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What is Flow Cytometry Assay Development?

Flow cytometry affords outstanding statistical power to detect rare cell populations and quantify cellular phenotypes in biological samples for pre-clinical and clinical applications. However, flow cytometric methods can be challenging to develop and validate, especially given the complexity of the measurements involved and the lack of standardized cellular reference materials.

Flow cytometry-based assays, when designed appropriately, can provide a wealth of information. The investment in Flow Cytometry Assay Development is critical at an early stage so that correlations can be made between processes and the manufactured products.

The process of flow cytometry assay development encompasses all aspects of the workflow:

  • Evaluation of Sample Types
  • Assessment of Conjugates
    (Clones and Fluorophore Combinations)
  • Sample Processing Techniques
  • Data Acquisition Analysis Strategies

The first step is to any Assay Development initiative is to fully understand the research applications for the assay. This ensures that the sample matrix, flow cytometry panels, and gating strategies can be designed and optimized accordingly, and assure adherence to all regulatory guidelines and principles (Good Laboratory Practice GLP, Good clinical Laboratory Practice, GCLP).

Once an assay has been custom developed per the sponsor’s needs, the validation stage can begin to ensure the assay is robust and reliable.


Flow Cytometry Assay Development typically includes these aspects:

  • Panel Design IconPanel Design
  • Antibody Titration IconAntibody Titration
  • Panel Optimization IconPanel Optimization
  • Determination of Proper Controls IconDetermination of Proper Controls
  • Assessment of Gating Strategy & Reportable Parameters IconAssessment of Gating Strategy & Reportable Parameters
  • Quality Control Specifications IconQuality Control Specifications

What is Flow Cytometry Assay Validation?

Assay validation is performed to demonstrate that the adopted method displays the required performance characteristics over the designated range of conditions of the study. Unlike many other platforms, flow cytometry lacks target standards from regulatory agencies, so validation strategies and approaches differ across laboratories. However, since flow cytometry is considered an analytical method, the FDA requires that clinical flow cytometric studies meet specific analytical method validation criteria to confirm that the process is fit for its intended use:

  • Specificity
  • Stability requirements
  • Linearity
  • Accuracy
  • Detection limits
  • Range
  • Precision
  • Limits of quantitation
  • Robustness
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Pillars of FlowMetric’s Flow Cytometry Assay Development Platform
  • Adoption of Appropriate Controls and Counting Beads (Compensation, FMO, Isotype) Icon
    Adoption of Appropriate Controls and Counting Beads (Compensation, FMO, Isotype)
  • Understanding Lineage and Functional Markers for Efficient Panel Design Icon
    Understanding Lineage and Functional Markers for Efficient Panel Design
  • Assay Optimization & Validation-Fit-For-Purpose Test Scripts Icon
    Assay Optimization & Validation-Fit-For-Purpose Test Scripts
  • Instrument Set-Up, Maintenance, Harmonization Icon
    Instrument Set-Up, Maintenance, Harmonization
  • Data Analysis-Validated Templates, Tech QC Icon
    Data Analysis-Validated Templates, Tech QC

Table 1. Example of Validation Parameters for Clinical Flow Cytometry Panel.

Parameter Assessment Acceptance Criteria
Antibody Titration ≥ 5 dilution of antibody Optimal staining index- optimal and stable separation of positive and negative populations.
Precision Intra-Assay Inter-Analyst ≥ 5 samples
≥ 3 replicates
CV ≤ 20% (30% for low frequency populations)
Day-to-Day Variability ≥ 5 samples collected over 3 days CV ≤ 20% (30% for low frequency populations)
Specificity Isotype Matched Controls Low signal
Antibody Interaction FMO controls The FMO panel provides comparable signals to the full panel when one antibody is removed, while a low signal is measured in the empty channel
Stability ≥ 3 samples (for each sex)
Samples are processed and acquired as possible or samples stained after X hours, acquired after Y hours
Comparison with fresh sample CV ≤ 20%
Parameter

Antibody Titration

Assessment


≥ 5 dilution of antibody

 

Acceptance Criteria


Optimal staining index- optimal and stable separation of positive and negative populations.

 

Precision Intra-Assay Inter-Analyst

Assessment


≥ 5 samples
≥ 3 replicates

 

Acceptance Criteria


CV ≤ 20% (30% for low frequency populations)

 

Day-to-Day Variability

Assessment


≥ 5 samples collected over 3 days

 

Acceptance Criteria


CV ≤ 20% (30% for low frequency populations)

 

Specificity

Assessment


Isotype Matched Controls

 

Acceptance Criteria


Low signal

 

Antibody Interaction

Assessment


FMO controls

 

Acceptance Criteria


The FMO panel provides comparable signals to the full panel when one antibody is removed, while a low signal is measured in the empty channel

 

Stability

Assessment


≥ 3 samples (for each sex)
Samples are processed and acquired as possible or samples stained after X hours, acquired after Y hours

 

Acceptance Criteria


Comparison with fresh sample CV ≤ 20%

 


Why is Assay Development and Validation Important?

