Effects of lactate and acetate on the determination of serum ethyl glucuronide by CZE

The analysis of ethyl glucuronide (EtG), a marker of recent alcohol consumption, in serum with an optimized CZE assay is reported. The method uses a 0.1-mm id fused-silica capillary of 50 cm effective length that is coated with linear polyacrylamide, a pH 4.4 nicotinic acid/epsilon-aminocaproic acid (EACA) BGE, reversed polarity and indirect analyte detection. The assay is based on a 1:1 dilution of serum with deionized water and has LODs for EtG, lactate and acetate of 3.8 x 10(-7) M, 2.60 x 10(-6 )M and 2.18 x 10(-6 )M, respectively. Separation of EtG from endogenous macro- and microcomponents (anionic serum components of high and low concentration, respectively) and its quantification are shown to be possible for a wide range of lactate (stacker) and acetate (destacker) concentrations, macrocomponents that have an impact on the CZE behavior of EtG and that change after intake of ethanol. The assay has been successfully applied to the analysis of EtG, lactate and acetate in (i) sera of volunteers that ingested known amounts of alcohol and (ii) samples of patients that were classified (teetotalers and social drinkers vs. alcohol abusers) via analysis of carbohydrate-deficient transferrin.

Prospective study of fetal DNA in serum and disease activity during pregnancy in women with inflammatory arthritis

OBJECTIVE: Rheumatoid arthritis (RA) usually improves during pregnancy and recurs postpartum. Fetal cells and cell-free DNA reach the maternal circulation during normal pregnancy. The present study investigated dynamic changes in levels of fetal DNA in serum from women with RA and inflammatory arthritis during and after pregnancy to test the hypothesis that the levels of circulating fetal DNA correlate with arthritis improvement. METHODS: Twenty-five pregnant patients were prospectively studied. A real-time quantitative polymerase chain reaction panel targeting unshared, paternally transmitted HLA sequences, a Y chromosome-specific sequence, or an insertion sequence within the glutathione S-transferase M1 gene was used to measure cell-free fetal DNA. Results were expressed as fetal genomic equivalents per milliliter (gE/ml) of maternal serum. Physical examinations were conducted during and after pregnancy. RESULTS: Levels of fetal DNA in women with improvement in or remission of arthritis were higher than those in women with active disease, especially in the third trimester. Overall, an inverse relationship between serum fetal DNA levels and disease activity was observed (P < 0.001). Serum fetal DNA increased with advancing gestation, reaching median levels of 24 gE/ml (range 0-334), 61 gE/ml (range 0-689), and 199 gE/ml (range 0-2,576) in the first, second, and third trimesters, respectively, with fetal DNA clearance observed postpartum. Arthritis improvement was initially noted in the first trimester for most patients, increased further or was sustained with advancing gestation, and was active postpartum. CONCLUSION: Changes in serum fetal DNA levels correlated with arthritis improvement during pregnancy and recurrence postpartum. Immunologic mechanisms by which pregnancy might modulate RA activity are described.

Is Pelvic Lymph Node Dissection Necessary in Patients with a Serum PSA<10ng/ml Undergoing Radical Prostatectomy for Prostate Cancer?

OBJECTIVE: Controversy persists concerning the role of pelvic lymph node dissection (PLND) in patients with preoperative PSA values <10ng/ml undergoing treatment for prostate cancer with a curative intent. The aim of this study was to determine the incidence of lymph node metastasis in this subgroup of patients. METHODS: Patients with clinically localized prostate cancer and a serum PSA<10ng/ml, without neoadjuvant hormonal or radiotherapy, with negative staging examinations who underwent radical retropubic prostatectomy with bilateral extended PLND and with >/=10 lymph nodes detected by the pathologist in the surgical specimen, were included in the study. RESULTS: A total of 231 patients with a median serum PSA of 6.7ng/ml (range 0.4-9.98) and a median age of 62 years (range 44-76) were evaluated. A median of 20 (range 10-72) nodes were removed per patient. Positive nodes were found in 26 of 231 patients (11%), the majority of which (81%) had a Gleason score >/=7 in the surgical specimen. Of the patients with a Gleason score >/=7 in the prostatectomy specimen 25% had positive nodes, whereas only 3% with a Gleason score serum PSA<10ng/ml and a Gleason score >/=7 in the prostatectomy specimen was 25% after extended PLND. It seems that in this patient group extended PLND, including removal of nodes along the internal iliac vessels, is warranted.

