Digital pathology evaluation in the multicenter Nephrotic Syndrome Study Network (NEPTUNE)

Abstract

Pathology consensus review for clinical trials and disease classification has historically been performed by manual light microscopy with sequential section review by study pathologists, or multi-headed microscope review. Limitations of this approach include high intra- and inter-reader variability, costs, and delays for slide mailing and consensus reviews. To improve this, the Nephrotic Syndrome Study Network (NEPTUNE) is systematically applying digital pathology review in a multicenter study using renal biopsy whole slide imaging (WSI) for observation-based data collection. Study pathology materials are acquired, scanned, uploaded, and stored in a web-based information system that is accessed through a web-browser interface. Quality control includes metadata and image quality review. Initially, digital slides are annotated, with each glomerulus identified, given a unique number, and maintained in all levels until the glomerulus disappears or sections end. The software allows viewing and annotation of multiple slide sections concurrently. Analysis utilizes "descriptors" for patterns of injury, rather than diagnoses, in renal parenchymal compartments. This multidimensional representation via WSI, allows more accurate glomerular counting and identification of all lesions in each glomerulus, with data available in a searchable database. The use of WSI brings about efficiency critical to pathology review in a clinical trial setting, including independent review by multiple pathologists, improved intraobserver and interobserver reproducibility, efficiencies and risk reduction in slide circulation and mailing, centralized management of data integrity and slide images for current or future studies, and web-based consensus meetings. The overall effect is improved incorporation of pathology review in a budget neutral approach.

Figure 1.

NEPTUNE digital pathology protocol. Scanning and uploading of pathology material can occur at the local enrolling centers (local scanning) or centrally (central scanning). For local scanning, glass slides are scanned into whole slide images and locally uploaded to the web-based pathology information management system by a password-protected access together with the electron microscopy and immunofluorescence digital images and PDF of the pathology report. For central scanning, all pathology material is mailed to the image management coordinating site at the National Institutes of Health, where glass slides are scanned into whole slide images and uploaded into the web-based pathology information management system together with the rest of the pathology material. Once cases pass quality control processes they are ready for triage, annotation and analysis. Analysis of whole slide images is performed with a two-step process: first the pathologist annotates all glomeruli in all levels available (multi-level representation of glomeruli). Reader pathologists score each case by using the NEPTUNE Pathology Scoring System. The entire process utilizes digital pathology rather than conventional light microscopy. NEPTUNE, Nephrotic Syndrome Study Network.

Figure 2.

Web-based pathology information management system organization. Access to the web-based information managing system organization is password protected. The system is organized into three categories of folders: the major study categories (MCD/FSGS, MGN, and others), the cases ready for triage, and the cases that fail quality control review (A). Within each of these major folders, there are individual patient folders labeled with the study PID (B). Each patient folder contains whole slide images labeled with the PID, slide level, and stain (C–E), digital images of electron microscopy (F), and of immunofluorescence (G) in addition to the PDF of the original pathology report (not shown). MCD, minimal change disease; MGN, membranous GN; PID, patient identification code.

Figure 3.

Simultaneous visualization of multiple virtual slides. The software allows simultaneous visualization of multiple sections, which facilitates annotation and scoring of glomerular lesions. The glomeruli are identified by the blue annotations. Annotation is generally initiated in the section with most glomeruli, as is evident in A. As the annotation process proceeds to other biopsy levels, new glomeruli can appear or disappear. The number of glomerulus decreases moving through the biopsy levels in A–D. However, a new glomerulus does appear in C.

Figure 4.

Multi-level representation of glomeruli. Using simultaneous section visualization and retrieval by unique glomerular identifiers, annotated glomeruli can be easily followed through the various levels. This allows determination of all lesions in a multi level representation of each glomerulus, rather than assessing single level representations. Level 1 (hematoxylin and eosin stain) shows two glomeruli annotated as glomerulus 1 and 2. Glomerulus 1 is histologically unremarkable and remains such throughout all levels here represented (level 2, silver methenamine; level 6, hematoxylin and eosin; level 7, periodic acid–Schiff; level 12, hematoxylin and eosin). Glomerulus 2 appears entirely sclerotic on level 1; however, in all other levels, it would be counted as a segmentally sclerotic glomerulus with an area of solidification of the tuft and adhesion to the Bowman’s capsule. No hyalinosis or foam cells are detected in any of the represented levels.

Figure 5.

The NEPTUNE Pathology Scoring System: Examples of descriptors. (A) Multi-level representation of a glomerulus annotated as glomerulus 3 throughout various levels. Although on level 1 there are no lesions of segmental sclerosis or collapse and this glomerulus would be counted as nonsclerotic if viewed on one level only, the same glomerulus reveals two distinct lesions of segmental sclerosis with hyalinosis on levels 8 and 9 (periodic acid–Schiff and silver stain, respectively). There is one segmental lesion containing hyalinosis on level 11 (hematoxylin and eosin) and the glomerulus has no segmental lesions again on level 12 (hematoxylin and eosin). The histologic profiling of this glomerulus includes the following descriptors: segmental sclerosis with hyalinosis cannot determine location ×2, and hyalinosis cannot determine location. (B) This glomerulus stained with silver is characterized by three descriptors: segmental sclerosing tip lesion, hyalinosis away from the vascular pole ×2, and mid-glomerular hyalinosis. (C) Three descriptors are used to profile this glomerulus (silver stain): segmental proliferative collapse, segmental podocyte hyperplasia, and segmental podocyte hypertrophy. (E) Descriptors are applied to this case of membranous glomerulopathy with segmental sclerosis. Both features of sclerosis and membranous are scored (silver stain). (F) A case of membranous glomerulopathy is illustrated (silver stain on the left and trichrome on the right). Descriptors used to profile this glomerulus include the following: diffuse spikes/hopes and global podocyte hypertrophy. (D and G) Descriptors are also applied to electron microscopy analysis. D shows that there is 100% effacement; condensation of the actin-based cytoskeleton and microvillous transformation are also scored. Primary processes are scored as presented. G shows subepithelial electron dense deposits. NEPTUNE, Nephrotic Syndrome Study Network.