Postmortem stability of laboratory parameters for HIV, HCV and HBV
I. Abstract p.1
II. Introduction p.1-3
III. Materials and Methods p.3-6
IV. Results p.6-8
V. Discussion p.8-11
VI. References p.11-13
I. Abstract
Safety and reliability of serology testing procedures is a crucial issue for allogenic tissue transplantation as transmission of viral and non-viral infectious pathogens remains its most serious potential adverse effect. The EU directive 2006/17/EC stipulates that the time slot for cadaveric blood specimens taken for serology testing must not exceed 24 hours post-mortem for HIV, HBV and HCV. Extending the postmortem interval for collection of samples to 48h would significantly improve availability of tissue donations and reduce the waiting lists, especially for corneal tissue. However, there is little significant data on the stability of infectious serology assays for anti-HIV, anti-HCV, HBsAg and anti-HBc for time slots greater than 24 hours post-mortem. In this prospective study, serum samples of deceased persons were taken upon admission to the Institute of Forensic Medicine, and after a further 12, 24, 36 and 48 hours post-mortem. All samples were measured twice using the Abbott Axsym® system (MEIA) in the UKE, and after 9-month storage at -70°C using the BEP-III®-System (ELISA) in the Charité Berlin. 6/6 samples of deceased with a pre-mortal HIV history were reactive at 0, 12, 24, 36, 48 h. 16/17 samples from deceased with a pre-mortal HCV history were reactive up to 48 h and 1/17 samples was reactive 36 h post mortem (48 h sample was not available). For HBV, 5/5 samples were initially positive for HBsAg and remained positive up to 48 h. 8/9 samples were reactive for anti-HBc up to 48 h and 1/9 sample up to 36 h post mortem (48 h sample was not available). Indeterminate or false negative results did not occur for neither of samples for HIV, HCV, and HBV. Data suggests that infectious serological testing may be extended for blood samples collected up to 48 hours post-mortem to detect antibodies or antigens for HIV, HBV and HCV of potential tissue donors.
II. Introduction
Postmortem stability for HIV, HBV and HCV has been investigated in regards to safety concerns in mortuaries or during autopsies, whereas today the most important issue is the transmission of viral infectious pathogens in allogenic tissue transplantation. About 30,000-35,000 allogenics tissues are transplanted each year in Germany (Pruss et al, 2011). The spectrum of tissues from cadaveric donors covers musculoskeletal tissues (bone, cartilage, ligaments, fascia), corneas and cardiovascular allografts (aortic and pulmonary valve, pericardium, veins, arteries). According to Yao et al (2007), donors rates of HIV/HCV/HBV infection are lower than in normal population (1:2 in corneas, 1:1 to 1:10 in cardiovascular transplants, 1:40 in musculoskeletal tissues ) but higher than in first-time blood donors. Access to donor health history is often problematic due to emotion in family members after death. Pre-mortal blood samples of cadaveric donors are usually not available, especially when they are not multi-organ donors (e.g. donors from Forensic Medicine Institutes) (Püschel et al., 1991). In the case of a hospital stay, the pre-mortal blood samples are either no longer available or older than 7 days. Moreover, information from multi-tranfusion donors is not reliable because of hemodilution.
EU directives 2004/23/EC and 2006/17/EC defined basic requirements of viral safety in tissue donation in general and state that cadaveric blood specimens for serology must be taken at a post-mortem interval of less than 24 hours. Infectious serological tests (anti-HIV 1/2, anti-HCV, HBsAg, anti-HBc, TPHA) are mandatory while nucleic acid testing (NAT) is not required. However, if samples within 24 hours post-mortem are not available, donation may not occur, resulting in a loss of potential donors. Moreover, in unexplained or unnatural deaths, the legal time of death is not known but only the time of discovery.
Few studies have investigated postmortem behaviour of antibodies for time slots greater than the legal interval of 24h. Pepose et al (1987) tested premortem and postmortem samples at intervals as high as 36h and obtained a sensitivity ranging from 94 to 97% and a specificity of 99%, but false positive or negative results did not seem to correlate with the length of post-mortem interval. Püschel et al in 1989 demonstrated persistence of HIV in cadavers stored under non-refrigerated conditions up to 36h after death in 13 of 2581 fatalities tested for HIV using ELISA. Miédougé et al (2002) investigated sera from potential organ (n=113) and cornea donors (n=368) for intervals enlarged to 48 hours after death and found a high rate of false positive results in cornea donors. Nyberg et al (1989) studied postmortem stability for HIV at intervals ranging from 1 to 6 days and obtained 100% sensibility for blood specimen, whereas 2/5 false negative results occurred for tissues. Heim et al., (1999) compared 33 pre-mortal and 33 post-mortal samples of the same donors at postmortem intervals as long as 60 hours. The authors found 16 false positive results for HBsAg, and 1 false negative result for anti-HCV However discrepant results did not correlate with enlarged postmortem intervals. In 2006 Challine et al. tested 565 cornea donors for anti-HIV, anti-HCV, HBsAg, anti-HBc at intervals of <12h, 12-24 h, 24-36 h and> 36 h and obtained discrepant or equivocal initial result in 12,1% cases.
