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Cancer Epidemiology, Biomarkers & Prevention
Cancer Epidemiology, Biomarkers & Prevention
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Research Articles

Evaluation of Multiplexed Cytokine and Inflammation Marker Measurements: a Methodologic Study

Anil K. Chaturvedi, Troy J. Kemp, Ruth M. Pfeiffer, Angelique Biancotto, Marcus Williams, Stella Munuo, Mark P. Purdue, Ann W. Hsing, Ligia Pinto, J. Philip McCoy and Allan Hildesheim
Anil K. Chaturvedi
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Troy J. Kemp
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Ruth M. Pfeiffer
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Angelique Biancotto
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Marcus Williams
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Stella Munuo
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Mark P. Purdue
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Ann W. Hsing
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Ligia Pinto
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J. Philip McCoy
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Allan Hildesheim
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DOI: 10.1158/1055-9965.EPI-11-0221 Published September 2011
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  • Figure 1.
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    Figure 1.

    The proportion of samples with detectable levels for 67 markers on Bio-Rad (A) and 97 markers on Millipore (B) across 3 specimen types—serum (open circles), heparin plasma (filled circles), and EDTA plasma (open squares).

  • Figure 2.
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    Figure 2.

    The CVs for 40 blinded duplicates, 20 placed within the same batch, and 20 placed across different batches. Results are shown separately for 67 Bio-Rad markers (A–C) and 97 Millipore markers (D–F) across 3 specimen types–heparin plasma, serum, and EDTA plasma.(D–F) across 3 specimen types—serum, heparin plasma, and EDTA plasma.

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    Figure 3.

    Spearman's correlation coefficients are shown for comparisons of the rank order of marker concentrations between T0 serum and T0 heparin plasma on Bio-Rad (A) and on Millipore (B). Comparisons between Bio-Rad and Millipore for T0 serum (C) and T0 heparin plasma (D) are also shown. T0 denotes baseline visit in the PLCO study.

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    Figure 4.

    Differences in ICCs between T0 heparin plasma versus T0 Serum for Bio-Rad (A) and for Millipore (B). Differences in ICCs between Millipore versus Bio-Rad for T0 serum (C) and T0 heparin plasma (D) are also shown. ICCs were estimated using variance components analyses.

Tables

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  • Table 1.

    Summary of results for evaluation of multiplexed inflammation marker assays

    Bio-Rad number of markers (%)Millipore number of markers (%)
    Total67 (100.0)97 (100.0)
    Markers with >25% detection
    Serum56 (83.5)89 (91.7)
    Heparin plasma63 (94.0)89 (91.7)
    EDTA plasma64 (95.5)89 (91.7)
    Markers with <20% CV for across-batch duplicatesa
    Serum51 (76.2)75 (77.3)
    Heparin plasma52 (77.6)69 (71.1)
    EDTA plasma47 (70.1)78 (80.4)
    ICCs for across-batch duplicates
    Serum
     <0.506 (8.9)5 (5.1)
     0.50–0.8029 (43.3)32 (33.0)
     0.80–0.9012 (17.9)19 (19.5)
     >0.9011 (16.4)34 (35.1)
    Heparin plasma
     <0.503 (4.5)9 (9.3)
     0.50–0.8038 (56.7)31 (31.9)
     0.80–0.9015 (22.3)25 (25.8)
     >0.907 (10.4)28 (28.9)
    EDTA plasma
     <0.505 (7.5)7 (7.2)
     0.50–0.8049 (73.1)25 (25.8)
     0.80–0.905 (7.5)24 (24.7)
     >0.905 (7.5)37 (38.1)
    Markers with acceptable performanceb45 (67.2)71 (73.2)
    • aCVs and ICCs were calculated for 20 blinded duplicate samples for each specimen type that were placed across different batches.

    • bAcceptable performance was defined as: (i) being detectable in greater than 25% of the 100 samples on all 3 specimen types and (ii) across-batch CVs of less than 20% for blinded duplicates on at least 2 of the 3 specimen types.

  • Table 2.

