WO2012030740A2

WO2012030740A2 - Method for collecting and analyzing data from a flow cytometer

Info

Publication number: WO2012030740A2
Application number: PCT/US2011/049598
Authority: WO
Inventors: Collin A. Rich; Jack T. Ball; Grant C. Howes
Original assignee: Accuri Cytometers Inc
Current assignee: Accuri Cytometers Inc
Priority date: 2010-08-30
Filing date: 2011-08-29
Publication date: 2012-03-08
Anticipated expiration: 2013-02-28
Also published as: WO2012030740A3

Abstract

A method 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; storing an initial data set of the full dynamic range of the input signals; allowing a first modification of at least a portion of the initial data set to create a first modified data set; permitting analysis of the first modified data set under a first test; allowing a second modification of at least a portion of the initial data set to create a second modified data set; and permitting analysis of the second modified data set under a second test. The method may further include displaying, saving, and/or exporting at least one of the initial data set, the first modified data set, and the second modified data set.

Description

METHOD FOR COLLECTING AND ANALYZING DATA

FROM A FLOW CYTOMETER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of US Provisional Application number

61/378,233, filed 30-AUG-2010, which is incorporated in its entirety by this reference.

TECHNICAL FIELD

[0002] 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.

BACKGROUND

[0003] A typical flow cytometer detector has a limited collection range. In quantitative terms, 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. In simple terms, 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.

[0004] As shown in FIGURE l, 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. Observing data signals outside of the pre-set collection range is only possible if (1) the user changes the gain level settings prior to the sample run or (2) the user is able to run an additional test sample that is relatively homogenous to the previous sample(s) and is temporally stable. [0005] The limitations of typical flow cytometer systems have at least four disadvantages: (1) the expenditure of valuable user time spent on the gain level-setting process to ensure it is set correctly; (2) the requirement of having significantly more sample available to determine the proper gain level settings in the trial-and-error process (e.g. more sample may be used in pilot samples for setting the gain level than is actually used in the data collection run), (3) the potential loss of valuable data in the data collection run because the user incorrectly anticipated the actual signal range, resulting in at least a portion of the input signals lying outside the user-set "active" collection range and being lost or not collected, and (4) the inability to observe and "undo" changes in user-set gain level/scaling settings without running additional samples. These limitations are particularly disadvantageous in applications that involve multiple samples, particularly samples that are in limited supply and/or are time sensitive.

[0006] Thus, there is a need in the flow cytometry field to create an improved method for collecting and analyzing data from a flow cytometer in the flow cytometry field This invention provides such an improved method for collecting and analyzing data from a flow cytometer field.

BRIEF DESCRIPTION OF THE FIGURES

[0007] FIGURE 1 is a schematic block diagram of a flow cytometer user interface of the prior art;

[0008] FIGURE 2 is a schematic of the method of a preferred embodiment; and [0009] FIGURES 3A and 3B are example variations of the method of a preferred embodiment.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The following description of preferred embodiments of the invention is not intended to limit the invention to these preferred embodiments, but rather to enable any person skilled in the art to make and use this invention.

[0011] In a preferred embodiment, as shown in FIGURES 2 and 3, the method

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. In some variations, 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. In some variations, 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. For instance, 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). For example, 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. Alternatively, the first and second tests using the first and second modified data sets may be similar or identical. For example, 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.

[0012] In a preferred embodiment, 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. With the method 100, modifications may be adjusted or undone without the need to re-run samples, which saves time, reduces the amount of sample required, and eliminates the potential of lost data due to incorrect gain level settings. In particular, 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). Furthermore, 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.

[0013] 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. For example, 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. In a second variation, 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. In a third variation, the samples are additionally and/or alternatively distinguished from one another by being different fluid types (e.g. a variety of blood, lymph, or other fluids) and/or sourced from different patients or other sample sources. However, the samples may additionally and/or alternatively be distinguished from one another in any suitable manner. Furthermore, 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. Furthermore, 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). However, 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.

[0014] 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). In a preferred embodiment, 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). Although 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. In an alternative embodiment, the collecting step S110 may be performed by a user interface in electronic communication with a composite of several narrower dynamic range flow cytometers.

[0015] 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. In a preferred embodiment, 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.

[0016] 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). In a "local" variation, the initial data set may be stored to an electronic storage unit coupled to the flow cytometer system and/or user interface. In a second "remote" variation, the initial data set may be stored to a server, network, or other electronic storage medium. [0017] Allowing a first modification of at least a portion of the initial data set S130 and allowing a second modification of at least a portion of the initial data set S150 function to permit the user to manipulate the collected data to form first and second modified data sets separate from the initial data set. 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.

[0018] 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. In another example, modifications may include adjusting the bounds of the data. However, modifications may alternatively be applied automatically based on an appropriate algorithm. In this alternative embodiment, 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.

[0019] 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. For instance, 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. Alternatively, 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. Alternatively, 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. Furthermore, 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.

[0020] The user interface may be virtual and/or physical with adjustable virtual and/or physical instrument settings. In a virtual user interface, 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.

[0021] 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. In a preferred embodiment for displaying at least one of the data sets, the user interface utilizes one or more graphical, menu-driven formats that can accept and display data sets. In an alternative embodiment, 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. In one variation, 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. In another variation, 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. In yet another variation, 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.

[0022] 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. In this manner, 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. Alternatively, 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. As shown in FIGURE 3A, in a first variation, 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. As shown in FIGURE 3B, in a second variation, 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.

[0023] 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). Similar to the step of storing the initial data set, 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.

[0024] 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. [0025] Further variations of 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.

