A Novel Quantitative Approach for Eliminating Sample-To-Sample Variation Using a Hue Saturation Value Analysis Program

Objectives: As computing technology and image analysis techniques have advanced, the practice of histology has grown from a purely qualitative method to one that is highly quantified. Current image analysis software is imprecise and prone to wide variation due to common artifacts and histological limitations. In order to minimize the impact of these artifacts, a more robust method for quantitative image analysis is required. Methods and Results: Here we present a novel image analysis software, based on the hue saturation value color space, to be applied to a wide variety of histological stains and tissue types. By using hue, saturation, and value variables instead of the more common red, green, and blue variables, our software offers some distinct advantages over other commercially available programs. We tested the program by analyzing several common histological stains, performed on tissue sections that ranged from 4 µm to 10 µm in thickness, using both a red green blue color space and a hue saturation value color space. Conclusion: We demonstrated that our new software is a simple method for quantitative analysis of histological sections, which is highly robust to variations in section thickness, sectioning artifacts, and stain quality, eliminating sample-to-sample variation

Genesis and growth of extracellular vesicle-derived microcalcification in atherosclerotic plaques

Clinical evidence links arterial calcification and cardiovascular risk. Finite-element modelling of the stress distribution within atherosclerotic plaques has suggested that subcellular microcalcifications in the fibrous cap may promote material failure of the plaque, but that large calcifications can stabilize it. Yet the physicochemical mechanisms underlying such mineral formation and growth in atheromata remain unknown. Here, by using three-dimensional collagen hydrogels that mimic structural features of the atherosclerotic fibrous cap, and high-resolution microscopic and spectroscopic analyses of both the hydrogels and of calcified human plaques, we demonstrate that calcific mineral formation and maturation results from a series of events involving the aggregation of calcifying extracellular vesicles, and the formation of microcalcifications and ultimately large calcification zones. We also show that calcification morphology and the plaque’s collagen content – two determinants of atherosclerotic plaque stability - are interlinked

Quantification of calcified particles in human valve tissue reveals asymmetry of calcific aortic valve disease development

Recent studies indicated that small calcified particles observable by scanning electron microcopy (SEM) may initiate calcification in cardiovascular tissues. We hypothesized that if the calcified particles precede gross calcification observed in calcific aortic valve disease (CAVD), they would exhibit a regional asymmetric distribution associated with CAVD development, which always initiates at the base of aortic valve leaflets adjacent to the aortic outflow in a region known as the fibrosa. Testing this hypothesis required counting the calcified particles in histological sections of aortic valve leaflets. SEM images, however, do not provide high contrast between components within images, making the identification and quantification of particles buried within tissue extracellular matrix difficult. We designed a new unique pattern matching-based technique to allow for flexibility in recognizing particles by creating a gap zone in the detection criteria that decreased the influence of non-particle image clutter in determining whether a particle was identified. We developed this flexible pattern particle labeling (FpPL) technique using synthetic test images and human carotid artery tissue sections. A conventional image particle counting method (pre-installed in ImageJ) did not properly recognize small calcified particles located in noisy images that include complex extracellular matrix structures, and other commonly used pattern matching methods failed to detect the wide variation in size, shape and brightness exhibited by the particles. Comparative experiments with the ImageJ particle counting method demonstrated that our method detected significantly more p<2*10-7 particles than the conventional method with significantly fewer p<0.0003 false positives and false negatives p<0.0003. We then applied the FpPL technique to CAVD leaflets and showed a significant increase in detected particles in the fibrosa at the base of the leaflets (p<0.0001), supporting our hypothesis. The outcomes of this study are two-fold: 1) development of a new image analysis technique that can be adapted to a wide range of applications; 2) acquisition of new insight on potential early mediators of calcification in CAVD

<i>CSH</i> is a powerful tool for a variety of stains.

<p>Common histological stains, displaying the fidelity of <i>CSH</i>. (Top panels): A mouse aorta stained with alkaline phosphatase (ALP, red) for detection of early calcification, with Gill's hematoxylin as counterstaining (purple), which depicts advanced calcification. ALP stain is scarlet red (denoted “A” in the top left panel), while hematoxylin is a shade of purple (denoted “H” in the top left panel). Visually, the hematoxylin interferes with the ALP, making it difficult to see where the ALP stain begins and ends. We analyzed the section for ALP-positive area using both CSH and an RGB-based method. (Bottom panels): A mouse liver stained with picrosirius red staining visualized using polarized light microscopy for detection of fibrosis. We analyzed the section using both CSH and an RGB-based method. The RGB method was unable to register the brightest parts of the stain as positive (gray), and falsely interpreted stain artifacts as positive areas (green in both “Merge” images).</p

<i>CSH</i> processing on an infarcted mouse heart.

