Standardization flow cytometry

Schwartz, A. Quantitative Fluorescein Microbead Standards Flow Cytometry Standards Corporation Research Triangle Park, NC 27709, [1985] technical brochure. 

A limitation of the flow cytometric binding assay has been the precise determination of the receptor affinity and calculation of the receptors per cell. This limitation appears to have been overcome by the development of fluorescein and phycoerythrin compensation-calibration standards (Flow Cytometry Standards Corp., Research Triangle Park, NC). These standards have made it possible to quantify the fluorescence intensity of samples labeled with fluorescein or phycoerythrin, and relate the intensity to molecules of equivalent soluble fluorochrome. These standards have been utilized in quantitative studies of neutrophil chemoattractant-ligand interaction (4). 

Read samples for CD4 surface levels by standard flow cytometry using a medium-maintained OM-10. 1 culture as a positive for surface expression (>90% CD4+) and a TNF-a-treated OM-10.1 culture as a negative for surface expression (<15% CD4+ by 24 h). 

The problem has now been solved, and it is possible to measure the phase angle of the probe as the cells pass through the laser beam.09,40) While these measurements have not yet been applied to Ca2+, the method has been validated with standard beads and stained cells. In our opinion, this new flow cytometry parameter, and our increasing knowledge of lifetimes of probes, will result in the increased use of flow cytometry for studies of intracellular physiology, in addition to the current emphasis in immunology, cell activation, and ploidy. 

The light scatter assay may be used to determine absolute numbers of viable cells if flow cytometry data from cell suspensions of known concentration are used to construct a standard curve. For that purpose, cell concentrations should be determined in a series of graded, standard cell suspensions with the use of a Coulter counter. A plot of those standard concentrations versus the number of events (Hght scatter signals) acquired during a specified acquisition interval in the flow cytometer may then be used to interpolate cell concentrations for test samples that have been assayed by the light scatter procedure. 

In another experimental setting in vitro data on the cell surface antigen expression were obtained for PBMCs isolated from chronic lymphocytic leukemia (CLL) patient blood samples and cultured in the presence of DIMS (according to standard procedures). Cultured cells were collected and stained with the corresponding antibodies for detection of the expression of cell surface antigens. Subsequendy they were analyzed by flow cytometry. 

Refining the standardized measurement referred to above to provide a calibrated scale is a more difficult task, although calibration beads of various types now available facilitate the process. Beads with manufacturer s reported equivalent fluorescein content have been available for some time (Flow Cytometry Standards Corporation, San Juan, Puerto Rico), and these are useful for calibration of measurements using fluorescein-labeled antibodies, although... 

Calibrator beads with known fluorescence intensity defined in terms of equivalent molecules of soluble fluorochrome (e.g., Quantum Beads, Flow Cytometry Standards Corporation). 

A similar method is used for beads with defined fluorochrome content (e g, Quantum beads, Flow Cytometry Standards Corporation), except that the beads are not subjected to the antibody-staining procedure The number of fluorochrome molecules per cell is deduced from the standard curve, and is converted to bound antibody molecules from knowledge of the fluorochrome protein ratio of the conjugate. 

Roe, R., Robins, R. A., Laxton, R R., and Baldwin, R W (1985) Kinetics of divalent monoclonal antibody binding to tumor cell surface antigens using flow cytometry—standardization and mathematical analysis Mol Immunol. 22, 11—21... 

One way around this problem is to compare cell fluorescence not with unstained cells but with the fluorescence of an external standard. This can be done by the use of fluorescent beads. In brief (this is an insiders joke you would be amazed at how much has been written about the use of beads in flow cytometry), there are commercially available polystyrene beads ( microspheres ) that have standardized... 

CV The coefficient of variation (CV) is defined as the standard deviation of a series of values divided by the mean of those values (expressed as a percentage). It is used in flow cytometry to describe the width of a histogram peak. Whereas in some proto-... 

FCS format Flow Cytometry Standard is a file format for flow cytometric data storage. Adherence to this format facilitates the programming for analysis of results. Manufacturers of cytometers have finally begun to embrace this standard. 

In sum, EMPs have emerged as a preferred direct method for assessing EC injury in different disorders. EMP analysis could provide insight into the actual status of the endothelium in vivo by a simple blood analysis. However, there is a need for refinement and standardization of the assay method. Overall, most groups have relied on flow cytometry for the measurement of EMPs nevertheless, other methods such as ELISA are available and may be an option in the future. The main challenge remains in the selection of specific and sensitive monoclonal antibodies that may yield consistent results between different laboratories. In addition, the protocols for sample handling and storage need to be clearly delineated. The assay is still a... 

Figure 9.3 Mean total lymphocyte count after one course of treatment. Peripheral blood was collected from individuals administered test article (circles) or placebo (squares) on a weekly basis, and subject to flow cytometry analysis to determine mean lymphocyte counts. A standard panel of fluorochrome-conjugated antibodies was used to identify the various lymphocyte sub populations. The solid bar indicates the dosing interval.

Interesting studies were also performed based on a suspension of beads in conjunction with flow cytometry measurements [23,24], Flow cytometry, which was the standard methodology for cell population study during the last 20 years, has now begun to serve for in vitro microspheres analysis [25]. Such systems were described as multiplex microsphere bead assays and were used to detect different nucleic acid sequences hybridized on beads having different properties (size, fluorescent label). 

The practical limitation on library oversampling depends on screening instrumentation, for which flow cytometry must be considered the gold standard. Commercially available flow cytometers analyze multicolor fluorescence signals and sort desired cells within user-defined gates at a rate of 50,000 cells per second [44], A salient comparison would be to microplate-based robotic screening. Considering each cell in a combinatorial library to be functionally equivalent to a microplate well, a flow cytometer can screen the equivalent of several million 1536-well microplates per day. 

The difficulty of separation is highly dependent on peak spreading, as shown in Fig. 6.5. It is therefore critical to minimize the peak width as far as possible. This would be difficult for cell display methods if only single color fluorescent labeling were used, because the primary source of variability is biological. Flow cytometry instrumentation point spread functions generally contribute below 2 % to the overall coefficient of variance (CV = standard deviation/mean), but typical overall CVs for yeast display are approximately 50 - 100 % for the logarithmic fluorescence intensity. 

Flow cytometry wash buffer standard phosphate-buffered saline (PBS, pH 7.4) containing 0.2% sodium azide, 0.1 % bovine serum albumin, and 2% human AB+ serum. Filter by suction through a 0.45-pM filter. 

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