Isolation of exfoliated colonocytes from human stool as a new technique for colonic cytology

Abstract

Cell exfoliation in the gut is an important cell renewal mechanism. To approach its investigation we applied a novel immunomagnetic technique for isolation of exfoliated cells from human stool. Exfoliated colonocytes were isolated from 168 stool samples. The cells were assessed microscopically using conventional stains and immunohistochemistry. The technique allowed us to obtain well-preserved colonocytes displaying characteristic features of well-differentiated colonic epithelium and positive immunostaining for cytokeratin 5/8. No mucin-producing cells were found. Exfoliated cells did not produce inducible nitric oxide synthase, albeit cultured colon carcinoma cells HT-29 analysed in parallel showed strong immunostaining. Analysis of exfoliated cell numbers in consecutive stool samples from the same subjects revealed considerable interindividual variation. Overall exfoliated colonocyte numbers were relatively low, isolation being unaffected by addition during the procedure of excessive amounts of HT-29 cells. Apoptosis was extremely rare among exfoliated colonocytes. Well-preserved exfoliated colonocytes can be consistently isolated from human faeces using a simple procedure. Our findings suggest that the actual process of cell exfoliation in the human colon may be much less intense than is generally accepted. Exfoliated cell isolation from human stool constitutes a convenient non-invasive approach that can be used for diagnostic and research purposes.

Recovery of Exfoliated Cells From the Gastrointestinal Tract of Premature Infants: A New Tool to Perform “Noninvasive Biopsies?”

“…These cells are nonapoptotic, and markers in exfoliated cells originating from the tumor are actively studied as a potential tool for cancer screening and prevention (3,21). Although the recovery of exfoliated colonocytes from the stools of normal adults has been described (10), and exfoliated cells from the urinary tract of infants have been studied in the past (22), we are not aware of previous studies in preterm infants with this approach.…”

“…Gastric residual fluid aspirates (0.5-1.5 mL; 89 samples) and fecal samples (0.5-3 g; 10 samples) were anonymously collected at random (and only once per infant) from a total of 96 premature infants. Gestational ages were between 24 and 36 wk and postnatal ages ranged from 1 to 90 d. Samples were immediately processed to isolate exfoliated cells by immunocapture using a procedure adapted from Bandaletova et al (10). Briefly, gastric residual fluid aspirates or fecal samples were resuspended by vigorous hand shaking in 20 mL phosphate-buffered saline solution without Ca 2ϩ and Mg 2ϩ (PBS0) containing 1 g/L D-glucose and antibiotics (penicillin and streptomycin) with 0.05 mM dithiotreitol (DTT) at room temperature.…”

Cell exfoliation in the human colon: Myth, reality and implications for colorectal cancer screening

“…In our earlier studies we explored this phenomenon initially in a rat model of colon carcinogenesis, 48 and then developed a method for human exfoliated colonocyte isolation based on the use of surface washes from whole stool samples in combination with immunomagnetic bead-based cell separation. 19,30 The method allowed isolation of well-preserved colonocytes and was later employed by other groups for the identification of cancer-associated cellular markers 49,50 ; however, few colonocytes were usually obtained, whereas squamous cells of the anal canal epithelium often dominated the pool of isolated cells. In addition, the standardization of the technique was extremely difficult because of the necessity to use intrinsically variable whole stool samples.…”

“…Although some authors still believe that massive exfoliation is constantly going on with millions of colonocytes shed into the faecal stream daily, 14,15 there is accumulating evidence that the rate of cell exfoliation from normal human colonic mucosa is much lower than it was previously believed. [16][17][18][19] Apoptosis in situ followed by the engulfment of apoptotic cells by adjacent colonocytes or subepithelial macrophages appears to be the main pathway of cell death in healthy colonic epithelium, 16,17,20,21 colonocyte exfoliation being an important, but auxiliary mechanism of cell loss. This situation can, however, be completely reversed in neoplastic growth.…”

Isolation of Stool-Derived Mucus Provides a High Yield of Colonocytes Suitable for Early Detection of Colorectal Carcinoma

“…This was a substantially greater number than we previously observed using smears and magnetic microbeads (5). It is likely that other groups also obtained low yields using density gradients or microbeads, considering the available images and the lack of cell quantification data in most published studies (9,(20)(21)(22)(23). Our method also allowed us to obtain morphologically atypical cells from all 20 stools from CRC patients, including the subject with transverse colon malignancy.…”