Volume 43, Issue 8

Familial hypercholesterolemia is an autosomal dominant disease defined at the molecular level mainly by the presence of mutations in the low-density lipoprotein receptor gene and is characterized by elevated low-density lipoprotein cholesterol, tendon xanthomas and increased risk of early cardiovascular disease. The type of mutation in the low-density lipoprotein receptor gene has been associated with different phenotype expression and response to statins. Several studies have been undertaken to assess the efficacy of statins and evaluate the influence of mutations on the response to treatment with statins. Not all patients respond to statin therapy with a reduction in cardiovascular disease. In this review paper, we will discuss the results available to date that correlate the low-density lipoprotein receptor genotype to the response to statins, and the interest in developing diagnostic systems which will allow identification of patients at increased risk of adverse drug reactions or patients in which a therapeutic effect is lacking.

The rapid detection of brain injury (neuronal damage in general) is an important parameter in the management of cerebrovascular accidents, especially in hemorrhagic and/or ischemic events. Two types of 15-kDa cytoplasmic fatty acid-binding proteins (FABPs), brain-type FABP and heart-type FABP, have recently been postulated as novel markers for brain injury detection. Here we review the possible roles of these FABPs as rapid diagnostic markers for the detection of brain injury due to cerebrovascular accident, trauma or neurodegenerative diseases. The occurrence of brain- and heart-type FABPs in segments of the human brain is also described. Although only limited amounts of data are available, brain- and heart-type FABPs show higher sensitivities and specificities than protein S100 and neuron specific enolase in the rapid detection of brain injury in stroke, trauma and neurodegenerative diseases.

Large numbers of mutations are postulated to occur as early events in carcinogenesis. For certain types of human tumors (mutator phenotypes) these mutations can be a driving force in generating clonogenic, causative genetic changes leading to multistage carcinogenesis. These low-level mutational events are highly significant due to their potential use as molecular markers for early identification of genomic instability that can lead to cancer and to their potential influence on the ability of tumors to resist drug treatment and/or metastasize. Detecting the presence and diversity of such genetic changes in human tumors is desirable due to their potential prognostic value. However, identification of these low-frequency genetic changes is difficult, since most mutations exist at mutant/wild-type ratios of <10 −3 . We recently developed inverse PCR-based amplified restriction fragment-length polymorphism (iFLP), a new technology that combines inverse PCR, RFLP, and denaturing HPLC to allow scanning of the genome at several thousand positions per experiment for low-level point mutations. Using iFLP we previously demonstrated low-level mutations (mutation frequency <10 −3 ) in human colon cancer cells that harbor mismatch repair deficiency and in sporadic colon cancer surgical specimens. In the present work we investigated whether low-level mutations are also present in sporadic breast cancer surgical specimens. Using iFLP we identified widespread low-level mutations in two out of ten surgical specimens examined (20%). Examination of the microsatellite instability status of these samples demonstrated that the samples are stable (MSI-S). We conclude that low-level mutations are less frequent in breast cancer than in colon cancer; however, single nucleotide instability that generates such mutations may still be present in a fraction of breast cancers.

In order to investigate possible ethnic differences in genetic and environmental determinants, we investigated several cardiovascular disease-associated genetic variations in successful ageing populations of France (Nancy) and China (Hong Kong). Allelic frequencies of these genetic variations were compared between healthy elderly Chinese (n=103) and French populations (n=100). A multi-locus assay was used to genotype 15 genes for 29 biallelic sites, genes implicated in lipid and homocysteine metabolism, thrombosis, leukocyte adhesion, and blood pressure regulation. For most of the candidate markers within lipid metabolism genes, the less frequent alleles were more common in the Chinese population compared with the French population, while the less frequent alleles of the majority of the other markers were detected only or more commonly in the French population. In conclusion, polymorphisms in 13 genes exhibited statistically significant differences in allelic frequencies between the two populations. Since the two populations were selected as examples of successful ageing, we could hypothesise that genetic factors that could play a role in a successful ageing process may be different between the two populations.

