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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 10  |  Issue : 2  |  Page : 103-110

Role of anti-phospholipase A2 Receptor antibodies in patients with membranous nephropathy: A prospective study on Indian cohort


1 Department of Pathology, St. John's Medical College and Hospital, Bengaluru, Karnataka, India
2 Department of Nephrology, St. John's Medical College and Hospital, Bengaluru, Karnataka, India

Date of Submission10-May-2021
Date of Decision19-Jun-2021
Date of Acceptance22-Jul-2021
Date of Web Publication05-Apr-2022

Correspondence Address:
Dr. Usha Kini
Department of Pathology, St. John's Medical College, St. John's National Academy of Health Sciences, Bengaluru - 560 034, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ajim.ajim_50_21

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  Abstract 


Context: A search for a cause for membranous nephropathy (MN) is crucial to determine its treatment and management. Primary MN was a diagnosis of exclusion until the discovery of the target antigen, phospholipase A2 receptor (PLA2R). Lack of published data from the Indian population prompted this prospective study to determine the sensitivity and specificity of circulating anti-PLA2R antibodies in MN patients by using cell-based indirect immunofluorescence test (IIFT) and correlating with clinical–histopathology features and response to treatment. Settings and Design: This was a cross-sectional prospective study. Materials and Methods: MN cases (n = 34) diagnosed by renal biopsy and IIFT were evaluated along with 10 controls for serum anti-PLA2R antibodies using IIFT on biochip containing HEK 293 cell lines transfected with cDNA coded for PLA2R in this cross-sectional prospective study and simultaneously investigated to find the cause for MN. Positive cases treated with the Ponticelli regimen were followed up for 6 months with repeat testing for PLA2R. Statistics were performed using Statistical Package for Social Sciences version 18 (IBM). P < 0.05 considered significant. Statistical parameters were analyzed using the Chi-square test. Results: Anti-PLA2R antibodies-positive MN (primary MN) cases (n = 20) had higher 24-h proteinuria (10.09 ± 2.46 g) with 25% cases showing mesangial hypercellularity and basement membrane thickening in all (100%), while 50% of secondary MN cases showed mesangial hypercellularity with 7.17 ± 3.8 g of proteinuria. The sensitivity, specificity, and accuracy rate of anti-PLA2R antibodies for a diagnosis of primary MN were 70%, 100%, and 82%, respectively. Conclusion: Anti-PLA2R antibody in serum is a good reliable noninvasive diagnostic biomarker for primary MN and for monitoring its disease activity.

Keywords: Anti-PLA2R antibody, indirect immunofluorescence test, membranous nephropathy, podocyte


How to cite this article:
Karimkhan A, Kini U, Shenoy PM, Satish R, Puttegowda D. Role of anti-phospholipase A2 Receptor antibodies in patients with membranous nephropathy: A prospective study on Indian cohort. APIK J Int Med 2022;10:103-10

How to cite this URL:
Karimkhan A, Kini U, Shenoy PM, Satish R, Puttegowda D. Role of anti-phospholipase A2 Receptor antibodies in patients with membranous nephropathy: A prospective study on Indian cohort. APIK J Int Med [serial online] 2022 [cited 2022 May 26];10:103-10. Available from: https://www.ajim.in/text.asp?2022/10/2/103/342539




  Introduction Top


Membranous nephropathy (MN) is a noninflammatory organ-specific autoimmune disease affecting the glomerulus and resulting in the production of immune deposits. It is the leading cause of nephrotic syndrome in adults with 20%–30% of untreated patients progressing to end-stage renal disease.[1] In about 25% of patients, it is associated with an underlying disease when it is concluded as secondary MN, while in about 75%, until recently, no underlying cause could be identified and has been considered idiopathic/primary MN.[2] In some patients, MN can appear months or even years before a secondary cause is detected. In these patients, particularly those older than 65 years, great uncertainty exists about the clinical management, as an undetected malignant tumor could also be the cause. In some, primary MN and a potential secondary cause of MN may develop independently of each other and present chronologically within a short period. The diagnosis of primary MN is being made by exclusion of secondary causes, based on clinical history, physical findings, and a panel of laboratory tests until the recent discovery of phospholipase A2 receptor (PLA2R) as the target antigen in primary MN.

