Lens culinaris agglutinin-reactive {alpha}-fetoprotein, an alternative variant to {alpha}-fetoprotein in prenatal screening for Down's syndrome

Ritsu Yamamoto1,3, Masaki Azuma1, Nobuhiko Hoshi, Tatsuro Kishida1, Shinji Satomura2 and Seiichiro Fujimoto1

1 Department of Obstetrics and Gynecology, Hokkaido University School of Medicine, Sapporo 060-8638, 2 Osaka Research Laboratories, Wako Pure Chemical Industries, Ltd., Amagasaki, 661-0963, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Three serum tests, {alpha}-fetoprotein (AFP), human chorionic gonadotrophin and unconjugated oestriol, are now widely used for screening for Down's syndrome. Lens culinaris agglutinin-reactive {alpha}-fetoprotein (AFP-L3) is a variant of {alpha}-fetoprotein with {alpha}1->6 fucose appended to the reducing terminal N-acetylglucosamine. It is the most prominent AFP detected in the serum of patients with hepatocellular carcinoma. METHODS: We investigated microheterogeneities of the carbohydrate chain on AFP in fetal liver tissues, amniotic fluids and maternal sera obtained from pregnancies with Down's syndrome using lectin affinity electrophoresis with four lectins. The percentages of AFP-L3 in maternal sera from 22 Down's syndrome and 227 unaffected pregnancies were determined. RESULTS: Unlike the case with AFP concentration, the percentage of AFP-L3 in maternal serum and amniotic fluid was similar, and apparantly not influenced by membrane permeability. Knowing the percentage of AFP-L3 in maternal serum was effective for discriminating between Down's syndrome-affected pregnancies and unaffected pregnancies. The percentage of AFP-L3 in maternal serum identified 55% of Down's syndrome cases with a 5% false-positive rate. CONCLUSIONS: AFP-L3 should be an effective replacement for AFP in prenatal Down's syndrome screening.

Key words: {alpha}-fetoprotein/AFP-L3/Down's syndrome/Lens culinaris agglutinin/prenatal screening


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Down's syndrome (DS) is caused by a chromosomal disorder resulting in the trisomy of chromosome 21 (Jacobs and Morton, 1977Go). During fetal life, DS can be diagnosed using amniocentesis followed by karyotyping of the fetal cells. This procedure is performed on women in the second trimester of pregnancy who are considered to be in a high risk group because of age or family history; however, the overall number of DS births is smaller in the high-risk group of women and greater in younger women (Lam, 1998Go; Spencer, 1999Go; Jou, 2000Go).

An association between trisomy 21 and low levels of {alpha}-fetoprotein (AFP) in maternal serum in mid-trimester was reported in the 1980s (Cuckle et al., 1984Go; Merkatz et al., 1984Go; Nicolini et al., 1988Go; Suzumori et al., 1997Go). In the early 1990s, biochemical markers such as human chorionic gonadotrophin (HCG) (Spencer et al., 1997Go), unconjugated oestriol (uE3) (Cheng et al., 1993Go; David, 1996Go), inhibin A (Van Lith et al., 1992Go), and pregnancy-associated plasma protein (Brambati et al., 1993Go) were studied to determine their potential for DS screening. Recently, Cole et al have focused on the presence of variant N-linked oligosaccharides on HCG-related molecules in DS pregnancies, and those may be an alternative to HCG (Cole et al., 1997Go; Cole, 1999Go; Bahado-Singh, 2000Go). There continues to be a need for more reliable screening marker for DS.

