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S.L. Stramer, R.A. Porter, J.P. Brodsky, K.J. Waldman, R.Y. Dodd, T.A. Armstrong and G.D. Griffin

Replacement of HIV-1 p24 Antigen Screening with HIV-1 RNA Nucleic Acid Testing (NAT) for Whole Blood Donations. S.L. Stramer, R.A. Porter, J.P. Brodsky, K.J. Waldman, R.Y. Dodd, T.A. Armstrong and G.D. Griffin. Background.

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S.L. Stramer, R.A. Porter, J.P. Brodsky, K.J. Waldman, R.Y. Dodd, T.A. Armstrong and G.D. Griffin

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  1. Replacement of HIV-1 p24 Antigen Screening with HIV-1 RNA Nucleic Acid Testing (NAT) for Whole Blood Donations S.L. Stramer, R.A. Porter, J.P. Brodsky, K.J. Waldman, R.Y. Dodd, T.A. Armstrongand G.D. Griffin

  2. Background • Since the implementation of p24 HIV-1 antigen (Ag) screening in March, 1996, only 5 Ag positive window-case donations have been identified • Due to low Ag yield and the improved sensitivity of NAT screening for HIV-1, replacement of Ag with HIV-1 NAT should be possible

  3. HIV-1 p24 Antigen Testing Experience During 2 years of testing 14 million donations (ARC), 136 samples were HIV-1 p24 Ag confirmed positive • 58 were false positive (based on RNA negativity, lack of seroconversion, and nonreproducible Ag neutralization) = 1:241,379 • 74 were HIV Ab positive • 4 were recently infected seroconverting donors • As of November 1999, 5 seroconverting donors identified in 23 million donations • 1:4.6 million (ARC) • 1:9.2 million (US)

  4. Samples for Testing • p24 Ag and HIV NAT reactive yield samples (N=4) • Tested undiluted and diluted 1:128 in RNA-negative defibrinated plasma • Commercial seroconversion samples (N=25) • Tested undiluted and diluted 1:128 • Diluted p24 Ag “External Control” sample that is tested on all NAT runs (N=437) • 3 Ag+/Ab– units (S/CO 1-2 Coulter) pooled • Pool diluted 1:16

  5. Comparison of HIV-1 p24 Ag Positive Window-Case U.S. Blood Donors HIV-1/HIV-2 Ab S/CO Sample Collection(days) RNA Copies/mL p24 Ag S/CO PercentNeut. SC WB 1 Index 6 x 105*14.0* 95 0.1 12 3 x 1041.2 95 2.3 POS – minus p31 22 1 x 105 0.3 5.6 POS – all bands 2 Index 2 x 106*28.5* 94 0.8 20 1 x 105 0.7 1.6 POS – minus p17, p31 43 1 x 104 0.1 5.3 POS – all bands 3 Index 1 x 1058.1 95 0.1 13 5 x 105*26.5* 90 8.8 POS – minus p31 4 Index 1 x 1052.4 95 0.0 13 1 x 106*17.5* 89 2.7 IND – p66 75 5 x 104 0.1 17.4 POS – all bands 5 Index 3 x 10518.6* 99 0.3 10 4 x 105* 0.2 4.3 POS – minus p31 17 1 x 105 0.9 12.5 POS – minus p31 24 3 x 104 0.7 8.5 POS – minus p31 31 2 x 104 0.1 13.1 POS – minus p31

  6. Detection of HIV-1 p24 Ag and NAT Yield Samples by HIV-1 NAT (1:128 Dilution) Index RNAConc. Undiluted(copies/mL) Multiplex1:128*(S/CO) IndexDetected By Case ARC 2 Ag 6x105 + (16.08) ARC 3 Ag 1x105 + (16.06) ARC 5 Ag 3x105 + (29.59) Milwaukee NAT 2x104 + (7.86) * Diluted in RNA screened negative defibrinated plasma

  7. HIV Panel 6240 – Virologic/Serologic Profile S/CO HIV PCR Quantitation 2 Days 5 Days Days PCR E992750C 7

  8. HIV Panel 6240 – Virologic/Serologic Profile S/CO HIV PCR Quantitation 2 Days 1:128 7 Days Neat 5 Days Days PCR E992750C 8

