Protocol for: l_mpimn07_2005_m_0802s QC Report and Summary for Panel 7 Set (3/28/13) 1. Description P07-CVD is a multiplexed of Luminex assay used to quantify 5 protein targets (Hemopexin, Tetranectin; Ceruloplasma; AGP-1 and Apo A-1 ) from human plasma in a 96-well microplate format. Samples are diluted at 1/100,000 and incubated with capture antibody coated beads for 2 hours at 25°C. After washing to remove unbound proteins, beads are mixed with a cocktail of five biotin-labeled detection antibodies and then reacted with streptavidin-phycoerythrin. The bead mounted antibody-protein complex is then measured with Streptavidin-Phycoerythrin using a BioPlex-200 reader. Bio-Plex Manager V 6.0 software was used to create standard curves and calculate the concentrations of seven proteins from all unknown samples. 2. Set QC Ranges to normalize and track assays in 239 plates QC ranges of each protein target in the panel were determined and used for normalizing the data from 239 assay plates containing 7643 FHS samples.      Two Quality Control (QC) samples were prepared, each QC sample contains 5 protein targets in the panel, QC1 is at low concentration and QC2 is at high concentration. Each QC sample was measured 20 times by five scientists on different dates and each time the QC1 and QC2 samples were measured in quadruplicate, with a total of 80 replicates. The average of each quadruplicate measurement of QC1 and QC2 was calculated. The mean of the 20 assays was determined and a standard deviation (SD) was then calculated. The acceptable range for FHS assay plates was the mean ± 3SD. If the QC1 or QC2 value of any assay plate was out of the range (see Table 1), the plate was repeated. For FHS sample testing, each 96 well assay plate included: 1) the standards in triplicate; 2) the QC1 and QC2 in quadruplicate; and 3) 32 FHS individual plasma samples in duplicate. P7-CVD QC1 QC2 HPX (44) TN (62) AGP-1 (26) CP (63) ApoA1 (12) Mean 2907.41 19.85 3168.99 5599.66 2334.62 SD 223.70 1.68 256.83 525.52 697.30 Mean 32589.77 207.53 33568.96 60184.57 36476.64 SD 1814.44 12.02 2785.92 3206.38 4271.85 QC1 and QC2 were used to monitor the variability of the 239 assay plates. See Trend Charts below 3. Duplication: Each of the 7643 FHS plasma samples was tested in duplicate. See table below for the average CV% See Charts below for the distribution of CV% among 7643 FHS 4. Precision : Two QC samples (QC1 at low level and QC2 at high level) are used to measure both intra-assay CV% and inter-assay CV%. For every test, quadruplicate of QC1 and QC2 samples are run in each assay for twenty times by four scientists at different dates (N=20). Intra-assay CV% Inter-assay CV% HPX (44) 4.66 3.39 7.69 5.57 QC1 QC2 QC1 QC2 TN (62) 5.35 2.77 8.47 5.79 AGP-1 (26) 4.63 5.72 8.10 8.30 CP (63) 5.00 3.48 9.38 5.33 ApoA1 (12) 7.11 7.25 29.87 11.71 5. Standard Curve: For each assay plate, a 7 point standard curve prepared in triplicate was used. A best fit of the three standard curves was plotted with the Bio-Plex ManagerTM software. All measurements of FHS samples and of QC1 & QC2 were derived from the optimized standard curve. 