Validating a stream cytometry (CF) assay will be difficult. Unlike the soluble analytes which might be detected in different immunoassays, within the case of stream cytometry these are normally mobile targets shaped by compounds with extra complicated traits and, subsequently, a few of the validation parameters could also be completely different. to these established for different analytical strategies.

In this entry we analyze the primary parameters to bear in mind when validating a stream cytometry assay .


Establish the sensitivity of an analytical technique includes know each the detection restrict and the decrease restrict of quantitation of the identical. In the case of stream cytometry, the methods for establishing these limits rely on the format of the assay being carried out.

When the studying is the fluorescence depth, each the utmost fluorescence depth of a constructive inhabitants and the utmost separation between constructive and unfavorable populations will immediately affect the sensitivity of the assay.


In most stream cytometric analyzes, it isn’t doable to find out the accuracy of the assay, since there isn’t any reference customary mobile materials out there for every particular examine.

One of probably the most fascinating benefits of stream cytometry over different analytical strategies is the pliability it presents to detect numerous mobile subgroups and intracellular antigens, and it’s exactly this flexibility that makes it inconceivable to develop reference mobile supplies for a number of trials.


Robustness defines the flexibility of a way to not be altered by small deliberate adjustments in its parameters, offering an thought of ​​its reliability beneath the traditional circumstances by which the check is carried out.

Factors that may be altered to review the robustness of the strategy embody testing antibody for detection of various heaps or performing the assay by completely different investigators.


Stability research ought to mirror the identical circumstances to which samples will likely be subjected throughout testing and evaluation.

The stability of an analyte in a sure matrix should be decided beneath particular circumstances to be able to establish the bounds of stability of the analytes together with the analysis, amongst others, of the time between the gathering of the pattern and the evaluation of the identical, the storage circumstances or freeze-thaw cycles .


To decide the specificity of a stream cytometry assay, it’s needed to guage the components that immediately influence it, comparable to:

  • Phenotype of the inhabitants of curiosity
  • Selection of monoclonal antibodies
  • Antibody and Fluorochrome Panel Design
  • Equipment Configuration

This parameter is important to evaluate within the case of quantitative strategies . In this case, it’s endorsed to guage no less than 5 concentrations that cowl the analytical measurement vary.