HCV-RNA and HIV-RNA detection in small test pools of cadaveric samples for viral safety of tissue transplants

1
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, AmsterdamBIS-
• Foundation, Leiden, The Netherlands

2
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

3
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).

4
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
  (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.

5
Validation 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).

6
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 (ODlt1.5) one invalid, one false
  negative and 3 IC signal 0.2 and 1.5.

7
On-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.

8
On-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
9
Analysis 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
10
Conclusions

• 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.