Challenges for Blood Donor Confirmatory Testing Algorithms Susan L. Stramer, Ph.D. American Red Cross, for HIV Diagnostics: New Developments and Challenges Meeting, Orlando, FL; Feb 28-March 1, 2005

Background HIV-1 algorithms utilize immunoassays for confirmation of repeat reactive blood donors Most commonly used: Western Blot (WB) Contains electrophoresed whole HIV lysate HIV-1 Immunofluorescence (also uses viral lysate) Not widely used in blood centers due to difficulty in interpretation HIV-2 confirmation also required for HIV-1 WB ind/neg samples Licensed HIV-2 EIA => Research based HIV-2 WB (CA) 6 or fewer HIV-2 confirmed pos blood donors in the US reported since 1992 No confirmation for HIV-1 type O

Background Issues with HIV confirmatory assays Poor performance Unreadable, uninterpretable or invalid results generated Misclassification of donors False positive and false negative results High rates of indeterminate results (neither pos or neg) in healthy individuals Donor deferral, anxiety, and use of complicated donor reentry algorithms, when available Mixed message, “You are healthy and not HIV infected, but you cannot donate blood”

Background Issues with HIV confirmatory assays, cont’d High costs Inconsistent availability No new products for “antibody confirmation” on the market for HIV-1 or HIV-2 Screening tests ongoing improvements since 1985 Costs associated with new products extremely high Can alternate algorithms be validated? HIV RNA (NAT), use of high/low screening S/CO ratios, dual EIA algorithm

Can Alternate Algorithms be Implemented Kits will require labeling for intended use 21 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, more specific) test has been approved for such use by FDA….” FDA’s current thinking is that an antibody RR must be confirmed by an antibody-specific assay Variances have been granted to exempt RNA pos samples from requiring WB Minipool or single donor RNA neg samples still require WB

Reactivity with 50 HIV-1 Commercial Seroconversion Panels Genetic SystemsTM HIV-1/HIV-2 plus O EIA May 2004, Package Insert (FDA License 1109) HIV-1/HIV-2 plus O Equivalent > Sensitive < Sensitive vs Licensed Kit #1 (EIA) 12/46* (26%) 34/46* (74%) (0%) vs Licensed Kit #2 (EIA) 35/50 (70%) 9/50 (18%) 6/50 (12%) vs Licensed Western Blot 13/50 37/50 * 4 panels not tested (no longer available)

Cambridge Biotech Human Immunodeficiency Virus Type 1 (HIV -1) Western Blot Kit March 16, 2000 BPAC 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).

HIV-1 Blood Donor Screening/Supplemental Test Results (ARC); March 16, 2000 BPAC 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%)

HIV-1 Blood Donor Screening/Supplemental Test Results (ARC) (Cont HIV-1 Blood Donor Screening/Supplemental Test Results (ARC) (Cont.); March 16, 2000 BPAC 1998-1999 5,161 Indeterminate (46.6%) Multiple Viral Bands (p24, gp41, gp120/160, but +/- intensity) 46 (1%) One Viral Band Multiple Viral Bands Background Only (obscures reading) Non- Viral Bands 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

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

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

HIV-1/HIV-2 EIA Repeat Reactives (ARC testing of 25 HIV-1/HIV-2 EIA Repeat Reactives (ARC testing of 25.6 million donations from 9/8/99 to 6/30/03) Perform WB N = 17,090 Pos Ind Neg HIV NAT HIV NAT HIV NAT Reactive Individually 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 HIV Not Infected HIV Not Infected 4.4% 51% 44.2% 0.4% 757 HIV Infected? Early seroconversion 8,704 HIV Infected? Early seroconversion False Rx NAT 7,562 61 <0.1% 0% 6

HIV-1/HIV-2 EIA Repeat Reactives (ARC testing of 25 HIV-1/HIV-2 EIA Repeat Reactives (ARC testing of 25.6 million donations from 9/8/99 to 6/30/03) Review HIV-1 NAT Results N = 17,090 Pool or Individual Unit NAT NonRx (16 dtns ® HIV-1/HCV TMA) Individual Unit NAT Rx (dHIV TMA) 96% 4% 16,327 763 Perform WB No Further Testing HIV Infected Pos Ind Neg 99.2% PPV 0.4% 53.3% 46.3% 757 WB Pos 61 8,704 7,562 92.5% Sensitivity

