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

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

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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

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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

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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%)


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

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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%)


(p24, gp41,(p24, gp41,gp120/160,gp120/160,

but +/- intensity)but +/- intensity)

11 (1.4%)

OneOneViralViralBandBand

460 (55.8%)


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

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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

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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

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Cambridge Biotech HIV-1 Western BlotCambridge Biotech HIV-1 Western Blot


p66p66

p55/51p55/51

gp41gp41

p31p31

p24p24

p17p17

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p66p66

p55/51p55/51gp41gp41

p31p31

p24p24

p17p17

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p66p66p55/51p55/51

gp41gp41

p31p31

p24p24

p17p17

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p66p66p55/51p55/51

gp41gp41

p31p31

p24p24

p17p17

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p66p66p55/51p55/51

gp41gp41

p31p31

p24p24

p17p17

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p66p66p55/51p55/51

gp41gp41

p31p31

p24p24

p17p17

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p66p66p55/51p55/51

gp41gp41

p31p31

p24p24

p17p17

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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

Documents

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