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Liquid biopsy-based detection of circulating and exfoliated cholangiocarcinoma tumor cells from blood and bile using heparan sulfate octasaccharides on integrated microfluidic systems.

Early diagnosis of cholangiocarcinoma (CCA) remains challenging because existing diagnostic approaches often lack sufficient sensitivity for reliable detection of early-stage disease. Circulating tumor cells (CTCs) in blood and exfoliated tumor cells (ETCs) in bile represent valuable targets for liquid biopsy-based detection; however, their low abundance and the complexity of clinical sample analysis pose substantial technical challenges for reliable enrichment and identification. Herein, we present a reproducible workflow for isolating and identifying CCA tumor cells from blood for CTCs and bile for ETCs using synthetic cell-surface heparan sulfate (HS) octasaccharide-functionalized magnetic beads (MBs) on integrated microfluidic systems. The method combined sample pre-processing, magnetic bead-based enrichment, controlled low-shear mixing and immunofluorescence-based identification into a unified workflow compatible with distinct clinical sample types. Key operational parameters, including MB concentration, mixing frequency, and pressure settings, were detailed to facilitate consistent performance. Using this workflow, tumor cell capture rates of approximately 70% in bile (for ETCs) and blood (for CTCs) were achieved, with a total processing time of 60-90 min per sample under clinically relevant low-abundance conditions. The platform enables reliable detection of as few as 1 tumor cell per mL of blood or bile. This method provides a practical and adaptable strategy for glycosaminoglycan-mediated liquid biopsy applications and may be extended to other tumor-cell enrichment workflows involving heterogeneous cell-surface interactions.

Humans

Pulse cytophotometric measurements on tumor cell suspensions and exfoliative material.

By impulse cytophotometry (ICP) it is possible to get a DNA-distribution curve of nuclei in cell suspensions. The ICP curve directly represents the ploidy pattern of the investigated cell population and, more indirectly the proliferation intensity. For practical tumor diagnosis in cytology by ICP as a first step we must know, wether the ICP is able to detect differences between normal and tumor cells by simple criteria. Together with our clinical coworkers we had investigated cell suspensions from carcinomas of the cervix, ovary, breast, larynx, stomach and malignant melanomas. Approximately one half of these tumors were poly- or aneuploid. In the remaining diploid tumors some did not show signs of strong proliferation in the form of high 4c-peaks in the ICP; for these the chances of detecting by ICP in exfoliative material are low. This is in agreement with our results on vaginal smears. Here we had a good correlation between the mean 4c-height of the ICP curves and the Papanicolaou grading. But mass screening is not the main application of ICP because the security of Pa-grading is more sufficient. In individual problematic cytological cases with repeated investigation ICP is a very valuable diagnostic aid.

Cell Division

Ploidy and proliferation in human bladder tumors as measured by flow-cytofluorometric DNA-analysis and its relations to histopathology and cytology.

Biopsies from bladder tumors of 41 patients were investigated by flow-cytofluorometric DNA analysis and compared with exfoliated cells. The degrees of ploidy and proliferation were determined. Good agreement was found between the degrees of ploidy and proliferation in the biopsies and the exfoliated cell material. Tumors Grade I-II were either euploid or aneuploid. All Grade III tumors were aneuploid. The S-phase fractions were about 6% in the diploid tumors and 17% with large variations in the aneuploid tumors. The histological grading was well correlated to the number of S-phase cells and the occurrence of aneuploidy. When the Grade II tumors were divided into two groups having lesser and more pronounced atypia, the two groups differed significantly with regard to their degrees of proliferation. In addition to aneuploidy as an important criterium for malignancy, the degree of proliferation appears to be of major biological significance.

Aneuploidy

Studies on the use of mumps virus for treatment of human cancer.

Purified mumps virus (Urabe strain) was given mainly by intravenous injection to a total of 200 patients with cancer. The only adverse clinical reaction was transient mild fever in about half the patients. The beneficial clinical effects were as follows; decrease or disappearance of ascites and edema of the lower limbs at high rates (26/37 and 4/4, respectively), usually within a week after treatment: decrease or stoppage of cancerous bleeding in 30 of 35 patients: decrease or disappearance of pain in most of the patients: and tumor regression in 26 patients with cancer of the breast, rectum, ileocaecum, thyroid gland, uterus, skin, etc. Histologically, the virus-treatment caused shrinkage of nuclei and vacuolization of the cytoplasma of tumor cells, but the degenerative changes of tumor cells were not so great as those after chemotherapy or radiotherapy. Infiltration of lymphocytes, fibrosis and collagenesis occurred around tumor tissues, where necrosis or exfoliation of tumor cells was frequently observed.

