Summary
Objective
Commercial available NAT systems are usually not validated for screening of post-mortem blood samples. NAT testing might be challenging due to inhibitory substances in the cadaveric blood sample that cause false-negative test results. Validation studies have to be performed to show the performance characteristics of the NAT assays for testing cadaveric blood.
Methods
A set of 32 post-mortem serum and plasma samples from cornea donors and 40 control samples from blood donors, serologically and NAT negative for all investigated parameters, were spiked with defined concentrations of WHO reference material and tested for HIV-1, HCV, HBV, and HAV by NAT using DRK Baden-Württemberg-Hesse CE PCR kits. Analytical sensitivity, analytical specificity and reproducibility/precision were validated and compared with each other in both groups of samples.
Results
The analytical sensitivity was 100% for control and post-mortem specimens when spiked with virus standards at concentrations of 3 × level of detection (LOD). Invalid results did not occur. The analytical specificity rate for all assays was 100%. Intra-assay variation was analyzed as a function of sample material and sampling time post mortem. Values of % coefficient of variation (%CV) were comparable for serum and plasma but slightly higher for post-mortem samples especially for those samples collected more than 24 h post mortem.
Conclusion
Based on the presented validation, postmortem donor samples can be tested with the automated DRK Baden-Würtemberg-Hesse NAT system.
KeyWords: NAT, Post-mortem blood, Tissue donation, Validation, HCV, HIV, HBV, HAV
Zusammenfassung
Hintergrund
Kommerzielle NAT Assays sind üblicherweise nicht für das Screening von Post-mortem-Blut validiert. Die Durchführung der NAT-Testung ist aufgrund inhibitorischer Substanzen im Blut Verstorbener schwierig und kann zu falsch-negativen Testergebnissen führen. Um die Leistungsmerkmale der NAT-Assays für die Testung von Post-mortem-Blut zu bestimmen, müssen Validierungsuntersuchungen durchgeführt werden.
Methoden
Plasma-und Serumproben von insgesamt 32 Hornhautspendern (Cornea) und 40 Kontrollproben von Blutspendern wurden serologisch und mittels NAT negativ auf HIV-1, HCV, HBV und HAV getestet. Aliquots dieser Proben wurden mit definierten Konzentrationen von WHO-NAT-Standardpräparationen gespickt und die Leistungsmerkmale der Assays wie Sensitivität, Spezifität und Reproduzierbarkeit bzw. Präzision validiert.
Ergebnisse
Die analytische Sensitivität betrug 100% sowohl für die Kontrollproben als auch für die Post-mortem-Proben. Alle Proben, die mit einer Viruskonzentration des dreifachen der Nachweisgrenze gespickt worden waren, wurden NAT-positiv detektiert. Die analytische Spezifität der NAT-Assays lag bei jeweils 100%. Es kam zu keinen invaliden Testergebnissen. Die Intra-Assay-Präzision war bei Testung der Kontrollproben am höchsten. Ein höherer % Variationskoeffizient (%CV) wurde für Post-mortem-Proben im Vergleich zu den Kontrollproben ermittelt und zeigte sich insbesondere bei Proben, die später als 24 h post mortem entnommen wurden.
Schlussfolgerung
Die validierten DRK-Baden-Würtemberg-Hessen-PCR-Assays sind für das automatisierte Screening von Post-mortem-Spenderproben geeignet.
Introduction
A high standard of viral safety of tissues for transplantation is guaranteed when using well validated test systems for viruses like HIV, HCV, HBV, and HAV. Serological screening of tissue donors is mandatory by EU and German law (EU directives 2006/17/EC and TPGGewV). While NAT have been established for blood donor screening since 1996 in Germany [1, 2], the detection of viral genomes in blood samples from tissue donors (post and pre mortem) is not described, neither in the requirements of the EU nor in the German Transplant Act.
Currently most tissue donors are only tested using immunoassays to detect antigens or antibodies that appear much later in the infection cycle than viral genome [3].
