Band 29, Heft 2

Relevant blood transfusion-transmitted viruses are still HBV, HCV and HIV. As newly emerged infectious agent, the vCJD prion is relevant as well and will cause difficulties within the next decade, since there is no diagnostic tool applicable to blood donation testing. Still, the most effective measure for the virus safety of blood is donor selection followed by antibody and nucleic acid testing, followed by derichment and inactivation performed to the extent possible. For selected recipients, relevant viruses are CMV and Parvovirus B19. Diagnostic testing for both viruses is no challenge. The prevalence of HTLV-I is very low in Germany but higher in many other countries. Therefore epidemiological surveillance is sufficient to control the spreading of this retrovirus in Germany. As the West Nile fever virus and the SARS coronavirus demonstrate, old viruses may newly enter the donor population and the efficient way to prevent these viruses from spreading is still the usual quarantine for donors who have visited contaminated areas. The history of blood transfusion shows that by taking appropriate measures safety will be increased, but also that new infectious agents will enter the donor population in future years.

The diagnosis of HCV infection is often hampered by false positive reactivities in antibody screening assays. Therefore, confirmatory assays are necessary. Since most unspecific reactivities are only slightly above the cut-off value, the intensity of the reactivity should always be considered. Particularly in groups with low HCV risk such as blood donors, positive reactivities obtained by one test system can often be ruled out by re-testing with another format (e.g. an enzyme immunoassay instead of a microparticle enzyme immunoassay). Most confirmatory assays are based on a recombinant immunoblot assay (RIBA); these tests should always be done in patients diagnosed as HCV positive for the first time. Additionally, in many cases, tests for HCV RNA are necessary. Based on these results, an evaluation of the risk of transmission to household contacts is possible and in patients receiving antiviral treatment, the success can be monitored. A negative PCR result in patients showing high positive antibody reactivities does not necessarily represent loss of the virus since even in chronic carriers, viral replication can be intermittently very low. Immuno-compromised patients often show prolonged seroconversion so that antibody screening assays remain negative for a long time while the patients have high level viraemia. Therefore, we recommend regular RT-PCR in populations at risk such as patients on chronic dialysis. To evaluate patients for antiviral treatment, the determination of the genotype is necessary. The HCV genotype also has an influence on the course of liver disease in chronic carriers. Possible transmission of HCV, e.g. by blood products, can be elucidated by sequence analysis of the high variance region (HVR) of the virus.

Several test systems are available for the diagnosis of infection with the hepatitis B virus (HBV). In most cases, reliable results lead to the detection of acute infection or chronic HBV infection or immunity due to past infection or active immunization. In some cases, however, results may be difficult to interpret or even misleading and special assays are necessary to answer more specific questions. During acute or chronic HBV infection, not only the qualitative detection of HBsAg is necessary, but also the quantitative determination may allow for the diagnosis of activity of the disease. In most cases, HBeAg closely correlates to viraemia and infectivity, but mutations within the pre-core region of the HBV genome may lead to a loss of HBeAg and to seroconversion to anti-HBe while the virus concentration in serum is high and patients are severely ill. Anti-HBc assays often give false positive test results which can be ruled out by re-testing a serum dilution. IgM antibodies against HBcAg are not only found during acute infection but may become positive again during reactivation in chronic virus carriers. Different genotypes of the virus are prevalent in different parts of the world and multiple infections are possible; these may lead to the simultaneous detection of HBsAg and anti-HBs. Viral DNA can be detected by several methods with different lower detection limits. Screening of blood donors to rule out HBsAg-negative virus carriers is done by polymerase chain reaction, since this method has the highest sensitivity. Infectivity of HBsAg-positive carriers may also be determined by other assays such as branched DNA or molecular hybridization. Patients on treatment with nucleoside analogs may show an increase in DNA concentration after an initial decrease or even loss. This may be due to mutation within the YMDD-motif of the DNA polymerase which can be detected by sequence analysis. In this overview, hints will be given on how to evaluate positive results in the different assays. In addition, it gives advice on how to establish a suitable diagnosis for a variety of clinical questions. Several suspicious constellations are discussed as well as the possibilities to clarify them.

European manufacturers of plasma products and German blood transfusion services were the first to introduce nucleic acid amplification testing (NAT) of blood products in the mid-1990s. Their primary goal was to increase the safety of blood by closing as far as possible the diagnostic window, which exists after the onset of viral infection until the appearance of the first detectable antibodies. Sample preparation, transport and storage are crucial steps in a quality-controlled PCR. Sensitivity and contamination rates highly depend on the sample preparation and storage techniques. Anticoagulants must be selected carefully because some may inhibit the PCR. Dilution of samples by pooling needs to be considered and should be compensated for by subsequent virus enrichment procedures, e.g. centrifugation. The whole process of sample preparation, pooling and virus enrichment must be validated and quality control measures must be implemented. Reagents for the extraction of viral nucleic acids should not pose any risk to the laboratory staff. Nevertheless, the reagents should be highly efficient in liberating viral nucleic acids at high yield and purity for the following amplification reactions. At this critical stage, quality control measures should guarantee an efficient extraction process and contain potential sources of contaminations. Several methods are available for the amplification of nucleic acids. PCR is the most common, especially in in-house assays. The amplification of nucleic acids should be performed as far as possible in a closed system, which may be guaranteed best by real-time PCR approaches. Reaction tubes need never be opened during the amplification because detection can be performed through the closed tube. Amplicons that could contaminate the following PCR reactions will not be released. It is of great importance to blood transfusion services to guarantee that negative results un-equivocally indicate virus negative blood donations. Therefore, internal control sequences should be implemented in each individual PCR reaction in order to monitor that the individual PCR has worked correctly. Besides internal control sequences, external negative and positive controls should be implemented in each PCR run to demonstrate false positive reactions as well as to monitor pre-PCR processes like virus enrichment and extraction. The whole process needs to be validated according to the criteria set in national guidelines or by national authorities. External quality assessment programs are highly recommended.

