Title: HCV-RNA and HIV-RNA detection in small test pools of cadaveric samples for viral safety of tissue transplants
1HCV-RNA and HIV-RNA detection in small test pools
of cadaveric samples for viral safety of tissue
transplants
- Maarten Koot, Theo Cuijpers, Marco Koppelman,
- Mirjam de Waal, Arlinke Bokhorst, Noor
Holsboer. - Sanquin Diagnostic Services, AmsterdamBIS-
- Foundation, Leiden, The Netherlands
2Tests on blood borne infections for tissue release
- Bone, ossicle
- Anti-HIV-1/2/antigen (Combo)
- HIV p24 antigen discont.
- HIV-1 DNA
- Anti-HTLV-I/II
- HBsAg
- Anti-HBc total
- Anti-HCV
- HCV/HIV-RNA test pools
- IgG anti-CMV
- TPPA
- Tissue cornea, skin, cardiac valve
- Anti-HIV-1/2/antigen (Combo)
- HIV p24 antigen discont.
- Anti-HTLV-I/II
- HBsAg
- Anti-HBc total (pos. anti-HBs)
- Anti-HCV
- HCV/HIV-RNA test pools
- TPPA
3Testing tissue donors in test pools of 4 for
HCV/HIV-RNA NAT
- NucliSens extractor (BioMerieux), AmpliScreen HCV
test vs 2.0., Ampliscreen HIV test vs 1.5. - System for blood screening of Sanquin.
- 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 ml donor plasma
- HCV-RNA 95 hit rate 840 geq/ml (520-1760 95
CI) 165 IU/ml - HIV-RNA 95 hit rate 1280 geq/ml (760-3040 95
CI) 806 IU/ml - Calculated on dilution factor based on
analytical validation Jongerius et al.
Transfusion 42, 792-797 (2002) and Koppelman et
al. 89, 193-200 (2005).
4Validation of testing tissue donors in test pools
of 4 for HCV-RNA NAT.
- Robustness on frozen samples, retrospectively
analyzed.320 non-selected samples from tissue
donors.-Acceptable level of invalid results 0.6
of the test pools. Responsible individual
sample can be traced. -Robust test, 95 of the
pools have a saturated signal for the IC
(ODgt2.5). - Sensitivity.Spiking of representative test pools
with 300 geq (58.8 IU) HCV-RNA -Level of 6000
geq/ml (1175 IU/ml) for the individual sample is
guaranteed. Nearly all spiked samples saturated
wild-type signals.
5Validation of testing tissue donors in test pools
of 4 for HIV-RNA NAT (1).
- Robustness on frozen samples, retrospectively
analyzed166 non-selected samples from tissue
donors 36 test pools. Samples frozen as test
pool of four. - 13/36 (36 ) test pools significantly reduced IC
signal (ODlt1.5).
6Validation of testing tissue donors in test pools
of 4 for HIV-RNA NAT (2).
- Sensitivity.Spiking of 27 test pools with 280
geq (176 IU) HIV-RNA. Samples frozen as test
pools of four. 5 pools with significantly reduced
WT signal (ODlt1.5) one invalid, one false
negative and 3 IC signal 0.2 and 1.5.
7On-line analysis of test pools HIV-/HCV-RNA (1)
- Sampling and centrifugation (asap) in Plasma
Preparation Tubes (BD). - Shipment and storage at 4-8 oC.
- HCV-RNA period (03-2005 / 05-2006) 202 test
pools, 808 donors. - HIV-RNA period (12-2005 / 05-2006) 166 test
pools, 664 donors. - No reactive donors for HIV-RNA and HCV-RNA. No
confirmed serological pos. donors for HIV and
HCV. - 4/808 (0.5) donors invalid result HCV-RNA
- 3/664 (0.5) donors invalid result HIV-RNA
- 3/664 (0.5) donors invalid as result of HCV/HCV
NAT testing.
8On-line analysis of test pools HIV-/HCV-RNA (2)
HCV-RNA HCV-RNA HIV-RNA HIV-RNA
Result test pool Result breakdown Result test pool Result donor
Positive WTgt0.2 1 0 0 n.a.
Invalid IClt1.5 8 4 7 3
Negative WTlt0.2 ICgt1.5 193 n.a. 159 n.a.
2 test pools no break down. 28 donors
negative 17 donors negative
9Analysis of test pools HIV-/HCV-RNA Robustness
- HCV
- IClt1.5 4
- IC1.5 and lt2.5 3
- IC2.5 93
- Total 202
- HIV
- IClt1.5 4
- IC1.5 and lt2.5 8
- IC2.5 88
- Total 166
HCV
HIV
10Conclusions
- Spiking experiments on test pools of 4 with
inhibiting substances Increase of Cut-off value
IC signal invalid/negative OD1.5 - Testing in pools of 4 with 50 ul of plasma per
tissue donor for HCV-/HIV-RNA yields an
acceptable level of invalid results (0.5 ) for
the individual donor. - The number of false positive results for test
pools is low (0.5 for HCV-RNA, 0 for
HIV-RNA). - The NucliSens extractor AmpliScreen HCV/HIV
assay performed on test pools of 4 tissue donor
is a robust test, 93 of the pools have a
saturated signal for the HCV-IC 88 for
HIV-RNA. - The HCV-/HIV-RNA assay in test pools is applied
for routine tissue release since February 2006.