Ina Wilkemeyer

A prospective time-course study on serological testing for human immunodeficiency virus, hepatitis B virus and hepatitis C virus with blood samples taken up to 48 h after death

The transmission of viral and non-viral infectious pathogens continues to be the most serious of the potential adverse effects of allogenic tissue transplantations. EU Directive 2006/17/EC stipulates that cadaveric blood specimens for serology testing in the context of post-mortem tissue donation must be taken not later than 24 h post-mortem. An expanded time slot would significantly improve the availability of tissue donations, but there are no significant data on the stability of infectious serology assays for anti-human immunodeficiency virus (HIV), anti-hepatitis C virus (HCV), hepatitis B virus (HBV) surface antigen (HBsAg) and anti-HBC core antigen (HBc) in samples collected more than 24 h post-mortem. In this prospective study, serum samples of 30 deceased persons were taken upon admission to the Institute of Forensic Medicine (University Hospital Hamburg-Eppendorf, Germany) and at 12, 24, 36 and 48 h post-mortem. All samples were measured twice, first using the Abbott AxSYM system, and then after ~9 months of storage at -70 °C using the BEP III System with Siemens and Ortho reagents. For HIV, six deceased persons with a pre-mortem HIV history were included. All samples (at committal and at 12, 24, 36, 48 h) were reactive. Indeterminate or false-negative results did not occur. For HCV, 17 deceased persons with a pre-mortem HCV history were included; 16 samples were reactive up to 48 h and one was reactive at 36 h post-mortem (48 h sample was not available). Indeterminate or false-negative results did not occur. For HBV, nine deceased persons were included: five samples were initially positive for HBsAg and remained positive up to 48 h, and eight of the samples were reactive for anti-HBc up to 48 h and one up to 36 h post-mortem (48 h sample was not available). Indeterminate or false-negative results did not occur. These data suggest that infectious serological testing may be extended for blood samples of potential tissue donors collected up to 48 h post-mortem to detect antibodies or antigens for HIV, HBV and HCV.

Validation of the Microbiological Testing of Tissue Preparations Using the BACTEC™ Blood Culture System

Background: Since blood culture bottles are validated by the manufacturer for blood only, an additional validation for the use with fluids of tissue preparations is necessary. Methods: Two 10-ml samples of cornea culture medium, histidine-tryptophan-ketoglutarate (HTK) solution, or Ringer solution at the end of femur head thermo-disinfection were given into blood culture bottles (BD BACTECTM Plus Aerobic/F, Anaerobic/F for cornea culture medium and BD BACTECTM Standard Aerobic/ Anaerobic for HTK and Ringer solution) and subsequently spiked with 10–100 colony forming units (CFU) of bacteria or fungi (aerobic bacteria: Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa; anaerobic bacteria: Clostridium sporogenes; fungi: Candida albicans, Aspergillus brasiliensis) according to the European Pharmacopoeia Chapter 2.6.1. Results: All tested bacteria and fungi could be detected in all solutions. All positive and negative controls were tested correctly. Compared to the positive controls, the microbial growth was delayed in the antibiotic-containing cornea culture medium, and negative in two cases of B. subtilis spiking. Conclusion: The use of BACTECTM blood culture bottles seems to be a suitable method for microbiological testing of HTK solution, Ringer solution, and, with limitations, also for testing of the antibiotic-containing cornea culture medium.

Comparison of in situ Corneoscleral Disc Excision versus Whole Globe Enucleation in Cornea Donors Regarding Microbial Contamination in Organ Culture Medium – a Prospective Monocentric Study over 9 Years

Background: Corneas needed for keratoplasty can be harvested using two techniques: whole globe enucleation and in situ excision of the corneoscleral disc. This study evaluates the rate of microbial contamination of the donor cornea organ culture medium according to the method of retrieval. Methods: All donor corneas of our cornea bank received between January 1, 2001 and December 31, 2009 put into organ culture and microbiologically tested were prospectively analyzed for microbial contamination of the organ culture medium. Results: 2,805 donor corneas could be included in this study in total. 975 of them were retrieved by whole globe enucleation (group 1) and 1,830 by in situ corneoscleral disc excision (group 2). 15 corneas of group 1 (1.5%) and 46 corneas of group 2 (2.5%) showed a contamination of the organ culture medium. The difference was shown not to be statistically significant (p = 0.082). Conclusion: The rate of microbial contamination in organ-cultured donor corneas does not seem to be dependent on the method of their retrieval.

