3. Sampling <ul><li>1. Adequate sample size. </li></ul><ul><li>18-19G small, narrow samples. Often inadequate representation of vessels. </li></ul><ul><ul><li>For focal lesions involving a small number of glomeruli, 25 glomeruli may be needed for LM examination to have > 95% chance of detecting those lesions. </li></ul></ul><ul><ul><li>Minimum sample size for diagnosis varies greatly with the specific diagnosis; </li></ul></ul><ul><ul><ul><li>Membranous glomerulonephritis can be diagnosed from a single glomerulus. </li></ul></ul></ul><ul><ul><ul><li>Transplant diagnoses are most accurate when the sample includes a minimum of 7 glomeruli. </li></ul></ul></ul><ul><ul><li>For most LM assessment to adequately assess severity and distribution of lesions 8 to 10 glomeruli. </li></ul></ul><ul><li>2.Sample Location— </li></ul><ul><ul><li>Juxtamedullary Versus Cortical/Subcapsular cortical samples have overrepresentation of global sclerosis related to aging/hypertension and non-specific scarring. </li></ul></ul><ul><ul><li>Juxtamedullary glomeruli are the earliest to be involved with segmental sclerosis in FSGS. </li></ul></ul><ul><ul><ul><li>This region should be included in the sample for optimal detection. </li></ul></ul></ul>
4. Allocation and Fixatives <ul><li>An adequate assessment of native renal biopsies includes light microscopy (LM), immunofluorescence microscopy (IF), and electron microscopy (EM). </li></ul><ul><li>For transplant biopsy, LM and IF are considered the standard, with repeat biopsies only needing LM in many cases. </li></ul><ul><li>• LM: For most differential diagnoses, the largest portion of cortex should be placed in fixative for LM. </li></ul><ul><ul><li>These fixatives include formalin, paraformaldehyde, or less commonly used alcoholic Bouin’s or Zenker’s. </li></ul></ul><ul><li>• IF: IF tissue should include a small piece of cortex, usually 3 to 4 mm. </li></ul><ul><ul><li>Tissue for IF can be directly frozen, or placed in tissue transport media such as Michel’s, and transported to the laboratory. </li></ul></ul><ul><ul><li>Tissue is stable at room temperature for express mailing to central laboratories in this media. </li></ul></ul><ul><li>• EM: Small, 1-mm tubes of cortex are allocated for EM, and optimally are placed directly in glutaraldehyde. </li></ul>
5. <ul><li>1.Dividing the Tissue </li></ul><ul><li>A dissecting microscope can be used or one can blindly remove 1-mm cubes from each end of each core and place in glutaraldehyde for EM, divide each remaining core into 2 nearly equal pieces, placing the larger of each core in fixative for LM, and the smaller section of each core in tissue-transport media for IF. </li></ul><ul><li>2.Handling of Tissue </li></ul><ul><li>◦ No forceps, manipulate with thin wooden stick to avoid crush artifact. </li></ul><ul><li>◦ Avoid touching tissue with a fixative-contaminated scalpel or razor blade (this contaminates the tissue for IF). </li></ul>
6. <ul><li>Inadequate tissue: </li></ul><ul><ul><li>tissue is frozen for IF stains—the remaining frozen tissue may be fixed in formalin and processed for LM. </li></ul></ul><ul><ul><li>EM study can be done by processing remaining tissue from the LM sample from the paraffin block. </li></ul></ul><ul><ul><li>Tissue that has not been in paraffin blocks too long can sometimes give satisfactory results for immunofluorescence done on fixed, paraffin-embedded tissue. </li></ul></ul><ul><li>LM: Tissue for LM is processed, dehydrated, and placed in paraffin block, and multiple serial sections are obtained and stained. </li></ul><ul><ul><li>Usual stains include hematoxylin & eosin, periodic acid-Schiff (PAS), silver methenamine (Jones’), and Masson trichrome. </li></ul></ul><ul><ul><li>Additional unstained slides are produced to allow additional special studies as needed. </li></ul></ul><ul><ul><li>Five hours of processing, sectioning, and staining time are typically needed to produce LM slides. </li></ul></ul><ul><li>IF: Tissue for IF is surrounded with OCT compound and