SoGAT 14-06-2006

21 apr 2023

HCV-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, Amsterdam;*BIS-Foundation, Leiden, The Netherlands

Tests 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

Testing 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).

Validation 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 (OD>2.5).

• 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.

Validation of testing tissue donors in test pools of 4 for HIV-RNA NAT (1).

Robustness on frozen samples, retrospectively analyzed:166 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 (OD<1.5).

0.000

0.500

1.000

1.500

2.000

2.500

3.000

3.500

4.000

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35

Pools

OD

IC

HIV

Validation 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 (OD<1.5): one invalid, one false negative and 3 IC signal 0.2 and 1.5.

0.000

0.500

1.000

1.500

2.000

2.500

3.000

3.500

4.000

4.500

1 3 5 7 9 11 13 15 17 19 21 23 25 27

Pools

OD IC OD

WT OD

On-line analysis of test pools HIV-/HCV-RNA (1)

• Sampling and centrifugation (asap) in Plasma Preparation Tubes (B&D).

• 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.

On-line analysis of test pools HIV-/HCV-RNA (2)

HCV-RNA HIV-RNA

Result test

pool

Result

breakdown

Result test

pool

Result

donor

Positive

WT>0.2

1 0 0 n.a.

Invalid

IC<1.5

8 4* 7& 3#

Negative

WT<0.2

IC>1.5

193 n.a. 159 n.a.

&2 test pools no break down.*28 donors negative#17 donors negative

Analysis of test pools HIV-/HCV-RNA; Robustness

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

1 16 31 46 61 76 91 106 121 136 151

Pool nr

OD

HIV

_IC

0.00.51.01.52.02.53.03.54.04.5

1 16 31 46 61 76 91 106 121 136 151 166 181 196

Pool nr

OD

HC

V_I

C

HCV

IC<1.5 4 %

IC≥1.5 and <2.5 3 %

IC≥2.5 93 %

Total 202

HIV

IC<1.5 4 %

IC≥1.5 and <2.5 8 %

IC≥2.5 88 %

Total 166

HCV

HIV

Conclusions• Spiking experiments on test pools of 4 with inhibiting

substances: Increase of Cut-off value IC signal invalid/negative OD=1.5

• 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.