA in the eluting solution on lead recovery from 6-month-old dried blood spots on filter paper. Open circles represents the lead optical density of samples eluted from blanks, i.e. 6.3 mm paper discs punched as close as possible to dried blood spot, full squares of samples eluted from 6.3 mm paper discs punched from dried blood spots.
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Table 2 Optical density median value at lead 283.3-nm wavelength of samples prepared by elution of 6-month-old dried blood spots and respective blanks in the presence or in the absence of 5 mmol/L EDTA and statistical significance of differences Samples (no. 40)
Optical density at 283.3 nm (median value)
Statistical significance of differences at Pb0.001
1 2 3 4
0.064 0.044 0.033 0.050
P 12b0.001 P 34b0.001 P 24=0.136
DBSs+EDTA BLANKS+EDTA DBSs EDTA BLANKS EDTA
From dried blood spots (DBSs), 6.3 mm paper discs were punched and eluted with matrix modifier solution (see Material and methods) with or without 5 mmol/L EDTA. Blanks were 6.3 mm paper discs punched as close as possible to whole spots and eluted under the same conditions as for dried whole blood discs.
To evaluate the performance of methods using DBS on filter paper to determine lead blood, a proficiency testing program has been carried out [5]. The results of this program do not eliminate the doubts about the validity of filter paper collected blood samples to screen lead levels in children. Two reasons support this conclusion: (1) unacceptable results according to the criteria of Clinical Laboratory Improvement Amendments of 1988 (CLIA ’88) were obtained from three of six laboratories participating to the program [5]; (2) the blood used for the proficiency testing program was collected in tubes containing EDTA as anticoagulant, then spotted onto filter paper and dried at room temperature, in contrast, blood as dried spots for routine screening is collected without EDTA and it retains intact properties of clotting. Five Laboratories participating in the proficiency testing program have extracted lead from DBS, the sixth laboratory used a Delves cup flame atomic absorption with a preashing step. This last laboratory along with other two extracting lead from DBS with a HNO3-Triton X-100 solution disagreed with CLIA ’88 criteria because of substantial negative bias in the slope of their regressions. The bias was probably due to factors not depending on the extraction procedure. Among the Laboratories respecting the CLIA ’88 criteria, two extracted blood spots with (NH4)H2PO4-Triton X 100 solution, the remaining one added also HNO3 to the extracting solution. Although we extracted lead from DBS by reproducing identical conditions of this last Laboratory, at lead wavelength absorption of 283.3 nm, the optical
density of the sample solution obtained by eluting lead from 3- (not shown) and 6-month-old (Fig. 3) DBSs was lower than the blank solution and near the background. When we extracted lead in the presence of 5 mmol/L Na2EDTA (Fig. 4), optical density of DBS was significantly higher ( Pb0.001) than the blanks (Table 2). This result allows us to measure lead concentration in DBS sample to carry out epidemiological investigations. In the absence of EDTA in the eluting solution, the difference between blanks and blood samples decreased significantly as the storage time increased; the difference between blanks and sample has a low statistical significance ( P=0.038) after 2 weeks of storage and no significance ( P=0.522) after 3 months; after 6 months, the blanks show optical density significantly higher ( Pb0.001) than the samples. Stanton et al. [5] suggested, as a possible cause for negative bias, a negative effect of EDTA on the efficiency of extraction with HNO3 in the blood samples, in the absence of (NH4)H2PO4. According to our results, the negative bias was probably due to the inability of EDTA to bind lead fixed on DBS, at low pH. The addition of 5 mmol/L EDTA in the extracting solution containing 0.2% v/v HNO3 and 2% m/v (NH4)H2PO4 increases the lead recovery (Fig. 1). The presence of EDTA at 5 mmol/L (Fig. 2, Table 1) enabled total recovery: the lead level was substantially the same for samples eluted from DBSs in presence of EDTA and liquid whole blood samples; differences between the mean values were not statistically significant ( P=0.975). At lead level higher than 10 Ag/dL (Fig. 2), values were comparable for whole blood and untreated EDTA DBSs; these results were