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Keywords:

  • ADAMTS13;
  • von Willebrand factor;
  • ratio;
  • antigen;
  • activity

Summary

  1. Top of page
  2. Summary
  3. Design and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The plasma metalloprotease ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 motif 13) cleaves prothrombotic ultralarge multimers of the platelet-adhesive protein von Willebrand factor (ULVWF) into less active multimers that promote haemostasis in injured blood vessels. When the enzyme is dysfunctional or undetectable, circulating ULVWF may cause massive intravascular aggregation of platelets and thrombotic thrombocytopenic purpura. This study compared ADAMTS13 antigen and activity in a large set of plasmas collected from subjects with various conditions of health and disease, most of which were associated with an increased thrombotic tendency. Pathological conditions were liver cirrhosis (n = 90), inflammatory bowel disease (n = 44) and cardiac surgery (n = 30). Healthy conditions were pregnancy (n = 42), oral contraceptive intake (n = 33) and the neonatal state (n = 41). Normal individuals of different ages were taken as controls (n = 132). The antigen assay showed less variability than the collagen binding-based activity assay. Antigen values correlated well with activity in normal individuals, but were discrepant to various degrees in neonates, pregnancies of later maternal age and cardiac surgery. No discrepancies were noted in liver cirrhosis and inflammatory bowel disease, which were both associated with low-plasma levels of ADAMTS13. The parallel measurement of ADAMTS13 activity and antigen provides a new tool for understanding the behaviour of the VWF cleaving protease in health and disease.

Thrombotic thrombocytopenic purpura (TTP) is a rare but life-threatening disease involving at least three determinants; von Willebrand factor (VWF), blood platelets and A Disintegrin And Metalloprotease with ThromboSpondin type 1 motif 13 (ADAMTS13). VWF is a multimeric plasma glycoprotein that bridges exposed subendothelium to flowing platelets when a blood vessel is damaged. It is released from endothelial cells in an ultra large form (ULVWF), which distinguishes itself not only by molecular weight but also by the ability to aggregate platelets in conditions of high shear stress (Arya et al, 2002). Proteolysis of the VWF 1605Y-1606M peptide bond by ADAMTS13 produces the naturally occurring multimeric VWF ladder in plasma. When ADAMTS13 is absent or dysfunctional, TTP may occur because ULVWF can circulate freely in plasma and potentially cause intravascular platelet aggregation and disseminated microvascular thrombosis (Lammle et al, 2005). This mechanism fits with pathological observations of VWF-rich thrombi and with the observed presence of ULVWF in TTP plasma. However, a number of TTP patients have detectable and even normal levels of plasma ADAMTS13 activity in vitro (Veyradier et al, 2001) and so, whether or not severe ADAMTS13 deficiency is specific for TTP is still a matter of debate (Remuzzi, 2003; Tsai, 2003).

ADAMTS13 activity was previously measured in a large set of plasmas obtained from donors in well-defined physiological or pathological states, most of which were associated with an increased thrombotic tendency (Mannucci et al, 2001). Significantly reduced levels of ADAMTS13 activity were found in physiological states, such as pregnancy and the neonatal period, as well as in several pathological conditions, in spite of the absence of clinically overt thrombotic events. At that time no tools were available to measure the immunoreactive protease in these conditions. In this study, a monoclonal antibody-based ADAMTS13 antigen assay (Feys et al, 2006) was employed to compare the immunoreactive protein with the corresponding functional activity in a freshly collected set of samples comparable to those of the previous study (Mannucci et al, 2001).

Design and methods

  1. Top of page
  2. Summary
  3. Design and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Blood collection and reference standard

Blood was collected from the antecubital vein in sodium citrate-containing vacutainers and centrifuged twice at 1500 g. Plasma was recovered and frozen at −80°C in aliquots. Normal pooled plasma, used as a reference for both the assays throughout the whole study, was collected from 50 healthy men and women, not pregnant and not taking oral contraceptives. Subjects contributing to the pool were different from those included in the group of normal individuals of different ages (see below). Reference plasma was set to contain 100% of ADAMTS13 activity and antigen, the latter roughly corresponding to 1 μg/ml (Majerus et al, 2005; Feys et al, 2006). All samples were collected at the Angelo Bianchi Bonomi Haemophilia and Thrombosis Centre of the University of Milan. Informed consent for blood sampling was obtained from normal individuals and patients (see below) or their custodians.

