Transcript HIV/STD Prevalence, Risks, & Missed Opportunities for

Validating 16 Member Pooled APTIMA®
HIV-1 RNA testing
Ethridge Steven F, Sullivan T, Bennett B, Parker
M, Hanson D, Hilliard J,
Hart C, Patel P
Diagnostic Applications Team
Behavioral and Clinical Surveillance Branch
Division of HIV/AIDS Prevention, Surveillance
& Epidemiology
Centers for Disease Control and Prevention, Div. of HIV/AIDS
Prevention, Laboratory Branch, Clinical Support Team
Clyde Hart and Joslyn Hilliard
Florida Bureau of Laboratories, Retrovirology Unit
Petrice Stephens
Olanike David
Berry Bennett
Sally Fordan
New York State Health Department Laboratory at Wadsworth,
Diagnostic HIV Laboratory, Molecular Testing Unit
Thomas Miller
Kurt Dunn
Philip Rivenburg Monica Parker
Tim Sullivan
Presentation outline
Background: Procleix ® HIV-1/HCV and
Aptima ® HIV-1 RNA Qualitative assays
 Objectives for validation
 Procedure for pooled testing
 Obtaining a standard for validation
 Preparing and processing validation standards
 Results of testing
 Lessons learned
 Next steps
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Background
 Procleix
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® HIV-1/HCV Assay
Intended for blood product testing only
Detects HIV-1 and HCV
FDA Labeled for 16 member pooling
Can be used as an aid in the diagnosis of
HIV-1 infection when using the
discriminatory assay.
Background: Labeled use of the
APTIMA® HIV-1 RNA Qualitative Assay
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Use as a aid to diagnosis of acute HIV-1
infection.
Use as an additional more specific test with
specimens found to be repeated reactive
using an EIA test.
Not labeled for specimen pooling as the
Procleix HIV-1/HCV testing system used for
blood product testing.
Highlights of the Procleix ® and Aptima ® tests
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High complexity CLIA tests
Single tube; 3 steps.
Internal control
EDTA plasma or whole blood can be
refrigerated for up to 8 days before testing.
Plasma tolerates up to 3 freeze thaw cycles.
Reliably detect HIV-1 RNA down to 33
copies/ml.
Tests reported as: reactive, non-reactive, or
invalid.
Background:
Reporting a positive result in a pooled format
Reporting a positive Aptima® HIV-1 RNA test
result isolated from pooled testing is
achieved by testing positive individual
specimens in duplicate as directed by the
product label.
Background:
Reporting a negative result in a pooled format
FDA requires a lower limit of detection of 5,000
copies/ml of HIV-1 RNA per screening pool for blood
product testing, documentation of sensitivity based on
pool size, and verification of no sample carryover or
degradation during the pooling process.
Objectives for validation
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Repeatedly challenge sensitivity of test at
lower detection limits in a 16 member pooled
format.
Demonstrate detection was not affected by
run to run variation, pooling size, sample
degradation or carryover during pooling.
Demonstrate that this can be done in a public
health laboratory.
Methods: Pooling procedure
New York: automated pooling with Hamilton AT Plus 2
Florida: manual pooling with standard laboratory pipettes
1-Stage Pooling
16 Individual specimens
A B C D E
F G H I J K L M N O P
16 member master Pool
Methods: Obtaining a HIV-1 RNA standard
Reagent: HIV-1 VQA RNA Quantification
Standard
Catalog Number: 3443 Release
Category: DLot
Number:200207029
Provided:1.25 ml, 150,000 copies/HIV-1 RNA/ml
spiked into negative plasma.
Methods: Preparing Standards
Estimated
Standard Calculated copies/ml in
copies/ml 16 member
pool
(undiluted)
CDC measured
Copies/ml
(undiluted)
Actual
copies/ml in
16 member
pool
Level 1
1070
67
n=9 (s.d.)
900 (200)
Level 2
2130
133
2500 (1300)
156
Level 3
5330
333
6100 (1200)
381
56
Note: Mean and standard deviation calculated by using the mean
of means model in SAS and rounding off the measurements to two
significant digits due to limitations with measuring viral loads.
Methods: Processing Standards
CDC prepares
55 vials
of each Secondary
Standard
25 Vials of each
standard
shipped
to New York
Laboratory
CDC retains
5 vials of
each standard
for Analysis
25 vials of each
standard shipped
to
Florida Laboratory
Methods:
Analysis of 3 level Standards in both
laboratories with the same reagent lot
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New York: vials 1-18 in separate
runs, and vials 19-24 in pairs for
n=24
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Florida: vials 1-24 in pairs for 12
separate runs, n=24
Results: Analysis of 3 low level HIV-1 RNA standards in
16 member pools in two AHI laboratories
Stand.
HIV-1 RNA
copies/ml
mean
FBOL
S/CO ratio
NYSDOH
S/CO ratio
mean SD CV%
mean
SD
CV%
900
10.9
2.3 20.8
12.4
5.1
40.7
2500
14.6
4.0 27.5
17.3
5.4
30.9
6100
19.3
3.5 18.2
22.0
4.6
20.8
Each Standard was run 24 times and repeated to
verify reactive result per product insert instructions.
Results: Analysis of 3 low level HIV-1 RNA standards in
16 member pools in two AHI laboratories
Stand.
HIV-1
RNA
copies/ml
mean
FBOL
Analyte RLU
mean
900
NYSDOH
Analyte RLU
SD
CV%
mean
SD
419,645
91,540
21.8
418,629 170,175 40.7
2500
558,915
145,131 26.0
568,719 183,368 32.2
6100
739,775
131,377 17.8
737,530 132,587 18.0
Each Standard was run 24 times and repeated to
verify reactive result per product insert instructions.
CV%
Lessons Learned
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Not necessary to run more than one low level
standard for validation or repeat samples that
are reactive for validation purposes.
Not possible to obtain more than one lot
number for validation.
Test is relatively easy to validate for 16
member pooling.
Check with CLIA before validating this pooling
procedure in your laboratory.
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Conclusion:
Our estimated lower limit of detection
(sensitivity) of the Aptima® HIV-1
RNA test in a 16 member pool of 900
(2 SD 500-1300) copies per ml met the
FDA-required lower limit of detection
for a screening pool.
There was no significant difference in
using automated pooling or manual
pooling for testing performance during
this validation period.
Next Steps
 We will use one level standard for the 32
member pool validation with each
laboratory running 12 runs each due to the
success of the 16 member pooled validation.
 Both laboratories believe that 16 and 32
member pools are the most practical pool
sizes to use in this application.