Volume 44, Issue 5

Venous thromboembolism is a well-known complication of oral contraception and hormonal replacement therapy. Inherited thrombophilia is viewed as an important determinant in modulating the effects of estrogens on thrombotic risk. An increasing number of kits for thrombophilic mutations [factor V Leiden, G20210A prothrombin and methylenetetrahydrofolate reductase ( MTHFR ) C677T genes] are becoming commercially available, and screening for inherited thrombotic risk is among the most requested genetic tests in molecular diagnostic laboratories. However, the question of routine genetic screening for thrombophilia before prescribing hormones is still a matter of debate. The purpose of this article is to discuss the usefulness and practical applications of thrombotic genetic testing to identify which women should be tested to improve both the safety and efficacy of individualized estrogen therapy.

Immunoglobulin light chains are components of antibodies, but some exist in a free form in serum and urine as a result of their excess production over heavy chains. Free light chain (FLC) levels are of the order of milligram per liter in normal serum and urine, but marked increases have been observed in various disease conditions. It has now been established that the measurement of FLC levels contributes to diagnosis and clinical management in monoclonal gammopathies. Recent developments in FLC assays have been adapted to several automated platforms and they have now become available in laboratories. There have, however, been some concerns regarding the analytical aspects. The current assay specificity appears to be insufficient to prevent the influence of intact light chains of several orders of magnitude greater than FLCs in serum. Moreover, the heterogeneous nature of light chains makes accurate quantification unreliable. FLC assays have never been standardized because of the lack of an international reference calibrator. In this review, we summarize the reports on FLC measurements and examine the specificity of anti-FLC antibodies and the reliability of FLC assays. We also discuss difficulties in the standardization and setting of normal reference intervals for FLC assays.

Background : Mutations in the retina-specific ABC transporter ( ABCA4 ) gene are associated with different types of macular degeneration, including Stargardt disease, cone-rod dystrophy, Fundus flavimaculatus, Retinitis pigmentosa and probably age-related macular degeneration. Methods : Screening for mutations in the ABCA4 gene was performed using denaturing high-performance liquid chromatography and direct sequencing. Results : We describe the identification of a new de novo 44-bp deletion in an Italian patient affected by cone-rod dystrophy. The mutation, located in intron 48 of the ABCA4 gene, is predicted to cause exon 49 skipping, resulting in loss of the C-terminus of the ABCA4 protein. Interestingly, exon 49 also codes for a highly conserved VFVNFA motif, which has been demonstrated to be essential for the activity of ABCA1 , another gene of the ABC transporter family. The presence of CT repeats at the breakpoints might have facilitated the generation of the deletion through a slippage mispairing mechanism. Conclusions : The new 6730–16del44 deletion is the first de novo mutation associated with cone-rod dystrophy and may contribute to a better understand-ing of the role of ABCA4 mutations in macular dystrophies.

The squamous cell carcinoma antigen (SCC-Ag) has been widely applied as a serum marker in different kinds of cancer and was reported as a target gene for the detection of tumor cells in peripheral blood in cervical cancer. Nucleic acids released into the circulation are non-invasive diagnostic tools for cancer detection. The objective of this study was to determine the utility of SCC-Ag mRNA as a cancer detection marker in blood of cancer patients. For this purpose, 77 blood samples from five gastric cancer, 23 laryngeal cancer, 31 lung cancer, nine esophageal, and nine cervical cancer patients were analyzed. The SCC gene was amplified by reverse transcription-polymerase chain reaction. SCC-Ag mRNA was detected in two patients with gastric cancer, six patients with laryngeal cancer, 17 patients with lung cancer, three patients with esophageal cancer, and two patients with cervix cancer. The detection rate was highest (54.83%) in patients with lung cancer. SCC-Ag transcripts were not detectable in the control group, indicating that molecular analysis showed no false positives. Our results indicate that this approach could be useful in a considerable number of patients and could improve the lower diagnostic yield of conventional tests.

Background : An in vitro study was conducted to determine whether haptoglobin phenotypes differed in their protective effect against oxidative stress induced by extracellular hemoglobin on red blood cells. Methods : Conjugated dienes and thiobarbituric acid-reactive substances (TBARS) were determined in human red blood cell membranes in the presence of hemoglobin and various concentrations of each type of purified haptoglobin. In addition, the release of K + and lactate dehydrogenase from red blood cells was measured. Results : A protective effect of haptoglobin was observed in terms of results obtained for the four parameters examined, with significant differences (p<0.001) between the three haptoglobin types; type 1-1 was the most active and type 2-2 the least active. A proportion of oxidative damage was not sensitive to haptoglobin, but to desferrioxamine (an iron chelator), indicating the participation of two actors, hemoglobin and free iron, in the oxidative stress of membrane lipids. Conclusions : The antioxidant role of haptoglobin and the phenotype dependence were confirmed for preventing possible oxidative damage induced by free hemoglobin and iron release during its catabolism.