NAT systems are able to shorten the diagnostic window period to a minimum and to increase blood safety to the highest standard [4, 5, 6, 7]. The presence of inhibitors due to hemolysis of the samples may lead to false-negative results or a reduced analytical sensitivity of the NAT system. An efficient extraction procedure is therefore essential for the performance of the NAT.
This article describes the validation of the DRK Baden-Würtemberg-Hesse PCR test system in combination with the extraction on the Zelos x100 platform for the detection of HIV-1, HBV, HCV, and HAV in blood samples of deceased cornea donors.
To assess the performance, we determined the analytical sensitivity, the analytical specificity, and the reproducibility/precision by testing cadaveric samples compared to control (living) blood donor specimens.
Material and Methods
Study Design
In order to validate the performance characteristics of a NAT for cadaveric indication, the optimal choice is to use matched pairs of pre- and post-mortem specimens for the experiments as recommended by Paul Ehrlich Institute.
Due to the fact only 8 pre-mortem/post-mortem matched pairs of specimens were available, we have used plasma and serum specimens from living donors (blood donors) as control too. Aliquots of control and cadaveric specimens were used in spiking experiments in order to evaluate assay features like sensitivity, specificity, and reproducibility.
Samples
Sets of 20 control serum and 20 plasma aliquots were taken from blood donor samples after routine screening for viral markers. Whole blood was centrifuged with 3,500 × g for 15 min, serum or plasma was removed and then stored at 2–8 °C for up to 24 h before testing.
A set of 16 post-mortem serum and 16 plasma blood samples were obtained from donors between 11 and 54 h (mean 31.5 h) after death and were provided by the University Tissue Bank of the Charité University Berlin. Eight matched pairs of pre-mortem/post-mortem samples were included in the study. Samples were treated as described elsewhere [8].
All samples were serologically tested negative for anti-HIV-1/2, anti-HCV and HBsAg using Enzygnost® Anti-HIV-1/2 Plus (Siemens, Munich, Germany), HCV-version 3.0 ELISA with Enhanced Save (Ortho Clinical Diagnostics, Neckargemünd, Germany), Enzygnost HBsAg version 6.0 and Enzygnost anti-HBc monoclonal assays on the Siemens BEP III Automatic System (post-mortem specimens), or using ABBOTT PRISM HIVAg/Ab Combo Assay, ABBOTT PRISM HCV Assay Kit, ABBOTT PRISM HBsAg Assay Kit and ABBOTT PRISM HBcore Assay Kit on the ABBOT Prism System (control specimens) (all Abbott GmbH & Co. KG, Wiesbaden, Germany).
Non-spiked aliquots of control and post-mortem specimens were first tested by NAT to verify their non-reactivity for HIV-1, HCV, HBV, and HAV.
Spiking with WHO International Standards
A second aliquot of each specimen was spiked with a dilution of a WHO standard preparation of the respective virus at 3 × level of detection (LOD) as recommended by the Paul Ehrlich Institute. Dilutions of standard material were done using NAT and serologically negative human plasma.
A list of WHO NAT standards that have been used for spiking experiments is shown in table 1.
Table 1.
List of WHO NAT standards
| Virus | WHO international standard / NIBSC –code |
|---|---|
| HCV | HCV 06/102 |
| HBV | HBV 10/264 |
| HAV | HAV 00/560 |
| HIV | HIV 97/650 |
Extraction of Nucleic Acids
Nucleic acid extraction was performed on a Zelos x100 platform using the chemagic viral DNA and RNA kit special (PerkingElmer, chemagen Technologie GmbH, Baesweiler, Germany). Extraction procedure is described elsewhere [9]. This method in combination with DRK PCR assays is applied routinely for blood donor screening for HIV-1, HCV, HBV, HAV, and parvo-virus-B19 in minipools of 96 plasma samples and was used for preparation of viral nucleic acids from single cadaveric samples in this study too.
