News Column

Patent Application Titled "Methods and Systems for Assessing Sample Behavior in a Flow Cytometer" Published Online

August 26, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- According to news reporting originating from Washington, D.C., by NewsRx journalists, a patent application by the inventors Trotter, Joseph (La Jolla, CA); Iyer, Sujata (San Jose, CA), filed on January 31, 2014, was made available online on August 14, 2014 (see also Becton Dickinson And Company).

The assignee for this patent application is Becton Dickinson And Company.

Reporters obtained the following quote from the background information supplied by the inventors: "This disclosure relates to relates generally to the field of flow cytometry and more particularly to sample analysis methods.

"Particle analyzers, such as flow and scanning cytometers, are analytical tools that enable the characterization of particles on the basis of optical parameters such as light scatter and fluorescence. In a flow cytometer, for example, particles, such as molecules, analyte-bound beads, or individual cells, in a fluid suspension are passed by a detection region in which the particles are exposed to an excitation light, typically from one or more lasers, and the light scattering and fluorescence properties of the particles are measured. Particles or components thereof typically are labeled with fluorescent dyes to facilitate detection. A multiplicity of different particles or components may be simultaneously detected by using spectrally distinct fluorescent dyes to label the different particles or components. In some implementations, a multiplicity of photodetectors, one for each of the scatter parameters to be measured, and one for each of the distinct dyes to be detected are included in the analyzer. The data obtained comprise the signals measured for each of the light scatter parameters and the fluorescence emissions.

"Cytometers may further comprise means for recording the measured data and analyzing the data. For example, data storage and analysis may be carried out using a computer connected to the detection electronics. For example, the data can be stored in tabular form, where each row corresponds to data for one particle, and the columns correspond to each of the measured parameters. The use of standard file formats, such as an 'FCS' file format, for storing data from a flow cytometer facilitates analyzing data using separate programs and/or machines. Using current analysis methods, the data typically are displayed in 2-dimensional (2D) plots for ease of visualization, but other methods may be used to visualize multidimensional data.

"The parameters measured using a flow cytometer typically include the excitation light that is scattered by the particle along a mostly forward direction, referred to as forward scatter (FSC), the excitation light that is scattered by the particle in a mostly sideways direction, referred to as side scatter (SSC), and the light emitted from fluorescent molecules in one or more channels (range of frequencies) of the spectrum, referred to as FL1, FL2, etc., or by the fluorescent dye that is primarily detected in that channel. Different cell types can be identified by the scatter parameters and the fluorescence emissions resulting from labeling various cell proteins with dye-labeled antibodies.

"Both flow and scanning cytometers are commercially available from, for example, BD Biosciences (San Jose, Calif.). Flow cytometry is described in, for example, Landy et al. (eds.), Clinical Flow Cytometry, Annals of the New York Academy of Sciences Volume 677 (1993); Bauer et al. (eds.), Clinical Flow Cytometry: Principles and Applications, Williams & Wilkins (1993); Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); and Practical Shapiro, Flow Cytometry, 4th ed., Wiley-Liss (2003); all incorporated herein by reference. Fluorescence imaging microscopy is described in, for example, Pawley (ed.), Handbook of Biological Confocal Microscopy, 2nd Edition, Plenum Press (1989), incorporated herein by reference.

"Fluorescence-activated cell sorting or particle sorting is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of particles into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It records fluorescent signals from individual cells, and physically separates cells of particular interest. The acronym FACS is trademarked and owned by Becton Dickinson.

"The particle suspension is placed near the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that on the average (Poisson distribution) there is a large separation between particles relative to their diameter. A vibrating mechanism causes the stream of particles to break into individual droplets. The system is adjusted so that there is a low probability of more than one particle being in a droplet. Just before the stream breaks into droplets the flow passes through one or more laser intersects where the fluorescent character of interest of each particles are measured. If a particle is to be collected, a charge is applied to the flow cell during the period of time one or more drops form and break off from the stream. These charged droplets then fall through an electrostatic deflection system that diverts droplets into target containers based upon the charge applied to the droplet.

"A sample can include thousands if not millions of cells. Cells may be sorted to purify a sample to the cells of interest. The sorting process can generally identify three varieties of cells: cells of interest, cells which are not of interest, and cells which cannot be identified. In order to sort cells with high purity (e.g., high concentration of cells of interest), droplet generating cell sorters typically abort cells that are too close to another unwanted cell electronically and thereby reduce contamination of the sorted populations. Highly discriminate sorting can lead to a reduced number of cells for analysis. In addition, the time needed to perform the detailed sorting may be higher than less strict sorting.

"Furthermore, in samples of adherent cells, or antigen presenting cells such as monocytes and dendritic cells or those that have been processed in a manner that increases cell to cell interaction, this can often lead to poor recovery. In a well dispersed sample, the distribution of cells is random and follows the statistics of a Poisson distribution. As noted by Lindmo (1981) by measuring the time interval between events it is possible to evaluate the deviation from the ideal distribution. Accordingly there is a need to assess in 'real time' sample behavior to determine whether a sample is well distributed."

In addition to obtaining background information on this patent application, NewsRx editors also obtained the inventors' summary information for this patent application: "The systems, methods, and devices of the disclosure each have several innovative aspects, no single one of which is solely responsible for the desirable attributes disclosed herein.

"In one innovative aspect, a method for generating an entrainment factor for a flow cytometer sample is provided. The method includes flowing the sample comprising a series of particles through the flow cytometer wherein each particle is associated with a signal. The method includes detecting a series of events with a detection system in a first time interval from a stream formed from the flow cytometer. The method further includes calculating an expected frequency of events in the first time interval based on a characterizing distribution such as a homogenous Poisson distribution. The method also includes measuring an observed frequency of events within a second time interval, wherein the second time interval is within the first time interval. The method includes generating the entrainment factor based at least in part on a ratio of the observed frequency to the expected frequency.

