The pathological diagnosis of renal biopsy is an indispensable diagnostic method to clarify the lesion site (glomerulus, tubules, interstitium, renal vessels), lesion nature (inflammatory and non-inflammatory, active or inactive), lesion extent (focal or diffuse, globular or segmental), and is the main basis for deciding the treatment plan and prognosis of renal diseases.
The pathological diagnosis of renal biopsy is to infer the whole renal lesion from the lesion of some renal tissues, and the description of its pathology sometimes cannot make the final diagnosis of the disease independently; it must be combined with clinical manifestations and examination results for systematic analysis in order to make the correct diagnosis of the disease. Therefore, it is the basic skill of nephrologists to understand the information about the severity of renal lesions from pathological examination reports and to combine them with clinical findings so as to grasp the disease comprehensively and accurately.
Immunofluorescence examination and light microscopy are the most commonly used methods of renal pathology examination, so nephrologists must master the skills of reading the diagnostic reports of these two examinations.
I. Immunofluorescence examination
Immunofluorescence antibody technique is to use the principle that an antibody can only bind specifically to a corresponding antigen, and fluorescein is labeled on the known antibody, and the known antibody labeled with fluorescein reacts with the kidney tissue to be examined. The complex is observed under a fluorescence microscope with an ultraviolet light source, and the fluorescein as an indicator is excited to emit bright fluorescence, which means that the kidney tissue under examination has the corresponding antigen present at the site where the fluorescence appears.
Immunofluorescence examination is an indispensable method in the pathological examination of glomerular diseases. Care should be taken to have a full range of known antibodies and complement species to ensure that one to two glomeruli are present. The following antigens can be revealed by immunofluorescence examination.
1, globulin class: Using immunofluorescence technique, human immunoglobulins (IgG, IgA, IgM) on kidney tissue sections are routinely examined to determine the type of globulin involved in the immune response;
2, complement class: such as C3, C1q, to confirm the pathway in which complement is activated during the immune response, i.e. classical or bypass pathway; plasma protein class: such as plasma proteins (fibrinogen), to determine the activity of renal lesions; specific antigens: such as HBsAg, HbcAg, alpha chain of type IV collagen, etc., to detect nephritis-associated antigens.
3. The following should be noted when looking at the immunofluorescence test report.
What kind of components are deposited: IgG, IgA, IgM, C3, C1q, FRA; when all are deposited, it is called “full brightness”.
Deposition site: such as in the glomerular capillary wall, thylakoid area, renal capsule, tubular basement membrane, interstitial vascular wall, etc.
Morphology: continuous linear, discontinuous granular, clumped or short linear, irregular, etc.
Intensity of fluorescence display: (-) not shown under both high and low magnification; (±) faintly visible under high magnification; (+) visible under high magnification, faintly visible under low magnification; (++) clearly visible under high magnification, visible under low magnification; (++++) dazzling under high magnification, clearly visible under low magnification; (++++) harsh under high magnification, dazzling under low magnification.
Distribution of fluorescence in the form of: focal, diffuse and segmental.
In thylakoid proliferative nephritis, the glomerular thylakoid zone shows deposits of one or several immunoglobulins or complements of varying intensity; in primary intracapillary proliferative nephritis, high-intensity IgG and complement C3 are deposited in coarse granules in the capillary wall and thylakoid zone; in primary membranous nephropathy, IgG and complement C3 are deposited in fine granules along the glomerular capillary wall; in atypical membranous nephropathy, IgG, IgA IgM, C3, C1q and FRA are deposited in the thylakoid area and capillary wall with high intensity, i.e., “full-tongued”.
Light microscopy
Light microscopic observation of glomerular lesions is different from ordinary pathological examination, which only observes HE staining and PAS staining, and PASM staining and Masson staining are more important.
HE (hematoxylin eosin stain): observe the nucleus, distinguish the cell type, and indicate the thickness of basement membrane.
PAS (Schiff’s periodate stain): observe GBM, TBM and extracellular matrix (ECM), but the thylakoid matrix (MM) stained wider than actual.
The stain is wider than the actual one, and the stain is rarely used to observe GBM.
PASM (hexosamine silver stain): The main focus is on GBM, but MM is also clear.
Masson: Observe the site of complexophilic hemoglobin (mostly suggesting immune complexes) deposition, the degree of interstitial fibrosis (collagen fibers are green, nuclei, immune complexes, thrombi, and plasma are red), etc.
1. Pathological terms.
Diffuse: more than 50% of the glomeruli have lesions/sections ;
Focal: less than 50% of the glomeruli have lesions/sections;
Sphericity: more than 50% of glomeruli are lesioned;
Segmental: less than 50% of glomeruli have lesions;
Hyperplasia: increased number of glomerular lamina propria cells;
Endothelial cell hyperplasia: 2 or more endothelial cells in one section of the capillary lumen or endothelial cells are seen in most of the capillary lumen;
Thylakoid hyperplasia: 4 or more thylakoid cells within the thylakoid zone in one section;
Sclerosis: Highly nodular hyperplasia of the thylakoid stroma, collapse of capillary loops, thickening and wrinkling of the GBM;
Fibrosis: proliferation of fibroblasts in the mesenchyme, secretion of collagen I and collagen III;
A complete pathology report of renal tissue light microscopy must be integrated with HE, PAS, PASM and Masson staining to describe glomerular, tubular, interstitial and small arterial lesions in a comprehensive manner and provide clinicians with more detailed information on the pathological changes in the kidney. When nephrologists read the diagnostic pathology report, they should pay special attention to the following information.
