Volume 43, Issue 11

PCR is a promising method for detection of human papillomavirus (HPV), the high-risk forms of which are responsible for cervical cancer. PCR primers that target the L1 or E1 region can be unreliable and may miss more advanced disease, whereas those directed at the E6 or E7 regions, which encode oncogenic products, are preferable because 1) the LI/E1 regions, but never the E6/E7 regions, are lost during integration of viral DNA into host genomic DNA, a process that can represent an integral component of progression from infection to tumorigenesis; and 2) the E6/E7 nucleotide sequence exhibits less nucleotide variation. The choice of region used for PCR has implications for HPV screening strategies in the clinical diagnosis and management of cervical cancer.

Background: Isotretinoin (Iso) is currently indicated for the treatment of cystic acne (CA) and is related to marked teratogenicity. Aim: The aim of the study was to evaluate the relationship between total antioxidant status (TAS) and a serum marker of DNA oxidative damage, 8-hydroxy-2-desoxyguanosine (8-OHdG), in patients on Iso treatment. Patients and methods: Patients with CA (n=18) were evaluated before and 45days after Iso (0.5mg/kg per day) treatment and non-diseased controls (n=22) were tested only once. Plasma TAS levels and 8-OHdG were measured spectrophotometrically and with an immunoassay, respectively. Liver biochemical parameters and muscle enzymes were measured on a blood chemistry analyzer. Results: TAS levels were significantly (p<0.0001) lower in patients before treatment (921±124μmol/L) compared with those after treatment (1335±93μmol/L) and in controls (1536±126μmol/L). In contrast, 8-OHdG serum levels were two-fold higher in patients after treatment (0.21±0.03ng/mL) than before treatment (0.11±0.02ng/mL) and three-fold higher than in controls (0.07±0.01ng/mL; p<0.0001). Negative correlations were found between TAS and 8-OHdG (r=−0.754, p<0.0001) in patients before therapy and positive correlations were found between creatine kinase (CK) and 8-OHdG (r=0.488, p<0.001) and liver enzymes after Iso treatment. Conclusions: High serum levels of 8-OHdG in patients on Iso therapy may be due to a direct effect of Iso on liver, muscle and skin epidermal cells. Regular evaluation of 8-OHdG in sera of patients, especially of women of reproductive age, on Iso treatment could be a sensitive follow-up biomarker of DNA oxidation.

In this work we evaluate the proteolytic state of plasma α 2 -macroglobulin in acute pancreatitis. In addition, the plasma activity of matrix metalloproteinase-2 (MMP-2), MMP-9 and serine proteinases were analyzed. A total of 33 patients with acute pancreatitis were studied, of whom 16 were diagnosed as having mild and 17 as having severe acute pancreatitis. In the latter group, three patients progressed to multi-organ failure and died as a consequence of these complications. The proteolytic fragmentation of α 2 -macroglobulin was evaluated by Western blotting, whereas the plasma activity of MMP-2, MMP-9 and serine proteinases was evaluated by gelatin zymography. Enhanced fragmentation of α 2 -macroglobulin was detected in severe acute pancreatitis patients with multiple organ failure and lethal complications. In this same patient group, increased plasma activity of the active forms of MMP-2 and MMP-9, as well as serine proteinases, was apparent. In addition, we demonstrate that chymotrypsin-like proteinases could be the principal cause of α 2 -macroglobulin degradation in this group of patients. Our results indicate that secondary proteolysis of α 2 -macroglobulin promotes impaired control of extracellular proteolytic activity, leading to local and distant tissue injuries during severe acute pancreatitis. Finally, the structural evaluation of plasma α 2 -macroglobulin could be used as a prognostic marker of the severity of acute pancreatitis.

In multicentre studies of malignant and inflammatory diseases, whole blood, cell or tissue samples are often collected for analyses of gene expression to predict or monitor treatment effects. For correct analysis, sample stability during handling and transport is crucial. In developing the logistics for multicentre studies in malignant melanoma and inflammatory bowel disease, we found poor stability of a number of transcripts using the PAXgene™ Blood RNA System, which was advertised to maintain RNA stability for several days at room temperature. The results indicate that general statements on sample stability are not reliable and have to be verified for the specific transcripts of interest.

