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Abnormal metabolic network activity in Parkinson's disease: test-retest reproducibility.

Parkinson's disease (PD) is associated with an abnormal pattern of regional brain function. The expression of this PD-related covariance pattern (PDRP) has been used to assess disease progression and the response to treatment. In this study, we validated the PDRP network as a measure of parkinsonism by prospectively computing its expression (PDRP scores) in (15)O-water (H(2)(15)O) and (18)F-fluorodeoxyglucose (FDG) positron emission tomography (PET) scans from PD patients and healthy volunteers. The reliability of this measure was also assessed within subjects using a test-retest design in mildly affected and advanced PD patients scanned at baseline and during treatment with levodopa or deep brain stimulation (DBS). We found that PDRP expression was significantly elevated in PD patients (P<0.001) relative to controls in a prospective analysis of brain scans obtained with either H(2)(15)O or FDG PET. A significant correlation (R(2)=0.61; P<0.001) was evident between PDRP scores computed from H(2)(15)O and FDG images in PD subjects scanned with both tracers. Test-retest reproducibility was very high (intraclass correlation coefficient (ICC)>0.92) for PDRP scores measured both within PET session and between sessions separated by up to 2 months. This high reproducibility was observed in both early stage and advanced PD patients scanned at baseline and during treatment. The within-subject variability of this measure was less than 10% for both unmedicated and treated conditions. These findings suggest that the PDRP network is a reproducible and stable descriptor of regional functional abnormalities in parkinsonism. The quantification of PDRP expression in PD patients can serve as a potential biomarker in PET intervention studies for this disorder.

Aged↗

Predicting cognitive decline in Alzheimer disease. Role of platelet amyloid precursor protein.

An altered pattern of platelet amyloid precursor protein (APP) forms, consisting of a reduced ratio of the upper (130 kDa) to the lower (110 to 106 kDa) immunoreactivity band (APPr), is associated with Alzheimer disease (AD), although in the early stages of AD this pattern shows high variability. To explore whether APPr values at baseline may predict the rate of cognitive decline, we evaluated patients with mild AD before and after 1 year of treatment with acetylcholinesterase inhibitors. Lower APPr at baseline was the only predictor of progressive cognitive decline, suggesting that this biomarker might be a useful indicator of prognosis for patients with AD.

Age of Onset↗

Eosinophilic dysplasia of the cervix: a newly recognized variant of cervical squamous intraepithelial neoplasia.

A proportion of cervical squamous intraepithelial lesions encountered in surgical pathology practice contain both metaplastic features and some degree of atypia [so-called eosinophilic dysplasia (ED)] that defy classification according to established criteria. To elucidate the nature of these lesions, we compared 44 cases of ED to 20 classic high-grade squamous intraepithelial lesions (HSILs) and 10 squamous metaplasias using a panel of biomarkers and human papillomavirus (HPV) testing. EDs were defined as 1) lack of normal maturation; 2) relatively abundant eosinophilic cytoplasm and distinct cell borders compared with conventional HSIL; 3) mildly to moderately increased nuclear to cytoplasmic ratio; and 4) focal dysplastic nuclei showing nuclear enlargement, hyperchromasia, variable nuclear membrane irregularities, and appreciable nucleoli. Expression of p16 (p16), MIB-1 (Ki-67) labeling index, and HPV DNA detection and typing were performed on each case. The majority of EDs showed more than three atypical cells in an entire lesion but lack of apparent features of HSIL. It was common to find neighboring cervical squamous metaplasia and/or conventional SILs (either HSIL or low-grade squamous intraepithelial lesion [LSIL]). Among the 44 cases, 18 (45%) ED lesions were found to be associated with HSIL, 15 (34%) with LSIL or condylomatous lesions, and 13 (30%) EDs were seen without any SILs in the entire specimens. Area of benign squamous metaplasia was found in all ED cases. High levels of p16 and MIB-1 expression were seen in 41 (93%) of 44 ED cases with degrees of immunoreactivity closely resembling those seen with HSIL. Of 16 EDs tested, 13 (81%) were positive for HPV DNAs. Among 10 HPV-positive cases subtyped, 9 (90%) cases contained intermediate- and/or high-risk HPVs and 1 case contained a novel HPV. In the follow-up of pure ED cases, the majority showed presence of dysplastic lesions of either HSIL or LSIL on either loop electric excision procedures or Papanicolaou test samples after a 6- to 10-week period. Therefore, ED represents an unrecognized and potentially clinically significant subgroup of cervical intraepithelial lesions. Based on the unique histologic appearance of ED, its association in some cases with HSIL, the overall immunohistochemical findings, frequent association of ED with intermediate- and/or high-risk HPV infection, and limited follow-up data, we believe that ED represents a variant of HSIL (CIN 2). Since ED possesses histologic features of both dysplasia and metaplasia, we speculate that it may arise from metaplastic cervical squamous epithelium that has subsequently become infected with intermediate- or high-risk HPV.

