1. E0304206A 1 Supplemental Testing for HIV-1 and HCV Blood Products Advisory Committee Meeting September 18, 2003 Susan L. Stramer, Ph.D. National Testing and Reference Laboratories American Red Cross Jaye P. Brodsky Quality Analytics

2. E0304206A 2 Background uHIV-1 and HCV algorithms utilize immunoassays for confirmation –HIV-1 uses Western Blot (WB) containing electrophoresed whole HIV lysate –HCV uses Recombinant Immunoblot Assay (RIBA) containing painted recombinant HCV antigens uIssues with supplemental assays –Poor performance Unreadable or invalidUnreadable or invalid False positivity; false negativityFalse positivity; false negativity High rates of indeterminate results (neither pos or neg) in healthy individualsHigh rates of indeterminate results (neither pos or neg) in healthy individuals

3. E0304206A 3 Background uIssues with supplemental assays –High costs –Inconsistent availability (HIV) –Is all the testing necessary, or is redundant testing being performed? uCan alternatives be validated?

4. E0304206A 4 Alternative Algorithms uHCV Alternative – 1 –Use of HCV NAT in supplemental algorithms Already being performed as a screening test for all donationsAlready being performed as a screening test for all donations NAT results integrated into donor counselingNAT results integrated into donor counseling Sensitivity high relative to RIBA even though NAT is performed in pools (16-24)Sensitivity high relative to RIBA even though NAT is performed in pools (16-24) Specificity high (false-positive donor rates less than 1:40,000)Specificity high (false-positive donor rates less than 1:40,000) Already recommended for diagnostic use by CDCAlready recommended for diagnostic use by CDC –If NAT reactive, RIBA not needed uHCV Alternative – 2 –Use of S/CO to further reduce the need for RIBA in HCV NAT nonreactive individuals

5. E0304206A 5 Alternative Algorithms uHIV Alternative – 1 –Use of NAT and/or S/CO as proposed for HCV uHIV Alternative – 2 –Use of a second EIA where discordant EIA reactives (EIA-1 Rx and EIA-2 nonRx) are not further tested –Feasibility for HIV versus HCV Anti-HCV licensed screening assays use same recombinant antigens, same assay format; overlapping populations of false positivesAnti-HCV licensed screening assays use same recombinant antigens, same assay format; overlapping populations of false positives Anti-HIV licensed screening assays use different antigens (viral lysate, recombinants and peptides); populations of false positives are uniqueAnti-HIV licensed screening assays use different antigens (viral lysate, recombinants and peptides); populations of false positives are unique

6. E0304206A 6 Alternative Algorithms uIssues for HIV or HCV alternatives –Potential misclassification of donors Current algorithms containing RIBA or WB in combination with NAT (and screening S/CO value) provide specific counseling messagesCurrent algorithms containing RIBA or WB in combination with NAT (and screening S/CO value) provide specific counseling messages –Kits will require labeling for intended use (supplemental claims to NAT assays or EIAs) 21 CFR 610.40 Subpart E21 CFR 610.40 Subpart E –“You must further test each donation, including autologous donations, found to be reactive by a screening test …whenever a supplemental (additional, most specific) test has been approved for such use by FDA….”

7. E0304206A 7 Current: HCV EIA 2.0 (Abbott) or 3.0 (Ortho) Repeat Reactive Perform RIBA 3.0 SIA N = 34,656 IndPos Reactive Individually Neg NonRx in Pool or Individually Reactive Individually NonRx in Pool or Individually Reactive Individually NonRx in Pool or Individually HCV Infected HCV Infected? Resolved infection (≈20%) Viral load < NAT cutoff False Pos RIBA 38% 13,198 HCV Not Infected HCV Infected? Early seroconversion False Rx NAT HCV Infected? 17.2% 5,967 0.15% 50 34.5% 11,963 HCV Infected Early seroconversion 0.5% 159 9.6% 3,319 HCV NAT

