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 Table of Contents  
ORIGINAL ARTICLE
Year : 2012  |  Volume : 37  |  Issue : 4  |  Page : 262-267

The reliability of endothelial progenitor cells (CD34+, KDR+, and CD34+/KDR+) in the diagnosis of coronary artery disease and its severity


1 Department of Internal Medicine, Assiut University Hospital, Assiut, Egypt
2 Department of Clinical Pathology, Assiut University Hospital, Assiut, Egypt

Date of Submission12-Jan-2012
Date of Acceptance04-Feb-2012
Date of Web Publication21-Jun-2014

Correspondence Address:
Eman M. Sewify
Department of Internal Medicine, Assiut University Hospital, P.O. Box 71526, Assiut
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.7123/01.EJH.0000419284.25275.82

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  Abstract 

Background

Bone marrow-derived endothelial progenitor cells (EPCs) play an integral role in the regulation and protection of the endothelium, as well as new vessel formation. Changes in EPC number and function during coronary heart disease allow their use as a biomarker. However, so far, there is no suggested definite level at which we can diagnose coronary artery disease (CAD) or determine the severity of the disease.

Aim of the work

To assess the sensitivity, specificity, and accuracy of the circulating EPCs count to diagnose coronary heart disease and to predict the severity of the disease.

Patients and methods

The percentage and count of circulating EPCs (CD34+KDR+, CD34+, and KDR+) were measured by flow cytometry in 52 patients who underwent diagnostic angiography. The correlation between the level of the EPCs on the one hand and the presence or absence of CAD and the Gensini score calculated for each patient on the other was determined.

Results

Thirty-eight patients were found to have CAD and 14 had normal coronaries. For those with CAD, 22 had single-vessel disease and 16 had multiple vessel disease. It was found that EPCs (CD34+KDR+% and count and CD34+%) were significantly lower in patients with CAD compared with those with normal coronaries. CD34+KDR+% had an AUROC of 0.846 to diagnose CAD, with a sensitivity of 45.5%, a specificity of 100%, a positive predictive value (PPV) of 100%, a negative predictive value (NPV) of 64.3%, and an accuracy of 72.75%. CD34+% and count and CD34+KDR+ count were negatively correlated with the Gensini score. CD34+% has an AUROC of 0.802 to diagnose the Gensini score of more than 6, with a sensitivity of 100%, a specificity of 55.2%, a PPV of 69%, an NPV of 100%, and an accuracy of 77.6%.

Conclusion

EPCs decrease in patients with CAD compared with those without CAD. CD34+KDR+% can diagnose CAD with a sensitivity, specificity, PPV, NPV, and accuracy of 45.55, 100, 100, 64.3, and 72.75%. CD34% can help to exclude the presence of severe CAD with a sensitivity, specificity, PPV, NPV, and accuracy of 100, 55.2, 69, 100, and 77.6%, respectively.

Keywords: coronary heart disease, endothelial progenitor cells, flow cytometry, severity


How to cite this article:
Sewify EM, Afifi O, Soliman MM, Ziada AI. The reliability of endothelial progenitor cells (CD34+, KDR+, and CD34+/KDR+) in the diagnosis of coronary artery disease and its severity. Egypt J Haematol 2012;37:262-7

How to cite this URL:
Sewify EM, Afifi O, Soliman MM, Ziada AI. The reliability of endothelial progenitor cells (CD34+, KDR+, and CD34+/KDR+) in the diagnosis of coronary artery disease and its severity. Egypt J Haematol [serial online] 2012 [cited 2019 Dec 9];37:262-7. Available from: http://www.ehj.eg.net/text.asp?2012/37/4/262/134975


  Introduction Top


In recent years, evidence has accumulated in support of the existence of circulating progenitors for several lineages important for the cardiovascular system, including endothelial progenitor cells (EPCS), smooth muscle, and cardiomyocyte progenitor cells. EPCs are, by far, the most extensively characterized of these circulating progenitors 1. They are a scarce population of bone-derived cells that play an important role in neoangiogenesis after tissue ischemia has occurred 2.