Adolescent↗

Is 1-hydroxypyrene a reliable bioindicator of measured dietary polycyclic aromatic hydrocarbon under normal conditions?

Five healthy volunteers consumed similar amounts of identical foods for 5 consecutive days. The concentration of pyrene and of benzo(a)pyrene was determined in each of the 15 meals by a short analytical method that included sample saponification, solvent extraction, and HPLC analysis. The volunteers also provided three daily total volume 8-h urine samples for the duration of the study for the assessment of 1-hydroxypyrene, a biomarker of pyrene and polycyclic aromatic hydrocarbon (PAH) exposure. Mean recoveries were 83 and 75%, respectively, for pyrene and benzo(a)pyrene in food. Daily dietary pyrene doses varied from 0.7 to 3 microg. Excluding two outliers consisting of meals containing charbroiled pork and beef, pyrene content in the meals estimated from the published literature data was correlated to the measured pyrene, but overestimated the actual concentration by ca. 70%. Despite the identical ingested doses of pyrene, there was a 50-76% (coefficient of variation) interindividual variability in the daily-excreted amount of 1-hydroxypyrene. Urinary excretion of this metabolite was not correlated with ingested dose of pyrene under the normal feeding conditions used in this study. Bioavailability, enzymatic polymorphism, and differences in enterohepatic cycling of the metabolite may contribute to the observed variability. It was calculated that dietary pyrene intake accounts for between 87.5 and 99.8% of the sum of dietary and inhalation intake. From the presented data, unless the above-mentioned factors are taken into account, 1-hydroxypyrene might not be a reliable bioindicator of ingested pyrene (PAHs) under normal feeding conditions.

Biomarkers↗

Exposures among pregnant women near the World Trade Center site on 11 September 2001.

We have characterized environmental exposures among 187 women who were pregnant, were at or near the World Trade Center (WTC) on or soon after 11 September 2001, and are enrolled in a prospective cohort study of health effects. Exposures were assessed by estimating time spent in five zones around the WTC and by developing an exposure index (EI) based on plume reconstruction modeling. The daily reconstructed dust levels were correlated with levels of particulate matter < or = 2.5 microm in aerodynamic diameter (PM2.5; r = 0.68) or PM10 (r = 0.73-0.93) reported from 26 September through 8 October 2001 at four of six sites near the WTC whose data we examined. Biomarkers were measured in a subset. Most (71%) of these women were located within eight blocks of the WTC at 0900 hr on 11 September, and 12 women were in one of the two WTC towers. Daily EIs were determined to be highest immediately after 11 September and became much lower but remained highly variable over the next 4 weeks. The weekly summary EI was associated strongly with women's perception of air quality from week 2 to week 4 after the collapse (p < 0.0001). The highest levels of polycyclic aromatic hydrocarbon-deoxyribonucleic acid (PAH-DNA) adducts were seen among women whose blood was collected sooner after 11 September, but levels showed no significant associations with EI or other potential WTC exposure sources. Lead and cobalt in urine were weakly correlated with sigmaEI, but not among samples collected closest to 11 September. Plasma OC levels were low. The median polychlorinated biphenyl level (sum of congeners 118, 138, 153, 180) was 84 ng/g lipid and had a nonsignificant positive association with sigmaEI (p > 0.05). 1,2,3,4,6,7,8-Heptachlorodibenzodioxin levels (median, 30 pg/g lipid) were similar to levels reported in WTC-exposed firefighters but were not associated with EI. This report indicates intense bystander exposure after the WTC collapse and provides information about nonoccupational exposures among a vulnerable population of pregnant women.