Primary MN is treated with an immunosuppressive regimen while the treatment is targeted toward the underlying disease in cases with secondary MN. Hence, it is mandatory and of great clinical importance to differentiate primary from secondary MN as investigative protocol and therapeutic modalities are different, thereby only the group of secondary MN patients may be subjected to the battery of investigations and not subject them otherwise to immunosuppressive therapy unlike patients with primary MN.

The landmark discovery of PLA2R as a target antigen in primary MN detectable in the serum of up to 70% of patients by Beck et al. in 2009 was a landmark that made it possible to differentiate primary MN from secondary MN.[3] This prospective study is aimed at evaluating anti-PLA2R antibodies in serum of Indian patients with a histologic diagnosis of MN as a noninvasive diagnostic biomarker to distinguish primary MN from secondary MN.


  Materials and Methods Top


This is a prospective 2-year cross-sectional study conducted in a tertiary medical center on 34 cases of newly detected MN along with 10 controls.

Patients with nephrotic syndrome of either sex diagnosed with MN on renal biopsy, confirmed with immune fluorescence (positive with IgG and C3), and followed up for a minimum of 6 months were selected for this study. Patients of MN on immunosuppressive therapy and/or not worked up for secondary causes and/or not followed up were excluded. Serum samples from healthy donors were taken as controls. The study was approved by the Institutional Ethical Board (No 3/13-II (1)/13).

Selected patients with nephrotic syndrome were evaluated as per the flowchart shown in [Figure 1]. Renal biopsy was performed using an 18 gauge needle fixed in 10% buffered neutral formalin and processed for histopathology using the standard protocol. The tissue sections were routinely stained with hematoxylin and eosin, periodic acid–Schiff (PAS), periodic acid methenamine silver stain (PAS-M), and Masson trichrome. Fresh renal core biopsies received in Michel's transport medium for immunofluorescence (IF) study were frozen and the sections were obtained in a cryostat for direct IF using the standard protocol. They were stained for deposits of IgG, IgA, IgM, C3, and C1q (Caps, Denmark) in dilution of 1:30 and interpreted.

Serum separated from fresh whole blood samples drawn from these selected patients for detection of PLA2R antibodies by indirect IF test (IIFT) was coded and stored at 4°C if testing was planned within 72 h or at − 20°C if testing was planned after 3 days. The test was carried out on biochips with HEK 293 cell line transfected with cDNA coded for PLA2R and nontransfected cells as negative control available commercially as kits from Euroimmun AG, Germany.
Figure 1: Flowchart to illustrate the methodology

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PLA2R-IIF was performed on substrate slides containing 3 wells each, with a biochip containing HEK 293 cell line transfected with c DNA coded for PLA2R and nontransfected cells as the negative control. Serum samples diluted 1:10 in phosphate-buffered saline (PBS) were overlaid onto the wells of substrate slide and stained for PLA2R. All PLA2R stained wells were evaluated by two pathologists using a fluorescence microscope with an LED light source under 40, 100, and 400 magnification. A test was read as PLA2R positive (primary MN) if the culture cell line showed membranous fluorescence positivity [Figure 2] and PLA2R negative (secondary MN) if there was no fluorescence.
Figure 2: Note the distinct membranous staining of HEK 293 cell line transfected with c DNA coded for phospholipase A2 receptor indicating positivity with indirect immunofluorescence in a patient with primary membranous nephropathy (×400)

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Simultaneously, the patients underwent tests for detection of secondary causes, as listed out in [Figure 1]. Pathologists reading the immune fluorescence tests were blinded to the outcome of these results. Serum PLA2R antibody results along with follow-up data were correlated after the patient was evaluated in detail for a secondary etiology for MN.