It has been suggested that soluble CD30 (sCD30) serum levels in chronic hepatitis C are correlated with the activity of the disease and with the outcome of interferon (IFN) treatment. In this study, sCD30 serum levels in 25 patients with chronic hepatitis C, before and after treatment with IFN-2α, were measured. A total of 20 healthy subjects were used as controls. High sCD30 levels in serum were found in 36% of patients and in 5% of controls. In patients with sCD30 levels above or within the normal range, no significant differences in age, gender, serum transaminases and histology activity index were found. In relation to IFN treatment, only responder patients had serum sCD30 higher than controls, although the difference between responders and non-responders was not significant. No changes from baseline values were observed after treatment. Although high, sCD30 serum levels in chronic hepatitis C are not correlated with the disease activity, are not affected by IFN treatment and are not predictors of response to IFN treatment.

Nucleic acid amplification testing for hepatitis C virus (HCV) RNA has become an essential tool for the prevention and clinical management of hepatitis C. We describe the development, validation and evaluation of a homogenous reverse transcriptase-initiated HCV-PCR assay with competitive internal control that is applicable to both the quantitative detection of HCV genomes in single patient samples and the screening of blood donations by mini-pool testing. For the implementation of a positive run control, a HCV RNA-positive plasma sample was calibrated against an international HCV RNA standard preparation. For quantification purposes, an in vitro-transcribed RNA calibrator sequence was used. The detection limit of the assay (95% positive cut-off) was determined by probit analysis and was calculated as 114IU/mL. Comparable sensitivity to different HCV template sequences was verified for HCV genotypes 1–5. Quantitative test results correlated well with viral loads that had been previously determined by the Bayer VERSANT HCV RNA 3.0 bDNA assay (n=53, R=0.943, p<0.001). During more than 5years of blood donation testing, the specificity of the assay was found to be 99.51%. All assay components showed constant performance during this time period. In conclusion, we introduce a well-proven method that allows fast and reliable quantification of HCV genomes.

Adriamycin (ADR), a cytotoxic antineoplastic drug, is used in the treatment of various solid tumors. However, its efficacy continues to be challenged by significant toxicities including nephrotoxicity. In the present study, the effects of N-acetyl cysteine (NAC) and vitamin E, known antioxidants, were investigated on ADR-induced peroxidative damage in rat kidney. Adult male albino rats of Wistar strain were administered ADR as a single dose (10mg/kg body weight, i.v.). Histopathological studies indicated that ADR-treated kidney sections show focal tubular necrosis and casts. ADR-injected rats showed a significant decline in the activities/levels of enzymic antioxidants (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione-S-transferase) and non-enzymic antioxidants (thiols, vitamin C and vitamin E) with high malondialdehyde levels. The extent of nephrotoxicity was evident from the increased activities of urinary marker enzymes (alkaline phosphatase, lactate dehydrogenase and γ-glutamyltransferase). Treatment with NAC and vitamin E (50mg/kg b.w., i.p.) 1day prior to ADR administration maintained near normal activities of the enzymes, significantly reduced lipid peroxidation and prevented the necrosis caused by ADR, thereby proving to be an effective thiol replenishing agent and antioxidant.

Ethidium bromide (EB) is a mutagen and toxin that is widely used in the laboratory for visualization of nucleic acids. Safer nucleic acid stains, such as SYBR ® Gold, SYBR ® Green, GoldView™, GeneFinder ® , and GoldStar ® , have been developed. However, there has been no systematic comparative analysis of the staining efficiencies of these dyes. In the present study, SYBR ® Gold, SYBR ® Green I, GoldView™ and EB were compared. Although both SYBR ® Gold and SYBR ® Green alter electrophoretic mobility and thus DNA size estimates, they are cost-effective alternatives to EB. SYBR ® Gold was more sensitive than SYBR ® Green I at detecting short fragments, but 50-bp bands were clearly visible using either dye when visualized with a long integration time. SYBR ® Gold or SYBR ® Green I are sensitive and relatively safe alternatives to EB. In our laboratory, the SYBR ® Gold method is now used routinely by all members of our group with great consistency and success.