Adult↗

Influence of initiation of maintenance hemodialysis on biomarkers of inflammation and oxidative stress.

BACKGROUND: Chronic inflammation and oxidative stress are highly prevalent in patients with chronic kidney disease and end-stage renal disease (ESRD). These conditions contribute to high mortality rates associated with cardiovascular disease, the leading cause of death in this patient population. To our knowledge, no prospective studies have examined how initiation of maintenance hemodialysis (MHD) affects biomarkers of inflammation and oxidative stress status. METHODS: This was a prospective cohort study evaluating time-dependent changes in C-reactive protein (CRP), interleukin-6 (IL-6), interleukin-10 (IL-10), and protein carbonyl content before and after initiation of MHD over a 12-month period. Fifty incident hemodialysis patients [57.6 +/- 17.2 years, 60% male, 38% Caucasians, 32% insulin-dependent diabetes mellitus (IDDM) were studied, with 50 healthy subjects for comparison. The study variables were assessed before the initial outpatient hemodialysis treatment, and every 3 months thereafter for 12 months. RESULTS: At baseline, CRP, IL-6, and carbonyl content levels were significantly higher in MHD patients compared with healthy subjects (P < 0.001 for each). After initiation of MHD, there were no significant changes in any of the study variables. Patients who initiated MHD with the highest levels of all the study variables had a significant decrease over the next year, but the variables were still higher than normal at the end of the 12-month study period. CONCLUSION: Our data show that initiation of MHD does not have significant influence on plasma concentrations of CRP, IL-6, and IL-10, as well as plasma protein carbonyl content. These findings suggest that MHD is ineffective in controlling inflammation and oxidative stress in uremia.

Adult↗

Prediction of survival in 70-year olds.

The purpose of this study was to find predictors of 10-year survival among 70-year olds and to show that there might be more than one good set of predictors. All variables found to be significantly related to survival in univariate tests (predictors) are given a risk value (0 = no risk, 1 = risk), and classified into high-order domains. The number of domains containing predictors with risk values are then counted, giving the individual a certain risk index. The method was applied on data from the longitudinal population study of 70-year-old people in Gothenburg, Sweden, the H70-study. In a random sample of 474 men and 562 women predictors for survival over a 10-year period, from the age of 70, were selected for men and women, respectively. The predictors were classified into seven domains: socio-economic status, life style, social networks, need of help, biomarkers of ageing, physical health and cognitive function. The analysis revealed a consistent pattern in which survival was related to the number of risk domains. In the no-risk sub-sample 12.5% of the men were deceased after 10 years, in the sub-sample with five or more risk domains involved 78.0% were deceased. The corresponding figures for women were 10.0% and 60.0%. The results show that it is possible to find variables that can predict survival. However, there is not only one set of good predictors. As an alternative to use of specific variables as predictors the presumptive predictors can be grouped into a few main domains and good predictions be made on the basis of these domains.

Journal Article↗

Biological markers in nasal secretions.