Extraction was performed with 100 μl sample material that was mixed with 4.7 ml phosphate buffered saline (PBS) to simulate a sample volume similar to a size of a minipool. Nucleic acids were eluted in 100 μl of elution buffer.
NAT Tests for the Detection of HIV-1, HCV, HBV and HAV
Serum and plasma samples were analyzed using DRK PCR kits (German Red Cross Blood Service Baden-Württemberg-Hesse, Frankfurt/M., Germany) which are CE marked real-time PCR assays for blood donor screening in minipools up to a maximum pool size of 96 samples per pool. Test kits are listed in table 2.
Table 2.
List of NAT test kits
| Test kit | 95% LOD, IU/ml |
|---|---|
| DRK HIV-1 PCR kit | 8.9 |
| DRK HCV PCR kit | 6.8 |
| DRK HBV PCR kit | 0.6 |
| DRK HAV PCR kit | 0.65 |
Statistics
Standard deviation, coefficient of variation (CV) and Wilcoxon signed-rank test were calculated using Excel software (Microsoft Corp., Redmont, WA, USA).
Results
Analytical Sensitivity
In order to validate the analytical sensitivity of the assays, control and post-mortem samples were spiked with WHO standard preparations at 3 × LOD of the respective virus. The positivity rate of all DRK-PCR assays was 100% for both control and post-mortem samples (table 3).
Table 3.
Sensitivity data of the DRK PCR assays in cadaveric and control donor specimens
| Donor | Number of samples | Validity rate, % | Reactivity rate, % |
|---|---|---|---|
| HIV | |||
| Control | 40 | 100 | 100 |
| Cadaveric | 32 | 100 | 100 |
| HCV | |||
| Control | 40 | 100 | 100 |
| Cadaveric | 32 | 100 | 100 |
| HBV | |||
| Control | 40 | 100 | 100 |
| Cadaveric | 32 | 100 | 100 |
| HAV | |||
| Control | 40 | 100 | 100 |
| Cadaveric | 32 | 100 | 100 |
Analytical Specificity
Non-spiked aliquots of samples from blood donors (control) and post-mortem donors tested negative by all NAT assays. Specificity of NAT assays was 100% for both control and postmortem specimens for all parameters (table 4).
Table 4.
Specificity data of the DRK PCR assays in cadaveric and control donor specimens
| Donor | Number of samples | Validity rate, % | Specificity rate, % |
|---|---|---|---|
| HIV | |||
| Control | 40 | 100 | 100 |
| Cadaveric | 32 | 100 | 100 |
| HCV | |||
| Control | 40 | 100 | 100 |
| Cadaveric | 32 | 100 | 100 |
| HBV | |||
| Control | 40 | 100 | 100 |
| Cadaveric | 32 | 100 | 100 |
| HAV | |||
| Control | 40 | 100 | 100 |
| Cadaveric | 32 | 100 | 100 |
Reproducibility/Precision
We have analyzed mean threshold cycle, SD and %CV of control samples versus post-mortem samples. The %CV of postmortem samples did not exceed the value of 4.94 for the virus signal and 3.30 for the IC signal and is slightly higher than the %CV of the control samples (table 5). These findings could be confirmed in a comparison with 8 pre-mortem/post mortem sample pairs (table 6).
Table 5.
