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Improved reproducibility and quality of GvHD biomarker assay: application of multiplex microfluidic channel system

Bone Marrow Transplantation volume 51, pages 1615–1616 (2016)Cite this article

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A major limitation to GvHD research is that diagnosis relies on clinical features and biopsy of the involved tissues. A major advance in the field of GvHD diagnosis and prediction has been the identification and validation of a series of GvHD biomarkers that promise to revolutionize the ability to manage GvHD. Proteomic studies have identified serum or plasma protein markers to predict non-relapse mortality and acute GvHD.1 Especially ST2,2, 3 Reg3α4 and TNFR15 are promising biomarkers to predict transplant outcomes in multiple cohorts of allogeneic stem cell transplantation (allo-SCT).6, 7 However, before biomarker measurements can be applied widely as a reproducible diagnostic tool, reliable and standardized biomarker assays are needed as suggested by the NIH consensus.8 An ideal GvHD diagnostic, prognostic or predictive test for routine use should provide a high level of discrimination between patients with and without GvHD, have adequate sensitivity, high specificity and reproducibility. In addition, the assay should be rapid, easy to perform, widely available and affordable. Here, we evaluated a new multiplex microfluidic channel assay system adapted to measure GvHD biomarkers and compared it with a conventional ELISA.

In a single institution retrospective study, we analyzed plasma samples collected from 21 patients with hematologic malignancies who underwent T-cell depleted HLA-matched sibling allo-SCT with an ex vivo CD34+ selected graft and survived beyond day 100 post transplant (Protocol 13-H-0144, ClinicalTrials.gov Identifier: NCT01866839). The conditioning regimen included cyclophosphamide, fludarabine and myeloablative TBI. Low-dose, short-course cyclosporine was used as GvHD prophylaxis for 21 days. Plasma samples were collected at day 14 and day 28 post transplant. The concentrations of ST2, Reg3α and TNFR1 in plasma samples were measured using two protein quantification strategies: an automatic multiplex microfluidic channel system (Simple Plex, Protein Simple, San Jose, CA, USA) and a single-analyte ELISA (ST2, TNFR1, R&D, Minneapolis, MN, USA; Reg3α, MBL, Woburn, MA, USA) under similar conditions. The coefficient of variation (CV) was calculated as the ratio of the SD to the mean to measure the repeatability and reproducibility of each assay. Pearson’s correlation was used to assess the association between the assays. Mann–Whitney test was used to compare the assay levels between subgroups of patients. All tests were two-sided and a P-value <0.05 was considered to be statistically significant. Data analyses were performed using Prism Version 6 software (GraphPad, Inc., La Jolla, CA, USA) and R statistical software 3.2.3 (R Foundation for Statistical Computing, Vienna, Austria).

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References

  1. Paczesny S . Discovery and validation of graft-versus-host disease biomarkers. Blood 2013; 121: 585–594.

    Article  CAS  Google Scholar 

  2. Ponce DM, Hilden P, Mumaw C, Devlin SM, Lubin M, Giralt S et al. High day 28 ST2 levels predict for acute graft-versus-host disease and transplant-related mortality after cord blood transplantation. Blood 2015; 125: 199–205.

    Article  CAS  Google Scholar 

  3. Vander Lugt MT, Braun TM, Hanash S, Ritz J, Ho VT, Antin JH et al. ST2 as a marker for risk of therapy-resistant graft-versus-host disease and death. N Engl J Med 2013; 369: 529–539.

    Article  CAS  Google Scholar 

  4. Ferrara JL, Harris AC, Greenson JK, Braun TM, Holler E, Teshima T et al. Regenerating islet-derived 3-alpha is a biomarker of gastrointestinal graft-versus-host disease. Blood 2011; 118: 6702–6708.

    Article  CAS  Google Scholar 

  5. Choi SW, Kitko CL, Braun T, Paczesny S, Yanik G, Mineishi S et al. Change in plasma tumor necrosis factor receptor 1 levels in the first week after myeloablative allogeneic transplantation correlates with severity and incidence of GVHD and survival. Blood 2008; 112: 1539–1542.

    Article  CAS  Google Scholar 

  6. Levine JE, Braun TM, Harris AC, Holler E, Taylor A, Miller H et al. A prognostic score for acute graft-versus-host disease based on biomarkers: a multicentre study. Lancet Haematol 2015; 2: e21–e29.

    Article  Google Scholar 

  7. Ponce DM, Hilden P, Mumaw C, Devlin SM, Lubin M, Giralt S et al. High day 28 ST2 levels predict for acute graft-versus-host disease and transplant-related mortality after cord blood transplantation. Blood 2014; 125: 199–205.

    Article  Google Scholar 

  8. Paczesny S, Hakim FT, Pidala J, Cooke KR, Lathrop J, Griffith LM et al. National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: III. The 2014 Biomarker Working Group Report. Biol Blood Marrow Transplant 2015; 21: 780–792.

    Article  Google Scholar 

  9. Le RQ, Tian X, Jain NA, Lu K, Ito S, Draper DA et al. Clinical comorbidity predictive measures in ex vivo T-cell-depleted allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant 2015; 50: 1138–1140.

    Article  CAS  Google Scholar 

  10. Sorror ML, Maris MB, Storb R, Baron F, Sandmaier BM, Maloney DG et al. Hematopoietic cell transplantation (HCT)-specific comorbidity index: a new tool for risk assessment before allogeneic HCT. Blood 2005; 106: 2912–2919.

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Rajiv Pande, Berendia Jackson and Danielle Svancara (Protein Simple, San Jose, CA USA) for their technical support. We also thank all patients who donated the samples to this study. This research was supported by the Intramural Research Program of the National Heart, Lung, and Blood Institute at the National Institutes of Health.

Author contributions

Study concept and design (PA, ND, AJB and SI); in vitro experiment and data collection (PA, FC, CRC and NH); analysis and interpretation of data (PA, XT, CRC, SP, AJB and SI); drafting of the manuscript (PA, XT, CRC, MB, SP, AJB and SI); statistical analysis (PA, XT, AJB and SI); clinical data collection (PA, ND, PM, EK, DD, SM, JS, MB, AJB and SI); and obtained funding and study supervision (AJB).

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Authors and Affiliations

  1. Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA

    P Anandi, F Chinian, C R Cantilena, N Dunavin, N Hensel, D Draper, E Koklanaris, S Maxwell, J Superata, P Muranski, M Battiwalla, A J Barrett & S Ito

  2. Office of Biostatistics Research, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA

    X Tian

  3. Department of Pediatrics, Herman B wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA

    S Paczesny

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  1. P Anandi
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Correspondence to S Ito.

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Competing interests

Dr SP has a patent on ‘Methods of detection of graft-versus-host disease’ licensed to Viracor-IBT Laboratories. The remaining authors declare no conflict of interest.

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Supplementary Information accompanies this paper on Bone Marrow Transplantation website

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Anandi, P., Tian, X., Chinian, F. et al. Improved reproducibility and quality of GvHD biomarker assay: application of multiplex microfluidic channel system. Bone Marrow Transplant 51, 1615–1616 (2016). https://doi.org/10.1038/bmt.2016.226

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