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PubMed1990

An automated method to prepare cell suspensions from human biopsy samples for immunophenotyping by flow cytometry.

A comparison was made between a manual and automated method for preparing single cell suspensions from various types of human biopsy samples. The automated method uses the shearing action of fluid movement generated by a reciprocating paddle system. When compared on 11 samples, the automated instrument always isolated cells in less time, usually requiring only 15-30 seconds in contrast to 10-15 minutes for the manual method. The time comparisons involved "set up" time of the required minor equipment as well as the time to make a complete single cell suspension from the portion of the biopsy sample sent to the Flow Cytometry Lab so extra cells could be used for other purposes and cryogenically stored for future reference. Cells isolated by the automated method from various B-lymphocytic and T-lymphocytic malignancies were still viable and could be successfully immunophenotyped by flow cytometry. The immunophenotyping results were compared for both cell isolation methods on seven of these samples, and the results were comparable. The automated technique has now been used to satisfactorily immunophenotype more than 50 biopsy samples. The automated method should represent a significant aid for clinical flow cytometry laboratories performing immunophenotyping tests by efficiently preparing single cell suspensions in a few seconds instead of minutes. Furthermore, the automated method uses a closed sterile bag system that helps minimize the exposure of personnel to potential infectious material present in biopsy samples and prevents external contamination of cell suspensions. The automated technique proven successful for immunophenotyping may also be helpful for related procedures involving hematopoietic malignancies such as DNA content analysis and cell functional assays by flow cytometry, as well as other assays such as tissue typing, gene probe, and in vitro chemosensitivity assays.

Antigens, CD↗

PubMed2004

Performance characteristics of five automated serum cortisol immunoassays.

BACKGROUND: Serum cortisol measurements are used to help diagnose conditions of cortisol deficiency and excess. Automated, nonisotopic immunoassays are used in most clinical laboratories. METHODS: Linearity, imprecision, and method comparison studies for serum cortisol assays were performed with the Access, Advia Centaur, AxSYM, Elecsys 2010, and IMMULITE 2000 analyzers. RESULTS: All methods demonstrated acceptable linearity with the AxSYM method showing the least deviation from target values. The Elecsys 2010 method was the most precise for all three levels of quality control material. Method comparison studies demonstrated good concordance among methods except for two patient samples that gave results on the Elecsys 2010 that were significantly higher than the other methods. Differences in assay calibration were observed. CONCLUSIONS: Overall, all methods performed well. Additional calibration standardization efforts are required to truly harmonize the results from each assay. The Elecsys 2010 method is less specific than other automated methods based on two discordant patient results.

Autoanalysis↗

PubMed2000

High-level, stable expression of blood group antigens in a heterologous system.

The detection and identification of blood group antibodies in patients is crucial for successful allogeneic blood transfusions. Current methods are highly subjective and rely on red blood cells (RBCs), which simultaneously express many blood group antigens, have a short shelf-life, and carry potential biohazard risks. To overcome these problems, we have used the approach of expressing individual blood group antigen-bearing proteins in a heterologous system. We report here the high-level surface expression of type I (Knops), type II (Kell), and type III/multi-pass (Duffy) membrane proteins that carry blood group antigens in mouse erythroleukaemic (MEL) cells using a vector containing the beta-globin locus control region. Importantly, the antigens expressed were detected specifically by a panel of patients' sera containing alloantibodies at sensitivities that are comparable to antigen-positive RBCs. Furthermore, in contrast to other mammalian expression systems, antigen expression was stable following freezing and thawing of the cell lines. Thus, this system has the potential both to replace the current use of RBCs by providing a one step method to detect and identify blood group antibodies and to allow the automation of antibody identification for the clinical laboratory.

Animals↗

PubMed1992

Postangioplasty restenosis rate between segments of the major coronary arteries.

Conflicting data have been published regarding the rate of postangioplasty restenosis observed in diverse segments of the coronary tree. However, these studies may be criticized for their biased selection of patients, methods of analysis, and definitions of restenosis. In the present study, 1,353 patients underwent a successful coronary dilatation of greater than or equal to 1 site. In all, 1,234 patients (91%) had a follow-up angiogram after 6 months, or earlier when indicated by symptoms. All films were processed and analyzed at the thoraxcenter core laboratory with the coronary angiography analysis system (automated contour detection). Restenosis was considered present if the diameter stenosis at follow-up was greater than 50%. No differences in restenosis rates were observed between coronary segments using this categorical definition. A continuous approach was also used; absolute changes in minimal luminal diameter adjusted for vessel size were used in order to allow comparison between vessels of different sizes (relative loss). No significant differences were observed between the coronary segments with this continuous approach. These results suggest that restenosis is a ubiquitous phenomenon without any predilection for a particular site in the coronary tree.

