Chapter Outline
SAFETY AND COMPLICATIONS OF BIOPSIES, 915
PERFORMING THE BIOPSY, 918
BIOPSY SPECIMEN HANDLING, 919
LIGHT MICROSCOPY, 919
Staining for Light Microscopy, 919
Examination of the Biopsy Specimen by Light Microscopy, 920
Terminology for Description of Glomerular Disease, 920
IMMUNOHISTOCHEMICAL ASSAY, 921
ELECTRON MICROSCOPY, 922
OTHER STUDIES PERFORMED ON THE KIDNEY BIOPSY SPECIMEN, 923
BIOPSY SPECIMENS FROM TRANSPLANTED KIDNEYS, 923
SIZE OF THE BIOPSY SPECIMEN, 923
BIOPSY REPORT, 924
CONCLUSIONS, 924
The kidney biopsy has become a fundamental component in the management of renal disease. Before its routine use, only autopsy material was available to investigate the pathophysiology of kidney disease, limiting antemortem diagnosis. However, its development and refinement since the late 1950s has been fundamental for the diagnosis and definition of clinical syndromes and the discovery of new pathologic entities. Through the critical analysis of kidney biopsies taken at different disease time points, key pathophysiologic features of kidney disease have been discovered, which have in turn helped to establish new paradigms in nephrology and have led to considerable alterations in patient management. This is true for biopsies of both native kidneys and renal transplants. In addition, much is still being learned regarding disease pathogenesis through the study of kidney biopsy material, which not only remains a gold standard for disease diagnosis, but has allowed the development of novel biopsy markers, which have revolutionized our concepts of pathologic mechanisms.
The first percutaneous kidney biopsies were performed over 50 years ago using a liver biopsy needle and intravenous pyelograms for screening, with the patient either sitting or supine. Their success in obtaining renal tissue and in aiding management confirmed the benefit of the procedure. Many innovations, including using real-time ultrasonography, which allows visualization of the needle entering the kidney, spring-loaded needles or needle holders, and careful preoperative evaluation of the patient, have improved the rate of obtaining renal tissue while minimizing the risks of the procedure. Consequently, this has placed percutaneous kidney biopsy at the very center of modern clinical nephrology. The range of diagnoses for a group of 2219 native kidney biopsies performed at the authors’ institution over a 5-year period is shown in Figure 29.1 .
Safety and Complications of Biopsies
Although generally considered safe, there is morbidity and a small, but measurable, mortality associated with the procedure, and it is therefore imperative to subject to these risks only those patients in whom there will be a potential benefit. Indications for kidney biopsy may vary from center to center, but accepted indications are listed in Table 29.1 . The significant complications related to the procedure are hemorrhage, development of arteriovenous fistulas, and to a lesser extent sepsis. Bleeding with macroscopic hematuria and the development of perinephric hematomas may be minor and self-resolving or major and require intervention in the form of blood transfusions, embolization, or rarely surgery. There is a risk for formation of arteriovenous fistulas, which may be asymptomatic and spontaneously resolve or lead to a significant vascular steal syndrome, compromising the rest of the kidney through ischemia. Finally, there is the risk for sepsis following the procedure, through the introduction of a septic focus or its dissemination. Overall, the risks for complication vary from center to center and between practitioners but can be estimated to be between 3.5% and 13%, with the majority being minor complications (approximately 3% to 9%). Mortality from the procedure is generally as a result of undiagnosed bleeding with significant hematoma formation and was reported in up to 0.2% of cases from some of the larger biopsy series, although other studies suggest that it represents an extremely rare adverse event. Some degree of bleeding is common, with approximately half of patients showing a drop in hemoglobin level after biopsy and a third developing some hematoma, but bleeding is significant and requires intervention in only a minority (up to 7%). Complications appear to be more common in biopsies of native than transplanted kidneys and in patients with more advanced renal impairment, prolonged bleeding times, or lower hemoglobin levels (11 ± 2 vs. 12 ± 2 g/dL). One prospective study identified the only risk factors for bleeding complications as female gender, younger age (35 ± 14.5 years vs. 40.3 ± 15.4 years), and a prolonged partial thromboplastin time. Interestingly, needle size, number of passes, blood pressure, and renal impairment were not different between those with bleeding complications and those without. However, in this study all patients with prolonged bleeding times received desmopressin (DDAVP) to correct the abnormality, and 75% of patients had serum creatinine values of less than 132 µmol. Conversely, others using retrospective univariate analysis have reported blood pressure greater than 160/100 mm Hg or a serum creatinine level of more than 2 mg/dL more than doubled the risk for bleeding. Overall, however, no effective means has been established to identify those individuals at risk for developing clinically significant complications. In one small series, the results of ultrasonography performed within an hour after biopsy had a 95% negative predictive value for predicting clinically significant hemorrhagic complications, meaning that the absence of a hematoma on the postbiopsy scan was very suggestive of an uncomplicated clinical course. Debate