Non-invasive diagnosis of endometriosis based on a combined analysis of six plasma biomarkers.

BACKGROUND: Lack of a non-invasive diagnostic test contributes to the long delay between onset of symptoms and diagnosis of endometriosis. The aim of this study was to evaluate the combined performance of six potential plasma biomarkers in the diagnosis of endometriosis. METHODS: This case-control study was conducted in 294 infertile women, consisting of 93 women with a normal pelvis and 201 women with endometriosis. We measured plasma concentrations of interleukin (IL)-6, IL-8, tumour necrosis factor-alpha, high-sensitivity C-reactive protein (hsCRP), and cancer antigens CA-125 and CA-19-9. Analyses were done using the Kruskal-Wallis test, Mann-Whitney test, receiver operator characteristic, stepwise logistic regression and least squares support vector machines (LSSVM). RESULTS: Plasma levels of IL-6, IL-8 and CA-125 were increased in all women with endometriosis and in those with minimal-mild endometriosis, compared with controls. In women with moderate-severe endometriosis, plasma levels of IL-6, IL-8 and CA-125, but also of hsCRP, were significantly higher than in controls. Using stepwise logistic regression, moderate-severe endometriosis was diagnosed with a sensitivity of 100% (specificity 84%) and minimal-mild endometriosis was detected with a sensitivity of 87% (specificity 71%) during the secretory phase. Using LSSVM analysis, minimal-mild endometriosis was diagnosed with a sensitivity of 94% (specificity 61%) during the secretory phase and with a sensitivity of 92% (specificity 63%) during the menstrual phase. CONCLUSIONS: Advanced statistical analysis of a panel of six selected plasma biomarkers on samples obtained during the secretory phase or during menstruation allows the diagnosis of both minimal-mild and moderate-severe endometriosis with high sensitivity and clinically acceptable specificity.

Carrier-mediated translocation of uridine diphosphate glucose into the lumen of endoplasmic reticulum-derived vesicles from rat liver.

Radiolabeled UDPGlc incubated with rough endoplasmic reticulum (RER)-derived microsomes from rat liver became associated with the vesicles. This microsomal uptake of nucleotide sugar was time and temperature dependent. Analysis of the molecular species containing radiolabel revealed that initial uptake represented entry of predominantly intact UDPGlc in the microsomes. Conclusive evidence for proper translocation of UDPGlc across the microsomal membrane into the intravesicular space was obtained by demonstrating that UDPGlc was transported into an osmotically sensitive compartment. Microsomal uptake of UDPGlc exhibited features characteristic of carrier-mediated transport including saturation, specificity, and countertransport. Inhibition and trans-stimulation studies showed that other uridine-containing nucleotide sugars and 5'-UMP were substrates of the postulated microsomal carrier system for UDPGlc, while cytosine- or guanosine-containing nucleotides and non-5'-uridine monophosphates were, at best, very poor substrates. UDPGlc translocation activities were lower in smooth microsomal fractions than in the RER-derived vesicles, indicating that contamination with Golgi membranes could not be responsible for microsomal transport of UDPGlc. Our findings suggest that rat liver endoplasmic reticulum possesses a carrier system mediating proper translocation of UDPGlc and 5'-uridine-substituted structural analogues across the membrane.

Mechanism of bilirubin diglucuronide formation in intact rats: bilirubin diglucuronide formation in vivo.

Although it is well established that bilirubin monoglucuronide is formed in the liver from bilirubin by a microsomal bilirubin uridine diphosphate (UDP)-glucuronosyltransferase, the subcellular site of conversion of monoglucuronide to diglucuronide and the molecular mechanism involved in diglucuronide synthesis have not been identified. Based on in vitro studies, it has been proposed that two fundamentally different enzyme systems may be involved in diglucuronide synthesis in rat liver: (a) a microsomal UDP-glucuronosyltransferase system requiring UDP-glucuronic acid as sugar donor or (b) a transglucuronidation mechanism that involves transfer of a glucuronosyl residue from one monoglucuronide molecule to another, catalyzed by a liver plasma membrane enzyme. To clarify the mechanism by which bilirubin monoglucuronide is converted in vivo to diglucuronide, three different experimental approaches were used. First, normal rats were injected with either equal amounts of bilirubin-IIIalpha [(14)C]monoglucuronide and unlabeled bilirubin-XIIIalpha monoglucuronide, or bilirubin-XIIIalpha [(14)C]monoglucuronide and unlabeled bilirubin-IIIalpha monoglucuronide. Analysis of radiolabeled diglucuronide excreted in bile showed that [(14)C]glucuronosyl residues were not transferred between monoglucuronide molecules. Second, in normal rats infused intravenously with dual-labeled [(3)H]bilirubin [(14)C]monoglucuronide, no transfer or exchange of the [(14)C]glucuronosyl group between injected and endogenously produced bilirubin monoglucuronide could be detected in the excreted bilirubin diglucuronide. Third, in homozygous Gunn rats, injected (14)C-labeled or unlabeled bilirubin mono- or diglucuronides were excreted in bile unchanged (except that diglucuronide was hydrolyzed to a minor degree). This indicates that Gunn rats, which lack bilirubin UDP-glucuronosyltransferase activity, are unable to convert injected monoglucuronide to diglucuronide. Collectively, these findings establish that a transglucuronidation mechanism is not operational in vivo and support the concept that bilirubin diglucuronide is formed by a microsomal UDP-glucuronosyltransferase system.

