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Spot Counting and Analysis – What Is a Real Spot?

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Each spot represents the ‘cytokine signature’ of a single cell. Due to diffusion properties, a true spot has a densely colored center which fades toward the edges; the size and/or color intensity of the spots is determined by the amount of cytokine released.

Due to differences in analyte measured, incubation time, antibody concentration, enzyme activity, substrates and other materials used as well as the functional state of the cytokine-secreting cells, spot size and density can vary greatly. Artifactual spots may appear and can be caused by the aggregation of antibodies or the incomplete removal of cells and cellular debris. Morphologically, these spots can be differentiated from ‘true’ spots by their homogeneity in color intensity and sharper (nonrounded) edges.

Automatic Spot Reading

From the above description, manual spot counting by light microscopy would be classified as a highly subjective process, fraught with a great degree of inter-user variability. Further, when considering the sheer number of wells that may need to quantified in a standard vaccine trials, the task of ELISpot data analysis becomes a far too laborious task for human eyes. The availability of sophisticated ELISpot readers offers a complete solution for precise evaluation of spot data. These instruments include features to overcome problems with variable background intensity and the ability to distinguish true single cell spots from artifacts. The latter capability relies upon the use of minimum and maximum threshold values for spot size and intensity, permitting the exclusion of weak bystander responses and clusters containing multiple cells, respectively. Beyond speed, spot analysis software offers process standardization, a critical component when studies are performed across sites, such as is the case for diagnostic testing and vaccine trials. Moreover, ELISpot readers and analysis software open the door for more precise measurements of spots, permitting the quantitation of secretion of multiple cytokines on a per-cell basis.

Harmonization: Manual and Automated Plate Evaluation

Significant user training and cross validation as part of a harmonization program is warranted to ensure that ELISpot reading is standardized. It may be misleading to think that because automated evaluation is accomplished by machine algorithm the results are without bias. However consider this:
  • An ELISpot reader counts within its capability according to how we program it to pick up spots
  • Variability in programming can still introduce bias
  • Automated ELISpot evaluation is therefore operator-dependent and still requires harmonization. 
  • Harmonization SOPs and response definition tools are available
Watch ELISpot expert Dr. Sylvia Janetzki's (ZellNet Consulting) webinar on strategies for enhancing ELISpot/Fluorospot testing in research and clinical trials