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Abstract: TH-PO977

Realization and Future Applications of LCM from Renal Formalin-Fixed Paraffin-Embedded Slices

Session Information

Category: Pathology and Lab Medicine

  • 1501 Pathology and Lab Medicine: Basic

Authors

  • Alfieri, Carlo M., Fondazione IRCCS Ca' Granda Ospedale Policlinico Milan, Milan, Italy
  • Mattinzoli, Deborah, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milano, Italy
  • Ikehata, Masami, Fondazione IRCCS Ca' Granda Ospedale Policlinico Milan, Milan, Italy
  • Cresseri, Donata, Fondazione IRCCS Ca' Granda Ospedale Policlinico Milan, Milan, Italy
  • Moroni, Gabriella, Fondazione IRCCS Ca' Granda Ospedale Policlinico Milan, Milan, Italy
  • Vaira, Valentina, University of Milan, Milan, Italy
  • Pallotti, Francesco, Fondazione IRCCS Ca' Granda Ospedale Policlinico Milan, Milan, Italy
  • Messa, Piergiorgio, Fondazione IRCCS Ca' Granda Ospedale Policlinico Milan, Milan, Italy
Background

Kidney transplantation(KTx) damage surveillance is relevant in clinical practice. Renal biopsy(RBx) is essential to guide a specific treatment. Laser capture microdissection(LCM) combining molecular biology and histology is a novel approach. Using LCM, we aim to test the expression of some pathologic markers in different renal compartments starting from formalin-fixed paraffin-embedded(FFPE) samples.

Methods

The principal difficulties present in LCM realization were the poor quantity and quality of the FFPE samples related to not RNA suitable sample procession. The late housekeeping Ct coupled with the uncertain RNA amount, caused by infiltrating inflammatory cells and by the fibrosis presence, lead us to opt for a cDNA pre-amp. Since RNA quality has a significant impact on reference gene stability, samples quality was evaluated by means of the housekeeping stability. Among the housekeeping examined,RPL4 had the best Ct and stability.

Results

After verifying the reliability of the selected SsoAdvanced PreAmp Supermix Bio-Rad kit, the method was first used in two different RBx (RBx-A: biopsy without histological alteration; RBx-B: biopsy from a KTx patient with initial transplant glomerulopathy). Then, the most promising biomarkers evidenced were validated in two RBx from the same KTx patient in distinct CKD progression phase (RBx-C1: no injuries;RBx-C2: initial chronic allograft nephropathy, associated to severe interstitial fibrosis)
Renal fibrosis: our result suggested a DDR1-TGFβ-COLIII-COLI- chronological formation of the fibrotic matrix in all RBx-B compartments. The same gene expression trend was observed in the RBx-C1 with a later collagens protein formation in the RBx-C2.
Predictive biomarkers: Among the biomarkers, Klotho resulted the earliest with a downregulation in RBx-B and RBx-C1. Periostin, although later than Klotho, showed a great damaged compartment discrimination capacity resulting higher in both RBx-B and RBx-C2 glomeruli.

Conclusion

The method presented permits to obtain reliable results in LCM from FPPE samples. Furthermore, it is reproducible, easy and fast. In the future, a study in KTx-RBx of the relationship between RNA-expression at different renal sites and clinical and biochemical parameters and of its predictive role of KTx outcome will be possible.