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 transmit viruses such as HBV, HCV and HIVBlood derived products have the potential to transmit viruses such as HBV, HCV and HIV Donations are currently tested for a variety of different viruses using antibody and antigen detection methodologies (EIA)Donations are currently tested for a variety of different viruses using antibody and antigen 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 pool testing system utilizing RT-PCR to accurately detect donations infected with HIV prior to p24 testing of individual donations.Demonstration of the ability of a plasma pool testing system utilizing RT-PCR to accurately detect donations infected with HIV prior to p24 testing of individual donations.

HCV and HIV Study Design This prospective study included analysis of 342,729 donations from approximately 48,000 randomly selected plasma donors.This prospective study included analysis of 342,729 donations from approximately 48,000 randomly selected plasma donors. Individual samples were also tested for anti- HCV, HIV and HIV p24 and HBV s Antigen as well as ALT levelsIndividual samples were also tested for anti- HCV, HIV and HIV p24 and HBV s Antigen as well as ALT levels Samples from new donors were tested bySamples from new donors were tested by RT-PCR only after passing standard screening Samples from repeat donors were tested bySamples from repeat donors were tested by RT-PCR concurrently with standard screening

PCR Analytical Sensitivity

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 Y3 Z3 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 OnePrimer Pair Two Re-Hybridized with the Internal Control Probe First Reaction Second Reaction

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

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

PCR vs. HIV p24 Antigen (n = 258) PCR of 1:512 Dilutions Coulter or Abbott p24 Ag

Clinical Trial Period Entry CriteriaEntry Criteria –HCV RT-PCR positive/anti-HCV negative/ALT normal (22 eligible/ 13 enrolled) –HIV (4 eligible / 2 enrolled) Donors were followed for six months and monitored for seroconversion or P24 positivity.Donors were followed for six months and monitored for seroconversion or P24 positivity.

Summary of Matrix Testing Total Matrices Tested = 12,278 (~6,000,000 Donations)

Summary of Prevalence

STUDY FINDINGS F PCR+ Non-Seroconverting Donors - Female2 - First HCV PCR test resultsPositive - Duration of HCV PCR positive testsPositive > 176 days - Viral load by Quantitative PCR140 K million - Duration of HCV bDNA positive testsPositive > 176 days - Duration of normal ALT valuesNormal > 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 resultsPositive

Contributors National Genetics InstituteNational Genetics Institute –Andrew Conrad, Ph.D –Peter Schmid, M.D./Ph.D –Richard Smith, Ph.D –Jeff Albrecht, Ph.D. –Larry Blatt D.PH. Alpha Therapeutic CorporationAlpha Therapeutic Corporation –Charles Heldebrant, Ph.D. –Lorraine Peddada, Ph.D