Background : Specific gene rearrangements are used for minimal residual disease (MRD) assessment, but are frequently lacking in leukaemias. In these cases, the quantification of PRAME (preferentially expressed antigen of melanoma) transcripts could be useful. Methods : PRAME transcripts were quantified by real-time RT-PCR in normal and leukaemic samples, and the results were compared with those of conventional RT-PCR. Basal expression of PRAME was determined in 25 blood samples and 25 bone marrow samples from healthy donors, as well as in 12 haematological cell lines (Jurkat, K562, HL60, DOHH2, IM9, Daudi, CEM, KG1, DG75, 8226, U937, Raji). Results : In paediatric acute myeloid leukaemia (AML) (n=22) and acute lymphoblastic leukaemia (ALL) (n=17), and in adult AML (n=20), abnormal PRAME expression was found in 41%, 35% and 40% of cases, respectively. To assess the sensitivity of PRAME for monitoring MRD, PRAME -positive t(8;21) AML samples with detectable AML1/ETO expression by conventional RT-PCR (n=17) were assessed for quantitative expression of AML1/ETO and PRAME . The expression of these genes was closely correlated. To confirm that PRAME expression was correlated with clinical data, the expression of PRAME was also sequentially followed in patients (n=13) from onset to cytological remission or relapse. The cytological and molecular data were highly correlated in all patients. Conclusions : Our data confirm that PRAME quantification by real-time RT-PCR appears suitable for monitoring MRD in PRAME -positive leukaemia.

Background : Type 2 diabetes mellitus is associated with increased cardiovascular risk. One laboratory marker for cardiovascular risk assessment is high-sensitivity C-reactive protein (hsCRP). Methods : This cross-sectional study attempted to analyze the association of hsCRP levels with insulin resistance, β-cell dysfunction and macrovascular disease in 4270 non-insulin-treated patients with type 2 diabetes [2146 male, 2124 female; mean age ±SD, 63.9±11.1years; body mass index (BMI) 30.1±5.5kg/m 2 ; disease duration 5.4±5.6years; hemoglobin A 1c (HbA 1c ) 6.8±1.3%]. It consisted of a single morning visit with collection of a fasting blood sample. Observational parameters included several clinical scores and laboratory biomarkers. Results : Stratification into cardiovascular risk groups according to hsCRP levels revealed that 934 patients had low risk (hsCRP <1mg/L), 1369 patients had intermediate risk (hsCRP 1–3mg/L), 1352 patients had high risk (hsCRP >3–10mg/L), and 610 patients had unspecific hsCRP elevation (>10mg/L). Increased hsCRP levels were associated with other indicators of diabetes-related cardiovascular risk (homeostatic model assessment, intact proinsulin, insulin, BMI, β-cell dysfunction, all p<0.001), but showed no correlation with disease duration or glucose control. The majority of the patients were treated with diet (34.1%; hsCRP levels 2.85±2.39mg/L) or metformin monotherapy (21.1%; 2.95±2.50mg/L hsCRP). The highest hsCRP levels were observed in patients treated with sulfonylurea (17.0%; 3.00±2.43mg/L). Conclusions : Our results indicate that hsCRP may be used as a cardiovascular risk marker in patients with type 2 diabetes mellitus and should be evaluated in further prospective studies.

Background : Previously, we found high urinary glycosaminoglycan (GAG) concentration, together with an altered electrophoretic pattern, in normoalbuminuric type 1 diabetic subjects with hemoglobin A 1c (HbA 1c ) ≥8.0%. The purpose of this study was long-term evaluation of GAG excretion variations in these patients compared to those with HbA 1c <8.0% at baseline who maintained better metabolic control over time. Methods : We enrolled 26 normotensive, normoalbuminuric type 1 diabetic patients and divided them into two groups according to mean HbA 1c levels during the follow-up period. GAGs were isolated from 24-h urine samples on two separate occasions, at baseline and after a mean (±SD) follow-up of 6.8±1.1years. Results : All patients remained normoalbuminuric at follow-up, and had normal urinary α 1 -microglobulin levels. In patients with HbA 1c <8.0%, total GAG levels and low sulfated chondroitin sulfate-proteoglycan/chondroitin sulfate ratio were almost unchanged during the follow-up period. In contrast, these increased in patients with HbA 1c ≥8.0% and were significantly related to both HbA 1c levels and the duration of poor glycemic control. Conclusions : Our results show a strong influence of hyperglycemic environment on GAG metabolism in diabetes and indicate that the distribution pattern of urinary GAGs, besides their total concentration, may be predictive of altered GAG metabolism in type 1 diabetes.