The aims of this study were to diagnose iron-restricted erythropoiesis (functional iron deficiency) in patients with classic iron deficiency (ID), anemia of chronic disease (ACD) and the combined state of ID/ACD with the use of two hematological methods for the measurement of reticulocyte hemoglobinization. In comparison, the biochemical markers of iron status were determined. We studied 474 anemic patients admitted to hospital with a broad spectrum of diseases. We measured indicators of reticulocyte hemoglobinization. CHr was determined on an Advia 120 hematology analyzer. A Sysmex XE-2100 hematology analyzer was used to determine RET-Y, the forward scatter of fluorescence-labeled reticulocytes, which can also be expressed as the reticulocyte hemoglobin equivalent (RET-H e ), as well as RBC-Y, the forward scatter of fluorescence-labeled erythrocytes, which can be expressed as the erythrocyte hemoglobin equivalent. Ferritin, soluble transferrin receptor (sTfR) and the sTfR/log ferritin ratio (sTfR-F index) were used as biochemical markers. The comparison of RET-Y with CHr demonstrated an excellent curvilinear relationship between the two parameters. The normal reference range for Ret-Y was 1630–1860 arbitrary units (AU); mathematical transformation to RET-H e gave a range of 28.2–35.7 pg. Correlations of biochemical iron markers with RET-H e were as weak as with CHr in patients with ACD and acute phase response. In a diagnostic plot to identify iron status, RET-H e could replace CHr without any loss of sensitivity or specificity. Patient mismatch analysis between RET-H e and CHr in the diagnostic plot demonstrated agreement for 449 of 474 patients (94.4%). Patient specific anemia mismatches were 2.9–6.2%. According to our results, the indicators of reticulocyte hemoglobinization, RET-H e and CHr, measure the same phenomenon. RET-H e is as valuable as CHr for the diagnosis of iron-restricted erythropoiesis. The combination of RET-H e and the sTfR-F index in a diagnostic plot offers an attractive tool for the evaluation of iron status and identification of the progression of ID.

The purpose of this study was to ascertain whether analysis of the physical properties of ejaculate also has any diagnostic potential for evaluating the function of these accessory sex glands. Diverse normal and abnormal states of coagulation, liquefaction, volume, viscosity and pH were studied with regard to the levels of biochemical markers of the seminal vesicles (fructose and inorganic phosphorus) and prostate (calcium, zinc and acid phosphatase). Fructose and inorganic phosphorus were significantly decreased in samples with absent or poor coagulation (p<0.001), volume <2.0mL (p=0.009 and p<0.001, respectively), hypoviscosity (p=0.013 and p<0.001), hyperviscosity (p=0.006 and p<0.001) and pH ≤7.1 (p=0.018 and p<0.001). Also, fructose and inorganic phosphorus were significantly decreased in samples with liquefaction >120min (p=0.003) and pH >8.0 (p<0.001), respectively. Calcium, zinc and acid phosphatase activity were significantly increased in samples with absent or poor coagulation (p<0.001), and significantly decreased in samples with volume >5.0mL (p=0.007, p=0.034 and p=0.011) and pH >8.0 (p<0.001). Also, calcium and zinc were significantly increased in hypoviscous samples (p=0.012 and p=0.003), whereas the zinc concentration was significantly lower in hyperviscous samples (p=0.026). Using receiver operating characteristic (ROC) curve analysis, pH showed the highest predictive power to identify prostate dysfunction (83.6%) and simultaneous prostate and seminal vesicle dysfunction (98.8%). Physical analysis of ejaculate was also found to be clinically useful for evaluating the secretory activity of the seminal vesicles and prostate. Abnormal coagulation, liquefaction, volume, viscosity and pH strongly suggest gland dysfunction.

The aim of this study was to investigate the specificity of the 3 H-triolein assay and to investigate the recovery of highly purified pancreatic lipase and pancreatic lipase in the form of pure non-activated pig pancreatic juice. Blood plasma from pigs was analysed for pancreatic lipase activity using the 3 H-triolein substrate assay, with a method specific for lipoprotein lipase and with a method specific for hepatic lipase. The recovery of pancreatic lipase from pancreatic juice was approximately 100%, while the recovery of highly purified pancreatic lipase in plasma or whole blood was found to be approximately 1%. Preparations of highly concentrated, purified lipoprotein lipase showed activity in the 3 H-triolein assay designed for pancreatic lipase, but the activity did not exceed 1% of the activity of this enzyme measured in an assay specific for lipoprotein lipase (samples containing physiological levels of lipoprotein lipase did not show any activity in the assay). Hepatic lipase was not measurable under the conditions of the 3 H-triolein assay. In conclusion, the 3 H-triolein assay showed pronounced specificity for pancreatic lipase compared with lipoprotein lipase or hepatic lipase.

The concentration of neuron-specific enolase (NSE) in serum and cerebrospinal fluid (CSF) has been used as a biomarker in some cancers and, more recently, in neurodegenerative diseases. Pre-analytical conditions are very important for the quality of returned results. In this study, we evaluated the effects of storage conditions (temperature and duration of storage) and hemolysis on the concentration of NSE in serum and CSF. Our results demonstrate that samples for NSE measurement may be stored at −80°C for no more than 6 months in the case of CSF and 9months in the case of serum samples. Even invisible hemolysis may increase NSE levels in samples. Consequently, an index of hemolysis should be determined before deciding whether or not to perform NSE measurement.