It is well known that AFP is a glycoprotein produced by the fetal liver, hepatocellular carcinoma and yolk sac tumours. The carbohydrate structure of human AFP has been analysed using preparations purified from ascites fluid of a patient with hepatocellular carcinoma (Yoshima et al., 1980Go) and a yolk sac tumour (Yamashita et al., 1983Go). The sugar chain microheterogeneities of AFP, especially Lens culinaris agglutinin-reactive AFP (AFP-L3), have been studied using lectin affinity electrophoresis in relation to hepatocellular carcinoma (Breborowicz et al., 1981Go; Taketa et al., 1993Go; Yamashita et al., 1996Go). The relationship between AFP glycoforms in maternal serum at the second trimester and DS-affected pregnancies has only been determined using Concanavalin A (Gembruch, 1987; Kim, 1990Go; Los et al., 1995Go). In this study, we determined the percentage of AFP variants that react with lectins as a way to find out whether the analysis of the carbohydrate chain microheterogeneities of maternal serum AFP could be useful for prenatal DS screening.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Clinical materials
Maternal serum, amniotic fluids and fetal liver tissues were obtained at Hokkaido University Hospital. The serum and amniotic fluids were stored at –20°C, and the liver tissues were stored at –80°C. Between 1989 and 1998 maternal sera were collected from 22 pregnant women (maternal age 38.8 ± 4.3 years, age of gestation 16.3 ± 0.9 weeks, maternal weight 55.0 ± 4.8 kg, mean ± SD) who were diagnosed as carrying a fetus with a trisomy 21 based on amniocentesis followed by karyotyping of the fetal cells. Maternal sera samples obtained before amniocentesis from 227 pregnant women (maternal age 35.6 ± 5.2 years, age of gestation 16.4 ± 1.1 weeks, maternal weight 56.0 ± 8.5 kg, mean ± SD) whose fetuses were diagnosed as karyotypically normal were also collected. All women gave informed consent for amniocentesis and abortion at their own request. Pregnancies complicated with diabetes mellitus, hepatitis or neural tube defects and multifetal pregnancies were excluded from this study.

Measurement of total AFP, HCG, and uE3
Total AFP, HCG and uE3 concentrations were determined routinely using commercially available kits (AFP; Abbott Laboratories, IL, USA; HCG; Wallak Oy., Turku, Finland; uE3; Diagnostic Products Corp., CA, USA). The multiple of the median (MoM) of the AFP, HCG and uE3 in this clinical study was calculated based on 4256 Japanese unaffected pregnancies at 14–20 weeks gestation (Suzumori et al., 1997Go).

Lectin-affinity electrophoresis analysis of AFP
The percentage of AFP reactive with Lens culinaris agglutinin (LCA) was obtained by lectin-affinity electrophoresis coupled with antibody-affinity blotting (AFP Differentiation Kit L; Wako Pure Chemical Industries, Ltd., Amagasaki, Japan) (Shimizu et al., 1993Go). The percentage of other lectin-reactive AFPs was measured similarly using Concanavalin A (Con A), erythroagglutinating phytohemagglutinin E4 (EPHA) and Ricinus communis agglutinin-120 (RCA) in agarose gels (Shimizu et al., 1996Go). Band intensities separated by a lectin-affinity electrophoresis are expressed as percentages of the total band intensity. Faint bands that were not detectable by densitometer were expressed as <0.5% and the minimum detection level of AFP in one band was 2 ng/ml in a sample (8 pg/band) (Shimizu et al., 1993Go). The AFP band 1 had no affinity for lectins because the mobility was identical to that on gels without lectin, and bands numbered 2 and higher had correspondingly higher affinities for lectins. The correspondence between separated AFP bands and the estimated structure of the carbohydrate chain has been described elsewhere (Shimizu et al., 1996Go). Both AFP-L2 and AFP-L3 have an {alpha}1->6 fucose residue, and the sum of them was evaluated, although AFP-L2 appears generally in amniotic fluids.

Extraction of AFP from liver tissue
Liver tissue samples ~3x3 mm were washed with ice-cold 10 mmol/l phosphate buffer including 0.15 mol/l NaCl, pH 8.0, to remove blood. The tissue weight was measured after removal of the buffer, and then it was homogenized with 0.5 ml of buffer using a glass homogenizer in an ice-bath. AFP in the fetal liver was obtained from the supernatant by centrifugation (1200 g, 10 min), and it was analysed by lectin affinity electrophoresis.