Background : Recently revised diagnostic criteria for diabetes mellitus and the lack of universal agreement on the methodology for the screening and diagnosis of gestational diabetes mellitus (GDM) still generate inconsistency in execution of the oral glucose tolerance test (OGTT). The aim of the present survey was to evaluate the adherence of Italian laboratories to the internationally accepted guidelines in carrying out the OGTT for the diagnosis of diabetes in the general population and for the screening of GDM. Methods : A questionnaire was designed to investigate the following issues related to the OGTT: 1) the relationship between laboratories and diabetes centres for the definition of standard protocols; 2) the amount of glucose administered; 3) the number and timing of blood samples; 4) the procedures used for the screening and diagnosis of GDM; and 5) reference to WHO guidelines for the interpretation of the results. The questionnaire was administered to 400 specialists in laboratory medicine working in public or private laboratories nationwide participating in the “Italian External Evaluation of Quality in Laboratory Medicine” Study Group. Results : The survey was completed in the period from June to September 2003. In the observation period, 241 questionnaires were returned by specialists working in laboratories scattered throughout 15 out of the 20 Italian regions. Only 50% of the laboratories performed the OGTT according to protocols defined in agreement with local reference diabetes centres. OGTT using 75g of glucose in adults and 1.75g/kg for children as recommended by WHO was performed by 87.1% of the laboratories. WHO indications to collect samples at baseline and at 120min were followed by 33.2% of the centres. Higher variability was highlighted with respect to the methodology for GDM screening: 49.8% of the laboratories always adopted the two-step procedure consisting of a glucose challenge test (GCT) and subsequent OGTT in positive cases; 4.9% performed the 100-g OGTT with four blood samples; 1.6% the 75-g OGTT with two blood samples; and 2.7% the 75-g OGTT with four blood samples. More than 30% of the centres referred to different diagnostic schemes, 62% of which used individually chosen procedures amongst those reported above, 19% used only the GCT and no subsequent OGTT in positive cases, and 18.4% used a variety of completely different, arbitrarily chosen methods. Finally, only 25.6% of the laboratories referred to the WHO limits for interpretation of the results. Conclusions : For the Italian laboratories investigated, relevant variability was highlighted for performance of the OGTT in general and GDM screening in particular. A variable relationship between laboratories and diabetes centres was also detected, which might represent a relevant indicator for the need for rationalisation or standardisation of the method for performing an OGTT. These data highlight the need for greater collaboration between these different bodies. We suggest that other similar investigations should be carried out in other countries within the framework of the IFCC Global Campaign on Diabetes Mellitus.

Background : Oxidative modification of lipoproteins has been reported in β-thalassemia and has been suggested to relate to atherogenesis-risk. This study focused on the change in cholesteryl esters in plasma lipoproteins under oxidative stress resulting from iron overload in β-thalassemia/hemoglobin E (β-thal/Hb E) patients. Methods : Markers of oxidative damage and cholesteryl esters (CEs) were measured in plasma and lipo-proteins from 30 β-thal/Hb E patients and compared to those from 10 healthy volunteers. CEs in plasma, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were separated and identified using HPLC. Results : β-Thal/Hb E patients presented iron overload, a precipitous decrease in α-tocopherol and increased lipid peroxidation (thiobarbituric acid-reactive substances; TBARs) in both plasma and lipoproteins. Cholesteryl linoleate, the most abundant CE in lipoproteins, showed a reduction of 70% in LDL, while other CEs showed a lower reduction (50%). An inverse relationship between the cholesteryl linoleate/cholesteryl oleate ratio (CL/CO) and the degree of clinical severity suggested that the CL/CO ratio is an index of damaged lipoproteins and could be used as a pathologic marker of underlying iron overload. Good correlation of non-transferrin-bound iron (NTBI) and TBARs (r=0.8, p<0.01) in LDL strongly supported the contention that iron overload is responsible for initiating the lipid peroxidation in β-thal/Hb E. Conclusions : This study suggests that cholesteryl linoleate is the primary target of oxidative modification induced by NTBI in β-thal/Hb E patients and that reduction in cholesteryl linoleate in lipoproteins could be used as a severity index for β-thal/Hb E.