The Processes of Assay Development and Validation represent the bedrock of robust clinical assay performance. Initial assay development addresses the clinical significance of the assay endpoints as well as identifies limitations of the assay, including the limits of detection and the interference of sample components, along with an understanding of the natural variation within clinical samples.

Before use in clinical applications or across multiple sites, validation of the assay enables the robustness of the assay to be defined. For clinical assays, the validation should employ test scripts that demonstrate the reproducibility of the assay across screening days, analysts, and instrumentation. Since many clinical assays support high throughput levels of sample analysis, the quality of the assay must be maintained when transferred to an HTS platform. This is defined as the Z’-factor and can be determined using a number of different ways and is typically captured within the validation test scripts.

Cost is also a factor for many clinical assays, and therefore building efficiency into a panel design, reagent requirements, and acquisition and analysis time are all factors that are considered.


How FlowMetric Supports Assay Development

The FlowMetric team works with all types of samples for flow cytometry assay developments and validations. FlowMetric offers in-house sample procurement, preparation, and stimulation per client needs. Some of the samples FlowMetric routinely works on are listed below.

  • Whole blood Icon
    Whole blood
  • PBMC (fresh or cryopreserved) Icon
    PBMC (fresh or cryopreserved)
  • Bone Marrow aspirate Icon
    Bone Marrow aspirate
  • Dissociated Tumors/Tissue Icon
    Dissociated Tumors/Tissue
  • Cells Icon
    Cells

Some of the routinely performed Flow cytometry assay development and validation services at FlowMetric per client needs are listed below.

  • Cell profiling and subsetting Icon
    Cell profiling and subsetting
  • Rare cell detection and quantification Icon
    Rare cell detection and quantification
  • Receptor occupancy (RO) measurement Icon
    Receptor occupancy (RO) measurement
  • Intracellular staining (ICS) Icon
    Intracellular staining (ICS)
  • Cytokine measurement Icon
    Cytokine measurement
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How FlowMetric Supports Assay Validation

Understanding the importance of Flow Cytometry can protect your research investment. By leveraging FlowMetric expert insight, you can avoid pitfalls and reveal new opportunities to maximize your research. The FlowMetric Validation Process follows a three-step approach (Fig.1):

The establishment of the Validation Plan or Protocol  |  The Experimental Phase  |  The generation of the Validation Report The establishment of the Validation Plan or Protocol  |  The Experimental Phase  |  The generation of the Validation Report

Technology Transfer Validation is also conducted routinely at FlowMetric. This process is adopted when an assay is transferred from one laboratory to another in order to ensure that comparable results are achieved in the laboratory where the initial validation was performed (Comparative Method Lab) and at FlowMetric lab (Test Method Lab).

At FlowMetric, assay validation is performed on qualified and maintained instruments in accordance with GLP guidelines.

Role Of Quality In Assay Validation

Once a Flow Cytometry method is validated, the next step is to establish a quality control system for monitoring the assay performance. QC for FC requires the expertise of experienced and technically proficient scientists and technicians who are familiar with QC procedures and documentation as well as the nuances of FC protocols. This level of experience can be found in FlowMetric, where QC and validation are integral to the daily routine of these labs.

Flow cytometry assay validation can be performed in as little as two to four weeks for a simple 4-8 color assay or up to three months for highly complex 18+ multiparameter assays. If you are performing work that requires assay validation, it is a good investment to spend time working with experts in assay validation as you plan your overall process. You may also need input from regulatory experts to make sure you are examining all the proper criteria. Validation involves extensive documentation, so this is something else you’ll need to plan for and consider. In some situations, you may need to carry out inter-assay and inter-laboratory assessments of reproducibility and robustness.

Fig. 1. Flow Cytometry Method Validation Workflow Overview

Validation Workflow Diagram
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Method Validation/ Bioanalytical Report

At the completion of the validation study, FlowMetric will provide a method validation/bioanalytical report. The validation report contains items presented in the below table.

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Table 2. Minimal Requirements for validation and analytical reports.