Evidence has been accumulating that 7-hydroxyepimers of dehydroepiandrosterone may act as locally active immunomodulatory and immunoprotective agents, counteracting the actions of glucocorticoids. 7α- and 7β-hydroxylated metabolites of dehydroepiandrosterone are present in near nanomolar concentrations in human blood. Limited data have been available on its changes during the last decades of human life. We examined 213 healthy Central European males in age groups from 50 to 91years and determined both 7-hydroxyepimers, 7α- and 7β-hydroxy-dehydroepiandrosterone, in sera using specific radioimmunoassays. The age dependence of steroid levels was evaluated using one-way ANOVA. In men, a distinct decline in 7α-hydroxy-dehydroepiandrosterone with age was observed. The curves for age dependence of 7-hydroxy metabolite levels in males resembled those previously determined for unconjugated dehydroepiandrosterone. The levels of both 7-hydroxylated epimers correlated excellently with each other.

Undiagnosed thyroid disease is a common problem with significant public health implications. This is especially true during pregnancy, when the health of both the mother and the developing child can be adversely affected by abnormal maternal thyroid function. Measurement of serum thyroid stimulating hormone (TSH) and thyroid peroxidase antibodies (TPO-Ab) are two common ways to assess maternal thyroid status. The objective of our study was to determine the prevalence of abnormal TSH and TPO-Ab tests in a population of pregnant women in the Samara region of the Russian Federation. Serum samples were obtained from 1588 pregnant women as part of their routine antenatal care. TSH and TPO-Ab were measured, and trimester-specific reference values for TSH (2.5–97.5 percentiles) were calculated using TPO-Ab-negative women. TSH results outside these ranges were considered abnormal; TPO-Ab levels outside the manufacturer's reference range (>12IU/mL) were considered abnormal. Overall, the prevalence of abnormal results was 6.3% for TSH and 10.7% for TPO-Ab. High TSH (>97.5 trimester-specific percentile) and TPO-Ab-positive results were most common in the first trimester (5.7% and 13.8%, respectively). TSH levels were associated with gestational age and TPO-Ab status, and with maternal age in TPO-Ab-negative women. TPO-Ab status was associated with both maternal and gestational age. Women with TSH >2.5mIU/L had a significantly increased risk of being TPO-Ab-positive, and this risk increased with age. Based on our data, we conclude that abnormal TSH and TPO-Ab are common in pregnant women of the Samara region. Given the association of thyroid dysfunction to adverse pregnancy outcomes, screening of this population for abnormal thyroid function should be considered.

Purpose: The aim of our study was to measure the inter-assay variation and accuracy of serum creatinine assays and to assess the effect of standardized calibration procedures on this variability. Methods: We analyzed 30 human sera and three reference materials, using 17 creatinine assays (12 colorimetric, 4 enzymatic and 1 HPLC). We compared two standardized calibration procedures, using either a reference material or secondary standards, to that recommended by the manufacturers. Results: For assays calibrated according to the manufacturers' recommendations, the median inter-assay coefficient of variation (CV) was 14.2% for 20 low samples (45–150μM), and 7.7% for 10 high samples (250–350μM). The CV was significantly influenced by the calibration procedure, but none of the standardized calibration procedures significantly improved the inter-assay variability. However, a significant decrease in CV was noted within each type of assay method (colorimetric or enzymatic) when the standardized calibration used standards of level(s) close to the concentrations to be measured. Only the compensated Jaffe technique and the amido-hydrolase assay showed bias of less than 10%. Conclusions: Standardizing calibration procedures is unlikely to decrease the analytical variability of creatinine assays enough to allow uniform and reliable use of the equations for estimation of glomerular filtration rate.

The objective of the present study was to determine the efficacy of anti-cyclic citrullinated peptide (anti-CCP) antibody detection in the early diagnosis of rheumatoid arthritis (RA), as well as to compare three commercially available enzyme-linked immunosorbent assay (ELISA) kits used to detect such antibodies. We analysed the presence of anti-CCP antibodies in the sera of 78 patients who had been newly referred from primary healthcare centres to the Early Polyarthritis Unit. We also included in the study a group of 50 healthy controls. None of the patients had previously received treatment for the disease. After 1-year follow-up, the diagnosis of RA was confirmed in 53 of these patients. The ELISA kits under study were IMMUNOSCAN RA (Euro-Diagnostica AB), QUANTA Lite™ CCP IgG ELISA (INOVA Diagnostic) and DIA-STAT™ Anti-CCP (Axis-Shield Diagnostics); the sensitivity obtained was 52.8%, 58.5% and 52.8%, respectively, with 100% specificity for all three kits. Anti-CCP antibodies detected the presence of RA in 26% of patients without positive rheumatoid factor (RF). The sum of anti-CCP antibodies or the presence of RF gave a sensitivity of up to 67%, with specificity ranging between 94 and 97%. Anti-CCP antibodies show high specificity for the diagnosis of RA. The three ELISAs analysed offer the same degree of diagnostic accuracy.

