Volume 28, Issue 1

There is an increasing demand for a rational diagnosis of patients with thrombophilia by clinical laboratories. The protein C pathway plays an important part in this system. To rationalize the diagnostics, methods have been introduced to test the anticoagulant capacity of the whole protein C pathway. These screening assays should reliably detect a factor V Leiden mutation, protein C and S deficiency. These assays differ in detection methods, interfering factors and even in their responsiveness to protein S. The methodological approach is based on the activation of endogenous plasma protein C by a snake venom extract. Clincial evaluations demonstrated the validity of these tests to screen for a factor V Leiden mutation and protein C deficiency. Protein S deficiency cannot be identified sufficiently. The assays, however, seem to have additional positive effects. They could be used to assess the risk for thrombosis in the future. Initial studies confirm this hypothesis. Hence, a positive test result in protein S-deficient patients correlated with a higher risk for thrombosis.

APC resistance, defined as a poor anticoagulant response to activated protein C (APC), was described in February 1993 as a novel and important inherited risk factor of thrombosis. An avalanche of research activities was triggered that during the past eleven years has elucidated not only the molecular mechanisms of APC resistance, but also general pathogenetic mechanisms of thrombosis. In 1994, factor V (FV) was identified as the factor associated with APC resistance, and a point mutation in the FV gene (Arg506Gln or FV Leiden) was found to be the genetic cause of APC resistance. This mutation eliminates one of three APC-cleavage sites in FV, which not only results in impaired APCmediated degradation of FVa, but surprisingly also of FVIIIa. The dual effects of FV Leiden were explained by the discovery of a novel property of FV, namely that FV has the potential to express anticoagulant activity in addition to being a procoagulant protein. As an anticoagulant, FV functions as an APC cofactor in synergy with protein S in the regulation of FVIIIa in the tenase complex. Studies of APC resistance and FV Leiden performed in many laboratories world-wide have given insights into clinical, epidemiological as well as molecular details. This review focuses on what we have learned since 1993 about the molecular mechanisms of APC resistance and the involvement of FV in the regulation of blood coagulation by the anticoagulant protein C system.

Patients with the highest risk of recurrent thromboses due to inherited or acquired plasmatic factors can be identified by laboratory testing, the so-called "thrombophilia diagnostics". This review summarizes the advantages of thrombophilia diagnostics in organ transplant recipients for the primary prevention of vessel occlusion and transplant dysfunction. On the basis of published studies, the primary prevention of the transplant dysfunction of the kidney is a useful indication for thrombophilia diagnostics while this testing is only of limited use in heart transplant recipients. In patients with a proven hypercoagulable state, appropiate immunosuppressive agents should be chosen with respect to prothrombotic effects and prophylactic anticoagulation should be considered.

Women with thrombophilic defects have been shown to be at increased risk, not only of pregnancy associated thromboembolism but also of other vascular complications of pregnancy, including preeclampsia and fetal loss. First trimester fetal loss is associated with factor V Leiden mutation, activated protein C resistance without factor V Leiden mutation and prothrombin G20210A mutation. Late nonrecurrent fetal loss is associated with factor V Leiden mutation, prothrombin mutation and protein S deficiency. Concerning acquired thrombophilia, recurrent fetal loss is a well-documented finding in patients with antiphospholipid antibodies. Associations between thrombophilia polymorphisms and an increased risk of intrauterine growth restriction have been discussed in small series of cases but could not be confirmed in large scale studies. Frequencies for anticardiolipin antibodies or lupus anticoagulants and antinuclear antibodies were significantly higher in women with infants small for gestational age compared to controls. Concerning preeclampsia, gestational hypertension and thrombophilia, a number of studies have examined these relationships with conflicting results. For factor V Leiden, MTHFR C677T and prothrombin mutation, no association with preeclampsia was observed, when severe cases were excluded. If studies were restricted to those of severe preeclampsia, an association with the factor V Leiden mutation was apparent and, to a lesser extent, with the MTHFR-mutation. For antithrombotic therapy, it was shown that in women with antiphospholipid syndrome and recurrent pregnancy loss, unfractionated heparin plus lowdose aspirin results in significantly better gestational outcome than lowdose aspirin alone. Concerning therapy of women with inherited thrombophilia and pregnancy loss, only small, uncontrolled studies are available, demonstrating improved pregnancy outcome when low molecular weight heparin (LMWH) is used for treatment. In conclusion, heritable thrombophilia and the antiphospholipid-syndrome are major causes of fetal loss after exclusion of other underlying pathologies like chromosomal abnormalities, and screening should be recommended. LMWH with or without aspirin may be used for treatment. There is little value in antenatal screening for prothrombotic polymorphisms to predict the development of small for gestational age infants, preeclampsia or gestational hypertension.