PLA2R-positive MN cases were treated as per Ponticelli regimen. At the end of 6 months, the patients were further categorized clinically into those with complete remission, partial remission, and no remission based on reduction in proteinuria as shown below.[4]

  • Complete remission: Urinary protein excretion 0.3 g/d (uPCR 300 mg/g or 30 mg/mmol), confirmed by two values at least 1 week apart, accompanied by a normal serum albumin concentration, and a normal SCr
  • Partial remission: Urinary protein excretion 3.5 g/d (uPCR 3500 mg/g or 350 mg/mmol) and a 50% or greater reduction from peak values, confirmed by two values at least 1 week apart, accompanied by an improvement or normalization of the serum albumin concentration and stable SCr
  • No remission: No reduction in proteinuria.


Statistical analysis

The data were collected and analyzed using SPSS Inc (2009. PASW Statistics for Windows, Version 18.0. Chicago) and P < 0.05 was considered significant. Mean and standard deviation were obtained for continuous variables. Categorical variables were subjected to number and percentages. Sensitivity and specificity were computed.


  Results Top


In this 2 years prospective study, 425 of the 1008 renal cases evaluated, 8% (34/425) patients diagnosed with MN ranged from 6 years to 75 years of age with a median age of 38.5 years and a male-to-female ratio of 2.7:1 were evaluated for serum PLA2R. These patients with the nephrotic syndrome had varying ranges of severe proteinuria (mean of 8.34 ± 7.2), albuminuria with mean serum albumin of 1.70 (±1.64), and mean serum creatinine of 1.05 (±1.26) [Table 1].
Table 1: Biochemical parameters in patients with membranous nephropathy at the time of initial evaluation (n=34)

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Renal biopsies showed thickening of the glomerular basement membrane distinctly noted in 97.05% (33/34) [Figure 3]a. Glomerular sclerosis was noted in 47% (16/34) with associated focal tubular atrophy in 32% (11/34). The spectrum of histopathological features is summarized in [Table 2]. At the time of this prospective study, the PLA2R antibody for demonstration by tissue immunohistochemistry was not yet available in the market for correlation.
Table 2: The spectrum of histopathological features seen in renal biopsy of cases with membranous nephropathy in this study (n=34)

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PLA2R-positive MN (primary MN) cases (20/34) had higher 24-h proteinuria (10.09 ± 2.46 g) with BM thickening and lower incidence of mesangial hypercellularity/endocapillary proliferation versus 7.17 ± 3.8 g proteinuria in secondary MN showing higher incidence mesangial hypercellularity and endocapillary proliferation [Table 3].
Figure 3: (a) High-power magnification to show thickened basement membrane in a case of membranous nephropathy (H and E, ×400). (b) Direct immunofluorescence to show 3 + peripheral granular positivity for IgG (×400)

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Table 3: The comparison of clinicopathologic features between primary and secondary cases of membranous nephropathy in this study

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All the renal biopsies showed positivity for IgG (100%) with varying degree of positivity for C3 (47.05%), IgM (26.47%), IgA (23.52%), and C1q (20.58%) [Table 3]. The deposits of IgG were peripheral in 85% of cases (29/34) [Figure 3]b, and both peripheral and mesangial in 15% (5/34) of cases with varying intensities. IgG was seen in combination with other immunoglobulins and complements in 67% of cases (23/34), the most common combination being IgG with C3 alone in 20% of cases (7/34). Full-house positivity was seen in 2.94% of the cases (1/34). A demonstrable etiology for MN was found in 41% (14/34) during the clinical work-up of the case, as shown in [Table 4].
Table 4: List of conditions associated with secondary membranous nephropathy in this study (n=14)

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Among the 34 test cases, serum PLA2R antibodies were positive [Figure 4] in 41% (14/34) of cases. About 41.2% (14/34) of cases showed demonstrable causes for MN (secondary MN) [Table 4]. About 30% of patients (6/34) who were negative for anti-PLA2R antibodies were also negative with all diagnostic modalities for causative agents (considered as equivocal cases) and no secondary cause could be demonstrated in them even after extensive workup. These six cases were treated with the Ponticelli regimen; they showed a good clinical response and went into the remission phase. Hence, these six cases were considered as false negative for PLA2R antibodies [Figure 4].
Figure 4: Distribution of MN cases based on phospholipase A2 receptor positivity in the present study

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Of the 29 cases which were suspected to be primarily based on IIFT (only peripheral deposits), 19 were diagnosed as primary MN and ten as secondary MN. In five cases suspected as secondary based on IIFT (peripheral and mesangial deposits), four were secondary MN and one was primary. The sensitivity, specificity, and accuracy rate of 70%, 100%, and 82% were noted, respectively, with the negative and positive predictive values of 70% and 100% for patients with primary MN using anti-PLA2R antibodies.