<p>A) An infarcted mouse heart section cut 8 µm thick. Muscle tissue is scarlet red, while collagen fibers appear blue, and necrotic regions are purple-black. Insets show enlarged areas of muscle, collagen and necrotic region. B) The same mouse heart, post-processing by <i>CSH</i>. The areas that <i>CSH</i> determined as collagen are blue, and the areas that <i>CSH</i> determined as muscle are red. The background is yellow. C) A plot of the pixels from the original heart image mapped to HSV space. The gray arrows indicate the direction from which this 3-D graph will be displayed in the following 2-D images. D) A plot of the pixels from the original image in the Hue-Saturation plane. The borders collagen and the muscle rectangular thresholds are visible at Hue = {200, 300, 385}. E) A plot of the pixels from the original image in the Hue-Value plane. F) A plot of the pixels from the original image in the Value-Saturation plane. This graph most clearly shows the different shapes of the collagen peak (blue) and the muscle peak (red).</p

Analytic performance across diverse section thicknesses.

<p>A) A section of infarcted mouse heart cut to 4 µm and stained with Masson's trichrome. Descending from the original image, we see the RGB binary image, the HSV binary image, a density map of the pixels mapped to the RGB color space, and a density map of the pixels mapped to the HSV color space. B) A section of infarcted mouse heart cut to 6 µm and stained with Masson's trichrome. C) A section of infarcted mouse heart cut to 8 µm and stained with Masson's trichrome. D) For each of four experimental hearts and each of the three section thicknesses, the area identified as muscle is plotted next to the area identified as collagen using the RGB method. Because each heart has a different size infarction, these results for each heart are normalized as a percentage of the measured area in the 6 µm sample. As the section thickness increases, RGB analysis decreases the perceived collagen area, despite analyzing adjacent sections of heart. E) For each of four experimental hearts and each of the three section thicknesses, the area identified as muscle is plotted next to the area identified as collagen using the HSV method. Because each heart has a different size infarction, these results for each heart are normalized as a percentage of the measured area in the 6 µm sample. There is no discernible change in perceived muscle or collagen area as the section thickness increases when using the HSV method.</p

<i>CSH</i> is consistent between individuals.

<p>Apoe−/− mouse innominate arteries stained with MAC3 antibody for detection of macrophages. RGB1(threshold1) was optimized for Cross Section 1, and overestimates the positive area when applied to Cross Section 2. RGB2(threshold2) was optimized for Cross Section 2, and underestimates the positive area when applied to Cross Section 1. <i>CSH</i> was able to effectively use a single HSV threshold on both cross sections. In the overlays between the HSV and RGB1 and RGB2, yellow area shows where there is agreement between the HSV method and the RGB method. Green area in the overlays may indicate false positive area reported by the RGB method, while red area in the overlays may represent false negative area reported by the RGB method.</p

Macrophage-Derived Matrix Vesicles: An Alternative Novel Mechanism for Microcalcification in Atherosclerotic Plaques

RATIONALE: We previously showed that early calcification of atherosclerotic plaques associates with macrophage accumulation. Chronic renal disease (CRD) and mineral imbalance accelerates calcification and the subsequent release of matrix vesicles (MVs) — precursors of microcalcification. OBJECTIVE: We tested the hypothesis that macrophage-derived MVs contribute directly to microcalcification. METHODS AND RESULTS: Macrophages associated with regions of calcified vesicular structures in human carotid plaques (n=136 patients). In vitro, macrophages released MVs with high calcification and aggregation potential. MVs expressed exosomal markers (CD9 and TSG101), and contained S100A9 and annexin V (Anx5). Silencing S100A9 in vitro and genetic deficiency in S100A9(−/−) mice reduced MV calcification, while stimulation with S100A9 increased calcification potential. Externalization of phosphatidylserine (PS) after Ca/P stimulation and interaction of S100A9 and Anx5, indicated that a PS-Anx5-S100A9 membrane complex facilitates hydroxyapatite nucleation within the macrophage-derived MV membrane. CONCLUSIONS: Our results support the novel concept that macrophages release calcifying MVs enriched in S100A9 and Anx5, which contribute to accelerated microcalcification in CRD