    Summary of performance of multiplexed markers on Bio-Rad and Millipore

    Bio-Rad markers with acceptable performanceaBio-Rad markers with <25% detectabilitybMillipore markers with acceptable performanceMillipore markers with acceptable performanceaMillipore markers with <25% detectabilityb
    A2MB_NGFAmylin_TotalMIP_1DEotaxin 3
    CRPGM_CSFBCA_1PPGhrelin
    CTACKIFNA2CCL19_MIP3BPYYIL_20
    C PeptideIL_12P40CCL20_MIP3ASAAIL_21
    EotaxinIL_15CKINESAPIL_28a
    FerritinIL_1ACRPSCD30IL_3
    FGF_BasicIL_2CTACKSCD40LIL_4
    FibrinogenIL_3CXCL11_I_TACSCFIL_5
    GhrelinLIFCXCL6_GCP2SDF_1AM_CSF
    GIPMCP_3CXCL9_MIGSEGFRXCL1_Lympho
    GLP_1TNFBC_PeptideSGP130Millipore markers
    GlucagonEGFSILRIIwith >20% CVc
    GROBio-Rad markersENA_78SIL_1RI
    Haptoglobinwith >20% CVcEotaxinSIL_2RAGM_CSF
    HGFEotaxin_2SIL_4RIL_10
    IFNGB_NGFFGF_BasicSIL_6RIL_12P70
    IL_16GM_CSFFIT_3_LigandSRAGEIL_13
    IL_18G_CSFFractalkineSTNFRIIL_15
    IL 1RAIL_10GIPSTNFRIIIL_17
    IL 2RAIL_12P70GLP_1SVEGFR1IL_1B
    IL_6IL_13GlucagonSVEGFR2IL_1RA
    IL_8IL_15GROSVEGFR3IL_2
    IL 9IL_17G_CSFTARCIL_21
    InsulinIL_1AIFNA2TNF-αIL_23
    IP_10IL 1BIFNGTPOIL_28A
    LeptinIL_2IL_11TRAILIL_3
    MCP_1MCIL_4IL_12P40TSLPIL_4
    MIFIL_5IL_16VEGFIL_5
    MIGIL_7IL_1AIL_6
    MIP_1BLIFIL_29_IFNG1IL_7
    M_CSFMCP_3IL_33IL_9
    PAI_1MIP 1AIL_8I_309
    PCTTNF_BINSULINM_CSF
    PDGF_BBTPAIP_10TGFA
    RantesLEPTINTNF-B
    ResistinLIFXCL1_Lympho
    SAAMCP_1
    SAPMCP_2
    SCFMCP_3
    SCGF_BMCP_4
    SDF_1AMDC
    TNF-αMIP_1A
    TRAILMIP_1B
    VEGF
    Visfatin
    • aAcceptable performance was defined as (i) being detectable in greater than 25% of the 100 samples on all 3 specimen types and (ii) across-batch CVs of less than 20% for blinded duplicates on at least 2 of the 3 specimen types.

    • bMarkers with less than 25% detectability on at least 1 of 3 (serum, heparin plasma, and EDTA plasma) specimen types.

    • cMarkers with CVs for across-batch duplicates greater than 20% on 2 or more of 3 specimen types. CVs were calculated for 20 blinded duplicate samples for each specimen type that were placed across different batches.

  • Table 3.

    Comparison of assay performance across specimen types and kitsa

    Comparison% Detectability number of markersbMedian concentration number of markersc
    Bio-Rad (45 markers)
    T0 serum vs. T0 heparin plasma
     Significantly higher in serum716
     Significantly higher in plasma1019
     Similar2110
     Not evaluabled7
    T3 EDTA plasma vs. T3 heparin plasma
     Significantly higher in EDTA27
     Significantly higher in heparin212
     Similar3226
     Not evaluable9
    Millipore (71 markers)
     T0 serum vs. T0 heparin plasma
     Significantly higher in serum418
     Significantly higher in plasma513
     Similar5140
     Not evaluable11
    T3 EDTA plasma vs. T3 heparin plasma
     Significantly higher in EDTA53
     Significantly higher in heparin130
     Similar4938
     Not evaluable16
    Bio-Rad vs. Millipore (23 markers)
     T0 Serum
     Significantly higher in Bio-Rad215
     Significantly higher in Millipore27
     Similar101
     Not Evaluable9
    T0 heparin plasma
     Significantly higher in Bio-Rad416
     Significantly higher in Millipore47
     Similar60
     Not evaluable9
    T3 EDTA plasma
     Significantly higher in Bio-Rad315
     Significantly higher in Millipore38
     Similar70
     Not evaluable10
    • aComparisons were restricted to markers with acceptable performance—45 Bio-Rad markers, 71 Millipore markers, and 23 markers measured using both kits.

    • bComparisons of percentage of detectability across specimen types and kits were conducted using the McNemar's test.

    • cComparisons of median observed concentrations across specimen types and kits were conducted using the Wilcoxon signed-rank test. Observations below the assay's lower limit were excluded.

    • dMcNemar's P value could not be evaluated because one of the groups had 100% detectability.

Additional Files

  • Figures
  • Tables
  • Supplementary Data, Chaturvedi, et al

    Files in this Data Supplement:

    • Supplementary Figures 1 and 2
    • Supplementary Table 1
    • Supplementary Table 2
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Cancer Epidemiology Biomarkers & Prevention: 20 (9)
September 2011
Volume 20, Issue 9
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Evaluation of Multiplexed Cytokine and Inflammation Marker Measurements: a Methodologic Study
Anil K. Chaturvedi, Troy J. Kemp, Ruth M. Pfeiffer, Angelique Biancotto, Marcus Williams, Stella Munuo, Mark P. Purdue, Ann W. Hsing, Ligia Pinto, J. Philip McCoy and Allan Hildesheim
Cancer Epidemiol Biomarkers Prev September 1 2011 (20) (9) 1902-1911; DOI: 10.1158/1055-9965.EPI-11-0221

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Evaluation of Multiplexed Cytokine and Inflammation Marker Measurements: a Methodologic Study
Anil K. Chaturvedi, Troy J. Kemp, Ruth M. Pfeiffer, Angelique Biancotto, Marcus Williams, Stella Munuo, Mark P. Purdue, Ann W. Hsing, Ligia Pinto, J. Philip McCoy and Allan Hildesheim
Cancer Epidemiol Biomarkers Prev September 1 2011 (20) (9) 1902-1911; DOI: 10.1158/1055-9965.EPI-11-0221
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