Conformational distribution of surface-adsorbed fibronectin molecules explored by single molecule localization microscopy.

Adsorbed proteins that promote cell adhesion mediate the response of cells to biomaterials and scaffolds. As proteins undergo conformational changes upon surface adsorption, their functional display may be significantly affected by surface chemistry or solution conditions during the adsorption process. A high-resolution localization microscopy technique is extended here to probe the conformation of individual fibronectin (Fn) molecules at the glass-water interface under physiological buffer conditions. To map distances, four available cysteines located on the modules FnIII7 and FnIII15 of dimeric Fn were site-specifically labeled with Cy3B, and their relative positions were determined by stepwise photobleaching with nanometer precision. The four labels on single Fn molecules did not show a uniform or linear arrangement. The distances between label positions were distributed asymmetrically around 33 nm with a tail towards higher distances. Exposure of Fn to denaturing solution conditions during adsorption increased the average distances up to 43 nm for 4 M guanidinium HCl, while changing the solution conditions after the adsorption had no effect, indicating that the observed intra-molecular distances are locked-in during the adsorption process. Also surface coatings of different hydrophobicity altered the conformational distribution, shifting label distances from a median of 24 nm on hydrophilic to 49 nm on hydrophobic surfaces. These results further highlight that the conformation of macromolecules at interfaces depends on the adsorption history. While illustrated here for surface adsorbed Fn, the power of localization-based microscopy extends the repertoire of techniques to characterize biomolecules at interfaces.

An algorithm for testing and reporting serum choriogonadotropin at clinically significant decision levels with use of "pregnancy test" reagents.

We present an algorithm for monitoring the concentration of human choriogonadotropin (hCG) in serum at various clinical decision levels with use of fast, simple, and cost-effective qualitative pregnancy test reagents for hCG in serum. Based on correlation between laboratory data and clinical observations described in the literature, the following concentrations of hCG in serum can be considered as clinically significant decision levels: 5 int. units/L to exclude or "rule out" the presence of increased hCG; 25 int. units/L for "confirming pregnancy" or confirming the presence of increased hCG from sources other than the trophoblast; and 6500 and 82 500 int. units/L to enhance the sonographic diagnoses of ectopic pregnancies and molar pregnancies, respectively. We used Tandem Icon II (Hybritech) pregnancy test reagents and evaluated the reagents for possible "false-positive" findings at the 25 int. units/L limit of hCG detectability by analyzing 100 post-menopausal and more than 4000 premenopausal serum specimens. The performance of the reagents was validated also at 5 int. units/L and at limits of hCG detectability greater than 25 int. units/L.

Clinically useful limits (CUL) criteria as a basis for quality control including minimal and optimal goals for quality control.

Quality control goals for quantitative clinical chemistry assays are reviewed. Recommendations for clinically useful limits (CUL) criteria as minimally acceptable +/- 2 SD for run-to-run and/or day-to-day technical reliability are presented in terms of biologic variations for different levels and clinical applications. A review of the literature and the questionnaire response of a medical school staff presented in this study reveal striking agreement of acceptable analytic goals based on physicians' opinions, biologic variation, and practical analytic feasibility. The current state-of-the-art in clinical laboratories can approach excellence because actual within-laboratory 2-SD variability estimates are somewhere between acceptable CUL criteria and twice as good. Assessing the reliability of laboratory reports based on accuracy and precision or total analytic error is also discussed.

Applicability of the Ektachem 400 analyzer for assaying analytes in miscellaneous body fluids.

The Ektachem analyzer, a slide-film chemistry technology, demonstrated applicability to non-serum, non-urine stimulated and actual clinical specimens having a wide range of protein concentration and pH. The simulated specimens studied had pH and protein that ranged from 2 to 9 and 0 to 88 g/L, respectively. Actual patients' specimens studied, which had protein concentrations ranging from 0.1 to 60 g/L, included peritoneal fluid, vitreous fluid, synovial fluid, pericardial fluid, and cerebrospinal fluid. The analytes studied included glucose, urea nitrogen, uric acid, creatinine, calcium, phosphorus, sodium, potassium, chloride, and carbon dioxide.

Highly sensitive qualitative methods for serum choriogonadotropin (hCG): clinical specificity studies.