The severe acute respiratory syndrome (SARS) first occurred in southern China at the end of 2002 and has caused more than 8000 illnesses in total with over 700 fatalities as of end of August 2003. A unique collaborative effort, led by the World Health Organization (WHO) and uniting several laboratories from around the world, allowed the identification of the causative agent within only one month. It is a hitherto unknown novel coronavirus (provisionally termed SARS-associated coronavirus or SARS-CoV) that fulfills Kochs postulates. The virus is easily propagated in cell culture (under suitable highsecurity laboratory conditions) which should facilitate further studies into its stability as well as the development of antivirally active substances and vaccines. Although diagnostic laboratory tests, particularly for the detection of viral nucleic acid and virusspecific antibodies, were quickly available, the case definition for SARS continues to be based on clinical and epidemiological criteria. In view of the threat of a (seasonal) recurrence of the infection it is urgently necessary to evaluate and to further improve the available laboratory methods. SARS is a good example of how rapidly an infectious disease may spread through international travel activities; but it also demonstrates the importance of a wellcoordinated international cooperation in such a case. Through the use of the latest advances together with more traditional laboratory methods and the constant exchange of (interim) results, patient samples and reagents, an unprecedented collaborative effort soon achieved a breakthrough. This should provide encouragement in the struggle against other novel infectious agents.

The genetic variability of hepatitis B virus (HBV) represents a challenge for the sensitivity of immunodiagnosis, especially for the detection of surface antigen (HBsAg). There are two types of variants of HBV. Naturally occurring variants are the results of random changes selected over years of population pressure. These variants include HBV genotypes and unusual sequences, which may be poorly detected by immunoassays. The selected variants are mutants that arise in individuals under medically (vaccine, hepatitis B immune globulin and antiviral therapy) or naturally (chronic hepatitis B) induced immune pressure. HBV S-gene mutants have been identified in successfully immunized people worldwide. Based on the assumption that current vaccines containing S protein do not cross-protect against S gene mutants, a mathematical model predicts the disappearance of wild-type HBV in areas with HBsAg endemicity and the emergence of S gene mutants in approximately 100 years as a consequence of universal HBV vaccination. Mutant viruses may escape detection by commercial HBsAg kits. There are several reports on HBsAg negative carriers (HBV-DNA positive) of S gene mutants with immunosilent infection or "unusual" serologic constellations. Although S gene mutants have been found to be associated with a more severe clinical course of HBV infection and hepatocellular carcinoma, the clinical significance of the genetic variability of HBV genotypes and HBsAg mutants needs to be further investigated. Detection of HBsAg needs to be improved by the introduction of new HBsAg assays able to recognize S gene mutants described so far and with a lower detection threshold than current immunoassays in order to detect smallest amounts of HBsAg in low-level carriers. There is also a need for more complete epidemiological data on the prevalence of HBsAg mutants in Western Europe and assays for the (differential) screening of mutants need to be developed and evaluated.

Periodic evaluation of suppliers is an integral component of all relevant quality management systems established in medical laboratories. Performing such evaluation is always laborious, but rarely entails a perceptible benefit for laboratories and their suppliers. Therefore, we worked out 19 criteria for the assessment of suppliers and a model for the analysis of results in cooperation with 5 representative manufacturers for in vitro diagnostics and 11 medical laboratories. The criteria consider the special needs of medical laboratories combined with an effective procedure for continual quality improvement. In a pilot scheme the model was evaluated for its suitability in practical use. Various methods for data analysis enable suppliers to get relevant information about individual and collective customer satisfaction. In the case of centralised data collection and analysis a suitable tool for benchmarking is available.

Since 1994, efforts to maintain quality assurance in Swiss medical laboratories have had a legal basis in Switzerland which allows the enforcement of measures to improve efficiency in all types of laboratories. Proof of an adequate quality assurance program is a prerequisite for all laboratories which are reimbursed by the social security system. The first phase defined measures with regard to quality assurance and quality control. In the second phase, a continuous education program was established which allowed people with different educational backgrounds PhD, MD, pharmacist) to enter the program. In Switzerland, half of the analytical volume of medical diagnostics is carried out in the practitioners laboratory. By establishing a continuous education program especially designed for practitioners and by enlarging the number of hours of training for the students in medical school, the basis of laboratory medicine should become familiar to care providers as well. Using random spot checks, the laboratories are checked annually to see if they comply with legal requirements. Laboratories which do not meet the minimum requirements are subject to sanctions: these are primarily of educational nature, but can result in the suspension of remuneration by the social security system. More than 100 laboratories from different disciplines of laboratory medicine were accredited on a voluntary basis by the end of 2003. The number of newly accredited laboratories has been declining since 2001, since most of the labs which initially wanted to implement a quality management system are already accredited. As the next step, the authorities intend to improve the quality management of the pre-analytical phase.

