CCQM-P199: Interlaboratory comparability study of HIV-1 RNA copy number quantification

Published

April 13, 2024

Author(s)

Alison Devonshire, Eloise Busby, Gerwyn Jones, Denise O'Sullivan, Ana Fernandez-Gonzalez, Sachie Shibayama, Shin-ichiro Fujii, Megumi Kato, John Emerson Leguizamon Guerrero, Claudia Patricia Tere Peña, María Mercedes Arias Cortes, Roberto Becht Flatschart, Marcelo Neves de Medeiros, Antonio Marcos Saraiva, Young-Kyung Bae, Inchul Yang, Hee-Bong Yoo, Alexandra Bogožalec Košir, Mojca Milavec, Lianhua Dong, Chunyan Niu, Xia Wang, Phattaraporn Morris, Sasithon Temisak, Megan Cleveland, Peter Vallone, Daniel Burke, Michael Forbes-Smith, Jacob McLaughlin, Samreen Falak, Martin Hussels, Rainer Macdonald, Andreas Kummrow, Burhanettin Yalcinkaya, Sema Akyurek, Muslum Akgoz, Maxim Vonsky, Andrey Runov, Simon Cowen, Stephen Nyangoma, Stephen Ellison, Clare Morris, Neil Almond, Jim Huggett

Abstract

RNA is measured for a range of applications including clinical virology, disease-related gene expression biomarkers, biotechnology, cell therapy (viral vectors) and vaccines. For applications measuring viruses with RNA genomes, such as clinical viral load or viral vector titre determination, RNA copies, determined by measuring specific gene targets, is frequently the measurand, which can vary over many orders of magnitude from trace single copy levels to over 1010/mL (of original biological specimen such as plasma). Interlaboratory study CCQM-P199 applied the model of HIV-1 gene quantification and was conducted under the auspices of the Consultative Committee for Amount of Substance (CCQM) Nucleic Acid analysis Working Group (NAWG), with the aims of supporting its members' development of the capacity and evaluating candidate reference measurement procedures for applied viral nucleic acid measurements. Thirteen National Metrology Institutes (NMIs) and Designated Institutes (DIs) participated in Pilot study CCQM-P199 "HIV-1 RNA copy number quantification". Participants were requested to report the RNA copy number concentration, expressed in copies per microliter, of the HIV-1 group specific antigen (gag) gene (Measurand) of in vitro transcribed RNA molecules at low (103/L) and high concentration (109/L) (Study Materials 1 and 2, linked by gravimetric dilution) and purified genomic RNA from cultured virus (Study Material 3). Study Materials 1 and 3 contained a background human total RNA, whilst the matrix of Study Material 2 was buffered solution without additional human total RNA, making it suitable for analysis by "orthogonal methods" which are non-sequence specific and would be unable to distinguish between target viral and human derived sequences. Study Materials 1 and 3 were measured by participants using one-step RT-dPCR (Bio-Rad reagents) and/or two-step RT-dPCR with alternative cDNA synthesis reagents. The majority of laboratories used the QX100/200 dPCR system (Bio-Rad) and two laboratories used the Quantstudio 3D dPCR system (Thermo Fisher Scientific). Study Material 2 was measured by both RT-dPCR (one-step) (n = 4) and orthogonal methods: single molecule flow cytometric counting (n = 2), high performance liquid chromatograph (HPLC) (n = 1) and isotype dilution-mass spectrometry (ID-MS) (n = 1). Interlaboratory reproducibilities (expressed as %CV) were 21.4%, 15.3% and 22.0% for Study Materials 1, 2 and 3 respectively. Analysis of overdispersion showed that the interlaboratory variation for all three Study Materials was not accounted for in their reported uncertainties indicating additional uncharacterized sources of variation remain. Although the mean values of RT-dPCR and orthogonal method results were in agreement, the extrapolated mean Study Material 2 results were higher than mean Study Material 1 results (1196 vs. 808/L; p < 0.05). Follow-up analysis of Study Material 2 purity by ultra-performance liquid chromatography (UPLC) indicated higher MW impurities constituting of 16.6% of the molecules, which are hypothesised to be the cause of the HPLC and ID-MS results being higher than the majority of Study Material 1 and 2 results. Reproducible measurement of RNA templates was achieved by metrology laboratories, illustrating the potential of RT-dPCR combined with complimentary orthogonal approaches to support traceability and stability of diagnostic and biotechnogical application of RNA quantification. This study also highlighted that detailed characterization of RNA materials and sources of bias affecting measurements such as RT efficiency is needed to establish RT-dPCR as a primary reference measurement procedure for RNA copy number quantification.

Citation

Being submitted to WERB because they want to publish on BioRxiv (no journal selected yet)

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Keywords

dPCR, CCQM, nucleic acid

Citation

Devonshire, A. , Busby, E. , Jones, G. , O'Sullivan, D. , Fernandez-Gonzalez, A. , Shibayama, S. , Fujii, S. , Kato, M. , Leguizamon Guerrero, J. , Tere Peña, C. , Arias Cortes, M. , Becht Flatschart, R. , Neves de Medeiros, M. , Saraiva, A. , Bae, Y. , Yang, I. , Yoo, H. , Bogožalec Košir, A. , Milavec, M. , Dong, L. , Niu, C. , Wang, X. , Morris, P. , Temisak, S. , Cleveland, M. , Vallone, P. , Burke, D. , Forbes-Smith, M. , McLaughlin, J. , Falak, S. , Hussels, M. , Macdonald, R. , Kummrow, A. , Yalcinkaya, B. , Akyurek, S. , Akgoz, M. , Vonsky, M. , Runov, A. , Cowen, S. , Nyangoma, S. , Ellison, S. , Morris, C. , Almond, N. and Huggett, J. (2024), CCQM-P199: Interlaboratory comparability study of HIV-1 RNA copy number quantification, Being submitted to WERB because they want to publish on BioRxiv (no journal selected yet), [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=957040 (Accessed May 18, 2025)