Reproducibility/precision (intra-assay) data of the DRK PCR assays in cadaveric and control donor specimens
| Analyte | Control donor specimen (n = 40) |
Cadaveric donor specimen (n = 32) |
||||
|---|---|---|---|---|---|---|
| mean Ct | SD | %CV | mean Ct | SD | %CV | |
| HIV | ||||||
| IC | 24.32 | 0.30 | 1.20 | 24.80 | 0.43 | 1.74 |
| Virus | 24.80 | 0.50 | 1.89 | 24.54 | 0.98 | 3.98 |
| HCV | ||||||
| IC | 24.62 | 0.20 | 0.80 | 24.53 | 0.24 | 0.98 |
| Virus | 26.60 | 0.47 | 1.76 | 26.34 | 0.86 | 3.25 |
| HBV | ||||||
| IC | 23.30 | 0.41 | 1.70 | 23.96 | 0.79 | 3.30 |
| Virus | 27.60 | 0.25 | 1.10 | 27.51 | 0.84 | 3.07 |
| HAV | ||||||
| IC | 21.61 | 0.83 | 3.18 | 22.89 | 0.57 | 2.49 |
| Virus | 23.82 | 0.19 | 0.80 | 23.65 | 0.42 | 1.79 |
Table 6.
Reproducibility/precision (intra-assay) data of the DRK PCR assays from matched pre mortem / post mortem sample pairs
| Analyte | Pre-mortem specimen (n = 8) |
Post-mortem specimen (n = 8) |
||||
|---|---|---|---|---|---|---|
| mean Ct | SD | %CV | mean Ct | SD | %CV | |
| HIV | ||||||
| IC | 24.82 | 0.31 | 1.24 | 24.72 | 0.23 | 0.92 |
| Wild type | 25.49 | 0.71 | 2.80 | 25.53 | 1.26 | 4.94 |
| HCV | ||||||
| IC | 24.52 | 0.19 | 0.75 | 24.7 | 0.19 | 0.75 |
| Wild type | 25.25 | 0.40 | 1.55 | 25.43 | 0.29 | 1.14 |
| HBV | ||||||
| IC | 23.97 | 0.28 | 1.16 | 24.19 | 0.25 | 1.07 |
| Wild type | 28.15 | 0.49 | 1.73 | 28.04 | 0.49 | 1.74 |
| HAV | ||||||
| IC | 22.18 | 0.22 | 1.00 | 22.47 | 0.16 | 0.73 |
| Wild type | 23.74 | 0.53 | 2.24 | 23.71 | 0.53 | 2.22 |
Additionally we compared serum and plasma post-mortem samples as a function of sample material (table 7) and of sampling time post mortem (table 8).
Table 7.
Reproducibility/precision (intra-assay) data of the DRK PCR assays in cadaveric serum and plasma specimens
| Analyte | Post-mortem serum (n = 16) |
Post-mortem plasma (n = 16) |
||||
|---|---|---|---|---|---|---|
| mean Ct | SD | %CV | mean Ct | SD | %CV | |
| HIV | ||||||
| IC | 25.00 | 0.47 | 1.89 | 24.64 | 0.32 | 1.30 |
| Virus | 24.46 | 0.75 | 3.07 | 24.63 | 1.18 | 4.80 |
| HCV | ||||||
| IC | 24.51 | 0.25 | 1.02 | 24.67 | 0.57 | 2.29 |
| 2.29 | ||||||
| Virus | 26.34 | 0.70 | 2.67 | 26.35 | 1.01 | 3.83 |
| HBV | ||||||
| IC | 24.15 | 1.07 | 4.42 | 23.74 | 0.30 | 1.27 |
| Virus | 27.62 | 1.06 | 3.86 | 27.40 | 0.56 | 2.03 |
| HAV | ||||||
| IC | 23.00 | 0.57 | 2.47 | 22.90 | 0.71 | 3.10 |
| Virus | 23.69 | 0.35 | 1.47 | 23.62 | 0.50 | 2.10 |
Table 8.