This article deals with the situation of quality management in Austrian medical laboratories. Starting in 1994 after several rounds of discussion, the Society for Good Analytical und Laboratory Practise (GALP) was founded by the two major societies for laboratory medicine und clinical chemistry. This society had the purpose to work out strategies for quality management for Austrian medical laboratories. The major outcome of the activities was the adoption of ISO 9002:1994 as the model for quality management in medical laboratories and the publication of a guideline for the implementation of ISO 9002:1994 in the medical laboratories. This guideline was the basis for many medical laboratories in Austria to seek ISO 9001/2:1994 certification. Currently most medical laboratories (except those in hospitals, where certification is not so prevalent) are ISO 9000 certified. The next major development in quality management was the adoption of the third medical device guideline of the European Union (IVD-Guideline) and its national implementation in the Austrian Law on Medical Devices. This law includes several clauses, which can be used by the ministry of health to require quality management systems in medical laboratories and to establish rules for certification agencies in this field. However, the implementation of these clauses in the law is weak due to the lack of resources on side of the ministry. Nevertheless, European Union demands will lead to more effective measures as they did in the field of clinical studies. The development of EN ISO 15189:2003, which will be published in German language in December 2003, will lead to a change from certification to accreditation, at least in the high quality segment of medical laboratories. This will bring Austria in line with the international situation. Currently, quality does not have any impact on reimbursement of medical laboratories in Austria, so quality matters only in terms of cost but not in terms of higher earnings. Therefore quality is motivated by idealistic factors which tend to lose when economic pressures rise. In the future, changes in this field will be necessary too.

We have evaluated the cardiac troponin I (cTnI) enzyme immunoassay AIA-PACK cTnI 2G (Tosoh Bioscience). The interassay precision at different cTnI levels (0.30 6.70 ng/ml-1) showed coefficients of variation (CV) between 5.00% and 4.71% on the AIA 600 II instrument. These data are in accordance with the functional sensitivity (0.04 ng/ml-1) and analytical sensitivity (0.02 ng/ml-1) specified by the manufacturers. Comparative troponin I measurements in serum samples were performed on the AIA-600 II analyser (Tosoh Bioscience) and the Dimension RxL analyser (Dade Behring). The cTnI concentration of serum samples (n=226) ranged from 0 ng/ml-1 to 114 ng/ml-1. Regression analysis of the cTnI values obtained showed a close correlation between both results (RxL=0.96 AIA+0.38; r=0.960). To induce proteolytic in vitro degradation of the cTnI molecule, patient serum samples (n=20) were incubated at 37C for 24 h and the cTnI concentration was determined before and after the incubation with both analysers. The cTnI concentration measured with the AIA-600 II analyser showed nearly identical values before and after the incubation period. However, cTnI measured with the Dimension RxL analyser showed decreased cTnI values in a range of 10 to 50% after the incubation period. The AIA-PACK cTnI 2G assay uses two different monoclonal antibodies against two epitopes in the proteolytic stable region of the cTnI molecule (between position 40 and 90 of the cTnI amino acid sequence). Therefore, measurement of cTnI in serum remains precise and specific in spite of in vitro proteolysis.

Infections with hepatitis C virus (HCV) are pandemic. The WHO estimates a worldwide prevalence of 3%. For monitoring of chronic hepatitis C, assays for direct virus detection are employed. In this study, an improved version of the Ortho® trak-C assay™ (Ortho-Clinical Diagnostics, Raritan, NJ) for detection of the HCV core antigen was evaluated. Precision, interassay, and intraassay variation were determined and a total of 115 clinical specimens were investigated. When precision of the Ortho® trak-C assay™ was evaluated, the assay gave positive results in those samples above the expected detection limit. Interassay coefficients of variation ranged between 6–25%, while intra-assay coefficients of variation were found to be between 19%. When clinical samples were tested with the Ortho trakC assay and compared with results obtained with the Cobas Ampli Prep/Cobas Amplicor HCV Monitor Test (Roche Molecular Systems, Inc., Branchburg, NJ), 66% of all positive values were found to be within a +-0.5 log difference of the level obtained with the molecular assay. In conclusion, the Ortho® trak-C™ assay showed a good test performance and proved to be suitable for the routine-diagnostic laboratory.