Mean and variance rules for quality control are more powerful than rules based on individual values. An algorithm for applying such rules is described that controls type I errors (false alarms), while allowing for multiple levels of quality control samples, correlation between levels, small numbers of preliminary values, replication of samples and autocorrelation arising from random effects. Based on ANOVA and empirical approximations for small samples, the algorithm maintains a low per-batch probability of type I errors. Three statistics are computed, z m , z b and z w , which are shown by simulations to be primarily sensitive to a concordant shift in the quality control values, a discordant shift in the values, and an increase in random variability, respectively. Simulations also show that for a Gaussian distribution of analytical errors, the per-batch probability of a type I error is likely to be within the range 0.0045–0.0071 for two to four levels where there are 20–100 preliminary batches and the inter-level correlations are between zero and 0.8. This partial separation of out-of-control alarms into three components provides more assistance with trouble-shooting than do multivariate quality control schemes based on Hotelling's T 2 , while retaining comparable power.

The humoral immune responses of 69 active tuberculosis (TB) patients against three major mycobacterial lipid antigens, monoacyl phosphatidylinositol dimannoside (Ac-PIM 2 ), trehalose 6,6′-dimycolate (TDM-T) and trehalose 6-monomycolate (TMM-T) from Mycobacterium bovis BCG Tokyo 172, were examined by ELISA. IgG antibodies from active TB patients were reactive against each of the three lipid antigens (Ac-PIM 2 , TDM-T and TMM-T), giving positive results of 42.0–59.4%. If tests were combined and an overall positive was scored when any of the three tests was positive, sensitivity was 71.0%, showing better discrimination between patients and normal subjects. Although this value is not satisfactory for the clinical diagnosis of active TB, it is still higher than values for Determinar TBGL (56.5%) and MycoDot (31.9%) test results, both of which are commercially available. IgG antibody responses to particular lipid antigens in an individual patient differed diversely between patients. Positive IgG antibody rates and IgG antibody levels to lipid antigens were mostly paralleled by the amount of mycobacteria excreted and by the severity of pathological lesions (size and cavity formation). Serologically positive responsiveness was shown in 60.0% of tuberculin skin test (TST)-negative TB patients. Furthermore, seropositivity for multiple-antigen ELISA in active TB patients was demonstrated in other possible immune-suppressed cases, such as senile, diabetes mellitus and fulminant TB patients. Therefore, in contrast to tests based on cellular immune responses such as the TST, the humoral immune responses of TB patients against mycobacterial lipid antigens were disease-specific and were shown to be useful for the early diagnosis of active TB disease in conjunction with smear and cultivation tests, even if cellular immune responses are decreased.

Methods to quantify B-type natriuretic peptide (BNP) and N-terminal-propeptide (NT-proBNP) in plasma or serum samples are well established. We assessed the analytical performance of the Dimension RxL NT-proBNP method (Dade-Behring). Evaluation of different sample types was carried out. Controls and heparin plasma pools were used to determine the detection limit, precision, and linearity. Sample stability and the effect of interfering substances on the NT-proBNP concentrations were evaluated. Agreement between Dimension RxL and Elecsys 2010 (Roche Diagnostics) NT-proBNP methods was assessed. The influence of age and sex on NT-proBNP concentrations was evaluated in healthy subjects. Heparin plasma should be the matrix of choice. The detection limit was 2.0ng/L. The total imprecision was 2.6–3.6% for concentrations from 231 to 9471ng/L; mean NT-proBNP concentrations of 21 and 15ng/L were associated with coefficients of variation of 9.9% and 14.7%, respectively. The method was linear up to 32,650ng/L. There was no effect of temperature, freeze-thaw cycles and interfering substances. A bias was detected when Dimension RxL and Elecsys 2010 NT-proBNP methods were compared. Age and sex were significantly and independently related to NT-proBNP concentrations. The Dimension RxL NT-proBNP method, like the Elecsys 2010, is suitable for routine use in the diagnosis of heart failure.

Intestinal alkaline phosphatase (ALP) is more prevalent in individuals of blood group O or B, and increases after a meal, especially on a high-fat diet. We did not realize that clinicians could underestimate the importance of fasting for ALP measurement until one healthy adult showed a huge difference in ALP levels before and after a meal. We report a case of transient hyperphosphatasemia resulting in unnecessary workup due to intestinal ALP in a healthy adult of blood group O.