"In another innovative aspect, a method for generating sample behavior information for a flow cytometer sample is provided. The method includes flowing the sample comprising a series of particles through the flow cytometer wherein each particle is associated with an event and the particles are distributed in a fluidic stream. The method includes detecting a series of events and a corresponding arrival time for each event. The method includes determining a series of inter-arrivals times relative to a previous event within the series and an associated probability of occurrence of the inter-arrival times relative to a length of a specified time interval. The method also includes calculating an expected frequency of events over the series of inter-arrival times for a portion of the fluidic stream having a length equal to the specified time interval, the calculating based on a predetermined distribution such as a Poisson distribution. The method includes measuring an observed frequency of events over the series of inter-arrival times within the portion of the fluidic stream. The method also includes generating the sample behavior information based at least in part on a ratio of the observed frequency to the expected frequency of events over the series of inter-arrival times for the portion of the fluidic stream.

"In either of the innovative methods described, the stream may be comprised of regularly spaced droplets. In some implementations of the methods, the method may include comparing the entrainment factor to a predetermined value (e.g., greater than 1) and actuating a corrective action in a control system operationally connected to the flow cytometer based on a result of the comparing. An example of a corrective action includes halting the flow of the sample in the flow cytometer.

"Some implementations of the method may include sorting particles in the stream. In such implementations, the corrective action may include adjusting the sorting based on the entrainment factor.

"The innovative methods may generate the entrainment factor while the sample is flowing in the flow cytometer.

"Time intervals in accordance with the innovative methods may be measured in a variety of ways. One example time interval unit is fractions of a droplet.

"In some implementations, the entrainment factor is one of a plurality of entrainment factors calculated during successive time intervals for a flow cytometer sample.

"The sample may include multiple labeled particles. For example, the sample may include peripheral blood cells wherein the peripheral blood cells as are comprised of a first and second component labeled with a first and second label. In implementations with multiple components, it may be desirable to generate an entrainment factor for each component. In such implementations, the method may include generating the entrainment factor for the first component and another entrainment factor for the second component.

"In a further innovative aspect, a flow system for providing a sample entrainment factor is provided. The system includes a fluidics system. The fluidics system may include a sample tube and a moving fluid column within the sample tube in which particles of a sample move along a common sample path. The system includes a detection system for collecting a signal from each particle as it passes one or more detection stations along the common sample path. Each signal is assigned a signal value to form a data point for each particle. The detection system collects a succession of such data points in a first time interval. The system includes a control system operationally associated with the fluidics system. The control system is configured to generate a calculated signal frequency for at least a portion of the first time interval based on a Poisson distribution and the number of data points collected by the detection system during the first time interval. The control system is further configured to generate an experimental signal frequency based on the number of data points in the portion of the first time interval. The control system is further configured to compare the experimental signal frequency with that of a calculated signal frequency or a predetermined signal frequency. The control system is also configured to provide an entrainment factor for the sample, said entrainment factor based on said comparing.

"The flow system may compare the experimental signal frequency by determining a ratio of the experimental signal frequency to that of the calculated signal frequency or the predetermined signal frequency.

"In some implementations, the control system may, upon determining that the entrainment factor deviates from a predetermined value, actuate a corrective action. Corrective actions may include interrupting collection of signals by the detection system, purging the sample tube by said fluidics system, altering collection of signals via acoustic focusing based on the entrainment factor, and resuming collection of the signals by the detection system. Resuming collection of the signals may occur after initiating dispersion of the sample.

"In some implementations, the control system includes a first sorting configuration providing a high collection yield and a second sorting configuration providing a high collection purity. The control system may be configured to select one of the first sorting configuration or the second sorting configuration based on the entrainment factor.

"In a further innovative aspect, a non-transitory computer readable medium including instructions executable by a processor of an electronic device may be provided. The instructions, upon execution by the processor, may cause the electronic device to perform one or more of the innovative methods described above.

BRIEF DESCRIPTION OF DRAWINGS

"Several advantages of the present invention will be apparent upon consideration of the following detailed description taken in conjunction with the accompanying drawings, in which:

"FIG. 1 shows a functional block diagram for one example of an electronic device for processing cytometry data behavior information.

"FIG. 2 shows a functional block diagram for one example of a sample behavior analyzer.

"FIG. 3 shows a process flow diagram of an example method of sample behavior analysis.

"FIG. 4 shows plot diagrams for an example set of event data which does not exhibit dumpiness.

"FIG. 5 shows plot diagrams for an example set of event data which exhibits dumpiness.

"FIG. 6 shows a functional block diagram for one example of a sorting device including an entrainment factor feedback flow controller.

"FIG. 7 shows a reconstruction of event distribution in a portion of a stream which illustrates how the particles may be distributed within the portion of the stream.

"FIG. 8 shows a functional block diagram of a flow system for providing a sample entrainment factor."

For more information, see this patent application: Trotter, Joseph; Iyer, Sujata. Methods and Systems for Assessing Sample Behavior in a Flow Cytometer. Filed January 31, 2014 and posted August 14, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=7496&p=150&f=G&l=50&d=PG01&S1=20140807.PD.&OS=PD/20140807&RS=PD/20140807

Keywords for this news article include: Fluorescent Dyes, Luminescent Agents, Laboratory Chemicals, Becton Dickinson And Company.

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Source: Life Science Weekly


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