Five hepatitis viruses (A-E) cause more than 80% of cases of viral hepatitis. However, the fact that nearly 20% of individuals with acute hepatitis test negative for all known hepatitis viruses, as do up to 10% of patients with transfusion-associated hepatitis, suggests the existence of other viral hepatitis agents. Hepatitis G virus (HGV) and TT virus (TTV) were originally proposed as potential causative agents. The SEN virus (SENV) belongs to a recently discovered group of DNA viruses whose members (SENV D and SENV H) are associated with post-transfusion hepatitis. Eight different strains of SENV have been identified and provisionally classified as members of the Circoviridae family, a group of small, single-stranded, non-enveloped circular DNA viruses which includes, in addition to SENV, the TT virus (TTV), TUS01, SANBAN, PMV, and YONBAN viruses. SENV is a single-stranded circular, non-enveloped DNA virus of ~3.600 to ~3.900 nucleotides with at least three open reading frames (ORFs). The prevalence in different populations shows great variability with marked differences between different countries and groups. Although parenteral transmission is very likely, other routes of transmission are not excluded. Mother-to-infant transmission has been demonstrated. Data on other acute and chronic liver diseases are sparse. Further studies are needed to define the pathogenesis and clinical importance of SENV infection.

In practical use, the experiences of single marker interpretation influence the analysis of tumor marker profiles. From a mathematical point of view, there are different possibilities to link individual measured data. When comparing such methods of resolution, significant differences in their efficiency are observed. An increase in sensitivity generally results in a loss of specificity. Therefore, the choice of the mathematical process is decisively influenced by subjective expectations. The goal of the analysis of tumor marker profiles is raising the sensitivity while maintaining a high specificity. Based on the study of frequently used strategies of data evaluation, effects are described from which a universally valid concept of data evaluation can be derived. Special attention is given to the independence of the mathematical processes from specified cut-offs and thereby from diagnostic testing methods. Instead of quantitative measured data, qualitative valuations of decision security are handled. This not only enables a rise in sensitivity but also allows a plausible interpretation of results from the user's view. Thus the analysis of tumor marker profiles seems (fuzzy) logical and opens up new avenues to make far-reaching contributions to the clarification of clinical problems by laboratory analysis.

The past decade witnessed an increasing interest in assessing circulating DNA in the plasma and serum of patients with malignant and non-malignant diseases. This might be due to the availability of new and sensitive methods for the determination of qualitative and quantitative changes in circulating DNA. As, previously, tumor-specific mutations or epigenetic modifications have been detected predominantly in tissue specimens, the appealing possibility to use less invasive though specific methods for tumor diagnosis was a noticeable incentive for the exploration of circulating DNA. A considerable part of the circulating DNA, which is mostly present in serum and plasma as nucleosomal DNA, is released during apoptotic cell death. Because the rate of apoptosis is deregulated in many pathological situations such as degenerative, traumatic, ischemic, inflammatory, and malignant diseases, and because many cytotoxic therapies aim at reducing the cancer cell number by apoptosis, the cell death product “circulating DNA” might serve as an attractive and appropriate biochemical correlative. In this review, the physiological and pathophysiological background of the arrangement of DNA as nucleosomes and of its release into circulation is shown. Further, the metabolism of circulating DNA in plasma and serum and its role in the pathogenesis of various diseases is discussed. Finally, the diagnostic relevance of qualitative and quantitative changes in circulating DNA for screening, differential diagnosis, prognosis, monitoring of systemic therapies, early prediction of therapy response and detection of recurrence in malignant diseases is reviewed. Concluding, some methodical considerations regarding the measurement of circulating DNA are given.

One hospital-based laboratory and one laboratory serving a number of hospitals prospectively studied 3.907 blood culture bottles of the BACTEC™ 9000 System (BD Diagnostics, Heidelberg, Germany) (1.888 aerobic bottles, 1.880 anaerobic bottles and 139 pediatric bottles). Information on media type, blood volume, time of inoculation, entry into the system, anti-microbial treatment, time to detection, identification to the species level and positive rate compared to terminal subculture were recorded and analysed. Twenty-seven bottles were classified as false negative, seven of these had a positive cohort bottle ( Staphylococcus aureus , Enterobacter cloacae , Enterococcus faecalis , Candida albicans, Burkholderia cepacia and two strains of Pseudomonas aeruginosa ), four out of these seven were not expected to grow in anaerobic media, five patients were under antibiotic treatment and one bottle had a transport time >48 h and is also mentioned in this group. Fifteen of the false negative bottles had a transport time of >48 h. Eleven patients underwent antibiotic therapy, six out of 27 false negative bottles had a transport time <48 h, two patients had antibiotic therapy. Out of these six isolates, one C. glabrata did not grow in the anaerobic bottle. The rate of clinically relevant false negative blood cultures (pathogen not detected in cohort bottle) in bottles that were entered into BACTEC 9000 within 48 h after inoculation is 0,15%. All efforts need to be directed towards expediting the transportation of material to the microbiology laboratory.