Present research suggests that postmortem screening of infectious pathogens is not fully reliable. However, in all cases discrepant results did not correlate with enlarged postmortem intervals and false negative results remain exceptional, whereas false positive results lead to discarding of organs, tissues, and corneas.
My research is based on Pruss et al's (2011)'s study. Postmortem behaviour of serological laboratory in HIV, HBV or HCV parameters was investigated for the first time over regular time intervals of 12, 24, 36 and 48 hours. A post-mortem stability of these parameters over an extended interval would exclude an enlarged interval to be a cause of discrepancy in pre- and postmortem results. The number of possible donors would thus increase, thus reduce the waiting list in Europe, especially for cornea transplants, as the postmortem explantation delay was fixed at 72h after death. However no further prospective time course research exists nowadays.
III. Materials and Methods
Collection of samples
Serum samples were obtained from 6 HIV-positive deceased, 17 HCV-positive deceased and 9 HBV-positive deceased and a control group from cornea donors without these infections, of whom the relatives had compulsorily given informed content. Information was based on certificates of death from a hospital doctor or given by the relatives to the emergency doctor in forensic cases. If a non-natural cause of death was reported on the death certificate, the release by the legal authorities was also required before the collection of blood samples. Upon arrival in the forensic medicine department of the UKE, the time of death was determined in accordance with the forensic standards, as well as the acquisition of the infection history. The average time between death and delivery time was 5.9 hours (minimum of 2.0 h, maximum 13.0 h). None of the deceased showed signs of decomposition. The deceased were stored for the study duration in the cooling chambers at the Institute of Forensic Medicine under monitored temperatures of 2-8 °C. The study was carried out after examination and approval by the Ethics Committee of the Hamburg Chamber of Physicians (AZ: WF-024/09).
Serum samples of deceased persons were taken upon admission to the Institute of Forensic Medicine, and at 12, 24, 36 and 48 hours post-mortem. Due to technical or logistical reasons (e.g. low blood volume, delayed approval by the relatives, forensic issues) it was not possible to collect blood from all deceased at all time points. Also the control testing in the Charité was not in all cases possible. After disinfection of the blood sampling puncture site 20 ml of whole blood was extracted from peripheral large vessels (A./V. femoralis, A./V. subclavia) using sterile TSK-Supra hollow needles 2.00 x 100 (TSK) and sterile 20-ml syringes (BD Discardit) then transferred into labelled 10 ml test tube (Harre Company) and followed immediately by centrifugation and serum separation. Drawing blood from the heart was necessary in some cases. One part of the serum was then stored at 2-6 °C and measured within 12 hours in the Institute for Medical Microbiology and Virology at UKE. The second part of the serum was frozen immediately, stored at -80 °C, and tested at regular intervals (approx. 9 months later) in the Institute of Transfusion Medicine at the C in Berlin. All serum samples showed macroscopically a minimal hemolysis after 36 hours post-mortem and a moderate to severe hemolysis after 48 hours post-mortem. The serum samples of committal, 12 hours post-mortem and 24 hours post-mortem were macroscopically without pathological findings.
Measurements:
History of tests
Specific serologic assays that detect the presence of viral targets were developed 30 years ago and have progressed from manual, labor-intensive radio immunoassay and enzyme immunoassay procedures to procedures that use automated batch-processing analyzers and, most recently, to procedures that use random-access systems apt to process a variety of tests simultaneously. The traditional approach to the diagnosis of viral targets has been to collect a serum sample and to test it for the presence of surface antigen or antibodies to the core of viral target by solid phase radio- or enzyme immunoassays.All methods rely on the Antigen-Antibody complex formation.