Biological markers in nasal secretions provide valuable information on nasal pathophysiology. However, published data on biomarker concentrations in nasal fluids are remarkably inconsistent, and the bias due to different sampling techniques, has not yet been systematically evaluated. Concentrations of various protein were repeatedly determined in nasal secretions of 16 healthy volunteers. The proteins were detected by using: 1) alpha2-macroglobulin as a marker for plasma contamination; 2) lactoferrin as a marker for glandular secretion; 3) lactate dehydrogenase as a marker for tissue injury; and 4) interleukin (IL)-1beta, IL-8, tumour necrosis factor-alpha, and eosinophil cationic protein and tryptase as indicators for tissue inflammation. A total of four different sampling methods, including nasal lavage (NL) and a new polyurethane foam sampler technique (PFST) were employed. Analyte concentrations in NL were approximately 10-times lower than in specimens obtained by PFST. Due to the unpredictable dilution during NL, various analytes were below the detection limit of the high sensitivity assays employed. With PFST, concentrations below the detection limit rarely occurred. The specimens did not significantly differ regarding plasma contamination, glandular secretion or tissue injury. The considerable variability of reported analyte concentrations in nasal secretions mainly results from different sampling techniques. To collect nasal secretions, samplers are considered superior to nasal lavage techniques.

Adolescent↗

Monitoring of the impact of Prestige oil spill on Mytilus galloprovincialis from Galician coast.

In November 2002, the Prestige oil tanker was wrecked in front of Galician coast (NW of Spain), spilling near 63,000 tons of heavy oil until February 2003. Contamination produced was very extensive (70% of Galician beaches were reached by the oil) but heterogeneous, alternating intensely affected zones with neighbour locations where the repercussion was minimal. The objective of this study was to monitor sea environment contamination caused by Prestige oil spill during an 11-month period (August 2003-June 2004, nine samplings) in two locations of Galician coast with different geographical properties (Lira and Ancoradoiro beaches), by means of chemical determination of total polycyclic aromatic hydrocarbons (TPAH) in seawater, and using as exposure biomarker TPAH content in mussel (Mytilus galloprovincialis) tissues, and as effect biomarker DNA damage in mussel gills, evaluated by the comet assay. In addition, recovery ability of the mussels was determined after a 7-day stay in the laboratory. TPAH contents in seawater were very high in the earliest samplings, but then they maintained below 200 ng L(-)(1), similar to reference seawater. However, TPAH levels in mussel tissues were more variable: they increased again from January 2004, probably due to the adverse meteorological conditions that turned over the sea bottom and dispersed the oil accumulated in sediments. In most samplings, these levels decreased during the recovery stage. DNA damage in oil-exposed mussels was significantly higher than in reference mussels, both before and after the recovery phase, but they did not differ to one another. Comet tail length was slightly reduced during the recovery stage, indicative of a certain DNA repair in exposed mussels. This study showed up the importance of monitoring sea contamination events during an extended time, not only in evaluating the presence of the contaminants in the environment but also in determining their bioaccumulation and their effect on the exposed organisms.

Accidents↗

Cancer biomarker validation: standards and process: roles for the National Institute of Standards and Technology (NIST).

Rigorous validation of biomarkers for early detection of cancer differs at the National Institute of Standards and Technology (NIST) from similar processes common among research laboratories. As a newly discovered biomarker assay makes the transition from a research setting to the clinical diagnostic laboratory, it should progress through defined stages of assay confirmation. The first task of a validation laboratory is evaluation of research assay technology, performance, and specifications (analytical validation). However, the ultimate goal is initial validation of the test to identify early stage cancer (clinical validation). Upon technical and clinical confirmation, assays are moved systematically toward a standardized, reproducible, high-throughput format for clinical diagnostic implementation. With laboratory performance rigorously established, the clinical variables can subsequently be analyzed to define limitations, applications, and clinical utility. The role of NIST in technology evaluation for early cancer testing is described in the context of similar programs and prior experience at NIST. Here we conceptualize the validation steps of cancer test development and examine how NIST activities impact health care through institutional focus on measurement, technology, and standards development programs.