Validation of the Serological Testing for Anti-HIV-1/2, Anti-HCV, HBsAg, and Anti-HBc from Post-mortem Blood on the Siemens-BEP-III Automatic System

Background: Some properties of blood are modified post mortem. These modifications might give false-negative or false-positive results in infectious disease testing. Most CE-marked test equipment for infectious serology testing is not validated for testing post-mortal blood. Validation, however, is obligatory, if the results are used for the release of tissues for transplantation. Methods: Samples of pre- and post-mortem sera were obtained from 20 cornea donors, and the results were compared for anti-HIV-1/2, anti-HCV, HBsAg, and anti-HBc on the Siemens-BEP-III Automatic System. Negative post-mortem sera were spiked with standard sera (PEI anti-HCV IgG, PEI HBsAg ad 1000 standard, anti-HBc IgG (WHO) NIBSC 95/522, PEI anti-HIV-IV) in concentrations which give low- and high-positive results for the respective marker. Results: All pre-mortem sera were negative for all markers. None of the post-mortem samples was false-positive. None of the spiked post-mortem samples was false-negative. Technical errors occurred during the validation process but could be detected and eliminated. Serum samples should be centrifuged immediately after collection, and it must be taken into account that post-mortem serum could rarely lead to blockage of pipetting systems due to clotting phenomena. Conclusion: There is no indication that post-mortem samples give false-negative or false-positve results with the test system and test kits used. The procedure described might serve as a model for validating other test kits on post-mortem samples.

Validation of Virus NAT for HIV, HCV, HBV and HAV Using Post-Mortal Blood Samples

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.

Virus NAT for HIV, HBV, and HCV in Post-Mortal Blood Specimens over 48 h after Death of Infected Patients – First Results

Objective: According to EU regulations (EU directive 2006/17/EC), blood specimens for virologic testing in the context of post-mortal tissue donation must be taken not later than 24 h post mortem. Methods: To verify validity of NAT in blood specimens collected later, viral nucleic acid concentrations were monitored in blood samples of deceased persons infected with HIV (n = 7), HBV (n = 5), and HCV (n = 17) taken upon admission and at 12 h, 24 h, 36 h and 48 h post mortem. HIV and HCV RNA were quantified using Cobas TaqMan (Roche), HBV DNA was measured by in-house PCR. Results: A more than 10-fold decrease of viral load in samples taken 36 h or 48 h post mortem was seen in one HIV-infected patient only. For all other patients tested the decrease of viral load in 36hour or 48-hour post-mortal samples was less pronounced. Specimens of 3 HIV- and 2 HBV-infected patients taken 24 h post mortem or later were even found to have increased concentrations (>10-fold), possibly due to post-mortem liberation of virus from particular cells or tissues. Conclusion: Our preliminary data indicate that the time point of blood collection for HIV, HBV and HCV testing by PCR may be extended to 36 h or even 48 h post mortem and thus improve availability of tissue donations.

Validation of Serological Testing for Anti-Treponema pallidum from Postmortem Blood on the Siemens-BEP®-III Automatic System

SummaryBackground: Infectious disease marker testing is obligatory for the release of human tissue for transplantation. Most CE-marked tests are not validated for postmortem blood. In a previous study we have validated the testing for anti-HIV-1/2, anti-HCV, HBsAg, and anti-HBc. Here, we present the validation of testing for antibodies against T. pallidum, which is the last marker obligatory for tissue release for transplantation. Methods: 17 samples of postmortem sera and 10 samples of both pre- und postmortem sera were obtained from cornea donors and tested for anti-T. pallidum on the Siemens-BEP-III-System. These sera were spiked with anti-T. pallidum-positive standard sera in concentrations which give low- and high-positive results at the respective dilution. Results: Two of the unspiked postmortem sera were false-positive most likely due to intense hemolysis (free hemoglobin > 50 mg/dl). Of the 25 negative postmortem sera, none of the spiked samples was false-negative after 0, 24 and 60 h. Conclusion: There is no indication that postmortem samples give false-negative or false-positive results with the test system and test kits used in cases of low hemolysis. The procedure described might serve as a model for validating other test kits on postmortem samples.

Contents of Forthcoming Issues · Themenvorschau

Spinal cord chimeras were produced by replacing a small fragment of neural tube of a 2-day-old White Leghorn chicken embryo with a similar fragment from a Japanese quail embryo. The embryo mortality was 61%, and 72% of hatched birds were ‘cripples’ and had to be sacrificed within 5 days after hatching. Forty-nine chimeras, 10.9% of the total number of operated embryos, were alive for more than 3 weeks. For at least 17 days after hatching, all birds behaved like normal chicks, and the grey quail-like feathers were the only manifestations of their chimerism. Initial neurological symptoms of unsteady walking and drooping of the wings were noted in all birds except for 1 that died an accidental death before it became sick. Advanced symptoms characterized by paralysis of the legs forcing the bird to lie on its side were noted in 40 birds. The chimeras could be divided into two groups, each consisting of 24 birds. The short-survival (SS) chimeras of the first group became terminally ill and had to be sacrificed within 3 months. The long-survival (LS) chimeras of the second group showed more protracted disease, in that only 16 of them showed symptoms of the advanced disease, and the majority showed partial or complete recovery. Ten of the LS birds were kept alive for more than 8 months. Furthermore, many LS chimeras lost their grey feathers. The hallmarks of neurohistological manifestations were mononuclear cell infiltrates, demyelinization with preservation of axons and scar formation. These lesions were restricted to the quail fragment of the spinal cord except for 2 birds in which distant cellular infiltrates were observed. Direct immunofluorescence tests for chicken IgG were positive in spinal cords of most SS chimeras but only of some LS chimeras.