Simultaneous flow cytometric analysis of surface markers and nuclear Ki-67 antigen in leukemia and lymphoma

Area of Science:

• Immunology
• Cell Biology
• Hematology

Background:

• The Ki-67 antibody detects an antigen expressed in proliferating cells (G1, S, G2, M phases).
• Traditional Ki-67 immunostaining is effective but flow cytometry detection was limited by the antigen's nuclear location.
• Assessing tumor growth fraction is crucial for diagnosing and managing hematological malignancies.

Purpose of the Study:

• To develop and validate a flow cytometry method for quantifying the tumor growth fraction using Ki-67.
• To enable simultaneous analysis of cell surface markers and Ki-67 expression for phenotype characterization of proliferating cells.
• To apply this method to pathological blood and bone marrow samples from patients with leukemia and non-Hodgkin's lymphoma.

Main Methods:

• Cells were fixed using periodate-lysine-paraformaldehyde (PLP) at -10°C for 15 minutes to permeabilize the plasma membrane.
• Ki-67 antibody labeling was performed, followed by flow cytometric analysis.
• Double immunofluorescence studies combining Ki-67 with cell surface markers were conducted.

Main Results:

• The PLP fixation method successfully allowed Ki-67 detection via flow cytometry.
• The technique enabled the identification of the phenotype of proliferating cells through double immunofluorescence.
• Analysis of 91 patients with leukemia and non-Hodgkin's lymphoma revealed significant variability in Ki-67 values within each disease entity.

Conclusions:

• This optimized flow cytometry method using Ki-67 and PLP fixation is a valuable tool for quantifying proliferating cells.
• The method facilitates the assessment of tumor growth fraction in hematological malignancies.
• It allows for detailed phenotyping of actively dividing cells, aiding in disease characterization.