Background : Qualitative and quantitative analyses of glycosaminoglycans (GAGs) in serum obtained from Graves' disease (GD) patients without extrathyroidal complications were carried out to provide a clearer understanding of the role of these macromolecules in the disease pathogenesis. Methods : GAGs were isolated from serum of 17 GD patients before treatment and after attainment of the euthyroid state, as well as from 20 healthy individuals. GAGs were quantified using the hexuronic acid assay and subjected to electrophoretic fractionation. Results : Increased amounts of total GAGs were found in GD patients. Attainment of euthyroidism led to a decrease in, but not normalization of, total serum GAGs level. Electrophoretic analyses of GAGs before and after treatment identified the presence of chondroitin sulfate (CS), heparan sulfate/heparin (HS/H) and dermatan sulfate (DS) in serum from healthy subjects and GD patients. CS was the predominant serum GAG constituent in all subjects investigated. Enhanced CS levels in both GD patient groups were accompanied by increased structural heterogeneity of these compounds. Normalization of thyroid function did not change CS levels. DS levels in serum of untreated GD patients were elevated in comparison to healthy subjects. Anti-thyroid treatment led to a significant decrease in DS to levels below those in controls. DS in all serum samples investigated displayed a similar structure. HS/H levels in serum of untreated GD patients was seven-fold higher than in healthy subjects. In addition, HS/H in untreated GD patients were characterized by higher structural heterogeneity than those isolated from control subjects and euthyroid GD patients. Anti-thyroid therapy led to a decrease in HS/H concentrations towards normal values. Conclusions : Our results indicate that in the course of GD, the metabolism of particular types of GAGs is regulated by different mechanisms, including a hyperthyroid state and immunological abnormalities. Furthermore, qualitative and quantitative changes in serum GAGs seem to reflect GD-associated systemic changes in extracellular matrix properties.

Background : Overproduction of nitric oxide (NO) and hydrogen peroxide (H 2 O 2 ) may be an important factor in the pathogenesis of amyotrophic lateral sclerosis (ALS). Owing to their ability to permeate through biological membranes, excess NO and H 2 O 2 may be present in the media surrounding motor neurones. Anti-oxidative defence enzymes (ADEs) in erythrocytes are capable of detoxifying reactive oxygen species (produced endogenously or exogenously), but may also be structurally modified and inactivated by reactive oxygen and nitrogen species. Both balanced and coordinated ADE activities are of utmost importance for their correct physiological function. Methods : We determined activity of the following ADEs: copper-zinc superoxide dismutase (CuZn SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GR) in erythrocytes from sporadic ALS patients [SALS (−/+)], familial ALS patients with the Leu144Phe mutation in the SOD1 gene [FALS (+/+)], asymptomatic carriers with the Leu144Phe mutation in the SOD1 gene (+/−), and control subjects (−/−). We also examined the in vitro effect of diethyldithiocarbamate (DDC) on CuZn SOD activity in erythrocytes from FALS patients, SALS patients and control subjects. Results : The influence of the Leu144Phe mutation and/or disease was apparent for ADE activities measured in all three patient groups. The SOD1 gene mutation decreased CuZn SOD and GSH-Px activity (two-way ANOVA, significant mutation effect). We noted that the disease also contributed to decreased CuZn SOD activity in SALS patients in comparison with the control group (two-way ANOVA, mutation and disease effect). The disease also influenced CAT and GR activity. CAT activity was decreased in both SALS and FALS patients. In all three patient groups, GR activity was higher than in the control group. Finally, DDC inhibited CuZn SOD activity in erythrocytes from control subjects, FALS (Leu144Phe) patients and SALS patients; however, its effect was more pronounced and significant in FALS patients. Conclusions : Changes in erythrocyte ADE activities suggest that oxidative stress, involved in the motor neurone pathogenesis of SALS and FALS, also has systemic effects. Differences in ADE systems between the study groups revealed the presence of different types of oxidative pressure, indicating the potential additional benefit of individually designed anti-oxidant cocktail therapies.

Background : The immunoglobulin superfamily receptor translocation-associated 2 ( IRTA2 ) gene encodes both membrane and secreted forms of the B-cell differentiation antigen (also identified as FcRH5). The membrane form is highly expressed on the surface of hairy cell leukemia (HCL) cells from patients. This study aimed to develop immunoassays for soluble IRTA2/FcRH5 proteins in human serum. Methods : Two sandwich ELISAs for soluble IRTA2/FcRH5 were designed using two pairs of monoclonal antibodies specific to four different epitopes on IRTA2/FcRH5. Results : In both ELISAs, the lower limit of quantitation for soluble IRTA2/FcRH5 in human serum was 30ng/mL. The analytical recovery of 0.3–2.1ng/mL of IRTA2/FcRH5-Fc used as the standard, from a 100-fold dilution of IRTA2/FcRH5-free sera, was 94–106% for ELISA #1 and 89–97% for ELISA #2. Between-assay coefficients of variance were 7.7–17.6% for ELISA #1 and 7.7–32.7% for ELISA #2. Both ELISAs detected soluble IRTA2/FcRH5 protein in sera from normal donors (median 169ng/mL in ELISA #1 and 146 ng/mL in ELISA #2, n=123) without correlations to gender or age. A marked increase in soluble IRTA2/FcRH5 levels was observed in samples from patients with HCL (medians 719ng/mL in ELISA #1 and 754ng/mL in ELISA #2, n=44). Conclusions : These ELISAs may be useful for monitoring soluble IRTA2/FcRH5 in HCL and other B-cell malignancies.