6. Linearity of Dilution: A pool of 10 individual human plasma samples is diluted at 2 fold serials starting from 1/100,000 down to 1/50,000 and up to 1/400,000 in sample diluent. The concentration at each dilution point is calculated from standard curves with dilution factor (DF). Dilution linearity is expressed as percentage of recovery at each dilution point against the start sample (1/100,000) Dilution Linearity(% start) Dilution factor HPX (44) 102.0 1/50,000 100.0 1/200,000 101.3 1/400,000 101.1 Average TN (62) 98.7 101.2 104.3 101.4 AGP-1 (26) 96.3 101.6 101.2 99.7 CP (63) 101.7 97.8 97.5 99.0 ApoA1 (12) 104.4 98.3 87.9 96.9 7. Standard Recovery (%) A set of seven standards is run with samples in each assay plate. The percentage of recovery is the measured concentration over the expressed concentration at each standard point. Standard recovery(%) STD7 STD6 STD5 STD4 STD3 STD2 STD1 HPX (44) TN (62) AGP‐1 (26) CP (63) ApoA1 (12) 91.0 105.0 97.0 102.0 98.0 102.0 >OOR 103.0 98.0 100.0 101.0 99.0 100.0 100.0 100.0 101.0 99.0 102.0 96.0 105.0 >OOR 100.0 101.0 98.0 103.0 98.0 101.0 100.0 100.0 101.0 97.0 106.0 97.0 90.0 122.0 8. Calibration of Standards The standard mixture of P07-CVD is prepared from human plasma. The concentration of standard in P07-CVD panel is been calibrated on individual purified proteins listed below. Target HPX (44) Vendor Cat # Sigma H9291 TN (62) R & D Systems Sigma AGP1 (26) CP (63) ApoA1 (12) Athens Reserch and Tech Purity (%) >95% Type of STD purified protein 5170‐CL‐050 Lot# SLBC9915 RHU0212031 >95% purified protein A1614-90029 16-16-030518 2422504 CP2011-01 >98% purified protein 95% purified protein 3710-1H-6 28 (Kit lot) ND ELISA kit STD Mabtch Calibrated concentration of P07-STD (pg/mL) 818675.9 5281.0 849877.4 1515812.5 924173.4 9. Stability Study of Reagents All assay reagents were tested with control samples at proper storage conditions. For P07-CVD panel, the standard mixture is stored at -80°C with liquid format. The stability of P07-CVD reagents was tested with final assay format at real time manner for 80 days at -80°C, a time frame to cover screening 7643 FHS samples. The reagent stability is expressed with percentage of concentration recovery of four plasma samples at day 80 over the day 1 (see table below). Recovery % (Day 80 over Day 1) Sample HPX (44) S1 102.22 S2 100.75 S3 98.97 S4 94.85 Average(%) 99.20 TN (62) 104.94 102.30 99.76 94.44 100.36 AGP1 (26) 106.62 99.08 96.74 94.50 99.24 CP (63) 96.82 96.41 102.99 100.16 99.10 ApoA1 (12) 105.77 99.62 100.37 101.24 101.75 10. Detection limit (LLOQ and ULOQ) Detection limits of standard curves are automatically determined by Bio-Plex Manager software based on standard recovery and CV% of replicates at each standard point. The sample reportable range is calculated from the product of standard range and sample dilution factor. Samples being out of reportable range are reported as OOR< or OOR>. Standard range (pg/mL) HPX (44) TN (62) AGP-1 (26) CP (63) ApoA1 (12) LLOQ 1.82E+02 1.30E+00 2.07E+02 3.69E+02 2.25E+02 ULOQ 2.09E+05 5.28E+03 2.24E+05 1.51E+06 1.13E+06 Sample detection range at 1/100,000 dilution (pg/mL) HPX (44) TN (62) AGP-1 (26) CP (63) ApoA1 (12) LLOQ 1.82E+07 1.30E+05 2.07E+07 3.69E+07 2.25E+07 ULOQ 2.09E+10 5.28E+08 2.24E+10 1.51E+11 1.13E+11 11 Assay Results Detailed assay results including the concentration for individual FHS plasma sample and CV% of sample duplication are reported in NIH website. Data Summary in Charts- (The Measurement of 7643 FHS Plasma Samples) 12 Discussion: The plasma dilution factor for this panel is 1:100,000. Apo A-1 has been repeated in this panel with this new dilution factor resulting in more measurement within the linear range as compared with the measurement in Panel I in which a plasma dilution fator is 1:20,000 was used. We use Apo A1 in panel 7 to differentiate from Apo A I in panel I but they are the same target.