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E001372A 1E001372A 1
ARC Experience with ARC Experience with Indeterminate Blood DonorsIndeterminate Blood Donors
Susan L. Stramer, Ph.D.Susan L. Stramer, Ph.D.National Confirmatory Testing LaboratoryNational Confirmatory Testing Laboratory
American Red CrossAmerican Red Cross
E001372A 2
OutlineOutline
Definition of HIV-1 Western Blot Definition of HIV-1 Western Blot Indeterminate and “Non-Viral” BandsIndeterminate and “Non-Viral” Bands
Regulatory Consequences of Ignoring Regulatory Consequences of Ignoring “Non-Viral” Bands“Non-Viral” Bands
Current Frequency of Indeterminates and Current Frequency of Indeterminates and “Non-Viral” Bands (1998-1999 ARC Data)“Non-Viral” Bands (1998-1999 ARC Data)
Integration of NAT Results with Indeterminate Integration of NAT Results with Indeterminate InterpretationsInterpretations
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Cambridge BiotechCambridge BiotechHuman Immunodeficiency Virus Type 1 (HIV -1)Human Immunodeficiency Virus Type 1 (HIV -1)
Western Blot KitWestern Blot Kit
Any bands present but pattern does not meet criteria Any bands present but pattern does not meet criteria for POSITIVE = INDETERMINATEfor POSITIVE = INDETERMINATE
Non-viral bands have been observed with certain Non-viral bands have been observed with certain specimens. These bands are not usually accompanied specimens. These bands are not usually accompanied by any of the major viral bands of diagnostic by any of the major viral bands of diagnostic significance (p24, gp41/120/160). The non-viral bands significance (p24, gp41/120/160). The non-viral bands appear to be cell related with the most common in the appear to be cell related with the most common in the molecular weight range of 70K, 51-55K (possible molecular weight range of 70K, 51-55K (possible HLA-DR) and 43K (possible HLA-ABC).HLA-DR) and 43K (possible HLA-ABC).
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APHL 14th Annual Conference on Human APHL 14th Annual Conference on Human Retrovirus Testing – Conference Report, 1999Retrovirus Testing – Conference Report, 1999
The interpretation of indeterminate should The interpretation of indeterminate should notnot include include
reporting non-viral bands (e.g., p70). The rationale for this reporting non-viral bands (e.g., p70). The rationale for this
is that since 1991 no individual exhibiting non-viral is that since 1991 no individual exhibiting non-viral
banding has been associated with either seroconversion, banding has been associated with either seroconversion,
detection of different HIV-1 subtypes or other disease detection of different HIV-1 subtypes or other disease
agents. agents. Only viral bands that are specified in the Only viral bands that are specified in the
package insert ranging from molecular weights package insert ranging from molecular weights
p17 to gp160 should be reported.p17 to gp160 should be reported.
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Regulatory Consequences of Regulatory Consequences of Ignoring “Non-Viral” BandsIgnoring “Non-Viral” Bands
Between March 1989 and September 1993, a total of Between March 1989 and September 1993, a total of 621 samples were reported as negative by ARC when 621 samples were reported as negative by ARC when there was evidence of a band p70there was evidence of a band p70– 460 repeat reactive index donations (HIV-1 EIA)460 repeat reactive index donations (HIV-1 EIA)– 161 reentered donors161 reentered donors
Resulted in multimillion dollar recall of Resulted in multimillion dollar recall of “safe” products“safe” products
None of reentered donors (with hundreds of None of reentered donors (with hundreds of subsequent donations over the four year period) subsequent donations over the four year period) demonstrated any evidence of HIV seroconversiondemonstrated any evidence of HIV seroconversion
E001372A 6
HIV-1 Blood Donor Screening/SupplementalHIV-1 Blood Donor Screening/SupplementalTest Results (ARC)Test Results (ARC)
12.4 Million Donations Screened12.4 Million Donations Screened
PositivePositive791 (7.1%)791 (7.1%)
NegativeNegative5,128 (46.3%)5,128 (46.3%)
IndeterminateIndeterminate5,161 (46.6%)5,161 (46.6%)
1998-19991998-1999
11,080 Repeat Reactive (0.09%)11,080 Repeat Reactive (0.09%)HIV-1 WB (Cambridge Biotech)HIV-1 WB (Cambridge Biotech)
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0
100
200
300
400
500
600
700
800
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
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
Month (1999)
Num
ber
HIV-1/2 Testing by Month – 1999HIV-1/2 Testing by Month – 1999
E001372A 8
HIV-1 Blood Donor Screening/SupplementalHIV-1 Blood Donor Screening/SupplementalTest Results (ARC) Test Results (ARC) (Cont.)(Cont.)
