S.L. Stramer, R.A. Porter, J.P. Brodsky, K.J. Waldman, R.Y. Dodd, T.A. Armstrong and G.D. Griffin
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Transcript of S.L. Stramer, R.A. Porter, J.P. Brodsky, K.J. Waldman, R.Y. Dodd, T.A. Armstrong and G.D. Griffin
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E992750C 1
Replacement of HIV-1 p24 Antigen Replacement of HIV-1 p24 Antigen Screening with HIV-1 RNA Nucleic Acid Screening with HIV-1 RNA Nucleic Acid
Testing (NAT) for Whole Blood DonationsTesting (NAT) for Whole Blood Donations
S.L. Stramer, R.A. Porter, J.P. Brodsky, S.L. Stramer, R.A. Porter, J.P. Brodsky, K.J. Waldman, R.Y. Dodd, T.A. ArmstrongK.J. Waldman, R.Y. Dodd, T.A. Armstrong
and G.D. Griffinand G.D. Griffin
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E992750C 2
BackgroundBackground
Since the implementation of p24 HIV-1 antigen Since the implementation of p24 HIV-1 antigen (Ag) screening in March, 1996, only 5 Ag positive (Ag) screening in March, 1996, only 5 Ag positive window-case donations have been identifiedwindow-case donations have been identified
Due to low Ag yield and the improved sensitivity Due to low Ag yield and the improved sensitivity of NAT screening for HIV-1, replacement of Ag of NAT screening for HIV-1, replacement of Ag with HIV-1 NAT should be possiblewith HIV-1 NAT should be possible
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E992750C 3
HIV-1 p24 Antigen Testing ExperienceHIV-1 p24 Antigen Testing Experience
During 2 years of testing 14 million donations (ARC), During 2 years of testing 14 million donations (ARC), 136 samples were HIV-1 p24 Ag confirmed positive136 samples were HIV-1 p24 Ag confirmed positive 58 were false positive (based on RNA negativity, 58 were false positive (based on RNA negativity,
lack of seroconversion, and nonreproducible Ag lack of seroconversion, and nonreproducible Ag neutralization) = 1:241,379neutralization) = 1:241,379
74 were HIV Ab positive74 were HIV Ab positive 4 were recently infected seroconverting donors4 were recently infected seroconverting donors As of November 1999, 5 seroconverting donors identified As of November 1999, 5 seroconverting donors identified
in 23 million donationsin 23 million donations– 1:4.6 million (ARC)1:4.6 million (ARC)– 1:9.2 million (US)1:9.2 million (US)
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E992750C 4
Samples for TestingSamples for Testing
p24 Ag and HIV NAT reactive yield samples (N=4)p24 Ag and HIV NAT reactive yield samples (N=4)– Tested undiluted and diluted 1:128 in RNA-negative Tested undiluted and diluted 1:128 in RNA-negative
defibrinated plasmadefibrinated plasma Commercial seroconversion samples (N=25)Commercial seroconversion samples (N=25)
– Tested undiluted and diluted 1:128Tested undiluted and diluted 1:128 Diluted p24 Ag “External Control” sample that is Diluted p24 Ag “External Control” sample that is
tested on all NAT runs (N=437)tested on all NAT runs (N=437)– 3 Ag+/Ab– units (S/CO 1-2 Coulter) pooled3 Ag+/Ab– units (S/CO 1-2 Coulter) pooled– Pool diluted 1:16Pool diluted 1:16
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E992750C 5
Comparison of HIV-1 p24 Ag Positive Comparison of HIV-1 p24 Ag Positive Window-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 p17, p31POS – minus p17, 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
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E992750C 6