in agreement with conclusion of Srivuthana et al. [18] and Shen et al. [6], who found that the filter method for blood analysis is convenient to examine children with elevated blood lead levels. Our results offer suggestions useful to the lead blood screening proposed by Shen et al. [6]. These authors obtained good correlation between lead levels in matched samples of venous liquid blood collected in vacutainer tube containing EDTA and dried blood absorbed on filter paper from finger. Furthermore, they tested lead stability on filter paper after 4 weeks, and they concluded that blood collection on filter paper represents an optimal solution to send blood sample to specialized labo-
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ratory for lead analysis from remote regions of China. In this regard, we feel that the conditions for lead screening should be different from those described by Shen et al. In fact, blood on filter paper was dried for 10 min, then transported in hermetically closed plastic bag to the laboratory for lead analysis, the extracting step was started in the presence of 5% HNO3. As the arrival time from remote region of China could be longer and consequently also the contact of blood with filter paper, the Authors carried out test to verify lead stability, i.e. lead recovery, after 1, 2, 3 and 4 weeks from blood spotting on filter paper. About 50% of older samples show lower lead levels compared to lead levels in 1-week-old blood spot. The recovery was more efficient for sample with high lead level, according to our results indicating similar value for whole blood, EDTA-treated and -untreated DBS (Fig. 2). In the test performed by Shen et al. on 30 venous blood samples spotted on filter paper, 24 samples showed lead concentration N15 Ag/dl. According to our results, EDTA effect is stronger when lead concentration is less than 10 Ag/dl (Fig. 2), that is considered cut-off value for normal children [19]. EDTA at 5 mmol/L concentration gave optimal results in lead extraction from old DBSs as indicated in Fig. 1, showing the dose response of EDTA on lead extraction. The rapid decrease of the absorbance at 5 mmol/L could be due to the accumulation of carbon residues in the graphite tube at the level of atomization. This condition was suggested by Nakajima et al. [20], who observed irreproducible absorbance due to accumulation of carbon residues, at high EDTA concentrations, when lead, eluted with increasing EDTA concentrations from a lead selective resin, was injected in the graphite furnace. We observed that, at 283.3 nm, the absorbance of a solution containing lead in MMS, at concentration similar to most concentrated samples described in Fig. 1, was almost constant, ranging from 0.062 to 0.055, with 11.2% decrease, at EDTA concentrations between 0 and 5 mmol/L. On the other hand, a 50% decrease in the absorbance was observed at higher EDTA concentrations between 5 and 10 mmol/L, thus suggesting that the decrease in the absorbance, at EDTA concentration higher than 5 mmol/L, does not depend on the extraction efficiency but on lead
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atomization. The almost overlapping absorbances for EDTA concentrations above 6 mmol/L, measured for both samples (Fig. 2), suggest that EDTA does not specifically affect lead mesurement. The role of EDTA in lead recovery from blood absorbed on filter paper is based on the well-known chelant properties of EDTA, which forms a very stable complex with lead, log K stability=18 [21], in aqueous solution at pH as low as 3. Because of these chelant properties, EDTA is used as therapeutic agent in case of lead intoxication [11–13], or as solubilizing agent to eliminate soil pollution [14] or as analytical reagent in complexometry [22]. In our study, EDTA contributed to solubilize lead blocked on filter paper through protein matrix, as better as the storage time increases. The results of our study confirm that residual DBSs, stored after laboratory tests for neonatal screening of endocrine and metabolic diseases, could be useful tool for epidemiological survey of lead pollution. However, the lead must be extracted from DBS in the presence of optimal amounts of EDTA. Further investigations are needed to verify whether DBSs can be used for diagnostic results of lead intoxication.
Acknowledgements This work was supported by grant of Regione Calabria. We thank Prof. Nicola Perrotti for reviewing the English language of this text.