Alkaline phosphatase (ALP) conjugated to antibodies is often used in enzyme immunoassays (EIAs). These assays are notably sensitive to experimental conditions. A possible source of interference is bacterial ALP, which is released when bacterial contamination occurs in clinical analyzers. Preliminary experiments led to the selection of a detection kit, ALP source, and specific types of tubes for collecting water samples and performing assays. The release of ALP from various strains of bacteria identified in pure water was demonstrated (10–30×10 6 cfu/mL released 6–10μU/μL). It was shown that ultrafiltration is totally efficient in removing ALP from water, while residual ALP activity (2.21μU/μL after filtration of an ALP solution of 6.22μU/μL) was observed after filtration using a 0.22-μm filter.

Quantitative measurement of serum monoclonal protein (M-protein) is one of the most important tools for monitoring disease activity in monoclonal gammopathies. The aims of this study were to evaluate serum M-protein quantification by capillary zone electrophoresis (CZE) and to compare results with those obtained by densitometric scanning of high-resolution agarose gel electrophoresis (HRE-AGE). The evaluation was carried out on 82 samples from patients with various monoclonal gammopathies. All the suspected M-proteins were confirmed and characterised by immunofixation on agarose gel (IFE). CZE was performed on a Paragon CZE™ 2000 system (Beckman Coulter). Passing-Bablok regression was: y (CZE)=1.27×(HRE-AGE)–5.21g/L. The correlation coefficient was 0.92. Bland-Altman analysis demonstrated a mean difference of −1.83g/L (95% CI −0.76 to −2.90) with clear evidence of a concentration-related bias. Densitometry gave higher values at low M-spikes (<20g/L), whereas CZE gave higher values at large M-spikes (>20g/L). The concentration-related bias was found to be independent of the immunoglobulin isotype. In conclusion, to compare previous results obtained by M-protein densitometric scanning with those obtained by direct measurement of CZE peaks, the calculation of a univocal transforming factor appears to be unreliable.

Background: Saliva can be used as a diagnostic fluid in medicine. Components of saliva proposed as disease markers include enzymes (alkaline phosphatase, esterase, glucuronidase, aminopeptidase), immunoglobulins (IgA, IgG), and hormones (steroid hormones). Many of these salivary components appeared to be useful biochemical markers of the evolution of periodontal disease, for which salivary analysis can offer a cost-effective approach for monitoring the disease. The salivary components proposed as markers for periodontal disease activity are aspartate aminotransferase (AST), alkaline phosphatase (ALP), aminopeptidases, and glucuronidases. The purpose of our study was to illustrate the influence of periodontal disease on the level of salivary AST, alanine aminotransferase (ALT) and ALP. Methods: All clinical periodontal examinations were performed by the same periodontist. All patients included in the study presented a probing depth >5mm, bleeding on probing and alveolar bone loss >40%. Salivary AST, ALT and ALP activities were measured using DiaSys analysis kits from Diagnostic Systems. The methods were adapted for saliva. Results: Salivary AST activity in patients with periodontal disease was significantly increased (p<0.01) (median 81.75±23U/L) compared with controls (15.25±10.5U/L). Salivary ALT activity was not significantly modified in saliva from patients with periodontal disease compared with the control group. Our results showed a significant (p<0.01) increase in salivary ALP activity (34.38±1.5U/L) in patients with periodontal disease compared with controls (6.6±4.2U/L). Conclusions: Our results revealed that periodontal destruction such as periodontal pockets, gingival bleeding and suppuration are related to higher ALP and AST levels in saliva. Salivary AST could be used as a useful marker for monitoring periodontal disease. The increase in salivary ALP activity in periodontitis demonstrated could be associated with alveolar bone loss, a key feature of periodontal disease. More studies are necessary to evaluate which specific clinical, microbiological and histological characteristics of periodontal disease are associated with elevated levels of AST and ALP in saliva.