Adenocarcinoma

Sarcoma of the vagina an unusual cytologic diagnosis.

The reported case, exceptional in our oncological material, illustrates the cytologica features of sarcoma of the vagina. We believe that the diagnosis of sarcoma of the cervix or other parts of the female genital system is not difficult providing that the tumor is exfoliating cells. Sarcomatous cells should not be mistaken either with those from keratininzing epitheliomas with desquamated fiber cells or snake cells. Such differentiation should not be a problem for an experienced cytologst. In spite of the low frequency of this malignancy, the microscopical appearance of the cells is so different from the cells of routine gynecological material that when present we should, at least, suspect a sarcoma.

Cervix Uteri

Cytomorphology of carcinoid tumors.

This report is based on a review and study of carcinoid tumors as seen in smears prepared from exfoliative or aspiration smears. During a period of eight years (1970 to 1978), 236 cytologic specimens were examined from 64 patients treated for carcinoid tumors at Memorial Hospital. Thirty-eight cytologic specimens from 18 patients were interpreted as either suspicious or positive for malignant cells. Tumor cells were identified most often in tracheobronchial specimens, effusions and percutaneous aspirates. A striking similarity in cell morphology was found between exfoliated and aspirated tumor cells. Certain specific or suggestive cytologic features were recognized. The histogenesis of carcinoids and the role of intracytoplasmic neurosecretory granules in the differential diagnosis is discussed together with the clinicopathologic implication of positive cytologic findings.

Biopsy, Needle

The cyto-histopathology of a pseudomesotheliomatous carcinoma of the lung.

A cyto-histopathologic study of a pseudomesotheliomatous carcinoma of the lung has been presented. The cytologic preparations of the early morning and post-bronchoscopy sputum specimens, bronchial washings and pleural fluid contained tumor cells similar to those exfoliated from a terminal bronchiolar carcinoma, of which this neoplasm is considered a variant.

Adenocarcinoma

Cell viability studies on the exfoliated colonic cancer cell.

Suspensions of desquamated colonic cancer cells were obtained from patients with cancer of the large bowel by colonic exfoliative cytology and from resected specimens of colonic cancer by an exfoliative technique. In addition, a tumor suspension was obtained from the resected specimens. Cell viability studies were performed on these cell suspensions. Whereas 23 of the 25 tumour homogenate cell suspensions were shown to exclude trypan blue, none of the exfoliated colonic cancer cell suspensions had viable cells. This finding would cast some doubt on the hypothesis that suture line recurrence following large bowel cancer surgery is due to the implantation of cells desquamated from the surface of the growth.

Cell Separation

The cytopathology of mesothelioma.

Fifty-seven cases of mesothelioma from the Toronto General Hospital during the period 1965 to 1976 have been reviewed. Pleural effusions or ascites were present in 34 patients. The value of cytologic examination of effusion specimens was assessed, and the criteria for cytologic diagnosis were elucidated. It appears that patients with predominantly fibrous or sarcomatous mesothelioma were not prone to develop pleural effusions or ascites; few tumor cells, if any, from these mesotheliomas were exfoliated into effusions. Cytologic examinations of effusion specimens were positive in 12 of 14 cases of carcinomatous mesothelioma and in three of four cases of undifferentiated mesothelioma. However, only four of seven casee of benign mesothelioma (mostly epithelial type) showed positive results, as did two of four cases of sarcomatous mesothelioma. It appears that cytologic diagnosis of mesothelioma is more useful for the carcinomatous and undifferentiated types. The cytomorphologic features of the various types of mesothelioma are different, and by cytologic examination of effusion specimens, typing of mesothelioma is possible and correlates well with the histologic typing.

Adult

Cerebrospinal fluid cytology in patients with brain tumors; a simple method using the cell culture technique.

A simplified cytologic method using the cell culture technique was employed in 71 cases with brain tumor. Neoplasitc cells were demonstrated in 17 cases (24%), that is in 11 out of 30 cases of glioma, four out of ten cases of metastatic brain tumor, and two out of 17 cases of meningioma. None of 14 other miscellaneous tumors proved positive. Identification of glioma cells could be easily made because they usually showed characteristic morphology and good proliferation in vitro. However, for other types of tumor, conventional methods were considered to be superior to the culture method because their exfoliated cells usually underwent rapid degeneration without showing characteristic morphology during the culture. Some of the illustrative cases were presented.