5,161 Indeterminate (46.6%)5,161 Indeterminate (46.6%)
MultipleMultipleViralViralBandsBands
(p24, gp41,(p24, gp41,gp120/160,gp120/160,
but +/- intensity)but +/- intensity)
46 (1%)
OneOneViralViralBandBand
2,752 (53.3%)
MultipleMultipleViralViralBandsBands
589 (11.4%)
BackgroundBackgroundOnlyOnly
(obscures(obscuresreading)reading)
1,050 (20.3%)
Non-Non-ViralViralBandsBands
724 (14%)
16 (35%)16 (35%)“ENV” only“ENV” only
1,925 (70%)1,925 (70%)p24 “GAG” onlyp24 “GAG” only
464 (79%) 464 (79%) “GAG” reactivity “GAG” reactivity with or without with or without other bandingother banding
569 (79%)569 (79%)“p70”“p70”
65.7% Viral Banding65.7% Viral Banding
1998-19991998-1999
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HIV-1 Blood Donor Screening/Supplemental/NATHIV-1 Blood Donor Screening/Supplemental/NATTest Results (ARC)Test Results (ARC)
PositivePositive NegativeNegativeIndeterminateIndeterminate
9/8/99 – 12/31/999/8/99 – 12/31/99
1,836 Repeat Reactive (0.09%)1,836 Repeat Reactive (0.09%)HIV-1 WB (Cambridge Biotech)HIV-1 WB (Cambridge Biotech)
2.0 Million Donations Screened2.0 Million Donations Screened
71 (3.9%) 824 (44.9%) 941 (51.2%)
67 (94.4%)67 (94.4%) 1 (0.1%)1 (0.1%) 00No. HIVNo. HIVNAT Rx:NAT Rx:
4 HIV NAT 4 HIV NAT Neg (“ENV” Neg (“ENV” Only WB)Only WB)
p24 Only WB p24 Only WB S/CO EIA = 11.62S/CO EIA = 11.62S/CO NAT = 18.23S/CO NAT = 18.23
E001372A 10
HIV-1 Blood Donor Screening/Supplemental/NATHIV-1 Blood Donor Screening/Supplemental/NATTest Results (ARC) Test Results (ARC) (Cont.)(Cont.)
824 Indeterminate (44.9%)824 Indeterminate (44.9%)
MultipleMultipleViralViralBandsBands
(p24, gp41,(p24, gp41,gp120/160,gp120/160,
but +/- intensity)but +/- intensity)
11 (1.4%)
OneOneViralViralBandBand
460 (55.8%)
MultipleMultipleViralViralBandsBands
91 (11.0%)
BackgroundBackgroundOnlyOnly
(obscures(obscuresreading)reading)
154 (18.7%)
Non-Non-ViralViralBandsBands
108 (13.1%)
341 (74.1%)341 (74.1%)p24 “GAG” onlyp24 “GAG” only
72 (79%) 72 (79%) “GAG” reactivity “GAG” reactivity with or without with or without other bandingother banding
78 (72%)78 (72%)“p70”“p70”
00No. HIVNo. HIVNAT Rx:NAT Rx: 11 00 00 00
9/8/99 – 12/31/999/8/99 – 12/31/99
HIV-1 RNA Concentrations During SC Based on HIV-1 RNA Concentrations During SC Based on NGI’s PCR of 28 Plasma Donor Panels (N=221) 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
6Median Days
6
100
1,000
10,000
100,000
1,000,000
PCR+/Ag+/–/Ab+
(n=20)
0 7 3 4
HIV
-1 R
NA
Cop
ies/
mL
E001372A 11
E001372A 12
HIV-1 Viral Load in Indeterminate SamplesHIV-1 Viral Load in Indeterminate SamplesFrom Seroconverting DonorsFrom Seroconverting Donors
41 samples, all NAT reactive,41 samples, all NAT reactive,no non-viral bands reportedno non-viral bands reported
p24 Ag Reactivityp24 Ag Reactivity
29 (+)29 (+) 19 (–)19 (–)
4,7504,7503,500-75,0003,500-75,000
(4 samples = 200-400 copies/mL)(4 samples = 200-400 copies/mL)
90% (37/41) detected with pool testing 90% (37/41) detected with pool testing at a cutoff of 1,600-2,400 copies/mLat a cutoff of 1,600-2,400 copies/mL
Median copies/mL:Median copies/mL: 800,000800,000Range:Range: 5,500-1,000,0005,500-1,000,000
4,7504,7503,500-75,0003,500-75,000
(4 samples = 200-400 copies/mL)(4 samples = 200-400 copies/mL)
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Comparison of HIV-1 p24 Ag PositiveComparison of HIV-1 p24 Ag PositiveWindow-Case U.S. Blood DonorsWindow-Case U.S. Blood Donors
11 IndexIndex 6 x 106 x 1055** 14.0*14.0* 9595 0.10.11212 3 x 103 x 1044 1.21.2 9595 2.32.3 POS – minus p31POS – minus p312222 1 x 101 x 1055 0.30.3 5.65.6 POS – all bandsPOS – all bands
22 IndexIndex 2 x 102 x 1066** 28.5*28.5* 9494 0.80.82020 1 x 101 x 1055 0.70.7 1.61.6 POS – minus p31POS – minus p314343 1 x 101 x 1044 0.10.1 5.35.3 POS – all bandsPOS – all bands
33 IndexIndex 1 x 101 x 1055 8.18.1 9595 0.10.11313 5 x 105 x 1055** 26.5*26.5* 9090 8.88.8 POS – minus p31 POS – minus p31
44 IndexIndex 1 x 101 x 1055 2.42.4 9595 0.00.01313 1 x 101 x 1066** 17.5*17.5* 8989 2.72.7 IND – p66IND – p667575 5 x 105 x 1044 0.10.1 17.417.4 POS – all bandsPOS – all bands
55 IndexIndex 3 x 103 x 1055 18.6*18.6* 9999 0.30.31010 4 x 104 x 1055** 0.20.2 4.34.3 POS – minus p31POS – minus p311717 1 x 101 x 1055 0.90.9 12.512.5 POS – minus p31POS – minus p312424 3 x 103 x 1044 0.70.7 8.58.5 POS – minus p31POS – minus p313131 2 x 102 x 1044 0.10.1 13.113.1 POS – minus p31POS – minus p31
SCSC
Sample Sample CollectionCollection
(days)(days) WBWBRNA RNA
Copies/mLCopies/mLp24 Ag p24 Ag S/COS/CO
PercentPercentNeut.Neut.