Multiple variations have been developed in means to create a signal for viral targets, mostly using enzymes or secondary antibodies. The most widely used laboratory method that enablesto detect the presence of anantibodyor anantigenin a sample is the Enzyme-linked immunosorbent assay(ELISA). It was developed in 1971 by Peter Perlmann and Eva Engvall independantly and has played a major role in clinical diagnostic and in quality control for many industries and was the first screening test widely used for HIV because of its high sensitivity. Protein-preparations of viruses are immobilized to a solid surface using microtiter plates with 96 cavities and made off processed polysterene. Serum is then pipetted in the cavities so that if they contain the specific antibody they will form antigen-antibody complexes. Those Ag-Ab complexes are then detected using either a secondary antibody, or a covalently linked enzyme, which will typically produce a visible signal (fluorescence, colour change, etc) which will indicate the presence (qualitative ELISA) or the quantity of viral target in the sample and will be typically quantified with a spectrometer. Between each step, the plate is washed with a mild detergentsolution to remove any proteins or antibodies that are not specifically bound.
The test has many variations including indirect ELISA, which uses a secondary antibody linked to an enzyme in addition to a primary antibody. Another variant is the so-called sandwitch ELISA, a technique in which two antibodies can bind to two different epitopes on the same antigen. The "capture antibody" is immobilized on a microtitre well while the other antibody is labeled with an enzyme. Sample containing the antigens will react with the immobilized antibody, then the labeled antibody is added and will react with the bound antigen. In competitive ELISA the enzyme-linked secondary antibody compete with the sample antigen which is associated with the primary antibody. Free antigen and antibody are incubated to form antigen-antibody complex then bound to antigen-coated surface in the assay plate. The unbound antibody-antigen complex is first washed off then the enzyme-linked secondary antibody against the primary antibody is added before the substrate. Antigen concentration can then be determined by the signal strength elicited by the enzyme-substrate reaction. Competitive ELISA enables to use crude or impure samples and still specifically bind its antigens. Newly developed Multiplex ELISA uses a solid phase made up of an immunosorbent polystyrene rod with 8-12 protrudingogives, immersed in a test tube containing the collected sample. The ogives are then dipped in microwells of standard microplates pre-filled with reagents. Multiplex ELISA allows simultaneous detection of different antibodies and/or different antigens for multi-target assays.
Micro-particle enzyme immunoassays (MEIA) detects viral targets using fixation of the antigen between antigen-specific, enzyme-labeled antibodies and antibodies bound to a solid-phase support (liquid suspension of micro-particles). Antigen-Antibody complexes are then detected and quantified by analysis of fluorescence from the enzyme-substrate interaction.
Tests confront routine limits such as limited stability, variable specificity but allow wide range of analysis for in vivo preparation.
Analysis Device
All samples were measured twice. First, using a MEIA device from Abbott Axsym® in the UKE, and approximately 9-month later (storage at -70°C) using ELISA-test provided by BEP-III®-System in the Charité Berlin.
UKE used following analysis device: micro-particle enzyme immuno assays (MEIA) with test kit HIV Ag/Ab Combo (Abbott), HCV-Version 3.0, HbsAg V2 (Abbott) and Core (Abbott) for anti-HBc from Abbott Axsym system (MEIA). Th edevice used in UKE is intended for the qualitative detection of HBV core antigen (IgM anti-HBc) in serum or plasma, HIV-Ag/Ab complexes as well as HbsAg and antibodies to HCV. The tests were performed according medical laboratory standards, as well as the specifications of the manufacturer. The test laboratory met all the requirements for internal and external quality controls.
Charité (Berlin) used following analysis device: ELISA test with test kit Enzygnost Anti-HIV ½ plus for anti-HIV, HCV 3.0 ELISA with enhanced SAVE (Ortho) for anti-HCV, Enzygnost HbsAg 6.0 for Hbs1g and anti-HBc monoclonal was used for anti-HBc.The BEP® III System (ELISA) is used for automated microtitration plate processing which incorporates washing, dispensing of reagents,incubating and result evaluation of microtitration plates. The tests were performed according medical laboratory standards, as well as the specifications of the manufacturer. The test laboratory met all the requirements for internal and external quality controls.
The minimum control requirement for Axsym and BEP-III assays was a single sample of each of the Negative and Positive Controls tested at 12, 24, 36 and 48 hours postmortem for each reagent lot as a means of evaluating the assay calibration. Controls may be placed in any position in the Sample Carousel.
Statistical analysis was performed using software STATA for Windows, Version 10 (Stata Corp., College Station, TX, USA). Using a regression analysis by ANOVA (analysis of variance) the 48-hours results were compared with the results of the time points committal, 12 h, 24 h and 36 h.