frozen. </li></ul><ul><ul><li>Sections are produced and stained with fluorescein-tagged antibodies against IgG, IgA, IgM, complements C3 and C1q, κ and λ light chain. </li></ul></ul><ul><ul><li>Complement product C4d may also be stained on frozen tissue, with more technical difficulty in staining on paraffin-block tissue. </li></ul></ul><ul><ul><li>1-2 hours of processing, sectioning, and staining time are needed for production of IF slides. </li></ul></ul><ul><li>EM: EM tissue is processed and embedded in a plastic, hard media, and scout sections (so-called thick sections). </li></ul><ul><ul><li>Stained with toluidine blue to identify the specific area to be cut for thin sections to be placed on a grid for EM examination. </li></ul></ul><ul><ul><li>Typically 2 working days are needed to process and produce EM sections for ultrastructural examination. </li></ul></ul>
8. LM- PAS <ul><li>Periodic acid-Schiff (PAS): </li></ul><ul><ul><li>To identify glycogen in tissues. </li></ul></ul><ul><ul><li>Reaction of periodic acid selectively oxidizes the glucose residues, creates aldehydes that react with the Schiff reagent and creates a purple-magenta color. </li></ul></ul><ul><ul><li>A suitable basic stain is often used as a counterstain. </li></ul></ul><ul><ul><li>Mainly used for staining structures containing a high proportion of carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans), typically found in eg. connective tissues, mucus, and basal laminae. </li></ul></ul>
9. LM- Masson's trichrome <ul><li>Masson's trichrome: </li></ul><ul><ul><li>Three-color staining protocol. </li></ul></ul><ul><ul><ul><li>Red keratin and muscle fibers, </li></ul></ul></ul><ul><ul><ul><li>Blue or green collagen and bone, </li></ul></ul></ul><ul><ul><ul><li>Light red or pink cytoplasm, and </li></ul></ul></ul><ul><ul><ul><li>Dark brown to black cell nuclei. </li></ul></ul></ul><ul><ul><li>The trichrome is applied by immersion of the fixated sample into Weigert's iron hematoxylin, and then three different solutions, labeled A, B, and C: </li></ul></ul><ul><li>* Weigert's hematoxylin is a sequence of three solutions: ferric chloride in diluted hydrochloric acid, hematoxylin in 95% ethanol, and potassium ferricyanide solution alkalized by sodium borate. It is used to stain the nuclei. </li></ul>
10. <ul><li>* Solution A, also called plasma stain, contains acid fuchsin, Xylidine Ponceau, glacial acetic acid, and distilled water. Other red acid dyes can be used, eg. the Biebrich scarlet in Lillie's trichrome. </li></ul><ul><li>* Solution B contains phosphomolybdic acid in distilled water. </li></ul><ul><li>* Solution C, also called fibre stain, contains Light Green SF yellowish, or alternatively Fast Green FCF. It is used to stain collagen. If blue is preferred to green, methyl blue, water blue or aniline blue can be substituted. </li></ul><ul><li>A common variant is Lillie's trichrome. </li></ul>
11. LM- H&E <ul><li>H&E stain, HE stain or hematoxylin and eosin stain: </li></ul><ul><ul><li>Most widely used stain in medical diagnosis </li></ul></ul><ul><ul><li>Application of the basic dye hematoxylin, which </li></ul></ul><ul><ul><ul><li>colors basophilic structures with blue-purple hue, and </li></ul></ul></ul><ul><ul><ul><li>alcohol-based acidic eosin Y, which colors eosinophilic structures bright pink. </li></ul></ul></ul><ul><li>The basophilic structures - containing nucleic acids, such as the ribosomes and the chromatin-rich cell nucleus, and the cytoplasmatic regions rich in RNA. </li></ul><ul><li>The eosinophilic structures- intracellular or extracellular protein. The Lewy bodies and Mallory bodies are examples of eosinophilic structures. Most of the cytoplasm is eosinophilic. Red blood cells are stained intensely red. </li></ul><ul><li>Some structures do not stain well. </li></ul><ul><ul><li>BM: PAS stain or some silver stains </li></ul></ul><ul><ul><li>Reticular fibers: silver stain. </li></ul></ul><ul><ul><li>Hydrophobic structures also tend to remain clear; these are usually rich in fats, eg. adipocytes, myelin, and Golgi apparatus membranes. </li></ul></ul>