Sialidase digestion of AFP obtained from fetal liver
A volume of 5 µl of 150 IU/ml sialidase (Streptococcus sp.) in 0.1 mol/l 2-(N-morpholinoethanesulphonic acid/NaOH buffer, pH 6.0, was added to 45 µl of the supernatant of fetal liver tissue and incubated at 37°C for 24 h, and the resulting solution was analysed by lectin affinity electrophoresis.

Statistical analysis
Statistical significance was determined by Student's t test, Welch t-test and the Mann–Whitney U-test. To determine the clinical accuracy of the percentages of lectin-reactive AFP bands and other markers, the area under the receiver operating characteristics (ROC) curve was compared by Z score testing.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Comparison of the percentage of lectin-reactive AFPs of fetal liver tissues, amniotic fluids and maternal sera in pregnancies with DS by lectin affinity electrophoresis
Table IGo shows the lectin affinity electrophoresis results obtained from sialidase-digested AFP in fetal liver tissue using four different kinds of lectins. Con A binds to biantennary structure, and LCA binds an {alpha}1-6 fucose residue at N-acetylglucosaminne at the reducing end. Almost all the carbohydrate chain of fetal liver AFP has biantennary structure (AFP-C3, >99.5%) with ~30% of {alpha}1->6 fucose residue (AFP-L3) and 70% without fucose residue (AFP-L1). RCA binds to a galactose residue at the non-reducing end, and AFP-R0, AFP-R2f and AFP-R3f in Table IGo reveal the percentages of agalactosyl biantennary chain (41–46%), mono-galactosyl biantennary chain (5–7%) and di-galactosyl biantennary chain (47–54%) obtained after sialidase digestion, respectively. AFP-L2 possesses a ß1–4 N-acetylglucosaninine residue (bisecting N-acetylglucosamine) or branching (tri- or tetra-anternary) chain in addition to fucosylated AFP. Neither the branching structure nor the bisecting N-acetylglucosamine in fetal liver AFP with DS was measured during this analysis.


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Table I. Analyses of lectin-reactive AFPs in fetal liver of pregnancies with trisomy 21 after sialidase digestion
 
The lectin affinity electrophoresis results obtained from AFPs in fetal liver tissues, amniotic fluids and maternal sera derived from three pregnancies with DS using four different kinds of lectins without sialidase-digestion are compared in Table IIGo. In the lectin affinity electrophoresis using Con A and RCA, percentages of both AFP-C3 and AFP-R0 from the liver tissue extracted fluid decreased in both amniotic fluid and maternal serum to a level <0.5%. Almost all the carbohydrate chain of both amniotic fluid and maternal serum AFP does not have the asido-biantennary structure. According to LCA affinity electrophoresis, AFP-L2 was detected only in amniotic fluid. On the other hand, AFP-C1 was also detected in the amniotic fluid. The branching or bisecting N-acetylglucosamine interfere with Con A affinity and they were separated into the AFP-C1 band. The presence of both AFP-L2 and AFP-C1 indicate either or both parts of the branched structure and bisecting N-acetylglucosamine in the amniotic fluid AFP, which was not measured in fetal liver AFP, AFP-P5, band has bisecting N-acetylglucosamine residues and was found in both amniotic fluid and maternal serum.


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Table II. Analyses of lectin-reactive AFPs in fetal livers, aminotic fluids and maternal sera of pregnancies with a trisomy 21 fetus, without sialidase-digestion
 
Regression analyses of AFP concentration and the percentages of AFP-L3 between amniotic fluid and maternal serum
AFP concentration and the percentage of AFP-L3 in both maternal serum and amniotic fluid of 17 pregnancies with DS and 49 unaffected pregnancies were measured. Regression analyses revealed a positive correlation between the percentage of AFP-L3, in amniotic fluids and maternal sera of pregnancies with DS (n = 17, r = 0.848, P < 0.0001). No such correlation was observed in the amniotic fluids or maternal sera of unaffected pregnancies (n = 49, r = 0.255, P = 0.077; Figure 1Go). No correlation was observed between AFP concentration in amniotic fluids and maternal sera of pregnancies with DS (n = 17, r = 0.116, P = not significant) and that of unaffected pregnancies (n = 49, r = 0.099, P = not significant), and no relationships were discovered between AFP concentration and the percentage of AFP-L3 in maternal sera of pregnancies with DS (n = 17, r = 0.435, P = not significant) and that of unaffected pregnancies (n = 49, r = 0.055, P = not significant).