Analysis of Variance↗

PubMed1985

Clinician use of a pediatric cardiology database on a microcomputer.

A pediatric cardiology database was designed and implemented on a microcomputer using the MEDUS/A (Harvard School of Public Health) database management system. This database has potential utility in the areas of clinical research, patient management, and administration. Initial uses of the system include a study of ventricular septal defects, and automation of pacemaker clinic and cardiac catheterization laboratory records. Clinicians with little or no programming experience can alter the database structure and tailor complicated applications to their particular needs. Preliminary evaluation of the system is presented.

Cardiac Catheterization↗

PubMed1985

Drug level monitoring of antiasthmatic drugs.

In general assays pertaining to drug level monitoring (DLM) of antiasthmatic agents (except theophylline), published during the period 1978-1983, used mostly high-performance liquid chromatographic (HPLC) methodology (approximately 45%) with mass spectrometric (MS) based assays in second place (approximately 30%) followed by immunochemical techniques (approximately 25%). Whenever nanogram or subnanogram antiasthmatic drug concentrations had to be measured such as for the adrenergic stimulants or for the prophylactic agents, then both HPLC-and MS-based methodologies were employed with about equal frequency. The trend in DLM for the phosphodiesterase inhibitor class (theophyllines) seemed to be shifting towards the HPLC methodologies. In part, this was justified by the need for improved selectivity. This criterion appears to have been better satisfied by HPLC, but for all practical purposes the immunochemical methods are and will probably continue to prevail in the clinical laboratory setting until HPLC procedures become truly automated. In the case of DLM of corticosteroids used for the asthmatic, the situation is in our opinion still unclear. This is caused by the presence of endogenous corticosteroids and metabolites, the levels of which in man are known to vary. The current immunochemical procedures offer a facile but less selective option. The future for selective routine corticosteroid assays may well be in HPLC or gas chromatography coupled with MS.

Adrenal Cortex Hormones↗

PubMed1988

Rapid identification of Bacteroides species by high-performance liquid chromatography.

High-performance liquid chromatography was evaluated for the rapid identification of Bacteroides species of clinical interest. Each isolate was inoculated into a defined chemical medium containing primarily carbohydrates and was incubated aerobically at 37 degrees C for 1 h. After centrifugation, the supernatants were placed on ice to stop further enzymatic reactions. Specimens were injected into an Aminex HPX-87H column in order to determine carbohydrates and acid metabolic products. Peak areas of carbohydrates for each isolate were compared with those for uninoculated medium. As the utilization indexes of raffinose, lactose and arabinose were found to be particularly significant, the patterns of carbohydrate utilization could be used for the identification of Bacteroides species. This method can be adapted for diagnostic laboratory use and has good potential for automated microbial identification.

Bacteroides↗

PubMed2003

Use of methotrexate in sarcoid-associated optic neuropathy.

OBJECTIVE: To demonstrate the possible beneficial effects of methotrexate (MTX) therapy for patients with sarcoid-associated optic neuropathy (SAON). DESIGN: Retrospective, noncomparative, interventional case series. PARTICIPANTS: Three patients diagnosed with SAON who received MTX. Two patients had tissue biopsies consistent with sarcoidosis, and 1 patient had clinically diagnosed sarcoidosis based on laboratory and radiographic studies. All 3 patients developed side effects with corticosteroid treatment of their optic neuropathy. INTERVENTION: Patients were treated with weekly doses of oral MTX and monitored with neuro-ophthalmic, medical, and laboratory examinations. MAIN OUTCOME MEASURES: Visual acuity, automated perimetry, and reduction of oral prednisone therapy. RESULTS: After initiation of MTX, all 3 patients showed an improvement or stabilization of visual acuity. All patients had a decrease in their corticosteroid requirements, and all had improved or stabilized visual field deficits. One of the 3 patients developed leukopenia that necessitated a reduction of the methotrexate dose. CONCLUSION: Methotrexate may be effective for SAON as an adjunct to corticosteroid therapy or as an alternative for corticosteroid-intolerant patients. Oral MTX reduced the corticosteroid requirements of 3 patients with SAON, and all 3 demonstrated stable or improved visual function.

Adult↗

PubMed1997

Pericranial muscle tenderness and exteroceptive suppression of temporalis muscle activity: a blind study of chronic tension-type headache.

