PROCESSING OF RENAL SAMPLES
Daily we receive renal tissue samples obtained
by needle, open biopsies (wedge renal subcapsular cortex), or nephrectomy. Owing
to the nature of the renal diseases, the sample of renal tissue needs to be
examined with optimal methods to allow for a complete evaluation, including
light microscopy (LM), immunofluorescence (IF) or immunoperoxidase (IP), and,
frequently, electron microscopy (EM). The correct diagnosis requires a well-trained
renal pathologist with thorough knowledge of not only renal pathology but also
renal medicine in order to correlate intricate tissue-derived information with
detailed and sometimes subtle clinical data in order to provide the best possible
clinicopathologic diagnoses (Walker PD, et al Mod Pathol. 2004; 17:1555
The specimen must be processed carefully, avoiding to compress it or to fragment it. With wedge biopsies or nephrectomies there is no problem to select the fragments for LM, IF, and EM. In some cases and centers it is usual to take, in addition, frozen tissue for molecular studies, although the remaining tissue of that frozen for IF can be useful.
If the biopsy cores are sent to the pathology laboratory for division and processing, they may be placed in a special transport fluid, physiologic saline-moistened gauze, or moistened filter paper carefully folded over the tissue.
When biopsy cylinders, by needle, are the specimen received, we must identify, with the dissecting microscope, the cortex to select sample with glomeruli for IF and EM. The medulla shows parallel structures that correspond to collecting ducts and peritubular capillaries. The cortex appears irregular and the glomeruli usually are visible as small round structures with capillaries (Figure 1, below), or as red points. Sometimes, these glomeruli are pale or irregular, but they can be identified with relative certainty. In cases with extensive glomerular histologic alterations or circumferential crescents can be more difficult to identify them.
For IF it is recommendable to take at least a fragment of cylinder with several glomeruli or a piece of identified cortex. For EM should be taken two small fragments, of several millimeters, with glomeruli. The remaining tissue is placed in fixative for LM. Avoid touching tissue with a fixative-contaminated scalpel or razor blade because this contaminates the tissue for IF.
If there is only one core, it can be cut in half by crosssectioning and the larger piece placed in formalin or another fixative suitable for LM; the smaller portion is saved for immunofluorescence evaluation. If a second core is obtained, the ends should be taken for EM and the specimen again divided almost in half with the larger tissue core now kept for IF and the smaller for LM. Alternatively, if both cores contain cortex, one core can used for LM and one core for IF. If tissue is limited, the clinical differential diagnosis may drive the division of material, for example, in cases of recurrent hematuria in an adult patient, we must attempt obtain sufficient material for IF (IgA nephropathy is the more probable diagnosis). Frozen tissue used for IF can be subsequently processed for either LM or EM; although artefacts will be unavoidable, useful information can still be obtained. If there is not dissecting microscope the best method is to remove 1mm cubes from the ends of each cylinder and to place them in cooled glutaraldehyde or other fixative suitable for EM, and the remainder cylinder(s) is (are) divided for LM and IF. Fragments of 2 mm, from the remaining ends, can also be taken for IF (Walker PD, et al. Practice guidelines for the renal biopsy. Mod Pathol. 2004;17:1555 [PubMed link]).
In cases with enough renal tissue (wedge biopsies or nephrectomies) it is better to keep frozen more tissue that just the necessary: it is preferable to have suitably processed tissue and does not need it that need it and does not have it.
For LM the tissue must be fixed, and the most commonly used fixative is buffered, 10% formalin (formaldehyde). Some laboratories prefer alcoholic Bouin’s, Duboscq-Brasil or Zenker’s fixatives that provide better preservation of certain morphologic details; we use Bouin’s due to it fixes more quickly and it allows to see better the cellular characteristics. However, these fixatives limit recovery of material for EM, immunohistochemistry and molecular studies. Some laboratories prefer 4% paraformaldehyde to optimize tissue suitability for LM, IHC, EM, and in situ hybridization (ISH) studies. Methacarn, a modified Carnoys fixative (methyl alcohol 60%, chloroform 30%, glacial acetic acid 10%), provides good fixation for LM and EM and should provided recovery of mRNA (Walker PD, et al. Practice guidelines for the renal biopsy. Mod Pathol. 2004;17:1555 [PubMed link]). The material processed for LM can serve as reserve material for IHC or EM if either of these other modalities is found lacking glomeruli. Formalin fixation and paraffin embedding is not an impediment to electron microscopic evaluation and rapid reprocessing is a relatively simple procedure, although in general the quality of the images will be inferior and and there may be more artifacts. (Wang NS, Minassian H. The formaldehyde-fixed and paraffin-embedded tissues for diagnostic transmission electron microscopy: a retrospective and prospective study. Hum Pathol 1987;18:715–727 [PubMed Link]; Widehn S, Kindblom LG. A rapid and simple method for electron microscopy of paraffin-embedded tissue. Ultrastruct Pathol 1988;12:131–136[PubMed Link]).