Unconjugated bilirubin and an increased proportion of bilirubin monoconjugates in the bile of patients with Gilbert's syndrome and Crigler-Najjar disease.

Bilirubin pigments were studied in the bile of 20 normal adults, 25 patients with Gilbert's syndrome, 9 children with Crigler-Najjar disease, and 6 patients with hemolysis, to determine how a deficiency of hepatic bilirubin UDP-glucuronosyltransferase would affect the end products of bilirubin biotransformation. In the bile from patients with Gilbert's syndrome, a striking increase was found in the proportion of bilirubin monoconjugates (48.6+/-9.8% of total conjugates) relative to that in normal bile (27.2+/-7.8%). This increase was even more pronounced in children with Crigler-Najjar disease, in whom, even in the most severe cases, glucuronide could always be demonstrated in the bile. Furthermore, unconjugated bilirubin-IXalpha was unquestionably present in the bile of these children and amounted to 30-57% of their total bilirubin pigments (<1% in the controls). It was not possible to predict from the biliary bilirubin composition whether a child would respond to phenobarbital therapy or not. Bile composition was normal in patients with hemolysis, except when there was associated deficiency of hepatic glucuronosyltransferase. Therefore, the observed alterations were not a simple consequence of unconjugated hyperbilirubinemia. The present findings suggest that Crigler-Najjar disease represents a more pronounced expression than Gilbert's syndrome of a common biochemical defect. Hepatic bilirubin UDP-glucuronosyltransferase deficiency leads to decreased formation of diconjugates with an ensuing increase in the proportion of bilirubin monoconjugates in bile; in the most severe cases, an elevated content of biliary unconjugated bilirubin is also found.

Detection of single nucleotide polymorphisms in the mannose-binding lectin gene using minor groove binder-DNA probes.

Structural point mutations in exon 1 at codons 52, 54 and 57 and a promotor polymorphism at -221 bp of the mannose-binding lectin (MBL) gene are associated with increased susceptibility to various infectious diseases. We developed a genotyping method based on the 5' nuclease (TaqMan) assay in combination with the use of minor-groove-binder (MGB) probes in screening for these mutations/polymorphisms. In contrast to conventional probes, MGB probes have a short length and can be used for detection of mutations that are in close proximity to each other, as is the case for the structural mutations in exon 1 of the MBL gene. Results obtained with the 5' nuclease assay using MGB probes were identical with results obtained with classical techniques such as restriction fragment length polymorphism, allele-specific PCR, and sequencing. In conclusion, the 5' nuclease assay using MGB probes is useful for large-scale screening of point mutations/polymorphisms, even when they are in close proximity.

Influence of shielding blocks on the output of photon beams as a function of energy and type of treatment unit.

The influence of field-defining shielding blocks on the output of a cobalt unit and of seven different accelerators (one with dual energy output) has been investigated. The quality indices range from 0.57 (cobalt-60) to 0.79. The loss in output due to shielding blocks has been calculated taking into account loss in phantom scatter only. Comparison with experimental results shows that the calculation algorithm is correct in most of the clinical conditions. However, for quality indices of 0.70 and higher, for blocks close to the central beam axis, an overestimation of the output by the algorithm has been found. The maximum deviation observed is about 5% for the highest energy and for block positions corresponding to those applied, e.g. for inverted Y-fields with narrow lumbo-aortic block spacing.

Are port films reliable for in vivo exit dose measurements?

