WO2012030740A2 - Procédé de collecte et d'analyse de données provenant d'un cytomètre de flux - Google Patents

Procédé de collecte et d'analyse de données provenant d'un cytomètre de flux Download PDF

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Publication number
WO2012030740A2
WO2012030740A2 PCT/US2011/049598 US2011049598W WO2012030740A2 WO 2012030740 A2 WO2012030740 A2 WO 2012030740A2 US 2011049598 W US2011049598 W US 2011049598W WO 2012030740 A2 WO2012030740 A2 WO 2012030740A2
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Prior art keywords
data set
initial data
allowing
user
modified
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PCT/US2011/049598
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English (en)
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WO2012030740A3 (fr
Inventor
Collin A. Rich
Jack T. Ball
Grant C. Howes
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Accuri Cytometers, Inc.
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Publication of WO2012030740A2 publication Critical patent/WO2012030740A2/fr
Publication of WO2012030740A3 publication Critical patent/WO2012030740A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1429—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its signal processing

Definitions

  • This invention relates generally to the flow cytometry field, and more specifically to an improved method for collecting and analyzing data from a flow cytometer in the flow cytometry field.
  • a typical flow cytometer detector has a limited collection range.
  • the collection range for a conventional flow cytometer with a photomultiplier tube is approximately four decades, although the signal range of objects under detection in a typical sample may span more than five decades across experiments.
  • the collection range of a typical flow cytometer is smaller than the signal range of objects under detection. For this reason, the typical flow cytometer is supplied with a user-controlled gain level for the photomultiplier tube and/or an amplifier.
  • Detectors typically collect data relative to an object's size (light scatter) and/or brightness (fluorescence), but an appropriate gain level is often difficult to set.
  • Gain level is preferably increased to collect signals from small or faint objects, but the increased gain level results in detected signals from large or bright objects that are too intense to be collected. Conversely, gain level is preferably decreased to collect signals from large or bright objects, but the decreased gain level results in detected signals from small or faint objects that are too weak to be collected.
  • the typical flow cytometer user interface involves the preparation and running of a pilot sample in order to appropriately set the gain control and define the user-set collection range, which is typically limited to approximately four decades or less.
  • the preparation involves the steps of (1) setting the gain control to a level that the user predicts will provide the desired collection range, (2) running the pilot sample through the flow cytometer, (3) viewing the pilot data and signal collected from the pilot sample, (4) identifying the extent to which, if any, the gain setting should be modified to achieve a more suitable collection range, and (5) repeating steps (i)-(4) in a trial-and-error process until the desired collection range is achieved such that a subsequent experiment with a full sample collects desired Since the typical flow cytometer detector is unable to obtain useable data from signals beyond the collection range of the detector, and since the typical flow cytometer detector requires a pre-set gain level before data acquisition, for a particular sample run data beyond the pre-set collection range is lost.
  • FIGURE 1 is a schematic block diagram of a flow cytometer user interface of the prior art
  • FIGURE 2 is a schematic of the method of a preferred embodiment
  • FIGURES 3A and 3B are example variations of the method of a preferred embodiment.
  • 100 for collecting and analyzing data from a flow cytometer includes: collecting a full dynamic range of input signals from at least one flow cytometer sample S110; storing an initial data set of the full dynamic range of input signals S120; allowing a first modification of at least a portion of the initial data set to create a first modified data set S130; permitting analysis of the first modified data set under a first test S140; allowing a second modification of at least a portion of the initial data set to create a second modified data set S150; and permitting analysis of the second modified data set under a second test S160.
  • the method may further include displaying, storing, and/or exporting one or more of the data sets, including the initial data set, first modified data set, and/or second modified data set.
  • the method may further include displaying, storing and/or exporting the analysis of at least one of the first and second modified data sets.
  • the first and second modifications may each cater to different or similar tests.
  • the first and second tests using the first and second modified data sets may be performed on different samples or different subset portions of the samples, in which the different samples or subsets of samples include different markers specific to different tests, or are otherwise objects of differing sizes (resulting in different amounts of light scatter) and/or brightness (differing levels of fluorescence).
  • the initial data set may include data for different subsets of samples of blood from a patient, in which each subset of samples is designated for a different blood test.
  • the first and second tests using the first and second modified data sets may be similar or identical.
  • both the first and second tests may involve similar analyses for a particular marker on a specific kind of cell in the different samples, in which each sample comes from a different patient or other sample source.
  • the method 100 is performed with a user interface in a flow cytometer that extracts data from the full dynamic range of a flow cytometer in a single data collection run of one or more flow cytometer samples, and then allows modifications such as manipulation of scaling and/or culling factors across the full dynamic range after the data has been collected.
