Digital scanning and diagnostic scoring of kidney biopsies from children with steroid resistant nephritic syndrome

<p><strong>Background:</strong> Digital pathology is an attractive new tool for multicenter research. Practical improvements on conventional pathology include parallel evaluation by several experts in distant locations and reproducible identification of individual lesions. Also, digital image analysis allows more objective measures of cell staining and 3D reconstruction of glomeruli. </p> <p> </p> <p><strong>Design:</strong> We are establishing a digital pathology archive within WP2 of EURenOmics. Kidney biopsies from children with steroid resistant nephrotic syndrome (SRNS) enrolled in the PodoNet registry are collected. Analysis will be harmonized with digital pathology scoring developed by the NEPTUNE study (Nephrotic Syndrome Study Network) in the US.</p> <p> </p> <p><strong>Methods:</strong> The material is scanned at high resolution in Heidelberg at the Hamamatsu Tissue Image and AnalysisCenter using a Hamamatsu Nanozoomer. Digital images of existing electron microscopy and immunofluorescence scans and original pathology reports are also collected. After anonymization and manual quality controls (e.g. checking correct focus in all areas) data is stored centrally for remote review.</p> <p>Digital images are first annotated, i.e. each glomerulus is given a number which can be tracked across different section levels. Histopathological scoring of each glomerulus in each section level will then be performed in an unbiased fashion by independent blinded pathologists. These scores will be correlated to clinical and genetic information, conventional histopathological diagnoses and molecular profiles obtained by multi-omics profiling. </p> <p> </p> <p><strong>Progress:</strong> So far 83 kidney biopsies have been collected and at least another 180 are expected. Test runs of different stain types have produced good quality images. Bulk scanning and annotations are commencing, so that pathology reviews will start in spring 2014. </p> <p> </p> <p><strong>Outlook:</strong> The new digital pathology archive for children with SRNS will enable standardized review and objective scoring of histopathological findings. We hope that this will improve correlations of histological findings with clinical outcomes and biomarkers. Setting up the relevant infrastructure will allow extension of the project to other registries within WP2.</p

Morphometry Predicts Early GFR Change in Primary Proteinuric Glomerulopathies: A Longitudinal Cohort Study Using Generalized Estimating Equations

<div><p>Objective</p><p>Most predictive models of kidney disease progression have not incorporated structural data. If structural variables have been used in models, they have generally been only semi-quantitative.</p><p>Methods</p><p>We examined the predictive utility of quantitative structural parameters measured on the digital images of baseline kidney biopsies from the NEPTUNE study of primary proteinuric glomerulopathies. These variables were included in longitudinal statistical models predicting the change in estimated glomerular filtration rate (eGFR) over up to 55 months of follow-up.</p><p>Results</p><p>The participants were fifty-six pediatric and adult subjects from the NEPTUNE longitudinal cohort study who had measurements made on their digital biopsy images; 25% were African-American, 70% were male and 39% were children; 25 had focal segmental glomerular sclerosis, 19 had minimal change disease, and 12 had membranous nephropathy. We considered four different sets of candidate predictors, each including four quantitative structural variables (for example, mean glomerular tuft area, cortical density of patent glomeruli and two of the principal components from the correlation matrix of six fractional cortical areas–interstitium, atrophic tubule, intact tubule, blood vessel, sclerotic glomerulus, and patent glomerulus) along with 13 potentially confounding demographic and clinical variables (such as race, age, diagnosis, and baseline eGFR, quantitative proteinuria and BMI). We used longitudinal linear models based on these 17 variables to predict the change in eGFR over up to 55 months. All 4 models had a leave-one-out cross-validated R² of about 62%.</p><p>Conclusions</p><p>Several combinations of quantitative structural variables were significantly and strongly associated with changes in eGFR. The structural variables were generally stronger than any of the confounding variables, other than baseline eGFR. Our findings suggest that quantitative assessment of diagnostic renal biopsies may play a role in estimating the baseline risk of succeeding loss of renal function in future clinical studies, and possibly in clinical practice.</p></div

Pearson correlations among fractional cortical areas.

<p>Pearson correlations among fractional cortical areas.</p

Observed eGFR progression patterns over follow-up stratified by quartiles of FATA.

<p>Linear fits to observed eGFR values stratified by FATA from lowest (red) to highest (purple). Initial eGFR is highest for the lowest FATA quartile and decreases with each quartile. Compared to the reference category of the lowest FATA quartile, testing for the differential eGFR slopes of the second, third and fourth quartiles yielded P values of 0.15, 0.25 and <0.001, respectively. The apparent more negative slope of the lowest quartile may be due to a smaller number of long follow-up points.</p

Illustration of the point counting method on a section of cortex (PAS stain).

<p>Illustration of the point counting principle for assessing cortical compartments using SlidePath software. The hand-written numbers (10, 11, 12, 13 and part of 20) were added by the slide annotator, to keep track of individual glomeruli. Cross points of the red grid box were used other than the upper and right-side lines (equivalently, using the bottom-left corner point of each grid sub-box). Starting at the top-left, the first point ‘hits’ an intact glomerulus (#12). Moving right, the second point hits an intact tubule. The third hits an intact glomerulus (#11). Twenty-five points per grid are evaluated.</p

Digital annotation of whole slide images modestly improves accuracy of global glomerular sclerosis enumeration.

<p>(A,B) Box plot of annotated percent (%) globally sclerotic glomeruli and reported %globally sclerotic glomeruli in cases reporting (A) total number of glomeruli (n = 139) or (B) average number of glomeruli (n = 138). (C,D) Box plot of difference between number of annotated and reported % globally sclerotic glomeruli stratified by % globally sclerotic glomeruli in cases reporting (C) total number of glomeruli (n = 139) or (D) average number of glomeruli (n = 138).</p

Digital review and annotation of whole slide images in the NEPTUNE digital pathology repository.

<p>A) Example of multiple levels visualized at the same time. The software allows overlapping and orientation of the sections to facilitate multilevel reconstruction and representation of the biopsy levels. B) Digital annotation of whole slide images. Two levels are shown in alignment. Glomeruli in the left panel are annotated in red. In a subsequent level (right panel), two of the red-annotated glomeruli are still present (3 and 4), while one has disappeared (6). Three additional glomeruli, including one adjacent to glomerulus 3 and 6 have appeared in the deeper level (annotated in blue).</p

Difference in glomerular count of number of annotated versus reported glomeruli stratified by number of glomeruli.

<p>Difference in glomerular count of number of annotated versus reported glomeruli stratified by number of glomeruli.</p

Digital annotation of whole slide images improves accuracy of glomerular enumeration.

<p>(A,B) Scatter plot of annotated glomerular number versus reported glomerular number by diagnosis in cases reporting (A) total number of glomeruli or (B) average number of glomeruli per level. Dotted diagonal line shows where annotated = reported, and highlights the increase in number of glomeruli found by annotation. (A: n = 139; B: n = 138). (C,D) Box plot of annotated glomerular number and reported total glomerular number stratified by diagnosis in cases reporting (C) total number of glomeruli (n = 139) or (D) average number of glomeruli (n = 139).</p

Number of annotated versus reported glomeruli stratified by diagnosis.

<p>Number of annotated versus reported glomeruli stratified by diagnosis.</p