Download Real and perceived problems with Nucleic Acid

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
of Window
by NAT
RNA
Detection
Ab
Detection
HIV-1
11
22
50%
HCV
23
82
72%
Ab Detection
Days
Schreiber et al. N Engl J Med. 1996;334:1685.
RNA Detection
Reduction
......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?
4
Proper Assay Selection
PCR
Polymerase Chain Reaction
• DNA amplicons
• Thermal cycles
• Two separate
test/runs
• 6hrs
vs.
TMA
Transcription Mediated
Amplification
• RNA amplicons
• Isothermal
• Multiplex (HIV1/HCV)
• 4 hrs
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
2005
2006
2007
2008
2009
Total
288
946
1023
883
2122
2990
8252
0.69%
0.85%
2.73%
1.47%
1.97%
1.27%
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
Nonrepeatable
~1.8%
-repeat run with “virgin”
specimen
-retrain operators
-proper algorithm
Specimen with <1.0%
inhibitors
-proper algorithm
-dilute with PBS
False Positive
Results
-interpret NAT results together
with serology
-obtain risk factors
<0.1%
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
2004
285
2005
% of Invalids due to
Not Enough
Calibrators
10%
Rule
Tech
Error
Other Assay
Problems
16
31.9
55.3
10.7
2.1
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
*August 2006, 10% Rule removed from software.
Most Common Problems (3)
Non-repeatable results ~1.6%
Year
Time
Range
NonRepeatable
%
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%
57
8252
131
1.59%
Total
Total
Total
Months Tested
*Data consists of Organ and Tissue Donor, periods of lab contamination have been excluded