  • the data of the full dynamic range is collected and stored in raw or unmodified form and the user interface may display the unmodified data and/or modified data. Because modifications can be applied to the data after data acquisition is complete, the method facilitates real-time comparisons between the initial data and the modified data.
  • the method 100 enables efficient flow cytometry applications such as clinical applications in which a limited number of tissue samples from a patient are available, multiple gain level setting profiles are typically required for a variety of tests on the samples, and/or quick turnaround time for diagnostics is desirable (e.g., for increased throughput and for time-sensitive situations such as with samples having a short viable lifespan).
  • the method 100 can include modifications to selected portions of the initial data set such that, for example, in clinical applications, the selected portions of modified data from a single data collection run are used for different tests that would otherwise (in conventional flow cytometers) require multiple runs using different gain level settings. Furthermore, by collecting a full dynamic range of data and allowing modifications and analysis of the modified data without re-running samples, the method 100 permits subsequent, additional analyses on the collected data such as for verification or other retesting purposes, by the user and/or a third-party user.
  • the method 100 may be used for clinical diagnostic applications, drug development, industrial processes, or any other suitable clinical and/or research applications involving a flow cytometer or other suitable diagnostic and/or analysis system.
  • the steps of collecting a full dynamic range of input signals from at least one flow cytometer sample Siio functions to facilitate the collection (i.e., acquisition) of the full dynamic range of input signals from the flow cytometer sample.
  • Collecting a full dynamic range of input signals Siio may capture data from multiple samples in a single data collection run (e.g. each of multiple samples in a separate well in a 96-well plate, or multiple samples contained in another suitable sample vessel), or may capture data from one sample in a single data collection run.
  • the data collection run may gather data from multiple samples that are distinguished from one another in one or more variations. In a first variation, the samples are distinguished by being designated by the user for a particular test.
  • a portion of blood samples from a patient may be designated for one test, while another portion of blood samples from the patient may be designated for another test.
  • the samples are distinguished by being identified with different markers, such as different fluorescent tags. For example, in a particular data collection run, one portion of the samples may be marked with a fluorescent tag that provides a relatively faint input signal for detection, and another portion of the samples may be marked with a fluorescent tag that provides a relatively bright input signal for detection, such that the collection of input signals for the data collection run spans a full dynamic range.
  • the samples are additionally and/or alternatively distinguished from one another by being different fluid types (e.g.
  • samples may additionally and/or alternatively be distinguished from one another in any suitable manner.
  • some of the samples may be grouped to form subsets in the group of samples for the data collection run. Each subset may be distinguished from the other subsets by one or more of the above variations for distinguishing samples.
  • some or all of the subsets of the samples may overlap such that a sample is included in more than one subset of samples (e.g. a particular sample may be designated for multiple different tests and/or tagged with multiple different markers).
  • multiple samples and/or subsets of samples in a particular data collection run may be distinguished in any suitable manner.
  • Multiple samples and/or subsets of samples may be separated by a gas or fluid buffer that marks the end of one sample and/or subset and the beginning of another subset or sample.
  • the delineation between subsets and/or samples in the initial data set may additionally and/or alternatively be apparent to the user based on the difference in magnitude of the input signal in the data set.
  • Collecting a full dynamic range of input signals Sno is preferably performed with a user interface coupled to a flow cytometer (or other suitable diagnostic and/or analysis system).
  • the user interface is in electronic communication with an advanced flow cytometer that has a collection range approaching the total detected object signal range (e.g. broad or full dynamic range flow cytometers).
  • the advanced flow cytometer may be any suitable flow cytometer system
  • the collecting step Sno is preferably performed by a flow cytometer similar to that described in U.S. Patent Application 2006/0219873, entitled "Detection system for a flow cytometer", and/or with a user interface similar to that described in U.S.
  • Patent Application 2008/0228444 entitled "User interface for a flow cytometer system", which are each incorporated in its entirety by this reference.
  • the collecting step S110 may be performed by a user interface in electronic communication with a composite of several narrower dynamic range flow cytometers.
  • the full dynamic range of input signals is preferably defined as the range of input signals that provides at least a 1:100,000 ratio, and more preferably at least 1:1,000,000 ratio (e.g. 1:16,000,000) ratio between the faintest objects and the brightest objects.
  • the full dynamic range of an electrical subsystem in the flow cytometer is preferably similar, if not identical, to the full dynamic range that can be handled by the optical subsystem of the flow cytometer.
  • the full dynamic range of the input signals is preferably captured by a 24-bit process, which translates to approximately 16,700,000 levels of information, but the full dynamic range of input signals may alternatively be captured by any suitable process.
