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 Table of Contents  
ORIGINAL ARTICLE
Year : 2013  |  Volume : 38  |  Issue : 4  |  Page : 130-135

DNA nucleosomal fragments have a therapeutic significance in adult acute myeloid leukemia


1 Cytogenetic Unit, Clinical Pathology Department, Faculty of Medicine, Zagazig University, Egypt
2 Medical Oncology Department, Faculty of Medicine, Zagazig University, Egypt

Date of Submission16-Apr-2013
Date of Acceptance16-Jul-2013
Date of Web Publication19-Jun-2014

Correspondence Address:
Nashwa M.A. Alazizi
Cytogenetic Unit, Clinical Pathology Department, Faculty of Medicine, Zagazig University
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.7123/01.EJH.0000434282.48881.59

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  Abstract 

Aim

Nucleosomal DNA levels in plasma and serum can be correlated with the extent of cell death at a specific time point. The aim of this study was to evaluate the serum level of nucleosomal DNA fragments as an early predictive marker for acute myeloid leukemia (AML) therapy.

Materials and methods

This study included 77 participants: 14 healthy volunteers, included in the control group; they ranged in age between 20 and 55 years, median age 36.5 years. Routine investigations such as liver functions, blood urea, serum creatinine, and complete blood count were performed to confirm their healthy state. The patient group included 63 de-novo AML patients.

Results

In the patient group, serum nucleosome levels ranged between 25 and 760 AU, with a mean±SD of 203.7±197 and a median of 220. In the control group, serum nucleosome levels ranged between 38 and 99 AU, with a mean±SD of 72.8±20.8 and a median of 80; a statistically significant difference was found between nucleosome levels in patients and its level in the control group (P<0.001). In the patient group, the lactate dehydrogenase (LDH) level ranged between 189 and 2088 U/l, with a mean±SD of 714±413 and a median of 829. In the control group, the LDH level ranged between 100 and 430 U/l, with a mean±SD of 231±125 and a median of 195; a statistically significant difference was found between LDH levels in patients and its level in the control group (P<0.001).

Conclusion

It would be valuable to include these markers together with the kinetics of circulating nucleosomal DNA fragments in prospective trials to elucidate their potential additive role in the early prediction of response to therapy. In AML patients, the changes in nucleosomal DNA during the initial phase of induction chemotherapy are valuable markers for the early estimation of therapy response and should be validated in further prospective trials.

Keywords: acute myeloid leukemia, nucleosome, therapy


How to cite this article:
Alazizi NM, Hefny AM. DNA nucleosomal fragments have a therapeutic significance in adult acute myeloid leukemia. Egypt J Haematol 2013;38:130-5

How to cite this URL:
Alazizi NM, Hefny AM. DNA nucleosomal fragments have a therapeutic significance in adult acute myeloid leukemia. Egypt J Haematol [serial online] 2013 [cited 2019 Dec 8];38:130-5. Available from: http://www.ehj.eg.net/text.asp?2013/38/4/130/134779


  Introduction Top


Chronic acute myeloid leukemia (AML) is a heterogeneous group of hematological disorders characterized by a block in the terminal differentiation of particular hematopoietic cell lineage; it is the most common type of acute leukemia in adults 1.

Several prognostic parameters play a major role in predicting prognosis in newly diagnosed AML patients. In particular, age, cytogenetics, and early blast clearance have been found to be significantly related to complete remission rate and survival 2. High amounts of bone marrow (BM) blasts before therapy are associated with adverse outcome as well as incomplete elimination during the course of chemotherapy. Lactate dehydrogenase (LDH) and leukocyte number are further markers that are relevant for prognosis. However, additional and more precise markers are warranted to estimate the response to therapy as early as possible 3.

The apoptotic process by cytotoxic drugs is started by direct damage of DNA, interference in the cell cycle 4, or by the activation of apoptotic receptors such as Fas/Apo1/CD95. Subsequently, a cascade of enzymes is activated that results in the degradation of the cellular structure and cleavage of the chromatin into mononucleosomes and oligonucleosomes by various caspases and nucleases 5.

