High throughput flow cytometry system and method

Abstract

The invention provides systems, compositions, kits and methods for automated processing of biological samples and analysis using a flow cytometer.

1 .- 58 . (canceled) 
     
     
         59 . A method for automated or semi-automated processing of samples and multiparametric analysis of the processed samples in a cytometer, comprising:
 a. providing a system for processing samples to produce processed samples prior to analyzing the processed samples in the cytometer, comprising:
 1. at least one microplate and microplate holder; 
 2. a first reagent container containing a first detectable binding element that is specific for an activation state of a first activatable element, a second reagent container containing a second detectable binding element that is specific for a first cell surface marker, wherein the first and second detectable binding elements are differentially detectable by the cytometer, a third and fourth reagent containers containing a first and second modulator, respectively, wherein the detectable binding elements and the modulators are in liquid solution or suspension in their respective reagent containers; 
 3. a dispensing head configured to withdraw liquid from one of said reagent or sample containers and dispense it to a well in said microplate, and/or to withdraw liquid from a first well in the microplate and dispense it to a second well in the same or different microplate; 
 4. a microplate handling apparatus for moving said microplate, wherein:
 i. said microplate handling apparatus is configured to position said microplate near said dispensing head so that said dispensing head can dispense liquid from a reagent container into a well in said microplate and/or withdraw liquid from a well in said microplate during the production of a processed sample or to position said microplate so that it can be manually moved to be near said dispensing head; and 
 ii. said microplate handling apparatus is configured to either relocate said microplate to said cytometer for transfer of the processed sample to, and analysis of the processed sample by, the cytometer, or to position said microplate so that it may be relocated manually to said cytometer for transfer of the processed sample to, and analysis of the processed sample by the cytometer; 
 
   b. providing a system for analysis and management of the processed samples in step a, comprising:
 1. said cytometer, wherein the cytometer is configured to differentially detect a plurality of differentially detectable binding elements bound to activatable elements and cell surface markers in single cells present in said samples; and 
 2. a computer system including
 i. a processor, 
 ii. a laboratory information management system (LIMS) configured to track and record an experiment, inventory management and experimental design and to automate sample tracking from wherever the sample is initially input into the automation system through to data analysis and completion, and 
 iii. a memory unit configured to control, and operably connected to, said microplate holder, said tracking mechanism, said microplate handling apparatus, said dispensing head, and said cytometer, wherein the memory unit and the LIMS are configured to communicate with the cytometer by instrument control and image processing application software to inform the cytometer as to plate layout, including the presence or absence of the first or second differentially detectable binding elements, or combination thereof, for each well in the plate, wherein there are potentially at least  6  differentially detectable binding elements and each well of the plate may contain one or more different binding elements compared to other wells, as well as the presence or absence of one or more modulators, with corresponding modulation time, so as to direct proper analysis of each of the samples in each well of the plate by the cytometer, in a manner compatible with the cytometer software, wherein the computer system is configured to consolidate elements of data or metadata to a single file; 
 
   c. withdrawing and dispensing liquids from said reagent containers into one or more wells of the at least one microplate to perform a protocol, wherein said protocol comprises:
 1. preparing a master modulator plate in an automated process wherein the first modulator is removed from the third reagent container and dispensed into a first well in the microplate and the second modulator is removed from the fourth reagent container and dispensed into a second well in the microplate; 
 2. providing a second microplate comprising a first well and a second well, each of which contains a biological sample comprising cells, and dispensing the first modulator from the first well in the first microplate to the first well in the second microplate and the second modulator from the second well in the first microplate to the second well in the second microplate, so that the modulators and the cells are in contact, wherein said dispensing may be manual or automated or a combination of manual and automated; and 
 3. determining an activation level of at least said first activatable element in single cells in said one or more samples, wherein said first activatable element is an element in a pathway that is modulated by the first modulator or the second modulator, by said cytometer, wherein said activation level is determined by a process comprising: contacting said at least one biological sample with the first detectable binding element which is specific to an activation state of said first activatable element, wherein said contacting comprises: withdrawing said first detectable binding element from said first reagent container and dispensing said first detectable binding element to the well in the microplate containing said sample such that said first detectable binding element contacts the cells in said sample and binds to the first activatable element, if present, and detecting said bound first detectable binding element in said cells on a single cell basis in the cytometer; and 
 4. determining a level of at least said first cell surface marker in said one or more samples on a single cell basis by said cytometer, wherein said first cell surface marker level is determined by a process comprising: contacting said at least one biological sample with the second detectable binding element which is specific to the first cell surface marker, wherein said contacting comprises: withdrawing said second detectable binding element from said second reagent container and dispensing said second detectable binding element to the well in the microplate containing said sample such that said second detectable binding element contacts the cells in said sample and binds to said first cell surface marker, if present, and detecting said bound second detectable binding element in said cells on a single cell basis in the cytometer. 
   