Physiological conditions

A total of 132 individuals not taking oral contraceptives and not pregnant at the time of sampling, were arbitrarily divided in four age categories: below 35 or 17–35 years (n = 33), 36–50 years (n = 35), 51–65 years (n = 32) and above 65 or 65–85 years (n = 32). They were ostensibly healthy and chosen from those used as controls for other laboratory measurements at the Haemophilia and Thrombosis Centre, and were usually spouses or friends of patients referred for investigation of thrombotic and bleeding disorders. We also investigated 42 women in the third trimester of a normal pregnancy, who attended routine clinical visits and 41 full-term healthy newborns of both sexes within 4 d of birth. Samples from healthy women (n = 33) taking third-generation oral contraceptives were also examined.

Pathological conditions

Similar to the study performed by Mannucci et al (2001), we investigated three clinical conditions associated with organ involvement and/or acute inflammation. The group of 90 patients with liver cirrhosis was divided according to the Child score (Pugh et al, 1973) into 33 class A (least severe), 32 class B and 25 class C patients (most severe). Patients with inflammatory bowel disease (IBD) (n = 44) were classified according to the serum level of C-reactive protein (CRP) as an index for active inflammation: a threshold of 10 mg/l was arbitrarily chosen to divide the group into 12 patients with and 32 without disease activity. Twenty-seven patients were diagnosed with Crohn's disease and 17 with ulcerative colitis. Plasma from 30 patients undergoing open-heart surgery (18 were operated on-pump and 12 off-pump) was obtained prior to surgery, during the operation and 4 d later (corresponding to T0, T1 and T4 as published previously) (Mannucci et al, 2005). All donor samples included in this study were different from those previously analysed (Mannucci et al, 2001), except for those of cardiac surgery, where ADAMTS13 activity was measured afresh in parallel with antigen in the previously stored samples.

ADAMTS13 antigen assay

ADAMTS13 antigen was measured as reported previously with slight modifications (Feys et al, 2006). The murine monoclonal antibody 20A5 was used as catching antibody and biotinylated murine antibodies (13F7 and 5C11) as detecting antibodies. All antibodies were generated by multiple injections of a vector encoding ADAMTS13 cDNA and of recombinant ADAMTS13 protein. These antibodies exert higher affinities than those previously published (except for 13F7) and were preferred for that reason. Peroxidase-labeled streptavidin (Roche Diagnostics, Basel, Switzerland) was used to bind biotin molecules. Colorimetric development was with o-phenylenediamine dihydrochloride (OPD) (Sigma, St Louis, MO, USA) in 50 mmol/l phosphate-citrate buffer supplemented with 0·03% (m/v) H2O2, pH 5.0, for 15 min. The detection limit of this assay is 4% or lower. The inter- and intra-assay coefficients of variation were 8·3% and 4·4% respectively.

ADAMTS13 activity assay

All samples were analysed for ADAMTS13 activity using the collagen-binding method of Gerritsen et al (1999), with small modifications as previously described (Mannucci et al, 2001). The most recent laboratory evaluation of assay reproducibility yielded inter- and intra-assay coefficient of variation of 12% and 9% respectively. Previous analyses have shown that increased VWF antigen (VWF:Ag) levels do not influence to a major degree the ADAMTS13 activity data when obtained using this method (Mannucci et al, 2001).

Other assays

VWF:Ag was measured by enzyme-linked immunosorbent assay (ELISA) using a commercial polyclonal anti-VWF antibody as primary and as secondary antibody (Dako, Glostrup, Denmark). CRP was measured with a commercial kit (Instrumentation Laboratory, Milan, Italy).

Statistical analysis

All data were analysed for normal distribution using Shapiro-Wilk calculation. Means were compared using the Student's t-test (parametric) and the Mann–Whitney U-test (non-parametric). Association was assessed by calculating phi and its significance by chi-square analysis. A probability P < 0·05 was taken as significant for rejection of null hypotheses. All statistical applications used origin v7.5 software (OriginLab, Northampton, MA, USA).