Biomarkers, Tumor↗

Leucopenia is an independent predictor in cancer patients requiring invasive mechanical ventilation: a prognostic factor analysis in a series of 168 patients.

OBJECTIVE: To determine prognostic factors predicting success of invasive mechanical ventilation in medical cancer patients admitted to ICU for a complication, in terms of extubation and ICU and hospital discharges. DESIGN: Retrospective study SETTING: Medical ICU of an European cancer hospital. SUBJECTS: A total of 168 consecutive cancer patients who were admitted to ICU for an acute medical complication requiring immediate mechanical ventilation or who later needed mechanical ventilation. MEASUREMENTS: Variables related to the demographic, cancer, scores and complication characteristics. Extubation rates, ICU and hospital mortalities and duration of survival were measured. RESULTS: Respectively, 26%, 22% and 17% of the patients were extubated, discharged from the ICU and discharged from hospital. For weaning from mechanical ventilation, a higher APACHE II score and leucopenia were poor prognostic factors in univariate analysis, but leucopenia remained the only significant one in multivariate analysis. For ICU mortality, no significant prognostic feature was identified. For hospital mortality, leucopenia was the only significant factor in univariate as well as in multivariate analyses. CONCLUSION: Leucopenia appeared to be the only independent poor prognostic factor for both extubation and hospital discharge. None of the variables related to the cancer disease process was shown to be a predictor of success.

Adult↗

Immunocytochemical study with an anti-transferrin binding protein serum: a marker for avian oligodendrocytes.

We have investigated immunocytochemically the localization of a transferrin binding protein (TfBP) in adult CNS of avian and mammalian species using a polyclonal antibody raised against the protein purified from hen oviduct membranes (alpha OV-TfBP). TfBP has recently been shown to be HSP108. An overall strong immunoreactivity was revealed in most parts of the avian brains, especially in the white matter. The main immunoreactivity originated in small, intensely reacting cells interpreted as oligodendrocytes. The density of TfBP-labeled oligodendrocytes of the avian brains was generally proportional to the degree of myelination. There were no marked differences in TfBP-immunostaining pattern between avian species (chick, pigeon and lovebird). On the other hand, in rat, rabbit and cat brains we could not find any TfBP-immunoreactivity. Immunoelectron microscopy has further revealed that TfBP is present in the light and medium types of oligodendrocytes which are known to have high metabolic activities. TfBP reaction product was homogeneously dispersed throughout the perinuclear cytoplasm and fine processes of oligodendrocytes. The intracytoplasmic organelles such as mitochondria and Golgi apparatus were devoid of reaction product. The presence of TfBP in oligodendrocytes implies that this protein may play an important role in transferrin-mediated iron metabolism in the CNS. The complete lack of cross-reactivity between alpha OV-TfBP and mammalian tissues suggests that there is species variability in TfBP structure. We conclude that this chick TfBP antiserum will prove useful in studies of oligodendrocytes and myelination in the avian CNS.

Animals↗

Clinical performance of immunoreactive tartrate-resistant acid phosphatase isoform 5b as a marker of bone resorption.