These nucleosomes are packed into apoptotic bodies and are engulfed by neighboring cells and macrophages. However, a considerable part is released into circulation, particularly in high rates of cell death 6. Thus, nucleosomal DNA levels in plasma and serum can be correlated with the extent of cell death at a specific time point 7.

Many studies on circulating nucleic acids in serum and plasma showed their high potential for the diagnosis of cancer and measurement of circulating DNA fragments can be performed in plasma and serum by various methods including real-time PCR and enzyme-linked immuosorbent assay (ELISA) 8. Healthy individuals and patients with benign diseases show lower levels of nucleosomal DNA in their circulation than individuals with malignant diseases 8.

The main aim of this study was to evaluate the serum level of nucleosomal DNA fragments as an early predictive marker for AML therapy.


  Materials and methods Top


This study was carried out in the Clinical pathology Department and Hematology and Medical Oncology Unit of Internal Medicine Department, Faculty of Medicine, Zagazig University Hospital, between 2009 and 2011. The study included 77 participants, 14 healthy volunteers as controls and 63 patients, and they were classified into a control group (which included 14 apparently healthy volunteers ranging in age between 20 and 55 years, median age 36.5 years, and routine investigations such as liver functions, blood urea, serum creatinine, and complete blood count were performed to confirm their healthy state) and a patient group (which included 63 de-novo AML patients who were admitted at the Hematology & Medical Oncology Department ranging in age between 21 and 58 years, median age 41.6 years, 41 men and 22 women, male to female ratio 1.9 : 1). All the patients studied were subjected to assessment of complete medical history and complete clinical examination, with a special focus on age, sex, presence of leukemia-associated symptoms and signs (fever, pallor, bleeding tendency, and bone ache), onset of illness, and previous chemotherapy exposure. The presence and extent of leukemic involvement including purpuric eruptions, size of liver and spleen, lymphadenopathy, and central nervous system infiltration was also assessed.

Routine laboratory investigations were performed (complete blood count, ALT, AST, albumin, bilirubin, total protein, PT/INR, PTT, blood urea, serum creatinine), BM aspiration was performed by Leishman-stained smears, immunophenotyping by a flow cytometer using fluorescent activate cell-sorting, and cytogenetic analysis by karyotyping using the G-banding technique. Nucleosomal DNA fragments were assessed by quantitative determination of nucleosomes in serum using cell death detection ELISA (plus) supplied by Roche Diagnostic (Roche Diagnostics Inc., Mannheim, Germany). Blood samples were taken 1 day before the application of chemotherapy, and then 1 day after the chemotherapy and day 14 after chemotherapy 8. The above methods were approved by the local ethical committee and informed consent from patients was taken before the study.

Statistical analysis

The data were tabulated and statistically analyzed using Epi-INFO (Centers for Disease Control and Prevention, Atlanta, Georgia, USA)(2005) and statistical package for social sciences (SPSS Inc., Chicago, Illinois, USA).


  Results Top


This study included 63 de-novo AML patients ranging in age from 21 to 58 years, mean±SD 41.6±10.9, male : female ratio 1.9 : 1, and 14 healthy volunteers as controls ranging in age between 20 and 55 years, mean±SD 38.9±10.5, during the period from 2009 to 2011 [Table 1].
Table 1: Demographic data of the studied groups

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FAB classification and cytogenetics

There were different types of AML according to the FAB classification; M2 was seen in 18 cases (28.6%), M4 was seen in 30 cases (47.6%), and M5 was seen in 15 cases (23.8%). There were different cytogenetics; 33 cases (52.4%) had normal karyotyping but hyperploidy was seen in nine cases (14.3)%), trisomy 22 was seen in six cases (9.5)%), trisomy 8 was seen in three cases (4.7%), and it failed in 12 cases (19.1%) [Table 2]. M3 cases were excluded initially from the study.
Table 2: FAB and cytogenetic data of AML patients

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Serum nucleosomes

In the patient group, serum nucleosome levels ranged between 25 and 760 AU, with a mean±SD of 203.7±197 and a median of 220. In the control group, serum nucleosome levels ranged between 38 and 99 AU, with a mean±SD of 72.8±20.8 and a median of 80; a statistically significant difference was found between nucleosome levels in patients and their levels in the control group (P<0.001, [Table 3].
Table 3: Comparison of nucleosomes and LDH between the patient and the control group

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Lactate dehydrogenase

In the patient group, the LDH level ranged between 189 and 2088 U/l, with a mean±SD of 714±413 and a median of 829. In the control group, the LDH level ranged between 100 and 430 U/l, with a mean±SD of 231±125 and a median of 195; a statistically significant difference was found between LDH levels in patients and their levels in the control group (P<0.001, [Table 3].