Results

  1. Top of page
  2. Summary
  3. Design and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Table I summarises all data for VWF:Ag, ADAMTS13 activity and ADAMTS13 antigen, including the calculated ratio of ADAMTS13 activity over antigen.

Table I.   ADAMTS13 activity, antigen and VWF antigen (VWF:Ag) in physiological and pathological conditions.
ConditionnVWF:AgADAMTS13 activity (%)ADAMTS13 antigen (%)Activity to antigen ratio
  1. All values are expressed in percentage of normal pooled human plasma.

  2. Results are given as mean ± SD.

  3. For each condition and measurement, statistically significant differences (shown in italics) are shown in comparison with normal healthy controls.

  4. *P < 0·001.

  5. P < 0·01.

  6. P < 0·05.

Healthy individuals
 Age, years <3533118 ± 35‡117 ± 24107 ± 171·10 ± 0·20
 36–5035140 ± 54110 ± 30106 ± 191·05 ± 0·25
 51–6532142 ± 46113 ± 32104 ± 191·10 ± 0·30
 >6532149 ± 4186 ± 20†90 ± 15*0·98 ± 0·24
Neonates
 Full term41112 ± 2743 ± 20*73 ± 9*0·59 ± 0·25*
Pregnancy
 Third trimester42290 ± 102*85 ± 3692 ± 140·90 ± 0·32
Oral contraceptive intake
 –33141 ± 42103 ± 2697 ± 231·09 ± 0·26
Liver cirrhosis
 Child A33295 ± 80*93 ± 4194 ± 350·99 ± 0·29
 Child B32319 ± 108*89 ± 3490 ± 321·05 ± 0·33
 Child C25442 ± 262*67 ± 38*63 ± 35*1·13 ± 0·39
Inflammatory bowel disease
 CRP <132139 ± 4999 ± 2997 ± 151·04 ± 0·33
 CRP >112167 ± 6682 ± 2882 ± 201·02 ± 0·33
Cardiac surgery
 Baseline30146 ± 6480 ± 24*95 ± 150·87 ± 0·21*
 During surgery30174 ± 6553 ± 21*67 ± 14*0·81 ± 0·34*
 After 4 d30350 ± 95*48 ± 15*72 ± 14*0·71 ± 0·31*

Physiological conditions

Normal individuals.  ADAMTS13 activity was lower in healthy individuals aged 65 years or older than in younger individuals (P = <0·001) (Table I). The same was true for ADAMTS13 antigen values, which correlated positively with activity (R = 0·52, P < 0·001). When ratios of ADAMTS13 activity to antigen were calculated, no differences between individuals of different ages were observed. Although VWF:Ag increased slightly with age, there was no statistically significant inverse correlation between VWF:Ag and either ADAMTS13 measurement.

Neonatal period.  Both ADAMTS13 activity and antigen were lower in neonates than in healthy individuals. The activity to antigen ratio was very low and significantly lower than in normal controls (P < 0·001) (Fig 1).

image

Figure 1.  Ratio of ADAMTS13 activity to antigen in the neonatal period and healthy adults. The activity to antigen ratio is shown in the ordinate, and data are expressed as statistical box-whisker plots. Healthy adults were divided into four age categories, as indicated. The box indicates the interquartile range, divided by the median (central horizontal line), the mean is shown by the central square and the standard deviation by the error bars. *The difference of the mean ratios comparing neonates with all categories of healthy adults was significant (P < 0·001).