Background : The role of measurement of reticulocytes and their parameters is growing in sports medicine. The use of reticulocyte counts in protocols for evaluating and screening for the suspected abuse of hormones that stimulate the bone marrow is an example. Reticulocytes are also important for evaluation of the performance and general health status of athletes, especially for monitoring therapies and diets. The current availability of fully automated haematological systems that can measure reticulocyte numbers and characteristics (volume, density) increases the potential use of these parameters in laboratory and sports medicine. Few studies have considered the application of these parameters in athletes and a lack of specific reference ranges means that their valid clinical use is difficult. Methods : Using a Coulter LH700 instrument, we measured reticulocyte count (Retics), mean reticulocyte volume (MRV), immature reticulocyte fraction (IRF), and mean sphered cell volume (MSCV) in 106 male professional elite athletes (football and rugby players and skiers). Reference intervals for the athletes were compared with the intervals found for a control group of 73 age-matched males. Results : We calculated the following reference intervals: 0.30–1.54% for Retics, 93.1–114.8fL for MRV, 0.18–0.39% for IRF, and 76.8–94.5fL for MSCV. Conclusions : No statistically significant differences were observed for Retics, MRV, IRF, and MSCV between elite athletes and controls. Significant differences were observed for haemoglobin (Hb), erythrocytes, haematocrit (Ht), and mean corpuscular volume. Moreover, no statistical differences were observed among different sports, whereas differences were remarked in football and rugby players between the samples drawn before the start of competitive season and the samples drawn during the season, demonstrating that reticulocyte counts and parameters are useful for monitoring sportsmen.

Background : Over the past decade, it has been well established that elevated total serum homocysteine (tHcy) in adults is associated with increased risk of cardiovascular and thromboembolic diseases. Since risk factors for such diseases are established at a young age, the aim of the present study was to measure serum tHcy levels in 134 (71 boys, 63 girls) randomly selected healthy preschool children aged 4–6years (mean 5.1), and to investigate possible correlation with paraoxonase 1 (PON1) activity, an antioxidant enzyme that contributes to the antiatherogenic properties of high-density lipoprotein (HDL). Methods : tHcy was determined using an IMX tHcy assay (FPIA). PON1 was measured by a spectrophotometric method at 412nm. Results : Mean serum tHcy was 7.71±2.35μmol/L. A relatively significant percentage (15.6%) of boys and girls had elevated serum tHcy levels (>10μmol/L). tHcy levels were slightly higher in girls compared to boys (8.20±2.80 vs. 7.29±1.79μmol/L, respectively; p<0.11). There was no significant interaction between age and tHcy levels. Mean PON1 activity was 124.86±66.62U/L. No statistical difference in enzyme activity was observed between boys and girls (126.81±69.99 vs. 121.74±64.78U/L) was observed. On the contrary, a weak negative relationship between tHcy concentration and PON1 activity was detected, with Pearson's correlation coefficient of r=−0.27. Conclusions : The significant percentage of elevated tHcy levels observed in healthy preschool cases and the negative tHcy correlation with PON1 activity are reported for the first time. Since children with a family history of cardiovascular disease have higher levels of serum Hcy, tHcy screening in children, even of this age, in relation to other parameters, such the protective PON1, might prove a useful prevention procedure for the genetic risk of premature atherosclerosis.

Background : Homocysteine metabolism is mainly governed by serum concentrations of folate and vitamin B 12 , renal function and genetic factors. It is also well documented that endocrinological disturbances influence homocysteine metabolism. However, studies on the hormones of the hypothalamic-pituitary-adrenal axis have given conflicting results. Methods : A total of 30 healthy young males were randomised into three groups of equal size; one group received adrenocorticotrophic hormone (ACTH) 1–24 1mg i.m. daily for 4days, another group was treated with cortisol 50mg i.m. t.i.d. for 4days, while a control group was observed for 4days. Fasting blood samples were collected before and after treatment or observation. Results : The pattern of changes was the same for the ACTH and cortisol groups; there were significant decreases in serum concentrations of folate (23% and 24%) and cobalamines (13% and 19%) and decreases in plasma total homocysteine concentrations that did not reach significance. There were no changes in the control group. Conclusions : The virtually identical pattern of changes in both treatment groups suggests that the effects were mediated by cortisol. The reductions in serum concentrations of folate and cobalamines call for further research.