Adult AML in remission is defined as a normal peripheral blood cell count (absolute neutrophil count>1000 /mm3 and platelet count>100 000 /mm3), normocellular marrow with less than 5% blasts in the marrow, and none of them can have a leukemic phenotype (e.g. Auer rods), with no signs or symptoms of the disease and absence of extramedullary leukemia (e.g. central nervous system or soft tissue involvement) 9.

On the basis of these remission criteria, patients were classified according to their response into those who achieved a complete response and those who showed no response. In this study, 36 (57.2%) of 63 patients showed a complete remission in the peripheral blood and BM after induction chemotherapy. The remaining 27 (42.8%) AML patients showed no response to therapy and nine (14.3%) of them died because of treatment-induced BM hypoplasia and/or infection [Table 2].

In the overall analysis of variance, levels of nucleosomal DNA fragments were dependent on both variables, therapy response and time after start of chemotherapy, respectively. Significantly higher values of nucleosomal DNA were found on day 1 after therapy [Table 4] and [Figure 1].
Table 4: Levels of nucleosomal DNA in AML patients during the first week of induction therapy

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Figure 1: Difference between increased level of nucleosomes in responding (a) and nonresponding patients (b). There was a significant difference between increased level of nucleosomes in responding and nonresponding patients after therapy.

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In patients showing a complete response 36 (57.2%), DNA concentrations increased temporarily immediately after the start of the therapy, but the values decreased immediately in nonresponsive patients, 18 (28.5%). Thus, there was a significant difference between complete response and patients showing no response, and this difference was found in particular on day 1 after therapy (P<0.001) [Table 5], [Figure 1].
Table 5: Comparison of nucleosomal DNA fragment (AU) with therapy response

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Also, in the patients who showed a complete response, there was a highly significant difference between 1 day before therapy and 1 day after therapy, a significant difference between day 1 after therapy and day 7 after therapy, and no significant difference between 1 day before therapy and day 7 after therapy [Table 6]. Both patients showing a complete response and those who showed no response could be separated with a sensitivity of 66% and a specificity of 100% at day 1 after therapy.
Table 6: Paired t-test for comparison of nucleosomal DNA fragment (AU) in complete response patients

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Leukocytes showed an obvious dependence on time after start of chemotherapy but not on therapy response [Table 7] and [Table 8]. However, with respect to the percentual changes from 1 day before therapy to day 1 after therapy, a significant difference was observed (P<0.001) [Table 8].
Table 7: Comparison of peripheral leukocytic count, peripheral blasts, Hb, and platelet count during the first week of induction therapy

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Table 8: Comparison of LDH, TLC, and BM blasts with therapy response

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Peripheral blasts level showed a significant decrease during the first week (P<0.001) [Table 7]. Levels of LDH showed a significant change on day 1 after therapy (P<0.001) [Table 7] but not dependent on therapy response [Table 8].

The BM blast numbers on days 1 and 28 showed a significant decrease [Table 8], their percentage changes were comparable in both response groups, and showed a significant correlation with therapy response (P<0.001, Mann-Whitney=11.6) [Table 8].

Correlation study

A correlation study was carried out between serum nucleosomal levels and total leukocytic count (TLC), peripheral blasts, and BM blast. A positive correlation was found for the levels of nucleosomal DNA fragments with leukocytes at day 1 after therapy (P<0.05, r=0.45) as well as with the decrease of leukocytes from the day before therapy to day 1 after therapy (P<0.001). There was a negative significant correlation between serum nucleosome levels and peripheral blasts at day 7 after therapy (r=−0.15).

Nucleosomal DNA levels showed an inverse correlation with BM blast number after 28 days (r=−0.29; P=0.012). There was no significant correlation between serum nucleosome levels and LDH. Because cytogenetics and immunophenotype were highly heterogeneous, correlations with therapy response were not performed.


