Introduction Circulating tumor cells (CTCs) are detectable in most cancer patients and they can meet an existing medical need to monitor cancer patients during a course of treatment and to help determine recurrent disease. CTCs are rarely found in the blood of cancer patients and enrichment is necessary for sensitive CTC detection. Most CTC enrichment technologies are anti-EpCAM antibody based even though CTC identification criteria are cytokeratin positive (CK + ), CD45 negative (CD45 - ) and 4'6-diamidino-2-phenylindole (nuclear stain) positive (DAPI + ). However, some tumor cells express low or no EpCAM. Here we present a highly sensitive and reproducible enrichment method that is based on binding to anti-CK alone or a combination of anti-CK and anti-EpCAM antibodies. Methods Blood samples from 49 patients with metastatic breast cancer were processed using the CellSearch™ system (Veridex, LLC, Raritan, NJ, USA), in parallel with our CTC assay method. We used anti-CK alone or in combination with anti-EpCAM antibodies for CTC enrichment. Brightfield and fluorescence labeled anti-CK, anti-CD45 and DAPI (nuclear stain) images were used for CTC identification. The Ariol ® system (Genetix USA Inc, San Jose, CA, USA) was used for automated cell image capture and analysis of CTCs on glass slides. Results Our method has the capability to enrich three types of CTCs including CK + &EpCAM + , CK + &EpCAM -/low , and CK -/low &EpCAM + cells. In the blind method comparison, our anti-CK antibody enrichment method showed a significantly higher CTC positive rate (49% vs. 29%) and a larger dynamic CTC detected range (1 to 571 vs. 1 to 270) than that of the CellSearch™ system in the total of 49 breast cancer patients. Our method detected 15 to 111% more CTCs than the CellSearch™ method in patients with higher CTC counts (>20 CTCs per 7.5 ml of blood). The three fluorescent and brightfield images from the Ariol ® system reduced the number of false-positive CTC events according to the established CTC criteria. Conclusion Our data indicate that the tumor-specific intracellular CK marker could be used for efficient CTC enrichment. Enrichment with anti-CK alone or combined with anti-EpCAM antibodies significantly enhances assay sensitivity. The three fluorescent and brightfield superior images with the Ariol ® system reduced false-positive CTC events.

Atypical cells thought to be of endocervical glandular origin often cause diagnostic uncertainty in cervicovaginal smears. For this reason consecutive cases of endocervical glandular atypia diagnosed in smears were correlated with subsequent biopsy diagnoses and then retrospectively reviewed. Smears were originally diagnosed as "mild glandular atypia, probably reactive" or "severe glandular atypia, suggestive of adenocarcinoma in situ" (AIS). Biopsy follow-up was obtained on 34 of 58 patients diagnosed with severe endocervical glandular atypia. Nine patients (26%) had AIS, three with concomitant high-grade squamous intraepithelial lesions (HSIL) and tow with invasive adenocarcinoma. Eighteen patients (53%) had HSIL only. Seven had benign changes. Of 152 patients diagnosed with mild glandular atypia, biopsy follow-up was obtained on 40. One patient had AIS; 14 (35%) had HSIL; one had low-grade SIL (LSIL); and 24 (60%) had benign changes. Blinded review of these smears yielded results similar to those in the biopsy follow-up, that is, the prediction of AIS on smears included most cases of AIS, some invasive adenocarcinomas, a significant number of HSIL, cases and a few benign lesions. A review diagnosis of "reactive glandular cells" proved to be HSIL in 31% of cases and AIS in one case. We conclude that patients with a diagnosis of severe glandular atypia in smears may prove to have AIS or invasive adenocarcinoma, but often have HSIL without concomitant AIS. In addition, although "reactive" glandular atypia in smears usually reflects a benign condition, a significant minority of such patients prove to have HSIL.