SOGAT 25-05-2005

Validation of HCV-RNA detection in small test pools on cadaveric samples

Marco Koppelman#, Theo Cuypers#, Mirjam de Waal#, Maarten Koot#, Arlinke Bokhorst*, Noor Holsboer*.#Sanquin Diagnostic Services, Amsterdam;*BIS-Foundation, Leiden, The Netherlands

SOGAT 25-05-2005

Tests on blood borne infections for tissue release

Bone, ossicle

Anti-HIV-1/2

HIV p24 antigen

HIV-1 DNA

Anti-HTLV-I/II

HBsAg

Anti-HBc total

Anti-HCV

HCV-RNA

IgG anti-CMV

TPPA

Tissue: cornea, skin, cardiac valve

Anti-HIV-1/2

HIV p24 antigen

Anti-HTLV-I/II

HBsAg

Anti-HBc total

Anti-HCV (pos. HCV-RNA)

TPPA

SOGAT 25-05-2005

HCV-RNA: Bone-tissue and confirmation samples positive in serology

NucliSens extractor (BioMerieux), Amplicor HCV test vs 2.0

•1 ml plasma input: 95 % hit-rate 60 geq/ml (8.7 IU/ml) 15 % invalid results with neg. IC signal.

•0.1 ml plasma input with 0.9 ml negative human plasma: 95 % hit-rate 600 geq/ml (87 IU/ml)

Data from July 2001-August 2004 Result Bone and confirm.# Clinical samples

number % number %negative 300 94.34% 2368 62.17%positive 7 2.20% 1393 36.57%invalids 11 3.46% 48 1.26%Total 318 3809

#total number of tissues 5188

SOGAT 25-05-2005

Tests on blood borne infections for tissue release

Bone, ossicle

Anti-HIV-1/2

HIV p24 antigen

HIV-1 DNA

Anti-HTLV-I/II

HBsAg

Anti-HBc total

Anti-HCV

HCV-RNA

IgG anti-CMV

TPPA

Tissue: cornea, skin, cardiac valve

Anti-HIV-1/2

HIV p24 antigen

Anti-HTLV-I/II

HBsAg

Anti-HBc total (pos. anti-HBs)

Anti-HCV (pos. HCV-RNA)

HCV-RNA test pools

TPPA

SOGAT 25-05-2005

Testing tissue donors in test pools of 4 for HCV-RNA NAT

NucliSens extractor (BioMerieux), AmpliScreen HCV test vs 2.0. NAT Blood screening for HCV-RNA and HIV-RNA.

Tissues: testing in test pools of 4 samples. Input per donor sample 50 ul EDTA plasma. Filling up to 1 ml with 0.8 ml negative human plasma.

Sensitivity per donation*:

99 % hit rate 1600 geq/ml (880-4320 95 % CI) 313 IU/ml

95 % hit rate 840 geq/ml (520-1760 95% CI) 165 IU/ml

50 % hit rate 160 geq/ml (120-240 95% CI) 31 IU/ml*Calculated on dilution factor based on analytical validation Jongerius et al. 42, 792-

797 (2002).

SOGAT 25-05-2005

Validation of testing tissue donors in test pools of 4 for HCV-RNA NAT.

• Robustness.320 non-selected samples from tissue donors of period 6 weeksComposition of 80 test pools

-% invalid results, traceability sample(s) responsible for invalid result-% pools with inhibited IC-signal, distribution of IC signals

• Sensitivity.-Spiking of 26 test pools with 300 geq/ml HCV-RNA and analyses test pools with and without the HCV-RNA spike.

300 geq per extraction = load run control in standard HCV-RNA blood screening

SOGAT 25-05-2005

Acceptance criteria testing tissue donors in test pools of 4 for HCV-RNA NAT.

Robustness:

• Percentage invalid results in test pools < 5 %.

• Invalid test pools breakdown into one invalid donation and three donations with valid result.

• Total invalid results on tissue level < 2 %.

• > 90 % test pools OD/CO > 12.5, saturated signal.

Sensitivity:

• Non-spiked test pools negative for HCV-RNA, spiked test pools (300 geq per pool) positive.

SOGAT 25-05-2005

Results test pools of 4 tissue donors in HCV-RNA NAT

Internal control signals sorted OD/CO

0

5

10

15

20

25

1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77

number test pool

OD

/CO

IC

sig

nal

•78 test pools negative for HCV-RNA.

•2 test pools invalid results, with negative IC-signal

•2 test pools with inhibited IC-signal, one OD/CO ~ 5, one ~ 12.

SOGAT 25-05-2005

Retest and breakdown invalid test pools.

•Two invalid test pools composed once more. Re-test: one test pool invalid for HCV-RNA; the other one inhibited.

•Breakdown of the two invalid test pools in individual donations, one donor responsible for invalid result.

IC-RNA ResultSample OD/CO testPool 19 repeat composed 0.02 invalidPool 19-910679 0.01 invalidPool 19-910754 20 NegPool 19-910755 20 NegPool 19-910756 20 NegPool 23 repeat composed 5.26 NegPool 23-910864 20 NegPool 23-910865 0.01 invalidPool 23-910927 20 NegPool 23-910928 20 Neg

SOGAT 25-05-2005

Wild type and IC signal (OD/CO) test pools spiked with 300 geq/ml HCV-RNA

0

5

10

15

20

25

1 3 5 7 9 11 13 15 17 19 21 23 25

number

OD

/CO IC OD/CO

WT OD/CO

SOGAT 25-05-2005

Conclusions

• Testing in pools of 4 with 50 ul of plasma per tissue donor yields an acceptable level of invalid results of 2/320 (0.6 %) for the HCV-RNA NAT test in an unselected sample cohort.

• Test pools of 4 composed in this way yield a robust test, 95 % of the pools have a saturated signal for the IC.

• Based on the analytical validation, this test has a 95 % success rate for a positive result of 840 geq/ml (165 IU/ml) for samples tested in this dilution.

• Spiking experiment show that the level of 6000 geq/ml (1175 IU/ml) for the individual sample is guaranteed. Nearly all spiked samples have saturated wild-type signals.