References [1] Bannon DI, Murashchik C, Zapf CR, Farfel MR, Chisolm Jr JJ. Graphite furnace absorption spectroscopic measurement of blood lead in matrix-matched standards. Clin Chem 1994; 40:1730 – 4. [2] Morrow JC, Hannon WH, Henderson LO, Paschal D. Reliability of lead measurement of blood collected on filter paper and their potential application to epidemiological survey. In: Farriaux JP, Dhondt JL, editors. New horizons in neonatal screening. Amsterdam7 Elsevier; 1994. p. 289 – 92. [3] Yee HY, Holtrop TG. An improved capillary blood filter paper graphite furnace atomic absorption spectrometric method for lead screening. J Anal Toxicol 1997;21:1 – 7. [4] Holtrop TG, Yee HY, Simpson PM, Kauffmann RE. A community outreach lead screening program using capillary
150
[5]
[6]
[7]
[8]
[9] [10]
[11]
[12]
[13]
M.T. Di Martino et al. / Clinica Chimica Acta 350 (2004) 143–150 blood collected on filter paper. Arch Pediatr Adolesc Med 1998;152:455 – 8. Stanton NV, Maney JM, Jones R. Evaluation of filter paper blood lead methods: results of a pilot proficiency testing program. Clin Chem 1999;45:2229 – 35. Shen X-M, Zhang Y-W, Wu S-H, Yan C-H, Ying J-M, Li R-Q. Applicability of a filter paper method to measure blood lead levels in large populations of Chinese children. Clin Chim Acta 2003;328:99 – 104. Ewers U, Turfeld M, Jermann E. Lead. In: Herber RFM, Stoeppler M, editors. Trace element analysis in biological specimens. Amsterdam7 Elsevier; 1994. p. 371 – 84. Makowski GS, Davis ET, Hopfer SM. The effect of storage of Guthrie cards. Implication for deoxyribonucleic acid amplification. Ann Clin Lab Sci 1996;26:458 – 63. Moyer TP, Nixon DE, Ash OK. Filter paper-collected blood lead testing in children. Clin Chem 2000;46:1026 – 8. Markowitz ME, Bijur PE, Ruff H. Effects of calcium Disodium Versenate (CaNa2EDTA) chelation in moderate childhood lead. Pediatrics 1993;92:265 – 71. Guzman DD, Velez LI, Shepherd JG, Goto CS. Calcium EDTA and DMSA chelation of a neonate with congenital lead intoxication. J Toxicol Clin Toxicol 2000;38:548 – 9. Brownie CF, Aronson AL. Comparative effects of Ca ethylenediaminetetraacetic acid (EDTA), ZnEDTA and ZnCaEDTA in mobilizing lead. Toxicol Appl Pharmacol 1984;75:167 – 72. Kedziorek MAM, Dupuy A, Bourg ACM, Compere F. Leaching of Cd and Pb from a polluted soil during the percolation of EDTA: laboratory column experiments mod-
[14]
[15] [16] [17] [18]
[19]
[20]
[21]
[22]
elled with a non-equilibrium solubization step. Environ Sci Technol 1998;32:1609 – 14. Qiao H, Parsons PJ, Slavin W. Transferability of blood lead determination by furnace atomic absorption spectrophotometry and continuum background correction. Clin Chem 1995;41: 1451 – 4. Moyer TP, Nixon DN, Ash OK. Filter paper lead testing. Clin Chem 1999;45:2055 – 6. Vereby K. Filter paper-collected blood lead testing in children. Clin Chem 2000;46:1024 – 8. Stanton N, Maney J, Jones R. More on filter paper lead testing. Clin Chem 2000;46:1028 – 9. Srivuthana K, Yee HY, Bhambhani K, Elton RM, Simpson PM, Kauffmann RE. A new filter paper method to measure capillary blood lead level in children. Arch Pediatr Adolesc Med 1996;150:498 – 502. Centers for disease Control and Prevention. Preventing lead poisoning in young children. Atlanta, GA7 US Dept. of Health and Human Services, Public Health Service, CDC; 1991. Nakajima J, Hirano Y, Oguna K. Determination of lead in seawater by flow-injection on-line preconcentration electrothermal atomic absorption spectrometry after coprecipitation with iron (III) hydroxide. Anal Sci 2003;19:585 – 8. Ringbom A, Vanninen E. Theory of photoelectric complex formation titrations using metal indicators. Anal Chim Acta 1954;11:153 – 67. Skoog DA, West DM. Fundamentals of analytical chemistry. New York7 Holt, Rinehart and Winston; 1965.