8. E0304206A 8 Option 1: HCV EIA 2.0 (Abbott) or 3.0 (Ortho) Repeat Reactive Review HCV NAT Results N = 34,656 Pool or Individual Unit NonRx (16 dtns  HIV-1/HCV TMA, 24 dtns  HCV PCR) Individual Unit NAT Rx (dHCV TMA or HCV PCR) No Further Testing HCV Infected 38.7% 13,407 98.4% PPV 13,198 RIBA Pos 61.3% 21,249 Perform RIBA IndPosNeg 28.1% 5,967 15.6% 3,319 56.3% 11,963 79.9% Sensitivity

9. E0304206A 9 Option 2: HCV EIA 2.0 (Abbott) or 3.0 (Ortho) Repeat Reactive Review HCV NAT Results N = 34,656 Pool or Individual Unit NonRx (16 dtns  HIV-1/HCV TMA, 24 dtns  HCV PCR) Individual Unit NAT Rx (dHCV TMA or HCV PCR) No Further Testing HCV Infected 38.7% 13,407 98.4% PPV 13,198 RIBA Pos 61.3% 21,249 82.9% Sensitivity Perform RIBA IndPosNeg 31% 5,055 3.5% 566 65.5% 10,692 Review S/CO Values of EIA S/CO ≥ 3.8 23.2% 4,936 No Further Testing HCV Infected? 55.8% PPV 2,753 RIBA Pos S/CO < 3.8 76.8% 16,313 99.8% Sensitivity 13,177 S/CO ≥ 3.8

10. E0304206A 10 RIBA Result Correlation of NAT Screening with Supplemental HCV Serological Results (36,536 RR donations of which 34,656 (94.9%) had EIA and RIBA data) NAT Result Rx NonRx Total PosIndNeg 13,182 (13,198***) (80%) 3,319 16,502 (16,517***) (48%) 174* (159***) 5,967 6,141 (6,126***) 50** 11,963 12,013 (1.7%) Total 13,407 21,249 34,656 9/8/99 to 6/30/03 *140/174 (80%) dHCV and PCR Rx (<100-48,000,000 copies/mL) **16/50 (32%) dHCV and PCR Rx (<100-24,000,000 copies/mL) ***15 RIBA Ind due to  1 + hSOD (1:2310 in RR samples)

11. E0304206A 11 Identification of HCV RIBA-Positive, NAT-Nonreactive Samples 9/8/99 – 12/31/02 2,255 HCV RIBA Pos, NAT Neg Neat 812 Screened Neat, Neat Neg (36%) 730 29 Screened Pools, Pool Rx, Neat Neg (1%) 25 1,414 Screened Pools, Pool Neg (63%) 1,397 * A minimum of one of two reps positive Pos* 15 (2.1%) Neg 715 Pos 1 (3.7%) Neg 24 Pos* 29 (2.1%) Neg 1,368 Neat Retest: NGI x1, or Gen-Probe x2

12. E0304206A 12 Viral Loads of HCV RIBA-Positive Samples Screened NAT Nonreactive in Pools or Individually and that Retested NAT Reactive Copies/mL at NGI Screened in Pools (N=29) 20+ Quant/24 sent  100 (11) 1 * 380 (2) 4609101,500*7,700*9,400*34,000*5,200,000* Screened Individually (N=15) 10+ Quant/12 sent  100 (5) 1 * 200430*2,0007,400*12,000* * Of 30+ Quant, 10 (30%) tested PCR positive when diluted 1:16; frequency 8 of 2255 (1:282) total with viral loads >1000 copies/mL representing >16 million donations screened * Of 30+ Quant, 10 (30%) tested PCR positive when diluted 1:16; frequency 8 of 2255 (1:282) total with viral loads >1000 copies/mL representing >16 million donations screened

13. E0304206A 13 Relationship between HCV 3.0 EIA and RIBA All Samples 9/8/99 to 6/30/03 N = 34,656 S/CO HCV SIA (3.0) Results 2.433 1.637 1.274 3.079 1.876 >5.000 Neg N = 12,013 34.7% Ind N = 6,126 17.7% Pos N = 16,517 47.6% 1.024 - >5.000 95% Range 1.032 - >5.0003.077 - >5.000 1.354 4.943 4.583 0.00 1.00 2.00 3.00 4.00 5.00

