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  • Methodology
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Enabling methanol fixation of pediatric nasal wash during respiratory illness for single cell sequencing in comparison with fresh samples

Pediatric Research volume 95, pages 835–842 (2024)Cite this article

Abstract

Background

Lower respiratory tract infection (LRTI) including pneumonia, bronchitis, and bronchiolitis is the sixth leading cause of mortality around the world and leading cause of death in children under 5 years. Systemic immune response to viral infection is well characterized. However, there is little data regarding the immune response at the upper respiratory tract mucosa. The upper respiratory mucosa is the site of viral entry, initial replication and the first barrier against respiratory infections. Lower respiratory tract samples can be challenging to obtain and require more invasive procedures. However, nasal wash (NW) samples from the upper respiratory tract can be obtained with minimal discomfort to the patient.

Method

In a pilot study, we developed a protocol using NW samples obtained from hospitalized children with LRTI that enables single cell RNA sequencing (scRNA-seq) after the NW sample is methanol-fixed.

Results

We found no significant changes in scRNA-seq qualitative and quantitative parameters between methanol-fixed and fresh NW samples.

Conclusions

We present a novel protocol to enable scRNA-seq in NW samples from children admitted with LRTI. With the inherent challenges associated with clinical samples, the protocol described allows for processing flexibility as well as multicenter collaboration.

Impact

  • There are no significant differences in scRNA-seq qualitative and quantitative parameters between methanol fixed and fresh Pediatric Nasal wash samples.

  • The study demonstrates the effectiveness of methanol fixation process on preserving respiratory samples for single cell sequencing.

  • This enables Pediatric Nasal wash specimen for single cell RNA sequencing in pediatric patients with respiratory tract infection and allows processing flexibility and multicenter collaboration.

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Fig. 1: Nasal wash sample collection and processing.
Fig. 2: Histogram representing Quality Score per sequence showing the average quality score on the x-axis and the number of sequences with that average on the y-axis for three subject’s specimens processed by two methods.
Fig. 3: The violin plots show mitochondrial reads percentage of three subject’s specimens each processed by two methods.
Fig. 4: No significant difference in transcriptomic data between paired fresh and methanol-fixed samples.
Fig. 5: The frequency of identified immune cell types in paired Fresh and Methanol-Fixed samples is very similar, statistically indifferent.

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Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

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Acknowledgements

This project is supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number 5P20GM12134.

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Authors and Affiliations

  1. Environmental Influences on Health and Disease Group, Sanford Research, Sioux Falls, SD, USA

    Khaled A. Ismail & Santiago M. C. Lopez

  2. Functional Genomics and Bioinformatics Core, Sioux Falls, SD, USA

    Malini Mukherjee & Michael S. Kareta

  3. Genetics & Genomics Group, Sanford Research, Sioux Falls, SD, USA

    Michael S. Kareta

  4. Department of Pediatrics, Sanford School of Medicine-University of South Dakota, Sioux Falls, SD, USA

    Michael S. Kareta & Santiago M. C. Lopez

  5. Children’s Health Specialty Clinic, Sanford Children’s Hospital, Sioux Falls, SD, USA

    Santiago M. C. Lopez

Authors
  1. Khaled A. Ismail
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  4. Santiago M. C. Lopez
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Contributions

Each author has met the Pediatric Research authorship requirements. S.M.C.L. and K.A.I. completed the conception and design. K.A.I. and M.M. carried out the experiment. M.S.K., S.M.C.L. and K.A.I. completed acquisition of data, analysis and interpretation of data. S.M.C.L., K.A.I., M.M. and M.S.K. completed drafting the article and revising it critically for important intellectual content. S.M.C.L. completed the final approval of the version to be published.

Corresponding author

Correspondence to Santiago M. C. Lopez.

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Ismail, K.A., Mukherjee, M., Kareta, M.S. et al. Enabling methanol fixation of pediatric nasal wash during respiratory illness for single cell sequencing in comparison with fresh samples. Pediatr Res 95, 835–842 (2024). https://doi.org/10.1038/s41390-023-02780-2

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