Correlation of NAT with Supplemental HIV Serological Data (17,791 RR donations of which 17,090 (96.1%) had EIA and WB data) 9/8/99 to 6/30/03 Western Blot Result NAT Result Pos Ind Neg Total dHIV Rx NonRx Total 757 (99.2%) 61 (0.4%) 818 (4.8%) 6 8,704 8,710 (0.8%) 7,562 763 16,327 17,090 Of HIV WB neg or ind, 6/16,272 (1:2712 or 0.037%) infected with HIV

Characteristics of HIV-1 WB Pos/TMA Nonreactive Samples 9/8/99 – 8/31/00 Sample Pool Neat HIV-1/HIV-2 S/CO WB HIV PCR 1 NR 1.43 41, 120, 160 Neg 2 NR 1.03 41, 160 Neg 3 NR 1.62 41, 160 Neg 4 NR 1.11 41, 55, 160 Neg 5 NR 2.50 24, 41, 51, 61, 160 Neg 6 NR 20.18 all bands Neg 7 NR 1.22 24, 41, 160 Neg 8 NR 1.11 17, 41, 120, 160 Neg 9 NR 1.84 41, 160 Neg 10 NR 1.40 24, 41, 160 Neg 11 NR 1.70 17, 24, 41, 51, 160 Neg 12 NR 20.00 all bands Pos (200 copies/mL) 13 NR 17.84 all bands Neg All samples p24 Ag negative; lack of HIV infection in those weakly EIA Rx with follow up

Characteristics of HIV-1 WB Pos/TMA Nonreactive Samples 9/1/00 – 12/29/01 Sample Pool Neat HIV-1/HIV-2 S/CO WB HIV PCR 14 NR 8.73 17, 24, 160 Neg 15 NR 10.56 24, 55, 120, 160 Neg 16 NR 19.30 all bands Neg 17 NR 19.47 all bands Pos (200 copies/mL) 18 NR 1.04 41, 66, 120, 160 Neg 19 NR 4.72 41, 160 Neg 20 NR 1.50 24, 41, 160 Neg 21 NR 1.53 24, 41, 160 Neg 22 NR 1.60 24, 160 Neg 23 NR 17.60 all bands Neg 24 NR 19.30 all bands Neg All samples p24 Ag negative; lack of HIV infection in those weakly EIA Rx with follow up

Relationship between HIV-1/HIV-2 EIA and Western Blot NAT Reactive Samples 9/8/99 to 6/30/03 N = 763 (4% of total) Neg N = 0 Ind N = 6 0.8% Pos N = 757 99.2% 0.00 5.00 10.00 15.00 20.00 25.00 19.298 91.5% of WB pos/ NAT rxs had an S/CO ³ 15 18.182 17.837 16.373 EIA S/CO 11.208 5.683 3.632 – 18.966 8.349 – 20.952 95% Range Western Blot Results

Relationship between HIV-1/HIV-2 EIA and Western Blot NAT Nonreactive Samples 9/8/99 to 6/30/03 N = 16,327 (96% of total) Neg N = 7,562 46.3% Ind N = 8,704 53.3% Pos N = 61 0.4% 0.00 5.00 10.00 15.00 20.00 25.00 98.5% of WB neg/ind NAT nonrxs had an S/CO < 15 16.282 EIA S/CO 2.600 2.667 2.496 1.602 1.598 1.401 1.225 1.230 1.018 – 11.370 1.018 – 12.262 1.015 – 20.083 95% Range Western Blot Results

Relationship between HIV-1/HIV-2 EIA and Western Blot NAT Nonreactive Samples 9/8/99 to 6/30/03 N = 16,327 (96% of total) Neg N = 7,562 46.3% Ind N = 8,704 53.3% Pos, no p31 N = 39 0.2% Pos, with p31 N = 22 0.1% 0.00 5.00 10.00 15.00 20.00 25.00 18.867 98.5% of WB neg/ind NAT nonrxs had an S/CO < 15 16.675 16.137 EIA S/CO 2.600 2.667 2.390 1.602 1.598 1.523 1.225 1.230 1.228 1.018 – 11.370 1.018 – 12.262 1.000 – 15.520 6.023 – 20.183 95% Range Western Blot Results