For paraffin inclusion, tissues can be processed overnight using a protocol for small biopsy samples. Alternatively, rapid processing protocols are available, some employing microwave fixation that can be used for same-day processing (Rohr LR, et al. A comparison of routine and rapid microwave tissue processing in a surgical pathology laboratory. Quality of histologic sections and advantages of microwave processing. Am J Clin Pathol 2001;115:703–708 [PubMed link]). It is possible to include in plastic, which allows a better visualization of the microscopic structures; nevertheless, so processed tissue is not optimal for other techniques like immunohistochemistry or ISH. In addition, it is a complicated, laborious and expensive method.
On daily practice, thin cuts (2 - 3 microns of thickness) stained with hematoxylin-eosin, PAS, trichrome, and methenamine-silver, permit the diagnosis in most cases. In some cases other stains are used: Congo red, Wright, Giemsa, elastic, iron, phosphotungstic acid hematoxylin (PTAH), and so on, according to the clinical diagnosis or to the microscopic findings.
Ideally the tissue selected for IF must quickly be frozen, in isopentane or methylbutane, usually cooled in liquid nitrogen; the renal tissue is previously placed in OCT (Optimal Cutting Temperature compound). Some authors inform acceptable results performing fluorescence (non immunofluorescence) in samples routinely processed, but fixed in Hollande’s fixative, to detect immune deposits (McMahon JT, et al, Demonstration of immune complex deposits using fluorescence microscopy of hematoxylin and eosin-stained sections of renal Hollande's fixed biopsies. Mod Pathol. 2002; 15: 988-97 [PubMed Link])
There is no a minimum number of glomeruli indispensable for a suitable diagnosis. In global and diffuse lesions a single glomerulus can be sufficient. In segmental lesions or cases of minimal change disease many glomeruli may be needed. For focal lesions involving a small number of glomeruli, 25 glomeruli may be needed for LM examination to have a greater than 95% chance of detecting those lesions (Fogo AB. Approach to renal biopsy. Am J Kidney Dis. 2003;42:826-36 [PubMed link])
RENAL BIOPSIES PROTOCOL OF ROUTINE STUDY
IN OUR LABORATORY
In all native biopsies, and transplanted kidneys in which glomerular disease is suspected, the biopsy must be sent in fresh to be selected, in our department, the tissue for IF, LM, and, if required, EM. “In fresh” means without fixatives: formalin, Bouin’s, Zenker and so on. In general, is recommendable to place the sample in physiologic saline-moistened gauze, or moistened filter paper, or (no ideally) in a recipient with physiologic saline; the tissue must be not let dry. No use other papers to put the tissue: hygienic paper neither paper absorbent towels. If the sample will take more than 30 minutes in arriving at the laboratory it is recommendable refrigerate it putting the container with the biopsy in another one with ice cubes. Never put the sample in the freezer; the recommendable temperature is near to 4ºC.
The received material will be dissected (dissecting
microscope) to ensure that the fragments for IF and EM contain glomeruli (Figure
1 and 2). The remaining tissue will be fixed in 4% buffered formalin or Bouin's
for one hour and after transferred to formalin for later paraffin inclusion.
Figure 1. With the dissecting microscope, in this cylinder, there are, at least, four glomeruli (arrows). In some cases, as the one of the photography, the glomeruli highlight because there are blood capillary congestion. In other cases it is more difficult to identify them, mainly if there are severe alterations as glomeruloesclerosis or crescents. (In fresh cylinder of renal tissue observed with dissecting microscope, X32).
Figure 2. The renal medulla is easily recognized by the presence of tubules and peritubular capillary that appear like straight lines or slots. In this photography oblique lines or slots are seen crossing, almost transversally, the cylinder. The medulla fragments do not contain glomeruli and therefore they are not adequate for IF or EM. (In fresh cylinder of renal tissue observed with dissecting microscope, X32).
In all cases at least 7 histologic slides will be stained with H&E, trichrome (Masson or Gomori), P.A.S, and methenamine-silver. IF for IgG, IgA, IgM, C3, and C1q will be carried out in all cases. In selected cases, according to clinical or histologic features, kappa and lambda light chains, fibrinogen, and albumin will be also carried out.