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
Antibody Titration
Panel Optimization
Determination of Proper Controls
Assessment of Gating Strategy & Reportable Parameters
Quality Control Specifications
| 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
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.
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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