Real and perceived problems with Nucleic Acid Testing for organ and tissue donors 5 years experience Claudia Chinchilla, MB(ASCP)1, Dem Brucal, CLS1, James Schellenberg, MBA1, Tom Mone, MBA2, Helen Nelson3, Cindy Siljestrom4, Jill Stinebring5, Edwin Serna6, Tracy Schmidt7, Curt Kandra8, Wayne Dunlap9, Patricia Niles10 and Marek Nowicki, PhD1 1MNIT, Los Angeles, CA, 2OneLegacy, Los Angeles, CA, 3GSDS, Sacramento, CA, 4CTDN, Oakland, CA, 5LifeSharing, San Diego, CA, 6NDN, Las Vegas, NV, 7IMDS, Salt lake City, UT, 8PNTB, Portland, OR, 9LCNW, Bellevue, WA and 10NMDS, Albuquerque, NM.

Background Nucleic Acid Testing (NAT) can reduce “window” donations during the antibody negative phase of HCV & HIV-1 infection. Days of Infection to Reduction RNA Detection RNA Ab of Window Detection Detection by NAT Ab Detection HIV - 1 11 22 50% Reduction HCV 23 82 72% Days Schreiber et al. N Engl J Med. 1996;334:1685.

......But what about false positives? = How many donors are we going to defer/delay because of NAT false positive result?

“Amplicons” - short DNA or RNAs Why this is important?

Polymerase Chain Reaction Transcription Mediated Amplification Proper Assay Selection PCR Polymerase Chain Reaction DNA amplicons Thermal cycles Two separate test/runs 6hrs TMA Transcription Mediated Amplification RNA amplicons Isothermal Multiplex (HIV-1/HCV) 4 hrs vs.

Background Since Sep 2004 MNIT Laboratory has been screening organ donors for HIV-1 & HCV RNA by NAT. Aim To analyze and share our experience with NAT problems after 5306 runs testing 8252 donor specimens with Procleix® TMA NAT assay for HIV-1, HCV.

Methods Assay: Procleix HIV-1/HCV TMA (Genprobe, San Diego, CA) Testing: All serum specimens were tested: Real-time testing no batching Neat (undiluted) Diluted 1:5 with PBS (manufacturers recommendation) Discrimination step if needed, the second test is from untouched vial Evaluated population: 5306 NAT runs between Sep 2004 and Dec 2009

multiple NAT results compared with serology and donor risk factors MNIT NAT Algorithm multiple NAT results compared with serology and donor risk factors =

Most Common Problems (1) Invalid runs ~ 6-10% . Year No. runs % Invalid 2004 285 16 2005 892 17 2006 932 14 2007 925 8 2008 1147 7 2009 1125 6

Most Common Problems (2) Non-repeatable results ~1.6% Year Total Tested % 2004 288 0.69% 2005 946 0.85% 2006 1023 2.73% 2007 883 1.47% 2008 2122 1.97% 2009 2990 1.27% Total 8252 1.59%

Most Common Problems (3) Specimen with TMA Inhibitors ~ 1.0 % Some specimens gave un-interpretable NAT results likely due to TMA reaction inhibitors. The source of the TMA inhibition is unclear and most likely multifactorial. Only 1% of specimens were not resolved in a timely manner and affected Turn Around Time. Specimens with TMA inhibitors do occur, but majority of them are resolved when proper algorithm.

Most Common Problems (4) False positive results <0.1% False positive results: How to define? NAT reactive but seronegative? What is the “gold standard”? ...we do not transplant HIV+ and HCV + organs We had 5 donors with NAT positive result but with negative HIV and HCV serology. All of them occurred during initial NAT testing period Non-repeatable results: Specimen that initially tested reactive, when retested results were non-reactive.

Summary Problem Frequency Solution Invalid Runs 6-10% -work with assay manufacturer -retrain operators Non-repeatable ~1.8% -repeat run with “virgin” specimen -proper algorithm Specimen with inhibitors <1.0% -dilute with PBS False Positive Results <0.1% -interpret NAT results together with serology -obtain risk factors

Summary (2) Approx. 90% of donor specimens produced concordant and interpretable results for both neat and diluted replicate. Repeats of invalid runs or retesting of specimen with inhibitors caused delay in reporting and affected reporting time (TAT-Turn Around Time)

Other worries? Say NO to Contamination! Monthly swabbing Sticky mats Designated NAT lab coats Decontaminate after every run Decontamination log for each room UV light in each room Negative Air pressure

Conclusion Contrary to prevailing opinion that NAT produce many false positive results increasing loss of organs, these events are rare in a properly designed and QA lab with the proper chosen assay. Invalid runs and specimens with inhibitors were the only major problems we encountered during NAT testing and >99% were resolved in a timely manner. Most common problems were related to the implementation of a dramatically different technology and operators “learning curve”. Don’t relay on single NAT result - develop proper algorithm Evaluate NAT and serology together! To Date there have been NO organs defer simply because of false positive results from our lab.

Thank you! Acknowledgments MNIT for support & encouragement to perform the study MNIT lab staff collaboration Dem Brucal OPO’s OneLegacy – Maria Stadtler, Stephanie Collazo California Transplant Donor Network Golden State Donor Services Life Sharing Nevada Donor Network Intermountain Donor Services Pacific Northwest Transplant Bank Life Center North West New Mexico Donor Services

Most Common Problems (1) Invalid runs ~ 5-10% Year No. runs % Invalid % of Invalids due to Not Enough Calibrators 10% Rule Tech Error Other Assay Problems 2004 285 16 31.9 55.3 10.7 2.1 2005 892 17 43.3 48.7 4 2006 932 14 69 21* 8 2 2007 925 69.8 11.7 4.7 2008 1147 7 67.1 12.2 20.7 2009 1125 6 43.2 9 48.4 *August 2006, 10% Rule removed from software.

Most Common Problems (3) Non-repeatable results ~1.6% Year Time Range Total Months Total Tested Non-Repeatable % 2004 Sept-Dec 4 288 2 0.69% 2005 Jan-Dec 12 946 8 0.85% 2006 1023 28 2.73% 2007 Jan-Sep 9 883 13 1.47% 2008 May-Dec 2122 42 1.97% 2009 2990 38 1.27% Total 57 8252 131 1.59% *Data consists of Organ and Tissue Donor, periods of lab contamination have been excluded