HIV-1/HIV-1/HIV-2 Ab HIV-2 Ab
S/COS/CO
gp160gp160
p66p66
gp120gp120
p55/51p55/51
p31p31
p24p24
p17p17
gp41gp41
SC-1SC-1
PC
PC
NC
NC
We
ak
PC
We
ak
PC
Da
y 1
2D
ay
12
2222
E001372A 14
gp160gp160gp120gp120
p66p66
p17p17
p55/51p55/51
gp41gp41
p31p31
p24p24P
CP
C
PC
We
ak
PC
We
ak
NC
NC
Da
y 0
Da
y 0
2020
4343
SC-2SC-2 SC-3SC-3 SC-4SC-4
Da
y 1
3D
ay
13
Da
y 1
3D
ay
13 7575
E001372A 15
E001372A 16
Cambridge Biotech HIV-1 Western BlotCambridge Biotech HIV-1 Western Blot
gp160gp160gp120gp120
p66p66
p55/51p55/51
gp41gp41
p31p31
p24p24
p17p17
E001372A 17
Cambridge Biotech HIV-1 Western BlotCambridge Biotech HIV-1 Western Blot
gp160gp160gp120gp120
p66p66
p55/51p55/51gp41gp41
p31p31
p24p24
p17p17
E001372A 18
Cambridge Biotech HIV-1 Western BlotCambridge Biotech HIV-1 Western Blot
gp160gp160gp120gp120
p66p66p55/51p55/51
gp41gp41
p31p31
p24p24
p17p17
E001372A 19
Cambridge Biotech HIV-1 Western BlotCambridge Biotech HIV-1 Western Blot
gp160gp160gp120gp120
p66p66p55/51p55/51
gp41gp41
p31p31
p24p24
p17p17
E001372A 20
Cambridge Biotech HIV-1 Western BlotCambridge Biotech HIV-1 Western Blot
gp160gp160gp120gp120
p66p66p55/51p55/51
gp41gp41
p31p31
p24p24
p17p17
E001372A 21
Cambridge Biotech HIV-1 Western BlotCambridge Biotech HIV-1 Western Blot
gp160gp160gp120gp120
p66p66p55/51p55/51
gp41gp41
p31p31
p24p24
p17p17
E001372A 22
Cambridge Biotech HIV-1 Western BlotCambridge Biotech HIV-1 Western Blot
gp160gp160gp120gp120
p66p66p55/51p55/51
gp41gp41
p31p31
p24p24
p17p17
E001372A 23
Summary and ConclusionsSummary and Conclusions
Due to manufacturer’s requirements, samples with non-viral bands Due to manufacturer’s requirements, samples with non-viral bands must be reported as “indeterminate”must be reported as “indeterminate”
Non-viral bands are:Non-viral bands are:– ““Just that”Just that”– Do not indicate presence of any virus, past, present or futureDo not indicate presence of any virus, past, present or future
Donors with non-viral bands represent 14% of total indeterminates and Donors with non-viral bands represent 14% of total indeterminates and are otherwise “safe” donors (RNA negative)are otherwise “safe” donors (RNA negative)
HIV-1 viral loads in seroconverting individuals with viral indeterminate HIV-1 viral loads in seroconverting individuals with viral indeterminate patterns exceed the cutoff of pooled NAT testing (patterns exceed the cutoff of pooled NAT testing (90% of cases); 90% of cases); none with non-viral patternsnone with non-viral patterns
Patterns of non-viral bands do Patterns of non-viral bands do notnot resemble any pattern seen in early resemble any pattern seen in early HIV-1 seroconversionHIV-1 seroconversion