  Discussion Top


AML is the most common type of acute leukemia in adults 1. AML is a malignant neoplasm of hematopoietic cells that results from clonal transformation of hematopoietic precursors through the acquisition of chromosomal rearrangements and multiple gene mutations.

Response to induction therapy has long been recognized as a major independent prognostic factor in AML, predicting the risk of relapse and overall survival; thus, early response assessment may be required in investigational studies to evaluate the antileukemic efficacy of a novel agent or to guide the subsequent treatment 10.

Typical products of cell death are nucleosomes, complexes that are formed from a core particle of several histone components and DNA on the outside. Linker DNA connects the nucleosomes to a chain-like structure. During cell death, endonucleases bind preferentially to these easily accessible linking sites between the nucleosomes and cut the chromatin into multiple mononucleosomes and oligonucleosomes 11.

Previous investigations on the concentrations of nucleosomes in serum showed low levels in healthy individuals 8. In contrast, high amounts were found in patients with benign pathological conditions such as severe inflammations, trauma stroke, myocardial infarction, diabetes, and autoimmune diseases 12 and in patients with various malignant tumors such as solid tumors, leukemia, or lymphoma 13.

Nucleosomes can be used as an early marker for diagnosis of various tumors through an increase in the total amount of circulating DNA. Many studies on circulating nucleosomes in serum and plasma have shown their role in diagnosis such as Hashem 14, who found that serum nucleosomes show a significant increase in breast cancer patients as compared with healthy individuals, and it was the most sensitive marker but the least specific one versus CEA and CA15-3.

Hohaus et al. 15 observed that lymphoma patients frequently have elevated levels of cell-free circulating DNA at diagnosis and there is an association between DNA concentrations and a number of clinical parameters indicating a worse prognosis with older age, advanced stage of disease, and elevated LDH, suggesting that circulating DNA may reflect an active proliferating disease.

However, Gahan 16 reported that nucleosomes were less suitable for cancer diagnosis because of elevated levels of nucleosomes in patients with benign diseases.

Circulating nucleosomes can be used in monitoring treatment when DNA levels will return to normal upon treatment 16. Therapy outcome can be indicated by nucleosomal levels during the first week of chemotherapy and radiotherapy in patients with solid tumors as well as hematological malignancies. Thus, nucleosomes may play a strong role in early detection of therapy response 13.

The present study was carried out on 63 newly diagnosed AML patients (age range 21–58 years) and 14 apparently healthy individuals (age range 20–55 years) as controls. The de-novo AML patients received induction chemotherapy of a combination of an anthracycline and cytarabine (ara-C), with the anthracycline often administered for 3 days and ara-C administered for 7 days at 100 mg/m2/day as a continuous infusion, with daunorubicin administered at 45 mg/m2/day for 3 days, and this is considered the standard regimen and is referred to as the (3+7) regimen. Thirty-three patients of 63 one showed a complete response (57.2%) in the peripheral blood and BM after induction chemotherapy. The remaining 27 AML patients showed no response to therapy (42.8%) and nine of them died because of treatment-induced BM hyperplasia.

The concentration of nucleosomal DNA fragments was measured during the first week of induction therapy, at day 1 before therapy, day 1 after therapy, and day 7 after therapy using the ELISA technique, which is easy to handle and cost effective.

It was found that the serum values decreased significantly in all patients during this time frame. However, in patients who later achieved complete remission, levels increased at day 1 after therapy, followed by a rapid and continuous decrease compared with those who showed an insufficient therapy response as nucleosomal levels in nonresponders showed an early decline. This allows the prediction of a favorable therapy response at a sensitivity of 66% and a specificity of 100% at day 1 after induction therapy.

Our results are in agreement with those of Muller et al. 17, whose study included 25 patients and reported that the nucleosome levels were significantly higher during the initial phase of induction therapy, especially during days 2–4 in responder patients than in nonresponder patients, reflecting the immediate effect of therapy, and found that favorable therapy response could be predicted with a sensitivity of 56% and a specificity of 100% at days 2–4 after induction therapy.