14. E0304206A 14 Relationship between HCV 3.0 EIA and RIBA NAT Reactive Samples 9/8/99 to 6/30/03 N = 13,407 S/CO HCV SIA (3.0) Results 4.393 1.800 1.354 >5.000 4.763 4.221 >5.000 4.971 4.627 Neg N = 50 0.4% Ind N = 159 1.2% Pos N = 13,198 98.4% 1.026 - >5.000 95% Range 1.212 - >5.0004.145 - >5.000 0.00 1.00 2.00 3.00 4.00 5.00

15. E0304206A 15 Relationship between HCV 3.0 EIA and RIBA NAT Nonreactive Samples 9/8/99 to 6/30/03 N = 21,249 S/CO HCV SIA (3.0) Results 2.428 1.636 1.274 2.955 1.840 >5.000 Neg N = 11,963 56.3% Ind N = 5,967 28.1% Pos N = 3,319 15.6% 1.024 - >5.000 95% Range 0.00 1.00 2.00 3.00 4.00 5.00 1.031 - >5.0001.388 - >5.000 1.348 4.795 4.311

16. E0304206A 16 ARC HCV Testing 9/8/99 Through 6/30/03 NAT Reactive Specimens Only (N = 13,407) S/CO RIBA NEG RIBA IND RIBA POSTotal %RIBA POS 1.0 to 1.999271785215.4% 2.0 to 2.9998651926.3% 3.0 to 3.7991481361.5% 3.8 to 4.9998706,9547,03298.9% > 5.06626,2236,29198.9% Note: 13,323 of 13,407 (99.4%) NAT POS specimens had an S/CO ≥ 3.8; 13,177/13,323 (98.9%) specimens with S/CO ≥ 3.8 confirmed POS; 95% CI = 98.7 - 99.1 by binomial distribution 95% CI = 98.7 - 99.1 by binomial distribution Chi-square tests for independence were significant (p<0.0001)

17. E0304206A 17 S/CO RIBA NEG RIBA IND RIBA POSTotal %RIBA POS 1.0 to 1.9997,731330119811,2301.8% 2.0 to 2.9992,1911,2091813,5815.1% 3.0 to 3.7997705451871,50212.5% 3.8 to 4.9999536781,5223,15348.3% > 5.03182341,2311,78369.0% Note: 4,936 of 21,249 (23.2%) NAT NEG specimens had an S/CO ≥ 3.8; 2,753/4,936 (55.8%) specimens with S/CO ≥ 3.8 confirmed POS; 95% CI = 54.4 – 57.2 by binomial distribution Chi-square tests for independence were significant (p<0.0001) ARC HCV Testing 9/8/99 Through 6/30/03 NAT Nonreactive Specimens Only (N = 21,249)

18. E0304206A 18 PPV and Sensitivity of EIA and NAT vs RIBA RIBA POS NEG or IND TOTAL EIA RR, NAT Rx13,19820913,407 EIA RR, NAT NR3,31917,93021,249 TOTAL16,51718,13934,656 PPV = 13,198/13,407 = 98.4% Sens = 13,198/16,517 = 79.9%

19. E0304206A 19 PPV and Sensitivity of High S/CO (≥ 3.8) vs RIBA RIBA POS NEG or IND TOTAL EIA RR, S/CO ≥ 3.815,9302,32918,259 EIA RR, S/CO < 3.858715,81016,397 TOTAL16,51718,13934,656 PPV = 15,930/18,259 = 87.2% Sens = 15,930/16,517 = 96.4% Note:Increasing the S/CO limit to ≥ 5.0 increases the PPV to 92.3% and decreases the sensitivity to 45.1%

20. E0304206A 20 PPV and Sensitivity of High S/CO (≥ 3.8) vs RIBA Where NAT is Reactive RIBA POS NEG or IND TOTAL EIA RR, S/CO ≥ 3.813,17714613,323 EIA RR, S/CO < 3.8216384 TOTAL13,19820913,407 PPV = 13,177/13,323 = 98.9% Sens = 13,177/13,198 = 99.8%

