Celia, A. I. | Search bioRxiv

bioRxiv2026

Serial Immunohistochemistry for High-Dimensional Single-Cell Spatial Analysis of Human Kidney Biopsies

BackgroundTraditional immunohistochemistry (IHC) with chromogen detection has limited multiplex capacity, detecting at most 4 protein markers per tissue section simultaneously, thereby restricting comprehensive spatial analysis of valuable human biopsies. We developed and validated a robust serial IHC (sIHC) staining method to detect multiple antigens on a single kidney biopsy slide, maximizing data yield for diagnosing and studying complex kidney diseases. MethodsFormalin-fixed, paraffin-embedded kidney biopsy sections were subjected to repeated IHC/imaging cycles with antibody removal using an optimized sodium dodecyl sulfate-glycerol buffer stripping protocol. Images were then co-registered, and analysis was performed using a variety of methodologies, including color deconvolution, cell segmentation, and spatial clustering. ResultsThis optimized sIHC method successfully detected up to 20 antigens on a single slide. Combining image analysis and artificial intelligence software, for example with HALO (Indica Labs), the assay assembles high-dimensional images and enables quantitative histology and single-cell spatial analysis. Using this advanced method, we were able to identify rare cell populations, such as double-negative T cells, that are challenging to detect conventionally. ConclusionWe have developed a validated, high-capacity sIHC protocol that uses standard IHC procedures with commercially available, clinically validated off-the-shelf antibodies. This method is a valuable, cost-effective tool for obtaining extensive, high-dimensional single-cell-resolved spatial data from limited pathology samples, such as a human kidney biopsy.

pathology↗

bioRxiv2024

Inflammation in Areas of Fibrosis Precedes Loss of Kidney Function in Lupus Nephritis

BackgroundInterstitial fibrosis in lupus nephritis (LN) is often infiltrated by immune cells but typically regarded as nonspecific "scar reaction." This study aimed to investigate the relationship between inflammatory fibrosis and kidney disease progression in LN. MethodsInterstitial fibrosis and tubular atrophy (IFTA) were scored in 124 LN kidney biopsies. Inflammation in areas of IFTA (i-IFTA) was graded 0-3 according to the Banff Classification of Allograft Pathology. Significant glomerular filtration rate (GFR) loss was defined as a decline of >15 ml/min at 3 years from biopsy. Immune cell phenotype was defined by serial immunohistochemistry (13-plex). ResultsIFTA was observed in 88/124 (71%) biopsies, and i-IFTA was identified in 76/88 (86%) cases. The distribution of i-IFTA grades was heterogenous across all IFTA grades. In patients with moderate-to-severe IFTA (>25%), the degree of i-IFTA was associated with a higher risk of significant GFR loss: 0/2 (0%), 1/3 (33%), 3/4 (75%), and 7/9 (78%) for i-IFTA grades 0, 1, 2, and 3, respectively (p = 0.028). Multiplexed histology revealed that i-IFTA was mostly composed of CD163+ macrophages and CD4 T cells, followed by CD8 T cells and granulocytes. ConclusionI-IFTA is frequently observed in LN and is dominated by macrophages and T cells. For patients with baseline IFTA >25%, the degree of i-IFTA emerged as a predictor of GFR loss. These data support the routine scoring of i-IFTA in LN due to its prognostic implications and nominate i-IFTA as a potential therapeutic target. LAY SUMMARYScar tissue often contains immune cells, but we still do not fully understand their role. In lupus nephritis (LN), this is typically dismissed as "nonspecific inflammation". However, our study analyzed kidney biopsies from 124 people with LN and found that inflammation in scarred areas may predict future kidney function loss. Specifically, we identified a type of immune cell, CD163+ macrophages, that may contribute to scarring and kidney damage. Our findings suggest that routinely assessing inflammation in scarred areas could help predict kidney health in LN patients and highlight a possible new target for therapies to prevent kidney damage.

immunology↗