Background: Thyroidectomy followed by administration of large activities of 131 I is the treatment of choice for differentiated thyroid carcinomas. Due to its good prognosis, some authors argue that papillary thyroid cancer with a diameter of up to 10mm can be treated by surgery alone. In the new TNM classification started in 2002, the T1 group now encompasses all tumours with a diameter up to 20mm, which widens the indications for a conservative approach. In this instance, prognostic markers are needed to better select patients before planning non-aggressive treatment. Serum thyroglobulin plays a pivotal role in thyroid carcinoma management after thyroid ablation (i.e., surgery and radioiodine) but is of limited value before these treatments. However, thyroglobulin assay performed after surgery but before radioiodine treatment has been proven to be useful in predicting the presence/absence of distant metastasis. Patients and methods: Our study was undertaken in patients affected by pT1 papillary thyroid carcinoma to evaluate the predictive value of post-surgery thyroglobulin assay on 1) restaging immediately after radioiodine treatment and 2) restaging at 12months. We selected 156 patients affected by pT1 histologically proven papillary thyroid carcinoma, submitted to total thyroidectomy. Serum thyroglobulin was assayed by a specific immunoradiometric method 4weeks after surgery, just before radioiodine administration. Cut-off levels were selected by receiver operating characteristic curve analysis. Thyroglobulin levels were compared to the results of a post-radioiodine treatment scan and 12-month restaging. Results: Globally, 23 out of 156 patients showed persistent/recurrent disease (15%). Post-surgery thyroglobulin levels above 4.50μg/L identified 94% of patients with metastasis at post-dose scan, and a level below 3.20μg/L identified 86% and 93% of relapsed and disease-free patients at 12-month restaging, respectively. Multivariate analysis and Spearman rank correlation showed that the N-status and post-surgery thyroglobulin level are independent prognostic factors. Conclusions: The post-surgery thyroglobulin level could be systematically assayed in patients with pT1-papillary thyroid carcinoma and taken into account in planning treatment.

Few studies have evaluated cardiac troponin I (cTnI) as a marker for infarct size and left ventricular (LV) dysfunction. Here we investigated the ability of a single-point cTnI, measured with a second-generation assay (Access AccuTnI), to estimate infarct size and assess LV function in patients with a first myocardial infarction (AMI). cTnI measurements were performed 12 and 48h after admission in 63 consecutive AMI patients. LV function was evaluated by gated single-photon emission computed tomography (SPECT) and infarct size was estimated by CK-MB peak and SPECT myocardial perfusion. LV function and infarct size were evaluated by SPECT before hospital discharge. SPECT was also repeated 3months later. Significant correlations (p<0.001) were found between cTnI at 12 and 48h and both the peak CK-MB (r=0.61 and r=0.82, respectively) and the perfusion defect size at SPECT (r=0.55 and r=0.61, respectively). cTnI at 12 and 48h were inversely related (p<0.001) to LV ejection fraction (LVEF) assessed both early (r=–0.45 and r=–0.57, respectively) and 3months after AMI (r=–0.51 and r=–0.69, respectively). cTnI >14.8 μg/L at 48h predicted an LVEF <40% at 3months with a sensitivity of 100% [95% confidence interval (CI) 73.5–100%], specificity of 65% (CI 49–79%), and a negative predictive value of 100%. Our findings demonstrate that a single cTnI measurement 48h after admission is useful for ruling out impaired LV function in a routine clinical setting.

We determined the sensitivity, specificity, receiver operating characteristics and correlation between cystatin C (cysC) and two widely used markers of renal function, creatinine clearance and serum creatinine, in 244 patients (84 diabetics, 84 hypertensive and 76 healthy subjects). Renal failure was defined as creatinine clearance of less than either 80 or 60mL/min. Variables were evaluated for two definitions of renal failure and compared between patient groups. Correlation coefficients with cysC were −0.87 for creatinine clearance and 0.92 for creatinine in patients with hypertension; −0.90 for creatinine clearance and 0.97 for creatinine in diabetics; and −0.61 for creatinine clearance and 0.94 for creatinine in the control group. The receiver operating characteristic curves with a cut-off value of 60mL/min were similar for creatinine and cysC, while at 80mL/min they were 0.626 for creatinine and 0.813 for cysC levels. We classified the patients into three groups with respect to creatinine clearance (1, >80mL/min; 2, 60–80mL/min; 3, <60mL/min). Mean creatinine (p<0.0001) and cysC (p<0.0001) levels were significantly different between all the groups. Sensitivity, specificity and predictive values were higher for cysC levels, particularly in diabetics and hypertensive patients. The current study suggests that cysC is preferable for detecting temporal changes in renal function in the early stages of renal insufficiency.