Headings Validation Report Analytical Report
Aim of Study Description of assay and the intended use of data (exploratory versus decision-making)
  • Description of what is measured and what the intended use of data (exploratory vs. confirmatory) is.
  • Brief conclusion
Assay Procedure
  • Method description (stepwise)
  • Antibody Panel
  • Cytometer
  • Reagents (including donor batch, catalogue number for antibody, batch number)
  • Calibration beads/MESF beads
  • Flow cytometry apparatus/instrument
  • Analytical procedural/method description (stepwise)
  • Critical reagents (incl. batch no. calibration beads, MESF beads and antibodies)
  • Antibody panel
Acceptance Criteria Short description Short description of the acceptance criteria predicted in the validation report
Validation Parameters
  1. Precision
    – Intra-assay precision
    – Inter-assay precision
  2. Biological Variability
    – Intra-subject variability
    – Inter-subject variability (if possible in disease state; depends on intended use of data)
  3. Stability
    – Post-collection stability
    – Post-fixation stability
  4. Sensitivity
    – Blood volume for sample staining
    – Number of events for sample acquisition
N/A
Samples Description of how the samples should be taken incl. matrix and storage
  • Sample receipt (number expected, number received and number analyzed) and storage
  • Sample processing
  • Run overview including acceptance/rejection status
Data Processing and Storage N/A
  • Raw data acquisition and storage
  • Analysis of raw data
Gating and Analysis Procedures N/A Incl. gating strategy and reportable
Results Inclusion of summary tables and if acceptance criteria were met Incl. summary tables and If acceptance criteria were met
Conclusion Inclusion of reportable and how gating should be set Description of assay performance and if data are valid
Deviation Description of deviation and impact on study results Description of deviation and impact on study results
Appendix
  • Raw data tables (incl. exemplary plots/histographs)
  • Gating strategy
  • Analytical SOP
  • Data results tables (incl. exemplary plots/histographs)
  • Calibration beads
  • Analytical SOP
Headings

Aim of Study

Validation Report


Description of assay and the intended use of data (exploratory versus decision-making)

 

Analytical Report


  • Description of what is measured and what the intended use of data (exploratory vs. confirmatory) is.
  • Brief conclusion

 

Assay Procedure

Validation Report


  • Method description (stepwise)
  • Antibody Panel
  • Cytometer
  • Reagents (including donor batch, catalogue number for antibody, batch number)
  • Calibration beads/MESF beads

 

Analytical Report


  • Flow cytometry apparatus/instrument
  • Analytical procedural/method description (stepwise)
  • Critical reagents (incl. batch no. calibration beads, MESF beads and antibodies)
  • Antibody panel

 

Acceptance Criteria

Validation Report


Short description

 

Analytical Report


Short description of the acceptance criteria predicted in the validation report

 

Validation Parameters

Validation Report


  1. Precision
    – Intra-assay precision
    – Inter-assay precision
  2. Biological Variability
    – Intra-subject variability
    – Inter-subject variability (if possible in disease state; depends on intended use of data)
  3. Stability
    – Post-collection stability
    – Post-fixation stability
  4. Sensitivity
    – Blood volume for sample staining
    – Number of events for sample acquisition

 

Analytical Report


N/A

 

Samples

Validation Report


Description of how the samples should be taken incl. matrix and storage

 

Analytical Report


  • Sample receipt (number expected, number received and number analyzed) and storage
  • Sample processing
  • Run overview including acceptance/rejection status

 

Data Processing and Storage

Validation Report


N/A

 

Analytical Report


  • Raw data acquisition and storage
  • Analysis of raw data

 

Gating and Analysis Procedures

Validation Report


N/A

 

Analytical Report


Incl. gating strategy and reportable

 

Results

Validation Report


Inclusion of summary tables and if acceptance criteria were met

 

Analytical Report


Incl. summary tables and If acceptance criteria were met

 

Conclusion

Validation Report


Inclusion of reportable and how gating should be set

 

Analytical Report


Description of assay performance and if data are valid

 

Deviation

Validation Report


Description of deviation and impact on study results

 

Analytical Report


Description of deviation and impact on study results

 

Appendix

Validation Report


  • Raw data tables (incl. exemplary plots/histographs)
  • Gating strategy
  • Analytical SOP

 

Analytical Report


  • Data results tables (incl. exemplary plots/histographs)
  • Calibration beads
  • Analytical SOP

 


Regulatory Requirements for Assay Validation

The type of data and its intended use should be considered when a validation plan is designed. FlowMetric has extensive regulatory expertise in Assay validation. Speak with FlowMetric Regulatory Expert to discuss all of your regulatory needs for assay validation.


The FlowMetric Advantage

The FlowMetric team are industry experts in the development and validation of high-complexity flow cytometry panels. Our team includes scientific and quality management experts who specialize in efficient panel design and the adoption of fit-for-purpose validation test scripts. All performed with a commitment to quality to ensure that every aspect of the method, analytics, and reporting meet the regulatory requirements for your assay’s intended use.

Matrix selection (whole blood, PBMCs, tissue). GLP sample handling/ processing, sample stability assessment

Fit for Purpose High Dimensional Flow Cytometry Method Design, Optimization and Validation for clinical and diagnostic research programs

Quality Management Systems Regulatory Expertise: GLP, GcLP, CLIA, CAP

Cell profiling Advanced Analytics for Exploratory and Clinical EndPoints.

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