Histologic processing artifacts and inter-pathologist variation in measurement of inked margins of canine mast cell tumors

Abstract

Although quantitative assessment of margins is recommended for describing excision of cutaneous malignancies, there is poor understanding of limitations associated with this technique. We described and quantified histologic artifacts in inked margins and determined the association between artifacts and variance in histologic tumor-free margin (HTFM) measurements based on a novel grading scheme applied to 50 sections of normal canine skin and 56 radial margins taken from 15 different canine mast cell tumors (MCTs). Three broad categories of artifact were 1) tissue deformation at inked edges, 2) ink-associated artifacts, and 3) sectioning-associated artifacts. The most common artifacts in MCT margins were ink-associated artifacts, specifically ink absent from an edge (mean prevalence: 50%) and inappropriate ink coloring (mean: 45%). The prevalence of other artifacts in MCT skin was 4-50%. In MCT margins, frequency-adjusted kappa statistics found fair or better inter-rater reliability for 9 of 10 artifacts; intra-rater reliability was moderate or better in 9 of 10 artifacts. Digital HTFM measurements by 5 blinded pathologists had a median standard deviation (SD) of 1.9 mm (interquartile range: 0.8-3.6 mm; range: 0-6.2 mm). Intraclass correlation coefficients demonstrated good inter-pathologist reliability in HTFM measurement (κ = 0.81). Spearman rank correlation coefficients found negligible correlation between artifacts and HTFM SDs ( r ≤ 0.3). These data confirm that although histologic artifacts commonly occur in inked margin specimens, artifacts are not meaningfully associated with variation in HTFM measurements. Investigators can use the grading scheme presented herein to identify artifacts associated with tissue processing.

Keywords: Canine; histologic tumor-free margin; mast cell tumors.

Figure 1.

Tissue deformation in histologic sections of normal canine skin. A. Subgross image showing differential contraction of a surgical margin. Two edges of epidermis are positioned in a straight line, with a perpendicular line beginning at the epidermal margin edge. Artifact is considered present when the subjacent tissue deviates ≥+1 cm or ≤–1 cm from the perpendicular line. The arrow indicates skeletal muscle that has deviated ≥1 cm from the perpendicular line. B. Subgross image showing tissue deflection. There is deflected tissue directly apposed to the surgical margin, marked by the arrow.

Figure 2.

Ink-associated artifacts in histologic sections of normal canine skin. A. Ink dissection. Ink dissects between adipose and skeletal muscle, and is continuous with the margin for a width ≥1 complete 100× field. Arrows show dissection of ink that is continuous with the inked circumferential margin (*). B. Ink at an incorrect edge. Ink is on a tissue edge that does not represent a true margin (arrow). There is orange ink on the opposite end of the true margin (*). C. Ink absent from a tissue edge. The margin lacks ink (dotted line) for a length ≥ width of 1 complete 100× field. D. Ink separate from margin edge. Ink is contained within the body of tissue and not directly associated with the tissue edge or other artifacts. E. Inappropriate ink coloring. The margin has both green (black arrow) and black (gray arrow) ink.

Figure 3.

Sectioning artifacts in histologic sections of normal canine skin. A. Tissue folding. There is a slab of folded muscle, the width of which is ≥ the width of one 200× field. B. Tissue defect. There is a cavity within the body of the section (dotted square), leaving an empty space lacking identifiable tissue. The cavity is ≥ width of one 200× field. Asterisks (*) represent the green inked margin. C. Dissociated tissue. A discontinuous fragment of tissue (dense collagen) that has no direct anatomic association to the primary tissue section.

Figure 4.

Mean prevalence of processing artifacts in normal skin and mast cell tumor (MCT) radial sections. Data are expressed by mean ± standard error of the mean.

Figure 5.

Box and whiskers plot of histologic tumor-free margin (HTFM) measurements on 56 individual mast cell tumors by 5 blinded pathologists. The box extends from the 25th to 75th percentiles; the line in the middle is the median for each margin. Whiskers represent the range of HTFM measurements.