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Evaluation of the Sirscan automated zone reader in a clinical microbiology laboratory.

We compared readings of Kirby-Bauer plates by the Sirscan, an automated image analyzer that measures zone diameters, to those of experienced clinical microbiologists measuring zones with a hand-held caliper interfaced to a computer and with a ruler. To read plates of Escherichia coli, Morganella morganii, and Pseudomonas aeruginosa containing 12 antibiotic disks the Sirscan took 11 s; technologists took 28 s by caliper and 39 s by ruler. Reading times of four different technologists ranged from 22 to 44 s with the caliper and 10 to 12 s with Sirscan. Upon repeated testing zone size variation rarely exceeded 3 mm by caliper and 1 mm by Sirscan. Over a 4-month period, 368 clinical isolates were tested prospectively by both methods in the Clinical Microbiology Laboratory of the Miriam Hospital. There was good correlation of zone sizes for most antibiotics, but Sirscan zone diameter measurements tended to be 3 to 5 mm larger than caliper readings for ciprofloxacin, norfloxacin, aztreonam, erythromycin, clindamycin, and trimethoprim-sulfamethoxazole. Very major errors (resistant by caliper and susceptible by Sirscan) occurred with 10 of 3,770 readings (0.3%), mainly where breakpoint criteria lacked an intermediate zone. They occurred in testing staphylococci with amoxicillin-clavulanate (5 of 127 isolates, 3.9%), pseudomonas with piperacillin (1 of 28, 3.6%), coagulase-negative staphylococci with oxacillin (2 of 74, 2.7%), gram-negative bacilli with cefuroxime (1 of 209, 0.5%), and mixed species with trimethoprim-sulfamethoxazole (1 of 366, 0.3%). The Sirscan zone reader facilitates accurate, fully quantitative susceptibility testing in clinical microbiology laboratories.

Anti-Bacterial Agents↗

Analytical and laboratory evaluation of a new fully-automated third generation enzyme immunoassay for the detection of antibodies to the hepatitis C virus.

An analytical and laboratory evaluation of a newly-developed fully-automated third generation ELISA for the detection of anti-HCV (Cobas Core Anti-HCV EIA, Roche) was undertaken. Coefficients of variation (CVs) calculated on positive control and serum samples ranged from 5.9 to 9.8% in the intra-assay precision test and from 3.9 to 11.3% in the inter-assay evaluation. With regard to the study of clinical laboratory performance, five groups of sera pre-screened with two third generation ELISA (Ortho HCV 3.0 ELISA; Innotest HCV Ab III) were assayed: anti-HCV negative samples (n = 932); anti-HCV positive samples (n = 449); difficult sera of different origin (n = 113); sera with discrepant results in the two ELISAs (n = 50); sera with an indeterminate result in one or more confirmatory test (n = 34). The overall concordance between the Roche anti-HCV EIA and the two reference assays was 97.5 and 97.8% for the Ortho and for the Innogenetics assays, respectively. Although it is not possible to provide absolute figures for clinical sensitivity and specificity, the results of the study on discrepant samples show that the Cobas Core Anti-HCV gives a number of negative results with positive or indeterminate confirmatory anti-HCV tests, which is intermediate between the Ortho and the Innogenetics assay. In contrast, only 5% Cobas Core Anti-HCV reactive sera are not positive or clear-cut single band reactive by supplemental assays. The results show that the new fully-automated third generation anti-HCV test is a valid alternative to other commercially available assays for screening of antibodies to the hepatitis C virus.

Enzyme-Linked Immunosorbent Assay↗

Clinical evaluation of automated antibiotic susceptibility testing with the MS-2 system.

The MS-2 (Abbott Laboratories) system for automated antimicrobial susceptibility testing was evaluated for both accuracy and general utility in our clinical laboratory. A total of 984 fresh clinical bacterial isolates (745 gram-negative, 239 gram-positive) were tested with the MS-2 system, and results were compared directly with those from a conventional agar disk diffusion method. Discrepancies between the two methods were categorized as very major, major, and minor. For gram-positive isolates, full accord (all discrepancies considered) was 91.6%, and essential accord (minor discrepancies not included) was 96.2%. With gram-negative isolates, full accord was found to be 93.9%, with essential accord of 97.9%. Aggrement as a function both of organism group and of antimicrobial agent was determined. Full accord of 90% or more was found for all major organism groups tested, with the exception of enterococci, where discrepant results between the two methods were observed. Mean test time for all isolates tested was 4.3 h. The MS-2 was found to be an accurate and highly automated instrument which required minimal technician time and was readily adaptable to work flow in our clinical laboratory.