Adolescent

[Multicentric reticulohistiocytosis (lipoid dermato-arthritis). Apropos of a case with fatal outcome].

The authors report the case of a 37-year-old man affected by a seronegative polyarthritis associated at first with erythroderma and then with cutaneous and subcutaneous papulo-nodular structures and a xanthomatous eryption. Histological examination of the cutaneous nodules and of the synovial membrane confirmed the diagnosis of multicentred reticulohistiocytosis by showing the presence of lipid infiltrates in the histiocyte cells and of multinucleate giant cells. A study of the ultrastructure of the histiocytes revealed the presence of numerous intracytoplasmic vacuoles. In spite of antimalaria treatment, with cortisone and then with immuno-depressants, the outcome was fatal with a picture of acute reticulosis and neurological disorders. In discussing this case, the authors also make a general review of the subject.

Adult

Blastomatous tumors of the respiratory tract.

Two cases of blastomatous tumors of the respiratory tract are presented. The first is a pulmonary blastoma of an 81-year-old man, diagnosed as adenocarcinoma by cytologic examination, the cells being exfoliated from the large carcinomatous component. The patient died 1 year after manifestation of the symptoms. The second case is a tumor that developed in the nasopharynx of a 62-year-old man. This is the first reported case of a nasopharyngeal blastoma that presented a histology comparable to that seen in the pulmonary tumor. The presence of a hamartomatous benign mesenchymal component raises histogenetic considerations as to whether this was an independent part of an otherwise malignant tumor or whether it was induced by the malignant growth. Following incomplete surgical treatment and postoperative radiation, no recurrence was observed during the next 8 months.

Aged

Prostatic duct carcinoma: exfoliative cytology.

The cytologic findings in voided urine specimens from prostatic duct carcinoma are presented. Neoplastic cells from these unusual tumors were identified as well as the occasional red blood cell and histiocyte. The adenocarcinoma cells features irregular nuclei and their cytoplasm was occupied almost entirely by large secretory vacuoles. The differential diagnosis of this tumor from some other neoplasms and its association with transitional cell carcinoma as well as its histogenesis are discussed.

Adenocarcinoma

Cytopathologic manifestation of uterine leiomyosarcoma. Case report.

The presence of malignant tumor cells in a vaginal and cervical smear of a 53-year-old female with vaginal bleeding and with subsequent negative hsitology of the scraping material could be later correlated with a leiomyosarcoma of the myometrium that produced deep local invasion and pulmonary metastases. The findings that favor the cytologic diagnosis of this neoplasm are the presence of isolated cells or the side-by-side arrangement of the tumor cells, the elongated shape of the cytoplasm, and the ovoidal cigar-shaped nuclei with sparse, coarse chromocenters. Cytologic differential diagnosis of other malignant neoplasms capable of cellular exfoliation into the vagina is discussed.

Diagnostic Errors

Cytopathologic findings in pulmonary blastoma.

The exfoliative cytologic description of a pulmonary blastoma is reported. The debated histogenesis of these tumors is briefly reviewed. A review of all case reports recording cytopathology findings indicates that these rare tumors yield diagnostic cells in only a minority of cases. In the present case, as in previous reports, only the epithelial component (carcinoma) was recognized prior to the histopathologic diagnosis. The stromal cells were less numerous and not as conspicuous as the epithelial cells. The diagnosis of pulmonary blastoma should be considered whenever nonsquamous carcinomatous cells plus a separate population of smaller malignant cells are seen in a pulmonary cytology specimen.

Carcinoma

Characterization of urothelial carcinoma with respect to the content of carcinoembryonic antigen in exfoliated cells.

A sutstance immunologically similar to carcinoembryonic antigen (CEA) was demonstrated in urothelial bladder carcinoma cells. When indirect immunofluorescence with specific anti-CEA antisera was used, CEA-containing cells were seen in 18 out of 40 cases in 5% to 30% of the cells. Among patients with tumor cells of well differentiated morphology, 61% had CEA-containing cells, compared with only 24% of patients with a poorly differentiated tumor. Microfluorometry of single cells was performed in six cases to estimate the range of CEA content. The mean fluorescence intensity with anti-CEA antiserum was three to six times that of the same tumor cell population stained with non-immune rabbit sera. This fluorescence was decreased when the anti-CEA anti-sera were incubated with CEA but not with nonspecific cross-reactive antigen. The results show a wide range of CEA antigen content in exofoliated bladder tumor cells. In addition to proliferative status and differentiation, quantitative CEA measurements give further possibilities to study characteristics of tumor cell populations.

Carcinoembryonic Antigen