Previous immunoassays developed for the measurement of serum tartrate-resistant acid phosphatase (TRACP) have lacked specificity for osteoclastic TRACP, TRACP 5b, or have not shown satisfactory clinical performance. The aim of this study was to evaluate the clinical performance of a novel immunocapture activity assay for TRACP 5b, in comparison to telopeptide fragments of type I collagen. Within-subject variability and the effect of feeding on TRACP 5b and telopeptides of type I collagen were assessed in 20 healthy premenopausal women. Diurnal variation of TRACP 5b and serum beta C-terminal cross-linked telopeptide of type I collagen (sbetaCTX) was assessed in 12 healthy postmenopausal women. Renal clearance was assessed in 19 end stage renal failure patients undergoing routine haemodialysis. Response to antiresorptive treatment and calcium supplementation was assessed in osteoporotic postmenopausal women treated with alendronate and calcium (n = 16) or with calcium alone (n = 7) for 24 weeks.Within-subject variability (CVi) of TRACP 5b was 6.6%, lower than CVi of urinary and serum telopeptides. TRACP 5b decreased by 2.4 +/- 0.8%, in response to feeding (P < 0.05) compared to 7.0 +/- 2.6% to 7.9 +/- 3.7% for urinary telopeptides (P < 0.05 to < 0.01) and 8.5 +/- 1.7% to 17.8 +/- 2.6% for serum telopeptides (P < 0.0001). The amplitude of the diurnal rhythm for TRACP 5b was small compared to that of sbetaCTX, 14 +/- 4% vs. 137 +/- 14%. Haemodialysis did not have a significant effect on TRACP 5b but reduced sbetaCTX by 46 +/- 4% (P < 0.0001). In response to alendronate, TRACP 5b decreased by 39 +/- 4% compared to 49 +/- 4% to 69 +/- 5% for urinary telopeptides and 75 +/- 8% for sbetaCTX. We conclude that TRACP 5b shows an attenuated response to antiresorptive therapy in comparison with other markers of bone resorption, but that this may be offset by lower biological variability. TRACP 5b may provide useful additional information about bone resorption.

Acid Phosphatase↗

A decrease in glucose production is associated with an increase in plasma citrulline response to oral arginine in normal volunteers.

Acute arginine administration (30 g) increases insulin secretion and reduces glucose production (GP). A slower administration of L-arginine may have direct effect on the liver without increasing C-peptide or insulin secretion. We tested the direct effect of oral L-arginine on fasting GP in 15 normal-weight volunteers and compared these to a group of L-alanine-treated controls (placebo). Volunteers were admitted to the General Clinical Research Center for a 3-day stay. Three grams of freebase arginine or alanine was ingested hourly between 4 am and 2 pm. Neither arginine nor alanine had an effect on C-peptide or insulin concentration. Oral arginine, but not alanine, increased plasma arginine, citrulline, and ornithine concentrations. Arginine-treated volunteers had a greater fall in GP as compared to the alanine-treated group (16.2% +/- 1.9% v 9.7% +/- 3.6%, respectively; P<.05). Five volunteers treated with arginine had less than a 30% increase in citrulline concentration (26 +/- 2 to 32 +/- 2 micromol/L, mean +/- SEM) and 10 volunteers had equal to or greater than a 30% increase in plasma citrulline concentration (29 +/- 2 to 49 +/- 4 micromol/L, P<.05). Since citrulline is generated in the conversion of arginine to nitric oxide (NO), the failure of oral arginine to increase citrulline concentration suggests that NO generation may be varied in different individuals. The increased plasma citrulline group reduced GP by 18.2% +/- 1.9% over the final 4 hours of arginine administration (2.00 +/- 0.08 to 1.64 +/- 0.07 mg/kg/min; P<.01). In contrast, GP only decreased by 12.4% +/- 3.9% (1.97 +/- 0.13 to 1.73 +/- 0.13 mg/kg/min; not significant [NS]) in those who had little to no increase in plasma citrulline concentration. The 12% decrease in GP in the hyporesponders was similar to the 10% decrease seen in the alanine-treated normal volunteers (9.7% +/- 3.6%). Individuals may have a variable NO response from an oral arginine administration. GP is suppressed in those who have a greater increase in plasma citrulline concentration.

Adult↗

Elevated glycine betaine excretion in diabetes mellitus patients is associated with proximal tubular dysfunction and hyperglycemia.