Production of monoclonal antibodies specific for native equine IgE and their application to monitor total serum IgE responses in Icelandic and non-Icelandic horses with insect bite dermal hypersensitivity

Immunoglobulin E forms a minor component of serum antibody in mammals. In tissues IgE is bound by FcvarepsilonRI receptors on the surface of mast cells and mediates their release of inflammatory substances in response to antigen. IgE and mast cells have a central role in immunity to parasites and the pathogenesis of allergic diseases in horses and other mammals. This paper describes the production of several novel monoclonal antibodies that detect native equine IgE in immunohistology, ELISA and Western blotting. An antigen capture ELISA to quantify equine IgE in serum has been developed using two of these antibodies. The mean serum IgE concentration of a group of 122 adult horses was 23,523ng/ml with a range of 425-82,610ng/ml. Total serum IgE of healthy horses was compared with that of horses with insect bite dermal hypersensitivity (IBDH) an allergic reaction to the bites of blood feeding insects of Culicoides or Simulium spp. IBDH does not occur in Iceland where Culicoides spp. are absent, but following importation into mainland Europe native Icelandic horses have an exceptionally high incidence of this condition. In the present study Icelandic horses with IBDH had significantly higher total IgE than healthy Icelandic horse controls (P<0.05). By contrast in horses of other breeds the difference in total serum IgE between those affected with IBDH and healthy controls was not statistically significant. Total serum IgE was also monitored in a cohort of Icelandic horses prior to import into Switzerland and for a period of 3 years thereafter. High levels of serum IgE were present in all horses at the start of the study but dropped in the first year after import. Thereafter the total serum IgE remained low in Icelandic horses that remained healthy but rose significantly (P<0.05) in those that developed IBDH. These results support the conclusion that IBDH is a type I hypersensitivity response to insect allergens but indicate that IBDH in Icelandic horses may have a different pathogenesis from the same condition in other breeds.

Diagnostic significance of Neospora caninum DNA detected by PCR in cattle serum

A nested PCR that successfully detected Neospora caninum DNA in serum of cattle was used for investigation of selected abortion cases and in a study of healthy pregnant cows at an abattoir. N. caninum DNA was not detected in serum from antibody positive dams that aborted due to N. caninum, but was present in serum of some antibody negative dams that aborted due to other causes. N. caninum DNA was also found in the serum of about half of the animals that aborted of undetermined cause, but was not detected in cow sera from two beef cattle herds in Western Australia with no recent history of abortion. In the abattoir study of 79 dams and their foetuses N. caninum DNA was found in serum of 3 dams and in material from 11 foetuses. The majority of the cows and all foetuses were antibody negative. Our findings suggest that there is no obvious relationship between the presence or absence of N. caninum DNA in serum and the presence of antibodies to N. caninum in dams, the presence of N. caninum DNA in foetuses or abortion due to N. caninum. This is the first report of the detection of N. caninum DNA in serum of cattle rather than the white blood cell fraction. It indicates the presence of free tachyzoites and/or parasite DNA in circulation. The results suggest that persistent infection in the absence of antibodies is a possible outcome of N. caninum infection. Infection of foetuses in the absence of antibodies supports the possibility of persistent infection due to immunotolerance to an early in utero infection. It is therefore important to test for N. caninum DNA as well as antibodies for the detection of exposed and/or infected animals. However, the presence or absence of N. caninum antibodies or DNA did not support nor exclude N. caninum as the cause of abortion. Additional criteria are required for a positive diagnosis of abortion caused by N. caninum.

Serum levels of mannose-binding lectin and the risk of fever in neutropenia pediatric cancer patients

BACKGROUND: Fever in neutropenia (FN) is a frequent complication in pediatric oncology. Deficiency of mannose-binding lectin (MBL), an important component of innate immunity, is common due to genetic polymorphisms, but its impact on infections in oncologic patients is controversial. This study investigated whether MBL serum levels at cancer diagnosis are associated with the development of FN in pediatric cancer patients. PROCEDURE: Serum MBL was measured using ELISA. Frequency, duration, and cause of FN were assessed retrospectively. Association with MBL level was analyzed using uni- and multivariate Poisson regression taking into account both intensity and duration of chemotherapy. RESULTS: In 94 children, with a cumulative follow-up time of 81.7 years, 177 FN episodes were recorded. Patients with both very low MBL levels (<100 microg/L; risk ratio (RR), 1.93; 95% CI, 1.14-3.28; P = 0.014) and normal MBL levels (>/=1,000 microg/L; RR, P = 0.011) had significantly more frequent FN episodes than patients with low MBL levels (100-999 microg/L). Patients with very low MBL levels had significantly more episodes of FN with severe bacterial infection (bacteremia or pneumonia; RR, 4.49; 1.69 = 11.8; P = 0.003), while those with normal MBL levels had more FN episodes with no microbial etiology identified (RR, 1.85; 1.14 = 3.03; P = 0.014). CONCLUSIONS: Very low MBL levels are associated with more frequent FN episodes, mainly due to severe bacterial infections. The surprising finding that children with normal MBL levels had more frequent FN episodes than those with low MBL levels needs testing in prospective studies. Pediatr Blood Cancer (c) 2006 Wiley-Liss, Inc.