Also, it was confirmed by Schwarz et al. 18, who studied plasma DNA levels for the evaluation of minimal residual disease and as useful tools for prognostic purposes and for early detection of subclinical disease recurrence in children with acute lymphoblastic leukemia. This study observed that initially, plasma DNA was elevated upon treatment and decreased rapidly. This decrease paralleled the decline in peripheral blood leukocyte numbers.

Other studies were carried out to detect the role of nucleosomes in the prediction of therapy response in various solid tumors such as lung cancer, one of which was Holdenrieder and Stieber 8, who found that patients who went into remission had significantly lower nucleosome levels before therapy than patients with no change or progression. High levels and insufficient decreases in nucleosome levels during the course of chemotherapy indicated poor outcome; thus, monitoring of plasma nucleosome levels during the course of first-line chemotherapy could identify patients who are likely to have insufficient response to therapy and disease progression at an early stage.

This is in agreement with Holdenrieder and Stieber 8, who studied therapy response monitoring in various malignancies such as lung cancer, colorectal cancer, lymphoma, and other cancers and found that circulating nucleosomes increased rapidly 1–3 days after the start of therapy and decreased in the treatment-free period. Efficient reduction in tumor volume was correlated with a lower release of nucleosomes into the circulation with a rapid increase in the initial phase of therapy, but insufficient therapy of resistant or more aggressive tumors led to higher concentrations of nucleosomes at the end of the first cycle.

In this work, in terms of the correlation between serum nucleosomes levels and the different parameters studied in AML patients, there was a significant correlation between serum nucleosomes and total leukocyte number in the initial phase of therapy and this is in agreement with Mueller et al. 17, who identified a positive correlation between the level of nucleosomal DNA fragments and leukocytes at day 1 after therapy. Although there was no significant difference in the TLC between responsive and nonresponsive patients, the relative changes of TLC from day 1 before therapy to day 1 after therapy showed significant changes. Also, this is in agreement with Schwarz et al. 18, who found a positive correlation between the concentration of plasma DNA from children with acute lymphoblastic leukemia and the number of TLC.

In AML, malignant blasts are present in the blood circulation and BM and constitute the target for chemotherapeutic agents. High rates of apoptotic cells and thus high levels of nucleosomal DNA were passing directly into blood circulation, corresponding with sufficient response to the respective treatment, whereas low levels correlated with the relative resistance of target cells to undergo apoptosis. As nucleosomal DNA is already released 12–24 h after the apoptotic event and as they are removed rapidly from circulation under physiological conditions, the early increase, followed by a rapid decrease during the induction therapy might reflect the effective elimination of leukemic cells 19.

Serum nucleosomes also showed an inverse correlation with peripheral blasts at the end of the induction therapy. The peripheral blasts showed significant changes from 1 day before therapy to day 1 after therapy to day 7 and this is in agreement with Mueller et al. 17 Thus, it seems reasonable that the higher levels of circulating nucleosomal DNA fragments are mainly the result of effective blast reduction.

Comparison of circulating nucleosomal DNA with already established proliferation and cell death markers such as LDH, which is already in clinical use for follow-up of patients with leukemia or lymphomas, was carried out and showed slightly higher concentrations in patients with a complete response during chemotherapy, with no significant difference between complete response and no response.

Our results were in agreement with those of Mueller et al. 17, who found no significant association between therapy response and LDH concentration in de-novo AML patients. However, the presence of elevated levels of both pretheraputic LDH concentrations and circulating nucleosomes is associated with poor outcome. Similarly, Gautschi 19 found elevated LDH levels to be correlated with DNA plasma levels in patients with lung cancer; also, Hohaus et al. 15 found the same for lymphoma, indicating a common mechanism for the release of LDH and DNA from the tumor tissue.

In terms of the sensitivity and specificity of serum nucleosomes as a marker in early prediction of therapy response in newly diagnosed AML patients, our study showed that favorable therapy response can be predicted with sensitivity (66%) and specificity (100%) and this is in agreement with Mueller et al. 17, who found that favorable therapy response can be predicted with a sensitivity of 56% and a specificity of 100%.


