Establishing benchmarks and metrics for disruptive technologies, inappropriate and obsolete tests in the clinical laboratory.

Benchmarks and metrics related to laboratory test utilization are based on evidence-based medical literature that may suffer from a positive publication bias. Guidelines are only as good as the data reviewed to create them. Disruptive technologies require time for appropriate use to be established before utilization review will be meaningful.

Multicenter evaluation of the LightCycler MRSA advanced test, the Xpert MRSA Assay, and MRSASelect directly plated culture with simulated workflow comparison for the detection of methicillin-resistant Staphylococcus aureus in nasal swabs.

Rapid detection of nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA) followed by appropriate infection control procedures reduces MRSA infection and transmission. We compared the performance and workflow of two Food and Drug Administration-approved nucleic acid amplification assays, the LightCycler MRSA Advanced Test and the Xpert MRSA test, with those of directly plated culture (MRSASelect) using 1202 nasal swabs collected at three U.S.

Provider-performed microscopy.

Point-of-care testing (POCT) is defined as analytic testing performed outside the central laboratory using a device or devices that can be easily transported to the vicinity of the patient. This article discusses rules and regulations concerning POCT, especially those covering provider-performed microscopy (PPM). Types of PPM are also covered, including the fern test, tests for the presence on fecal leukocytes and pinworms, and examinations of urine sediment and seminal fluid.

Point-of-care testing and molecular diagnostics: miniaturization required.

Turnaround time for molecular diagnostic tests is critical in detecting infectious agents, in determining a patient's ability to metabolize a drug or drug class, and in detecting minimal residual disease. These applications would benefit from the development of a point-of-care device for nucleic acid extraction, amplification, and detection. The ideal device would have a low cost per test, use a disposable unit use device for all steps in the assay, be portable, and provide a result that requires no interpretation.

Molecular pathology: future issues.

Context: The field of molecular pathology is expanding in complexity. To achieve competency, vigilance is required.

Objective: To review the advances in clinically useful molecular biologic techniques and to identify their applications in clinical practice, as presented at the 13th Annual William Beaumont Hospital DNA Symposium.

Fatty acid synthase and its mRNA concentrations are decreased at different times following Hoechst 33342-induced apoptosis in BC3H-1 myocytes.

Fatty acid synthase (FAS) regulates the production of fatty acids and plays a role in regulating apoptosis. Hoechst 33342-induced apoptosis in BC3H-1 myocytes was used as a model to explore intracellular changes in FAS protein (Western blot) and FAS mRNA (RT-PCR). Total lipid and individual phospholipid synthesis was inhibited by a lethal dose of Hoechst 33342 (20 microg/ml) while total lipid and phospholipid degradation ([1-14C]-acetate pulse chase method) were not.

Point-of-care molecular diagnostic systems--past, present and future.

The field of molecular diagnostics has greatly decreased the time it takes to identify infectious agents and to test their antimicrobial resistance. Portable devices are currently in development that can easily identify a variety of nucleic acid targets (either DNA or RNA) from multiple sample types in under an hour. This is done by a variety of methods including real-time polymerase chain reaction, probe-based assays, bioluminescence real-time amplification, and microarray or micro-pump technologies.

Mitochondrial membrane potential change induced by Hoechst 33342 in myelogenous leukemia cell line HL-60.

Abstract. Hoechst 33342's effects on apoptosis and mitochondrial membrane potential (delta psi) were investigated in a myelogenous leukemia cell line, HL-60. Delta psi was detected with 2 lipophilic cationic fluorochromes: 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] or 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1).

Cytosine arabinoside substitution decreases transcription factor-DNA binding element complex formation.

Context: The pyrimidine nucleoside analog, cytosine arabinoside (Ara-C), is an effective therapeutic agent for acute leukemia. The phosphorylated triphosphate, cytosine arabinoside triphosphate, competes with deoxycytosine triphosphate as a substrate for incorporation into DNA. Once incorporated into DNA, it inhibits DNA polymerase and topoisomerase I and modifies the tertiary structure of DNA.

Effect of polymorphisms in the cytochrome P450 CYP2C9 gene on warfarin anticoagulation.

Context: Warfarin is a widely used anticoagulant with efficacy in treatment and prevention of thrombosis. Patient management, however, is difficult because of interindividual variation in response to standard doses due to significant differences in metabolic rates. Warfarin metabolism is under genetic control, involving primarily the CYP2C9 gene encoding the enzyme that catalyzes the conversion of warfarin to inactive metabolites.

The -omics era and its impact.

Objective: To review the advances in clinically useful molecular biologic techniques and to identify their applications, as presented at the 12th Annual William Beaumont Hospital DNA Symposium.

Data Sources: The 7 manuscripts submitted were reviewed and their major findings were compared with literature on the same or related topics.

Study Selection: Manuscripts address the use of molecular techniques in the detection of severe acute respiratory syndrome (SARS) and bacterial ribosome mutations, which may lead to ribosome-targeted drug resistance; pharmacogenomics as a clinical laboratory service and example of warfarin dosing using CYP2C9 mutation analysis; definition of the potential of cytosine arabinoside incorporation into DNA to disrupt transcription using an in vitro model of oligonucleotides; use of laser capture microdissection to isolate solid tumor cells free of nontumor cells; and molecular methods used to classify lymphomas.

Review: Glycosphingolipids in health and disease.

Glycosphingolipids are ubiquitous membrane constituents that are subdivided in neutral or acidic fractions (gangliosides and sulfatides). Their analysis requires extraction and separation by thin-layer chromatography or high-performance liquid chromatography. Ganglioside composition changes occur in response to variations in cellular morphology and function.