The Scientific Committee of Molecular Biology Techniques (C-MBT) in Clinical Chemistry of the IFCC has initiated a joint project in co-operation with the European Commission, Joint Research Centre, Institute of Reference Materials and Measurements to develop and produce plasmid-type reference materials (RMs) for the analysis of the human prothrombin gene G20210A mutation. Although DNA tests have a high impact on clinical decision-making and the number of tests performed in diagnostic laboratories is high, issues of quality and quality assurance exist, and currently only a few RMs for clinical genetic testing are available. A gene fragment chosen was produced that spans all primer annealing sites published to date. Both the wild-type and mutant alleles of this gene fragment were cloned into a pUC18 plasmid and two plasmid RMs were produced. In addition, a mixture of both plasmids was produced to mimic the heterozygous genotype. The present study describes the performance of these reference materials in a commutability study, in which they were tested by nine different methods in 13 expert laboratories. This series of plasmid RMs are, to the best of our knowledge, the first plasmid-type clinical genetic RMs introduced worldwide.

Control and standardization of preanalytical variability is a critical factor for achieving accuracy and precision in laboratory testing. Although venous stasis from tourniquet placement during venepuncture should be minimized, as it has been claimed to account for spurious and significant variations for several analytes in plasma, there is controversy surrounding its real impact on laboratory testing. The aim of the present study was the investigation of the influence of short-term venous stasis on routine biochemical testing, by measuring the plasma concentration of 12 common analytes, including proteins, protein-bound substances, enzymes and electrolytes, in plasma specimens collected either without venous stasis or following the application of standardized external pressure of 60mm Hg using a sphygmomanometer for 1 and 3min. Although the overall correlation between measures was acceptable, the pattern of change was mostly dependent on the length of stasis, size and protein-binding characteristics of the analytes, achieving clinical significance for albumin, calcium and potassium after 1-min stasis, and alanine aminotransferase, albumin, calcium, chloride, total cholesterol, glucose and potassium after 3-min stasis. Statistically significant differences could be observed in seven (alanine aminotransferase, albumin, calcium, total cholesterol, creatine kinase, iron and potassium) and ten (alanine aminotransferase, albumin, calcium, chloride, total cholesterol, creatine kinase, creatinine, glucose, iron and potassium) out of the 12 analytes tested, after 1- and 3-min venous stasis, respectively. The most clinically significant changes from standard venepuncture, when compared to the current analytical quality specifications for desirable bias, occurred for potassium (1-min stasis, −2.8%; 3-min stasis, −4.8%, both p<0.001), calcium (1-min stasis, +1.6%, p<0.05; 3-min stasis, +3.6%, p<0.001) and albumin (1-min stasis, +3.5%; 3-min stasis, +8.6%, both p<0.001). As most of these effects are dependent on the stasis time during venepuncture and biochemical or physiological characteristics of the analyte, these variations could likely be anticipated, allowing the most appropriate preventive measures to be adopted.

Portable meters for blood glucose concentrations are used at the patients bedside, as well as by patients for self-monitoring of blood glucose. Even though most devices have important technological advances that decrease operator error, the analytical goals proposed for the performance of glucose meters have been recently changed by the American Diabetes Association (ADA) to reach <5% analytical error and <7.9% total error. We studied 80meters throughout the Virgen Macarena Hospital and we found most devices with performance error higher than 10%. The aim of the present study was to establish a new system to control portable glucose meters together with an educational program for nurses in a 1200-bed University Hospital to achieve recommended analytical goals, so that we could improve the quality of diabetes care. We used portable glucose meters connected on-line to the laboratory after an educational program for nurses with responsibilities in point-of-care testing. We evaluated the system by assessing total error of the glucometers using high- and low-level glucose control solutions. In a period of 6months, we collected data from 5642 control samples obtained by 14 devices (Precision PCx) directly from the control program (QC manager). The average total error for the low-level glucose control (2.77mmol/l) was 6.3% (range 5.5–7.6%), and even lower for the high-level glucose control (16.66mmol/l), at 4.8% (range 4.1–6.5%). In conclusion, the performance of glucose meters used in our University Hospital with more than 1000 beds not only improved after the intervention, but the meters achieved the analytical goals of the suggested ADA/National Academy of Clinical Biochemistry criteria for total error (<7.9% in the range 2.77–16.66mmol/l glucose) and optimal total error for high glucose concentrations of <5%, which will improve the quality of care of our patients.