[0026] Furthermore, 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. For example, as shown in FIGURES 3A and 3B, storing and/or exporting the modified data sets may be performed before or after analysis of the modified data sets.

[0027] As a person skilled in the art will recognize from the previous detailed description and from the figures and claims, modifications and changes can be made to the preferred embodiments of the invention without departing from the scope of this invention defined in the following claims.

Claims

CLAIMS We Claim:

1. A method for collecting and analyzing data from a flow cytometer, comprising:

• collecting a full dynamic range of input signals from at least one flow cytometer sample;

• storing an initial data set of the full dynamic range of input signals;

• allowing a first modification of least a portion of the initial data set to create a first modified data set;

• permitting analysis of the first modified data set under a first test;

• allowing a second modification of at least a portion of the initial data set to create a second modified data set; and

• permitting analysis of the second modified data set under a second test.

2. The method of Claim l, wherein collecting a full dynamic range of input signals includes collecting an initial data set including a 1:100,000 ratio between the faintest objects and brightest objects.

3. The method of Claim 2, where collecting a full dynamic range of input signals includes collecting an initial data set including a 1:1,000,000 ratio between the faintest objects and brightest objects.

4. The method of Claim 1, wherein collecting the full dynamic range of input signals includes collecting the input signals without accepting a gain amplification level selection from a user.

5. The method of Claim 1, wherein allowing first and second modifications allows first and second modifications on mutually exclusive first and second portions of the data, respectively.

6. The method of Claim 1, wherein at least one of allowing a first modification of and allowing a second modification of at least a portion of the initial data set includes allowing a user to manipulate the initial data set across the full dynamic range of input signals from the flow cytometer and to create the modified data set.

7. The method of Claim 6, wherein allowing a user to manipulate the initial data set includes allowing the user to identify the modifications made to the initial data set.

8. The method of Claim 7, wherein allowing a user to manipulate the initial data set includes allowing the user to store settings used to generate a desired subset of data in the initial data set.

9. The method of Claim 8, wherein allowing a user to manipulate the initial data set includes allowing the user to generate at least one subset of data that corresponds to one or more sample populations contained within the initial data set.

10. The method of Claim 6, wherein allowing a user to manipulate the initial data set includes allowing the user to perform real-time comparisons between the initial data set and at least one of the first and second modified data sets for the flow cytometer sample.

11. The method of Claim 6, wherein allowing a user to manipulate the initial data set includes allowing the user to adjust or undo modifications on the initial data set, to make multiple adjustments on the same initial data set.

12. The method of Claim 6, wherein allowing a user to manipulate the initial data set includes providing adjustable virtual instrument settings.

13. The method of Claim 6, wherein allowing a user to manipulate the initial data set includes applying hierarchical scaling factors to independent data channels.

14. The method of Claim 1, wherein allowing a second modification to the initial data set includes allowing a second modification to the initial data set that is substantially different from the first modification to the initial data set, such that the first and second modified data sets are substantially different.

15. The method of Claim 6, wherein allowing a user to manipulate the initial data set includes allowing the user to cull data from the initial data set.

16. The method of Claim 15, wherein allowing a user to cull data from the initial data set includes allowing the user to adjust the bounds of the data.

17. The method of Claim 1, wherein permitting analysis of the first modified data set under a first test is performed before the step of allowing a second modification of at least a portion of the initial data set.

18. The method of Claim 1, wherein permitting analysis of the first modified data set under a first test is performed after the step of allowing a second modification of at least a portion of the initial data set.

19. The method of Claim 1, wherein permitting analysis of the second modified data set includes performing a second test on the second modified data set that is substantially different from the first test.

20. The method of Claim 1, further comprising displaying at least one of the initial data set, the first modified data set, and the second modified data set on a user interface.

21. The method of Claim 20, wherein displaying on a user interface includes permitting a user to observe the initial data set from the full dynamic range of input signals and permitting the user to identify appropriate modifications for the initial data set.

22. The method of Claim 20, wherein displaying on a user interface includes utilizing a graphical, menu-driven format configured to accept and display data sets.

23. The method of Claim 1, further comprising storing at least one of the first and second modified data sets.

24. The method of Claim 23, wherein storing at least one of the first and second modified data sets includes storing corresponding modification settings.

25. The method of Claim 1, further comprising storing the analysis of at least one of the first and second modified data sets.

26. The method of Claim 1, further comprising at least one of:

• exporting at least one of the first and second modified data sets from the flow cytometer system to a different medium; and

• exporting the analysis of at least one of the first and second modified data sets from the flow cytometer system to a different medium.

27. A method for collecting and analyzing data from a flow cytometer, comprising:

• storing an initial data set of the full dynamic range of input signals from the flow cytometer sample;

• displaying the initial data set on a user interface;

• allowing a first modification of least a portion of the initial data set to manipulate the initial data set across the range of input signals from the flow cytometer sample, thereby creating a first modified data set;

• allowing a second modification of at least a portion of the initial data set to manipulate the initial data set across the range of input signals from the flow cytometer sample, thereby creating a second modified data set, wherein the second modification is substantially different from the first modification; and

• saving the analysis of least one of the first and second modified data sets;

wherein collecting a full dynamic range of input signals includes collecting the full dynamic range of input signals to an initial data set that includes a 1:100,000 ratio between the faintest objects and brightest objects.

28. The method of Claim 27, further comprising storing and exporting at least one of the first and second modified data sets.

29. The method of Claim 27, further comprising permitting analysis of the first modified data set under a first test, and permitting analysis of the second modified data set under a second test.

30. The method of Claim 29, wherein permitting analysis of the second modified data set under a second test includes permitting analysis of the second modified data set under a second test substantially different from the first test.