Detection of HIV-1 p24 Ag and NAT Yield Detection of HIV-1 p24 Ag and NAT Yield Samples by HIV-1 NAT (1:128 Dilution)Samples by HIV-1 NAT (1:128 Dilution)
ARC 2ARC 2 AgAg 6x106x1055 ++ (16.08)(16.08)
ARC 3ARC 3 AgAg 1x101x1055 ++ (16.06)(16.06)
ARC 5ARC 5 AgAg 3x103x1055 ++ (29.59)(29.59)
MilwaukeeMilwaukee NATNAT 2x102x1044 ++ (7.86)(7.86)
CaseCaseIndexIndex
Detected ByDetected By
Index RNAIndex RNAConc. UndilutedConc. Undiluted
(copies/mL)(copies/mL)
MultiplexMultiplex1:128*1:128*(S/CO)(S/CO)
* Diluted in RNA screened negative defibrinated plasma* Diluted in RNA screened negative defibrinated plasma
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E992750C 7
HIV Panel 6240 – HIV Panel 6240 – Virologic/Serologic ProfileVirologic/Serologic Profile
DaysDays
S/C
OS
/CO
HIV
PC
R Q
uant
itatio
nH
IV P
CR
Qua
ntita
tion
PCR
100
1,000
10,000
100,000
1,000,000
0 10 20 30 40 50 600
5
10
15
20
25
30
35
HIV-1 p24 Antigen HIV 1/2 Antibody
5 5 DaysDays
2 2 DaysDays
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100
1,000
10,000
100,000
1,000,000
0 10 20 30 40 50 600
5
10
15
20
25
30
35
HIV-1 p24 Antigen HIV 1/2 Antibody Multiplex Neat Multiplex 1:128
HIV Panel 6240 – HIV Panel 6240 – Virologic/Serologic ProfileVirologic/Serologic Profile
DaysDays
S/C
OS
/CO
HIV
PC
R Q
uant
itatio
nH
IV P
CR
Qua
ntita
tion
2 Days 2 Days 1:1281:128
7 Days 7 Days NeatNeat
E992750C 8
PCR
5 Days5 Days
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PCR
Days
S/C
OH
IV P
CR
Qua
ntita
tion
100
1,000
10,000
100,000
1,000,000
0 20 40 60 80 100 120 140 160 180 2000
10
20
30
HIV-1 p24 Antigen HIV 1/2 Antibody
HIV Panel PRB932 –HIV Panel PRB932 –Virologic/Serologic ProfileVirologic/Serologic Profile
Cutoff
E992750C 9
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PCR
Days
S/C
OH
IV P
CR
Qua
ntita
tion
100
1,000
10,000
100,000
1,000,000
0 20 40 60 80 100 120 140 160 180 2000
10
20
30
HIV-1 p24 Antigen HIV 1/2 Antibody Multiplex Neat Multiplex 1:128
HIV Panel PRB932 –HIV Panel PRB932 –Virologic/Serologic ProfileVirologic/Serologic Profile
Cutoff
E992750C 10
14 Days 14 Days
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HIV Panel PRB943 –HIV Panel PRB943 –Virologic/Serologic ProfileVirologic/Serologic Profile
Days
S/C
OH
IV P
CR
Qua
ntita
tion
100
1,000
10,000
100,000
1,000,000
0 5 10 15 20 250
10
20
30
40
50
HIV-1 p24 Antigen HIV 1/2 AntibodyPCR
Cutoff
E992750C 11
12 Days 12 Days 2 Days 2 Days
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PCR
HIV Panel PRB943 –HIV Panel PRB943 –Virologic/Serologic ProfileVirologic/Serologic Profile
Days
S/C
OH
IV P
CR
Qua
ntita
tion
100
1,000
10,000
100,000
1,000,000
0 5 10 15 20 250
10
20
30
40
50
HIV-1 p24 Antigen HIV 1/2 Antibody Multiplex Neat Multiplex 1:128
Cutoff
E992750C 12
5 Days 5 Days 7 Days 7 Days 2 Days 2 Days
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E992750C 13
Clinical Sensitivity, Clinical Sensitivity, HIV Panels, Gen-ProbeHIV Panels, Gen-Probe
0
5
10
15
20
25
30
0 5 10 15 20 25 30
Multiplex Assay S/CO (Neat)
p24
Ant
igen
S/C
O
Cutoff
Lot 809355Lot 809355HIV Seronegative BleedsHIV Seronegative Bleeds
(92 samples from(92 samples from25 individuals)25 individuals)
29 NAT +/p24 Ag –29 NAT +/p24 Ag –
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E992750C 14
Cutoff
Clinical Sensitivity, Clinical Sensitivity, HIV Panels, Gen-ProbeHIV Panels, Gen-Probe
Lot 809355Lot 809355HIV Seronegative BleedsHIV Seronegative Bleeds
(92 samples from(92 samples from25 individuals)25 individuals)
Multiplex Assay S/CO (1:128)
p24
Ant
igen
S/C
O
0
5
10
15
20
25
30
0 5 10 15 20 25 30
