1. 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.

2. 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.

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

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

5. 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.

6. 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.

7. 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

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

9. 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

10. 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%

11. 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.

12. 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.

13. 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

14. 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)

15. 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

16. 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.

17. 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

18. 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.

19. 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