21. E0304206A 21 PPV and Sensitivity of High S/CO (≥ 3.8) vs RIBA Where NAT is Nonreactive RIBA POS NEG or IND TOTAL EIA RR, S/CO ≥ 3.82,7532,1834,936 EIA RR, S/CO < 3.856615,74716,313 TOTAL3,31917,93021,249 PPV = 2,753/4,936 = 55.8% Sens = 2,753/3,319 = 82.9%

22. E0304206A 22 HCV Alternate Algorithm Summary uUse of NAT as the first step of the supplemental test algorithm will reduce the amount of RIBA performed by approximately 40% uHCV NAT-reactive samples (even using pooled NAT for screening): –Sensitivity of 79.9% (detection of RIBA confirmed positives) Those not detected by NAT will be tested by RIBA (as true today)Those not detected by NAT will be tested by RIBA (as true today) –98.4% are RIBA pos (PPV) Small percent of NAT-reactive samples likely representing early seroconversion are RIBA ind or neg Small percent of NAT-reactive samples likely representing early seroconversion are RIBA ind or neg

23. E0304206A 23 HCV Alternate Algorithm Summary uUse of S/CO of > 3.8 can be applied following separation of RR population into NAT Rx and nonRx –Since NAT results are available for all donations, and is a specific test for HCV, S/CO introduced for NAT nonRx to further reduce the dependence on RIBA uS/CO > 3.8 does have high sensitivity and PPV relative to RIBA in populations where NAT is not performed –Sensitivity of 96.4% –PPV of 87.2% uHowever, use of a high S/CO for NAT nonRxs would only eliminate 23% of RIBAs performed with poor performance –Sensitivity of 82.9% –PPV of 55.8%

24. E0304206A 24 Cambridge Biotech Human Immunodeficiency Virus Type 1 (HIV -1) Western Blot Kit March 16, 2000 BPAC uAny bands present but pattern does not meet criteria for POSITIVE = INDETERMINATE uNon-viral bands have been observed with certain specimens. These bands are not usually accompanied by any of the major viral bands of diagnostic significance (p24, gp41/120/160). The non-viral bands appear to be cell related with the most common in the molecular weight range of 70K, 51-55K (possible HLA-DR) and 43K (possible HLA-ABC).

25. E0304206A 25 HIV-1 Blood Donor Screening/Supplemental Test Results (ARC); March 16, 2000 BPAC 12.4 Million Donations Screened Positive 791 (7.1%) Negative 5,128 (46.3%) Indeterminate 5,161 (46.6%) 1998-1999 11,080 Repeat Reactive (0.09%) HIV-1 WB (Cambridge Biotech)

26. E0304206A 26 HIV-1 Blood Donor Screening/Supplemental Test Results (ARC) (Cont.); March 16, 2000 BPAC 5,161 Indeterminate (46.6%) Multiple Viral Bands (p24, gp41, gp120/160, but +/- intensity) 46 (1%) OneViralBand 2,752 (53.3%) MultipleViralBands 589 (11.4%) Background Only (obscuresreading) 1,050 (20.3%) Non- Viral Bands 724 (14%)  16 (35%) “ENV” only  1,925 (70%) p24 “GAG” only  464 (79%) “GAG” reactivity with or without other banding  569 (79%) “p70” 65.7% Viral Banding 1998-1999

27. E0304206A 27 Calypte Biotech HIV-1 Western Blot gp160 gp120 p66 p55/51 gp41 p31 p24 p17

28. E0304206A 28 Calypte HIV-1 Western Blot gp160 gp120 p66 p55/51 gp41 p31 p24 p17

29. E0304206A 29 HIV-1 RNA Concentrations During SC Based on NGI’s PCR of 28 Plasma Donor Panels (N=221) Categories PCR–/Ag–/Ab– (n=19) PCR+/Ag–/Ab– (n=22) PCR+/Ag+/Ab– (n=22) PCR+/Ag+ / –/ IgM+/IgG– WB–/IND (n=19) PCR+/Ag+ / –/ IgM+/IgG– WB POS (n=12) 6 Median Days 6 100 1,000 10,000 100,000 1,000,000 PCR+/Ag+ / –/ Ab+ (n=20) 0 734 HIV-1 RNA Copies/mL E001372A 29

