Colon biopsies for evaluation of acute graft-versus-host disease (A-GVHD) in allogeneic bone marrow transplant patients

The colonic biopsies from 92 patients were evaluated retrospectively using archival material and were divided into three categories (group A- 44, group B- 27, and group C- 21). Group A included BMT cases with biopsy proven cutaneous A-GVHD. All cases in group A were allogeneic BMT and none received peripheral blood stem cell or cord blood transplantation. They received the same conditioning regimen (cyclophosphamide [Cytoxan], cytosine arabinoside, and total body irradiation). The type of A-GVHD prophylaxis used was post-transplatation cyclosporin A. Group B and C included negative controls.

Polyethylene glycol solution (GoLytely^®, Braintree Laboratories, Braintree, MA) was used for endoscopic preparation in all the groups. Due to known subtle differences between left and right side of the colon, the proportion of different sites in each group was matched for statistical correction. All colon biopsies were fixed in Bouin's fixative and processed routinely. Multiple levels of paraffin-embedded tissue sections stained with H&E were examined in blinded fashion without knowing the category.

Various histological features important for the differential diagnosis of A-GVHD were selected after review of literature [12‐14]. All H&E stained sections were evaluated qualitatively or semi-quantitatively for histomorphological features shown in table 2.

The mucosal thicknesses (Table 2, A) from the surface epithelium to the internal limit of muscularis mucosa in three places were measured in the area of least thickness and mean was calculated. Thickness > 500 microns was considered within normal limits [20]. Cryptitis and crypt abscess were defined respectively as infiltration of crypt epithelium with neutrophils and presence of neutrophils in crypt lumen (Table 2, F). Five or fewer intraepithelial lymphocytes per 100 epithelial cells in surface and crypt lining were considered to be within normal limits (Table 2, G and H) [21]. We classified 1–5 extravascular neutrophils per 20 inflammatory cells in the lamina propria (LP) as mild increase and more than five as marked increase (Table 2, L). Macrophages with mucin as faintly basophilic foamy cytoplasm in H&E stained sections were identified as muciphages. Five or more muciphages in LP per 10 crypts were considered significant (Table 2, P). Presence of five or more endothelial lined blood spaces larger than 50 microns for every 10 crypts was regarded as increased microvessel network (Table 2, Q). Apoptosis (defined as single cell death) was indentified in H&E stained sections as apoptotic cells with scattered karyorrhectic basophilic globular intracytoplasmic debris of apoptotic bodies (Figure 1). Apoptotic cells were counted in 10 high power field (HPF) at 40 × magnification with a field diameter of 0.35 mm. Mitotic figures were counted per 10 crypts (Table 3).

Each group was evaluated for the frequency of various histomorphological features shown in table 2. The findings in group A were compared with groups B and C using Fisher's exact test (Table 2). Number of apoptotic cells and mitotic figures in group A were compared with those in groups B and C using Wilcoxan Rank Sum Test (Table 3).

In order to compensate for the multiple testing and thus to avoid imparting spurious significance, we used a stringent significance level of 0.002 for each test. This was based on the Bonferroni correction factor for multiple testing to achieve an overall significance level of 5% [22]. Histomorphological features with statistically significant differences were separated for further analysis (Table 4).