Assessing intra-lab precision and inter-lab repeatability of outgrowth assays of HIV-1 latent reservoir size

Reservoir Assay Validation and Evaluation Network (RAVEN) Study Group

Research output: Contribution to journalArticle

Abstract

Quantitative viral outgrowth assays (QVOA) use limiting dilutions of CD4+ T cells to measure the size of the latent HIV-1 reservoir, a major obstacle to curing HIV-1. Efforts to reduce the reservoir require assays that can reliably quantify its size in blood and tissues. Although QVOA is regarded as a "gold standard" for reservoir measurement, little is known about its accuracy and precision or about how cell storage conditions or laboratory-specific practices affect results. Owing to this lack of knowledge, confidence intervals around reservoir size estimates-as well as judgments of the ability of therapeutic interventions to alter the size of the replication-competent but transcriptionally inactive latent reservoir-rely on theoretical statistical assumptions about dilution assays. To address this gap, we have carried out a Bayesian statistical analysis of QVOA reliability on 75 split samples of peripheral blood mononuclear cells (PBMC) from 5 antiretroviral therapy (ART)-suppressed participants, measured using four different QVOAs at separate labs, estimating assay precision and the effect of frozen cell storage on estimated reservoir size. We found that typical assay results are expected to differ from the true value by a factor of 1.6 to 1.9 up or down. Systematic assay differences comprised a 24-fold range between the assays with highest and lowest scales, likely reflecting differences in viral outgrowth readout and input cell stimulation protocols. We also found that controlled-rate freezing and storage of samples did not cause substantial differences in QVOA compared to use of fresh cells (95% probability of < 2-fold change), supporting continued use of frozen storage to allow transport and batched analysis of samples. Finally, we simulated an early-phase clinical trial to demonstrate that batched analysis of pre- and post-therapy samples may increase power to detect a three-fold reservoir reduction by 15 to 24 percentage points.

Original languageEnglish (US)
Pages (from-to)e1006849
JournalPLoS computational biology
Volume15
Issue number4
DOIs
StatePublished - Apr 1 2019

Fingerprint

Repeatability
human immunodeficiency virus
Human immunodeficiency virus 1
repeatability
HIV-1
Assays
assay
assays
Cell
Therapy
Aptitude
Bayes Theorem
Therapeutics
Blood
therapeutics
Freezing
Fold
Blood Cells
fold
Dilution

ASJC Scopus subject areas

  • Ecology, Evolution, Behavior and Systematics
  • Modeling and Simulation
  • Ecology
  • Molecular Biology
  • Genetics
  • Cellular and Molecular Neuroscience
  • Computational Theory and Mathematics

Cite this

Assessing intra-lab precision and inter-lab repeatability of outgrowth assays of HIV-1 latent reservoir size. / Reservoir Assay Validation and Evaluation Network (RAVEN) Study Group.

In: PLoS computational biology, Vol. 15, No. 4, 01.04.2019, p. e1006849.

Research output: Contribution to journalArticle

Reservoir Assay Validation and Evaluation Network (RAVEN) Study Group. / Assessing intra-lab precision and inter-lab repeatability of outgrowth assays of HIV-1 latent reservoir size. In: PLoS computational biology. 2019 ; Vol. 15, No. 4. pp. e1006849.
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abstract = "Quantitative viral outgrowth assays (QVOA) use limiting dilutions of CD4+ T cells to measure the size of the latent HIV-1 reservoir, a major obstacle to curing HIV-1. Efforts to reduce the reservoir require assays that can reliably quantify its size in blood and tissues. Although QVOA is regarded as a {"}gold standard{"} for reservoir measurement, little is known about its accuracy and precision or about how cell storage conditions or laboratory-specific practices affect results. Owing to this lack of knowledge, confidence intervals around reservoir size estimates-as well as judgments of the ability of therapeutic interventions to alter the size of the replication-competent but transcriptionally inactive latent reservoir-rely on theoretical statistical assumptions about dilution assays. To address this gap, we have carried out a Bayesian statistical analysis of QVOA reliability on 75 split samples of peripheral blood mononuclear cells (PBMC) from 5 antiretroviral therapy (ART)-suppressed participants, measured using four different QVOAs at separate labs, estimating assay precision and the effect of frozen cell storage on estimated reservoir size. We found that typical assay results are expected to differ from the true value by a factor of 1.6 to 1.9 up or down. Systematic assay differences comprised a 24-fold range between the assays with highest and lowest scales, likely reflecting differences in viral outgrowth readout and input cell stimulation protocols. We also found that controlled-rate freezing and storage of samples did not cause substantial differences in QVOA compared to use of fresh cells (95{\%} probability of < 2-fold change), supporting continued use of frozen storage to allow transport and batched analysis of samples. Finally, we simulated an early-phase clinical trial to demonstrate that batched analysis of pre- and post-therapy samples may increase power to detect a three-fold reservoir reduction by 15 to 24 percentage points.",
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AU - Bacchetti, Peter

AU - Stone, Mars

AU - Deng, Xutao

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