A PROSPECTIVE STUDY OF POLYMERASE CHAIN REACTION TESTING ON POOLED PLASMA VS. INDIVIDUAL DONATION HIV P24 TESTING.

A PROSPECTIVE STUDY OF POLYMERASE CHAIN REACTION TESTING

ON POOLED PLASMA VS. INDIVIDUAL DONATION HIV P24 TESTING

INTRODUCTION• Blood derived products have the potential to Blood derived products have the potential to

transmit viruses such as HBV, HCV and HIVtransmit viruses such as HBV, HCV and HIV• Donations are currently tested for a variety of Donations are currently tested for a variety of

different viruses using antibody and antigen different viruses using antibody and antigen detection methodologies (EIA)detection methodologies (EIA)

• HIV is screened for using the p24 antigen test kits. In an effort to avoid “window period” false negatives

• WE endeavored to conduct a study comparing the relative efficacy of pooled PCR vs. p24 testing.

OBJECTIVE

• Demonstration of the ability of a plasma Demonstration of the ability of a plasma pool testing system utilizing RT-PCR to pool testing system utilizing RT-PCR to accurately detect donations infected with accurately detect donations infected with HIV prior to p24 testing of individual HIV prior to p24 testing of individual donations.donations.

HCV and HIV Study Design• This prospective study included analysis of 342,729 This prospective study included analysis of 342,729

donations from approximately 48,000 randomly donations from approximately 48,000 randomly selected plasma donors.selected plasma donors.

• Individual samples were also tested for anti-HCV, HIV Individual samples were also tested for anti-HCV, HIV and HIV p24 and HBV s Antigen as well as ALT levels and HIV p24 and HBV s Antigen as well as ALT levels

• Samples from new donors were tested by Samples from new donors were tested by

RT-PCR only after passing standard screening RT-PCR only after passing standard screening

• Samples from repeat donors were tested by Samples from repeat donors were tested by

RT-PCR concurrently with standard screeningRT-PCR concurrently with standard screening

PCR Analytical SensitivityVirus RNA Copies/mL

MeanSensitivity

95% Hit Rate

HIV 3.1 5

Robotic Pooling Device

Steps to Build Pool a (8x8x8)512 Member Pool

The 8 Tecan pipettes draw from all (colored) ROWS in COLUMN Y1 in LAYER X1 and makes 3 deposits into secondary pool vials:

A) 8 individual aliquots into ROW (Z1-Z8=colors) pool vials

B) all 8 pipettes aliquots into the single COLUMN Y1 pool vial

C) all 8 pipettes aliquot into the single LAYER X1 pool vial

“Three Dimensional” Plasma Pool Sample Matrix

8X8X8 (512)

Primary Pool X1 Y3 Z3 Found Positive

Y3Z3

X1

“Three Dimensional” Plasma Pool Sample Matrix

8X8X8 (512)

• If the Master pool is found “Negative” then we conclude that all component samples are bellow assay cutoff or “Negative”

• If the master pool is found to be positive then the algorithm requires the testing of 25 more samples (8 rows, 8 columns and 8 layers + the implicated sample)

NGI UltraQual™ HBV PCR Raw Data

Primer Pair One Primer Pair Two

Re-Hybridized with the Internal Control Probe

FirstReaction

SecondReaction

HIV Results

• 18/348,000 (.005%) donations were found positive for HIV

• Of these donations 10 were both antibody and antigen Negative 8 were positive for either p24 or antibody or both.

• These 18 donations came from 4 donors for a rate of 4/48,000 (1in 12,000 or .008%)

Clinical Yield from >48,000 Donors

Number of Donations Found in PoolsVirus Subjects RNA (+)/Ab (-) Rate

HIV-1 4 eligible (Ab neg) ~1:12,000 donors

2 enrolled 4 2 not enrolled 6

HCV 22 eligible (Ab neg) ~1:2,200 donors

13 enrolled 60

9 not enrolled 25

UltraQual PCR undiluted National Genetics Institute - + + + + + + -

UltraQual PCR at 1:512 National Genetics Institute - - + + + + - -p24 Neutralizing

Coulter or Abbott - - - + + - - -Ab Repeat Reactive

Abbott - - - - + + + + Total

89 36 41 88 52 29 6 6 347

89

36 41

88

5229

6 6

HIV Marker Profile of 347 Potential HIV Window Period Plasma Samples

1998

0 10 20 30 40 50

HIV Ab

HIV RNA

p24 Ag

5288413689 29 6 6

cutoff for PCR undiluted

cutoff for 1:512 PCR, p24 and HIV Ab

Hypothetical Kinetics of HIV Infection

undiluted PCR - 252

1:512 PCR - 210

p24 Ag - 140

HIV Ab - 93

HIV Marker Profile of 347 Potential HIV Window Period Plasma Samples - 1998

True window period samples.

PCR vs. HIV p24 Antigen(n = 258)

+ -+ 140 0

- 70 48

PCR of 1:512Dilutions

Coulter or Abbott p24

Ag

Clinical Trial Period

• Entry CriteriaEntry Criteria

– HCV RT-PCR positive/anti-HCV negative/ALT HCV RT-PCR positive/anti-HCV negative/ALT

normal (22 eligible/ 13 enrolled)normal (22 eligible/ 13 enrolled)

– HIV (4 eligible / 2 enrolled) HIV (4 eligible / 2 enrolled)

• Donors were followed for six months and Donors were followed for six months and

monitored for seroconversion or P24 monitored for seroconversion or P24

positivity. positivity.

Summary of Matrix Testing

Total Matrices Tested = 12,278(~6,000,000 Donations)

TargetMatrices Tested

Positive Donations

HBV 664 13HCV 12,278 1380HIV 4,802 26

Summary of Prevalence

Virus PrevalenceHBV 11 positive donors in ~43,000 donorsHCV 32 positive donors in ~100,000 donorsHIV 4 positive donors in ~100,000 donors

STUDY FINDINGS

PCR+ Non-Seroconverting Donors- Female 2

- First HCV PCR test results Positive

- Duration of HCV PCR positive tests Positive > 176 days

- Viral load by Quantitative PCR 140 K - 4.5 million

- Duration of HCV bDNA positive tests Positive > 176 days

- Duration of normal ALT values Normal > 176 days

- Duration of negative antibody test

results (Abbot HCV EIA 2.0) Negative > 176 days

- (Ortho HCV EIA 3.0) test results at time

of first positive HCV PCR results Positive

Contributors

• National Genetics InstituteNational Genetics Institute– Andrew Conrad, Ph.DAndrew Conrad, Ph.D– Peter Schmid, M.D./Ph.DPeter Schmid, M.D./Ph.D– Richard Smith, Ph.DRichard Smith, Ph.D– Jeff Albrecht, Ph.D.Jeff Albrecht, Ph.D.– Larry Blatt D.PH.Larry Blatt D.PH.

• Alpha Therapeutic CorporationAlpha Therapeutic Corporation– Charles Heldebrant, Charles Heldebrant, Ph.DPh.D. . – Lorraine Peddada, Lorraine Peddada, Ph.DPh.D