Comparison of procedures for evaluating laboratory performance in external quality assessment schemes for lead in blood and aluminum in serum demonstrates the need for common quality specifications

BACKGROUND: The different scoring methods used by eight European External Quality Assessment Schemes (EQASs) for occupational and environmental laboratory medicine were compared to develop suitable quality specifications as a step toward harmonization. METHODS: Real results for blood lead and serum aluminum assays, reported by participants in Italian and United Kingdom EQASs, were evaluated according to individual scheme scoring criteria. The same results were then used to produce z scores using scheme-based between-laboratory SDs as the estimate of variability to determine whether simple performance-derived quality specifications produced better agreement among schemes. RESULTS: The schemes gave conflicting assessments of participants' performance, and participants judged to be successful by one scheme could be defined as performing inadequately by another. An approach proposed by Kenny et al. (Scand J Clin Lab Invest 1999;59:585), which uses clinical inputs to set targets for analytical imprecision, bias, and total error allowable, was then used to elaborate quality specifications. CONCLUSIONS: We suggest that the CLIA '88 recommendations for blood lead (+/- 40 micro g/L or +/- 10% of the target concentration, whichever is the greater) could be used as a quality specification, although a revision to +/- 30 micro g/L or +/- 10% is recommended. For serum aluminum, a suitable quality specification of +/- 5 micro g/L or +/- 20% of the target concentration, whichever is the greater, is suggested. These specifications may be used to compare laboratory performance across schemes.     

CYP3A induction by N-hydroxyformamide tumor necrosis factor-alpha converting enzyme/matrix metalloproteinase inhibitors use of a pregname X receptor activation assay and primary hepatocyte culture for assessing induction potential in humans.

A series of N-hydroxyformamide tumor necrosis factor-alpha converting enzyme (TACE)/matrix metalloprotease (MMP) inhibitors were evaluated for their potential to induce human cytochrome P450 3A (CYP3A). Two in vitro assays were used: 1) a cell-based reporter gene assay for activation of the pregnane X receptor (PXR), and 2) a primary "sandwich" culture of human hepatocytes. Approximately 50 TACE/MMP inhibitors were evaluated in the human PXR assay. A range of PXR activation was observed, 0 to 150% of the activation of the known human CYP3A inducer rifampicin. Three TACE/MMP inhibitors were evaluated in rat and human hepatocytes. Significantly higher PXR activation/CYP3A induction was observed in PXR/hepatocyte models, respectively, for (2R,3S) 3-(formyl-hydroxyamino)-2-(2-methyl-1-propyl)-4-methylpentanoic acid [(1S,2S)-2-methyl-1-(2-pyridylcarbamoyl)-1-butyl]amide (GW3333) compared with (2R,3S)-6,6,6-trifluoro-3-[formyl(hydroxy)amino]-2-isobutyl-N-[(1S,2R)-2-methoxy-1-[(1,3-thiazol-2-ylamino)carbonyl]propyl]hexanamide (GW6495) and (2R)-N-[(1S)-2,2-dimethyl-1-[(methylamino)carbonyl]-propyl]-2-[(1S)-1-[formyl(hydroxy)amino]ethyl]-5-phenylpentanamide (GI4023). The CYP3A induction level achieved with GW3333 at a concentration of approximately 10 microM in human hepatocytes was comparable to that achieved with rifampicin at a concentration of 10 microM. The extent of rodent CYP3A induction caused by GW3333 was confirmed in vivo after daily oral administration for 14 days to rats. In conclusion, GW3333 is a potential inducer of CYP3A expression in vivo in humans, but other N-hydroxyformamides are less likely to induce CYP3A.     

Preliminary studies of offspring exposure to phenylbutazone and ivermectin during the perinatal period in a Holstein cow-calf model

The pregnant Holstein cow and her newborn calf were evaluated as an animal model to study in utero and for lactational drug transfer and offspring exposure. A nonsteroidal antiinflammatory drug, phenylbutazone, and an antiparasitic drug, ivermectin, were tested in the model. Prior to parturition, pregnant cows were dosed orally to steady state with phenylbutazone at 4 g/day or given a single subcutaneous injection of 200 microg ivermectin/kg body wt. The level of drug transferred to calves exposed in utero, in utero combined with lactational exposure, and via lactational exposure only, was measured from days 1 through 7 postpartum. At birth the plasma level in phenylbutazone-exposed calves was approximately one-half the dam's steady-state level. For ivermectin-exposed calves, plasma levels were at or below the limit of quantitation (0.5 ng/ml) at birth, suggesting that placental transfer of ivermectin is limited in the cow. For both drugs, rapid accumulation of the drug in calf plasma occurred with lactational exposure to a mean daily dose of 2 microg ivermectin/kg body wt or 0.1 mg phenylbutazone/kg body wt/day for the first 7 days of life. The accumulation observed in the newborn calf is attributed to the lipid solubility and long elimination half-lives of these drugs. These results demonstrate that drug transfer and offspring exposure can be studied using the cow-calf model. The data also highlight the importance of considering not only the dose but also physicochemical characteristics and pharmacokinetics of the drug in the offspring when evaluating the safety of a newborn's exposure to a drug in breast milk.     