13. LM- Silver stain <ul><li>Silver stain: </li></ul><ul><ul><li>to show proteins (for example type III collagen) and DNA. </li></ul></ul><ul><li>Silver nitrate forms insoluble silver phosphate with phosphate ions. When subjected to a reducing agent, usually hydroquinone, it forms black elementary silver. This is used for study of formation of calcium phosphate particles during bone growth. </li></ul><ul><li>Silver staining is used in light microscopy. The metallic silver particles are deposited on sensitised reticulin fibres and are then easily seen in the microscopic preparations. </li></ul><ul><li>Silver stain aids in the perception of reticular fibers. </li></ul>
22. Types of hypercellularity <ul><li>Mesangial: Proliferating cells are located in the mesangium, with nothing or little diminution of the capillary lumens. The nuclei usually have a relatively homogenous aspect because proliferating cells are all, or almost all, from similar origin: mesangials. </li></ul><ul><li>Endocapillary: In this type of injury the proliferation is not only located into capillary lumens, but the cellular proliferation occupy the glomerular tuft and compress capillaries. The proliferating cells are from diverse origin: endothelial, mesangial, and inflammatory (lymphocytes, monocytes, and neutrophils), nevertheless, it is called “endocapillry” by convention (hypercellularity within the capillary tuft). The nuclei of the cells are more variable in form and size (because there are several types of cells) and tend to obstruct the capillary lumen. The proliferation is not limited to the mesangium. It can be global or segmental. </li></ul><ul><li>Extracapillary (crescents): Proliferation of cells in the Bowman’s space, with at least two layers of cells covering Bowman’s capsule. The cells that proliferate are parietal epithelial and infiltrating inflammatory cells, mainly monocytes. This type of proliferation is also known as: crescents. It can be circumscribed or circumferential. </li></ul><ul><li>When there is endocapillary and/or extracapillary cell proliferation we say that there is a “true glomerulonephritis”. </li></ul>
23. Other important terms in the description of glomerular changes: <ul><li>Global: A lesion involving the entire glomerulus. </li></ul><ul><li>Segmental: A lesion involving a part of the glomerulus, with al least a segment spared. </li></ul><ul><li>Diffuse: A lesion involving all or most of the glomeruli. </li></ul><ul><li>Focal: A lesion involving only some glomeruli. An exact number (or percentage) is not defined and accepted by all authors. For some it is <80% of glomeruli. In some classifications, like lupus nephritis, is called diffuse when there are 50% or more of glomeruli involved and segmental if <50%. </li></ul><ul><li>Hyalinosis: Homogenous, amorphous glomerular deposits composed mainly of proteinaceous material. It is observed reddish, or green or sometimes blue, according to the technique, with the trichrome stain, and they are positive with the PAS (periodic acid of Schiff) stain. Similar lesions can be observed in arterioles: hyaline arterioloesclerosis. </li></ul><ul><li>It must be differentiated from fibrinoid necrosis, that displays similar colour with these stains, but it is a more granular material, less homogenous and is accompanied by cellular debris and/or inflammatory cells. </li></ul><ul><li>Sclerosis: Is a glomerular scar produced with proliferation of type IV collagen (glomerular collagen). </li></ul><ul><li>Fibrosis: Scar produced with predominantly type I collagen (interstitial type collagen). </li></ul>
24. Normal Histology Glomerulus
26. Normal Histology
28. <ul><li>The network of capillaries, the mesangium, and the podocytes form the glomerular tuft. </li></ul><ul><li>This tuft is contained in the Bowman's capsule; this is formed by a basement membrane and a flat epithelium: parietal epithelial cells. </li></ul><ul><li>Between the Bowman’s capsule and the tuft is the Bowman’s space, which, in vivo, is a narrow space where parietal and visceral cells are almost united each other. </li></ul><ul><li>This space usually is seen large in the histologic sections due to retraction that undergo the capillaries during the tissue processing. </li></ul>
