Precision and comparability of Abuscreen OnLine assays for drugs of abuse screening in urine on Hitachi 917 with other immunochemical tests and with GC/MS.

Abuscreen OnLine assays for drugs of abuse screening in urine have recently been developed for use on Hitachi 917 analyzers (Roche Diagnostics GmbH). The assays are based on the kinetic interaction of microparticles as measured by changes in light transmission. Drug in a sample inhibits the formation of particle aggregates and diminishes absorbance change increases. It was the goal of this study to evaluate precision and comparability of the new asssys with CEDIA drugs of abuse tests on Hitachi 917 in different laboratories (three European and three US). The assays were calibrated in the nonlinear mode with four to six standards (semiquantitative application). Initial within-run (21 replicates, four labs) and between-day (10 days, two labs) imprecision studies using Abuscreen OnLine tests and commercial negative (0.5 x cut-off) and positive (1.5 x cut-off) controls revealed the following median CVs [withinrun neg./pos. control/between-day neg./pos. control]: amphetamines 1.9/1.3/3.4/2.4, barbiturates 3.0/1.6/3.9/3.1, benzodiazepines 4.7/1.5/6.3/3.0, cocaine metabolite 1.8/0.9/2.4/1.7, methadone 5.4/1.6/5.5/2.2, opiates 5.5/2.8/5.3/2.7, THC 8.9/4.8/21.8/12.1. CVs < 10% were obtained for the THC test using controls with concentrations closer to the cut-off. An identical set of 170 GC/MS analyzed urine samples was distributed to the six laboratories and measured with Abuscreen OnLine tests on Hitachi 917. The median values for each individual sample were calculated and compared with the results obtained on individual Hitachi 917 analyzers by Passing-Bablok regression analysis. A good agreement between the laboratories was found with less than +/- 11% slope deviation and intercepts below 7% of the cut-off except for benzodiazepines (one slope 17%, one slope--26%) and THC (one slope 34%, one slope--18%). The comparability with CEDIA tests was analyzed by concordance plots using randomized routine samples in three laboratories. The following results were obtained in one of the participating laboratories [cut-off ng/mL] (No. of positive/negative/discrepant samples): amphetamines [500] 2/147/0, barbiturates [200] 1/148/0, benzodiazepines [100] 52/91/7, cocaine metabolite [300] 17/129/3, methadone [300] 113/34/2, opiates [300] 31/114/4, THC [50] 66/81/2. GC/MS was performed for clarification of the discrepant results. In summary, Abuscreen OnLine tests on Hitachi 917 give precise results which compare well when analyzed in different laboratories. They can be rated as convenient and flexible methods for drugs of abuse screening in the routine.

Benzidine stain for the histochemical detection of hemoglobin in splinter hemorrhage (subungual hematoma) and black heel.

Minor nail trauma may cause bluish discoloration of the nail, while tangential skin trauma on the heel can result in a so-called black heel. To rule out melanoma in such clinical situations, a biopsy is needed to reveal homogeneous eosinophilic masses deposited under the nail plate or within it (transepidermal elimination). Most dermatopathologists attempt to demonstrate the presence of hemoglobin in these eosinophilic masses with Prussian blue stain, which typically remains negative. In our experience, these traumatically induced blood deposits are always situated in avascular spaces, devoid of degrading phagocytes. Consequently, a histochemical stain for these deposits should be directed specifically toward hemoglobin, not hemosiderin. In the dermatopathologic literature, the various techniques to detect hemoglobin deposits in tissue sections are not well-known. We would like to emphasize benzidine stain, a highly selective and efficient method to demonstrate the presence of hemoglobin deposits in histologic sections. To date, benzidine stain has not been utilized to characterize splinter hemorrhage (subungual hematoma). Of concern, the use of benzidine in histopathology laboratories is restricted because this agent is a known carcinogen, while the non-mutagenic derivative, 3,3',5,5'-tetramethylbenzidine, does not stain histologic sections. Patent blue V, a completely different and less specific agent, stains hemoglobin an intense blue-green.

Activation of methotrexate-alpha-alanine by carboxypeptidase A-monoclonal antibody conjugate.

Carboxypeptidase A (CP-A) and monoclonal antibody KS1/4 directed against an antigen on human lung adenocarcinoma cells (UCLA-P3) were derivatized by treatment with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and N-succinimidyl 3-(2-pyridyldithio)propionate, respectively. The derivatized proteins were reacted to produce thioether-linked enzyme-antibody conjugates. Sequential HPLC size-exclusion and DEAE chromatography separated the conjugate preparation from unreacted enzyme and antibody. On the basis of SDS-PAGE analysis and measurement of catalytic activity, the preparation contained approximately equal amounts of 1:1 and 2:1 (enzyme:antibody) conjugates; binding activity of the conjugate (1.8 x 10(5) molecules/cell) was similar to that of unreacted antibody. In vitro cytotoxicity studies with UCLA-P3 cells demonstrated the ability of cell-bound conjugate to convert the prodrug methotrexate-alpha-alanine (MTX-Ala) to methotrexate (MTX). In the absence of conjugate, ID50 values for MTX-Ala and MTX were 8.9 x 10(-6) and 5.2 x 10(-8) M, respectively. ID50 for the prodrug improved to 1.5 x 10(-6) M with cells containing bound conjugate. This potentiation of MTX-Ala cytotoxicity by conjugate-bound CP-A, which was at least 30-fold greater than that produced by a comparable amount of free enzyme, is attributed to enhanced effectiveness of MTX generated at the cell surface as opposed to the surrounding medium. Examination of the time course of cytotoxicity over a 96-h period showed that the conjugate-prodrug combination (at 2.5 x 10(-6) M) was nearly as effective as MTX in preventing cell replication. These results demonstrate the chemotherapeutic potential of carboxypeptidase-monoclonal antibody conjugates used in conjunction with MTX peptide prodrugs.