30. E0304206A 30 Comparison of HIV-1 p24 Ag Positive Window-Case U.S. Blood Donors 1Index6 x 10 5 *14.0*950.1 123 x 10 4 1.2952.3POS – minus p31 221 x 10 5 0.3 5.6POS – all bands 2Index2 x 10 6 *28.5*940.8 201 x 10 5 0.7 1.6POS – minus p31 431 x 10 4 0.1 5.3POS – all bands 3Index1 x 10 5 8.1950.1 135 x 10 5 *26.5*908.8POS – minus p31 4Index1 x 10 5 2.4950.0 131 x 10 6 *17.5*892.7IND – p66 755 x 10 4 0.1 17.4POS – all bands 5Index3 x 10 5 18.6*990.3 104 x 10 5 *0.24.3POS – minus p31 171 x 10 5 0.9 12.5POS – minus p31 243 x 10 4 0.7 8.5POS – minus p31 312 x 10 4 0.1 13.1POS – minus p31 SC Sample Collection (days) WB RNA Copies/mL p24 Ag S/CO Percent Neut. HIV-1/ HIV-2 Ab S/CO

31. E0304206A 31 Current: HIV-1/HIV-2 EIA (Abbott rDNA or GSC pEIA) Perform WB N = 17,090 IndPos Reactive Individually Neg NonRx in Pool or Individually Reactive Individually NonRx in Pool or Individually Reactive Individually NonRx in Pool or Individually HIV Infected HIV Infected? Viral load < NAT cutoff False Pos WB 4.4% 759 HIV Not Infected HIV Infected? Early seroconversion False Rx NAT HIV Not Infected? 51% 8,654 0.2% 37 (0) 44% 7,525 HIV Infected Early seroconversion 0.3% 56 (6) 0.3% 59 HIV NAT

32. E0304206A 32 Option 1: HIV-1/HIV-2 EIA (Abbott rDNA or GSC pEIA) Review HIV-1 NAT Results N = 17,090 Pool or Individual Unit NonRx (16 dtns  HIV-1/HCV TMA, 24 dtns  HIV PCR) Individual Unit NAT Rx (dHIV TMA or HIV PCR) No Further Testing HIV Infected 5% 852 (765) 89.1% PPV (99.2%) 759 WB Pos 95% 16,238 Perform WB IndPosNeg 53.3% 8,654 0.4% 59 46.3% 7,525 92.8% Sensitivity

33. E0304206A 33 Option 2: HIV-1/HIV-2 EIA (Abbott rDNA or GSC pEIA) Review HIV-1 NAT Results N = 17,090 Pool or Individual Unit NonRx (16 dtns  HIV-1/HCV TMA, 24 dtns  HIV PCR) Individual Unit NAT Rx (dHIV TMA or HIV PCR) No Further Testing HCV Infected 5% 852 (765) 89.1% PPV (99.2%) 759 WB Pos 95% 16,238 33.9% Sensitivity Perform WB IndPosNeg 53.3% 8,510 0.2% 39 46.5% 7,428 Review S/CO Values of EIA S/CO ≥ 15 1.6% 261 No Further Testing HIV Infected? 7.7% PPV 20 WB Pos S/CO < 15 98.4% 15,977 99.6% Sensitivity 694 S/CO > 15

34. E0304206A 34 Western Blot Result Correlation of NAT with Supplemental HIV Serological Data (17,791 RR donations of which 17,090 (96.1%) had EIA and WB data) NAT Result Rx NonRx Total PosIndNeg 759 (89.1%) 59 (0.4%) 818 (4.8%) 56* 8,654 8,710 37** 7,525 7,562 (10.9%) Total 852 16,238 17,090 9/8/99 to 6/30/03 *6/56 (11%) dHIV and PCR Rx (9,500-800,000 copies/mL) **0/37 (0%) dHIV and PCR Rx

