A multi-centre NAT evaluation study of run and trend control samples

Harry van Drimmelen1, Joe O’Donnellan2, Rene Bax1, Henrik Ullum3 and the Danish NAT study group and Wim Quint1

1. Biologicals Quality Control Unit, Delft Diagnostic Laboratory (DDL), the Netherlands, 2. Irish Blood Transfusion Service (IBTS), Dublin, Ireland

3. Rigshospitalet, Copenhagen, Denmark.

Background I Recalibration of standards in copies/ml

• HBV, HCV and HIV-1 analytical and infectivity standards1,2.3 have been recently recalibrated in copies/ml using multiple bDNA 3.0 assays4

• The recalibrated standards have been used for analytical sensitivity studies5,6 and TTI risk analysis7-12

virus Cps/IU Cps/CID50 50%-95% LOD range

ULTRIO TaqScreen

HBV 5.3 3.7 14.8 - 140 2.9 - 27

HCV 2.7 8.1 3.2 - 13 4.9 - 20

HIV-1 0.5 ~800 3.7 - 20 4.2 - 231.Lelie N. et al. Transfusion 2002:42;527. 2. Komiya K et al. Transfusion 2008;48:286 3. Katayama K et al, Intervirology, 2004, 47, 57 4. Van Drimmelen A.A.J. Vox Sang 96 Suppl1 ISBT abstract P075. 5. Assal et al. Transfusion 2009; 49:289 6. Assal et al. Transfusion 2009; 49:289 301. 7.Weusten J et al, Transfusion, accepted. 8. M. El Ekiaby et al. Vox Sang 96, Suppl1, ISBT abstract; 9 . El Ekiaby et al. Vox Sang 96, Suppl1, ISBT abstract; 17. 10. Vermeulen M et al, Transfusion 2009: in press. 11. Kleinman S et al Vox Sang 96, Suppl1, ISBT abstract; 12 . Goubran et al. Novartis, ISBT Satellite meeting, Cairo, 2009

..

Background II Recalibration of standards in copies/ml

63% HIV-RNA detection limits in ULTRIO

1,0

1,5

2,0

2,5

3,0

3,5

4,0

0 2 4 6 8 10 12 14

Study

63

% L

OD

in c

ps

/ml DDL standard

studies

WHO standard studies

theoretical limit is 1 HIV-RNA copy per TMA assay

63% LOD (cps/assay)

• Compare different HBV, HCV and HIV Quality Control standards in a multi-centre validation study of ULTRIO on TIGRIS in Denmark and Ireland

• Analyze TMA response values on QC standard dilutions to establish suitable run or trend control levels

Aim of the study

Methods IInactivation viral standards (DDL)

1. Lelie PN. , Reesink HW , Niessen J. , Brotman B and Prince AM. Inactivation of 1015 chimpanzee infectious doses of hepatitis B virus during preparation of an heat-inactivated hepatitis B vaccine. J. Med. Virol. 1987:23: 289-95

2. Dichtelmüller SW, Prince AM, Brotman B, Huima T. Inactivation of the Hutchinson strain of hepatitis non-A, non-B virus in intravenous immunoglobulin by beta-propiolactone. J Med Virol. 1988: 26:227-32.

3.. Lelie PN. Reesink HW and Lucas CJ. Inactivation of 12 viruses by heating steps applied during manufacture of a hepatitis B vaccine J. Med. Virol. 1987:23: 297-301.

virus Inactivation method viral reduction

RNA, DNA recovery

HBV 10 hours 65°C, 1:100 diluted in PBS1 >6 log 84%

HCV 24 hours incubation 37°C

with β-propriolacton2

>3 log 100%

HIV 2 hours 65°C, 1:10 diluted in PBS3 >5 log 62%

Methods IIIQC samples evaluated

• NIBSC working reagents

• Seracare-BBI Accurun range

• Acrometrix PeliSpy pro range

• ISS standards

• DDL bioQControl check and trend controls

• DDL inactivated standard dilutions

Methods IVCharacteristics QC samples

Supplier AcroMetrix. SeraCare NIBSC DDL

Matrix Defibr. delip. plasma

Defib plasma

Cryosuper-natant

EDTA plasma

Inactivation (insert)