EPC are defined by coexpression of immaturity (e.g. CD34) and endothelial [e.g. kinase domain receptor (KDR)] antigens 1. However, there is no single cell-surface marker available for the identification of EPCs 3 and there is still an ongoing debate about the definition of ‘true EPC’ as well as the availability of a reliable method to assess their quantity and quality 4.

Clinically, the number and function of EPCs may reflect the balance between endothelial integrity and repair, and both measures have been suggested as surrogate markers of endothelial function and cardiovascular diseases 5. Changes in the EPC number and function during coronary heart disease allow their use as a biomarker 3. However, so far, there is no suggested definite level at which we can diagnose coronary artery disease (CAD) or determine the severity of the disease.

Aim of the work

To assess the sensitivity, specificity, and accuracy of the count of EPCs (CD34+, KDR+, and CD34+KDR+) to diagnose CAD and to assess the severity of the disease.


  Patients and methods Top


Fifty-two patients undergoing diagnostic cardiac catheterization at Assiut University hospital were eligible for enrollment in the study. Patients’ clinical characteristics were recorded. Cardiac catheterization was carried out using standard techniques. Coronary angiograms were interpreted by attending cardiologists blinded to the results of the EPC assay.

Identification and quantification of circulating endothelial progenitor cell by flow cytometry

Three subpopulations of EPCs were determined by flow cytometry: CD34+, KDR+, and CD34+KDR+ cells.

Blood samples were drawn by venipuncture before coronary angiography. Fasting venous blood was collected in tubes with EDTA and processed within 2 h of collection. Peripheral blood mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation. Peripheral blood progenitor cells were analyzed for the expression of cell-surface antigens with direct two-color analysis using labeled monoclonal antibodies. Fluorescein isothiocyanate (FITC)-labeled anti-CD34 [immunoglobulin G1 (IgG1)-FITC isotype control] and phycoerythrin (PE)-labeled anti-KDR (IgG1–PE) were obtained from BD Biosciences (BD Biosciences, San Jose, California, USA).

For the analysis of the samples, 100 µl of peripheral blood mononuclear cells in PBS were incubated with anti-KDR-PE (10 µl) and anti-CD34-FITC (10 µl) for 15 min at room temperature. Flow cytometry measurement was carried out using the appropriate fluorescence compensation and setting excluding debris and platelets, and the numbers of CD34+, KDR+, and CD34+/KDR+ cells were analyzed in the lymphocyte and monocyte gates (mononuclear cells). At least 10.000 events were measured within the myelomonocytic gate. Respective PE-conjugated and FITC-conjugated isotype control antibodies from the same manufacturers served as controls. Cells were measured using appropriate fluorescence compensation and light scatter gating by FACS Calibur flow cytometry (BD Biosciences). Analysis was carried out using fluorescence-1/fluorescence-2 dot plot quadrant statistics and manual gating (Cell Quest Pro software, Becton Dickinson, BD Biosciences) using a blinded approach to patient characteristics. The percentage of positive cells was converted into absolute numbers of cells/mm3 using the white blood cell count. Formula: absolute cell count=EPC% total×WBC/100 6.

Evaluation of the severity of coronary artery disease

CAD was evaluated according to Gensini score 7. This depends on the degree of luminal narrowing and the importance of the site of coronary stenosis. According to the score, the narrowing of the lumen of the coronary arteries is graded as follows: 1 point: ⩽25%; 2 points: 26–50%; 4 points: 51–75%; 8 points: 76–90%; 16 points: 90–99%; and 32 points for total occlusion. In addition, this primary score is multiplied by a factor that takes into account the importance of the coronary artery containing the lesion (5 for the left main coronary, 2.5 for the proximal portion of the left anterior descending artery or the proximal left circumflex artery, and 1.5 for the middle region, 1 for the distal left anterior descending artery, and 1 for the mid-distal region of the left circumflex artery or the right coronary artery). The sum of the total score obtained was used for statistical analysis.

A patient was considered to have multivessel disease if he/she had a significant stenosis (≥75%) of 2 or more major coronary arteries or greater than 50% narrowing of the left main coronary artery 8.