35. E0304206A 35 Characteristics of HIV-1 WB Pos/TMA Nonreactive Samples All samples p24 Ag negative SamplePoolNeatHIV-1/HIV-2 S/COWBHIV PCR 1NR1.4341, 120, 160Neg 2NR1.0341, 160Neg 3NR1.6241, 160Neg 4NR1.1141, 55, 160Neg 5NR2.5024, 41, 51, 61, 160Neg 6NR20.18all bandsNeg 7NR1.2224, 41, 160Neg 8NR1.1117, 41, 120, 160Neg 9NR1.8441, 160Neg 10NR1.4024, 41, 160Neg 11NR1.7017, 24, 41, 51, 160Neg 12NR20.00all bandsPos (200 copies/mL) 13NR17.84all bandsNeg 9/8/99 – 8/31/00

36. E0304206A 36 Characteristics of HIV-1 WB Pos/TMA Nonreactive Samples All samples p24 Ag negative SamplePoolNeatHIV-1/HIV-2 S/COWBHIV PCR 14NR8.7317, 24, 160Neg 15NR10.5624, 55, 120, 160Neg 16NR19.30all bandsNeg 17NR19.47all bands Pos (200 copies/mL) 18NR1.0441, 66, 120, 160Neg 19NR4.7241, 160Neg 20NR1.5024, 41, 160Neg 21NR1.5324, 41, 160Neg 22NR1.6024, 160Neg 23NR17.60all bandsNeg 24NR19.30all bandsNeg 9/1/00 – 12/29/01

37. E0304206A 37 Characteristics of HIV-1 WB Pos/TMA Nonreactive Samples All samples p24 Ag negative SamplePoolNeatHIV-1/HIV-2 S/COWBHIV PCR 25NR1.0024, 120, 160Neg 26NR1.1941, 120, 160Neg 27NR17.43all bandsNeg 28NR1.3741, 160Neg 29NR1.4024, 41, 120, 160Neg 30NR1.4324, 160Neg 31NR15.2017, 24, 120, 160Neg 32NR18.80all bandsNeg 33NR15.93all bandsNeg 34NR1.3441, 160Neg 9/1/00 – 12/29/01

38. E0304206A 38 N = 17,090 Relationship between HIV-1/HIV-2 EIA and Western Blot-All Samples 9/8/99 to 6/30/03 EIA S/CO Western Blot Results 2.600 1.602 1.225 2.672 1.598 19.178 Neg N = 7,562 44.2% Ind N = 8,710 51.0% Pos N = 818 4.8% 1.018 – 11.370 95% Range 1.018 – 12.3291.513 – 20.952 1.231 18.030 16.008 0.00 5.00 10.00 15.00 20.00 25.00

39. E0304206A 39 Relationship between HIV-1/HIV-2 EIA and Western Blot NAT Nonreactive Samples 9/8/99 to 6/30/03 N = 16,238 (95% of total) Western Blot Results 2.602 1.602 1.225 2.672 1.599 16.268 Neg N = 7,525 46.3% Ind N = 8,654 53.5% Pos N = 59 0.4% 1.018 – 11.368 95% Range 0.00 5.00 10.00 15.00 20.00 25.00 1.018 – 12.2581.013 – 20.092 1.230 2.390 1.400 EIA S/CO

40. E0304206A 40 Relationship between HIV-1/HIV-2 EIA and Western Blot NAT Reactive Samples 9/8/99 to 6/30/03 Western Blot Results 2.545 1.578 1.314 2.560 1.581 19.298 Neg N = 37 4.3% Ind N = 56 6.6% Pos N = 759 89.1% 1.076 – 12.398 95% Range 1.012 – 18.3858.130 – 20.952 1.299 18.182 16.296 N = 852 (5% of total) 0.00 5.00 10.00 15.00 20.00 25.00 EIA S/CO

41. E0304206A 41 S/CO WB NEG WB IND WB POSTotal %WB POS 1.0 to 4.99935485885.7% 5.0 to 9.99912232688.5% 10.0 to 14.99913374190.2% 15.0 to 19.9990359159499.5% > 20.000103 100.0% Note: 697 of 852 (81.8%) NAT POS specimens had an S/CO ≥ 15.0; 694/697 (99.6%) specimens with S/CO ≥ 15.0 confirmed POS; 95% CI = 98.8 - 99.9 by binomial distribution Chi-square tests for independence were significant (p<0.0001) ARC HIV Testing 9/8/99 Through 6/30/03 NAT Reactive Specimens Only (N = 852)