Patients who were anti-PLA2R positive (14/34) were followed up for 6 months after the initiation of immunosuppressive treatment. About 72% (10/14) of patients showed complete remission, 14% (2/14) partial remission, and 14% (2/14) with no remission.

In the 8 patients who went into complete remission, 87.5% (7/8) were negative for PLA2R antibodies at the end of 6 months while 2 patients who failed to go into remission, 100% (2/2) continued to be positive for PLA2R antibodies at the end of 6 months.


  Discussion Top


Approximately 25% of MN cases are associated with variety of systemic diseases such as autoimmune (eg. SLE), infection (eg. Hepatitis B), Drugs (eg. NSAIDs) and malignancy. These cases are referred to as secondary MN, in contrast to ,primary MN which is idiopathic. Distinguishing primary from secondary MN is not always straightforward. Although long tables of secondary associations are listed in many textbooks and reviews, a true causal relationship is not often established and the possibility of two separate, but coincidental diseases can not be easily ruled out. These presenting as the nephrotic syndrome may very often lead to end-stage renal disease in many cases. This study was undertaken to study how PLA2R would help in differentiating primary from secondary MN and in further management.

In this study, 34 cases of MN were recruited from a total of 1008 kidney biopsies. About 58% were considered as primary MN with 70% PLA2R positive, while 41% were secondary MN with 100% PLA2R negative. Ten control samples namely, five healthy donors and five patients with nonmembranous nephrotic syndrome, were also negative for PLA2R. Interestingly, six cases that were PLA2R negative were also negative for all the secondary causes that the MN cases were investigated for. However, correlating the negative serum PLA2R status with negative detailed investigations for etiology and their clinical and laboratory response for standard treatment regime given to the primary MN patients, these cases were finally considered and treated as primary MN. They did respond like the primary MN. These patients are being followed up and tested out later to see if they show up positivity for anti-PLA2R antibodies.

The twenty patients who were diagnosed with primary MN with PLA2R were characteristically middle aged (40.5 years), predominantly male (70%), and had 24-h proteinuria exceeding 10 g. Similarly, patients with secondary MN were also middle aged, predominantly male (71.4%) but with 24-h proteinuria <10 g (7.17 ± 3.8 g). The finding of histopathology features typical of MN with a well-established secondary cause and classical histological features such as lupus is often enough to label that case secondary MN but may not be classical always as seen in this study. The five cases of autoimmune pathology diagnosed clinically had full-house positivity seen only in one case (2.94%) (lupus nephritis) wherein the classical teaching of a full-house pattern by IF (i.e., the presence of IgG, IgM, IgA, C3, andC1q) was very specific for secondary MN (lupus nephritis). Interestingly, out of seven cases positive for C1q, one (14.28%) was primary MN, while 6 (85.71%) were secondary MN.

IgG subclasses can also help to distinguish primary from secondary MN, although most renal pathology services do not routinely characterize the IgG subclasses. Primary MN is characterized by IgG4-rich deposits, especially IgG1. IgG1, IgG2, and IgG3 are the predominant forms in secondary MN deposits.[4],[5] With the inability of the IgG4 subclass to bind and activate the classic complement pathway, C1q usually is absent or found at very low levels in primary MN, whereas it more typically is present in the secondary disease.[4] Strangely, the one case of primary MN positive for PLA2R was also positive for C1q (2+) by IF.