     
     
         60 . The method of  claim 59  wherein the automated process of c.1 comprises
 (a) initiating a liquid handler program using automated software; 
 (b) system requesting an appropriate stock modulator to be loaded into automated system; and 
 (c) automated system automatically performs modulator dilutions to create working solutions for the first modulator in the third reagent container and the second modulator in the fourth reagent container, according to liquid handler program. 
 
     
     
         61 . The method of  claim 59  wherein said samples comprise intact viable cells and wherein said pre-processing comprises contacting said intact viable cells with the modulator for a predetermined time, and wherein the contacting of step c.2 further comprises contacting the cells and the modulator in such a way that the sample remains viable during the contacting for the predetermined time. 
     
     
         62 . The method of  claim 61  wherein the computer system is configured to control the contacting of the cells with the modulator for the predetermined time. 
     
     
         63 . The method of  claim 62  wherein the computer system controls the contacting of the cells with modulator for the predetermined time by controlling a process comprising fixing of the sample after treatment with the modulator. 
     
     
         64 . The method of  claim 63  wherein said process comprising fixing of the sample after treatment with the modulator comprises withdrawing the fixative from a reagent container or containers and dispensing the fixative to the wells of the at least one microplate containing the sample, or withdrawing said sample from a sample container and dispensing the sample to wells of the at least one microplate containing the fixative, such that the fixative contacts the cells of the sample. 
     
     
         65 . The method of  claim 64  wherein the computer system controls the contacting of the cells with the fixative for a predetermined time. 
     
     
         66 . The method of  claim 59  wherein the system further comprises at least one of a fifth reagent container containing a third detectable binding element specific to an activation state of a second activatable element, a sixth reagent container containing a fourth detectable binding element specific to an activation state of a third activatable element, or a seventh reagent container containing a fifth detectable binding element specific to an activation state of a fourth activatable element, wherein the third, fourth, and fifth detectable binding elements are differentially detectable from each other and from the first and second binding elements, and wherein each detectable binding element is contacted with cells in sample in at least one well of the at least one microplate and detected by the cytometer. 
     
     
         67 . The method of  claim 66  wherein the system comprises at least two of the fifth, sixth, and seventh reagent containers and their respective detectable binding elements. 
     
     
         68 . The method of  claim 67  wherein the system further comprises a reagent container containing a third modulator and wherein, in step c.1, the third modulator is removed from its reagent container and dispensed into a third well of the master modulator plate. 
     
     
         69 . The method of  claim 68  wherein the system further comprises a reagent container containing a fourth modulator and wherein, in step c.1, the fourth modulator is removed from its reagent container and dispensed into a fourth well of the master modulator plate. 
     
     
         70 . The method of  claim 69  wherein the system further comprises a reagent container containing a fifth modulator and wherein, in step c.1, the fifth modulator is removed from its reagent container and dispensed into a fifth well of the master modulator plate. 
     
     
         71 . The method of  claim 70  wherein the first, second, third, fourth, and fifth modulators are different from each other and the first, second, third, fourth, and fifth wells are different from each other. 
     
     
         72 . The method of  claim 59  wherein the cytometer is a flow cytometer. 
     
     
         73 . The method of  claim 59  wherein the cytometer is a mass cytometer. 
     
     
         74 . The method of  claim 59  wherein the single file is not duplicated. 
     
     
         75 . A method for automated or semi-automated processing of samples and analysis of the processed samples by a cytometer, comprising:
 a. providing a system for processing said samples prior to their analysis in a cytometer, comprising:
 1. at least one or more microplates and microplate holder; 
 2. a first reagent container containing a first detectable binding element that is specific for an activation state of a first activatable element, a second reagent container containing a second detectable binding element that is specific for a first cell surface marker, wherein the first and second detectable binding elements are differentially detectable, and either a third reagent container containing a first modulator or a sample container containing a biological sample comprising cells, or both, wherein the modulator, the detectable binding elements, and the biological sample comprising cells are in liquid solution or suspension in their respective reagent containers; 
 3. a dispensing head configured to withdraw liquid from one of said reagent or sample containers and dispense it to a well in said microplate and/or to withdraw liquid from a first well in the microplate and dispense it to a second well in the same or different microplate; 
 4. a microplate handling apparatus for moving said microplate, wherein;
 i. said microplate handling apparatus is configured to position said microplate near said dispensing head so that said dispensing head can dispense liquid from a reagent container into a well in said microplate and/or withdraw liquid from a well in said microplate during the production of a processed sample or to position said microplate so that it can be manually moved to be near said dispensing head; and 
 ii. said microplate handling apparatus is configured to either relocate said microplate to said cytometer for transfer of the processed sample to, and analysis of the processed sample by, the cytometer, or to position said microplate so that it may be relocated manually to said cytometer for transfer of the processed sample to, and analysis of the processed sample by the cytometer; 
 