Dual EIA Algorithm Feasibility 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 Used successfully for HTLV to eliminate >60% of repeat reactives requiring further testing by expensive, complicated, error prone and unavailable/unlicensed tests

HIV Dual EIA Algorithm Qualification Qualified 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) All HIV EIA repeat reactive samples from 1/1/00-3/31/02 having adequate volume for additional testing were evaluated (N=7884); all allogeneic donors Western Blot and NAT (TMA pools of 16) test-of-record data were used for analysis All 2nd EIA testing was performed centrally at Blood Systems Laboratories (BSL)

Application of HIV NAT/Dual EIA Algorithm in Routine Operations HIV Repeat Reactives (BSL + ARC; 1/1/00 – 3/31/02) 1,657 + 6,227 (7,884 RRs) Pool or Individual Unit NAT NonRx (16 dtns ® HIV-1/HCV TMA) 1,578 + 5,989 (7,567) Individual Unit NAT Rx (dHIV TMA) 79 + 238** (317) Perform 2nd EIA No Further Testing HIV Infected (4%) EIA NonRx 1,497 + 5,948* (7,445) EIA RR 81 + 41 (122) * Contains 16 false pos WB results ** Of 317: 316 WB pos, EIA concordant Rx; 1 WB ind, EIA concordant Rx No Further Testing HIV Not Infected (94.5%) Perform WB (1.5%) Pos 1 +13 (14) Ind 31 + 15 (46) Neg 49 + 13 (62)

N = 13, Abbott RR, WB Pos, GSC RR, NAT NR WB Banding Pattern Abbott S/CO GSC S/CO 1 24, 31, 41, 51, 66, 120, 160 17.43 5.80 2 17, 24, 120, 160 15.52 2.91 3 all bands 15.93 9.30 4 17.19 8.62 5 19.30 8.87 6 17, 24, 61, 160 2.40 7.06 7 20.95 4.39 8 24, 41, 120, 160 1.40 1.33 9 8.07 10 41, 66, 120, 160 1.04 9.38 11 17.60 8.59 12 17.84 8.93 13 18.80 11.12

N = 16, Abbott RR, WB Pos, GSC NR, NAT NR WB Banding Pattern Abbott S/CO GSC S/CO 1 17, 41, 160 1.53 0.17 2 1.50 0.20 3 41, 160 1.34 0.30 4 41, 120, 160 4.90 0.25 5 4.72 0.31 6 24, 120 1.52 0.29 7 4.92 8 24, 41, 160 1.00 0.38 9 1.19 10 24, 160 1.43 0.22 11 1.60 0.32 12 17, 24, 41, 120, 160 2.36 0.33 13 17, 41, 120, 160 1.11 14 24, 55, 120, 160 10.56 0.57* 15 17, 24, 160 8.73 0.62* 16 1.37 0.28 *Both samples PCR and repeat TMA (undilute) NR; one of two reported participation in an HIV vaccine trial; Repeat undilute PCR and follow up on others demonstrate no HIV infection

HIV Dual EIA Algorithm Findings/Conclusions Feasibility of NAT combined with the dual EIA algorithm for HIV confirmation was demonstrated Sensitivity = 100% (317/317); 95% CI 98.84-100% Specificity = 98.4% (7,445/7,567); 95% CI 98.11-98.68% No. WBs eliminated = 98.5% (7,762/7,884) No. indeterminate interpretations eliminated = 98.6% (3,388/3,434) Majority of WB false pos, selected by the use of one particular EIA, were eliminated Changes in EIAs to more sensitive/specific versions should not require extensive validations WB shown to be of little if any value!!!

Application of HIV NAT/Dual EIA Algorithm in Routine Operations AABB Proposal HIV Repeat Reactives Pool or Individual Unit NAT NonRx (16 dtns ® HIV-1/HCV TMA) Individual Unit NAT Rx (dHIV TMA) Perform 2nd EIA and Individual Unit NAT No Further Testing HIV Infected EIA NonRx and NAT Nonrx EIA RR Since most testing performed in pools, additional individual unit NAT significant (at max 7,445/7,567) No Further Testing HIV Not Infected Perform WB Pos Ind Neg