  • the captured data preferably includes an error rate induced by electric noise of less than one percent.
  • the full dynamic range is preferably maintained even at a high flow through, such as 10,000 events/second.
  • the data is collected in a raw, unmodified format without accepting a gain level amplification selection or adjustment from the user. Collecting the input signals in this manner may reduce the expenditure of valuable user time and reduce the potential loss of valuable data through misconfiguration of the system.
  • Storing an initial data set of the full dynamic range of input signals S120 functions to facilitate the electronic storage of the full dynamic range of input signals (in a raw, unmodified format) from the flow cytometer sample.
  • the initial data set may be stored to an electronic storage unit such as a computer hard drive (e.g. desktop, laptop, tablet, or mobile device), compact storage (e.g. USB drive) and/or server (e.g. local or network).
  • a computer hard drive e.g. desktop, laptop, tablet, or mobile device
  • compact storage e.g. USB drive
  • server e.g. local or network
  • the initial data set may be stored to an electronic storage unit coupled to the flow cytometer system and/or user interface.
  • the initial data set may be stored to a server, network, or other electronic storage medium.
  • the initial data set may be retrieved directly from the flow cytometer, and/or from an electronic storage medium or network.
  • the modification steps are preferably performed independently of the collecting step S110, and allow the user to perform real-time comparisons between the raw initial data set and at least one of the first and second modified data sets.
  • the modification steps may be adjusted, undone, and/or repeated without the need to re-run pilot samples, thereby allowing multiple adjustments on the same initial data set.
  • the modification steps preferably generate at least one subset of data (e.g. modified data set) that corresponds to one or more sample populations contained within the initial data set.
  • Modification steps S130 and S150 preferably include allowing a user to manipulate the initial data set across the full dynamic range of input signals from the flow cytometer, including scaling and/or culling at least a portion of the initial data set that corresponds to a desired sample population.
  • Scaling preferably includes amplifying or diminishing the signals at least a portion of the initial data set. For instance, scaling factors may be applied hierarchically based on forward scatter, which can be expanded to include any or all of the available data channels (e.g. scatter and fluorescence) in a progressive fashion.
  • Culling preferably includes isolating a subset of the data in a modified data set and may be one of several variations or any suitable variation, including: (1) storing the isolated subset while discarding the rest of the initial data set, (2) storing the isolated subset and storing the entire initial data set, (3) storing the isolated subset and storing the rest of the initial data set, and (4) discarding the isolated subset while storing at least a portion of the rest of the initial data set.
  • modifications may include adjusting the bounds of the data.
  • modifications may alternatively be applied automatically based on an appropriate algorithm.
  • the user interface may accept a user command that corresponds to, or identifies, the desired sample population. Allowing a user to manipulate the initial data set preferably further includes allowing the user to identify and/or store modifications and settings used to generate a subset of data.
  • the first and second modifications to the initial data set are preferably substantially different, such that the first and second modified data sets are substantially different.
  • the portion of the initial data set undergoing the first modification may correspond to a first subset of samples from the data collection run, and the portion of the initial data set undergoing the second modification may correspond to a second subset of samples from the data collection run.
  • the first and second modifications may be similar or identical, such that the first and second modified data sets are substantially similar.
  • the first and second modifications may be performed on distinct and mutually exclusive samples or subsets of samples, such that the first and second modifications do not compound on the data.
  • the first and second modifications may be performed on samples or subsets of samples that partially or wholly overlap, such that at least some of the samples or subsets of samples are subject to both the first and second modifications, and the first and second modifications compound on the overlapping samples or subsets of samples.
  • third, fourth, or additional modifications may similarly be performed, which may depend on the number of suitable samples or subsets of samples, and the number of different tests to perform on the samples. The number of modifications, as well as the characteristics of how none, some, or all of the modifications overlap and compound, preferably depends on the specific application of the method 100.
  • the user interface may be virtual and/or physical with adjustable virtual and/or physical instrument settings.
  • the modifications may be controlled with knobs, sliders, or other controls shown on a display, while in a physical interface the modifications may be controlled with knobs, sliders, or other controls in a physical unit.
  • the virtual and/or physical controls allow the initial data set to be modified in a step-wise, sequential fashion.
  • the user interface may additionally and/or alternatively allow the initial data set to be repeatedly or iteratively modified.
  • the user interface preferably allows each modification of the raw initial data set and the setting used to generate the modified data set to be individually stored, recorded, and/or identified.
  • the method 100 may further include displaying at least one of the initial data set, the first modified data set, and the second modified data set on a user interface S170.
  • the user interface utilizes one or more graphical, menu-driven formats that can accept and display data sets.