The aim of the present study was to examine the ability of pericranial muscle tenderness and the second exteroceptive suppression period to distinguish chronic tension-type headache sufferers, migraine sufferers, and controls in a young adult population utilizing a blind design. The second exteroceptive suppression periods were assessed using the methodology recommended by the European Headache Federation and were scored with an automated computer software program designed in our laboratory to provide reliable, standardized, and precise quantification of exteroceptive suppression periods and eliminate any influence of experimenter bias that may occur with manual scoring. Our sample consisted of 45 subjects diagnosed according to IHS criteria: 25 with chronic tension-type headache and 20 with migraine without aura. Twenty-three headache-free controls were recruited. Consistent with our previous findings, abnormalities in pericranial muscle tenderness, but not in the second exteroceptive suppression period distinguished chronic tension-type headache sufferers from controls. The chronic tension headache sufferers exhibited the highest pericranial muscle tenderness and the control group exhibited the lowest tenderness (P < .001). Pericranial muscle tenderness was quite successful in distinguishing recurrent headache sufferers from controls, but failed to distinguish chronic tension-type headache sufferers from migraineurs. Our findings raise the possibility that pericranial muscle tenderness is present early in the development of chronic tension-type headache and migraine without aura, and thus might contribute to the etiology of headache disorders. Our findings also indicate that a shortened second exteroceptive suppression period is not a reliable marker for chronic tension-type headache in young adults.

Adolescent↗

PubMed1992

Clinically significant heparin carryover on the ACL 300R.

Investigation of a patient with an unexpected prolonged activated partial thromboplastin time showed on repeat testing that the patient had normal coagulation times, but had been preceded on the automated coagulation analyzer run (ACL 300R--Instrumentation Laboratory, Lexington, MA) by a heparin-contaminated apheresis line sample taken from a different patient. Heparin carryover was suspected, and subsequent experiments showed that ex vivo heparinization of normal plasmas will produce similar effects in following normal plasma samples in a test series. This effect increases with the baseline activated partial thromboplastin time and the heparin concentration. It is recommended that coagulation analyzers that are susceptible to carryover be cleaned after testing of plasmas likely to have heparin concentration of more than 5 x 10(3) U/L. Furthermore, when using analyzers of this type, investigation of an unexplained activated partial thromboplastin time must include a review of the preceding samples tested, particularly when duplicate testing has been discontinued.

Blood Coagulation Tests↗

PubMed1988

Computer video acquisition and analysis system for biological data.

The BIAS (Biological Image Analysis System) was developed to: (i) permit accurate entry and image processing of biological data; (ii) minimize the need for specialized hardware; and (iii) aid in the human genome mapping and other projects. The first mouse/cursor key-driven module was designed to be user interactive and readily accessible to many laboratories. It contains the DRSNDS programs which automate the entering of data in a systematic format. The types of data that can be entered utilizing this module are DNA-RNA gels from either a positive or negative Polaroid image, autoradiograms or biotinylated images from Southern, Northern and dot or slot blot hybridization analyses. The image is acquired using a video camera and then digitized for subsequent analysis. During the analysis graphical representations of the intermediate results are provided to assure user confidence. At any point within the program the user may obtain on-line help with the current task. The output displays the mol. wt of each individual component in the appropriate context. The present version of the program produces results comparable with a human interpreter for some data. Band shifting and optical density calculations are in a prototype form to permit evaluation of various techniques. Future work is directed at expanding the system's capabilities to interpret data from other biological analyses including DNA sequencing gels.

Algorithms↗

PubMed2002

Analysis of duodenal drainage fluid after cholecystokinin (CCK) stimulation in healthy volunteers.