continues regarding the routine use of DDAVP to counteract uremic bleeding tendencies. In part this is because its use was previously reserved for only those patients with prolonged bleeding times, and numerous studies have since demonstrated that complication rates are no different if bleeding time estimation is omitted from the preoperative assessment, because it does not predict clinical complications. However, later data from a randomized double-blind trial suggested a significant benefit in preventing bleeding complications with few adverse events. In this trial 162 low-risk adult patients undergoing biopsy were enrolled and randomized to subcutaneous DDAVP (0.3 µg/kg) or placebo. The patients were normotensive and had preserved renal function with serum creatinine levels of less than 1.5 mg/dL (estimated glomerular filtration rate > 60 mL/min). The patients given desmopressin demonstrated a significant reduction in postbiopsy bleeding from 30.5% to 13.7% (relative risk [RR], 0.45), a significant reduction in hematoma size in those who did hemorrhage, and a reduction in duration of hospital stay. However, the drop in hemoglobin level after biopsy was minimal, and there were no major complications, leading some to question the benefit of reduction in clinically unimportant hematomas, which can be frequently found following biopsy if looked for. No thrombotic, hyponatremic, or cardiovascular events were recorded. Whether these data, in patients with preserved renal function, could be translated to those higher-risk patients with greater renal impairment is unclear and is a question worthy of a randomized trial.
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Guidelines on obtaining informed consent from patients and providing appropriate risk estimates have been produced by certain national renal groups, and one such example is provided in Table 29.2 . These estimates may err on the conservative side and should be adapted to local practice if adequate complication data are available. In addition to patients’ developing procedure-related complications, there is the chance that an inadequate core is obtained for diagnosis, containing too few glomeruli or insufficient cortical material, and this is reported in 1% to 5% of cases. The size requirements for accurate diagnosis are discussed later.
| Complication | Risk |
|---|---|
| Macroscopic hematuria | 1 : 10 |
| Bleeding that requires a blood transfusion | Less than 1 : 50 |
| Bleeding that may require urgent x-ray examination or even an operation to stop the bleeding | Less than 1 : 1500 |
| Severe bleeding necessitating nephrectomy | Less than 1 : 3000 |
| Deaths | Extremely rare |
* According to UK Renal Association. Available at: http://www.renal.org/information-resources/procedures-for-patients .
There are certain absolute contraindications that preclude percutaneous biopsy, whereas there are a number of relative contraindications ( Table 29.3 ) that may be circumvented depending on the importance of the biopsy, the operator’s experience, and the supportive facilities available. Ideally, all efforts should be made to deal with the relative contraindications; however, in the context of acute kidney injury this may not always be possible. The critical preoperative steps are to ensure that blood pressure is controlled, that the patient does not have a bleeding diathesis or a urinary tract infection, and that the kidneys are suitably imaged, with no evidence of obstruction, widespread cystic disease, or malignancy (although percutaneous biopsy is increasingly used to diagnose the nature of renal masses). As a result, preoperative assessment should allow those patients unsuitable for percutaneous biopsy to be referred for an alternative approach ( Figure 29.2 ). In these patients there are other means of obtaining renal tissue, which include open biopsies, laparoscopic biopsies, or transjugular biopsies. Each is associated with certain complications and has particular merits depending on the clinical scenario ( Table 29.4 ). Overall, these are generally required for only a minority of potential biopsy patients.
| Absolute Contraindication | Relative Contraindication |
|---|---|
|
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| Method | Advantage | Disadvantage |
|---|---|---|
| Transjugular approach | Can be of use in those with a bleeding diathesis and in patients receiving artificial ventilation, or if combined liver and kidney biopsy is required | Carries risk for capsular perforation Inadequate material retrieved in up to 24% |
| Open approach | Provides high yield of adequate tissue Hemostasis is more secure | Requires general/spinal anesthesia; recovery period is longer |
| Laparoscopic approach | Provides high yield of adequate tissue Hemostasis is more secure | Requires general/spinal anaesthesia; recovery period is longer |
The safe duration of observation following kidney biopsy has been investigated in a number of studies. Findings suggest that early discharge (after only 4 hours of observation) will result in a number of missed complications, with many more occurring between 8 and 24 hours after the procedure. Even after 8 hours, 23% to 33% of complications will be missed. However, an overnight stay will allow an extra 20% of complications to be identified before discharge, with between 85% and 95% of complication being identified at 12 hours and 89% to 98% following 24-hour observation. Some units practice a policy of day biopsies with a minimum 6-hour bed rest period, which is extended only if there is evidence of bleeding. Vigilant observation of blood pressure, pulse rate, and evidence of hematuria is required in all cases.
