Is the Optical Fractionator really assumption free and unbiased?

Questions

Is the Optical Fractionator really assumption free and unbiased?
There are a number of people who claim that the Optical Fractionator is not really assumption free and unbiased. They cite Benes and Lange, TINS 2001. Any comments?

Discussion

The term assumption free has been debated for a number of years. The term is used to indicate that the method does not make any of the following assumptions: size, shape, orientation or distribution of the objects. Of course, many assumptions are made as have been clearly stated in the articles describing these techniques. One assumption is that the researcher is able to understand what is being viewed in the microscope. Another assumption is that the researcher is able to measure the thickness of the sections.

Is stereology unbiased? The answer is yes. The TINS 2001 articles was written by Schmitz and Hof and refers to an article by Benes and Lange. Benes and Lange claim that the user must use very large counting frames. Schmitz and Hof dispute this. Benes and Lange also think that differential compression along the z-axis can bias the results. This is true. The issue is to determine how much of the z is being sampled. The number weighted section thickness addresses this issue. Please see Karl-Anton Dorph-Petersen et al, (Journal of Microscopy, Vol. 204, Pt 3, December 2001).

It appears that there is some confusion between bias and accuracy. An unbiased estimate is one in which the average of all of the estimates is the true value. This is good since more work improves the result. Biases cannot be filtered out of the data. Biases do not cancel out. An accurate estimate is one that is close to the true answer. An accurate estimate might be a biased estimate. If that is the case, it means doing more work makes it impossible to find the true answer.

In summary, the beauty of the Optical Fractionator is that it provides an unbiased estimate of particles regardless of size, shape, orientation, or distribution of the particles being counted.

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