  9. HIV Panel PRB932 –Virologic/Serologic Profile HIV PCR Quantitation S/CO Cutoff Days PCR E992750C 9

  10. HIV Panel PRB932 –Virologic/Serologic Profile 14 Days HIV PCR Quantitation S/CO Cutoff Days PCR E992750C 10

  11. HIV Panel PRB943 –Virologic/Serologic Profile HIV PCR Quantitation S/CO 12 Days 2 Days Cutoff Days PCR E992750C 11

  12. HIV Panel PRB943 –Virologic/Serologic Profile HIV PCR Quantitation S/CO 5 Days 7 Days 2 Days Cutoff Days PCR E992750C 12

  13. Clinical Sensitivity, HIV Panels, Gen-Probe Lot 809355HIV Seronegative Bleeds(92 samples from25 individuals) p24 Antigen S/CO 29 NAT +/p24 Ag – Cutoff Multiplex Assay S/CO (Neat)

  14. Clinical Sensitivity, HIV Panels, Gen-Probe Lot 809355HIV Seronegative Bleeds(92 samples from25 individuals) p24 Antigen S/CO 21 NAT +/p24 Ag – 1:128 Cutoff Multiplex Assay S/CO (1:128)

  15. Effect of Dilution on NAT Reactivity (Gen-Probe HIV-1/HCV Assay) HIV-1 (N=25 Panels) HCV (N=22 Panels) Undiluted 162 167 162 160 Diluted 1:128 145 (90%) 148 (89%) 151 (93%) 151 (94%) p24 Ag 82 (57%) 82 (55%) — — ALT (³60) — — 50 (44%)* 49 (43%)** Lot 355 Lot 356 Lot 355 Lot 356 * Of 114 tested;** Of 113 tested

  16. Gen-Probe Linked Study – Phase I External Control Panel TargetCopies/mL Member HIV-1 300 ± 150 HCV 300 ± 150 NEG — Weak HIV-1 p24 Ag(S/CO 1-2 Coulterdiluted 1:16) 6,800

  17. 0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 Gen-Probe Linked Study – Phase IExternal Control Distributions Control N Mean S/CO Range HIV-1 435 18.66* 8.9 – 37.6 HCV 438 8.27* 2.1 – 10.4 NEG 436 0.28 0.1 – 0.9 HIV-1/p24 Ag 437 20.06* 12.1 – 25.2 *p<0.05 (437 runs total) 200 150 Frequency 100 50 S/CO E992750C 17

  18. Conclusions • High RNA titers (»105 copies/mL) correspond with p24 Ag positivity • No p24 Ag reactive sample was found NAT nonreactive even using a pool size of 128 • NAT, even using pools of 128, is more sensitive than screening with the currently licensed tests for HIV-1 p24 Ag. According to these data, the Ag test could be replaced by the use of licensed pooled NAT

  19. 8 N = 146 R2 = 0.78 7 6 5 Log RNA Level [copies/mL] 4 3 2 1 -2 -1.5 -1 -.5 0 .5 1 1.5 2 Log p24 S/CO Linear Relationship Between HIV RNA Levels and p24 S/CO Ratios 4.03 log (596-190,108 copies/mL) = 1

  20. 9 8 N = 146 7 6 5 4 Log HIV RNA Level [copies/mL] 3 2 1 0 -1 -2 -2 -1.5 -1 -.5 0 .5 1 1.5 2 Log p24 S/CO 95% Prediction Limits for Relationship of HIV RNA Levels and p24 S/CO Ratios

  21. HIV-1 RNA Levels During “RNA Only” and “p24 Positive” Window Periods 8 P = <0.0001* 7 6 5 Log HIV RNA Level [copies/mL] 4 3 2 RNA Only (N=61) p24 Pos (N=85) 1 * Mann-Whitney Test

  22. Linear Relationship Between HIV-1 RNA Levels and p24 S/CO Ratios in 32 Cases 8 7 6 5 Log RNA Level [copies/mL] 4 3 2 1 -2 -1.5 -1 -.5 0 .5 1 1.5 2 Log p24 S/CO

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