These tests measure the quantity of various antigens associated with cell proliferation, not the actual rate of proliferation. Except for FCM, measurement is done by applying immunohistologic stains on microscopic tissue sections; either fresh tissue or with some methods, preserved and paraffin-embedded tissue. An antigen-antibody reaction is seen under the microscope by a color reaction in nuclei that contains the proliferation marker antigen. There are four types of cell proliferation marker tests:

1.FCM S-phase measurement: This is discussed in the section on FCM. Technical problems with S-phase measurement have led to research for other proliferation markers that are more easily and universally employed and do not require special equipment. However, it is still the reference method for proliferation markers.
2.Nuclear mitotic count (or index): The number of mitoses per microscope high-power field (usually 400Ч magnification) is the first of the cell proliferation markers, since the number of mitoses roughly correlates with tumor cell replication and with degree of tumor differentiation. The greater the number of mitoses, the more likely, in general, that the tumor will be less differentiated and more aggressive. However, this does not hold true in every tumor type nor is there a linear relationship with metastases or prognosis. Also, mitotic counts may differ in different areas of the same tumor and even in the same area are not as reproducible as would be desirable. This technique is used more often in soft tissue sarcomas.
3.Ki-67: This is a monoclonal antibody that detects a protein in cell nuclei that appears only in the growth phase of the cell proliferation cycle (G1, S, G2, and M phases). Detection begins in mid-G1 phase and lasts throughout the remainder of the proliferation phase. This is a measure of total tumor growth fraction. It correlated well with FCM S-phase measurements. This method requires fresh tissue and is performed on cryostat frozen tissue sections.
4.PCNA (Cyclin): This is a stable protein produced mostly during the proliferative phase of the cell cycle. It correlates directly with cell proliferation rate. In general, there is good correlation with flow cytometry S-phase measurements, but some discrepancies have been reported. Different commercial antibodies do not react with the same PCNA epitopes. The original method required cryostat-frozen fresh tissue sections, but at least one commercial kit will react with antigen in paraffin-embedded, formalin-fixed tissue. There is some evidence that PCNA production is greatest in the S-phase of the cell cycle.