Major histocompatibility complex-linked susceptibility or resistance to disease caused by a noncytopathic virus varies with the disease parameter evaluated.

The influence of major transplantation antigens on susceptibility to T cell-mediated disease caused by infection with the noncytopathic virus lymphocytic choriomeningitis virus (LCMV) was evaluated in B10 H-2-congenic mice. Susceptibility to early T cell-mediated liver cell destruction (day 7-9) and early mortality (before day 12) was H-2Dq linked and correlated directly with early (day 6-8) and high cytotoxic T cell activity. In contrast, susceptibility to become an LCMV carrier, inability to rapidly clear virus, or tendency to develop late hepatitis (day 14-17) was linked to Dk and correlated with absence of early cytotoxic T cell activity. Thus, H-2D-regulated T cell-immune responses controlling both virus spread and immunopathology may directly determine the type and severity of disease. The results illustrate that susceptibility to disease caused by one virus may be linked to distinct MHC alleles dependent upon the disease parameter studied.

T cell-mediated hepatitis in mice infected with lymphocytic choriomeningitis virus. Liver cell destruction by H-2 class I-restricted virus-specific cytotoxic T cells as a physiological correlate of the 51Cr-release assay?

A model for immunologically T cell-mediated hepatitis was established in mice infected with lymphocytic choriomeningitis virus (LCMV). The severity of hepatitis was monitored histologically and by determination of changes in serum levels of the enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH), and alkaline phosphatase (AP). Kinetics of histological disease manifestations, increases of liver enzyme levels in the serum, and cytotoxic T cell activities in livers and spleens all correlated and were dependent upon several parameters: LCMV-isolate; LCMV-WE caused extensive hepatitis, LCMV-Armstrong virtually none. Virus dose. Route of infection; i.v. or i.p. infection caused hepatitis, whereas infection into the footpad did not. The general genetic background of the murine host; of the strains tested, Swiss mice and A-strain mice were more susceptible than C57BL or CBA mice; BALB/c and DBA/2 mice were least susceptible. The degree of immunocompetence of the murine host; T cell deficient nu/nu mice never developed hepatitis, whereas nu/+ or +/+ mice always did. B cell-depleted anti-IgM-treated mice developed immune-mediated hepatitis comparably or even more extensively than control mice. Local cytotoxic T cell activity; mononuclear cells isolated from livers during the period of overt hepatitis were two to five times more active than equal numbers of spleen cells. Adoptive transfer of nylon wool-nonadherent anti-Thy-1.2 and anti-Lyt-2 plus C-sensitive, anti-L3T4 plus C-resistant lymphocytes into irradiated mice preinfected with LCMV-WE caused a rapid time- and dose-dependent linear increase of serum enzyme levels. This increase was caused by adoptive transfer of lymphocytes if immune cell donors and recipient mice shared class I, but not when they shared class II histocompatibility antigens. The donor cell dose-dependent increase of these enzymes was first measurable 6-18 h after transfer with 2 X 10(8) cells or 3 X 10(6) cells, respectively. The time-dependent increase caused by the adoptive transfer of 1-2 X 10(8) cells was strictly linear during a period of up to 25-40 h. These results indicate single-hit kinetics of liver cell death and suggest that effector T cells destroy infected liver cells via direct contact rather than via soluble toxic mediators. The results may represent the best in vivo correlate of the in vitro 51Cr-release assay that has been analyzed so far, and strongly support the view that antiviral cytotoxic T cells are directly cytolytic in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Analytical performance of the random access analyser Hitachi 737. A multicentre evaluation.

The selective multitest analyser Hitachi 737 was examined according to the ECCLS guidelines in a multicentre evaluation involving 4 laboratories. Twenty routine parameters, including the electrolytes, sodium, potassium and chloride, were measured at 37 degrees C. All of the measured values were included for evaluation without correcting for outliers. The trial, which lasted 4 months and involved over 70 000 analyses, basically yielded the following results: The precision can be termed very good. For the majority of the methods, the day-to-day coefficients of variation were below 2%. The highest coefficient of variation was 6.3% (for creatine kinase) and the lowest below 0.1% (for gamma-glutamyltransferase). The recovery of the assigned values of control sera was very good for most of the parameters (between 95% and 105%). Only with bilirubin, phosphate and chloride did greater deviations occur in a few control sera. Excellent agreement was found between the results obtained from the instruments used in the comparison: the Hitachi 705, a flame photometer and a chloride meter. No drift effects were observed. The same applied to carryover effects, provided that Boehringer Mannheim's recommendations concerning method combinations are observed. Because of the large measuring range, it is necessary to repeat analyses only in exceptional cases. During the entire period of the trial there were no stoppages due to the instrument malfunction. As a result of its reliability, the Hitachi 737 is well suited for routine operation and emergency analysis in medium to large-sized laboratories.