The possibility of using conventional port films as an in vivo method for obtaining relative exit dose distribution in patients is evaluated. Kodak "Verification" films in "Localization" cassettes are positioned in the usual clinical conditions behind an homogeneous polystyrene phantom as well as behind a phantom containing air, wood and aluminium inhomogeneities. Taking beam divergency into account the densitometric profiles are projected back to the exit side of the phantom. They are compared to the profiles obtained with an ionization chamber used under full backscatter conditions. The agreement between the off-axis ratios determined with either method are mostly better than 2% and never exceed 5%. These phantom measurements are completed by a comparison between off-axis ratios determined on a port film for a head and neck patient and those obtained by diode dosimetry applied on the patient at the exit side of the beam. A similar agreement as between film and ion chamber on the phantoms is obtained. These encouraging results illustrate the possibilities of using conventional port films for in vivo dosimetry.

Is it necessary to repeat quality control procedures for head and neck patients?

Using serial verification films for detection of localization errors and in vivo measurements of the delivered dose, a comparison was made of the information obtained from a single check on the first treatment session or from repeated checks in subsequent irradiations, leading to an assessment of the predictive value of a single check. A total number of 215 films and 261 entrance dose measurements have been performed on 34 fields for 10 head and neck patients. The patients are immobilized with individual plastic masks fixed on the couch and treated on a 6 MV linac, supplied with an automatic verification system excluding the couch parameters. The global results show Gaussian frequency distributions with standard deviations of 4 mm for port film measurements and 3.4% for the dose measurements. Large errors (greater than 5 mm displacement and greater than 4% deviation from the expected dose) have been detected in 16% in the cranio-caudal direction and 24% in the antero-posterior direction with port films and in 15% of the in vivo measurements. In order to identify the nature of the errors, which can be random or systematic, the first measurement is taken as the reference value and shows that consecutive measurements on the same field were reproducible with standard deviations of respectively 2.5 mm and 1.8%. This means that a large part of the spread of the global results can be explained by systematic errors in the treatment preparation chain. With the first check, 6 out of 10 systematic localization errors and 7 out of 7 systematic errors leading to erroneous dose delivery have been detected. Therefore, most of the systematic errors, which affect the overall quality of the treatment, can be identified with the first check. The four systematic localization errors, missed with the first film, were of rather limited size: only one of them showed a mean displacement larger than 7 mm. Because the first measurement is an acceptable indication of the overall quality of the treatment delivery, the authors propose a code of practice for checking the treatment quality at the patient level.

Disappointing dipstick screening for urinary tract infection in hospital inpatients.

AIM: To compare the performance of leucocyte esterase and nitrite dipstick tests with microscopic examination and culture of first morning urines (n = 420) of hospital inpatients. RESULTS: The sensitivity, specificity, and negative predictive value of the leucocyte esterase test for the cutoff of > 10 WBC/microliter were 57%, 94%, and 68%, respectively. For > 5 WBC per high power field (HPF) these variables were 84%, 90%, and 93%. For > 10(5) colony counts/ml, the sensitivity of the nitrite test was 27%, specificity 94%, and negative predictive value 87%. When either leucocyte esterase or nitrite positivity was accepted as a marker of urinary tract infection, the sensitivity was 78%, specificity 75%, and negative predictive value 94%, and there were 22% false negative results. Semiquantitative microscopic estimation of bacteria per HPF yielded 40% false positives. CONCLUSIONS: Leucocyte esterase and nitrite dipstick tests are not suitable for screening for urinary tract infections.

Serum tissue polypeptide antigen (TPA): monoclonal or polyclonal radio-immunometric assay for the follow-up of bladder cancer.

Tissue polypeptide antigen (TPA) is a serological tumor marker, measuring cytokeratin 8, 18 and 19, used in the follow-up of non squamous epithelium- and derived neoplasms. It has been demonstrated that TPA is reliable in the monitoring of the efficacy of a curative or palliative treatment of bladder cancer. Recently, a monoclonal antibody-based assay for TPA (TPA-M) has been developed, which seems to be equivalent to the polyclonal-based assay. The aim of the study was to assess the superiority of the monoclonal to the polyclonal test in patients with bladder carcinoma. The value of tissue polypeptide antigen was therefore measured both with TPA and TPA-M IRMA. A correlation coefficient of 0.96 was obtained. Precision testing showed a lower overall variability of TPA-M. Since both tests correlate well and TPA-M testing is more precise, faster and easy to perform, we conclude a superiority of TPA-M and advise the monoclonal test as best suited for clinical use in the follow-up of bladder cancer patients with poorly differentiated superficial, locally advanced or systemic disease after curative or palliative therapy.

Serum tissue polypeptide antigen (TPA) as tumor marker for bladder cancer.