2. The number of glomeruli in the punctured tissue.
The pathological diagnosis of renal biopsy is based on the changes in a certain number of glomeruli removed by kidney puncture to infer the pathology of the kidney tissue, i.e., to infer the whole from the local, so the kidney tissue specimens for pathological diagnosis must be guaranteed to have a sufficient number of glomeruli. According to statistics, the accuracy of using specimens containing 5 glomeruli to determine the state of all glomerular lesions is only 65%, while the accuracy of using specimens containing 15 glomeruli to infer the changes of all glomeruli is up to 95%, so the specimens for light microscopy should be more than 10 glomeruli.
3, the number of spherical sclerosis and ischemic spherical sclerosis.
Spherical sclerosis is seen in various types of glomerulonephritis and glomerulopathy; ischemic sclerosis without increased thylakoid matrix is commonly seen in renal ischemia caused by various causes. With age, a certain number of glomeruli may undergo spherosclerosis or ischemic sclerosis, and the number of physiologically sclerotic glomeruli accounts for ≤ [(age/2)-10] × 100% of the glomeruli in the puncture specimen, and ischemic sclerosis beyond this range is seen in ischemic kidney injury (such as ischemic kidney disease, hypertensive kidney injury); spherosclerosis beyond this range is mostly seen in focal Proliferative sclerosis (sclerotic glomeruli account for 25 to 50% of the glomeruli of the puncture specimen) glomerulonephritis, proliferative sclerosis (sclerotic glomeruli account for 50 to 75% of the glomeruli of the puncture specimen) glomerulonephritis, and sclerotic (sclerotic glomeruli exceed 75% of the glomeruli of the puncture specimen) glomerulonephritis.
The presence or absence of crescentic bodies: Crescent formation is seen in severe glomerular capillary wall damage due to various causes. According to the size of the crescent, there are large crescent (the volume of the crescent accounts for more than 50% of the renal capsule) and small crescent (the volume of the crescent accounts for less than 50% of the renal capsule).
The crescent is generally referred to as large crescent. According to the composition of the crescent, it is divided into cellular crescent, cellular fibrous crescent and fibrous crescent, which indicate the course of the disease and the degree of old and new lesions. Cellular crescent suggests acute inflammation, which should be treated actively; fibrous crescent suggests chronic lesions, and cellular fibrous crescent is between the two.
4. Proliferative changes of glomerular thylakoid cells and stroma.
Thylakoid cells are the most active reactive cells in the glomerulus, which are stimulated by many injury factors and harmful substances and activated, and then contract and proliferate, and undergo metabolic changes, synthesize and secrete a variety of inflammatory mediators and matrix components, leading to glomerular injury and sclerosis. According to the degree of proliferation, thylakoid cells are classified into mild, moderate and severe hyperplasia; according to the extent of hyperplasia, they are classified into diffuse and focal hyperplasia, and thylakoid cell hyperplasia (especially moderate and severe hyperplasia) indicates active lesions. Increased thylakoid stroma is the result of thylakoid cell hyperplasia, which mostly suggests that the lesion tends to be slowed down.
5, whether the basement membrane is thickened.
Glomerular lesions that only show basement membrane thickening are primary membranous nephropathy, which is divided into phase V according to the evolution of the lesion. The lesions with both basement membrane thickening and thylakoid cell hyperplasia and increased thylakoid stroma are atypical membranous nephropathy, mostly secondary glomerular lesions, and the immunofluorescence is often “full bright” and not staged.
The deposition site of eosinophilia: glomerular disease with thylakoid hyperplasia as the main lesion, IgA nephropathy, etc. eosinophilia is mainly deposited in the thylakoid region; primary membranous nephropathy eosinophilia is deposited in the subepithelium; secondary membranous nephropathy (atypical membranous nephropathy) eosinophilia is deposited in the subepithelium, thylakoid region, basement membrane and subendothelial multiple sites.
6. Renal tubular vacuolar degeneration and granular degeneration.
In massive proteinuria, renal tubular vacuolar degeneration and granular degeneration are present simultaneously. Vacuolar degeneration is also seen in osmotic nephropathy, acute cyclosporine nephropathy, hypokalemic nephropathy, etc.
7. Interstitial renal fibrosis.
Seen in inactive lesions in the late stages of various renal diseases.
8, Mild thylakoid proliferative glomerulonephritis with ischemic kidney injury or partial crescent formation.
When the glomerular lesions are mild and there is more ischemic glomerulosclerosis or more crescent formation, the milder glomerular lesions cannot explain the cause of the heavier lesions such as ischemic glomerulosclerosis or crescent, the mild thylakoid proliferative lesions are suggested to the clinician along with ischemic glomerulosclerosis or crescent to draw clinical attention so that the clinician can further search for the cause of ischemic glomerulosclerosis and other lesions. The report should be read in conjunction with the renal pathology report.
When reading the renal pathology report, the clinical manifestations, immunofluorescence examination and glomerular count and light microscopy must be integrated to have a more comprehensive understanding of the nature of the disease.