Assessment of Consistency in Assignment of Severe (Grade 3) Squamous Metaplasia to Human Bulbar Conjunctiva Impression Cytology Cell Samples

Nucleus-to-cytoplasm (N:C) characteristics of published images of human bulbar conjunctival cells designated as showing severe (grade 3) squamous metaplasia were retrospectively assessed. From publications over a 40-year period, measurements were made from images of cell and nucleus size (based on areas and dimensions) and four different calculations were made for nucleo-cytoplasmic (N:C) ratios. From 54 published images, the mean nucleus-to-cytoplasm area ratio (NU/CYT AREA ratio) was 0.145 +/- 0.077 (range 0.052 to 0.346), compared to two different reference set values of 0.069 +/- 0.017 and 0.080 +/- 0.021. Similarly, a nucleus-to-cytoplasm length ratio (as LNLONG) was 0.308 +/- 0.080 (range 0.191 to 0.475) compared to reference values of 0.226 +/- 0.032 and 0.236 +/- 0.034. Similar differences in reference values were obtained using two other N:C ratio calculations. A wide range of values was found for morphometric N:C indices from published images, especially those without a scale bar, indicating a high incidence of inconsistent grading assignments. Overall, only about 30% of the published images showed morphological features consistent with severe squamous metaplasia (i.e., with the rest neither enlarged nor showing substantially changed N:C ratios) with no substantial pictorial evidence indicating that cells from the human bulbar conjunctiva have pyknotic nuclei. Current evidence indicates that grade 3 squamous metaplasia cells should be substantially enlarged.