Performing the Biopsy
Following informed consent, the patient is positioned prone for biopsy of a native kidney or supine for biopsy of a renal transplant. A posterolateral approach is taken for biopsy of a native kidney. The procedure is performed under sterile conditions with disposable sterile ultrasonographic probe covers, allowing real-time visualization of the kidneys. The procedure is generally performed with the patient under light sedation and with local anesthesia. The lower pole of the left kidney is commonly the biopsy site, but the kidney that is best visualized and most accessible is preferable. After skin preparation, a small incision is made to accommodate the biopsy needle, which is advanced until it reaches the renal capsule. The patient is asked to hold his or her breath while the needle biopsy mechanism is deployed. Most operators now prefer spring-loaded Tru-cut needles or Biopty guns. Needle size varies from 14 gauge to 18 gauge, with many using 16 gauge as a compromise between obtaining a suitable core and increasing the risk for bleeding. Two cores are taken, which should be divided for different assessments as outlined later. In high-risk patients the needle tract may be plugged following the procedure with appropriate material such as Gelfoam. In these cases the biopsy is done through a coaxial introducer needle, and after biopsy the tract is plugged during removal of the coaxial introducer needle.
Careful postprocedure observations of vital signs are performed to detect early signs of bleeding, and all urine is tested by dipstick for blood.
Biopsy Specimen Handling
Detailed descriptions of methods of handling biopsies can be found in a number of publications including references.
A full assessment of the kidney biopsy specimen requires examination by light microscopy, immunohistochemical methods, and electron microscopy (EM), with the use of other tests in some circumstances. Therefore it is necessary for the biopsy specimen to be divided to provide material for each of these methods of examination. During this process it is extremely important that the biopsy not be damaged by handling or by drying, and that the tissue be fixed using an appropriate fixative as quickly as possible, ideally within minutes. This is best achieved by dividing the biopsy specimen at the bedside. Examination of the biopsy specimen with a dissecting microscope allows cortex, containing glomeruli, to be distinguished from medulla and thus facilitates assessment of the adequacy of the cores and division of the biopsy specimen so that glomeruli are present in the samples for each modality of examination. If a dissecting microscope is not available, then a standard light microscope can be used with the biopsy specimen placed in a drop of normal saline on a microscope slide. If it is not possible to examine the biopsy specimen in this way, then a standard approach to obtain material for EM is to take small fragments (approximately 1 mm in length) from each end of each core. In that way if there is cortex in the core, glomeruli should be sampled. The remainder of the cores can then be divided for light microscopy and for immunofluorescence. The part of the biopsy specimen for light microscopy is then placed in appropriate fixative and that for immunofluorescence is either snap frozen or transported to the laboratory in suitable transport medium such as that described by Michel and colleagues ; tissue placed in this medium can remain for several days at room temperature without loss of antigens. During division of the biopsy specimen it is important not to introduce artifacts due to crushing or stretching. Forceps should not be used to pick up the specimen; this can be done using either a needle or a small wooden stick such as a toothpick. The biopsy specimen should be cut using a fresh scalpel.
If the biopsy specimen has to be taken to the histology laboratory for division, this should be done as quickly as possible with the biopsy specimen wrapped in saline-moistened gauze or in tissue culture medium. Artifacts may be produced if the biopsy specimen is placed on dry gauze or gauze moistened with water, or if it is placed in ice-cold saline.
If the amount of material obtained at biopsy is limited, then it may be necessary to adapt the way in which it is divided, and the decision as to how this is done must depend on the clinical question. In most cases it is possible to omit frozen material for immunofluorescence and instead perform immunohistochemical examination on paraffin sections. However, if there is a suspicion of crescentic glomerulonephritis due to anti–glomerular basement membrane (anti-GBM) disease, immunofluorescent testing is more reliable for detecting the linear capillary wall staining. It may be possible to omit EM and perform it if necessary on material reprocessed from the paraffin block, but if this is done, it is not possible to obtain accurate measurements of glomerular capillary membrane thickness.
Light Microscopy
The most commonly used fixative for light microscopy is buffered 10% aqueous formaldehyde solution. This is actually a 10% solution of the 37% commercially available concentrated solution of formaldehyde, giving a final concentration of approximately 4%. This fixative is generally available in all histology laboratories, provides adequate fixation for light microscopy, and also allows the tissue to be used for immunohistochemical assay and EM.
