renal biopsy

Sample Size

 An adequate sample size must be obtained for diagnosis.

 Needle gauge has dramatic impact on sample obtained. Eighteen- or 19-gauge needles give very small, narrow samples and often may have inadequate representation of vessels.

 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.

 Minimum sample size for diagnosis varies greatly with the specific diagnosis; for instance, membranous glomerulonephritis can be diagnosed from a single glomerulus. Transplant diagnoses are most accurate when the sample includes a minimum of seven glomeruli.

 For most light microscopic assessment to adequately assess severity and distribution of lesions, 8 to 10 glomeruli are needed.

Sample Location-Juxtamedullary Versus Cortical

 Subcapsular cortical samples have overrepresentation of global sclerosis related to aging/hypertension and non-specific scarring.

 Juxtamedullary glomeruli are the earliest to be involved with segmental sclerosis in focal segmental glomerulosclerosis (FSGS). This region should be included in the sample for optimal detection.

Allocation and Fixatives

 An adequate assessment of native renal biopsies includes light microscopy (LM), immunofluorescence microscopy (IF), and electron microscopy (EM). For transplant biopsy, LM and IF are considered the standard, with repeat biopsies only needing LM in many cases.

 LM: For most differential diagnoses, the largest portion of cortex should be placed in fixative for LM. These fixatives include formalin, paraformaldehyde, or less commonly used alcoholic Bouin’s or Zenker’s.

 IF: IF tissue should include a small piece of cortex, usually 3 to 4 mm. Tissue for IF can be directly frozen, or placed in tissue transport media such as Michel’s, and transported to the laboratory. Tissue is stable at room temperature for express mailing to central laboratories in this media.

 EM: Small, 1-mm tubes of cortex are allocated for EM, and optimally are placed directly in glutaraldehyde.

Dividing the Tissue 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.

Handling of Tissue

 No forceps, manipulate with thin wooden stick to avoid crush artifact.

 Avoid touching tissue with a fixative-contaminated scalpel or razor blade (this contaminates the tissue for IF).

 If there is inadequate tissue in any of the media, sometimes results can still be obtained as follows:

 IF tissue is frozen for IF stains-the remaining frozen tissue may be fixed in formalin and processed for LM.

 EM study can be done by processing remaining tissue from the LM sample from the paraffin block.

 Tissue that has not been in paraffin blocks too long can sometimes give satisfactory results for immunofluorescence done on fixed, paraffin-embedded tissue.

 LM: Tissue for LM is processed, dehydrated, and placed in paraffin block, and multiple serial sections are obtained and stained. Usual stains include hematoxylin & eosin, periodic acid-Schiff (PAS), silver methenamine (Jones’), and Masson trichrome. Additional unstained slides are produced to allow additional special studies as needed. Five hours of processing, sectioning, and staining time are typically needed to produce LM slides.

 IF: Tissue for IF is surrounded with OCT compound and frozen, and sections are produced and stained with fluorescein-tagged antibodies against IgG, IgA, IgM, complements C3 and C1q, κ and λ light chain. Complement product C4d may also be stained on frozen tissue, with more technical difficulty in staining on paraffin-block tissue. One to 2 hours of processing, sectioning, and staining time are needed for production of IF slides.

 EM: EM tissue is processed and embedded in a plastic, hard media, and scout sections (so-called thick sections) are stained with toluidine blue to identify the specific area to be cut for thin sections to be placed on a grid for EM examination. Typically 2 working days are needed to process and produce EM sections for ultrastructural examination.

References

 Fogo AB. Approach to renal biopsy. Am J Kidney Dis. 2003 Oct;42(4):826-36. PMID: 14520635