  • the user interface additionally and/or alternatively utilizes a numerical display format, although representation of the initial and modified data sets may use any suitable format.
  • the user interface preferably permits the application of scaling and/or culling factors to the initial data set to modify its display representation.
  • the user interface may simultaneously present modified and raw representations of one data set, or of multiple data sets that can be simultaneously viewed, compared, and analyzed.
  • the user interface preferably allows the user to undo or otherwise alter the modifications of one or more of the data sets using the menu- driven options.
  • separate graphs are generated from the raw initial and modified data and are displayed in separate frames, which preferably represents a preview of the stored and/or exported version of the viewed data.
  • the raw initial and modified data are superimposed on one another in the same graph frame, with each data set preferably distinguished by color and/or shading.
  • the consequences of each modification applied to the raw initial data in the generation of the modified data set are represented in separate, independent planes of the same graph frame, and all modifications can be superimposed. Further variations of the displaying at least one of the initial data set and modified data sets may be similar to those described in U.S. Patent Application 2008/0228444, entitled "User interface for a flow cytometer system", or include any suitable displaying steps.
  • Permitting analysis of the first modified data set under a first test S140 and permitting analysis of the second modified data set under a second test S160 function to enable sample analysis from different samples or subsets of samples in the single data collection run.
  • the method allows a user to perform a variety of tests on samples from one or more patients, using an initial data set gathered from a single data collection run of the samples, without adjustment or calibration of gain level settings and other settings between the samples.
  • the first and second tests may be different, such as counting different types of cellular subpopulations and/or analyzing different kinds of tissue samples.
  • the first and second tests may be identical, such counting the same type of cellular subpopulation for samples from two different patients or counting the same kind of marker in different kinds of samples.
  • permitting analysis of the first modified data set S140 may be performed after allowing the first modification of at least a portion of the initial data set S130 and before allowing the second modification of at least a portion of the initial data set S150.
  • permitting analysis of the first and second modified data sets S140 and S160 may be performed after both allowing the first and second modifications of at least a portion of the initial data set S130 and S150.
  • Additional analyses of any of the modified data sets may include analyses under third, fourth, or more tests.
  • the number of analyses (or number of tests) may be equal to the number of modified data sets, or may be less than or greater than the number of modified data sets. In other words, in a first variation each modified data set may undergo analysis under a respective test; in a second variation one or more of the modified data sets may not undergo analysis; and in a third variation one or more of the modified data sets may undergo analyses under multiple tests.
  • the method 100 may further include storing at least one of the first modified data set and the second modified data set S180, which functions to permit the user to save the settings used to generate the modified data sets, thereby allowing the user to save the modified data sets and/or the corresponding modification parameters).
  • Storing at least one of the modified data sets S180 may include allowing the user to review one or more of the modifications of a modified data set, such as to verify the desired modifications prior to storing.
  • the storing step may save settings used to generate the data and other pertinent information regarding the sample (e.g. identification of the flow cytometer providing the initial data, sample identification).
  • one or more of the modified data sets may be stored to an electronic storage unit such as a computer hard drive (e.g. desktop, laptop, tablet, or mobile device), compact storage (e.g. USB drive) and/or server (e.g. local or network) in a "local" location or “remote” location.
  • the modified data set(s) may be stored to the same unit as the initial data set, or to a different unit than the initial data set.
  • the method may further include exporting the initial data set and/or at least one of the modified data sets S190, which functions to permit the user to transfer one or more of the initial or modified data sets to a different medium, such as a printout or an electronic file.
  • the exported data may be transferred to any suitable medium for subsequent viewing, analysis, and/or storage, and the settings used to generate the data and other pertinent information regarding the sample (e.g. identification of the flow cytometer providing the initial data, sample identification) may also be included for export.
  • the data is preferably saved in an fcs3.x (or greater) format, but may be saved in any suitable format.
  • the steps of storing and exporting at least one of the initial data set and modified data sets may be similar to those described in U.S. Patent Application 2008/0228444, entitled "User interface for a flow cytometer system". Additionally, the method may include displaying, storing, and/or exporting at least one of the analyses of any of the modified data sets, which are preferably similar to the variations of displaying, storing, and/or exporting at least one of the data sets.
  • variations of the method include any suitable permutation and combination of the above described steps, including collecting input signals, modifying the initial data set to create one or more modified data sets, permitting analysis of the one or more modified data sets, and any of the storing, displaying, and exporting steps.
  • storing and/or exporting the modified data sets may be performed before or after analysis of the modified data sets.

