Transcript Diapositiva 1

ITALIAN NATIONAL BLOOD CENTRE
CENTER FOR IMMUNOBIOLOGICALS
RESEARCH AND EVALUATION
Transfusion Safety Area
Biologicals Unit
NUCLEIC ACID AMPLIFICATION TECHNOLOGY
HCV-RNA / HBV-DNA / HIV-RNA
testing blood and blood components for transfusion
Italian External Quality Assessment Program, 2008
IT NAT EQA 2008
IT NAT EQA 2008 Study
This study was organized and promoted by the
Italian National Blood Centre in cooperation with
the Centre for Immunobiologicals Research and
Evaluation, Istituto Superiore di Sanità (ISS)
F.Luciani - SOGAT – Brussels 28-29 May 2009
IT NAT EQA 2008 - ISS
Scope and Participants
The IT NAT EQA 2008 study was organized for assessing the
analytical performance of the qualitative NAT assays/systems
currently used in Italian blood centers for HCV RNA, HIV RNA and
HBV DNA screening.
The study was extended, on a voluntary basis, also to other
european and international testing labs and blood products
manufacturers.
F.Luciani - SOGAT – Brussels 28-29 May 2009
IT NAT EQA 2008 - ISS
Participants
A total of 122 laboratories participated in the
IT NAT EQA 2008 study
Italy
98
Austria
1
Germany
3
United Kingdom 1
F.Luciani - SOGAT 28-29 May 2009
Greece
1
Spain
9
Switzerland
1
Lithuania
1
Thailand
7
IT NAT EQA 2008 - ISS
IT NAT EQA 2008 Study Timeline
1st phase
June 2008 to July 2008
2nd phase
Nov 2008 to Dec 2008
F.Luciani - SOGAT – Brussels 28-29 May 2009
IT NAT EQA 2008 - ISS
Design and rationale of the study (1)
It would not be possible to draw definitive conclusions using either:
-
low viral load (i.e. close to 95% DL): participants could miss the target
due to its random distribution in the plasma matrix, or
-
high viral load: it would produce 100% of correct results hiding any
occurrence of procedural mistakes.
Thus, panels were prepared taking into account the 95% detection
limit (DL) of the methods most commonly used by laboratories
involved in blood screening by NAT. (Pisani et al. Vox Sanguinis
2008, 95:8–12).
F.Luciani - SOGAT – Brussels 28-29 May 2009
IT NAT EQA 2008 - ISS
Panel sample
A load of 3x 95% DL should be found positive by all participant in
100% of the assays and any error in the procedure, even a minor
one, would be detected.
This concentration is also currently recommended to evaluate the
robustness of qualitative NAT methods in the context of validation
studies [Guidelines for validation of NAT for the detection of HCV
RNA in plasma pools (PA/PH/OMCL (98) 22, DEF)]
F.Luciani - SOGAT – Brussels 28-29 May 2009
IT NAT EQA 2008 - ISS
Assays
ASSAY
CODE
HCV RNA HIV RNA HBV DNA
IU/mL
IU/mL
IU/mL
DL
3xDL
DL
3xDL
DL
3xDL
TMA Ultrio Assay
TMA
3.0
10
20.2
60
10.4
30
COBAS Ampliprep/Taqman
CTM
10,7
33
49,0
150
3,7
12
COBAS Ampliscreen
AMP
28.2
85
78.4
235
5
15
F.Luciani - SOGAT – Brussels 28-29 May 2009
IT NAT EQA 2008 - ISS
International Standard
The majority of participating laboratories use commercial kits for NAT blood
screening for which the 95% DL was calculated by the manufacturers using
WHO standards.
Thus, WHO International Standards were selected for
the preparation of the panels.
F.Luciani - SOGAT – Brussels 28-29 May 2009
IT NAT EQA 2008 - ISS
Materials and Methods
Negative samples: they were prepared using a plasma pool made up of 25
donations tested negative for HCV, HIV and HBV by serological and NAT tests.
Positive samples: they were obtained spiking negative plasma pool with the
relevant WHO International Standard to obtain the following concentrations:
• 10, 33 and 85 IU/ml of the HCV RNA WHO IS 96/798, genotype 1
• 60, 150 and 235 IU/mL of the HIV RNA WHO IS 97/650, genotype B
• 12, 15 and 30 IU/ml of the HBV DNA WHO IS 97/746, genotype A
A total of 3000 vials, including negative and positive
samples, were prepared and stored at –80°C,
numbered from 0001 to 3000.
F.Luciani - SOGAT – Brussels 28-29 May 2009
IT NAT EQA 2008 - ISS
Panels
HCV
10
10
10
85
85
85
33
33
33
HIV
60
60
60
235
235
235
150
150
150
HBV
30
30
30
15
15
15
12
12
12
neg
TMA-panel
F.Luciani - SOGAT – Brussels 28-29 May 2009
AMP-panel
CTM-panel
IT NAT EQA 2008 - ISS
Design and rationale of the study (2)
•
The panel samples were tested in multiple runs
•
To better simulate a routine testing, participants were invited to
test four samples (one sub-panel) per day in a timeframe of 2–3
weeks, by different operators, where possible.
•
Two identical panels were sent to the participants, to be tested
separately in the 1st and 2nd phase of the study
•
Three samples with the same viral concentration were included in
the panel, to verify the consistency of the results obtained in
separate runs on different days.
