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ARC Experience with Indeterminate Blood Donors

ARC Experience with Indeterminate Blood Donors. Susan L. Stramer, Ph.D. National Confirmatory Testing Laboratory American Red Cross. E001372A 1. Outline. Definition of HIV-1 Western Blot Indeterminate and “Non-Viral” Bands Regulatory Consequences of Ignoring “Non-Viral” Bands

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ARC Experience with Indeterminate Blood Donors

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

  2. Outline • Definition of HIV-1 Western Blot Indeterminate and “Non-Viral” Bands • Regulatory Consequences of Ignoring “Non-Viral” Bands • Current Frequency of Indeterminates and “Non-Viral” Bands (1998-1999 ARC Data) • Integration of NAT Results with Indeterminate Interpretations

  3. Cambridge BiotechHuman Immunodeficiency Virus Type 1 (HIV -1)Western Blot Kit • Any bands present but pattern does not meet criteria for POSITIVE = INDETERMINATE • Non-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. 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. Regulatory Consequences of Ignoring “Non-Viral” Bands • Between 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 • Resulted in multimillion dollar recall of “safe” products • None of reentered donors (with hundreds of subsequent donations over the four year period) demonstrated any evidence of HIV seroconversion

  6. HIV-1 Blood Donor Screening/SupplementalTest Results (ARC) 1998-1999 12.4 Million Donations Screened 11,080 Repeat Reactive (0.09%)HIV-1 WB (Cambridge Biotech) Positive 791 (7.1%) Indeterminate 5,161 (46.6%) Negative 5,128 (46.3%)

  7. HIV-1/2 Testing by Month – 1999 Interp N % Avg/Month Neg 2,600 48.1 216.7 Ind 2,401 44.5 200.1 Pos 400 7.4 33.3 Total 5,401 450.1 Number Month (1999)

  8. MultipleViralBands (p24, gp41,gp120/160, but +/- intensity) 46 (1%) HIV-1 Blood Donor Screening/SupplementalTest Results (ARC) (Cont.) 1998-1999 5,161 Indeterminate (46.6%) One Viral Band Multiple Viral Bands BackgroundOnly (obscures reading) Non- ViralBands 2,752 (53.3%) 589 (11.4%) 724 (14%) 1,050 (20.3%) • 1,925 (70%)p24 “GAG” only • 464 (79%) “GAG” reactivity with or without other banding • 569 (79%)“p70” • 16 (35%)“ENV” only 65.7% Viral Banding

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

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

  11. 1,000,000 100,000 10,000 1,000 100 HIV-1 RNA Concentrations During SC Based on NGI’s PCR of 28 Plasma Donor Panels (N=221) HIV-1 RNA Copies/mL 0 6 6 7 3 4 Median Days 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) PCR+/Ag+/–/Ab+(n=20) Categories E001372A 11

  12. HIV-1 Viral Load in Indeterminate SamplesFrom Seroconverting Donors 41 samples, all NAT reactive,no non-viral bands reported p24 Ag Reactivity 29 (+) 19 (–) 4,7503,500-75,000(4 samples = 200-400 copies/mL) Median copies/mL: 800,000Range: 5,500-1,000,000 4,7503,500-75,000(4 samples = 200-400 copies/mL) 90% (37/41) detected with pool testing at a cutoff of 1,600-2,400 copies/mL

  13. Comparison of HIV-1 p24 Ag PositiveWindow-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 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

  14. SC-1 gp160 gp120 p66 p55/51 gp41 p31 p24 p17 22 PC NC Day 12 Weak PC E001372A 14

  15. SC-2 SC-3 SC-4 gp160 gp120 p66 p55/51 gp41 p31 p24 p17 75 20 43 PC NC Day 0 Day 13 Day 13 PC Weak E001372A 15

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

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

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

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

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

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

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

  23. Summary and Conclusions • Due to manufacturer’s requirements, samples with non-viral bands must be reported as “indeterminate” • Non-viral bands are: • “Just that” • Do not indicate presence of any virus, past, present or future • Donors 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 (³90% of cases); none with non-viral patterns • Patterns of non-viral bands do not resemble any pattern seen in early HIV-1 seroconversion

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