We screened six highly sensitive kits, designed for serum pregnancy tests, for false-positive results. The two best were then evaluated more extensively. The "BETA-hCG MAIA-clone" (I) and the "TANDEM ICON" (II) kits gave only negative results for 100 sera from men at 5, 10, and 25 int. units/L (1st IRP). Of 100 serum specimens from post-menopausal women three and 10 were hCG positive by the II and the I reagents, respectively, but only at the 5 int. units (1st IRP) of hCG per liter level of sensitivity. At 10 and 25 int. units/L, all specimens were negative by both tests. The manufacturers of these kits recommend pregnancy testing only at the 25 int. units/L level of analytical sensitivity. By quantitative methods, hCG concentrations in the II positive samples ranged from 6 to 20 int. units/L (1st IRP) and lutropin concentrations were between 130 to greater than 150 int. units/L. The medical records of the corresponding patients did not support the presence of trophoblast or any other hCG-secreting tissues. During 15 months of routine use of the II reagents at an analytical sensitivity of 25 int. units/L (1st IRP) hCG for pregnancy testing (greater than 4000 serum specimens from pre-menopausal women), the staff has not reported to us any suspected false-positive findings.

Enzyme-linked immunosorbent urine pregnancy tests. Clinical specificity studies.

The performance of six recently introduced highly sensitive enzyme-linked immunosorbent (ELISA) urine pregnancy test reagent kits was evaluated for false positive results using 100 male and 100 postmenopausal female urine specimens and the findings were compared with those of a qualitative radioimmunoassay (RIA) procedure. Based on the findings, the most suitable pregnancy test reagent kit was then selected for doing routine pregnancy testing of urine samples of premenopausal patients. The less sensitive PregnaSTICK ELISA method and Concept RIA procedure did not give any positive results. Positive results for postmenopausal female and male urine samples were obtained as follows: Testpack, 2 and 0 (greater than 50 mIU/mL [IU/L]); Icon, 2 and 0 (greater than 50 mIU/mL [IU/L]); Quest, 4 and 2 (greater than 50 mIU/mL [IU/L]); Nimbus, 17 and 4 (greater than 25 mIU/mL [IU/L]); and Sensi-Chrome, 33 and 19 (greater than 50 mIU/mL [IU/L]), respectively. The medical records of the patients whose urine samples gave positive results were examined for information that would have explained the positive results, but no clear-cut reasons were found. Comparison of the routine urinalysis findings showed that there was a correlation between the mucus content of female (but not of male) urine samples and the incidence of false positive human chorionic gonadotropin results. During 12 months of routine use of the Icon reagents for pregnancy testing of premenopausal urine samples, the University of Texas Medical Branch staff has not reported any suspected false positive findings to the authors.

A quality control system and its application to clinical chemistry.

A quality control system is presented that involves the Quality Control Log for documenting data, decisions, and corrective actions. It emphasizes the use of the Calibration Work Record, cusum, and comparison of means to monitor acute and chronic drift and method bias. It introduces clinically useful limits criteria for efficient and cost-effective quality control.

Interference by IgG paraproteins in the Jaffe method for creatinine determination.

A large (60-100%) negative interference in serum creatinine value of some (3 of 18 myeloma patients, all with monoclonal gammopathy of IgG type, was observed on several instruments that determine creatinine by the Jaffe reaction. No interference was observed in an enzymatic method. Serum electrophoresis showed a monoclonal band in all three samples. The interferent could be removed by acid precipitation or dithiothreitol preincubation, and partially isolated in the high molecular weight dialyzate from the serum. It was demonstrated that the interferent is not albumin or normal gammaglobulin. Kinetic analysis of the Jaffe reaction showed large initial decreases in absorbance, in agreement with the corresponding creatinine results. It is suggested that some monoclonal IgGs may react with the Jaffe picrate reagent, resulting in a false lowering of serum creatinine values.

Quality control for a 14- to 18-megohm-cm deionized water supply.

We studied the quality control for a 14- to 18-megohm-cm deionized water system with multiple outlets. The quality of water was determined by measuring specific resistance, performing a total colony count, trace metal analysis, and high-performance liquid chromatographic analysis. The specific resistivity of water was at the level of 17 to 18 megohms-cm at the recirculation loop and greater than 10 megohms-cm at the individual faucets. The microbial content was consistently less than 100 colony-forming units/mL. Periodic trace-metal analysis indicated less than 1 microgram/L of copper, bismuth, zinc, cadmium, mercury, thallium, and lead. High-performance liquid chromatographic analysis, as determined by the absorbance of the highest peak by ultraviolet and fluorescence spectroscopy, indicated less than or equal to 0.001 absorbance unit. Our experience suggests that maintaining laboratory water at 14 to 18 megohms using a plastic system with plastic faucets eliminates the likelihood of bacterial organic and trace-metal contamination of the water.

