NUCLEIC ACID AMPLIFICATION TECHNOLOGY HCV-RNA / HBV-DNA / HIV-RNA testing blood and blood components for transfusion Italian External Quality Assessment

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

CENTER FOR IMMUNOBIOLOGICALS CENTER FOR IMMUNOBIOLOGICALS RESEARCH AND EVALUATIONRESEARCH AND EVALUATION

Biologicals UnitBiologicals Unit

ITALIAN NATIONAL BLOOD CENTREITALIAN NATIONAL BLOOD CENTRE

Transfusion Safety AreaTransfusion Safety Area

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)

IT NAT EQA 2008 Study

F.Luciani - SOGAT – Brussels 28-29 May 2009 IT NAT EQA 2008 - ISS

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.

Scope and Participants


Participants

A total of 122 laboratories participated in the

IT NAT EQA 2008 study

Italy 98

Austria 1

Germany 3

United Kingdom

1

Greece 1

Spain 9

Switzerland 1

Lithuania 1

Thailand 7F.Luciani - SOGAT 28-29 May 2009 IT NAT EQA 2008 - ISS

1st phase June 2008 to July 2008

2nd phaseNov 2008 to Dec 2008

IT NAT EQA 2008 Study Timeline


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).


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)]

Panel sample


Assays


International Standard

The majority of participating laboratories use commercial kits for NAT blood screening for which the 95% DL was calculated by the manufacturers usingWHO standards.

Thus, WHO International Standards were selected for the preparation of the panels.


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.

Materials and Methods


HCV

HIV

HBV

neg

TMA-panelTMA-panel AMP-panelAMP-panel CTM-panelCTM-panel

Panels


10

30

60

10

30

60

10

30

60

85

15

235

85

15

235

85

15

235

33

12

150

33

12

150

33

12

150

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.


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)

Quality control


ShipmentPanels 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

Assays

ASSAY CODE 1st phase 2nd phaseTMA Ultrio Assay TMA 42 42

COBAS Ampliprep/Taqman CTM 41 54COBAS Ampliscreen AMP 40 29

In-House IH 3 2


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)

RESULTSSamples


Assay HCV-RNA % HIV-RNA % HBV-DNA % Negative %

TMA1 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

CTM 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

AMP 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

IH 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

TOTAL

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

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

RESULTS Protocol deviations


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

RESULTS Operators


A total of 28 errors (20 laboratories) occurred.

RESULTS

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

Errors


ParticipantParticipant CoordinatorCoordinatorResults sheet

Feedback

24-48 h

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.


Acknowledgments

CRIVIBCRIVIBBiologicals UnitBiologicals Unit

CNSCNSTransfusion Safety Transfusion Safety

AreaArea

Giulio Pisani

Karen CristianoFrancesco Marino

Claudio MeleGuillermo BissoAndrea GaggioliDaniela Adriani

Maria Wirz

Simonetta Pupella

Vanessa PiccininiSilvia Vitali

Claudio MeleMaria Wirz

Secretarial AssistanceSecretarial Assistance

Katia ColomboCristina Marra


Back-up slides

95% DL

3x 95% DL

40 IU/mL

100 IU/mL

20 IU/mL

HCV-RNA HIV-RNA HBV-DNA

95% DL

3x 95% DL

10 IU/mL

HCV-RNA HIV-RNA HBV-DNA

33 IU/mL

85 IU/mL

60 IU/mL

150 IU/mL

235 IU/mL

30 IU/mL

12 IU/mL 15 IU/mL

10 33 85

60 150 235

30 12 15

Documents

NUCLEIC ACID AMPLIFICATION TECHNOLOGY HCV-RNA / HBV-DNA / HIV-RNA testing blood and blood components for transfusion Italian External Quality Assessment