In an ambulatory population of diabetic subjects (Type 1 and Type 2), the urine excretion of the renal osmolyte, glycine betaine, was compared to known markers of glycemic control, renal dysfunction and to the excretion of related betaines, including trigonelline, proline betaine, carnitine and acetyl-carnitine. Of the 85 subjects, 20 patients had urine glycine betaine concentrations above the reference range for normal subjects. Plasma glycine betaine concentrations were within reference ranges for normal subjects. Patients with elevated glycine betaine excretion tended to have lower plasma glycine betaine concentrations, but this did not reach statistical significance. One way analysis of variance found excretion is independent of treatment, duration of diagnosed diabetes, blood pressure and body mass index (BMI). An association between glycine betaine excretion and glycemic control was observed with statistically significant correlations occurring with both plasma glucose (r = 0.43, P < 0.001) and glycated haemoglobin (HbA1c) (r = 0.35, P < 0.005). The excretion of carnitine, acetyl-carnitine and proline betaine were related to glycine betaine excretion (r = 0.49, P < 0.001; r = 0.40, P < 0.001; r = 0.27, P < 0.05, respectively). Urine carnitine and acetyl-carnitine concentrations were also related to plasma glucose concentrations (r = 0.30, P < 0.01). Increased urine retinol binding protein concentrations (RBP), a marker of proximal tubular dysfunction, correlated with elevated urine glycine betaine excretion and plasma HbA1c (r = 0.28, P < 0.01). These results suggest poor glycemic control is associated with the increase in urine glycine betaine, carnitine, acetyl-carnitine and RBP excretion in diabetic patients. However, < 50% of the observed increase in glycine betaine excretion has been accounted for by the variables measured, suggesting other unidentified processes may also be involved.

Adult↗

Quantitative reverse transcriptase-PCR amplification of cytokine mRNA in liver biopsy specimens using a non-competitive method.

Reverse transcriptase-PCR (RT-PCR) amplification of mRNA is often the only technique able to detect expression of cytokine mRNA in small samples. The aim of this work was to investigate the utility of a non-competitive RT-PCR which used external standards to quantitate TNF-alpha mRNA in liver biopsy specimens from liver transplant patients. It involved removal of aliquots from the PCR reaction at successive cycles, followed by dot-blotting of the samples onto nylon membrane and hybridization with a radioactively-labelled internal probe. Phosphorimage analysis of the labelled membranes allowed quantitation of the relative amount of PCR product at successive cycles. Plots of log(counts) versus cycle number showed straight lines in the exponential phase of amplification. The slopes of these lines showed the efficiency of amplification, which ranged from 76 to 87% for liver biopsy samples. Estimation of liver biopsy levels of TNF-alpha in two separate PCR amplifications showed low inter-assay variability (r2 = 0.98). Comparison of two separate cDNA syntheses also showed good correlation (r2 = 0.81, P < 0.0001), although not as good as for the PCR alone. This shows that variation in efficiency of cDNA synthesis is likely to contribute as much or more to variability of the analysis as variations in PCR amplification.

Biomarkers↗

Variability of Nt-proANP and C-ANP.

BACKGROUND: Natriuretic peptides are frequently measured in patients with chronic cardiac failure (CCF). We set out to compare the variability of atrial natriuretic peptide (C-ANP) and its precursor N-terminal pro-ANP (Nt-proANP) to decide which would be more suitable for routine use. METHODS: Ten males with compensated CCF (age range 62-76 years) were studied, with matched controls. Blood was withdrawn every 2 min for 90 min from a forearm vein, and plasma C-ANP and Nt-proANP were measured by radioimmunoassay. RESULTS: Levels were elevated in the patient group [C-ANP: median 268 (range 171-423) vs. 40 (28-56) ng L-1, P < 0.0002 Mann-Whitney U-test; Nt-proANP: 1955 (562-4451) vs. 621 (409-961) pmol L-1, P < 0.003]. A similar number of 'peaks' was observed in both groups with both peptides, about one every 10 min, and their relative height was similar in both groups. Variability was greater for C-ANP than for Nt-proANP in both patients [coefficient of variation of means 51 (range 36-70) vs. 3.6 (2.1-6.2)%, P < 0.01; sign test] and controls [65 (49-83) vs. 8.9 (4.7-13.5)%, P < 0.01]. CONCLUSION: Nt-proANP is less variable than C-ANP and hence more suited for diagnostic or prognostic use.