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Pregnacy and oral contraceptive intake.  Plasma from third trimester pregnancies contained slightly but significantly less ADAMTS13 activity than age-matched control women and women taking oral contraceptives (Table I, Fig 2, top). ADAMTS13 antigen was slightly higher than activity in pregnant women. Although the difference between activity and antigen was not as marked as in newborns, the corresponding ratio was significantly lower than normal (P < 0·05) (Table I). ADAMTS13 activity to antigen ratios were associated with age, because pregnant women aged 36–50 years had a significantly greater likelihood than younger women of having a ratio below 1 (OR = 4·7, CI: 1·3–17·7 and phi = 0·36, P < 0·05) (Fig 2, bottom).

image

Figure 2.  ADAMTS13 in pregnancy, women taking oral contraceptives and matched control women. (top) Both ADAMTS13 activity and antigen are shown as box-whisker plots and expressed as a percentage of the reference plasma. †The difference of the mean values comparing pregnancy and oral contraceptive intake with the matched controls was significant (P < 0·01), only for the pregnancy group. NS, not significant (P > 0·05) (bottom) ADAMTS13 activity to antigen ratio in matched controls (control women), pregnant women (pregnancy) and women taking oral contraceptives (OC intake) according to age, as indicated in the x-axis. The boxes indicate the interquartile range, divided by the median (central horizontal line), the mean is shown by the central square and the standard deviation by the error bars. There was no statistical difference between the mean ratios, but an association between maternal age and ADAMTS13 activity over antigen ratio was found.

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Pathological conditions

Liver cirrhosis.  Patients with all degrees of severity of impaired liver function were investigated. While VWF:Ag was much higher than normal in all cases (Table I), a decrease in both ADAMTS13 activity and antigen sets occurred when cirrhosis became more severe, with the most drastically reduced values observed in Child class C patients (Fig 3). ADAMTS13 antigen and activity correlated well in all the cirrhosis samples (R = 0·80, P < 0·0001), indicating the presence of fully active enzyme, even when liver function was markedly impaired. Despite the decrease of mean ADAMTS13 values, a number of samples had high levels of both ADAMTS13 activity and antigen, as shown by the wide range of values compared with those of normal controls (Fig 3).

image

Figure 3.  ADAMTS13 according to the degree of severity to liver cirrhosis. Both ADAMTS13 antigen and activity are shown as box-whisker plots and expressed as a percentage of the reference plasma. The normal ADAMTS13 antigen and activity ranges are shown by dotted and dashed horizontal lines respectively. The boxes indicate the interquartile range, divided by the median (central horizontal line), the mean is shown by the central square, (x) indicates the range of data and the standard deviation is shown by the error bars. All calculated means of both measured parameters differed significantly from those of normal controls, except for ADAMTS13 antigen in Child class A liver cirrhosis.

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Inflammatory bowel disease.  There was no difference between patients with the two main types of IBD (ulcerative colitis or Crohn disease). When the whole group was split in two subgroups using CRP levels (10 mg/l) as a criterion for active inflammation, patients above the threshold (CRP range 14–79 mg/l) had slightly increased levels of VWF:Ag (P = 0·12) when compared with the low CRP level subgroup. Both ADAMTS13 activity (P = 0·11) and antigen (P = 0·03) were lower in patients with high CRP levels compared to those with normal CRP levels, although only antigen levels reached statistical significance. Antigen levels corresponded well with activity, indicating the presence of a fully active enzyme.

Cardiac surgery.  ADAMTS13 activity fell in the postoperative period, and the same pattern was observed for antigen values (Fig 4). Furthermore, at all time-points, a significantly discrepant ratio was observed when compared with healthy controls (Table I), due to higher antigen than activity values. Four days after surgery (T4) a moderate rise in ADAMTS13 antigen, but not activity, was observed (Fig 4), resulting in a significantly lower ratio (P < 0·01) than found at baseline. No such statistical difference was observed for activity over antigen ratio between baseline (T0) and T1.

image

Figure 4.  VWF antigen (VWF:Ag) and ADAMTS13 activity and antigen at cardiac surgery. Mean ADAMTS13 antigen, activity and VWF:Ag levels are shown as box plots. The boxes indicate the interquartile range, divided by the median (central horizontal line), the mean is shown by the central square and the standard deviation by the error bars. Samples were obtained in 33 patients before (T0), during (T1) and 4 d after surgery (T4). The left y-axis refers to ADAMTS13 data and the right to VWF:Ag.

