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Background

Assay Calibration, Standardization, Proficiency Testing and Validation

 

Promoting "Best Practice Criteria Testing"

 

An assay that is calibrated and standardized helps to provide the user with the guarantee that the assay is working correctly and that the results can be trusted. 

 

All reagents used for an assay should be quality controlled, but that does not mean the assay is standardized. Assays can only be standardized if external standards and controls are be included. This, in turn, depended on the assay signal detection readout. In many instances external standards may not be available. Lack of standards and controls makes it virtually impossible to validate an assay.

 

Adenosine triphosphate (ATP) is a well established biochemical marker of the cell's energy source. The intracellular concentration changes of ATP observed as a result of cell proliferation or inhibition, cell number and metabolic integrity as a indicator of metabolic viability, have been known for many years.

 

 

 

The ATP standard curve; the basis of all HemoGenix ATP bioluminescence assays

 

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Calibration, Standardization and Validation of ATP Bioluminescence Assays

All HemoGenix® ATP bioluminescence assays (LIVEGlo™, STEMpredict™, HALO®, ImmunoGlo™MSCGlo™, STEMGlo™) include standards and controls. These provide the ability to:

  • Calibrate of the instrument.
  • Standardize the assay by interpolating non-standardized Relative Luminescence Units or RLU as the output of the luminescence plate reader, into standardized ATP concentrations (see diagram).
  • Ensure the reagents function as intended.
  • Controls pipetting error.
  • Ensures the assay is working correctly prior to measuring samples.
  • Directly compare results from samples over time without normalization.
  • Validate the assay according to regulatory guidelines.
  • Perform an internal proficient test prior to measuring samples.

 

Typical assay parameters and validation characteristics

Assay Characteristics

  • ATP Standard Curve Slope = 0.937 (range: 0.8-1.1)
  • Low ATP Control Range: 0.043µM - 0.057µM
  • High ATP Control Range: 0.595µM - 0.805µM
  • Extra High ATP COntrol Range: 1.488µM - 2.013µM
  • Lowest ATP value indicating unsuitable cell proliferation: =<0.04µM
  • ATP value below which cells are not metabolically viable: = < 0.01µM
  • Assay linearity: >4 logs
  • Assay cell sensitivity: 20-25 cells/well

 

Assay Validation Parameters

  • Accuracy: >95%
  • Sensitivity/Specificity (ROC curves): >0.75
  • Precision (reliability & reproducibility): =< 15%
  • Robustness: >95%
  • Z Factor for high throughput capability: >0.76

 

Proficiency Testing

A proficiency test is a test to evaluate the adequancy of testing methods and equiment and the competency of personnel performing the test. Essentially, it is a test that allows one laboratory to compare results against another laboratory. In simple terms, it is a method used to validate a particular measurement process.

 

The Assay Characteristics provided above show that specific assay measurements must be met in order to continue to process and measure samples. Every day an assay is performed, it provides the information that the instrument, reagents and the competency of the user are within the necessary ranges to perform the assay. Every ATP bioluminescence assay produced by HemoGenix® includes the reagents that in turn allow implementation of a proficiency test every time the assay is used.