2D-DIGE gels were scanned using Typhoon 9400 variable mode imager (GE Healthcare) at a 100 mm resolution employing suitable excitation/emission wavelengths for each of the CyDye [Cy3 (523/580 nm), Cy5 (633/670 nm) and Cy2 (488/520 nm)]. After scanning, gel images were cropped properly using Im-ageQuant software; version 5.0 (GE Healthcare) prior to importing in DeCyder 2D software; version 7.0 (GE Healthcare) for comparative analysis and relative protein quantification across the FM and control samples. Comparative analysis was performed using two different modules, differential in-gel analysis (DIA) and biological variation analysis (BVA) of the DeCyder software. Preliminary analysis was performed using DIA module to detect spots on a cumulative image derived from merging up to three individual images from an in-gel linked image set (malaria, controls and internal standard). It permits the pair-wise comparisons of each normal and malaria samples to the mixed standard present in each gel and offers spot-wise protein abundance as ratios. Further analysis was performed using BVA module to get the variation in protein expression levels between any of the two experimental groups (FM vs. VM, FM vs. HC and VM vs HC) across all the sets. Statistical significance of the average ratio of expressions was analyzed by Student's t-test. Protein spots exhibiting differential expression with reproducibly and statistical significance (p,0.05) were considered for further analysis. Bonferroni correction (for reducing Type I errors) of the p-values obtained from Student's t-test was performed using standard Bonferroni procedure to recognize those marker proteins which have very strong connection with the diseased state (remains significant after Bonferroni correction). Since Bonferroni correction is extremely conservative; comparatively less stringent false discovery rate (FDR) correction was also performed as detailed in Benjamini and Hochberg [38] .