Comparison of NET quantification methods based on immunofluorescence microscopy: Hand-counting, semi-automated and automated evaluations
Timo Henneck,
Christina Krüger,
Andreas Nerlich,
Melissa Langer,
Leonie Fingerhut,
Marta C. Bonilla,
Marita Meurer,
Sönke von den Berg,
Nicole de Buhr,
Katja Branitzki-Heinemann,
Maren von Köckritz-Blickwede
Affiliations
Timo Henneck
Institute of Biochemistry, University of Veterinary Medicine Hannover, Foundation, 30559, Hannover, Germany
Christina Krüger
Institute of Biochemistry, University of Veterinary Medicine Hannover, Foundation, 30559, Hannover, Germany
Andreas Nerlich
Institute for Microbiology, University of Veterinary Medicine Hannover, Foundation, 30173, Hannover, Germany
Melissa Langer
Institute of Biochemistry, University of Veterinary Medicine Hannover, Foundation, 30559, Hannover, Germany
Leonie Fingerhut
Institute of Biochemistry, University of Veterinary Medicine Hannover, Foundation, 30559, Hannover, Germany; Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Foundation, 30559, Hannover, Germany
Marta C. Bonilla
Institute of Biochemistry, University of Veterinary Medicine Hannover, Foundation, 30559, Hannover, Germany; Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Foundation, 30559, Hannover, Germany
Marita Meurer
Institute of Biochemistry, University of Veterinary Medicine Hannover, Foundation, 30559, Hannover, Germany; Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Foundation, 30559, Hannover, Germany
Sönke von den Berg
Institute for Zoology, University of Veterinary Medicine Hannover, Foundation, 30559, Hannover, Germany
Nicole de Buhr
Institute of Biochemistry, University of Veterinary Medicine Hannover, Foundation, 30559, Hannover, Germany; Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Foundation, 30559, Hannover, Germany
Katja Branitzki-Heinemann
Institute of Biochemistry, University of Veterinary Medicine Hannover, Foundation, 30559, Hannover, Germany; Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Foundation, 30559, Hannover, Germany
Maren von Köckritz-Blickwede
Institute of Biochemistry, University of Veterinary Medicine Hannover, Foundation, 30559, Hannover, Germany; Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Foundation, 30559, Hannover, Germany; Corresponding author. Department of Biochemistry, University of Veterinary Medicine Hannover, Foundation, Bünteweg 17, 30559, Hannover, Germany.
Formation of neutrophil extracellular traps was first described in 2004, showing that NETs are composed of decondensed chromatin fibers and nuclear and granule components. Free DNA is often used to quantify NETs, but to differentiate NETosis from necrotic DNA-release, immunofluorescence microscopy with NET-specific markers is required. Although evaluation by hand is time-consuming and difficult to standardize, it is still widespread. Unfortunately, no standardized method and only limited software tools are available for NET evaluation. This study provides an overview of recent techniques in use and aims to compare two published computer-based methods with hand counting. We found that the selected semi-automated quantification method and fully automated quantification via NETQUANT differed significantly from results obtained by hand and exhibited problems in detection of complex NET structures with partially illogical results. In contrast to that, trained persons were able to adapt to varying settings. Future approaches aimed at developing deep-learning algorithms for fast and reproducible quantification of NETs are needed.