29. <ul><li>The glomerular tuft is formed by lobules of capillaries. </li></ul><ul><li>The afferent arteriole gives origin to 4 - 8 capillaries, each one of which is subdivided to form a lobule. </li></ul><ul><li>In each lobule there are several mesangial areas: the portion of mesangium that support several capillaries. </li></ul><ul><li>It is very important to recognize the mesangial areas to determine when there is or non hypercellularity: cluster of three or more nuclei per mesangial area in thin 2 to 3 micron sections away from the vascular pole. </li></ul>
30. Image of a lobule of the glomerular tuft: red arrows indicate several mesangial areas in which there are 1 or 2 nuclei. Green arrows indicate nuclei of endothelial cells (Masson’s trichrome, X400).
31. <ul><li>The mesangial matrix is formed by different types of collagen (III, IV, V and VI), microfibrillar proteins, glycoproteins, proteoglycans and other components </li></ul><ul><li>The mesangial matrix, like the basement membranes of capillaries, Bowman’s capsule, and tubules are rich in type IV collagen, and has affinity by the methenamine-silver stain. See the irregular characteristic aspect of mesangial matrix (in black) in a normal glomerulus (Methenamine-silver, X.400). </li></ul>
32. The mesangial matrix also stains with the PAS, like the basal membranes, due to the affinity of PAS by type IV collagen (PAS, X300).
33. <ul><li>Vascular pole: site by where the afferent and efferent arterioles arrive and leave the glomerulus. </li></ul><ul><li>The two arterioles are separated for a space containing extracellular matrix and cells: extraglomerular mesangium. </li></ul><ul><li>The GBM has a variable thickness, between 240 and 340 nm in the adult, and is slightly more thickness in men than in women. </li></ul><ul><li>On the ultrastructural images it appears like a trilaminar structure, with a central zone: lamina densa, surrounded by less dense layers: lamina rara interna and lamina rara externa. </li></ul><ul><li>Conventional stains, GBM appears as a homogeneous dense layer directly adhered to epithelium and endothelium </li></ul><ul><li>Components of the GBM are type IV collagen, heparan sulphate, laminin, proteoglycans, entactin, and fibronectin </li></ul>
34. The GBM is seen perfectly smooth, without perpendicular irregularities nor projections (red arrows). The flat cytoplasm of the visceral epithelial cell can be seen; and in some points, it is possible also to see the cytoplasm of the endothelial cells. The nucleus of a podocyte appears pointed with the green arrow. The nuclei of the endothelial cells usually are found towards the mesangial portion of the capillary (blue arrow) (Methenamine-silver)
36. <ul><li>Mesangial/endothelial/podocytes: 2/3/1 </li></ul><ul><li>(JGA): formed by the terminal portion of the afferent arteriole, the first portion of the efferent arteriole, the extraglomerular mesangium (between both arterioles) and the macula densa (a plaque of very specialized and differentiated cells, in the distal straight tubule, that adheres to the vascular pole of glomerulus of the same nephron). </li></ul><ul><li>It has been suggested that the peripolar cells (cells located just at the transition of the parietal to the visceral epithelium) should also be included as part of JGA, nevertheless, its function is not well known. </li></ul>
38. JGA: The yellow arrows indicate the macula densa, see the apical nuclei. Almost in contact with macula densa cells is the extraglomerular mesangium indicated with the black arrows. The green arrow marks the efferent arteriole and the blue arrow the afferent arteriole. The Peripolar cells are located exactly in the angle in which parietal epithelium contacts visceral epithelium (H&E, X.400).
