Multi-photon microscopy for the evaluation of interstitial fibrosis in extended criteria donor kidneys: A proof-of-concept study

Introduction: To evaluate the initial use of label-free second harmonic generation (SHG) imaging with two-photon excitation (2PE) auto-fluorescence in multiphoton microscopy (MPM) for the quantification of collagen/fibrosis on preimplantation biopsies of extended criteria donors (ECD). Materials and methods: Twenty preimplantation core biopsies were extracted from 10 donor kidney samples, of which originated from seven donors. Kidney Donor Profile Index (KDPI) and Remuzzi scores of biopsies were calculated. Collagen parameters measured included quantification by the Collagen Area Ratio in Total Tissue (CART) and qualitative measurements by Collagen Reticulation Index (CRI). Results: Biopsies classified with > 85% KDPI scores had significantly higher CART ( p = .011) and lower CRI values ( p = .025) than biopsies with ≤ 85% KDPI scores. Increase in CRI values correlated significantly with rise in recipient creatinine levels 1-year post-transplant ( p = .027; 95% CI: 4.635-66.797). Conclusion: MPM is an evolving technology that enables the quantification of the amount (CART) and quality (CRI) of collagen deposition in unstained preimplantation biopsies of donor kidneys stratified by KDPI scores. This initial evaluation found significant differences in both parameters between donor kidneys with more or less than 85% KDPI.

spectrum of available ECDs. The dichotomous classification of single versus dual implantation has also reaffirmed the need to objectively determine the quality of a donor kidney. 2 Several frameworks exist to characterize the quality of donor kidneys. One approach is the procurement of a preimplantation renal biopsy for histopathological analysis. Remuzzi et al. pioneered a histological-based selection criteria to evaluate the quality of donor kidneys for single or dual implantation. Four variables are analyzed -glomerular global sclerosis, tubular atrophy, interstitial fibrosis, arterial and arteriolar narrowing. 3 In the same vein, The United Network for Organ Sharing Kidney Transplantation Committee 4 developed the Kidney Donor Profile Index (KDPI) score, which aims to incorporate clinical parameters to determine the quality of deceased donor kidneys.
As a transplant center located within Asia, we previously sought to clarify if clinical evaluation by KDPI was superior to histopathological assessment of donor kidneys. 5 It was concluded that KDPI per se was insufficient to stratify single versus dual implants, and that Remuzzi scoring demonstrated higher sensitivity, specificity, and positive predictive value in selecting kidneys for dual implants. Patients with KDPI > 1.1 were recommended still have routine histopathological biopsies to optimize stratification of ECD kidneys.
With the advancements of in-vivo histological techniques, there has been growing interest in multiphoton microscopy (MPM) as a step up from conventional histological staining methods. 6 It combines Second Harmonic Generation (SHG) and Two Photon Excitation (TPE) Fluorescence to image extracellular matrix architecture in intricate detail. 7 MPM has been previously utilized for deep optical sectioning of live human tissue and is exceptionally accurate at demonstrating specific contrast in tissue exhibiting fibrillar collagenous qualities. 8 Within the context of donor kidney classification, MPM may be used for the quantification of renal interstitial fibrosis. Since interstitial fibrosis comprises part of Remuzzi's variables, the aim of the present study was to employ MPM as a novel imaging technique to characterize and quantify interstitial fibrosis in preimplantation kidney biopsies.

Sample retrieval
The study protocol was approved by the National Healthcare Group

Sample preparation and multi-photon microscopy
The unstained de-paraffinized Formalin-Fixed Paraffin-Embedded tissues of 5-micron thickness were imaged using the commercially available laser-based Genesis200 Multi-Photon Microscopy system (Histoindex Pte. Ltd, Singapore). Image acquisition was performed at 2X and 20X objective with 512 × 512-pixel resolution. MPM is an imaging modality comprising SHG and TPE microscopy. SHG signals are detected when a laser of femtosecond range interacts with tissues that have unique, noncentrosymmetric structure within the ECM such as collagen fibers and elastin. When this SHG signal is combined with auto-fluorescence signal from the cells using TPE, MPM is able to penetrate deep into tissues to generate a high-resolution image that reflects both the architecture of the ECM and surrounding cellular structure.
On SHG microscopy, the fibrosis within the renal interstitium was  (Figures 1 and 2). Collagen F I G U R E 1 MPM imaging of renal preimplantation biopsy (S/N 5) with lowest CART value and corresponding hematoxylin and eosin (H&E) image F I G U R E 2 MPM imaging of renal preimplantation biopsy (S/N 9) with the highest CART value and corresponding hematoxylin and eosin (H&E) image content was measured quantitatively by CAR and CART, and collagen structure was measured by CRI.

Statistical analysis
Parametric continuous variables were presented as Mean ± Standard Deviation (SD

Donor baseline characteristics
Twenty preimplantation core biopsies were imaged from 10 donor kidney samples, of which originated from seven donors ( Table 1). All samples comprised of more than 10 glomeruli for histopathological evaluation. Banff and Remuzzi scorings of each biopsy specimen are provided in Table 2. Baseline characteristics are detailed in Table 3.

Comparison of recipient clinical outcomes post-transplant by KDPI
Recipient creatinine and eGFR levels at 1 and 3-years post-transplant did not differ significantly between 20-85% and > 85% KDPI scores (    [10][11][12][13][14] Of which includes fibrillar collagen, that is found within the extracellular matrix of the renal parenchyma. This is also coupled with TPE -fluorescence imaging of the fibrotic regions, undergoing extrinsic fluorescent labeling and auto-fluorescence with two-photon excitation sequence. 15 In fact, MPM is similarly evaluated within the fields of genito-urinary oncology, yielding some promising results. It could potentially serve as a biomarker for prostate tumor aggressiveness, 16 or even as a prognostic indicator of progression of nonmuscle invasive bladder cancer. 17 Most notably, a distinct advantage that it has over routine histological assessment is the ability to evaluate tissue samples without the need for any staining. 7 Pathologists have conventionally relied upon various methods (light microscopy, immunohistochemistry, and electron microscopy) for the fixation, processing, sectioning, and staining of a said tissue specimen. 18 Glomerulosclerosis, tubular atrophy, interstitial fibrosis, and vascular attenuation are the key points of interest to determine the classification, suitability, and type of transplantation to be performed. With the incorporation of MPM technology, shortcomings of histological assessment can be accounted for. 19 Ongoing research to establish the utility of MPM and artificial intel-

Strengths and limitations
To the best of our knowledge, this is the first study to evaluate the potential of MPM to characterize renal interstitial fibrosis in donor kidneys stratified by KDPI scores. However, several limitations ought to be acknowledged. First, the sample size is small, affecting the validity and increasing the margin of error of the aforementioned results. Second, this study is retrospective in nature and hence prone to selection bias. A larger number over a longer duration of the study period would allow us to correlate MPM-derived collagen characteristics with more confidence. Lastly, although CRI was a statistically significant predictor of recipient creatinine levels 1-year post-transplant, it should be noted that this is accompanied by correspondingly wide confidence intervals.
Hence, concluding that CRI was truly indeed an independent predictor should be validated with future studies.

CONCLUSION
In this proof-of-concept study, we reported noteworthy differences in MPM-derived collagen parameters between donor kidneys of varying KDPI scores. When pegged against validated histological and clinical frameworks in stratifying donor kidney biopsies, it is still an evolving technology in question. With further advancements in imaging techniques, future observational studies are eagerly awaited to support or challenge the reproducibility of our findings.