Immunochemical methods for measurement of transferrin in serum: effects of analytical errors and inappropriate reference intervals on diagnostic utility.

We estimated analytical errors of the Calbiochem, Kallestad, Hyland, Meloy, Helena, and Beckman immunochemical methods for serum transferrin. Intermethod biases were determined by analysis of the "Reference Preparation for Serum Proteins" of the College of American Pathologists and by analysis of 106 patients' serum samples. We judged the acceptability of errors by comparing confidence limits for total errors with 1/4 of the normal reference intervals. The transferrin status of each patient's sample was interpreted by comparing the result of each method with the normal reference interval claimed by the corresponding manufacturer. We found that the combined effects of medically unacceptable analytical errors and inappropriate normal intervals caused results of the tested methods not to be interchangeable. The Calbiochem method identified 61 serum samples (57%) as having abnormally high transferrin concentrations. In contrast, for the same specimen, with the Meloy method we found abnormally high transferrin concentrations for only two samples (1.8%).

Quality control log with CUSUM and clinically useful limits criteria.

We describe a quality control (QC) log including its advantages over the more commonly used Levey-Jennings and Shewart graphic charts. It represents a practical system of analytic quality control for quantitative assays in clinical chemistry. It utilizes cumulative sum (CUSUM) techniques and comparison of means to monitor acute and chronic analytic drift, respectively. Clinically useful limits (CUL) criteria for QC decisions are used. An algorithm to facilitate the description of, training for, and use of, the QC log system is provided.

Simultaneous occurrence of idiopathic lipemic tears and massive seborrhea.

A 42-year-old man had idiopathic lipemic tears and massive seborrhea. Recent investigations have shown that cholesterol and other lipids occasionally found in tears are produced by meibomian rather than lacrimal glands. The finding in this patient of hypersecretion by both sebaceous glands and meibomian glands, a sebaceous gland variant, suggests that a common mechanism may regulate secretory control of both of these glands.

Vapor pressure and freezing point osmolality measurements applied to a volatile screen.

This is a report of a rapid and precise screening procedure, developed for the determination of ethanol in serum using osmolality measurements. The osmolality of the patient is determined by freezing point method (freezing point osmometry) and dew point (water vapor pressure osmometry) method. The difference between freezing point osmolality and vapor pressure osmolality (delta osm) is due to the presence of volatiles in the serum, because the volatiles are not measured by vapor pressure osmometry. The amount of ethanol (mg/dL) in serum is estimated by multiplying delta osm by a factor of 4.2. As a comparison method, ethanol also is measured by a spectrophotometric alcohol dehydrogenase method. In addition, a significant difference between an osmometric alcohol assayed value and enzymatic spectrophotometric measurement indicates the presence of volatiles, other than ethanol. In addition to ethanol there is a linear relationship between osmolality and isopropanol or methanol when added in vitro to serum.

The storage stability of 3-hydroxybutyrate in serum, plasma, and whole blood.

The storage stability of 3-hydroxybutyrate in whole blood, serum, and plasma was evaluated. The levels of 3-hydroxybutyrate were measured using an enzymatic rate reaction of the specific 3-hydroxybutyrate dehydrogenase with the NAD-NADH coupled reaction. 3-Hydroxybutyrate was found to be a stable analyte in whole blood, plasma, and serum. The long-term stability of beta-hydroxybutyrate allows ample time for separation of blood components and offers storage of samples to meet quality control needs as well as the possibility of mailing specimens. Further studies indicate that NaF plasma, heparin plasma, and serum are the preferred specimens, because both EDTA and oxalate showed the most significant interference with the determination of 3-hydroxybutyrate.

[Concentrations of mezlocillin in cerebrospinal fluid in vira meningitis (author's transl)]. / Mezlocillinkonzentration im Liquor bei Virusmeningitis.

The concentrations of mezlocillin in the cerebrospinal fluid were measured in ten patients with viral meningitis after a single infusion of 5 g lasting 30 minutes. One to two hours after infusion the CSF concentrations were between 0.1 mcg/ml and 1.6 mcg/ml, and thus exceeded the minimal inhibitory concentrations of Neisseria meningitidis and Diplococcus pneumoniae, the most common pathogens in bacterial meningitis in adults. The minimal inhibitory concentration of ampicillin-sensitive strains of Haemophilus influenzae was also attained. Further investigations must be carried out, in order to ascertain whether higher CSF concentrations are attained in bacterial meningitis.