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PubMed2003

Preparation of a purified fibrinogen calibration material for Clauss method and turbidimetric immunoassay possessing biological activity and antigenicity.

Fibrinogen plays a major role in basic coagulation tests such as prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT). These show high interlaboratory variation because of inaccurate instrumental calibration. The same is true of the fibrinogen assay, despite its being a quantitative assay. Most medical laboratories use automated coagulometers and commercially available calibration materials (calibrators) to obtain an accurate fibrinogen value, but, when checked, calibrators have been found to deviate from the assigned value. The Japan Society of Laboratory Medicine (JSLM) has identified the need for a reliable plasma fibrinogen standard. To enhance the reliability of calibrator fibrinogen values and thereby remedy the poor precision and accuracy of plasma fibrinogen testing, we undertook the preparation of a standard calibration material applicable to both the Clauss method and turbidimetric immunoassay (TIA). The calibrator was prepared from fresh human plasma by glycine precipitation and virus inactivation followed by affinity chromatography to remove contaminated plasminogen. In the resulting product, clottable fibrinogen accounted for 95% of total protein and within-run precision showed a CV of less than 1.8%. We believe the preparation will become a candidate material for laboratory and manufacturer use in Japan.

Blood Coagulation Tests↗
PubMed1994

Lipid testing for the year 2000 and beyond.

Lipid testing has progressed from early measurements of total lipid by extraction and weighing to assess the fat content of the specimen. This nonspecific approach to lipid testing has been replaced in clinical laboratories by automated and quantitative procedures that avoid the extraction process. Instead, selective enzymes are utilized in reaction schemes to quantitate the individual lipid classes present in patient specimens. For example, cholesterol esterase and oxidase are used on a routine basis to measure total cholesterol in plasma and serum specimens. Similar use of other enzyme systems has permitted triglycerides and phospholipids to be measured by clinical laboratories. Lipid and lipoprotein measurements have advanced considerably from the early nonspecific extraction and gravimetric analysis schemes to the specific automated procedures that are commonly used today. However, as lipids and lipoproteins increased in their clinical usefulness as cardiovascular risk assessment tools, the search intensified for newer approaches to measure these entities more easily and more accurately. The influence of National Cholesterol Education Program has played a key role in highlighting the importance of lipids and lipoprotein analysis. Today, lipid testing is available outside the traditional laboratory environments - drugstores sell units that individuals can use at home to assess cholesterol levels. Lipid testing has come a long way, and we have only begun to experience some of the remarkable changes for the future.

Humans↗
PubMed2001

Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units.

Large-scale genotyping of Mycobacterium tuberculosis is especially challenging, as the current typing methods are labor-intensive and the results are difficult to compare among laboratories. Here, automated typing based on variable-number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) in 12 mammalian minisatellite-like loci of M. tuberculosis is presented. This system combines analysis of multiplex PCRs on a fluorescence-based DNA analyzer with computerized automation of the genotyping. Analysis of a blinded reference set of 90 strains from 38 countries (K. Kremer et al., J. Clin. Microbiol. 37:2607-2618, 1999) demonstrated that it is 100% reproducible, sensitive, and specific for M. tuberculosis complex isolates, a performance that has not been achieved by any other typing method tested in the same conditions. MIRU-VNTRs can be used for analysis of the global genetic diversity of M. tuberculosis complex strains at different levels of evolutionary divergence. To fully exploit the portability of this typing system, a website was set up for the analysis of M. tuberculosis MIRU-VNTR genotypes via the Internet. This opens the way for global epidemiological surveillance of tuberculosis and should lead to novel insights into the evolutionary and population genetics of this major pathogen.

Automation↗
PubMed1988

Accuracy and reproducibility of cholesterol assay in the western Cape.