Anti-Bacterial Agents↗

Accuracy of three automated compounding systems determined by end-product laboratory testing and comparison with manual preparation.

The accuracy of three parenteral nutrient (PN) automated compounding systems and the usefulness of end-product laboratory testing of PN solutions were evaluated. Ten identical PN solutions were prepared by the Nutrimix (Abbott), MicroMacro 23 (Baxa), and Automix-Micromix (Clintec) compounders and manually. Three samples were removed from each of the 40 solutions. Dextrose was measured by high-performance liquid chromatography using a modified sorbitol assay; sodium, potassium, calcium, and magnesium were measured by atomic absorption spectroscopy. Theoretical concentrations of ingredients in the PN solution were adjusted by the actual concentrations in the source containers. The solutions differed significantly in their concentrations of glucose, calcium, and magnesium but not in their concentrations of sodium and potassium. The mean glucose concentrations produced by the manual method and Nutrimix system matched the adjusted theoretical values and by the MicroMacro and Automix-Micromix compounders were within the manufactures' accuracy specifications. The sodium concentrations of all solutions were higher than the adjusted theoretical concentration but within the manufacturers' specifications. Potassium and magnesium concentrations were lower in all solutions than the adjusted theoretical concentrations. Calcium concentrations were above the adjusted theoretical value in all solutions. Differences in compounder accuracy, even when significant, were not consistent and not appreciable. Multiple samples of the same solution should be tested if laboratory analysis is part of quality control.

Automation↗

Automated counting of in situ hybridization dots in interphase cells of leukemia samples.

Twenty-seven samples (cell cultures prepared for routine cytogenetics) of leukemia patients with known cytogenetic abnormalities were stained by in situ hybridization for interphase cytogenetics with centromere specific probes for chromosome Nos 4, 6, 7, 8, 9, 12, 17, 18, X and Y. The number of hybridization domains per nucleus was quantified using a semi-automated system developed in our laboratory. Results of this automated counting procedure (with and without verification of the counting results by the operator) were compared with conventional cytogenetic data and with visual scoring of the number of hybridization dots. The findings show that the system is capable of analysing 1000 cell nuclei in less than 30 min, including the necessary verification of the results by the operator. Automated counting and visual scoring were in good agreement. Conventional cytogenetics and interphase cytogenetics agreed in only 50% of the cases, confirming other studies showing that conventional cytogenetic results are not always representative for the majority of the cell population.

Adolescent↗

Workflow improvement and impact of the new Beckman Coulter LH 1500 high throughput automated hematology workcell.

Our core laboratory at St. Joseph's Medical Center (SJMC) recently installed the new Beckman Coulter LH 1500 fully automated hematology workcell. Like many laboratories in North America and other parts of the world, we are under significant pressure to improve productivity, reduce costs, and improve service provision to our hospital and clinician bases. The difficulty of meeting this challenge is often further compounded by an increasing shortage of qualified technologist personnel. We recently reported on the significant productivity gains with the introduction of the LH 750 hematology system into the core laboratory, including a 23% reduction in slide review rate, automation of reticulocyte and nucleated red blood cell counts, and an 82% increase in the pass-through rate. Evolution to the next stage of automation has come much quicker than anticipated owing to a steep rise in workload and an ever increasing shortage of trained and experienced technologists to fill vacancies in the 24-hour core laboratory operation. We chose to upgrade our LH 750 analyzers to the newly introduced LH 1500 hematology workcell in order to cope with the increased workload and shortage of available experienced hematology technologists. The LH 1500 is a compact, high throughput, fully automated hematology workcell. It is designed to automate all manual process and handling steps associated with hematology analyses, including sample sorting, loading and unloading of cassettes, automatic rerun and reflex testing, sample storage, and tracking of samples. The LH 1500 is comprised of a minimum of 2, and up to 4, LH 750 instruments (with or without integrated slidemaker/stainers). The instruments are bidirectionally interfaced and linked to a fully automated conveyor track, specimen inlet and outlet stations, a sample stockyard, and a line controller. The configuration we chose at SJMC incorporates 1 LH 750 and 1 LH 755. We report that the upgrade to the LH 1500 was a relatively straightforward process and required no redesign of the core laboratory. Based on our evaluation, we estimate cost savings with the LH 1500 of between three quarters to 1 full-time equivalent, which eliminated or decreased many manual handling steps. In addition, this increase in efficiency has freed up our technologist resources to concentrate on the more difficult and clinically important samples. This is the first published report on the new LH 1500 hematology workcell. Our research indicates that the LH 1500 is the first hematology workcell to offer hematology laboratories the next level of automation and virtually eliminate preanalytical and postanalytical sample handling and sorting. For core laboratories and high-throughput hematology laboratories that face similar pressures to ours, the LH 1500 would appear as an attractive solution to maximize hematology productivity and improve service delivery.