   b. providing a system for, analysis and management of the processed samples in step a, comprising:
 1. said cytometer, wherein the cytometer is configured to differentially detect a plurality of differentially detectable binding elements bound to activatable elements and cell surface markers in single cells present in said samples; 
 2. a computer system, wherein said computer system comprises
 i. a processor, 
 ii. a LIMS configured to track and record an experiment design and to automate sample tracking from wherever the sample is initially input into said automation process and analysis through to data analysis and completion, and 
 iii. a memory unit configured to control, and operably connected to, said microplate holder, said barcode reader, said microplate handling apparatus, the timing of said microplate handling steps, said dispensing head, and said cytometer, wherein the memory unit and the LIMS are configured to communicate with the cytometer by instrument control and image processing application software to inform the cytometer as to plate layout, including the presence or absence of the first or second differentially detectable binding elements, or combination thereof, for each well in the plate, wherein there are potentially at least 6 differentially detectable binding elements and each well of the plate may contain one or more different binding elements compared to other wells, as well as the presence or absence of one or more modulators, with corresponding modulation time, so as to direct proper analysis of each of the samples in each well of the plate by the cytometer, in a manner compatible with the cytometer software, wherein the computer system is configured to consolidate elements of data or metadata to a single file; 
 
   c. withdrawing and dispensing liquids from said reagent containers into one or more wells of the at least one microplate to perform a protocol, wherein said protocol comprises:
 1. either:
 i. providing the at least one microplate, one or more of whose wells contains biological sample comprising cells, and dispensing modulator from said third reagent container into the wells so that the modulator and the cells are in contact; or ii. providing the at least one microplate, one or more of whose wells contains a modulator and dispensing at least one biological sample comprising cells from said sample container into the wells so that the modulator and the cells are in contact; or 
 iii. providing the at least one microplate and dispensing at least one biological sample from the sample container into one or more wells of the microplate and dispensing modulator from the third reagent container into one or more of the wells of the microplate, so that the modulator and the cells are in contact; and 
 
 2. contacting said samples with a fixative, wherein said contacting is performed by automation at a time determined by the LIMs, and a permeablization reagent; 
 3. determining an activation level of at least said first activatable element in single cells in said one or more samples wherein said first activatable element is an element in a pathway that is modulated by the modulator, by said cytometer, wherein said activation level is determined by a process comprising: contacting said at least one biological sample with the first detectable binding element which is specific to an activation state of said first activatable element, wherein said contacting comprises: withdrawing said first detectable binding element from said first reagent container and dispensing said first detectable binding element to the well in the microplate containing said sample such that said first detectable binding element contacts the cells in said sample and binds to the first activatable element, if present, and detecting said bound first detectable binding element in said cells on a single cell basis in the cytometer; 
 4. determining a level of at least said first cell surface marker in said one or more samples on a single cell basis by said cytometer, wherein said first cell surface marker level is determined by a process comprising: contacting said at least one biological sample with the second detectable binding element which is specific to the first cell surface marker, wherein said contacting comprises: withdrawing said second detectable binding element from said second reagent container and dispensing said second detectable binding element to the well in the microplate containing said sample such that said second detectable binding element contacts the cells in said sample and binds to said first cell surface marker, if present, and detecting said bound second detectable binding element in said cells on a single cell basis in the cytometer; 
   d. wherein the LIMS software is configured to direct experimental design, plate layout, sample tracking, and inventory management and one or more of the following purposes from the group consisting of instrument alignment; correct connections; motor operations; timing for sample handling; and data analysis.   
     
     
         76 . The method of  claim 75 , wherein the steps in the protocol are conducted in islands of automation. 
     
     
         77 . The method of  claim 75 , wherein one or more components of the system are in a thermo regulating component. 
     
     
         78 . The method of  claim 75 , wherein the microplate handling apparatus comprises a robotic arm, and further comprising moving the at least one microplate using the robotic arm. 
     
     
         79 . A method for processing of samples and analysis of the processed samples in a cytometer, comprising:
 (i) preparing a master modulator microplate, wherein the master modulator plate comprises at least a first well or tube containing a first modulator at a first concentration and a second well or tube containing a second modulator at a second concentration, each of which contains one of a plurality of modulators, each modulator being present at a predetermined concentration for that modulator, comprising
 (a) initiating a liquid handler program using automated software; 
 (b) loading stock modulator for each well into automated system according to a system request, wherein the loading may be automated or manual; 
 (c) automatically performing modulator dilutions by the automated system to create working solutions in working solution containers according to liquid handler program; 
 (d) automatically aliquoting working solutions into master modulator plate by automated system, thus preparing a master modulator plate according to liquid handler program; 
   (ii) providing a sample microplate, comprising a first well and a second well, each of which contains a biological sample comprising cells, and dispensing the first modulator from the first well in the master modulator microplate to the first well in the sample microplate and the second modulator from the second well in the master modulator microplate to the second well in the sample microplate, so that the modulators and the cells are in contact, wherein said dispensing may be manual or automated or a combination of manual and automated;   (iii) adding fixative to the first well of the sample microplate at a first time after modulator and cells come in contact and to the second well of the sample microplate at a second time after modulator and cells come in contact; and   (iv) analyzing single cells from the first well in a cytometer for the presence of a first activatable element and analyzing cells from the second well in the cytometer for the presence of a second activatable element.