Tissue polypeptide antigen is a differentiation and proliferation marker of non-squamous epithelium and derived neoplasms. No reliable tumor markers are available for bladder cancer. The value of tissue polypeptide antigen was therefore prospectively investigated. The serum tissue polypeptide antigen samples were obtained from 144 newly diagnosed transitional cell carcinoma patients and from 92 patients that were followed after treatment. The normal cut off value was defined at 95 units per liter. Nearly all TaT1 patients had normal TPA values, and 80% of the muscle invasive cancers had normal TPA levels. In those patients where TPA was elevated before treatment its monitoring proved to be a reliable predictor of tumor progression. Tissue polypeptide antigen is a useful marker not for the early detection of bladder cancer but for the monitoring of the efficacy of a treatment.

Sludge is calcium bilirubinate associated with bile stasis.

Biliary sludge is a frequent finding on abdominal sonography. It is most often found after prolonged stasis of gallbladder bile associated with other illness or mechanical obstruction of the common duct, and seldom indicates primary gallbladder disease. In most cases, sludge is a suspension of pigment precipitates in bile, and is at least in part calcium bilirubinate. Sludge may disappear with the return of normal gallbladder contractility. The ease with which this precipitate forms during stasis of gallbladder bile suggests a role for this process in the pathogenesis of cholelithiasis.

Monoclonal IgM: difficulties with correct measurement.

We describe three monoclonal IgM paraproteins for which nephelometric IgM quantification generated inaccurate results.

Screening for autoantibodies to SS-A/RO by indirect immunofluorescence using HEp-2000 cells.

We evaluated indirect immunofluorescence (IF) using HEp-2000 slides, which are transfected with SS-A cDNA, for screening for anti-SS-A antibodies, by comparing it with counterimmunoelectrophoresis (CIE). A total of 2427 specimens were screened for IF reactivity and for SS-A precipitins, of which 1033 (43%) were negative on both IF and CIE. There were 1271 SS-A precipitin-negative specimens (52%) which were IF-positive but lacked the distinctive SS-A staining pattern. One precipitin-negative serum was IF-positive with the distinctive SS-A pattern in the HEp-2000 system. One hundred and twenty-two specimens (5%) were positive for anti-SS-A precipitins on CIE, 107 showed the distinctive SS-A fluorescence staining pattern, whereas 15 of these precipitin-positive samples (12%) were IF-positive but did not display the distinctive SS-A pattern on the transfected cells. Fourteen of the 15 samples in which the distinctive SS-A pattern was not observed displayed other significant antinuclear antibody (titre equal or >1:320) patterns. In conclusion, the presence of the typical 'distinctive' SS-A pattern on IF using the HEp-2000 slides is highly specific for the presence of autoantibodies to SS-A and has a sensitivity of 88% for detecting these antibodies.

Time course of serum lipids and apolipoproteins after acute myocardial infarction: modification by pravastatin.

Pravastatin was administered to patients suffering an acute myocardial infarction starting with a dose of 10 mg/day at day 3 and continuing with a dose of 20 mg/day up to a period of 3 months. The study was performed on the basis of a randomized placebo-controlled double-blind trial. At the dosage used pravastatin significantly lowered the total cholesterol and LDL-cholesterol levels and increased the HDL-cholesterol levels compared to placebo. A prospective clinical trial in order to examine the possible clinical relevance of these findings is recommended.

The congenic normal R/APfd and jaundiced R/APfd-j/j rat strains: a new animal model of hereditary non-haemolytic unconjugated hyperbilirubinaemia due to defective bilirubin conjugation.

In this paper the production of the R/APfd-j/j strain which is congenic with the R/APfd strain is reported. The R/APfd-j/j completely lacks hepatic bilirubin UDP-glucuronyltransferase activity, as do our GUNNXR/Pfd-j/j rat strain and various other stocks of GUNN rats (j/j) described in the literature. Our recombinant inbred strain GUNNXR/Pfd-j/j was produced from non-inbred GUNN (j/j) rats. This GUNNXR/Pfd-j/j rat was used as a donor of the jaundice gene j, the R/APfd rat serving as the recipient. After eight backcross-intercross cycles (16 generations) the R/APfd-j/j strain was obtained which is congenic with the R/APfd strain. Congenicity was demonstrated by various techniques including transplantation of skin tissue, strain-specific tumour cells and hepatocytes, the mixed lymphocyte reaction, and comparison of biochemical markers. The potential of the novel inbred strain of jaundiced rat, R/APfd-j/j, and the corresponding control strain R/APfd for biochemical and clinical studies of bilirubin metabolism are briefly discussed.