Automation↗

Laboratory evaluation of the Miles H.3 automated reticulocyte counter. A comparative study with manual reference method and Sysmex R-1000.

OBJECTIVE: To evaluate the performance of the new commercial Miles H.3 RTX analyzer in counting reticulocytes. METHODS AND PATIENTS: The results from the counter were compared to those obtained from microscopic methods, following the National Committee for Clinical Laboratory Standards H44-P guidelines, and to the results from the Sysmex R-1000 counter. In total, 279 samples were analyzed in duplicate with each of the three methods. One hundred thirty-three samples were from healthy subjects, while 146 were from patients with various pathologies, 10 of whom presented with posttherapeutic aplasia of the bone marrow and 9 with iron-deficiency anemia. RESULTS: The reference intervals for the normal controls are different for each of the three methods (manual: 0.35-2.35%, 16 to 116 x 10(9)/L; Miles H.3: 0.65-2.30%, 35.1 to 112.0 x 10(9)/L; Sysmex R-1000: 0.6-1.85%, 28.0 to 85.0 x 10(9)/L). The overall imprecision was lower for the instruments than for the microscopic method (Miles H.3: coefficient of variation, 11.6%; R-1000: coefficient of variation, 4.2%; microscopic method: coefficient of variation, 24.2%). The Miles H.3 shows a good correlation with the other methods, yet it overestimated the low values with respect to both the microscopic method (intercept, 0.55; slope, 0.70) and the R-1000 (intercept, 0.44; slope, 0.78). This became particularly pronounced in patients with marrow aplasia. CONCLUSIONS: Miles H.3 can produce results with an acceptable degree of accuracy. The agreement with the dedicated fluorescence-based flow cytometer R-1000 at normal and high concentrations is also good. The possibility of providing reticulocyte indices as well as erythrocyte indices (mean volume, mean hemoglobin content, mean hemoglobin concentration) and the relative dispersion indices could be useful in understanding red cell pathophysiology in normal and iron deficient patients.

Anemia, Aplastic↗

Application of automation and information systems to forensic genetic specimen processing.

During the last 10 years, the introduction of PCR-based DNA typing technologies in forensic applications has been highly successful. This technology has become pervasive throughout forensic laboratories and it continues to grow in prevalence. For many criminal cases, it provides the most probative evidence. Criminal genotype data banking and victim identification initiatives that follow mass-fatality incidents have benefited the most from the introduction of automation for sample processing and data analysis. Attributes of offender specimens including large numbers, high quality and identical collection and processing are ideal for the application of laboratory automation. The magnitude of kinship analysis required by mass-fatality incidents necessitates the application of computing solutions to automate the task. More recently, the development activities of many forensic laboratories are focused on leveraging experience from these two applications to casework sample processing. The trend toward increased prevalence of forensic genetic analysis will continue to drive additional innovations in high-throughput laboratory automation and information systems.

Automation↗

High-throughput automated DNA sequencing facility with fluorescent labels at the European Molecular Biology Laboratory.

One of the aims of the facility is to develop and push the automated on-line DNA sequencing gel technology to its limit in sequence throughput, which may be somewhere around 100 kilobases of sequence per device per day. Key new developments were initiated and applied in operation on the European Molecular Biology Laboratory (EMBL) automated sequencer and its commercial version A.L.F. (Pharmacia). Sequencing speed was increased by a factor of 10-20, up to 1500 bases per hour per clone on ultrathin (about 100 microns) gels, while the resolution and reading length were extended to 1000 bases on gels with 50 cm separation length, using fluorescein-15-*dATP as the internal label. With our sequencing strategy, closing about 40% of the sequence with "walking" primers and F-15-*dATP as internal label, we sequenced both strands of a cosmid insert of 38.5 kb in length, each strand twice, in only 430 sequencing reactions and with average reading of 380 bases per reaction.

Base Sequence↗