Segmentation of the fixed structure.

<p>The color image (a) is segmented by relative RGB color planes (b, c). Some morphological operations (scrapping of small objects, closing) yield the blue stained area A_B (d).</p

Results of analysis of samples.

<p>(a) Relative density of CXCR3⁺ cells in neighborhood of capillaries and venules. (b) Relative density of CD56⁺ cells in neighborhood of capillaries and venules. (c) Relative density of CD56⁺ cells in neighborhood of spiral arteries. The data are individually normalized in relation to density in residual decidua = 1. (y axis, log scale.)</p

Definition of the endothelial neighborhood by morphological dilation.

<p>The segmented image of the endothelium (a) is morphologically dilated (b). (Here a neighborhood width of 30 µm is chosen.) The dilated image A_B + N (c) subtracted with the blue stained endothelial area A_B (d) yields the endothelial neighborhood area A_N (e). The total specimen area A_T (f) subtracted by the dilated area A_B + N (g) yields the residual decidua area A_D (h). A_N: Neighborhood area, A_D: Area of residual decidua, A_B: Area of endothelium, A_T: Area of total specimen.</p

Examples of microphotographs for automated analysis: Two-color immunohistochemistry of non-invaded first trimester decidua.

<p>(a) Red: (mostly uterine NK) cells stained with anti-CD56-HRP-AEC, blue: CD34⁺ vascular endothelium stained with anti-CD34-APAAP-NBT/BCIP. (b) Red: CD56⁺ cells, blue: tunica media of spiral arteries stained with anti-smooth muscle actin – APAAP-BCIP.</p

Quantile - quantile plot for neighborhood 10 µm.

<p>The dots (red: CXCR3⁺ cells in relation to endothelium, green: CD56⁺ cells in relation to endothelium, blue: CD56⁺ cells in relation to the tunica media of spiral arteries) represent the medians of patients' data of cell densities in the neighborhood (x-axis) versus in the residual decidua (y-axis). The black line represents the random distribution, the black bars the standard deviation of the random distribution.</p

Segmentation of the mobile cells.

<p>(In this example: CD56⁺ cells). The color image (a) is segmented by relative RGB color planes (b, c). Some morphological operations (closing, opening) yield the red stained area A_R (d).</p

Endothelial neighborhood (A_N) and area of residual decidua (A_D) with increasing neighborhood width.

<p>Endothelial neighborhood (A_N) and area of residual decidua (A_D) with increasing neighborhood width.</p

Artificial images with randomly scattered cell distributions.

<p>For the sake of better comparison of cell distributions at different overall cell densities a model of random distribution of artificial cells in relation to neighborhood areas and rest decidua of a specific sample was generated. Randomly distributed cells projected onto a sample object of the total specimen's area A_T (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015086#pone-0015086-g003" target="_blank">Figure 3b</a>). (a) Low cell density with 100 seeds, (b) high cell density with 500 seeds.</p

Definition of neighborhood area on the basis of a microphotograph of two-color immunohistochemistry of first trimester decidua (see Figure 1a).

<p>Generation of an area of vicinity of a size to be chosen (in our example, 3 sizes at distances of up to 10, 20, and 30 µm from the vascular endothelium are given).</p

Analysis of the effect of SALSA on coagulation.

<p>A pool of citrated plasma was added to SALSA-coated wells (1–5 μg/ml) and coagulation was initiated by adding BC Thrombin reagent at time point 0. The coagulation was followed by absorbance at 405 nm and compared to coagulation in wells without SALSA coating. We did not observe an effect of SASLA on coagulation. Displayed are averages ± SD’s from 5 different wells. A similar result was obtained when soluble SALSA (1–5 μg/ml) was added to the plasma prior to the coagulation test (data not shown).</p