The enumeration of absolute levels of cells and their subsets in clinical samples is of primary importance in human immunodeficiency virus (HIV)؉ individuals (CD4؉ T-lymphocyte enumeration), in patients who are candidates for autotransplantation (CD34؉ hematopoietic progenitor cells), and in evaluating leukoreduced blood products (residual white blood cells). These measurements share a number of technical options, namely, single-or multiple-color cell staining and logical gating strategies. These can be accomplished using single-or dual-platform counting technologies employing cytometric methods. Dual-platform counting technologies couple the percentage of positive cell subsets obtained by cytometry and the absolute cell count obtained by automated hematology analyzers to derive the absolute value of such subsets. Despite having many conceptual and technical limitations, this approach is traditionally considered as the reference method for absolute cell count enumeration. As a result, the development of single-platform technologies has recently attracted attention with several different technical approaches now being readily available. These single-platform approaches have less sources of variability. A number of reports clearly demonstrate that they provide better coefficients of variation (CVs) in multicenter studies and a lower chance to generate aberrant results. These methods are therefore candidates for the new gold standard for absolute cell assessments. The currently available technical options are discussed in this review together with the results of some cross-comparative studies. Each analytical system has its own specific requirements as far as the dispensing precision steps are concerned. The importance of precision reverse pipetting is emphasized. Issues still under development include the establishment of the critical error ranges, which are different in each test setting, and the applicability of simplified low-cost techniques to be used in countries with limited resources. Cytometry (Comm. Clin. Cytometry) 42:327-346, 2000.

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While the term flow cytometry refers to the measurement of cells, the approach of making sensitive multiparameter optical measurements in a flowing sample stream is a very general analytical approach. The past few years have seen an explosion in the application of flow cytometry technology for molecular analysis and measurements using microparticles as solid supports. While microsphere-based molecular analyses using flow cytometry date back three decades, the need for highly parallel quantitative molecular measurements that has arisen from various genomic and proteomic advances has driven the development in particle encoding technology to enable highly multiplexed assays. Multiplexed particle-based immunoassays are now common place, and new assays to study genes, protein function, and molecular assembly. Numerous efforts are underway to extend the multiplexing capabilities of microparticle-based assays through new approaches to particle encoding and analyte reporting. The impact of these developments will be seen in the basic research and clinical laboratories, as well as in drug development. q 2006 International Society for Analytical CytologyKey terms: microarray; systems biology; proteomics; protein array; high throughput screening; drug discovery; diagnostics A major goal for biomedical research in the 21st century will be to collect and integrate molecular information about genes, proteins, and numerous other biomolecules into the working models of cell and organism function from which predictions can be made. The rationale for pursuing such an ambitious goal stems from the very significant advances in molecular analysis that enable the sequencing of whole genomes, the highly parallel analysis of gene expression levels, and large scale identification of proteins in complex samples. These advances resulted from new molecular reagents and assay chemistries, new instrumentation with improved sensitivity and throughput, new computational tools, and a significant change in focus for experimental biology from one that focuses on individual molecules to one that considers the abundance and interactions of many different molecules as they function in networks of biochemical pathways in living systems.However, just as these new technologies have enabled the rapid acceleration of data collection and interpretation, continued progress toward transforming this information into biological understanding is dependent on continued improvement in analytical technologies. In particular, it is critical to augment qualitative analysis methods that allow the identification of important molecules with quantitative measurements of their abundance and function. The ability to make quantitative measurements of the concentrations of many individual proteins, their interactions and the formation of macromolecular assemblies, and the measurement of these assemblies in live cells and organisms represent major challenges in understanding the systems of molecular networks and pathways that underlie physiology and disease. These challenge...

Multiplexed assays using fluorescent microspheres is an exciting technique that has been gaining popularity among researchers, particularly those in the public health field. Part of its popularity is due to its flexibility, as both immunoassays and oligonucleotide hybridization assays can be developed on this platform. This report summarizes a workshop held by the Centers for Disease Control and Prevention that discussed issues surrounding these assays and the Luminex 100 xMAP instrument. Topics included instrumentation, assay design, sample matrix and volume, quality control, and development of commercial applications. Cytometry (Clin. Cytometry) 50:239 -242,

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Background: An independent African Regional External Quality Assessment Scheme (AFREQAS) was implemented from Johannesburg. The aim was to establish a network of CD4 laboratories supporting HIV/ AIDS anti-retroviral therapy programs and improve the quality of regional CD4 testing with EQA assessment, feedback, remedial action, and technical training. Results: Average participation was 91.5%. Overall AFREQAS between-laboratory reproducibility (trimmed %CV) was 10.5% and 9.1% for absolute CD4 and CD4%/Ly, respectively. For the respective CD4abs and CD4%/Ly values in the trials where ''normal'' material was shipped trimmed %CV of 10.9 and 7.3% were noted, and in ''low'' value shipments %CV of 13.8% and 12.4% were noted. Cumulative absolute CD4 SDI analysis revealed the best between-laboratory precision amongst FACSCount and PanLeucogating (PLG-CD4) users (both SD of SDI 5 <1.2 and %CV of 8%). Dual Platform or Single Platform algorithm-based systems and certain volumetric methods (laboratories who used Partec CyFlow instruments) had higher numbers of outlying laboratories (>12-25%CV and SD(SDI) > 2.2 noted), indicating that additional technical training and/or manufacturer support was required.Conclusions: Participation in an AFREQAS with feedback and remedial action improves the quality of CD4 testing. African laboratory professionals can easily master CD4 counting technologies. However, the introduction of the simplest and most cost-effective methodologies is required to take ownership, and enable the delivery of quality CD4 counts in vast numbers necessary to support expansion of African ART programs. q

Aims: The development and evaluation of a sensitive and specific TaqMan Ò real-time polymerase chain reaction (PCR) for the detection and identification of Pantoea stewartii on maize. Methods and Results: A TaqMan Ò -based real-time PCR assay targeting the cpsD gene enabling specific detection of P. stewartii in maize leaves and seeds was developed. Under optimal conditions, the selected primers and probe were specific for the detection of all 14 reference P. stewartii strains by real-time PCR. The 32 non-Panteoa and eight other Pantoea strains tested negative. The TaqMan Ò PCR assay detected 1 pg of purified DNA and 10 4 P. stewartii colony forming units per millilitre (10 cells per reaction) in pure cultures consisting of 92AE0% intact (viable) cells. Direct processing of leaf lesions and seeds by the real-time PCR detected 10 and 50 P. stewartii cells per reaction respectively. TaqMan Ò real-time PCR results were validated by dilution plating of macerates and PCR-based subcloning followed by DNA sequencing. Conclusions: The real-time PCR assay described is a rapid, reliable and more sensitive tool for the detection of P. stewartii. Significance and Impact of the study: This real-time PCR assay would avoid false-negative results and reduce the time required for certifying maize seed shipments.

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