Immunofluorescence Use and Techniques in Glomerular Diseases: A Review

Abstract

Background: Immunofluorescence (IF) studies play an essential role in the evaluation of medical renal biopsies. Particularly, in the study of renal glomerular diseases, where it provides fundamental data for the diagnosis, classification, and etiology of the glomerular pathologies. Diverse techniques may be used to optimize the utilization of IF studies, from variations on the test methodologies to expertise on the interpretation of the results and knowledge of potential pitfalls.

Summary: This manuscript presents a brief review on the history of IF and its utilization in kidney pathology, followed by a description of the IF methods, including the use of IF on paraffin-embedded tissue (paraffin IF), and other novel techniques. Guidelines on how to best report IF findings are reviewed, along with a description of antibodies commonly used in glomerular diseases, highlighting their distribution within the normal kidney and potential pitfalls in interpretation. Finally, the use and interpretation of IF are discussed in more detail in individual entities on a range of glomerular diseases.

Key messages: IF is crucial for interpretation of renal biopsies and diagnosis of glomerular diseases. Knowledge of IF techniques, alternative procedures, its use and proper interpretation is essential for optimal utilization of IF in renal pathology, and this review proposes to serve as a simplified and practical guide on this topic.

Keywords: Immunofluorescence; Kidney; Renal pathology; Review.

Fig. 1.

Common glomerular IF patterns and pearls on IF interpretation. From (a–d) the common patterns of IF in the glomeruli include linear (a), granular (b), smudgy (c), and punctate (d), here seen respectively in anti-GBM disease, postinfectious GN, fibrillary GN, and minimal change disease (MCD). Kappa and lambda light chains should show similar intensity in the tissue as illustrated in (e, f). Remarkable difference in intensity of staining between the light chains or heavy chains supports MGRS lesions, as illustrated by the difference between kappa (g) and lambda (h) light chains in this case of kappa light chain deposition disease. Certain stains are observed normally in kidney tissue and may serve as internal control, such as IgA in tubuli (i) and C3 in vascular poles (j). Other stains may be nonspecific or have no significant or known clinical impact as IgM in FGS lesion (k), and C1q in transplant (l).

Fig. 2.

Examples on IF use in glomerular disease. In podocytopathies, punctate IgG deposits in podocytes (a) may suggest anti-nephrin antibodies. PLA2R-positive MN has usually global and diffuse capillary loop staining with IgG (b), while in NELL1 positive MN, IgG may be segmental (c). Extraglomerular staining is more often seen in MN associated with SLE (d). Primary IgAN usually shows mesangial deposits (e) which may be accompanied by C3 (f), of same or less intensity. In IgA deposits secondary to MRSA, IgA (g) may be less intense than C3 (h) and extends into capillary loops. However, the IF features alone do not confidently distinguish primary IgA versus secondary causes. In images, a case of C3G associated with MGRS shows intense C3 (i), with background kappa predominance in proximal tubules (j) when compared with lambda (l) and paraffin IF negative for IgG (l) or other possible masked Igs, confirming C3G diagnosis.