39. Normal Histology Interstitium
40. <ul><li>Space between glomeruli, tubules, vessels and nerves. It is scant in the cortex and abundant in the medulla. </li></ul><ul><li>Composed of cells and extracellular fibrillar structures, proteoglycans, glycoproteins and fluid. </li></ul><ul><li>The cells are of two types: fibroblasts and cells of the immune system that have migrated (monocytes/macrophages, dendritic cells and lymphocytes). </li></ul><ul><li>The cortical fibroblasts produce erythropoietin. </li></ul>
41. <ul><li>The peritubular interstitium corresponds to the 7-9% of the cortical volume and usually is fused with basement membranes of the tubules and capillaries. </li></ul><ul><li>The periarterial interstitium surrounds the intrarenal arteries and finishes throughout the afferent arterioles, and contains lymphatic and nerves. In the periarterial interstitium the fibroblasts, apparently, do not synthesize erythropoietin. </li></ul><ul><li>The lymphatics are embedded in the periarterial tissue, they start in the vicinity of the afferent arterioles running the arterial course toward the hilium. </li></ul><ul><li>The intrarenal nerves run alongside the arteries in the periarterial connective tissue. There are independent autonomic nerves for the vessel walls, juxtaglomerular apparatus, and some fibres contact the pars convolute of the proximal tubule. </li></ul>
42. Cortex- Scant interstitium
43. Medulla- more interstitium
44. Nerves accompanying the vessels
45. Normal histology Tubules
46. <ul><li>Correspond to 90% of the renal cortex. </li></ul><ul><li>The tubular system of nephrons is divided in several segments: </li></ul><ul><ul><li>proximal tubule, </li></ul></ul><ul><ul><li>thin limb of Henle, </li></ul></ul><ul><ul><li>thick ascending limb of Henle (here is the macula densa), </li></ul></ul><ul><ul><li>distal tubule, </li></ul></ul><ul><ul><li>collecting duct. </li></ul></ul>
47. Proximal tubules are characterized to have an abundant, eosinophilic cytoplasm and a brush border easy to identify. The cytoplasmic size, the tall cells and the brush border (arrows) are more prominent in the proximal convoluted portion. (H&E, X400). <ul><li>Proximal tubule has been divided (morphologically and functionally) in two portions: the proximal convoluted portion (pars convoluta), which occupies the cortical labyrinth, and the straight portion (pars recta), in the medullary rays of the cortex and outer medulla. </li></ul>
48. Brush border of the proximal tubules has affinity by the reagents used in the periodic acid of Schiff coloration (arrows). (PAS, X200).
49. The silver stain emphasizes the basement membranes of tubules and it allows us to delineate the contours very well. The prominent eosinophilic cytoplasm of the proximal tubules contrasts with the clearer and less abundant cytoplasm of the distal tubules (asterisks). (Methenamine-silver stain, X200).