Exclusion criteria

Patients with malignant disease, peripheral vascular disease, recent (<2 months) acute coronary syndrome (ACS), myocardial infarction, inflammation, bleeding, or blood transfusion were not included in the study as these conditions and procedures could have influenced the number of EPCs. All patients enrolled provided an informed consent. The study was approved by the ethical committee of Assiut University Hospital.

Statistical analysis

Data are expressed in mean±SD. Values were considered statistically significant for P values up to 0.05. The difference in means was assessed using an independent two-sample student t-test. The correlation between the levels of each EPCs group and the Gensini score was examined by partial correlation adjusted for possible factors that may affect the EPCs level. To measure the sensitivity and specificity of CD34+KDR+, CD34+, and CDKDR+ cells to diagnose CAD, a conventional receiver–operator curve (ROC) curve was generated using individuals with normal angiography as controls. The area under curve (AUROC) was calculated to determine the quality of EPC values as a biomarker. An area of 0.5 is no better than expected by chance, whereas a value of 1.0 indicates a perfect biomarker. The SPSS version 10 (SPSS Inc., Chicago, Illinois, USA) software was used for statistical analysis.


  Results Top


Fifty-two patients of those who underwent diagnostic angiography were enrolled. Their mean age of the patients was 54.3±7.9 years (range: 30–66 years). Twenty-one patients were diabetic and 20 were hypertensive. Twelve of our enrolled patients were women. Angiography was found to be normal in 14 individuals (normal coronary artery group), whereas 38 patients had angiographic evidence of CAD (CAD group).

The mean±SD values of CD34+, CD34+KDR+, KDR+ percentages, and absolute counts of cells in patients with and without angiographic evidence of CAD are presented in [Table 1].
Table 1: Percentages and absolute counts of CD34+, CD34+KDR+, and KDR+ cells in patients with and without coronary artery disease

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Patients who were found to have CAD had a significantly lower percentage (lower by 60%) and count of CD34+KDR+ cells (lower by 38.6%) as well as a lower percentage of CD34+ (lower by 42%) compared with those with normal angiography [Figure 1].
Figure 1: Flow cytometry quantification of EPCs. (a) Control: UL (CDKDR+): 0.05%, UR (CD34+KDR+): 0.11, LR (CD34+): 0.12. (b) Patient: UL (CDKDR+: 0.01), UR (CD34+KDR+): 0.06, LR (CD34+): 0.05. EPCs, endothelial progenitor cells.

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The AUROC of CD34+KDR+ cells% to diagnose CAD was 0.846 [Figure 2], [Table 2]. At a cut-off of 0.025% or less of CD34+KDR+ cells, the diagnosis of CAD can be made with a sensitivity of 45.5% and a specificity of 100%. At a higher cut-off value (0.08%), a diagnosis of CAD can be made with a sensitivity of 100% and a specificity of 40%. The CD34+KDR+ cell count can diagnose CAD with an AUROC of 0.653 with a sensitivity of 33.3% and a specificity of 100% at a cut-off value of 1.8 cells/mm3 and a sensitivity of 70.8% and a specificity of 55.6% at a cut-off value of 3.3 cells/mm3.
Figure 2: AUROC for CD34+KDR+ cells percentage for diagnosing CAD (AUROC: 0.846). AUROC, area under the receiver–operator curve; CAD, coronary artery disease.

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Table 2: Diagnostic reliability of CD34+KDR+% and count in diagnosing patients with coronary artery disease

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From the above results, it is clear that a patient with a CD34+KDR+% of 0.025% or less may be diagnosed as having CAD with a positive predictive value (PPV) of as high as 100% and a percentage of CD34+KDR+ of more than 0.08% and may exclude the presence of CAD with a negative predictive value (NPV) of 100%.