Aged↗

Basal cell cocktail (34betaE12 + p63) improves the detection of prostate basal cells.

Antibodies against high molecular weight cytokeratin (34betaE12) and p63 are frequently used basal cell markers to aid in the diagnosis of prostate cancer (Pca). Absence of a basal cell marker in an atypical lesion histologically suspicious for cancer supports a diagnosis of Pca. However, absence of basal cells demonstrable by basal cell immunohistochemistry (IHC) is not always conclusive for PCa. Some benign prostatic lesions may have inconspicuous or even lack basal cell lining focally. Technical factors such as tissue fixation and antigen retrieval techniques may also make the detection of basal cells difficult. Improving the sensitivity of current basal cell markers is critical if these tests are being used to help make diagnostic decisions in conjunction with standard histology. In this study, we test the hypothesis that that inclusion of both 34betaE12 and p63 in the same IHC reaction (basal cell cocktail) is advantageous over either marker used alone. One thousand three hundred fifty glands from 9 trans-urethral resectioned of prostate specimens with benign prostatic hypertrophy were used to study the immunostaining intensity and pattern for 34betaE12, p63, and the basal cell cocktail. Basal cell marker expression was scored as strong, moderate, weak, or negative. Basal cell staining was considered complete if 75% of the gland's circumference was positive for the basal cell marker and partial if <25% of the circumference was stained. The mean staining intensity and variance were calculated for 34betaE12, p63, and the basal cell cocktail. A paired test was used to evaluate whether the overall basal cell staining was significantly different between 34betaE12, p63, and the basal cell cocktail. F-test was used to assess the variances for 34betaE12, p63, and the basal cell cocktail. A high-density tissue microarray (TMA) comprising prostate tissue from 103 tumors from men with clinically localized Pca and a separate TMA comprising metastatic hormone-refractory Pca samples from 23 rapid autopsy cases were used to study the aberrant expression of 34betaE12 and p63 in clinically localized and poorly differentiated Pca. The prostate glands in transition zone have variable basal cell staining intensity and pattern with 34betaE12, p63, or the cocktail. Histologically, benign glands lack basal cell lining in 2%, 6%, and 2% of glands with cocktail, 34betaE12, and p63 staining, respectively. The staining variance for the cocktail is significantly smaller than that for 34betaE12 (0.0100 vs 0.1559, p = 0.0008). It is also smaller than that for p63, although a statistical significance has not been reached (0.0100 vs 0.0345, p = 0.099). The basal cell cocktail stains the basal cell layers more intensely than either 34betaE12 or p63 alone, with complete and partial strong basal cell staining in 93% and 1% of benign glands, compared with 55% and 4% with 34betaE12 and 81% and 1% with p63. Complete and partial weak staining is seen in 0% and 0% of benign glands with basal cell cocktail, compared with 8% and 7% with 34betaE12 and 4% and 1% with p63 (p = 0.007 and 0.014 for cocktail vs 34betaE12 and cocktail vs p63, respectively). A total of 2.8% clinically localized Pca had positive 34betaE12 staining and 0.3% had positive p63 staining. Five (22%) of the metastatic Pca is positive for 34betaE12. However, none had p63 expression. The basal cell cocktail had a staining pattern identical to that of 34betaE12. IHC of the prostatic glands from the transition zone is subjected to staining variability that results in frequent variable and occasional negative basal cell staining in histologically benign glands; 34betaE12 is most susceptible, and basal cell cocktail is least susceptible to such variability. Basal cell cocktail not only increases the sensitivity of the basal cell detection, but also reduces the staining variability and therefore renders the basal cell immunostaining more consistent. We recommend this basal cell cocktail for routine Pca diagnostic work-up.

Biomarkers, Tumor↗