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Figure 1. Relationship between the percentage of AFP-L3 in amniotic fluids and maternal serum obtained from pregnancies with fetuses affected with Down's syndrome (DS) and unaffected pregnancies. • = DS affected pregnancies, {circ} = unaffected pregnancies.

 
The percentages of lectin-reactive AFPs in maternal sera and amniotic fluids in unaffected pregnancies and pregnancies with DS
The percentages of lectin-reactive AFPs with the use of three lectins in both maternal sera and amniotic fluids in unaffected pregnancies (n = 20) and DS pregnancies (n = 17) were compared for a pilot study. No significant differences were observed in maternal age or weeks of gestation between the unaffected pregnancies and the pregnancies with DS: unaffected pregnancies, maternal age 39.7 ± 3.3 years , age of gestation 16.6 ± 0.9 weeks; pregnancies with DS, maternal age 39.1 ± 4.7 years, age of gestation 16.2 ± 0.9 weeks, (mean ± SD). The percentage of AFP-L3 and AFP concentration in maternal sera and the percentages of AFP-L3 and AFP-P4 in amniotic fluids show a significant difference between unaffected pregnancies and pregnancies with DS, as shown in Table IIIGo. The percentages of AFP-L3 and AFP concentration in maternal serum were selected for further study.


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Table III. Percentages of lectin-reactive AFPs in unaffected and affected pregnancies
 
ROC analyses and diagnostic indices of the percentage of AFP-L3 in maternal serum
The mean and SD for MoM AFP, MoM HCG, MoM uE3 and the percentage of AFP-L3 in maternal serum and the P value for discrimination between 22 pregnancies with DS and 227 unaffected pregnancies are shown in Table IVGo. The percentage of AFP-L3 in maternal serum in pregnancies with DS was significantly higher than that in unaffected pregnancies (P < 0.00001), and three conventional markers show significant differences. Furthermore, the mean ± SD for the percentage of AFP-L3 in women >35 years old (n = 162) and <35 years old (n = 65) of unaffected pregnancies are 30.7 ± 8.3 and 29.5 ± 8.6 respectively. This suggested that the percentage of AFP-L3 in maternal serum was not affected by maternal age (P = not significant).


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Table IV. Mean and SD values of MoM AFP, MoM HCG, MoM uE3 and the percentage of AFP-L3 in maternal serum
 
The fractional areas under the ROC curves were 0.734, 0.763, 0.730 and 0.875 for MoM AFP, MoM HCG, MoM uE3 and the percentage of AFP-L3 respectively (Figure 2Go). The percentage of AFP-L3 in maternal serum revealed a significantly higher area than MoM AFP and MoM uE3 and it was revealed to have a tendency of a larger area than MoM HCG as shown in Table IVGo.



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Figure 2. Receiver-operating characteristic curves of the percentage of AFP-L3, MoM AFP, MoM HCG and MoM uE3 for 22 Down's syndrome pregnancies and 227 unaffected pregnancies.• = percentage of AFP-L3; {circ} = MoM AFP; {triangleup} = MoMHCG; {square} = MoM uE3.

