A PROSPECTIVE STUDY OF POLYMERASE CHAIN REACTION TESTING ON POOLED PLASMA VS. INDIVIDUAL DONATION...
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Transcript of A PROSPECTIVE STUDY OF POLYMERASE CHAIN REACTION TESTING ON POOLED PLASMA VS. INDIVIDUAL DONATION...
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