HBV-DNA Heat None 10h, 65°C

HCV-RNA Heat None None β-propiolacton

HIV-RNA Heat Defect strain

None 2h, 65°C

Quantification No No IU/ml cps/ml

* Defibrination; removal of cry precipitating proteins

Methods V multi-centre performance evaluation study

• Five laboratories from Denmark and Ireland tested multiple replicates of QC samples on TIGRIS in validation phase

• Inactivated viral standard dilutions were tested in multiple replicates (n=52-58) and 95% and 50% detection limits were determined by probit analysis

• QC samples were tested in 1:3 and 1:10 dilutions (n=30-33)

• Distribution of ULTRIO S/CO response values on inactivated standard dilutions were analyzed

• Longitudinal Procleix Duplex S/CO values of IBTS (Ireland) on HCV trend control sample (27 cps/ml) were analyzed

Results I detection limits on inactivated standards

virus Repli cates

ULTRIO detection limits in cps/ml

95% LOD (CI) 50% LOD (CI)

HBV 58 699 (337 - 2301) 55 (33 - 93)

HCV 52 31 (21 - 55) 3.9 (21 - 55)

HIV 52 20 (14 - 34) 2.9 (2.2 - 3.7)virus Studies,

replicatesdetection limits in cps/ml on native

standards (low - high)

95% LOD (L-H) 50% LOD (L-H)

HBV 5, 108 186 (96-241) 28 (18 - 37)

HCV 4, 86 27 (22-35) 3.3 (2.8 - 4.5)

HIV 4, 86 26 (19-57) 3.1 (1.9 -4.3)

Proportion of reactive ULTRIO tests (n=33) on diluted run control samples Run control dilution HBV HCV HIV

AcroMetrix PeliSpy Pro 3 88 % 100 % 91 %

AcroMetrix PeliSpy Pro 10 52 % 73% 61 %

CheckControl 125 cps/ml* 1 100 % 100 % 100 %

TrendControl 25 cps/ml* 5 93 % 94 % 91 %

NIBSC Working reagent 3 100 % 100 % 100 %

NIBSC Working reagent 10 79 % 100 % 85 %

Seracare Accurun series 150 3 97 % 97 %

Seracare Accurun series 150 10 93 % 85 %

ISS standard** ** 100 % 100 %

ISS standard** ** 97 % 94 %

* for HBV ULTRIO 1000 and 200 cps/ml

** for HCV 5700 IU/ml diluted to 40 and 12.5 IU/ml and for HIV 4000 IU/ml diluted to 100 and 33 IU/ml

Distribution S/CO results (n=52) for inactivated HCV-RNA standard dilutions

35

-5

0

5

10

15

20

25

30

0 1 2 3 4 5 6 7 8 9

ULTRIO S/CO range

nu

mb

er

345 cps/ml

116 cps/ml

35 cps/ml

12 cps/ml

cps/ml Mean S/CO

SD

345 7.0 0.7

116 6.9 0.6

35 6.2 1.3

12 4.2 3.0

0-1 2-31-2 3-4 4-5 5-6 6-7 7-8 8-9

saturated

Distribution S/CO results (n=778 runs) on HCV-RNA trend control (27 cps/ml)

0

20

40

60

80

100

120

140

160

0 1 2 3 4 5 6 7 8 9

Procleix Duplex S/CO range

Nu

mb

er

0-1 1-2 2-3 4-53-4 5-6 6-7 7-8 8-9 9-10

Averaged (50) proportion TMA reactive (n=778 HCV Procleix duplex runs)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0 100 200 300 400 500 600 700 800

Procleix duplex run

% r

eact

ive

% reactive (mean + 25 and – 25 runs)

% saturated (mean + 25 and – 25 runs)

Conclusions and discussion• Viral concentration in most QC samples appears to

be sufficiently critical for external run controls • Run controls should be quantified; consistent

concentrations between different batches • ULTRIO detection limits on untreated and inactivated

viral standards were comparable*• A trend control sample of ~25 cps/ml (around the

90-95% detection limit) is more instrumental for longitudinal monitoring of analytical sensitivity of TMA test runs**

* For HBV further studies required to prove the correct calibration in copies/ml

** Follow percentage TMA reactive and the distribution of S/CO values over time