Some more specialized fixatives such as Bouin’s or Zenker’s fixative provide better preservation of certain morphologic details, but in general the problems with handling these fixatives and the difficulties of subsequently using the material for immunohistochemical assay or EM, outweigh the advantages. For example, Bouin’s contains picric acid, which is explosive when dry. However, the authors do commonly use Bouin’s fixative for examination of mouse kidneys, in which the improvement in glomerular morphology is significant. Methacarn, a modified Carnoy’s fixative, also provides good fixation for light microscopy and EM and may allow the immunohistochemical detection of antigens that are not detected in formalin-fixed tissue. Details of the preparation of various fixatives can be found in the appendix of Churg and associates.
The standard method of processing tissue for light microscopy is by dehydration in graded alcohols, transfer to a clearing agent such as xylene, and embedding in paraffin wax. This is usually performed in an automated instrument but can be done by hand. Rapid processing schedules allow for same-day processing, and it is possible to obtain stained slides within 3 to 4 hours of receipt of the specimen in the laboratory.
It is important to have thin uniform sections for light microscopy. These should be cut as thin as possible—no more than 3 µm. It is often stated that kidney biopsy sections should be cut at 2 µm, but this may lead to problems in cutting with damage to the tissue. Because many pathologic lesions may be focal within glomeruli, interstitium, or vessels, it is essential that the biopsy be examined at multiple levels, and each laboratory will have its preferred way to achieve that. In general, serial sections should be cut with at least two placed on each slide. Multiple slides can then be stained with each stain, with some intervening unstained sections kept either for potential immunohistochemical examination or for other special stains as necessary.
Staining for Light Microscopy
Most renal pathologists employ a number of stains for light microscopy. The commonly used stains are hematoxylin and eosin (H&E), periodic acid–Schiff (PAS), silver methenamine, and a trichrome stain. The H&E stain is a good general histologic stain for studying the overall architecture of the kidney. It is good for studying the morphology of tubular cells and the morphology of interstitial infiltrates. With experience the different staining characteristics of hyaline, fibrin, and amyloid, all of which are eosinophilic, can usually be distinguished. However, the H&E stain does not distinguish staining of glomerular matrix and basement membrane from cell cytoplasm and therefore is less useful for the assessment of glomerular architecture. In the PAS reaction the mesangial matrix and basement membrane are stained purple, and this allows a good assessment of the amount of matrix and the thickness of the GBM. PAS also stains the tubular basement membranes and hyaline deposits. The silver methenamine stain is the best stain for studying the detailed morphology of the GBM and for highlighting the membrane spikes seen in membranous glomerulonephritis and the double contours seen in membranoproliferative glomerulonephritis. Its only drawback is that a satisfactory result is more technically demanding than the other stains. A trichrome stain, such as Masson’s trichrome, will stain the glomerular mesangial matrix and basement membrane and may also help in highlighting fibrin and immune complex deposits. Other stains are a matter of personal preference. The authors always use an elastin stain to demonstrate the elastic laminae of vessels, and this is counterstained with picrosirius red to stain fibrillar collagen in the interstitium. Amyloid is most specifically detected in a Congo red stain, and the authors feel it is prudent to perform this in all native kidney biopsy specimens. This is the exception to the requirement for thin sections; because the Congo red stain is relatively insensitive, a section cut at 10 µm should be used. Details of staining methods are given in the appendix of Churg and coworkers. Other stains that may be employed when necessary include the von Kossa stain, which demonstrates calcium deposition, and the Perls Prussian blue stain for iron.
Examination of the Biopsy Specimen by Light Microscopy
It is important to approach the examination of the biopsy systematically. Sections should first be assessed at low power to determine what parts of the kidney (or other structures in some cases) they contain, including whether there is cortex and/or medulla. A low-power view will also allow an assessment of the amount of chronic nephron damage, as demonstrated by tubular atrophy and interstitial fibrosis, and the presence of interstitial inflammatory infiltrates. It will also allow an assessment of interstitial expansion, most commonly due to either edema or fibrosis, but occasionally due to infiltration by, for example, amyloid. Examination should then proceed by studying the glomeruli, tubules, interstitium, and vessels, including arteries, arterioles, and veins, in more detail. Features that should be looked for in glomeruli and tubules are detailed in Tables 29.5 and 29.6 . Arterioles should be examined for the presence of hyalinosis, thrombosis, and necrosis. Arteries should be assessed for intimal thickening and whether it is accompanied by reduplication of the internal elastic lamina, thrombosis, necrosis, inflammation, and cholesterol emboli.
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