 
Relationship between the percentage of AFP-L3 and gestational week
The percentages of maternal serum AFP-L3 of 28 samples from 22 pregnancies with DS and 265 samples from 227 unaffected pregnancies were plotted against gestational week (Figure 3Go). The equation for the line of weekly median of unaffected pregnancies was as follows: AFP-L3 (%) = –0.31xgestational week + 35.5. The preliminary threshold points for four serum markers were determined to be equivalent to the 90th and 95th percentiles of the normal range, similar to 90 and 95% specificity respectively. Thus, 54.5% (12/22), 36.4% (8/22), 36.4% (8/22) and 45.5% (10/22) sensitivities were obtained using a threshold value of 40.5%, 0.64, 1.90 and 0.57 at a 10% false positive rate for AFP-L3, MoM AFP, MoM HCG and MoM uE3 respectively. And 54.5% (12/22), 27.7% (5/22), 27.7% (5/22) and 31.8% (7/22) sensitivities were obtained using a threshold value of 41.9%, 0.54, 2.18 and 0.50 at a 5% false positive rate for AFP-L3, MoM AFP, MoM HCG and MoM uE3 respectively. The percentages of AFP-L3 and MoM HCG were evaluated in combination by taking the results positive either alone or both became positive, the slightly increased sensitivity of 59.1% was obtained at a 5% false positive rate.



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Figure 3. Percentage of AFP-L3 in maternal serum from 28 samples from 22 Down's syndrome (DS) affected pregnancies and from 265 samples from 227 unaffected pregnancies. The line shows the 50th percentile of the unaffected pregnancies. • = DS affected pregnancies; {circ} = unaffected pregnancies.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our aim was to evaluate the potential value of examining the microheterogeneities of the AFP carbohydrate chain to discriminate between unaffected pregnancies and pregnancies associated with DS at the second trimester. In a normal pregnancy, it is known that the percentage of AFP-L3 in maternal serum and amniotic fluid gradually decreases as the pregnancy progresses (Taketa, 1995Go). This phenomenon is considered to be a reflection of the maturity of the fetal liver. In studies of hepatocellular carcinoma, fucosylation of AFP has been shown to be related to the de-differentiation of human hepatocytes through carcinogenesis, and less differentiated tumours show the greater increase in AFP-L3 (Taketa et al., 1993Go; Yamashita et al., 1993Go). There is no genetic template to code for a diverse carbohydrate chain structure; however, the percentage of AFP-L3 in patient serum with hepatocellular carcinoma reflects the level of cancerous differentiation. Fetal liver and hepatocellular carcinoma tissues are completely different, since the former change is differentiation and the latter is de-differentiation. We hypothesized that pregnancies with fetal chromosomal abnormalities had different AFP carbohydrate chain structures which reflect the de-differentiation of hepatocytes, and the livers of DS fetuses are less differentiated and less mature than those of normal fetuses at the same gestational week.

We first focused on any changes in the AFP carbohydrate chain structure during the processes ranging from the AFP production in the fetal liver to its presence in maternal serum. AFP is produced in the developing fetus by both the yolk sac and the fetal liver (Gembruch et al., 1987Go). At around 12 weeks gestation, the yolk sac degenerates and the fetal liver becomes the main site of synthesis. It was expected that AFP in very early amniotic fluid would mainly be of the yolk sac type sugar chain (Los et al., 1995Go). After 14 weeks gestation, AFP synthesis decreases with advancing gestation; therefore, maximum fetal plasma concentration of AFP is reached at 13 weeks gestation and declines exponentially thereafter (Blair et al., 1987Go). No significant difference was reported in the steady-state level of fetal liver AFP mRNA levels in trisomy 21 and 18 groups, and the decrease in maternal serum AFP concentration is unlikely to be the consequence of impaired transcription of the AFP gene by the fetal liver (Brizot et al., 1996Go). In maternal blood, AFP concentrations rise throughout the first and second trimesters and decline only after 32 gestational weeks. This increase is thought to be due to increased placental permeability to fetal plasma proteins with advancing gestation (Gitlin, 1975Go; van Lith et al., 1991Go). In the present study, we determined that the carbohydrate structure of AFP in the trisomy 21 fetal liver was simple and uniform in three cases, namely, almost all have biantennary structure with ~30% containing {alpha}1->6 fucose residue, 70% without fucose residue, and the reducing end is uneven, with ~44% of agalactosyl, ~6% mono-galactosyl and ~50% of the di-galactosyl biantennary chain.