Background: The aim of this study was to compare cotinine determinations in three biological fluids for assessing environmental tobacco smoke (ETS) exposure in female non-smokers (n=1605) in Italy. Methods: Information about ETS exposure at home, in the workplace, and in other places within the previous week was collected via questionnaire. Plasma, salivary and urinary cotinine levels were measured. Cotinine levels of ≥0.1ng/mL for plasma, ≥0.2ng/mL for saliva, and ≥0.5ng/mL for urine were used to determine biochemical exposure. Results: Median cotinine levels were significantly higher in exposed than in unexposed women (0.21 vs. 0.05ng/mL in plasma, 0.80 vs. 0.41ng/mL in saliva, and 9.74 vs. 5.30ng/mL in urine). Self-reported ETS exposure was significantly related to biochemical exposure [odds ratio 2.99, (95% CI 2.40–3.72) for plasma; 1.90 (1.51–2.39) for saliva; and 2.67 (2.14–3.34) for urine]. Cotinine significantly increased with increasing exposure level, regardless of the exposure source. Among self-reported exposed subjects, higher percentages of cotinine level above the cut-off, i.e., indicating exposure, were found in saliva (76%) and urine (75%) than in plasma (52%). Conclusions: In general, women correctly reported their ETS exposure status. Both non-invasive salivary and urinary cotinine determinations seem preferable in epidemiological studies, in view of their higher sensitivity, when compared to plasma cotinine.

Background : The aim of this study was to determine cut-off values for total serum immunoglobulin E (IgE) between non-atopic and atopic children with respiratory symptoms. Children of 0–16years of age were evaluated for respiratory symptoms of >4-week duration. Methods : Children were divided into two groups: non-atopic children (n=3355) who were non-IgE-sensitized with undetectable allergen-specific IgE (<0.35kIU A /L), and atopic children (n=4620) who were sensitized to ≥1 allergens (specific IgE ≥0.35 kIU A /L). Upper and lower centiles were determined and cut-off values calculated using receiver operating characteristic (ROC) analysis. Results : Serum total IgE increased with age in both groups, although at a variable level and rate, and reached a plateau at 9 and 10years in non-atopic and atopic children, respectively. Atopic children had on average 14-fold higher serum total IgE compared to non-atopic children. In both groups, the median was lower than the corresponding mean and the distribution skewness was always positive (group I, 0.87; group II, 0.91). In almost all age groups, the 95th percentile for non-atopic children corresponded to the calculated cut-off values, whereas the 10th percentile for atopic children corresponded to the respective cut-off values only until the age of 8years, after which greater differences between the cut-off values and the 10th percentile were recorded. Cut-off values for total serum IgE in children up to 16years were determined with diagnostic sensitivity, specificity and area under the ROC curve of 96%, 91% and 0.950, respectively. Conclusions : The 95th percentile for total IgE in non-atopic children and the 10th percentile in atopic children could be taken as cut-off values in children up to 8years of age, after which significant percentile discrepancies between non-atopic and atopic children were recorded. Since atopic subjects show a more irregular centile distribution, cut-off values are best determined by ROC analysis.

Background : Circulating human thyroglobulin (TG) measurement has a pivotal role in the management of patients affected by differentiated thyroid cancer (DTC). Undetectable thyrotropin (TSH)-stimulated serum TG after thyroid ablation (i.e., thyroidectomy and radioiodine) implies the absence of residual or relapsing DTC. Recently, high-cost recombinant human TSH (rhTSH) was proposed for TG stimulation to avoid uncomfortable thyroxine (T 4 ) withdrawal. However, only a small fraction of relapsing DTC patients showed undetectable TG under T 4 treatment (onT 4 -TG) by high-sensitivity assays. The present study was undertaken to compare onT 4 -TG with the rhTSH-stimulated TG assay (rhTSH-TG), 131 I scanning and neck ultrasound (US) with fine-needle aspiration biopsy. Methods : We enrolled 117 patients with histologically proven DTC treated by total thyroidectomy and radioiodine. Inclusion criteria were: complete tumour excision, no radioiodine uptake outside of the thyroid bed at post-treatment scan and undetectable onT 4 -TG 3months after primary treatment. At 1year after radioiodine treatment, all patients underwent onT 4 -TG assay, rhTSH-stimulated TG assay, 131 I scanning and neck US. Based on histology, clinical data and long-term follow-up, persistent/relapsing disease was confirmed in 14 patients. Results : onT 4 - and rhTSH-TG were positive in 10 and 12 patients, respectively and two patients converted from undetectable to detectable TG after rhTSH administration. Neck US was positive in 10 patients and a combination of US with onT 4 - and rhTSH-TG assays showed positivity in 13 and 14 out 14 patients, respectively. A radioiodine scan was positive in six patients, all with positive onT 4 - and rhTSH-TG levels. Globally, the negative predictive value of the onT 4 - and rhTSH-TG assays was 99% and 100%, respectively, and 104 rhTSH stimulations had to be performed to detect one local recurrence with negative onT 4 -TG. Conclusions : Our preliminary data need further confirmation on larger groups of patients, but seem to indicate that onT 4 -TG assay by a high-sensitivity method combined with neck US may avoid rhTSH stimulation in low-risk DTC patients after surgery and radioiodine thyroid ablation.