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Discussion

  1. Top of page
  2. Summary
  3. Design and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

A large series of plasmas was previously investigated to explore the behaviour of VWF cleaving activity in physiological or pathological conditions (Mannucci et al, 2001). At that time no antibodies for the determination of immunoreactive protease were available. Measuring antigen levels and comparing them with activity levels should help to understand whether or not the protease is fully enzymatically active, a situation expressed by values of activity to antigen ratios equal or close to 1. ADAMTS13 antigen levels were previously measured in a large number of TTP patients and higher values than those of activity were often found, indicating that a fraction of ADAMTS13 is non-functional in these patients (Peyvandi et al, 2006).

The present study involved a new group of healthy individuals and patients in whom ADAMTS13 activity and ADAMTS13 antigen levels were measured in parallel. VWF, the protease's substrate, was also measured immunologically in the same samples. In general, the antigen assay appeared to be less variable than the activity assay, as indicated by smaller standard deviations and coefficients of variation. The limitations of the indirect functional assays of ADAMTS13, and particularly of those measuring residual collagen binding of the substrate, have been a matter of debate ever since their initial description (Mannucci, 2003). Even though the FRETS-VWF73 assay (Kokame et al, 2005) offered a more direct measurement of ADAMTS13 through the assessment of the cleavage of a peptide containing the VWF cleavage sequence, the requirements for this assay in terms of equipment are quite demanding for the majority of unspecialised haemostasis laboratories. Hence the antigen assay used in this study may offer a good alternative, with acceptable coefficients of variation, classical setup (sandwich ELISA) and easy access.

The main finding of this study is that plasma levels of ADAMTS13 activity do not always coincide with those of the immunoreactive protease, as exemplified by the newborn group. Two reports claimed that ADAMTS13 activity was low in neonates (Mannucci et al, 2001; Takahashi et al, 2001) but another found normal levels (Tsai et al, 2002). In our study, neonatal plasma contained less ADAMTS13 antigen than that of young healthy adults taken as controls (between 17 and 35 years of age). Moreover, ADAMTS13 activity levels were even lower than antigen levels, indicating that a substantial amount of protease moieties were not active in vitro. Whether the metalloprotease is inactivated or consumed after VWF proteolysis at birth or whether there is an inhibition of its enzymatic activity remains to be understood. Large VWF multimers have been shown to be present in some newborn infants (Schmugge et al, 2004). Yet, previous studies have shown that high VWF levels do not affect the ADAMTS13 activity measurements by residual collagen binding (Mannucci et al, 2001).

We chose to measure ADAMTS13 in pregnancy because approximately two-thirds of all TTP cases occur in females, of which 12–25% occur during pregnancy (George, 2003). In agreement with our previous observations (Mannucci et al, 2001), Sanchez-Luceros et al (2004) reported a mean ADAMTS13 activity of 61·5% ± 14·5% (n = 71) in the late period of pregnancy. In our study, mean ADAMTS13 activity was only slightly reduced, with somewhat higher antigen values, particularly in a subset of older women aged above 35 years. Women taking oral contraceptives had normal and concordant levels of both ADAMTS13 activity and antigen, indicating that the hormonal changes induced by these drugs do not change ADAMTS13, at variance with pregnancy. Previous studies have shown that the high VWF:Ag levels, as observed in pregnancy, do not significantly alter the ADAMTS13 activity results when determined by residual collagen binding (Mannucci et al, 2001).