Demonstration of the presence of circulating autoantibodies directed against the M-type phospholipase A2 receptor (PLA2R1), an a180-kD transmembrane glycoprotein expressed on glomerular podocytes is well depicted in a wide range of specificity and sensitivity in the series of eight studies[3],[6],[7],[8],[9],[10],[11] as shown in [Table 5] while Svobodova et al[12] have shown the least specificity of 40%. In contrast, our study showed a sensitivity of 70% and specificity of 100% with an accuracy rate of 82% [Table 5].
Table 5: Sensitivity and specificity rates of serum anti - phospholipase A2 receptor antibodies (by indirect immunofluorescence) in primary membranous nephropathy in literature


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Not all patients with primary MN were PLA2R positive, as seen in six (30%) of our patients. This was also noted by Hoxha et al.[10] in 12 of their patients. This discrepancy was considered due to the probable existence of antigens other than PLA2R, which however cannot be confirmed at present as no definite test marker is available for the same. These groups of antigens have been assumed to be cytoplasmic such as aldose reductase, superoxide dismutase, and α-enolase. These enzymes are expected to be weakly expressed on the membrane of normal podocytes. Anti-PLA2R antibodies which were not detected in 41% (n = 14) of our cases were considered as secondary MN [Table 6] as they were found positive for diabetes mellitus in 28% (n = 4), drug induced (native medication) in 15% (n = 2), hepatitis in 7.14% (n = 1), retroposition in 7.14% (n = 1), carcinoma stomach in 7.14% (n = 1), and 35.71% cases (n = 5) of autoimmune etiology of which one was lupus nephritis, one case of rheumatoid arthritis, two cases of mixed connective tissue disease, and one case of autoimmune hemolytic anemia.
Table 6: Correlation of serum Anti - PLA2R antibodies with secondary causes of MN


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The meta-analysis done by Du et al.[13] concludes that the sensitivity and specificity of PLA2R in serum are 78% and 99%, respectively, while our study showed 70% and 100%, respectively, which also matches perfectly with the figures shown in the systematic review by Hu et al.[14] with sensitivity and specificity of 69% and 99%, respectively [Table 7].
Table 7: The comparison of results of various methods employed for detection of anti - phospholipase A2 receptor in literature


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This prospective study of serum PLA2R on biopsy-proven active cases of MN by IIF in this study is unique in Indian literature in comparison to those published earlier,[15],[16],[17],[18] by its correlation of follow-up data of serum PLA2R with response to treatment.

This study calls for correlation of serum PLA2R positivity with serum PLA2R antibody titers as well as demonstration of PLA2R antibodies on the glomerular basement membrane on tissue biopsies with immunohistochemistry which became available at the end of this study. Since PLA2R antibody is part of IgG4 and IgG4 being the dominant IgG subclass positive in patients with MN, it is good to correlate the renal biopsies with IgG4 in addition to thrombospondin type I domain-containing 7A antigenic target of autoantibodies leading to primary membranous glomerulopathy in cases of PLA2R-negative membranous glomerulopathy.[18]


  Conclusion Top


This prospective study establishes that testing for anti-PLA2R antibodies in serum of Indian patients with a working diagnosis of membranous nephropathy, is a good biomarker to distinguish primary MN from secondary MN and for monitoring of disease activity in such patients. We propose it to be a potential marker for a therapeutic target in future.

Financial support and sponsorship

This study was supported by Medical Education and Research Trust, Karnataka and Biocon, Bengaluru.

Conflicts of interest

The authors declare no conflict of interest.



 
  References Top

1.
Lai WL, Yeh TH, Chen PM, Chan CK, Chiang WC, Chen YM, et al. Membranous nephropathy: A review on the pathogenesis, diagnosis, and treatment. J Formos Med Assoc 2015;114:102-11.  Back to cited text no. 1
    
2.
Longo, Dan, Fauci AS, Kasper DL, Hauser SL, Jameson J, Loscalzo J.eds. Harrison's Principles of Internal Medicine 18th edition. New York, NY: Mc Graw Hill, 2012.  Back to cited text no. 2
    
3.
Beck LH Jr., Bonegio RG, Lambeau G, Beck DM, Powell DW, Cummins TD, et al. M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy. N Engl J Med 2009;361:11-21.  Back to cited text no. 3
    
4.
Cattran DC, Feehally J, Cook HT, Liu ZH, Fervenza FC, Mezzano SA, et al. Kidney disease: improving global outcomes (KDIGO) glomerulonephritis work group. KDIGO clinical practice guideline for glomerulonephritis. Kidney International Supplements. 2012;2:139-274.  Back to cited text no. 4
    