First trimester markers for pre-eclampsia: placental vs. non-placental protein serum levels

BACKGROUND/AIM: Parallel investigation, in a matched case-control study, of the association of different first-trimester markers with the risk of subsequent pre-eclampsia (PE). METHOD: The levels of different first trimester serum markers and fetal nuchal translucency thickness were compared between 52 cases of PE and 104 control women by non-parametric two-group comparisons and by calculating matched odds ratios. RESULTS: In univariable analysis increased concentrations of inhibin A and activin A were associated with subsequent PE (p < 0.02). Multivariable conditional logistic regression models revealed an association between increased risk of PE and increased inhibin A and translucency thickness and respectively reduced pregnancy-associated plasma protein A (PAPP-A) and placental lactogen . However, these associations varied with the gestational age at sample collection. For blood samples taken in pregnancy weeks 12 and 13 only, increased levels of activin A, inhibin A and nuchal translucency thickness, and lower levels of placenta growth factor and PAPP-A were associated with an increased risk of PE. CONCLUSIONS: Members of the inhibin family and to some extent PAPP-A and placental growth factor are superior to other serum markers, and the predictive value of these depends on the gestational age at blood sampling. The availability of a single, early pregnancy 'miracle' serum marker for PE risk assessment seems unlikely in the near future.

Clonal analysis of Inquilinus limosus isolates from six cystic fibrosis patients and specific serum antibody response

Inquilinus limosus is a novel Gram-negative bacterium of the subdivision alpha-Proteobacteria recently found in the airways of patients with cystic fibrosis (CF). Here, the authors report on the clinical courses of six CF patients colonized with I. limosus. Five patients suffered from either an acute respiratory exacerbation or a progressive loss of pulmonary function, whereas one patient was in a stable clinical situation. This study focused on two aims: (i) the clonal analysis of I. limosus isolates by random amplified polymorphic DNA (RAPD)-PCR, and (ii) the clarification of whether the presence of I. limosus in the respiratory tract is associated with a specific serum antibody response. Serum IgG was detected by immunoblotting using I. limosus whole-cell-lysate proteins as antigens. Sera from healthy blood donors (n=10) and from CF patients colonized with Pseudomonas aeruginosa (n=10) were found to be immunoblot negative. All six Inquilinus-positive patients raised serum IgG antibodies against various I. limosus antigens. Surprisingly, in one patient, a specific I. limosus serum antibody response was already detected 1 year prior to Inquilinus-positive sputum cultures. Two prominent antigens were characterized by MALDI-MS: a 23 kDa protein revealed homology to the outer membrane lipoprotein OmlA of Actinobacillus pleuropneumoniae, and an 18 kDa protein to a protein-tyrosine phosphatase of Burkholderia cepacia. In conclusion, detection of I. limosus is accompanied by a specific serum antibody response and may reflect the infectious/pathogenic potential of I. limosus. Moreover, IgG immunoblotting may be useful to detect early infection with I. limosus and may support the selective cultivation of this novel emerging pathogen.

Rapid measurement of S-100B serum protein levels by Elecsys S100 immunoassay in patients undergoing carotid artery stenting or endarterectomy

OBJECTIVES:: This study was designed to apply the rapid Elecsys(R) S100 immunoassay for real-time measurement of S100 protein serum levels indicating acute brain damage in patients undergoing carotid artery stenting (CAS) or endarterectomy (CEA). DESIGN AND METHODS:: Data of 14 CAS patients were compared to those of 43 CEA and 14 control patients undergoing coronary angiography (CA). S100 serum levels were measured by the full-automatic Elecsys(R) S100 immunoassay and compared to those obtained by the well-established LIA-mat(R) S100 system. RESULTS:: In contrast to CAS and CA patients, median S100 serum levels of CEA patients significantly increased to 0.24 ng/mL before declamping, but subsequently returned to baseline. Three CEA patients with neurological deficits showed sustained elevated S100 levels 6 h after extubation. Absolute S100 values were not significantly different between the two methods. Bland-Altman plot analyses displayed a good agreement, mostly indicating slightly smaller values applying the Elecsys(R) S100 system. CONCLUSIONS:: The Elecsys(R) S100 system appears to be suitable for rapid real-time detection of neurological deficits in patients undergoing CAS and CEA. Persistent elevations of Elecsys(R) S100 levels during CEA were associated with prolonged neurological disorders, whereas transient increases seem to represent impaired blood-brain barrier integrity without neurological deficits.