The accuracy and precision of cholesterol assay in the western Cape region is reported. The survey was carried out over 15 weeks utilising three human EDTA plasma pools with normal, borderline high and high cholesterol levels respectively. All 11 laboratories in the region providing a service to academic, provincial or military hospitals or to the private medical sector were included in the study. Ten of the 11 laboratories utilised automated enzymatic methods of cholesterol assay whereas 1 used a manual procedure based on the Liebermann-Burchard reaction. Methods were standardised by means of a variety of commercial calibrator material in all except 1 laboratory which used reference sera from the Centers for Disease Control, Atlanta. The performance of the 4 best laboratories met the standard of precision recommended for cholesterol assay, viz. total coefficient of variation of less than or equal to 2.5%. However, only 2 of the 11 laboratories achieved the optimum objective of an overall bias of less than 2.0% together with precision of less than or equal to 2.5%. Rational use of cholesterol assay for diagnosis and management will therefore require standardisation of cholesterol assay on a common reference material and greater attention to analytical factors influencing the reproducibility of results. Intrinsic biological variation also contributes uncertainty to the interpretation of a single value. Thus important clinical decisions must be based on two or more assays carried out using appropriate methodology.

Cholesterol↗
PubMed1994

Data management in the vascular laboratory.

As the clinical workload in the vascular laboratory increases and as the demand for additional documentation by hospital oversight committees and outside agencies grow, the need for computerized data management will become obvious. Although there are no generic, broadly applicable software programs to automate the laboratory's operations, applications can easily be developed using any of the current database programs to meet the needs of most laboratories. Fortunately, the intense competition in the microcomputer industry has recently made very powerful systems that are capable of providing the necessary computing support increasingly affordable. Such systems can be very simple or incredibly complex depending on the available local expertise and each laboratory's specific needs. In addition to facilitating the laboratory's daily operations, such a system will inevitably expedite the implementation of the laboratory's quality assurance program and will maximize utilization of existing personnel. This type of cost-effective solution to the ever-increasing demand for service will become increasingly important in maintaining the laboratory's fiscal viability. Although the prospects of undertaking such a task might seem daunting, especially to the computer novice, it is important to begin. Keep the system simple, at first, and allow it to develop as local expertise and confidence develop. The only prospect more frightening that sitting down to develop a computerized system for managing the laboratory's data, is the prospect of trying to continue without one!

Clinical Laboratory Information Systems↗
PubMed2003

Evaluation of automated RT-PCR to accelerate the laboratory diagnosis of foot-and-mouth disease virus.

Automated fluorogenic (5' nuclease probe-based) reverse transcription polymerase chain reaction (RT-PCR) procedures were evaluated for the diagnosis of foot-and-mouth disease (FMD) using suspensions of vesicular epithelium, heparinised or clotted blood, milk and oesophageal-pharyngeal fluid ('probang') samples from the United Kingdom (UK) 2001 epidemic and on sera from animals experimentally infected with the outbreak serotype O FMD virus strain. A MagNA Pure LC was initially programmed to automate the nucleic acid extraction and RT procedures with the PCR amplification carried out manually by fluorogenic assay in a GeneAmp 5700 Sequence Detection System. This allowed 32 samples to be tested by one person in a typical working day or 64 samples by two people within 10-12 h. The PCR amplification was later automated and a protocol developed for one person to complete a single test incorporating 96 RT-PCR results within 2 working days or for two people to do the same thing in around 12 h. The RT-PCR results were directly compared with those obtained by the routine diagnostic tests of ELISA and virus isolation in cell culture. The results on blood, probang and milk samples were in broad agreement between the three procedures but specific RT-PCR protocols for such material have to be fully optimised as perhaps have the positive-negative acceptance criteria. However, the automated RT-PCR achieved definitive diagnostic results (positive or negative) on supernatant fluids from first passage inoculated cell cultures and its sensitivity was greater than ELISA on suspensions of vesicular epithelium (ES) and at least equivalent to that of virus isolation in cell culture. The combined tests of ELISA, virus isolation in cell culture and RT-PCR might, therefore, only be required for confirmation of a first outbreak of FMD in a previously FMD-free country. Should a prolonged outbreak subsequently occur, then either ELISA plus RT-PCR or else RT-PCR alone could be used as the laboratory diagnostic tool(s). Either approach would eliminate the requirement for sample passage in cell culture and considerably advance the issue of laboratory diagnostic test results.

Animals↗
PubMed1998

[Blood culture: gold standard for definitive diagnosis of bacterial and fungal infections--from the laboratory aspect].