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-induced death. Somatic mutation of the p53 gene is the most common genetic abnormality so far described in human cancer and there is evidence that supports a high level of p53 alterations in HCC. The aim of this study was to investigate serum levels of p53 in Egyptian patients with HCC, and its relation to other prognostic factors such as tumor grade, α-fetoprotein (AFP), and liver function tests in an attempt to clarify their significance in the pathogenesis of the disease. Liver function tests were carried out and AFP and p53 levels were measured for all individuals studied. Our results show that detection of p53 increased the frequency of HCC prediction from 79.5% to 86.3%. Moreover, significant positive correlation between p53 and tumor size (cm) for tumor grade II and III was identified. In conclusion, serum concentration of p53 protein may be a convenient and useful non-invasive screening test for prediction of HCC.

Experienced physicians noted unexpectedly elevated concentrations of lactate dehydrogenase in some patient samples, but quality control specimens showed no bias. To evaluate this problem, we used a “latent reference individual extraction method”, designed to obtain reference intervals from a laboratory database by excluding individuals who have abnormal results for basic analytes other than the analyte in question, in this case lactate dehydrogenase. The reference interval derived for the suspected year was 264–530U/L, while that of the previous year was 248–495U/L. The only change we found was the introduction of an order entry system, which requests precise sampling volumes rather than complete filling of vacuum tubes. The effect of vacuum persistence was tested using ten freshly drawn blood samples. Compared with complete filling, 1/5 filling resulted in average elevations of lactate dehydrogenase, aspartic aminotransferase, and potassium levels of 8.0%, 3.8%, and 3.4%, respectively (all p<0.01). Microhemolysis was confirmed using a urine stick method. The length of time before centrifugation determined the degree of hemolysis, while vacuum during centrifugation did not affect it. Microhemolysis is the probable cause of the suspected pseudo-elevation noted by the physicians. Data-mining methodology represents a valuable tool for monitoring long-term bias in laboratory results.

Background: There is a lack of consensus regarding the most appropriate specimen type for analysis of many biochemistry analytes. The aim of this study was to compare renal and lipid analyte profiles and phenytoin values in plain serum (S), serum gel (G) and plasma (lithium heparin, P) tubes and to investigate the stability of these analytes after prolonged contact with cells or gel at room temperature (RT, 20°C) and as aliquoted and stored at 4°C. Methods: Primary specimens were centrifuged once, maintained at RT and analysed within 2h (T 0 ) and after 24 h (T 24 ) and 48h (T 48 ). For assessment of stability at 4°C, two cell-free aliquots were separated from each of the primary tubes and stored at 4°C and then analysed at T 24 and T 48 . Differences in analyte concentrations between tubes at T 0 and following storage (at T 24 and T 48 ) were evaluated for both statistical and clinical significance. Results : At T 0 all analytes, except potassium, demonstrated equivalence between serum, gel and plasma tubes. Potassium and creatinine were more stable in gel tubes than in serum/plasma tubes. In contrast, phentytoin was stable in plain serum and plasma up to T 48 at RT, but showed a progressive and clinically significant decrease in concentration in gel tubes at T 24 and T 48 at RT. All analytes except CO 2 were stable up to T 48 when aliquoted and stored at 4°C. Conclusions : We concluded that the serum gel tube has advantages over plain serum and plasma tubes for measurement of the analytes investigated in this study, with the exception of phenytoin. In practice, the gel tubes demonstrate enhanced analyte stability and reduce the need to aliquot specimens, with greater protection against possible contamination. Further investigation would be required to evaluate a broader range of analytes.

We have characterized the performance of a commercial two-site immunoradiometric assay for manual in vitro diagnostic measurement of plasma corticotropin from Scantibodies Laboratory. We compared the results with those of a similar commonly used assay from Nichols Institute Diagnostics that has recently been withdrawn from production. The lower detection limit, range of the standard curve, cross-reactivity, and intra-assay and inter-assay imprecision of the two assays are very similar. Measurement of clinical samples and a series of samples from an experimental subject demonstrate high correlations between the two assays. These factors, together with recent clearance by the United States Food and Drug Administration for manual in vitro diagnostic measurement, make the Scantibodies corticotropin immunoradiometric assay an appropriate replacement for the Nichols assay.