Objective

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in the blood, referred to as RNAemia, has been reported as a potential marker of severe coronavirus disease 2019 (COVID-19). In clinical studies, serum is commonly used for the detection of RNAemia; however, serum separation is often difficult in forensic samples because of postmortem changes. In this study, we aimed to establish an efficient method for extracting SARS-CoV-2 RNA from postmortem whole blood, and to evaluate whether whole blood is a suitable alternative specimen to serum.

Materials and Methods

SARS-CoV-2 culture supernatant was spiked into human whole blood, and viral RNA was extracted using three methods: acid guanidinium thiocyanate–phenol–chloroform (AGPC), spin column (SC), and magnetic bead methods. Viral RNA was quantified using digital PCR. Based on the results, a modified AGPC–SC protocol was developed that combines homogenized whole blood with AGPC extraction, followed by SC purification. The optimized protocol was subsequently applied to postmortem whole blood samples from 74 forensic external examination cases. Nasopharyngeal swabs obtained during all external examinations were positive for SARS-CoV-2. The results were compared with those of paired serum samples extracted using the SC method.

Results and Discussion

SARS-CoV-2 RNA was detected in whole blood using all three extraction methods, and the AGPC method had the highest RNA yield (15,315 copies/µL). The modified AGPC–SC protocol produced RNA yields (14,700 copies/µL) that were comparable to those obtained using the AGPC method alone, with improved precision, reducing the coefficient of variation from 13% to 2%. These findings suggest that the modified AGPC-SC method is suitable for viral RNA extraction from whole blood, and applicable to postmortem blood samples. In the 74 forensic cases, the qualitative results for whole blood and serum showed a positive percent agreement of 85.3% and a negative percent agreement of 92.5%. Viral loads in whole blood and serum were strongly correlated (Spearman’s ρ = 0.9014), and Bland–Altman analysis demonstrated minimal systematic bias (mean difference −0.053 log10). Postmortem whole blood may serve as an alternative to serum. Accordingly, the AGPC-SC method with homogenization provides a practical approach for SARS-CoV-2 RNA extraction from postmortem blood in forensic practice and may provide useful information for assessing antemortem COVID-19 severity.

Authors

• Kazuho Maeda (Yokohama City University Graduate School of Medicine, Japan)
• Noriko Ogawa (Yokohama City University Graduate School of Medicine, Japan)
• Momoka Tanabe (Yokohama City University Graduate School of Medicine, Japan)
• Hikaru Kuninaka (Yokohama City University Graduate School of Medicine, Japan)
• Moe Mukai (Yokohama City University Graduate School of Medicine, Japan)
• Ayako Nasu (Yokohama City University Graduate School of Medicine, Japan)
• Chiaki Fuke (Yokohama City University Graduate School of Medicine, Japan)
• Yosuke Usumoto (Graduate School of Medical Sciences Kyushu University, Japan)
• Yoko Ihama (Yokohama City University Graduate School of Medicine, Japan)