F.Luciani - SOGAT – Brussels 28-29 May 2009
IT NAT EQA 2008 - ISS
Quality control
To confirm the negative and positive status of the samples (homogeneity),
the selection of the vials to be tested was carried out randomly during the
filling.
For each dilution, 5 samples were tested before the study (t=0), 5 samples
were tested at the end of the study (t= 6 months)
Shipment
Panels were shipped in dry ice (48 hours delivery).
Participants were asked to check the integrity of the parcel, the presence of dry ice
and the status of the samples and to fax this information to the ISS using the
acknowledgement of receipt sheet.
Each participant subscribed a responsibility sheet to acknowledge that the samples
received were potentially infectious
F.Luciani - SOGAT – Brussels 28-29 May 2009
IT NAT EQA 2008 - ISS
Assays
ASSAY
CODE
1st phase
2nd phase
TMA Ultrio Assay
TMA
42
42
COBAS Ampliprep/Taqman
CTM
41
54
COBAS Ampliscreen
AMP
40
29
In-House
IH
3
2
F.Luciani - SOGAT – Brussels 28-29 May 2009
IT NAT EQA 2008 - ISS
RESULTS
Samples
A total of 2848 samples were tested and
the results returned to ISS by fax or e-mail:
HCV RNA (716 data)
HIV RNA
(713 data)
HBV DNA (699 data)
Negative samples (720 data)
F.Luciani - SOGAT – Brussels 28-29 May 2009
IT NAT EQA 2008 - ISS
Assay
TMA
HCV-RNA %
HIV-RNA %
HBV-DNA %
Negative %
phase 1
125/125
100
124/124
100
119/119
100
0/125
100
phase 2
120/121
99,2
121/121
100
115/115
100
0/121
100
phase 1
114/114
100
114/114
100
112/112
100
0/116
100
phase 2
151/151
100
151/151
100
151/151
100
1/151
99,3
phase 1
116/117
99,1
113/115
98,3
113/114
98,3
2/116
98,3
phase 2
73/74
98,6
74/74
100
73/74
98,6
1/74
98,6
phase 1
8/8
100
8/8
100
8/8
100
0/11
100
phase 2
6/6
100
6/6
100
6/6
100
0/6
100
phase 1
363/364
99,7
359/361
99,4
352/353
99,7
2/368
99.4
phase 2
350/352
99,6
352/352
100
345/346
99,7
2/352
99,4
phase 1+2
713/716
99,6
711/713
99,7
697/699
99,7
4/720
99,4
1
CTM
AMP
IH
TOTAL
RESULTS
Protocol deviations
36 deviations were observed (21 in EQA-1 and 15 in EQA-2),
classified as follows:
1) Time schedule not observed
2) One or more samples not tested
3) One sub-panel not tested
4) Dedicated panel (e.g. for TMA) tested with a different assay
F.Luciani - SOGAT – Brussels 28-29 May 2009
IT NAT EQA 2008 - ISS
RESULTS
Operators
A total of 247 different operators participated in EQA study testing one
or more sub-panels:
• 32 laboratories - 1 operator
• 55 laboratories - 2 operators
• 35 laboratories - 3 operators
F.Luciani - SOGAT – Brussels 28-29 May 2009
IT NAT EQA 2008 - ISS
RESULTS
Errors
A total of 28 errors (20 laboratories) occurred.
EQA Ph-1
EQA Ph-2
N° of laboratories who reported at least 1 error
11
9
Total errors observed
12
16
unspecified analitytcal error
4
3
analytical errors classified as contamination
2
6
pre/post analytical errors
6
7
F.Luciani - SOGAT – Brussels 28-29 May 2009
IT NAT EQA 2008 - ISS
Participant
Results sheet
Coordinator
24-48 h
Feedback
Laboratories failing to correctly identify the positive or negative
samples were invited to critically evaluate each single step in order to
identify the cause of the mistake.
CONCLUSIONS
•The study allowed participants to verify their performance.
Under these conditions, it was demonstrated that, despite
the high level of automation reached by NAT assays,
human errors can still occur.
•Laboratories reporting a failure are encouraged to better
comply with the good laboratory practice in terms of
operator training and control of contamination.
F.Luciani - SOGAT – Brussels 28-29 May 2009
IT NAT EQA 2008 - ISS
Acknowledgments
CRIVIB
Biologicals Unit
CNS
Transfusion Safety Area
Simonetta Pupella
Giulio Pisani
Karen Cristiano
Francesco Marino
Claudio Mele
Guillermo Bisso
Andrea Gaggioli
Daniela Adriani
Maria Wirz
F.Luciani - SOGAT – Brussels 28-29 May 2009
Vanessa Piccinini
Silvia Vitali
Claudio Mele
Maria Wirz
Secretarial Assistance
Katia Colombo
Cristina Marra
IT NAT EQA 2008 - ISS
Back-up slides
95% DL
3x 95% DL
100 IU/mL
40 IU/mL
20 IU/mL
HCV-RNA
HIV-RNA
HBV-DNA
95% DL
3x 95% DL
60
150
235
235 IU/mL
10 33 85
150 IU/mL
30
85 IU/mL
12 15
60 IU/mL
33 IU/mL
30 IU/mL
12 IU/mL 15 IU/mL
10 IU/mL
HCV-RNA
HIV-RNA
HBV-DNA