1. 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

2. New York State Health Department Laboratory at Wadsworth, Diagnostic HIV Laboratory, Molecular Testing Unit Petrice Stephens Berry Bennett Olanike David Sally Fordan Clyde Hart and Joslyn Hilliard Thomas Miller Kurt Dunn Philip Rivenburg Monica Parker Tim Sullivan Florida Bureau of Laboratories, Retrovirology Unit Centers for Disease Control and Prevention, Div. of HIV/AIDS Prevention, Laboratory Branch, Clinical Support Team

3. 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

4. Background Procleix ® 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.

5. Background: Labeled use of the APTIMA® HIV-1 RNA Qualitative Assay 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.

6. 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. Highlights of the Procleix ® and Aptima ® tests

7. 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 positive result in a pooled format

8. 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.

9. Objectives for validation 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.

10. 16 member master Pool 1-Stage Pooling 16 Individual specimens A B C D E F G H I J Methods: Pooling procedure New York: automated pooling with Hamilton AT Plus 2 Florida: manual pooling with standard laboratory pipettes K L M N O P

11. 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: Obtaining a HIV-1 RNA standard

12. Methods: Preparing Standards StandardCalculated copies/ml (undiluted) Estimated copies/ml in 16 member pool CDC measured Copies/ml (undiluted) n=9 (s.d.) Actual copies/ml in 16 member pool Level 1107067900 (200)56 Level 221301332500 (1300)156 Level 353303336100 (1200)381 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.

13. 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

14. Methods: Analysis of 3 level Standards in both laboratories with the same reagent lot New York: vials 1-18 in separate runs, and vials 19-24 in pairs for n=24 Florida: vials 1-24 in pairs for 12 separate runs, n=24

15. Stand. HIV-1 RNA copies/ml FBOL S/CO ratio NYSDOH S/CO ratio mean SDCV% mean SDCV% 90010.9 2.320.812.4 5.140.7 250014.6 4.027.517.3 5.430.9 610019.3 3.518.222.0 4.620.8 Results: Analysis of 3 low level HIV-1 RNA standards in 16 member pools in two AHI laboratories Each Standard was run 24 times and repeated to verify reactive result per product insert instructions.

16. Stand. HIV-1 RNA copies/ml FBOL Analyte RLU NYSDOH Analyte RLU mean SDCV% mean SDCV% 900419,64591,54021.8418,629170,17540.7 2500558,915145,13126.0568,719183,36832.2 6100739,775131,37717.8737,530132,58718.0 Results: Analysis of 3 low level HIV-1 RNA standards in 16 member pools in two AHI laboratories Each Standard was run 24 times and repeated to verify reactive result per product insert instructions.

17. Lessons Learned 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.

18. 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.

19. 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.