Blood taken immediately after fatal resuscitation attempts yields higher quality DNA for genetic studies as compared to autopsy samples

Abstract

Background: The out-of-hospital cardiac arrest (OHCA) in the young may be associated with a genetic predisposition which is relevant even for genetic counseling of relatives. The identification of genetic variants depends on the availability of intact genomic DNA. DNA from autopsy may be not available due to low autopsy frequencies or not suitable for high-throughput DNA sequencing (NGS). The emergency medical service (EMS) plays an important role to save biomaterial for subsequent molecular autopsy. It is not known whether the DNA integrity of samples collected by the EMS is better suited for NGS than autopsy specimens.

Material and methods: DNA integrity was analyzed by standardized protocols. Fourteen blood samples collected by the EMS and biomaterials from autopsy were compared. We collected 172 autopsy samples from different tissues and blood with postmortem intervals of 14-168 h. For comparison, DNA integrity derived from blood stored under experimental conditions was checked against autopsy blood after different time intervals.

Results: DNA integrity and extraction yield were higher in EMS blood compared to any autopsy tissue. DNA stability in autopsy specimens was highly variable and had unpredictable quality. In contrast, collecting blood samples by the EMS is feasible and delivered comparably the highest DNA integrity.

Conclusions: Isolation yield and DNA integrity from blood samples collected by the EMS is superior in comparison to autopsy specimens. DNA from blood samples collected by the EMS on scene is stable at room temperature or even for days at 4 °C. We conclude that the EMS personnel should always save a blood sample of young fatal OHCA cases died on scene to enable subsequent genetic analysis.

Keywords: Cardiomyopathy; Channelopathy; DNA stability; Emergency medical service; Molecular autopsy; Next-generation sequencing; Resuscitation.

Fig. 1

Study overview (compare also Table 1 for details)

Fig. 2

Violin plot of age distribution and postmortem intervals (PMIs) at autopsy. The median age of autopsy cases is 39 years. The median PMI was 64 h. Truncated violin plots showing medians as solid lines, quartiles are given as dotted lines

Fig. 3

Violin plot of gDNA extraction yields of solid tissues (left Y-axis) and blood (red, right Y-axis) derived from autopsies. gDNA isolated from autopsy blood provides by far the highest yields of gDNA (Kruskal-Wallis test for multiple comparisons; medians are given as solid, quartiles as dotted lines)

Fig. 4

DNA integrity plotted against the postmortem interval (PMI) for different tissues from autopsy biomaterial. There is no linear correlation between DNA integrity and PMI (maximum 6 days). Results from linear regression analysis are given as dotted lines (slopes are not significant from zero). Dashed lines indicate cutoff of DIN = 7. A Heart; B skeletal muscle; C lung; D kidney; E cartilage; F bone; G fibrous connective tissue; H blood. DIN relative DNA integrity number

Fig. 5

Comparison of the DNA integrity number (DIN) derived from autopsy samples including blood and blood samples collected by the emergency medical services (EMS). The DIN of the EMS blood samples from 14 fatal OHCA cases is significantly higher (p = 0.005) as compared to any other tissue except cartilage, bone, and skeletal muscle from autopsy. For the statistical evaluation, the Kruskal-Wallis test for multiple comparisons was used (box and whiskers plots: boxes represent the quartiles, whiskers extend from 10 to 90 percentiles, outliers are given as dots, medians as solid lines and means as crosses)

Fig. 6

Experimental comparison of the influence of different storage conditions (4 °C, room temperature (RT), 32 °C and 42 °C) on EDTA blood samples from donors or of blood collected during autopsy on DNA integrity, respectively. The DNA integrity number (DIN) is plotted against the blood sample incubation time or postmortem interval (PMI). Blood samples from autopsy are shown as means ± standard error. For donor blood samples stored at different temperatures, the means ± standard deviation is given (see also Supplements sFig. 4)