ADAMTS13 mRNA has been detected in many organs, but the stellate cell of the liver is a major site of production (Levy et al, 2001; Uemura et al, 2005). A model for injured liver in the rat indicated no significantly altered levels of ADAMTS13 in rat plasma, despite a substantial increase of protein transcription in the injured organ (Niiya et al, 2006). On the other hand, a very recent study performed in rats showed that, while the inhibition of hepatocyte function did not affect ADAMTS13 levels, specific inhibition of stellate cell function led to a decrease in ADAMTS13 activity (Kume et al, 2007). Whether this is also the case in human cirrhotic livers is poorly understood. Lisman et al (2006) reported that levels of ADAMTS13 activity were not significantly altered in the three Child-Pugh subclasses of liver cirrhosis Their data were very much spread, with some patients having over 300% of normal ADAMTS13 levels and others presenting with extremely low values. The present study involved 90 cirrhotics and although an important degree of dispersion was confirmed, none of the patients had activity nor antigen levels above 200%. At variance with Lisman et al (2006) a significant decrease of both enzymatically active and immunoreactive ADAMTS13 was found in Child classes B and C, with a more marked drop in those patients with more severe disease. The conflicting data between the different studies may be attributed to the use of different antigen assays. Lisman et al (2006) employed a commercial kit based on polyclonal anti-ADAMTS13 antibodies. This assay resulted in a lower correlation between activity and antigen (R = 0·39) compared to our study (R = 0·80), perhaps because of the overall lower variability in both our activity and antigen measurements. It has been suggested that high plasma levels of VWF, as those found in liver cirrhosis (Table I), may influence the collagen-binding assay of ADAMTS13 activity and give spuriously low levels. Our previous in vitro studies showed that this phenomenon is not prominent, even at high endogenous VWF levels similar to those found in liver cirrhosis (Mannucci et al, 2001). The reliability of the collagen-binding assay to measure ADAMTS13 activity in liver cirrhosis, even in the presence of high VWF in plasma, was indirectly confirmed by the close concordance between the low values of ADAMTS13 activity and antigen in this study, because the ADAMTS13 antigen assay is not influenced by VWF levels. On the whole, these data establish unequivocally that ADAMTS13 is lowered in severe liver cirrhosis (albeit not in all patients), consistent with the recent studies in rats (Kume et al, 2007).

ADAMTS13 activity was previously shown to be drastically reduced in patients with inflammatory diseases characterised by CRP levels higher than 50 mg/l (Mannucci et al, 2001). The current study involved only one such patient. Nevertheless, when comparing patients with moderate inflammation (CRP >10 mg/l) and those with normal CRP, significantly lower values for both activity and antigen were observed in the former. As in most other conditions, VWF:Ag was increased in the inflammation group, indicative for endothelial activation. Despite the presence of ADAMTS13 in endothelial cells (Shang et al, 2006; Turner et al, 2006), we found no specific rise of ADAMTS13 antigen nor activity. On the contrary, our observations of low activity and antigen values might imply that ADAMTS13 is not released from these cells or that it is rapidly cleared or consumed upon release.

In 2004, we showed that cardiac surgery drastically lowered ADAMTS13 activity (Mannucci et al, 2005). This is in contrast to recent findings of Lo et al (2007), who showed that ADAMTS13 activity towards the FRETS-VWF73 substrate was increased in off-pump cardiac surgery. Our current study extends our previous findings, showing that ADAMTS13 antigen levels were decreased after cardiac surgery (both off and on-pump cases were included), yet were higher than enzymatic activity at all investigated time-points, yielding a significantly discrepant ratio. The activation of coagulation during inflammation and cardiac surgery may result in inhibition of ADAMTS13 activity through proteolytic inactivation by thrombin (Crawley et al, 2005).

In conclusion, measuring both ADAMTS13 activity and antigen is preferred over measuring only one parameter, as exemplified by the novel information obtained in this study that investigated the same conditions reported by Mannucci et al (2001). Moreover, the antigen assay used here is less prone to artifacts than the most widely used activity assays. It remains to be explained and understood whether low levels of protease antigen found in newborns, after cardiac surgery and, to a lesser extent, during pregnancy are due to inhibition of enzymatic activity, consumption of the protease or increased clearance from plasma.

Acknowledgements

  1. Top of page
  2. Summary
  3. Design and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank all donors for their readiness to enroll in this study. We also thank Dr Marina Camera (IRCCS Fondazione Monzino, Milano) who provided and allowed us to study the samples from patients undergoing cardiac surgery, Dr Fabio Mosca for the newborn plasmas, Dr Alberto D'Alberton for the pregnancy and oral contraceptive plasmas and Dr Massimo Primignani for the IBD plasmas. H.B.F. is supported by grant 21934 from the Instituut voor de Aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen. K.V. is a postdoctoral fellow from the Fonds voor Wetenschappelijk Onderzoek Vlaanderen. FP is receiving a research grant from the Italo Monzino Foundation, PMM from the Italian Ministries of Health and University.

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  1. Top of page
  2. Summary
  3. Design and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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