Assay Development Topics
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 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:
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:
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% |
Assessment
Acceptance Criteria
Assessment
Acceptance Criteria
Assessment
Acceptance Criteria
Assessment
Acceptance Criteria
Assessment
Acceptance Criteria
Assessment
Acceptance Criteria
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.
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.
Some of the routinely performed Flow cytometry assay development and validation services at FlowMetric per client needs are listed below.
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):
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.
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.
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.
Headings | Validation Report | Analytical Report |
---|---|---|
Aim of Study | Description of assay and the intended use of data (exploratory versus decision-making) |
|
Assay Procedure |
|
|
Acceptance Criteria |
Short description | Short description of the acceptance criteria predicted in the validation report |
Validation Parameters |
|
N/A |
Samples |
Description of how the samples should be taken incl. matrix and storage |
|
Data Processing and Storage |
N/A |
|
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 |
|
|
Validation Report
Analytical Report
Validation Report
Analytical Report
Validation Report
Analytical Report
Validation Report
Analytical Report
Validation Report
Analytical Report
Validation Report
Analytical Report
Validation Report
Analytical Report
Validation Report
Analytical Report
Validation Report
Analytical Report
Validation Report
Analytical Report
Validation Report
Analytical Report
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 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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