21 NAT +/p24 Ag – 1:12821 NAT +/p24 Ag – 1:128
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E992750C 15
Effect of Dilution on NAT Reactivity Effect of Dilution on NAT Reactivity (Gen-Probe HIV-1/HCV Assay)(Gen-Probe HIV-1/HCV Assay)
Lot 355Lot 355HIV-1 (N=25 Panels)HIV-1 (N=25 Panels)
Lot 356Lot 356HCV (N=22 Panels)HCV (N=22 Panels)
Lot 355Lot 355 Lot 356Lot 356
UndilutedUndiluted 162162 167167 162162 160160
Diluted 1:128Diluted 1:128 145 (90%)145 (90%) 148 (89%)148 (89%) 151 (93%) 151 (93%) 151 (94%)151 (94%)
p24 Agp24 Ag 82 (57%)82 (57%) 82 (55%)82 (55%) —— ——
ALT (ALT (60)60) —— —— 50 (44%)*50 (44%)* 49 (43%)**49 (43%)**
* Of 114 tested;* Of 114 tested;** Of 113 tested** Of 113 tested
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E992750C 16
Gen-Probe Linked Study – Phase I Gen-Probe Linked Study – Phase I External Control PanelExternal Control Panel
MemberMember
HIV-1 300 ± 150
HCV 300 ± 150
NEG —
Weak HIV-1 p24 Ag(S/CO 1-2 Coulterdiluted 1:16) 6,800
TargetTargetCopies/mLCopies/mL
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Gen-Probe Linked Study – Phase IGen-Probe Linked Study – Phase IExternal Control DistributionsExternal Control Distributions
Freq
uenc
y
S/CO1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
50
100
150
200Control N Mean S/CO Range
HIV-1 435 18.66* 8.9 – 37.6
HCV 438 8.27* 2.1 – 10.4
NEG 436 0.28 0.1 – 0.9
HIV-1/p24 Ag 437 20.06* 12.1 – 25.2
*p<0.05 (437 runs total)
0
E992750C 17
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E992750C 18
ConclusionsConclusions
High RNA titers (High RNA titers (101055 copies/mL) correspond copies/mL) correspond with p24 Ag positivitywith p24 Ag positivity
No p24 Ag reactive sample was found NAT No p24 Ag reactive sample was found NAT nonreactive even using a pool size of 128nonreactive even using a pool size of 128
NAT, even using pools of 128, is more sensitive NAT, even using pools of 128, is more sensitive than screening with the currently licensed tests than screening with the currently licensed tests for HIV-1 p24 Ag. According to these data, the for HIV-1 p24 Ag. According to these data, the Ag test could be replaced by the use of licensed Ag test could be replaced by the use of licensed pooled NATpooled NAT
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E992750C 19
1
2
3
4
5
6
7
8Lo
g R
NA
Lev
el [c
opie
s/m
L]
-2 -1.5 -1 -.5 0 .5 1 1.5 2
Log p24 S/CO
N = 146R2 = 0.78
Linear Relationship Between HIV RNA Linear Relationship Between HIV RNA Levels and p24 S/CO RatiosLevels and p24 S/CO Ratios
4.03 log (596-190,108 copies/mL) = 1
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E992750C 20
-2-10123456789
Log
HIV
RN
A L
evel
[cop
ies/
mL]
-2 -1.5 -1 -.5 0 .5 1 1.5 2Log p24 S/CO
N = 146
95% Prediction Limits for Relationship of 95% Prediction Limits for Relationship of HIV RNA Levels and p24 S/CO RatiosHIV RNA Levels and p24 S/CO Ratios
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E992750C 21
1
2
3
4
5
6
7
8Lo
g H
IV R
NA
Lev
el [c
opie
s/m
L]
RNA Only (N=61) p24 Pos (N=85)
P = <0.0001*
* Mann-Whitney Test
HIV-1 RNA Levels During “RNA Only” HIV-1 RNA Levels During “RNA Only” and “p24 Positive” Window Periodsand “p24 Positive” Window Periods
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E992750C 22
1
2
3
4
5
6
7
8Lo
g R
NA
Lev
el [c
opie
s/m
L]
-2 -1.5 -1 -.5 0 .5 1 1.5 2
Log p24 S/CO
Linear Relationship Between HIV-1 RNA Levels Linear Relationship Between HIV-1 RNA Levels and p24 S/CO Ratios in 32 Casesand p24 S/CO Ratios in 32 Cases