Abstract

La présente invention concerne un procédé de collecte et d'analyse de données provenant d'un cytomètre de flux. Le procédé consiste à collecter une plage dynamique complète de signaux d'entrée provenant d'au moins un échantillon de cytomètre de flux ; à mémoriser un jeu initial de données de ladite plage de signaux d'entrée ; à autoriser une première modification d'au moins une partie du jeu initial de données afin de créer un premier jeu modifié de données ; à permettre l'analyse du premier jeu modifié de données lors d'un premier test ; à autoriser une seconde modification d'au moins une partie du jeu initial de données afin de créer un second jeu modifié de données ; et à permettre l'analyse du second jeu modifié de données lors d'un second test. Le procédé peut en outre consister à afficher, enregistrer et/ou exporter le jeu initial de données et/ou le premier jeu modifié de données et/ou le second jeu modifié de données.
PCT/US2011/049598 2010-08-30 2011-08-29 Procédé de collecte et d'analyse de données provenant d'un cytomètre de flux WO2012030740A2 (fr)

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Cited By (3)

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US8229684B2 (en) 2006-12-22 2012-07-24 Accuri Cytometers, Inc. Detection system and user interface for a flow cytometer system
US8229707B2 (en) 2005-08-22 2012-07-24 Accuri Cytometers, Inc. User interface for a flow cytometer system
US11320359B2 (en) * 2015-12-18 2022-05-03 Abbott Laboratories Flow cytometer for detecting types, volume, and concentration of cells

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US8779387B2 (en) 2010-02-23 2014-07-15 Accuri Cytometers, Inc. Method and system for detecting fluorochromes in a flow cytometer
WO2011159708A1 (fr) 2010-06-14 2011-12-22 Accuri Cytometers, Inc. Système et procédé de création d'un réseau de cytomètres de flux

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US20030148379A1 (en) * 2002-02-06 2003-08-07 Roitman Daniel B. Methods for making microbar encoders for bioprobes
US20080215297A1 (en) * 2006-12-22 2008-09-04 Clement James Goebel Detection system and user interface for a flow cytometer system
US20080228444A1 (en) * 2005-08-22 2008-09-18 David Olson User interface for a flow cytometer system

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US20030148379A1 (en) * 2002-02-06 2003-08-07 Roitman Daniel B. Methods for making microbar encoders for bioprobes
US20080228444A1 (en) * 2005-08-22 2008-09-18 David Olson User interface for a flow cytometer system
US20080215297A1 (en) * 2006-12-22 2008-09-04 Clement James Goebel Detection system and user interface for a flow cytometer system

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8229707B2 (en) 2005-08-22 2012-07-24 Accuri Cytometers, Inc. User interface for a flow cytometer system
US8229684B2 (en) 2006-12-22 2012-07-24 Accuri Cytometers, Inc. Detection system and user interface for a flow cytometer system
US11320359B2 (en) * 2015-12-18 2022-05-03 Abbott Laboratories Flow cytometer for detecting types, volume, and concentration of cells

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