We examined specimens obtained from fetal liver, amniotic fluid and maternal serum from three pregnant women with trisomy 21 fetuses since the presence in these different tissues may be affected by membrane-permeability, diffusion and active transport. The lectin affinity electrophoresis in these cases revealed relatively identical results. Lectin-reactive AFP bands AFP-L2, AFP-C1, AFP-P4 and AFP-P5 in the amniotic fluid, which have the additional branching sugar to a biantennary structure, indicated relatively greater values than those in both fetal liver and maternal serum. That is possibly because these AFPs having multi-antennary and/or branching sugars do not easily permeate through the membrane of the placenta, although the production in fetal liver is limited in quantity. We failed to detect AFP-L2, which has both {alpha}1->6 fucose residue and an additional branching sugar to a biantennary structure in the maternal serum, but we were able to detect it in the amniotic fluid. Even though AFP-L2 has extremely low permeability, the percentage of AFP-L3 in amniotic fluid and maternal serum was similar in the DS pregnancies, thereby suggesting that the presence of {alpha}1->6 fucose residue on N-acetylglucosamine at the reducing end does not generally affect the permeability; however the additional branching sugar to the biantennary structure does affect the permeability. AFP-C1 has an additional branching sugar or a multi-antennary carbohydrate chain, while AFP-C2+C3 shows a biantennary chain. Compared with the amniotic fluid and maternal serum, the biantennary chain ratio in the maternal serum tends to increase, indicating that permeability and/or transport at a biantennary chain in the placenta or fetal membrane is relatively high or that of a multi-antennary chain is relatively low.

Because of the special nature of the extracted fluid from the trisomy 21 fetal liver tissues, the difference between sugar-chain bands at different stages in the process by which AFP reaches the maternal blood is great, with the exception of AFP-L3. Looking at the barriers between the amniotic fluid and maternal serum, it is necessary to keep in mind the difference in fluid quantity of the two. The total AFP concentration in the amniotic fluid is 13 722 ± 6586 ng/ml, while that in the maternal serum is 42 ± 7 ng/ml, suggesting that the total AFP hardly permeates the membrane. No correlation was obtained between the AFP concentration in amniotic fluids and that in maternal serum in either pregnancies with DS or unaffected pregnancies, as shown elsewhere (Barford et al., 1985Go). However, the percentage of AFP-L3 in the cases of the pregnancies with a trisomy 21 fetus seems to be relatively unaffected by such factors as the permeability and/or transport of fetal membrane and placenta, or metabolism in the amniotic fluid and maternal tissues. These findings make AFP-L3 an appropriate choice for a trisomy 21 biochemical marker.

Our second focus was to determine the clinical utility of examining microheterogeneities of the AFP carbohydrate chain in maternal serum for prenatal screening for DS. All AFP bands in maternal serum and amniotic fluid were compared for discriminating efficiency between 17 DS affected pregnancies and 20 unaffected pregnancies, with only AFP-L3 showing a significant difference as a maternal serum biochemical marker. With ROC analysis using 22 DS-affected pregnancies and 227 unaffected pregnancies, the percentage of AFP-L3 in maternal serum was revealed to have a greater area under the curve than those of MoM AFP and MoM uE3, and it was revealed to have a tendency towards a larger area than that of MoM HCG.

This is a first report consisting of findings based on the study of fewer than 250 pregnancies at around 16 weeks gestation. Considering that AFP concentrations in the fetal liver peak at 13 weeks gestation (Brizot et al., 1996Go), a prospective study of the measurement of AFP-L3 in maternal serum at the end of first trimester to early in the second trimester of pregnancy is advisable. Due to the technical complexity of measurement, it is difficult to measure large numbers of samples in a short time. The DS screening utility of this potentially valuable marker with the use of an automatic analyser for measuring AFP-L3 (Katoh et al., 1998Go) will be tested further.


    Notes
 
3 To whom correspondence should be addressed. E-mail: rityam{at}med.hokudai.ac.jp Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on March 19, 2001; accepted on July 25, 2001.





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