Blood culture has been long recognized as a gold standard for definitive diagnosis of bacterial and fungal infections worldwide. However, due to the lack of this recognition, particularly among physicians, very few number of blood cultures have been tested in microbiology laboratories throughout Japan. Positive results from blood cultures would have a great impact on clinical decisions; initiation of antibiotic chemotherapy, change to effective chemotherapy, and more frequently, change to less expensive chemotherapy. The "Rules of Three"; nearly all instances of bacteremia will be recognized after the incubation of three blood cultures for three days, originally proposed by Young et al. is still applicable to the most clinical instances, and will increase the value of blood cultures. The key to assuring greater clinical impact from blood cultures is rapidity to detect positive by the laboratory. Several automated blood culture systems are now commercially available, and will enable us to detect positive cultures significantly earlier than before.

Bacterial Infections↗
PubMed1999

Comparison of the erythrocyte sedimentation rate measured in the eye casualty department by the Seditainer method with an automated system.

PURPOSE: To compare a new automated system for the measurement of erythrocyte sedimentation rate (ESR) with the established manual Seditainer method. METHODS: Two hundred and twelve patients undergoing investigation for giant cell arteritis or other systemic vasculitides had ESR measurements by both the established manual Seditainer and the new laboratory-based automated system. The results were compared by correlation coefficient and mean difference. The limits of agreement with confidence intervals were also calculated. RESULTS: Across the range of results from 1 to 120 mm/h, the correlation coefficient was 0.844. The automated method had a mean negative bias of -9.8 mm/h (95% confidence interval: -12.2 to -7.4 mm/h). The wide scatter of results produced limits of agreement (+/- 2 standard deviations) between the two methods of -45 to 26 mm/h. There were seven results that were underestimated by the automated system which were clinically significant. CONCLUSIONS: There is a wide degree of scatter between the two sets of results. The automated system has a negative bias when compared with the manual method. There is a propensity for the automated system to sporadically underestimate the true result, sometimes to a degree that is clinically significant. The authors therefore cannot recommend replacement of the manual Seditainer system at the present time.

Adolescent↗
PubMed1994

Haematocrit measurements during cardiopulmonary bypass surgery: comparison of three stat methods with a blood cell counter.

Monitoring packed cell volume (PCV) is essential to safeguard oxygen delivery to the tissues during cardiopulmonary bypass (CPB). We compared the results obtained using a PCV monitor, a blood gas analyser and a microcentrifuge with those from a laboratory-based automated blood cell counter. PCV was measured in 221 samples of 40 patients undergoing open-heart surgery. The limits of agreement (mean difference +/- 2 SD) was unacceptably wide when the results obtained with the PCV monitor and the blood gas analyser were compared with those of the automated blood cell counter. This was most pronounced when PCV was measured in low concentration samples. The microcentrifuge showed good limits of agreement regardless of sample concentration. The data suggests that measurement of PCV by either continuous monitoring or by a blood gas analyser is not an accurate alternative to an established laboratory method. The microcentrifuge is the most accurate method of the ones investigated to measure haematocrit during CPB.

Aged↗
PubMed1995

Accuracy of automated DNA sequencing: a multi-laboratory comparison of sequencing results.

A double-stranded (ds)DNA template of "unknown" sequence was distributed to approximately 80 core DNA sequencing laboratories by the Association of Biomolecular Resource Facilities (ABRF) for automated DNA sequence analysis. Forty-four different facilities responded with 83 usable sequence submissions. These sequences were grouped by both sequencing protocol (dye-primer or dye-terminator) and whether manually edited or not. The sequences were aligned with the known sequence, and the number of correct base calls, insertions, deletions, no-calls and miscalls were determined for each group. The dye-primer sequencing protocol provided the longest and most accurate sequence. The edited dye-primer data were > 95% accurate out to 400-450 bp, while the edited dye-terminator data could call only 300-350 bases at this accuracy. However, 75% of the laboratories in this sampling preferred the dye-terminator protocol, presumably because of its versatility and convenience. Laboratories that manually edited the automatically called data were able to obtain an additional 100 bases of good sequence when the dye-primer protocol was used. Surprisingly though, editing of dye-terminator results did not increase the amount of good sequence, although the dye-terminator protocol had a superior base-calling ability within the first 100 bases of called sequence.