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Ma H, Sandor DG, Beck LH Jr. The role of complement in membranous nephropathy. Semin Nephrol 2013;33:531-42.  Back to cited text no. 5
    
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Oh YJ, Yang SH, Kim DK, Kang SW, Kim YS. Autoantibodies against phospholipase A2 receptor in Korean patients with membranous nephropathy. PLoS One 2013;8:e62151.  Back to cited text no. 6
    
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Murtas C, Bruschi M, Candiano G, Moroni G, Magistroni R, Magnano A, et al. Coexistence of different circulating anti-podocyte antibodies in membranous nephropathy. Clin J Am Soc Nephrol 2012;7:1394-400.  Back to cited text no. 7
    
8.
Qin W, Beck LH Jr., Zeng C, Chen Z, Li S, Zuo K, et al. Anti-phospholipase A2 receptor antibody in membranous nephropathy. J Am Soc Nephrol 2011;22:1137-43.  Back to cited text no. 8
    
9.
Hoxha E, Harendza S, Zahner G, Panzer U, Steinmetz O, Fechner K, et al. An immunofluorescence test for phospholipase-A2-receptor antibodies and its clinical usefulness in patients with membranous glomerulonephritis. Nephrol Dial Transplant 2011;26:2526-32.  Back to cited text no. 9
    
10.
Hoxha E, Kneißler U, Stege G, Zahner G, Thiele I, Panzer U, et al. Enhanced expression of the M-type phospholipase A2 receptor in glomeruli correlates with serum receptor antibodies in primary membranous nephropathy. Kidney Int 2012;82:797-804.  Back to cited text no. 10
    
11.
Dähnrich C, Komorowski L, Probst C, Seitz-Polski B, Esnault V, Wetzels JF, et al. Development of a standardized ELISA for the determination of autoantibodies against human M-type phospholipase A2 receptor in primary membranous nephropathy. Clin Chim Acta 2013;421:213-8.  Back to cited text no. 11
    
12.
Svobodova B, Honsova E, Ronco P, Tesar V, Debiec H. Kidney biopsy is a sensitive tool for retrospective diagnosis of PLA2R-related membranous nephropathy. Nephrol Dial Transplant 2013;28:1839-44.  Back to cited text no. 12
    
13.
Du Y, Li J, He F, Lv Y, Liu W, Wu P, et al. The diagnosis accuracy of PLA2R-AB in the diagnosis of idiopathic membranous nephropathy: A metaanalysis. PLoS One 2014;9:e104936.  Back to cited text no. 13
    
14.
Hu SL, Wang D, Gou WJ, Lei QF, Ma TA, Cheng JZ. Diagnostic value of phospholipase A2 receptor in idiopathic membranous nephropathy: A systematic review and meta-analysis. J Nephrol 2014;27:111-6.  Back to cited text no. 14
    
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Ramachandran R, Kumar V, Kumar A, Yadav AK, Nada R, Kumar H, et al. PLA2R antibodies, glomerular PLA2R deposits and variations in PLA2R1 and HLA-DQA1 genes in primary membranous nephropathy in South Asians. Nephrol Dial Transplant 2016;31:1486-93.  Back to cited text no. 15
    
16.
Gopalakrishnan N, Abeesh P, Dineshkumar T, Murugananth S, Sakthirajan R, Raman GS, et al. Haris prevalence of serum anti Mtype phospholipase A2 receptor antibody in primary membranous nephropathy: A single center experience Indian J Nephrol 2016;26:257-61.  Back to cited text no. 16
    
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Gudipati A, Uppin MS, Kalidindi RS, Swarnalatha G, Das U, Taduri G, et al. Immunohistochemical analysis of antiphospholipase A2 receptor antibody on renalbiopsies: A single tertiary care center study. J Nephrol 2017;27:353-8.  Back to cited text no. 17
    
18.
Subramanian P, Kumar H, Tiwari B, Barwad A, Bagchi S, Bagga A, et al. Profile of Indian patients with membranous nephropathy. Kidney Int Rep 2020;5:1551-7.  Back to cited text no. 18
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

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



 

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