Relation between the vessel number and endothelial progenitor cells

Comparison of the count and percentage of EPCs in patients with single-vessel disease with that in patients with multivessel disease showed a statistically insignificant decrease in EPCs cells [Table 3] in patients with multivessel disease compared with those with single-vessel disease.
Table 3: Percentages and absolute counts of CD34+, CD34+KDR+, and KDR+ cells in patients with single-vessel and multivessel disease

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Relation between endothelial progenitor cells and the Gensini score

After controlling for age, hypertension, diabetes mellitus, and vessel number, the Gensini score was correlated negatively with CD34+% (r=−0.39, P=0.002), CD34+ count (r=−0.29, P=0.024), and CD34+KDR+% (r=−0.27, P=0.032). However, no correlation of significance was found between CD34+KDR+ count, KDR%, or count with the Gensini score.

AUROC of CD34+KDR+% for diagnosing a Gensini score of 20 or more is 0.746 and at a CD34+KDR+% level of 0.015% or less with a sensitivity of 75%, a specificity of 75%, a PPV of 75%, an NPV of 75%, and an accuracy of 75% [Table 4], [Figure 3]a.
Figure 3: Left (a) AUROC (0.746) for the CD34+KDR+% to diagnose a patient with a Gensini score of at least 20 right (b) AUROC (0.802) for the level of CD34% for the diagnosis of a Gensini score of at least 6. AUROC, area under the receiver–operator curve.

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Table 4: Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of CD34+KDR+% for diagnosing a Gensini score of 20 or more

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AUROC of CD34% for diagnosing a Gensini score of 6 or more is 0.802 and at a CD34% level of 0.025%, the diagnosis can be made with a sensitivity of 100%, a specificity of 55.2%, a PPV of 69%, an NPV of 100%, and an accuracy of 77.6%. At a CD34% level of 0.005%, the diagnosis can be made with a sensitivity of 50%, a specificity of 96.2%, a PPV of 93%, an NPV of 65.8%, and an accuracy of 73.1% [Table 5] and [Figure 3]b. This means that in a patient with a CD34% level above 0.025%, we can exclude severe CAD and with a level of 0.005% or less, the presence of CAD with a score of 6 or more can be detected with a PPV of 93%.
Table 5: Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of CD34+% for diagnosing a Gensini score of 6 or more

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  Discussion Top


In this study, we examined the ability of EPCs, namely, CD34+KDR+ cells, CD34+, and KDR+ cells, to diagnose the presence of coronary heart disease and to assess the severity of disease.

The current study showed that patients with CAD had significantly lower values of CD34+KDR+% and absolute count compared with those with normal angiography. CD34+% was also significantly lower in patients with CAD. This was generally in agreement with the previously reported results. Liu et al. 9 and Wang et al. 10 reported that the number of EPCs (CD133+KDR+) in patients with CAD was significantly lower than those with a normal coronary artery. Ruda et al. 11 evaluated the number of CD34+ cells in patients with ischemic heart disease. They found that patients with stable angina and ACS had significantly lower levels of circulating CD34+ cells compared with the controls. This observation was more evident in ACS. Bozdag-Turan et al. 12 suggested that the frequency of CD34+/45+ BM-derived EPCs in peripheral blood is impaired in patients with ischemic heart disease.

To our knowledge, the current study is the first to calculate a cut-off value of the EPCs to diagnose CAD. A cut-off value for CD34+KDR+% of 0.025% or less may diagnose CAD with 45.5% sensitivity and 100% specificity. This might be very valuable in limiting invasive diagnostic techniques, but these results still need confirmation by extrapolating the study to a higher number of patients.

Several potential explanations for the association between low EPC counts and CAD could be suggested. Ruda et al. 11 suggested that CD34+ cells contribute toward the repair of injured endothelium and the reduction in their numbers in the peripheral blood indicates an endothelial state. Kunz et al. 8 concluded that a low EPC count may be merely a marker for the presence of other cardiac risk factors and previous studies have reported an inverse association between EPC counts and traditional risk factors. Another explanation was that the same harmful processes that contribute to atherosclerosis (diabetes, aging, inflammation, etc.) also suppress bone marrow production of EPCs or shorten EPC survival in circulation; the observed association could be found without any actual causal relationship between EPC levels and CAD 8.

