1. E001372A 1 ARC Experience with Indeterminate Blood Donors Susan L. Stramer, Ph.D. National Confirmatory Testing Laboratory American Red Cross

2. E001372A 2 Outline uDefinition of HIV-1 Western Blot Indeterminate and “Non-Viral” Bands uRegulatory Consequences of Ignoring “Non-Viral” Bands uCurrent Frequency of Indeterminates and “Non-Viral” Bands (1998-1999 ARC Data) uIntegration of NAT Results with Indeterminate Interpretations

3. E001372A 3 Cambridge Biotech Human Immunodeficiency Virus Type 1 (HIV -1) Western Blot Kit 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).

4. E001372A 4 APHL 14th Annual Conference on Human Retrovirus Testing – Conference Report, 1999 The interpretation of indeterminate should not include reporting non-viral bands (e.g., p70). The rationale for this is that since 1991 no individual exhibiting non-viral banding has been associated with either seroconversion, detection of different HIV-1 subtypes or other disease agents. Only viral bands that are specified in the package insert ranging from molecular weights p17 to gp160 should be reported.

5. E001372A 5 Regulatory Consequences of Ignoring “Non-Viral” Bands uBetween March 1989 and September 1993, a total of 621 samples were reported as negative by ARC when there was evidence of a band p70 –460 repeat reactive index donations (HIV-1 EIA) –161 reentered donors uResulted in multimillion dollar recall of “safe” products uNone of reentered donors (with hundreds of subsequent donations over the four year period) demonstrated any evidence of HIV seroconversion

6. E001372A 6 HIV-1 Blood Donor Screening/Supplemental Test Results (ARC) 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)

7. E001372A 7 Interp N%Avg/Month Neg2,60048.1216.7 Ind2,40144.5200.1 Pos4007.433.3 Total5,401450.1 Month (1999) Number HIV-1/2 Testing by Month – 1999

8. E001372A 8 HIV-1 Blood Donor Screening/Supplemental Test Results (ARC) (Cont.) 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

9. E001372A 9 HIV-1 Blood Donor Screening/Supplemental/NAT Test Results (ARC) PositiveNegativeIndeterminate 9/8/99 – 12/31/99 1,836 Repeat Reactive (0.09%) HIV-1 WB (Cambridge Biotech) 2.0 Million Donations Screened 71 (3.9%)824 (44.9%)941 (51.2%) 67 (94.4%) 1 (0.1%) 0 No. HIV NAT Rx:  4 HIV NAT Neg (“ENV” Only WB)  p24 Only WB S/CO EIA = 11.62 S/CO NAT = 18.23

10. E001372A 10 HIV-1 Blood Donor Screening/Supplemental/NAT Test Results (ARC) (Cont.) 824 Indeterminate (44.9%) Multiple Viral Bands (p24, gp41, gp120/160, but +/- intensity) 11 (1.4%) OneViralBand 460 (55.8%) MultipleViralBands 91 (11.0%) Background Only (obscuresreading) 154 (18.7%) Non- Viral Bands 108 (13.1%)  341 (74.1%) p24 “GAG” only  72 (79%) “GAG” reactivity with or without other banding  78 (72%) “p70” 0 No. HIV NAT Rx: 1000 9/8/99 – 12/31/99

11. HIV-1 RNA Concentrations During SC Based on NGI’s PCR of 28 Plasma Donor Panels (N=221) 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) Categories 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 11

12. E001372A 12 HIV-1 Viral Load in Indeterminate Samples From Seroconverting Donors 41 samples, all NAT reactive, no non-viral bands reported p24 Ag Reactivity 29 (+) 19 (–) 4,750 3,500-75,000 (4 samples = 200-400 copies/mL) 90% (37/41) detected with pool testing at a cutoff of 1,600-2,400 copies/mL Median copies/mL:800,000 Range:5,500-1,000,000 4,750 3,500-75,000 (4 samples = 200-400 copies/mL)

13. E001372A 13 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

14. gp160 p66 gp120 p55/51 p31 p24 p17 gp41SC-1PC NC Weak PC Day 12 22 E001372A 14

15. gp160 gp120 p66 p17 p55/51 gp41 p31 p24 PC PC Weak NC Day 0 2043SC-2SC-3SC-4 Day 13 75 E001372A 15

16. E001372A 16 Cambridge Biotech HIV-1 Western Blot gp160 gp120 p66 p55/51 gp41 p31 p24 p17

17. E001372A 17 Cambridge Biotech HIV-1 Western Blot gp160 gp120 p66 p55/51 gp41 p31 p24 p17

18. E001372A 18 Cambridge Biotech HIV-1 Western Blot gp160 gp120 p66 p55/51 gp41 p31 p24 p17

19. E001372A 19 Cambridge Biotech HIV-1 Western Blot gp160 gp120 p66 p55/51 gp41 p31 p24 p17

20. E001372A 20 Cambridge Biotech HIV-1 Western Blot gp160 gp120 p66 p55/51 gp41 p31 p24 p17

21. E001372A 21 Cambridge Biotech HIV-1 Western Blot gp160 gp120 p66 p55/51 gp41 p31 p24 p17

22. E001372A 22 Cambridge Biotech HIV-1 Western Blot gp160 gp120 p66 p55/51 gp41 p31 p24 p17

23. E001372A 23 Summary and Conclusions uDue to manufacturer’s requirements, samples with non-viral bands must be reported as “indeterminate” uNon-viral bands are: –“Just that” –Do not indicate presence of any virus, past, present or future uDonors with non-viral bands represent 14% of total indeterminates and are otherwise “safe” donors (RNA negative)  HIV-1 viral loads in seroconverting individuals with viral indeterminate patterns exceed the cutoff of pooled NAT testing (  90% of cases); none with non-viral patterns uPatterns of non-viral bands do not resemble any pattern seen in early HIV-1 seroconversion