Autoanalysis↗
PubMedDate not supplied

Computerized recording and reporting system for cytology and colposcopy.

The computerized system for recording and reporting cytologic and colposcopic data at the Madrid Provincial Hospital is discussed. Cytodiagnostic codes based on standard international nomenclature, and yet open to constant updating, were created for the anatomic sites from which samples are currently examined (vagina and cervix, endometrium, vulva and breast), with areas allocated for expansion to other sites. A similar novel code was devised for colposcopic data. Specific forms were developed for each anatomic site; the cervicovaginal and endometrial forms are used for both cytology and colposcopy. The system has provided the laboratory with automation of its managerial, clerical and administrative tasks, including (1) record keeping, (2) reporting, (3) statistics, (4) correlation of clinical, cytologic, colposcopic and histopathologic data, (5) patient follow-up, (6) technical support in research programs and (7) quality control. Use of the system led to an improvement in the efficiency of the laboratory and resulted in reports of better quality. The program has been of help in our research and has not increased the total cost of cytologic studies.

Breast↗
PubMed1985

Laboratory equipment maintenance contracts.

The increasing level of technical sophistication and complexity found in clinical laboratory instrumentation today more than ever demands careful attention to maintenance service needs. The time-worn caution for careful definition of requirements for acquisition of a system should also carry over to acquisition of maintenance service. Guidelines are presented for specifications of terms and conditions for maintenance service from the perspective of the laboratorian in the automated clinical laboratory.

Commerce↗
PubMed2004

Position of immunological techniques in screening in clinical toxicology.

There is a continuing increase in the use of immunological techniques in the field of clinical toxicology. This is primarily due to the rapidity by which analytical results are now required, and can be obtained, following the testing of individuals for drug use. There has recently been an increase in the repertoire of assays now available to testing laboratories (e.g., buprenorphine and heroin metabolite assays), with the techniques themselves becoming increasingly more specific for the drugs and/or metabolites being monitored (e.g., methadone metabolite assays). The near patient testing (NPT), or point-of-care testing (POCT), devices are now several generations forward from their inception, with some tests now approaching the sensitivity and specificity of automated laboratory-based methods. This review has been collated from the literature to illustrate some of the possible reasons for the move towards the increasing use of immunological techniques, and to highlight some of the advantages and disadvantages associated with such drug screening methods. In particular, it has been shown that it is important to determine, monitor and review the knowledge and training of the individual using the technique. In addition, quality control and quality assessment are paramount to ensure the validity of any drug testing being performed. It has also been shown that it is vital to maintain and develop the relationships between the staff performing the testing, the laboratory (if the testing is performed using NPT devices), and the clinicians utilising the results obtained from drug testing. Without these links, interpretive errors could arise which could adversely affect the diagnosis and management of patients.

Drug Evaluation, Preclinical↗
PubMed2003

An automated feeding apparatus for in vitro maintenance of the human head louse, Pediculus capitis (Anoplura: Pediculidae).

An automated feeding apparatus was developed to maintain the human head louse (Pediculus capitis DeGeer) in vitro. With the use of valves and timers, banked human blood and saline from refrigerated reservoirs were pumped into and flushed out of the system every 7 d. During this rotational interval, bloodmeals were provided to head lice continuously and ad libitum through a stretched Nescofilm-silicone sandwich membrane. Compared with our previous in vitro human head louse-rearing apparatus, greater numbers of lice could be fed simultaneously with minimal human monitoring. Development of second to third instars and third instars to adults was significantly faster when lice were reared in vivo than on either of the in vitro rearing systems; there was no significant difference in the duration of the first instar. Although fecundity and hatch rates were significantly higher for female lice reared in vivo, similar trends have been observed for other membrane-fed arthropods. Body lice (Pediculus humanus L.) and bed bugs (Cimex lectularius [L.]) also completed most of their life cycle on this apparatus. Our automated mass-rearing system has broad applications for maintaining fluid-sucking ectoparasites and will facilitate various toxicological, behavioral, and disease-transmission investigations.

Animals↗