Kunz et al. 8 found that EPC counts were independently predictive of the severity of coronary disease after controlling for the known risk factors. In agreement with these authors and with Wang et al. 10, the present study showed a statistically significant negative correlation between the Gensini score and the levels of CD34+ and CD34+KDR+ cells. Furthermore, the current study suggested a cut-off value for the percentage of CD34+KDR+ and CD34+ that can diagnose a Gensini score of 20 or more and 6 or more, respectively. Using these values, the presence of severe CAD can be excluded with CD34% of more than 0.025% with an NPV of 100% (thus a lot of intervention may be saved) and a Gensini score of 6 or more is diagnosed with 93%PPV if CD34% is 0.005% or less.

Schmidt-Lucke et al. 2 reported a decrease of EPCs in patients with multivessel disease compared with those with single-vessel disease. In the current study, although a similar finding was obtained, the decrease was not statistically significant.

Jimenez-Navarro et al. 13 studied the number of EPCs CD34+, CD34+/133+, CD34+/KDR+, and CD34+/133+/KDR+/45+ (weak) and angiogenic cytokines in patients admitted for a first myocardial infarction with ST segment elevation. Higher number of EPCs and angiogenic cytokines were detected compared with the controls. The levels of these cell subsets were higher in the patients with single-vessel disease, although the release of endothelial progenitor growth factors was similar in the two groups. They showed the mobilization of a higher number of peripheral blood progenitor cells in patients who have a first acute myocardial infarction and single-vessel disease compared with patients with a first infarction and multivessel disease, as documented by coronary angiography. Jimenez-Navarro et al. 13 suggested that the lower number in EPC in patients with multivessel disease could be the consequence of the loss of capacity for vascular regeneration and the release of a sufficient number of EPC, described in patients with arteriosclerotic disorders

Kocaman et al. 6 concluded that EPCs can be mobilized from the bone marrow to induce coronary collateral growth in case of myocardial ischemia even in the presence of vascular risk factors and extensive atherosclerosis. Gunetti et al. 14 reported that the use of BM-derived CD34+ cells represents a promising strategy for a successful cell-based therapy of acute myocardial infarction. Losordo et al. 15 found that patients with refractory angina who received intramyocardial injections of autologous CD34+ cells experienced significant improvement in angina frequency and exercise tolerance. Quyyumi et al. 16 reported that an infusion of CD34+ cells into the infarct-related artery during the repair phase is associated with a significant improvement in perfusion that may limit deterioration in cardiac function after acute myocardial infarction.


  Conclusion Top


CD34+KDR+ decrease in patients who have CAD compared with those who do not have CAD. A cut-off value of 0.025% for the percentage of CD34+KDR+ or less can diagnose CAD with a sensitivity of 45.5%, a specificity of 100%, a PPV of 100%, an NPV of 64.3%, and an accuracy of 72.75%. CD34+KDR+% at a cut-off value of 0.015 or less can diagnose a Gensini score of 20 or more with a sensitivity of 75% and a specificity of 75%. CD34% can help to exclude the presence of severe CAD with a sensitivity of 100%, a specificity of 55.2%, a PPV of 69%, an NPV of 100%, and an accuracy of 77.6%.

Limitations of the study

The number of patients included in the study is small and this might be a limiting factor for statistical results. This study should be extended to include a larger number of patients to test the reliability of this cut-off value for diagnosing CAD and detecting its severity.[16]

 
  References Top

1.Fadini GP, Albiero M, Menegazzo L, Boscaro E, Agostini C, de Kreutzenberg SV, et al. Procalcific phenotypic drift of circulating progenitor cells in type 2 diabetes with coronary artery disease. Exper Diab Res. 2012;92:1685  Back to cited text no. 1
    
2.Schwartzenberg S, Afec A, Charach G, Rubinstein A, Ben-Shoshan Y, Kissil S, et al. Comparative analysis of the predictive power of different endothelial progenitor cell phenotypes on cardiovascular outcome. World J Cardiol. 2010;2:299–304  Back to cited text no. 2
    
3.Sen S, McDonald P, Coates P, Bonder C. Endothelial progenitor cells: novel biomarker and promising cell therapy for cardiovascular disease. Clin Sci. 2011;120:263–283  Back to cited text no. 3
    