42. E0304206A 42 S/CO WB NEG WB IND WB POSTotal %WB POS 1.0 to 4.9996,7697,7493514,5530.2% 5.0 to 9.99951560931,1270.3% 10.0 to 14.99914415212970.3% 15.0 to 19.99986133182377.6% > 20.011 2248.3% Note: 261 of 16,238 (1.6%) NAT NEG specimens had an S/CO ≥ 15.0; 20/261 (7.7%) specimens with s/co ≥ 15.0 confirmed POS; 95% CI = 4.7 – 11.6 by binomial distribution Chi-square tests for independence were significant (p<0.0001) ARC HIV Testing 9/8/99 Through 6/30/03 NAT Nonreactive Specimens Only (N = 16,238)

43. E0304206A 43 Western Blot POS NEG or IND TOTAL EIA RR, NAT Rx75993 (6)852 (765) EIA RR, NAT NR5916,17916,238 TOTAL81816,27217,090 PPV = 759/852 = 89.1% (759/765 = 99.2%) Sens = 759/818 = 92.8% PPV and Sensitivity of HIV-1/HIV-2 EIA and NAT vs Western Blot

44. E0304206A 44 Western Blot POS NEG or IND TOTAL EIA RR, S/CO ≥ 15.0714244958 EIA RR, S/CO < 15.010416,02816,132 TOTAL81816,27217,090 PPV = 714/958 = 74.5% Sens = 714/818 = 87.3% PPV and Sensitivity of High S/CO (≥ 15.0) vs Western Blot

45. E0304206A 45 Western Blot POS NEG or IND TOTAL EIA RR, S/CO ≥ 15.06943697 EIA RR, S/CO < 15.06590155 TOTAL75993 (6)852 (765) PPV = 694/697 = 99.6% Sens = 694/759 = 91.4% PPV and Sensitivity of High S/CO (≥ 15.0) vs WB Where NAT is Reactive

46. E0304206A 46 Western Blot POS NEG or IND TOTAL EIA RR, S/CO ≥ 15.020241261 EIA RR, S/CO < 15.03915,93815,977 TOTAL5916,17916,238 PPV = 20/261 = 7.7% Sens = 20/59 = 33.9% PPV and Sensitivity of High S/CO (≥ 15.0) vs WB Where NAT is Nonreactive

47. E0304206A 47 HIV Alternate Algorithm Summary uUse of NAT as the first step of the supplemental test algorithm will reduce the amount of HIV WB performed by only 5% uHIV NAT-reactive samples (even using pooled NAT for screening): –Sensitivity of 92.8% (detection of WB confirmed positives) Those not detected will be tested by WB; only 0.4% of positives remain and majority are false positive on WB (as indicated by repeat NAT nonRx, low EIA S/CO and weak WB banding patterns)Those not detected will be tested by WB; only 0.4% of positives remain and majority are false positive on WB (as indicated by repeat NAT nonRx, low EIA S/CO and weak WB banding patterns)

48. E0304206A 48 HIV Alternate Algorithm Summary uUse of S/CO > 15 following separation of RR population into NAT Rx and nonRx is not practical –Vast majority of HIV RR samples are not from individuals with HIV infection and consequently are NAT nonRx and have low S/CO values –Of those with an S/CO > 15, the PPV is 7.7% (20/261) –Of those with an S/CO < 15, it is likely that all WB pos are false pos uOther options –Dual EIA Algorithm

49. E0304206A 49 Dual EIA Algorithm uFeasibility based on the concept: –If two assays with comparable sensitivity are composed of differing rare reagents and have a different format, the false positive populations should have limited cross over; the more unique the tests, the greater the separation of false positive populations uUsed successfully for HTLV and HBsAg to eliminate >60% of repeat reactives requiring further testing by expensive, complicated, error prone and unavailable/unlicensed tests (e.g., HTLV)