50. In this image we can see distal tubules at both sides of a thin portion of the thin limb of Henle with a hyaline cast (arrow). In many cases it is very difficult, with light microscopy, to differentiate if small spaces in the medulla, like this here observed, are peritubular capillaries or thin portion of the limb of Henle. (H&E, X400). <ul><li>The thin limbs of Henle begins near the corticomedullary union, it has a descendent and ascending portions and it is formed by flat epithelium; </li></ul><ul><li>It also finishes near the corticomedullary union. Nephrons originated in juxtamedullary areas are long-looped type and penetrate until the inner medulla. </li></ul>
51. <ul><li>Distal tubule is divided in pars straight or thick ascending limb of loop of Henle, where it is the macula densa, and distal convoluted tubule. The epithelium of pars straight have tall cells that interdigitate each other. </li></ul><ul><li>There is an abrupt increase in the height of the epithelium in the transition of distal straight tubule and distal convoluted tubule. </li></ul>
52. Distal tubule and collecting duct cells have less eosinophilic cytoplasm than proximal tubule cells. See a clear halo surrounding the nucleus in many cells. (H&E, X400). <ul><li>The collecting duct has cortical and medullary portions. </li></ul><ul><li>Its epithelium changes when descending in the medulla, in more distal portions the cells are taller and have more complex unions. </li></ul><ul><li>The diameter of the ducts increases progressively. </li></ul><ul><li>There are two cell types: principal cells (collecting duct cells) with an important function in water reabsorption and Na and K transport; these cells also have vasopressin receptors. </li></ul><ul><li>And the intercalated cells , with darker cytoplasm that evidence high carbonic anhydrase activity, with an important role in acid-base balance. </li></ul>
56. <ul><li>The main renal artery arises from the aorta and it is divided, usually, into the anterior and posterior branches and sometimes also into an inferior division. This artery is divided to form the segmental arteries, usually four or five, although there are many variants. These arteries irrigate different segments of the kidney and they are end arteries: there is not significant collateral circulation between the territories supplied by them. The segmental arteries divide within the renal sinus to form the interlobar arteries which enter the kidney crossing the space between the calyces and adjacent cortical parenchyma, they continue by the space between pyramids (medulla) and the septa or columns of Bertin (the cortical tissue that surrounds pyramids). </li></ul><ul><li>The interlobar arteries divide, dichotomously, to originate the arcuate arteries that are located between cortex and medulla, tending to surround one half of a pyramid. This arrangement creates two sets of arcuate arteries between adjacent pyramids with each of the sets running between the septa of Bertin and the medullary tissue in the pyramids. Each one of these sets supplies a part of the septum proximate to it. This vascular pattern reinforces the concept that septa of Bertin are derived by the process of lobar fusion, whereby the cortical cap of the pyramid in each lobe fuses with its neighbour during fetal development </li></ul>
57. <ul><li>The arcuate arteries converge near the centers of the basal surfaces of the pyramids, but do not anastomose (end arteries). </li></ul><ul><li>The arcuates give off branches: the interlobular or cortical radial arteries, which are arranged radially over the basal surface of the pyramids, perpendicular to the renal surface. Few interlobular arteries reach the surface of the kidney where anastomose with capsular branches of suprarenal and gonadal arteries. </li></ul><ul><li>The interlobular arteries give off lateral branches at regular intervals: the afferent arterioles. Some afferent arterioles can form directly of the arcuate, or even directly of interlobars. </li></ul><ul><li>There are not arteries penetrating in the renal medulla. </li></ul><ul><li>The veins are originated in the cortex and follow parallel to the interlobular, arcuate, interlobar, and segmental arteries until forming the main renal vein. </li></ul>
58. <ul><li>The renal arteries and arterioles have the same histologic structure that arteries or arterioles elsewhere in the body. They are formed by endothelium, subendothelial connective tissue or intima, internal elastic lamella (difficult to identify in the small arteries), muscular media, and adventitia that fuses with the interstitial tissue. </li></ul>
59. The arcuate arteries run by the interstitial space between the cortex and medulla, and they are accompanied by lymphatic vessels, nerves and veins (asterisk) (H&E, X100).
60. The cortical radial or interlobular arteries are branches of the arcuate and originate the afferent arterioles. Usually they have, depending of the thickness of their wall, several layers of muscular cells (H&E, X300).