4.Schmidt-Lucke C, Fichtlscherer S, Aicher A, Tschöpe S, Schultheiss H, Zeiher A, Dimmeler S. Quantification of circulating endothelial progenitor cells using the modified ISHAGE protocol. PLoS One. 2010;5:e13790  Back to cited text no. 4
    
5.Hamed S, Brenner B, Roguin A. Nitric oxide: a key factor behind the dysfunctionality of endothelial progenitor cells in diabetes mellitus type-2. Cardiovasc Res. 2011;91:9–15  Back to cited text no. 5
    
6.Kocaman SA, Yalcin MR, Yagci M, Sahinarslan A, Turkglu S, Arslan U, et al. Endothelial progenitor cells (CD34+KDR+) and monocytes may provide the development of good coronary collaterals despite the vascular risk factors and extensive atherosclerosis. Anadolu Kardiyol Derg. 2011;11:290–299  Back to cited text no. 6
    
7.Montorsi P, Ravagnanil P, Gallil S, Rotatori F, Veglia F, Brignati A, et al. Association between erectile dysfunction and coronary artery disease. role of coronary clinical presentation and extent of coronary vessels involvement: the COBRA trial. Eur Heart J. 2006;27:2613–2614  Back to cited text no. 7
    
8.Kunz G, Liang G, Cuculi F, Gregg D, Vata K, Shaw L, et al. Circulating endothelial progenitor cells predict coronary artery disease severity. Am Heart J. 2006;152:190–195  Back to cited text no. 8
    
9.Liu YR, Chen JZ, Wang XX, Zhu JH, Xie XD, Sun J. Changes of endothelial progenitor cells from peripheral blood in patients with coronary heart diseases. Zhejiang Da Xue Xue BAo Yi Xue Ban. 2005;34:163–166  Back to cited text no. 9
    
10.Wang H, Gao P, Ji K, Shen W, Fan C, Lu L, Zhu D. Circulating endothelial progenitor cells, C-reactive protein and severity of coronary stenosis in Chinese patients with coronary artery disease. Hypertens Res. 2007;30:133–141  Back to cited text no. 10
    
11.Ruda MM, Arefeva TI, Tripoten MI, Balakhonova TV, Parfenova EV, Karpov IuA. Circulating endothelial progenitor cells and vascular endothelial dysfunction. Ross Fizio Zh Im I M Sechenova. 2009;95:545–562  Back to cited text no. 11
    
12.Bozdag-Turan L, Turan RG, Turan CH, Ludovicy S, Akin I, Kische S, et al. Relation between the frequency of CD34+ bone marrow derived circulating progenitor cells and the number of diseased coronary arteries in patients with myocardial ischemia and diabetes. Cardiovasc Diabetol. 2011;10:107–115  Back to cited text no. 12
    
13.Jimenez-Navarro M, Gonzalez F, Caballero-Borrego J, Marchal J, Rodriguez-Losada N, Carrillo E, et al. Coronary disease extension determines mobilization of endothelial progenitor cells and cytokines after a first myocardial infarction with ST elevation. Rev Esp Cardiol. 2011;64:1123–1129  Back to cited text no. 13
    
14.Gunetti M, Noghero A, Molla F, Staszewsky LI, de Angelis N, Soldo A, et al. Ex vivo-expanded bone marrow CD34(+) for acute myocardial infarction treatment: in vitro and in vivo studies. Cytotherapy. 2011;13:1140–1152  Back to cited text no. 14
    
15.Losordo DW, Henry TD, Davidson C, Sup Lee J, Costa MA, Bass T, et al. Intramyocardial autologus CD34+ ce; therapy for refractory angina. Circ Res. 2011;109:428–436  Back to cited text no. 15
    
16.Quyyumi AA, Waller EK, Murrow J, Esteves F, Galt J, Oshinski J, et al. CD34(+) cell infusion after ST elevation myocardial infarction is associated with improved perfusion and is dose dependent. Am Heart J. 2011;161:98–105  Back to cited text no. 16
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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