50. E0304206A 50 ARC HTLV Supplemental Results * * State of California Testing Initiated Month (2000-2002) Number

51. E0304206A 51 HIV Dual EIA Algorithm Qualification uQualified in both directions based on the two FDA licensed HIV-1/HIV-2 EIAs: –Abbott (EIA-1)  Genetic Systems (EIA-2) (ARC) –Genetic Systems (EIA-1)  Abbott (EIA-2) (BSL) uAll HIV EIA repeat reactive samples from 1/1/00-3/31/02 having adequate volume for additional testing were evaluated uWestern Blot and NAT (TMA pools of 16) test-of-record data were used for analysis uAll 2 nd EIA testing was performed centrally at BSL

52. E0304206A 52 HIV Dual EIA Algorithm Genetic Systems pEIA (EIA-1) at BSL N = 1,657 RR Samples (1/1/00 – 3/31/02) Ind 544 (32.8%) Pos 80 (4.8%) RR80 Neg 1,033 (62.4%) NR0RR31NR513RR49NR984 Tested by Western Blot (Bio-Rad) AbbottEIA-2AbbottEIA-2AbbottEIA-2 +79–1+0–31+0–513+0–49+0–984 NATNATNAT (all WB bands; high S/CO both EIAs)

53. E0304206A 53 HIV Dual EIA Algorithm Abbott rDNA EIA (EIA-1) at ARC N = 6,227 RR Samples (1/1/00 – 3/31/02) Ind 2,890 (46.4%) Pos 266 (4.3%) RR250 Neg 3,071 (49.3%) NR16RR16NR2,874RR13NR3,058 Tested by Western Blot (Calypte) GSCEIA-2GSCEIA-2GSCEIA-2 +237–13+1–15+0–2,874+0–13+0–3,058 NATNATNAT +0–16 (+/- 24, 160)

54. E0304206A 54 N = 13, Abbott RR, WB Pos, GSC RR, NAT NR WB Banding PatternAbbott S/COGSC S/CO 124, 31, 41, 51, 66, 120, 16017.435.80 217, 24, 120, 16015.522.91 3all bands15.939.30 4all bands17.198.62 5all bands19.308.87 617, 24, 61, 1602.407.06 724, 31, 41, 51, 66, 120, 16020.954.39 824, 41, 120, 1601.401.33 9all bands19.308.07 1041, 66, 120, 1601.049.38 11all bands17.608.59 12all bands17.848.93 13all bands18.8011.12

55. E0304206A 55 N = 16, Abbott RR, WB Pos, GSC NR, NAT NR WB Banding PatternAbbott S/COGSC S/CO 117, 41, 1601.530.17 217, 41, 1601.500.20 341, 1601.340.30 441, 120, 1604.900.25 541, 1604.720.31 624, 1201.520.29 741, 120, 1604.920.25 824, 41, 1601.000.38 941, 120, 1601.190.29 1024, 1601.430.22 1124, 1601.600.32 1217, 24, 41, 120, 1602.360.33 1317, 41, 120, 1601.110.17 1424, 55, 120, 16010.56 0.57* 1517, 24, 1608.73 0.62* 1641, 1601.370.28 * Both samples PCR and repeat TMA (undilute) NR; one of two reported participation in an HIV vaccine trial

56. E0304206A 56 GSC – BSL N = 1,657 No. Abbott RR No. NAT Rx No. WB:Pos = 80 (5%)80 (100%)79 (Bio-Rad)Ind = 544 (33%)31 (6%)0 Neg = 1,033 (62%)49 (5%)0 HIV Dual EIA Summary Abbott – ARC N = 6,227 No. GSC RR No. NAT Rx No. WB:Pos = 266 (4%)250 (94%)237 (Calypte)Ind = 2,890 (46%)16 (0.6%)1 Neg = 3,071 (49%)13 (0.4%)0

57. E0304206A 57 HIV Dual EIA Algorithm Findings Using NAT as the “gold standard” uSensitivity = 100% (317/317) –95% CI 98.84-100% uSpecificity = 98.4% (7,447/7,567) –95% CI 98.11-98.68% uNo. WBs eliminated = 98.4% (7,764/7,884) uNo. indeterminate interpretations eliminated = 98.6% (3,387/3,434)