INTRODUCTION AND AIMS: Hormonal stimulatory agents are used to assess pancreatic function. Biologically derived secretin, the most widely used pancreatic secretagogue, is no longer available in the United States. Existing secretory tests using cholecystokinin alone are cumbersome, requiring a unique dual tube (gastric and duodenal) collection system and constant perfusion of a nonabsorbable marker to calculate enzyme output (in international units [IU]). A simpler, quantitative cholecystokinin stimulation test that measures enzyme concentrations (in international units per liter [IU/L]) instead of total output would obviate need for marker perfusion/collection. The aim of our experiment was to study the secretory patterns of pancreatic enzyme concentration in duodenal fluid after cholecystokinin stimulation in healthy volunteers. METHODOLOGY: Healthy subjects had a Dreiling tube inserted endoscopically into the ligament of Treitz. Gastric and duodenal aspiration ports were connected to low intermittent suction. A 20-minute baseline was obtained to clear the gastric and duodenal lumina of residual fluid. Cholecystokinin was infused at a constant rate of 40 ng/kg per hour. Duodenal fluid was collected on ice for 80 minutes in four 20-minute aliquots. Aspirated fluid was analyzed for enzyme concentration with an automated chemistry analyzer in the hospital biochemistry laboratory. RESULTS: Nineteen healthy volunteers were studied. The mean volume (+/-SEM) of duodenal fluid collected was 85 +/- 4.4 mL (range, 48 to 118 mL). Fluid analysis revealed a significant rise in mean lipase concentration (+/-SEM) from a baseline of 595,680 +/- 11,930 IU/L to a peak of 1,778,847 +/- 171,204 IU/L (mean difference = 1,183,167 IU/L; 95% CI= 664,459 IU/L to 1,701,875 IU/L; < 0.001, Student test). Increases in amylase concentrations were markedly less pronounced and did not reach statistical significance. Mean peak lipase concentration occurred within 50 minutes of acinar cell stimulation. All patients tolerated tube placement, and there were no episodes of acute pancreatitis or abdominal pain. CONCLUSIONS: Pancreatic lipase concentrations in duodenal fluid increase nearly threefold after cholecystokinin stimulation in healthy volunteers. This magnitude of enzyme secretory response may be a marker of pancreatic function and could potentially lead to a more clinically useful and simpler pancreatic function test. This physiologic study serves as the basis for our further investigations of cholecystokinin-stimulated lipase concentrations as a new test in the assessment of pancreatic insufficiency.

Amylases↗

PubMed2002

Characterization of ciprofloxacin resistance in Neisseria gonorrhoeae isolates in Canada.

BACKGROUND: Ciprofloxacin (500 mg orally, single dose) is one of the recommended therapies for gonorrhea in Canada. In Canada, the first ciprofloxacin-resistant (CipR) Neisseria gonorrhoeae strain was isolated in 1993. Antimicrobial susceptibilities of N gonorrhoeae isolates were monitored as part of a national surveillance program to ensure efficacy of antimicrobial therapies. GOAL: The goal was to determine the characteristics of ciprofloxacin resistance in Canadian gonococcal isolates. STUDY DESIGN: Susceptibility testing was performed on gonococcal strains from different provinces in Canada to determine the prevalence of CipR strains and their distribution. The CipR strains were further differentiated according to auxotype (A), serotype (S), plasmid profile (P), and pulsed-field gel electrophoresis (PFGE) profile. DNA sequencing and DNA microarray technology were used to determine mutations in gyrA and parC. RESULTS: In Canada, between 1997 and 1999, 4.8% of resistant strains (130 of 2687 antibiotic-resistant N gonorrhoeae isolates) were CipR (MICs of 1-32 microg/l) and belonged to 48 A/S/P classes. Sixty-eight of the strains that were not differentiated by A/S/P were subtyped into 47 classes with PFGE. DNA sequencing and DNA microarray showed that the most common mutations had amino acid substitutions of Ser-->Phe at codon 91 and Asp-->Gly at codon 95 of the gyrA and Ser-->Arg at codon 87 of parC. CONCLUSION: The CipR strains isolated in Canada are phenotypically and genotypically diverse, indicating that they were imported from overseas and not endemic in Canada. Mutations in gyrA and parC previously only identified by DNA sequencing were successfully identified with DNA microarray technology. DNA microarray technology could be an alternative tool for identifying point mutations in resistance genes or other epidemiologic markers when clinical laboratories replace culture methods with rapid and automated molecular methods for diagnosis.

Anti-Infective Agents↗

PubMed1992

Detection of human DNA mutations with nonradioactive, allele-specific oligonucleotide probes.

We describe a method of detecting human DNA mutations with nonradioactive, biotinylated allele-specific oligonucleotide probes. This method can detect seven different mutations in the butyrylcholinesterase, cystic fibrosis, and N-acetyltransferase genes under identical assay conditions. This indicates that it may be used to detect mutations responsible for a wide variety of genetic diseases and pharmacogenetic conditions. The method involves first amplifying selected DNA fragments by the polymerase chain reaction and dot blotting the amplified DNA in duplicate onto small nitrocellulose squares. Each dot blot is then hybridized in individual wells containing a tetramethylammonium chloride solution with short biotinylated probes specific for either the normal or mutant allele. Successfully hybridized probes are detected by a simple colorimetric reaction using an avidin-alkaline phosphatase conjugate, which yields a very strong, clear signal. DNA from homozygous normal or mutant individuals hybridizes only to the normal- or mutant-specific probes respectively, while DNA from heterozygous individuals hybridizes equally well with both probes. These results can be easily interpreted to assign a genotype to the sample DNA. This method is amenable to automation, and may be useful in clinical laboratories for diagnosis of a wide variety of DNA mutations responsible for unusual reactions to drugs and environmental chemicals.

