Perls' Prussian Blue Intensity Quantification

Hi,

We have a project where the goal is to quantify Perls’ Prussian Blue Staining.

We have set a pipeline in QuPath to:

  • set the stain vectors for color deconvolution;
  • apply intensity based-thresholding to segment Perls’ staining;
  • extract the area of Perls’ staining.

For now, we were extracting the Perls’ stained area as an output for the quantification, but we were wondering if we could also quantify the staining intensity.

I read that "*

Stain intensity quantification is meaningful only when dye uptake is stoichiometric (e.g. Feulgen reaction for DNA)

*".

Landini, G., et al. (2020). “Colour Deconvolution – stain unmixing in histological imaging.” Bioinformatics.

Looking at the reaction formula for the detection of ferric iron in the Perls’ Prussian blue method, I think we could perform intensity quantifications.

Is it correct to perform intensity quantifications in Perls’ stained tissues?

If we could quantify the intensity of Perls’ staining, and taking into account the information available about Colour Deconvolution in Fiji should we use the 32-bit Absorbance output?

Thanks in advance,
Daniela

That a chemical reaction is stoichiometric does not necessarily mean that the concentration of the reaction product can be determined from its light attenuation or absorption.

Strictly speaking, the Beer-Lambert law and the associated linear relationship between absorbance and concentration only apply to low concentration staining solutions.

However, the reaction product of the Perls Prussian Blue staining is a pigment.
This calls into question the fulfillment of the Beer-Lambert relationship.

Moreover, the Beer-Lambert law applies only to monochromatic conditions.
In many cases, this condition is not fulfilled in the measuring and recording system.
(See section Validity)

I would be very skeptical whether the concentration of a Pearl’s Purssuan Blue stain is quantifiable.

See also this

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Thank you so much for the feedback @phaub

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