  Conclusion Top


It would be valuable to include these markers together with the kinetics of circulating nucleosomal DNA fragments in prospective trials to elucidate their potential additive role in the early prediction of therapy response. In AML patients, the changes in nucleosomal DNA during the initial phase of induction chemotherapy are valuable markers for the early estimation of therapy response and should be validated in further prospective trials.[19]

 
  References Top

1.Döhner K, Hartmut D.Molecular characterization of acute myeloid leukemia.Haematologica2008;93Issue 7976–982.  Back to cited text no. 1
    
2.Schlenk RF, Dohner K, Krauter J.Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia.N Engl J Med2009;358:1909–1925.  Back to cited text no. 2
    
3.Nannya Y, Kanda Y, Oshima K, Kaneko M, Yamamoto R.Prognostic factors in elderly patients with acute myelogenous leukemia: a single center study in Japan.Leuk Lymphoma2006;43:83–87.  Back to cited text no. 3
    
4.Stahnke K, Eckhoff S, Mohr A, Meyer LH, Debatin KM.Apoptosis induction in peripheral leukemia cells by remission induction treatment in vivo: selective depletion and apoptosis in a CD34+ subpopulation of leukemia cells.Leukemia2004;17:2130–2139.  Back to cited text no. 4
    
5.Wyllie AH, Bellamy CO, Bubb VJ, Clarke AR, Corbet S, Curtis L.Apoptosis and carcinogenesis.Br J Cancer2003;80S134–37.  Back to cited text no. 5
    
6.Kerr JFR, Winterford CM, Harmon BV.Apoptosis. Its significance in cancer and cancer therapy.Cancer2003;73:2013–2026.  Back to cited text no. 6
    
7.Butt AN, Swaminathan R.Overview of circulating nucleic acids in plasma/serum.Ann NY Acad Sci2008;1137:236–242.  Back to cited text no. 7
    
8.Holdenrieder S, Stieber P.Apoptotic markers in cancer.Clin Biochem2005;37:605–617.  Back to cited text no. 8
    
9.Dohner H, Estey EH, Amadori S, Appelbaum FR, Buchner T, Burnett AK, Dombret H.Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet.Blood2010;115:453–474.  Back to cited text no. 9
    
10.Grimwade D, Hills RK.Independent prognostic factors for AML outcome.Hematology Am Soc Hematol Educ Program2009;12:385–954.  Back to cited text no. 10
    
11.Kamakata RT, Biggins S.Histone variants: deviants?Genes Dev2005;19:259–310.  Back to cited text no. 11
    
12.Geiger S.Nucleosomes as a new prognostic marker in early cerebral stroke.J Neurol2007;254:617–623.  Back to cited text no. 12
    
13.Holdenrieder S, Burges A, Reich O.DNA integrity in plasma and serum of patients with malignant and benign diseases.Ann NY Acad Sci2008;1137:162–170.  Back to cited text no. 13
    
14.Hashem ME.Serum nucleosomes and HER-2 in breast cancer [Thesis for MD]. Egypt: Degree in Clinical Pathology, Faculty of Medicine Zagazig University; 2006. pp. 112–114.  Back to cited text no. 14
    
15.Hohaus S, Giachelia G, Massini A, Mansueto B, Vannata V, Bozzoli M, et al..Cell-free circulating DNA in Hodgkin’s and non-Hodgkin’s lymphomas.Ann Oncol2009;20:1408–1413.  Back to cited text no. 15
    
16.Gehan PB.Circulating nucleic acids in plasma and serum: diagnosis and prognosis in cancer.EPMA J2010;1:503–512.  Back to cited text no. 16
    
17.Mueller S, Holdenrieder S, Stieber P, Haferlach T, Schalhorn A, Braess J, et al..Early prediction of therapy response in patients with acute myeloid leukemia by nucleosomal DNA fragments.BMC Cancer2006;6:143–155.  Back to cited text no. 17
    
18.Schwarz AK, Stanulla M, Cario G, Flohr T, Sutton R, Möricke A, et al..Quantification of free total plasma DNA and minimal residual disease detection in the plasma of children with acute lymphoblastic leukemia.Ann Hematol2009;88:897–905.  Back to cited text no. 18
    
19.Gautschi O.Circulating deoxyribonucleic acid as prognostic marker in non-small-cell lung cancer patients undergoing chemotherapy.J Clin Oncol2004;22:4157–4164.  Back to cited text no. 19
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]



 

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