Alleles↗

PubMed1994

Actigraphic home-monitoring of the sleep patterns of in vitro fertilization children and their matched controls.

The sleep patterns of 35 in vitro fertilization (IVF) children aged 19 to 57 months and their matched controls were evaluated by actigraphic home-monitoring for three consecutive nights. Actigraphic data were analysed by an automated scoring procedure developed in the authors' laboratory. There were no significant differences in activity levels between IVF multiple-birth subjects and their matched controls or between IVF singletons and their matched controls. In contrast, multiple-birth subjects had higher activity levels during sleep and lower sleep efficiency than singletons in both IVF and control groups. Sleep measures showed night-to-night stability in all groups.

Age Factors↗

PubMed2006

Optimising workflow in andrology: a new electronic patient record and database.

AIM: To improve workflow and usability by introduction of a new electronic patient record (EPR) and database. METHODS: Establishment of an EPR based on open source technology (MySQL database and PHP scripting language) in a tertiary care andrology center at a university clinic. Workflow analysis, a benchmark comparing the two systems and a survey for usability and ergonomics were carried out. RESULTS: Workflow optimizations (electronic ordering of laboratory analysis, elimination of transcription steps and automated referral letters) and the decrease in time required for data entry per patient to 71%+/-27%, P<0.05, lead to a workload reduction. The benchmark showed a significant performance increase (highest with starting the respective system: 1.3+/-0.2 s vs. 11.1+/-0.2 s, mean+/-SD). In the survey, users rated the new system at least two ranks higher over its predecessor (P<0.01) in all sub-areas. CONCLUSION: With further improvements, today's EPR can evolve to substitute paper records, saving time (and possibly costs), supporting user satisfaction and expanding the basis for scientific evaluation when more data is electronically available. Newly introduced systems should be versatile, adaptable for users, and workflow-oriented to yield the highest benefit. If ready-made software is purchased, customization should be implemented during rollout.

Andrology↗

PubMed1986

Controls for flow cytometers in hematology and cellular immunology.

The necessity of using freshly prepared biological samples to control and calibrate flow cytometers has impeded the utilization of flow cytometry in the clinical laboratory where regulations demand careful control calibration and yet workload and technician training militate against the preparation of specialized control samples. For a number of years, cell preparations with stabilized properties have been used routinely in hematology laboratories to evaluate the daily performance of automated blood cell counters including optical flow cytometers. Unfortunately, the stabilized preparations do not necessarily duplicate many important aspects of whole blood, resulting in variable performance of a given control preparation on different types of instrumentation. The new generation of hematology analyzers measures a host of new parameters and will require more sophisticated controls for these enhanced parameters. Some practical aspects of present and future hematology controls will be discussed. Immunology, a new area for automated cell analysis, can benefit greatly from controls both for instrument performance and for cell staining. Up to this time there has been little availability of control materials suitable for clinical immunology. We have been evaluating fluorochrome-stained cell nuclei for use as an instrument control and for calibration of immunofluorescence analyses. Our development and use of this type of control will be described. We will also discuss approaches and experiments aimed at producing cells with stabilized antigenic properties which can be stained with monoclonal antibodies and can thus act as actual reagent controls.

Antibodies, Monoclonal↗

PubMed1981

Clinical laboratory evaluation of the Auto-Microbic system for rapid identification of Enterobacteriaceae.

The capability of the Auto-Microbic system (Vitek Systems, Inc., Hazelwood, Mo.) has been expanded to identify members of the family Enterobacteriaceae with the use of a sealed, disposable accessory card (the Enterobacteriaceae Biochemical Card) containing 26 biochemical tests. To judge the accuracy of the AutoMicrobic system's identification in a hospital laboratory, 933 Enterobacteriaceae isolates were studied. The AutoMicrobic system provided the correct identification for 905 of the isolates (97%). For Enterobacteriaceae excluding Escherichia coli (533 isolates not E. coli), 506 isolates were correctly identified (94.9%). The AutoMicrobic system integrates well into the clinical microbiology laboratory, providing a simple, accurate, and efficient automated system for the rapid identification of Enterobacteriaceae.

Bacteriological Techniques↗