Figure 3.

Improved method to identify tissue specimens containing lymphatic collecting vessels links them to accumulation of tertiary lymphoid organs (TLOs) in Crohn disease (CD) mesentery. In all parts with the exception of D, the images are positioned so that the intestinal border is toward the top of the figure. In D, the intestinal border is on the right of the image. A: Evaluation of methyl salicylate–cleared tissue slices using a light-emitting diode (LED) allows detection of lymph nodes (LNs), lymphoid follicles, and vessels that link them together. Three CD mesenteric specimens shown herein arise from patients with ileal disease that led to surgery because of ileal occlusion in one case, stenosis in another, and fistulizing, perforating disease in the third. Image on the left shows LN with collecting lymphatic vessel (arrowheads) leading back to intestinal wall, with smaller lymphoid collections along the route (arrows). Two images on the right show chains of TLOs that are too small to be visible in the tissue slice when examined by eye, but which are readily identified using LED light scanning. B: Whole mount samples as in A were processed to generate 5-μm paraffin-embedded sections that were immunostained to identify α-smooth muscle actin (α-SMA)–positive vessels and podoplanin (Podo). Data reveal collecting lymphatic vessels interrupted by lymphoid follicle structures (white arrows). Enlargement on the right allows a better view, in the absence of the strong α-SMA staining, of the weaker podoplanin staining that characterizes the lumen of collecting lymphatic vessels. C: Different patient mesenteric fat sample cut in thicker 100-μm sections showing a collecting lymphatic vessel running into a follicle, with some tissue damage generated during delicate preparation. White arrow points to lymphatic collecting vessel valve that defines the direction of flow as being toward the follicle. The lower image, the next 100-μm section in the sequence, shows lymphatic vessels as they wind their way through to the efferent side of the follicle. D: CD20 and CD3 staining to identify follicles as lymphocyte-rich structures. LN on the left, two follicles on the right. Collecting lymphatic vessel that links these structures in a chain is thin enough that it is missing in this 5-μm paraffin section. E and F: Staining of follicle structures for peripheral node addressin (PNAd; E) and CD68 (F). Asterisks in E denote adipocytes. G: Quantification of cell numbers in lymph nodes and TLOs. Each lymph node analysis comes from a different CD specimen. For the TLO analysis, we measured the size of multiple TLOs in the same specimen. The 41 TLO evaluations depicted in the panel arise from TLOs combined from 10 different patients. H: Tissues from nine of the patient samples were stained for CD20, and the fraction of TLO structures positive for CD20 is depicted. I: The TLO area in the mesentery of the same 10 CD patients was quantified relative to the total mesenteric area in cross sections. Each dot in H and I represents data from one patient. Horizontal lines represent the mean value in the respective data column. Scale bars = 300 μm.