Insulin increases the intracellular concentration of total oxalyl thiolesters in BC3H-1 myocytes: potential anti-insulin mediators.

Oxalyl thiolesters (RS-CO-COOH) may represent negative intracellular messengers for insulin action. Using a reverse-phase, ion-pair high pressure liquid chromatographic technique, total intracellular oxalyl thiolesters were measured in insulin-sensitive BC3H-1 myocytes after the addition of insulin. The total oxalyl thiolester concentration increased to a maximum of 2.

Hoechst 33342 alters luciferase gene expression in transfected BC3H-1 myocytes.

Background: Hoechst 33342 and Hoechst 33258 bind to the minor groove of DNA. Hoechst 33342 induces apoptosis in a variety of cell types by a mechanism that is associated with disruption of the formation of the TATA box-binding protein/DNA complex.

Objective: To further investigate the role of Hoechst 33342 in gene regulation using BC3H-1 myocytes transfected with 4 different pGL3 luciferase reporter vectors constructed with or without the SV40 promoter and/or enhancer regions or with 2 synthetic Renilla luciferase vectors (phRL-null and phRL-TK).

Genomics, transcriptomics, proteomics, and numbers.

Objective: To review the advances in clinically useful molecular biologic techniques and to identify their applications in clinical practice, as presented at the 11th Annual William Beaumont Hospital DNA Symposium.

Data Sources: The 8 manuscripts submitted were reviewed, and their major findings were compared with literature on the same or related topics.

Study Selection: Manuscripts address the use of molecular techniques in microbiology to evaluate infectious disease and epidemiology; molecular microbiology methods, including rapid-cycle real-time polymerase chain reaction; peroxisome proliferator-activated receptor gamma as a potential therapeutic target in inflammatory bowel disease or colon cancer; the effect of nonapoptotic doses of the bisbenizamide dye Hoechst 33342 on luciferase expression in plasmid-transfected BC3H-1 myocytes; the routine use of cystic fibrosis screening and its challenges; and the use of flow cytometry and/or chromosomal translocation in the diagnostic evaluation of hematopoietic malignancies.

Apoptosis: biochemical aspects and clinical implications.

Apoptosis and necrosis represent two distinct types of cell death. Apoptosis possesses unique morphologic and biochemical features which distinguish this mechanism of programmed cell death from necrosis. Extrinsic apoptotic cell death is receptor-linked and initiates apoptosis by activating caspase 8.

The postgenomic era: implications for the clinical laboratory.

Objectives: To review the advances in clinically useful molecular biological techniques and to identify their applications in clinical practice, as presented at the Tenth Annual William Beaumont Hospital DNA Symposium.

Data Sources: The 11 manuscripts submitted were reviewed and their major findings were compared with literature on the same topic.

Study Selection: Manuscripts address creative thinking techniques applied to DNA discovery, extraction of DNA from clotted blood, the relationship of mitochondrial dysfunction in neurodegenerative disorders, and molecular methods to identify human lymphocyte antigen class I and class II loci.

Disruption of replication protein A/single-stranded DNA complexes during apoptosis in HL-60 cells.

Replication protein A (RPA) is a single-stranded DNA-binding protein which plays a role in DNA replication, repair, and recombination. We used gel mobility shift, super gel mobility shift, and Western blot to determine the fate of RPA during Hoechst 33342-induced apoptosis in HL-60 cells. Multiple bands were detected by gel mobility shift after the incubation of single-stranded gamma-(32)P-labeled oligo(dT)(30) with the nuclear extracts of HL-60 cells.

Provider-performed microscopy.

The category of provider-performed microscopy was defined in the Federal Register to provide a unique regulatory approach for bright-field and phase-contrast microscopy performed by a physician, dentist, or midlevel practitioner examining labile specimens. A CLIA certificate for this category of testing is available, which also permits the performance of waived tests. Because these provider-performed microscopy procedures do not have quality-control materials available, special challenges are encountered in establishing and monitoring such testing within a hospital.

Nitric oxide measurements during endotoxemia.

Background: Excessive continuous NO release from inducible NO synthase over prolonged periods under pathological conditions, such as endotoxemia, contributes significantly to circulatory failure, hypotension, and septic shock. This NO production during endotoxemia is accompanied by superoxide release, which contributes to the fast decay of NO. Therefore, the amount of NO that diffuses to target sites may be much lower than the total amount released under pathological conditions.

Hoechst 33342-induced apoptosis is associated with decreased immunoreactive topoisomerase I and topoisomerase I-DNA complex formation.

Hoechst 33342, but not Hoechst 33258, induces apoptosis and inhibits topoisomerase 1 activity in vivo. Topoisomerase I relaxes superhelical DNA through a single strand breakage/rejoining reaction in which the active site tyrosine links covalently to a 3' phosphate at the break site, forming a transient intermediate called a cleavable complex. The fate of cellular topoisomerase 1 in Hoechst 33342-induced apoptosis is unknown.

Hoechst 33342-induced apoptosis is associated with intracellular accumulation of E2F-1 protein in BC3H-1 myocytes and HL-60 cells.

Context: Hoechst 33342 induces apoptosis, inhibits topoisomerase I, and disrupts TATA box-binding protein/TATA box element binding in BC3H-1 myocytes and HL-60 cells. In contrast, Hoechst 33258 does not have any of these actions.

Objective: To determine if Hoechst 33342 or Hoechst 33258 treatment of BC3H-1 myocytes or HL-60 cells is associated with the intracellular accumulation of the nuclear transcription factor E2F-1, known to induce apoptosis.