Real and perceived problems with Nucleic Acid Testing for organ and

tissue donors5 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.

Schreiber et al. N Engl J Med. 1996;334:1685.

RNA Detection

Ab Detection

Reduction

HIV-1 11 22 50%

HCV 23 82 72%

Days of Infection to

RNA Detection

Ab Detection

Reductionof Window

by NAT

Days

=

How many donors are we going to defer/delay because of NAT false positive result?

......But what about false positives?

4

“Amplicons” - short DNA or RNAs

Why this is important?

vs.

Proper Assay Selection

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

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.

SummaryProblem Frequency Solution

Invalid Runs 6-10% -work with assay manufacturer-retrain operators

Non-repeatable

~1.8% -repeat run with “virgin” specimen-retrain operators-proper algorithm

Specimen with inhibitors

<1.0% -proper algorithm-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?

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%

*August 2006, 10% Rule removed from software.

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 0

2006 932 14 69 21* 8 2

2007 925 8 69.8 0 11.7 4.7

2008 1147 7 67.1 0 12.2 20.7

2009 1125 6 43.2 0 9 48.4

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 Jan-Dec 12 1023 28 2.73%

2007 Jan-Sep 9 883 13 1.47%

2008 May-Dec 8 2122 42 1.97%

2009 Jan-Dec 12 2990 38 1.27%

Total 57 8252 131 1.59%*Data consists of Organ and Tissue Donor, periods of lab contamination have been excluded