Impact of cardiac magnetic resonance imaging on human lymphocyte DNA integrity.

Methods and results In 20 prospectively enrolled patients, peripheral venous blood was drawn before and after 1.5 T CMR scanning. After density gradient cell separation of blood samples, DNA DSBs in lymphocytes were quantified using immunofluorescence microscopy and flow cytometric analysis. Wilcoxon signed-rank testing was used for statistical analysis. Immunofluorescence microscopic and flow cytometric analysis revealed a significant increase in median numbers of DNA DSBs in lymphocytes induced by routine 1.5 T CMR examination.

Methods

Twenty consecutive patients referred for cardiac evaluation were included. After obtaining written informed consent, 10 mL of peripheral blood was drawn before and after undergoing routine contrast (gadobutrolum, Gadovist, Bayer Schering Pharma, Germany) enhanced CMR examination 18 on a 1.5 T MR scanner (Philips Achieva, Best, NL, USA) as approved by the local ethics committee (KEK-Nr. 849). PBMCs were obtained using density gradient separation (Histopaque 1077, Sigma-Aldrich) as previously established. 19 The clinical CMR protocol used in our daily routine has been recently reported in detail. 20 In brief, a commercially available MR scanner (Philips 1.5 T, Achieva, software release 3.2.1) equipped with a maximum gradient strength of 42 mT/m and a maximum gradient speed of 180 mT/m/ms was used. The following standard pulse sequences to generate images were used: gradient echo, steady-state free precession, FastSE, T 2 -weighted double-inversion black-blood spin-echo sequence for oedema imaging, balanced SSFP sequence for perfusion and inversion recovery segmented gradient echo sequence for late gadolinium enhancement. DSBs were detected by immunofluorescence microscopy using a rabbit-anti-human phospho-histone γ-H2AX and a goat-anti-rabbit-AlexaFluor-488 antibody (CST Cell Signalling Technology, adapted from May et al . 21 ). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories) and the γ-H2AX foci per lymphocyte were visualized on an inverse confocal microscope (CLSM-Model SP5, Leica Microsystems) and quantified by a blinded observer. With flow cytometry (FACScanto, BD Bioscience), DSBs were additionally quantified in T-lymphocytes 22 , 23 previously identified by a mouse-anti-human CD3-APC antibody (Life Technologies). Based on forward and side light scattering, PBMCs were gated for viable single-cell events and proper compensation controls were used in flow cytometric analyses to correct for spectral overlap.

Data from flow cytometric quantification

(MFI, geometric mean of fluorescence intensity of γ-H2AXpositive T-lymphocytes) was evaluated using FlowJo software (V10.0.2, Tree Star, Inc.). Based on a variation of γ-H2AX assessment at 20% as reported by Muslimovic et al ., 22 an average difference in γ-H2AX findings reported in ex vivo experiments, 4 aiming at alpha = 0.05 and a power (1 − β ) of 0.8, the number of patients necessary was calculated between 10 and 15. SPSS 20.0 (SPSS, Chicago, IL, USA) was used for all statistical analysis. The Shapiro–Wilk test was applied to exclude normal distribution of data sets. This was followed by testing for significant differences between DSBs before and after CMR examination by using the Wilcoxon signed-rank test. P -values of