Reproducibility/precision (intra-assay) data of the DRK PCR assays in cadaveric specimens as a function of sampling time post mortem
| Analyte | Up to 24 h post mortem (n = 8) |
Up to 57 h post mortem (n = 24) |
||||
|---|---|---|---|---|---|---|
| mean Ct | SD | %CV | mean Ct | SD | %CV | |
| HIV | ||||||
| IC | 24.66 | 0.19 | 0.77 | 24.84 | 0.47 | 1.90 |
| Virus | 24.69 | 0.60 | 2.43 | 24.53 | 1.03 | 4.21 |
| HCV | ||||||
| IC | 24.55 | 0.19 | 0.76 | 24.54 | 0.28 | 1.16 |
| Virus | 25.58 | 0.49 | 1.92 | 26.47 | 0.86 | 3.23 |
| HBV | ||||||
| IC | 23.81 | 0.43 | 1.79 | 24.18 | 1.25 | 5.16 |
| Virus | 27.83 | 0.81 | 2.91 | 27.48 | 0.86 | 3.23 |
| HAV | ||||||
| IC | 22.41 | 0.42 | 1.88 | 23.07 | 0.54 | 2.36 |
| Virus | 23.77 | 0.25 | 1.05 | 23.63 | 0.44 | 1.88 |
Variation coefficients of serum and plasma control samples and post-mortem samples were comparable, whereas %CV of samples collected more than 24 h post mortem were slightly higher than those of samples which were collected up to 24 h after death.
All NAT tests included internal controls that monitored each PCR reaction. No reaction was invalid. Although 9 of 32 postmortem samples were highly hemolyzed (dark red in appearance), they could be verified to be correctly negative (non-spiked) or positive when spiked with defined concentrations of virus reference material.
Statistically significant differences as a function of sample material and sampling time were not found by the Wilcoxon test (data not shown).
Discussion
Studies showed the additional benefit of NAT testing for the safety of tissue and organ transplants [10, 11, 12, 13].
By contrast, up to date the detection of viral genomes in blood samples from tissue donors (post and pre mortem) is not explicitly described, neither in the requirements of the EU nor in the German Transplant Act, and NAT testing is only voluntary.
In this study we validated the DRK Baden-Württemberg-Hesse PCR assays in combination with the extraction on the Zelos x100 for the detection of HIV-1, HCV, HBV, and HAV in cadaveric tissue donor samples.
As only a small number of paired pre-mortem/post-mortem samples was available, we decided to include control samples from blood donors that were tested negative by routine antigen/antibody and NAT screening.
Positivity rates of spiked sample aliquots (serum versus plasma, control versus post mortem, pre-mortem/post-mortem pairs) were highly consistent, indicating 100% sensitivity.
The rate of unspecific reactive results was equivalent for all examined samples at zero. The highly congruent specificity of 100% shows the advantages of NAT compared to sero-logic assays where false reactivity can be a serious problem [8].
We could find only a slight influence of sampling time post mortem on the precision of NAT assays. Samples with a sampling time > 24 h had an elevated CV in comparison to samples with a post-mortem sampling time < 24 h.
NAT testing of blood donors is routinely performed with plasma. For cadaveric indications NAT needs to be validated with both sample qualities. The DRK PCR assays are equally qualified for serum and plasma post-mortem samples.
The extraction method used is routinely utilized for the screening of high-volume minipool blood donor samples of up to 96 samples per minipool. Therefore, it has the capacity to reliably remove large amounts of PCR inhibitors such as hemoglobin. This could be demonstrated by the data of 9 highly hemolyzed post-mortem samples.
As a prolonged time slot for sampling would improve the availability of tissue donations [14], this method could be well suited for the testing of post-mortem samples collected later than 24 h from death when the amount of disturbing substances increases.
On the other hand, it is known that degradation of viral genomes is dependent on sampling time from death [15, 16]. In order to verify the usability of NAT for screening of samples collected later than 24 h post mortem, further data on stability of HIV-1, HCV, HBV, and HAV in cadaveric samples need to be assessed. Such data are presented in another paper of this special issue [17].
In view of the validation data described in this study, we conclude that the DRK PCR assays fulfill all requirements of the Paul Ehrlich Institute and guarantee a reliable and highly sensitive detection of HIV-1, HCV, HBV, and HAV in cadaveric serum and plasma samples.
Disclosure Statement
The authors declared no conflict of interest.
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