Dual-Parameter Immunoflow Cytometry – A New Technique in the Noninvasive Diagnosis of Bladder Cancer

Abstract

The introduction of monoclonal antibodies into the diagnosis of transitional cell carcinoma (TCC) has been a further step toward the use of biological parameters to support conventional cytological diagnosis of this tumor entity. Several investigators have demonstrated the high sensitivity and good specificity of immunocytology. Nevertheless this technique is hampered by the inconvenient and time-consuming microscopic analysis. The purpose of this study was to combine the advantages of immunocytology with the capabilities of an automated flow-cytometric system. Since all monoclonal antibodies (mAbs) currently used for immunocytology cross-react with granulocytes a preselection of urothelial cells becomes necessary for immuno-flow cytometry (immuno-FCM). Based on earlier analyses mAb Due AUT 2, reactive against urothelium and not against granulocytes, was chosen to preselect for urothelial cells. mAb Due ABC 3 was used to discriminate between normal and malignant urothelial cells. Initial experiments including 10 barbotage specimens from patients with histologically proven TCC and concomitant urinary tract infection yielded a high sensitivity (90%) of immuno-FCM. In 10 control patients with malignancies other than bladder TCC and benign diseases ABC 3 expression was not increased. Flow-cytometric examination of irrigation specimens from 10 patients with a history of bladder cancer but without cys-toscopic and cytologic evidence of tumor recurrence showed abnormal results in 6 patients. Rebiopsy and/or cystectomy confirmed tumor recurrence in 5 of these patients and in 1 patient with negative immuno-FCM. Ongoing prospective trials will further define the clinical impact of this approach in the diagnosis and follow-up of patients with bladder cancer.

Dual-Parameter Immunoflow Cytometry in Diagnosis and Follow-Up of Patients with Bladder Cancer

“…The examination of specimens from patients with endoscopically and pathologically proven TCC, irrespective of concomitant urinary tract infection, confirms the high sensitivity (95%) of immuno-FCM observed earlier in a smaller study population [4]. The detection of all 10 cases with G1 tumors is remarkable and confirms the results of earlier immunocytological observations [8, 9, 10, 11].…”

“…Evaluation of Lewis X antigen reactivity in bladder irrigation specimens from bladder cancer patients by Sheinfeld and associates [10]revealed that in 90% of the positive specimens the antigen was expressed in 25% or more of the epithelial cells. Our experience with immunocytology and earlier investigations showed that the 25% cutoff value of ABC 3-positive cells among all urothelial cells yielded an optimal discrimination between healthy donors and patients with transitional cell carcinoma [3, 4, 8, 9]. …”

“…According to our experience with immunocytology [8, 9]and the reports of others [10, 11, 12]a cutoff value was set. A value of 25% was chosen, since earlier experiments demonstrated that 95% of healthy donors have less than 25% ABC 3-positive urothelial cells [3, 4]. …”

“…The basic idea of this approach was to discriminate between urothelial and nonurothelial cells with a first mAb and subsequently identify antigenic changes within the urothelial cells by a second mAb. Based upon extensive preclinical studies mAb Due AUT 2, reactive with urothelial cells but not with granulocytes, was chosen to preselect for urothelial cells regardless of origin [3, 4]. mAb Due ABC 3 was used to determine the proportion of antigen-positive urothelial tumor cells.…”

“…Preliminary investigations have demonstrated the potential of DPI-FCM analyzing antigen expression in TCC cell lines even in the presence of white blood cells [3, 4]. In this paper the clinical experience using DPI-FCM for the investigation of exfoliated cells in the diagnosis and specifically in the follow-up of patients with bladder cancer is reported.…”