CCCardiol CroatCardiologia CroaticaCardiol. Croat.1848-543X1848-5448Croatian Cardiac SocietyCC 2023 18_11-12_314-510.15836/ccar2023.314Extended AbstractMiscellaneousX-ray phase contrast imaging of endomyocardial biopsy samples preserved in formalin and embedded in paraffin – a comparison of tissue preparation methodshttps://orcid.org/0000-0003-1730-8768ŠkrebNikola1*https://orcid.org/0000-0002-7865-1108LončarićFilip1https://orcid.org/0000-0001-5537-8682BonninAnne2https://orcid.org/0000-0003-2584-9812DejeaHector3https://orcid.org/0000-0003-1988-6684IlićIvana1https://orcid.org/0000-0002-2492-3702GašparovićHrvoje1https://orcid.org/0000-0001-5979-2346SkorićBoško1https://orcid.org/0000-0003-3130-6937BijnensBart45https://orcid.org/0000-0001-9101-1570MiličićDavor1https://orcid.org/0000-0003-0561-6704PlanincIvo1https://orcid.org/0000-0002-4772-5549ČikešMaja1University Hospital Centre Zagreb, University of Zagreb School of Medicine, Zagreb, CroatiaSwiss Light Source, Paul Scherrer Institute, Villigen, SwitzerlandEuropean Synchrotron Radiation Facility, Grenoble, FranceInstituto de Investigaciones Biomédicas August Pi i Sunyer (IDIBAPS), Barcelona, SpainCatalan Institution for Research and Advanced Studies (ICREA), Barcelona, SpainADDRESS FOR CORRESPONDENCE: Nikola Škreb, Klinički bolnički centar Zagreb, Kišpatićeva 12, HR-10000 Zagreb, Croatia. / Phone: +385-92-380-4962 / E-mail: nikola.skreb7@gmail.com0920231811-123143152009202327092023Croatian Cardiac Society2023Croatian Cardiac SocietyKEYWORDS: heart transplantationgraft rejectionsynchrotron imaginghistology
Background: Endomyocardial biopsy (EMB) is the gold standard in heart transplantation (HTx) follow-up, with samples commonly fixed with formalin, and then embedded in paraffin for histology analysis. Recently, EMB samples have been scanned with synchrotron X-ray phase-contrast imaging (X-PCI) to assess graft rejection. (1) We aim to compare imaging time efficiency and image quality between formalin-fixed and paraffin-embedded samples to determine the optimal scanning methodology.
Methods: Three adult patients undergoing EMB after HTx were included. EMB samples were initially stored in formalin and imaged by X-PCI at the Paul Scherrer Institute TOMCAT beamline (Villigen, Switzerland). On site samples were scanned in glass tubes in deionised, degassed water, and then embedded in paraffin, positioned on a holder, and scanned again using a multi-scale beamline set-up. Imaging time efficiency was measured by on-site sample preparation and scan time, and image quality was assessed with signal-to-noise ratio (SNR) and pixel resolution. Post-processing comparison included fibrosis quantification (using Ilastik for segmentation and Fiji for calculating the average percentage of collagen in 3 selected areas) and graft-rejection grading (assessed by two blinded observers based on the ISHLT 2004. criteria) (2).
Results: Scanning F1-F3 and P1-P3 samples produced the same imaging resolution, while F1-F3 samples exhibited higher SNR values (clearer sample visibility) (Table 1). On site preparation and scan time were shorter with P1-P3 samples. Fibrosis quantification produced similar results in all samples, with F1-F3 showing slightly higher collagen percentage compared to the corresponding P1-P3 samples (Table 1 and Figure 1). Samples F1 and F2 were graded as 1R, with others classified as 0R (ISHLT 2004.) (Table 1).
Imaging time (including preparation and scanning), technical parameters and imaging data analysis between the two sample preparation methodologies.
Imaging time efficiency
Technical image quality
Image post-processing analysis
Sample
Methodology
On-site preparation time (min:sec)
Scan time(min:sec)
SNR (dB)
Average percentage of collagen in 3 selected areas (%)
Rejection grading(ISHLT 2004. criteria)
F1
Formalin
3:58
49:08
112,16
0.34
1R
P1
Paraffin
0:17
6:34
72,86
0.21
0R
F2
Formalin
4:13
37:24
119,39
0.16
1R
P2
Paraffin
0:20
12:03
54,65
0.11
0R
F3
Formalin
4:21
49:08
112,19
0.37
0R
P3
Paraffin
0:32
12:08
56,72
0.12
0R
Left side of the figure showing X-PCI images of formalin samples and the right side of the figure showing X-PCI images of the same samples in paraffin. Both set of samples are marked with the corresponding tissue areas for the collagen segmentation and quantification (collagen shown in light blue).
Conclusion: Embedding EMB samples in paraffin is more time efficient in terms of on-site sample preparation and imaging. Results showed similar fibrosis quantification regardless of preparation methods, whereas rejection grading did not differ in clinically meaningful way. In conclusion, in initial testing using small sample number, no significant difference was found between the preparation methods.
Supported by the Croatian Science Foundation (project no. UIP-2020-02-5572).
LITERATUREPlanincIIlicIDejeaHGarcia-CanadillaPGasparovicHJurinHA Novel Three-Dimensional Approach Towards Evaluating Endomyocardial Biopsies for Follow-Up After Heart Transplantation: X-Ray Phase Contrast Imaging and Its Agreement With Classical Histopathology.Transpl Int. 2023 January 24;36:11046. 10.3389/ti.2023.1104636762268StewartSWintersGLFishbeinMCTazelaarHDKobashigawaJAbramsJRevision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection.J Heart Lung Transplant. 2005 November;24(11):1710–20. 10.1016/j.healun.2005.03.01916297770