In clinical laboratories, patient results can be obtained in two ways: (i) by direct determination of requested tests using various chemical methods, (ii) by calculation of unknown test results, based on relationships between measured tests. The reliability of measured tests can be checked by various quality control rules. However, no test is performed to check the reliability of calculated data. In this study we develop a method using Taylor series expansion and an alternative equation to obtain the standard deviation of calculated laboratory tests and discuss the reliability of calculated data. To obtain reliable test results by calculation instead of being measured by chemical methods, the standard deviation of each measured component of the equation must be thoroughly analyzed and then the standard deviation of the equation must be determined. We conclude that the analytical coefficient of variation of any measured component must be lower so as to obtain an acceptable analytical coefficient of variation for calculated tests. Otherwise we should measure the concentration of requested tests by chemical methods instead of calculation by equation using specified components.

We evaluated the analytical performance of a new, commercial, fully automated immunoturbidimetric assay for the determination of ferritin [FER-Latex(X2)CN SEIKEN, Denka Seiken, Japan] in serum on the Olympus AU2700 analyzer. The new assay is a latex-enhanced turbidimetric immunoassay with an analysis time of 10min. The linearity of the assay was confirmed up to 2505pmol/L (R 2 =0.999). The detection limit and the functional sensitivity were both 4.5pmol/L. The intra- and inter-assay imprecision (CV) at 67, 506, 2186pmol/L was <1.8% and <2.5%, respectively. Verification of the traceability to a WHO standard (80/578) showed a recovery of 102.6% (target value 449pmol/L). No hook effect was observed in samples containing up to 33,705pmol/L. The assay showed good correlation with the Beckman Immage nephelometric system (r=0.999). Hemoglobin (≤9.8g/L), total bilirubin (≤113μmol/L), conjugated bilirubin (≤109μmol/L) and rheumatoid factor (≤5.2×10 5 IU/L) did not interfere with the assay. The reference interval (2.5–97.5 percentile) was 72–521pmol/L for men and 27–267pmol/L for women. The reference interval in patients with anemia, malignant tumors and hemochromatosis was 5.6–52, 130–2436 and 1465–2903pmol/L, respectively. On the basis of the receiver operating characteristic curve, the 90% sensitivity cut-off value to distinguish between patients with and without iron deficiency was 40pmol/L. The new latex turbidimetric procedure for ferritin assay is an attractive alternative that avoids the need for dedicated instrumentation.

Assay of human chorionic gonadotropin (hCG) is mainly used for the detection and monitoring of pregnancy, and for the follow-up of trophoblastic tumors. The serum free β-hCG subunit (hCGβ) is also a tumor marker in many non-trophoblastic tumors, including gastrointestinal cancers. In this work, we compared the performance of several immunoassays for pregnancy exclusion before liver transplantation and in the follow-up of a woman with cholangiocarcinoma. Serum hCG was detected with the Abbott Testpack plus hCG-Combo and measured with four automated sandwich immunoassays: ADVIA-Centaur, ACS:180, AxSYM and Dimension. hCGβ was determined by an automated fluorescence sandwich immunoassay (Kryptor-Free β hCG) and with a specific immunoradiometric assay (ELSA-F β hCG, Schering). The expression of hCG was also evaluated by immunohistochemistry on sections of intrahepatic cholangiocarcinoma cells and on peritoneal metastases. Before transplantation, discordant results were observed for pregnancy exclusion. Qualitative Testpack and Dimension tests detected no hCG-like immunoreactivity, whereas the ADVIA-Centaur, ACS:180 and AxSYM tests revealed positive levels. The same discrepancy was obtained in follow-up of the patient after liver transplantation. hCGβ assay and immunohistochemical staining revealed tumor cell secretion of hCGβ. In conclusion, a specific serum immunoassay for intact dimeric hCG without cross-reaction with hCGβ should be adopted as routine policy for pregnancy exclusion before liver transplantation.