Intraoperative examination of sentinel lymph nodes by ultrarapid immunohistochemistry

The recently developed method of ultrarapid immunohistochemistry (IHC) was applied to the intraoperative examination of sentinel lymph nodes (SLNs) in breast cancer patients. In a prospective study of 50 patients with invasive breast carcinomas, a total of 60 SLNs were studied. Among them, 33 SLNs from 30 patients were studied intraoperatively using a direct immunoperoxidase method with anticytokeratin antibody clone MNF116. This technique has a turnaround time of less than 20 minutes. Ultrarapid IHC revealed 15 positive SLNs compared to 14 positive SLNs using hematoxylin and eosin (H and E) frozen sections. The one SLN missed in H and E frozen sections presented with cytokeratin-positive isolated tumor cells in the lymph node sinus. After paraffin embedding, H and E-stained serial step sections of the SLN specimens detected another two patients with isolated tumor cells. We also examined the remaining axillary lymph nodes (ALNs) by H and E-stained serial step paraffin sections. From 17 of the 30 patients with positive SLNs, 6 patients also had metastatic involvement of the ALNs of level I or II. Thus ultrarapid IHC was a very sensitive and rapid technique for the intraoperative detection of metastatic involvement of SLNs in breast cancer patients. This technique may be a useful complementary tool for the intraoperative study of SLNs, particularly in tumors that are a diagnostic challenge, such as lobular carcinoma.     

Cognitive impairment in SCA-19

The autosomal dominant cerebellar ataxias (ADCAs) are a heterogeneous group of neurodegenerative disorders characterised by progressive cerebellar dysfunction in combination with various associated features. Since 1993, ADCAs have been increasingly characterised in terms of their genetic mutation and are currently referred to as spinocerebellar ataxias (SCAs). The discovery of genetic abnormalities offers the opportunity to study the possible interaction between the identified gene mutation and cognitive function. In this study, we focus on the neuropsychological abnormalities in a Dutch ADCA family, in which a new locus was recently identified (SCA-19). The family members showed frontal-executive dysfunction, with global cognitive impairment occurring in some of the more severely affected patients. Interestingly, the neuropsychological profile of this new family seems to overlap that of individuals with various other SCAs. Apparently, similar pattern of neuronal degeneration in various SCA subtypes accounts for the neuropsychological dysfunction, which is thus not genotype specific.     

The serum protein alpha 2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification

Ectopic calcification is a frequent complication of many degenerative diseases. Here we identify the serum protein alpha2-Heremans-Schmid glycoprotein (Ahsg, also known as fetuin-A) as an important inhibitor of ectopic calcification acting on the systemic level. Ahsg-deficient mice are phenotypically normal, but develop severe calcification of various organs on a mineral and vitamin D-rich diet and on a normal diet when the deficiency is combined with a DBA/2 genetic background. This phenotype is not associated with apparent changes in calcium and phosphate homeostasis, but with a decreased inhibitory activity of the Ahsg-deficient extracellular fluid on mineral formation. The same underlying principle may contribute to many calcifying disorders including calciphylaxis, a syndrome of severe systemic calcification in patients with chronic renal failure. Taken together, our data demonstrate a critical role of Ahsg as an inhibitor of unwanted mineralization and provide a novel therapeutic concept to prevent ectopic calcification accompanying various diseases.     

Potential Relevance of Agmatine as a Virulence Factor of Helicobacter pylori

The polyamine agmatine is able to increasegastric acid secretion. Therefore, we investigatedwhether Helicobacter pylori is able to form and releaseagmatine in vitro and in the human stomach in vivo , and if so, whether a relationship exists amongagmatine concentration in gastric juice, H. pyloriinfection, and gastroduodenal lesions. Agmatine wasdetermined by means of HPLC. In the supernatant of H. pylori cultures, agmatine concentrations up to1500 ng/ml (~12 M) were determined, depending on thenumber of the bacteria in the individual cultures.Agmatine concentration in gastric juice from H. pylori-positive patients was higher than inthat from H. pylori-negative patients. Gastrin in bloodwas elevated in H. pylori-positive patients comparedwith H. pylori-negative patients. Agmatine concentration in gastric juice and serum gastrin levelappeared to be related. In conclusion, H. pylori is ableto form and to release agmatine in vitro and in vivo.This may be assumed to be relevant in vivo, since higher amounts of agmatine are present ingastric juice from H. pylori-positive than from H.pylori-negative patients. Accordingly, agmatine producedby H. pylori may be a virulence factor of this bacterium and may be involved in the pathogenesis ofgastroduodenal lesions.