Various indices of cellular proliferative activity have been investigated. Mitotic counts only detect cells in the M phase, are dependent on the period of time between surgical removal and fixation of the specimen, and suffer from heterogeneous distribution and confusion between mitoses and nuclear pyknosis and karyorrhexis. Counting nucleolar organiser regions (AgNORs) and 5-bromodeoxyuridine or tritiated thymidine labelling are also not practical in a routine situation. Ki-67 recognises a proliferation specific nuclear antigen10.

Immunohistochemical expression

Ki-67 is expressed by proliferating cells in late G1, S, G2 and M phases, but not in resting cells in G0. Staining is commonly nucleolar or perinucleolar. In conjunction with Ki-S2, it may be used to derive a cell cycling ratio.

The prototypic monoclonal antibody works only in frozen sections. Subsequently, antibodies against formalin-resistant epitopes, MIB-1 and NCL-Ki-67-MM1 (MM1), have been raised. Polyclonal antibodies such as NCL-Ki-67p and rabbit anti-human (Rah) Ki-67, are also immunoreactive in paraffin sections. Staining is nuclear and may be diffuse or granular, or a combination1. There are good correlations between immunoreactivity using monoclonal Ki-67 in frozen section, polyclonal antibodies in paraffin sections and other indices of cell proliferation. In one study1, MIB-1 gives a higher labelling index than other antibodies, with good inter-reading reproducibility, although this was not reproduced in a second study2, which showed considerable variation between readings. It is therefore important to identify the antibody used in every study.


prototypic Ki-67




Rah Ki-67


tonsillar squamous epithelium1


granular & diffuse

diffuse only

granular & diffuse

diffuse only

granular & diffuse

diffuse only

granular & diffuse

diffuse only

30.8 to 30.9%1

18.2 to 18.3%1

13.9 to 14.50%1

5.8, 5.8%1

20.2 to 21.2%1

7.2 to 7.2%1

16.6 to 17.9%1

7.7 to 7.8%1



6.1 to 9.4%1



5.2 to 10.9%1


Immunoreactivity has therefore been shown not to be fixation dependent, an advantage over PCNA.



pattern of staining with Ki-67


Resting lymphoid tissue

The proliferation rate is low. Distinction from follicular lymphoma may be made by the negativity of the follicles for CD10 and bcl-6. (bcl-2 may be positive.)

Follicular hyperplasia

Follicles show a high level of staining, with a polarity; there is almost 100% staining of nuclei in the dark zone, which tends to form a meniscus. The borders of the follicles are sharply demarcated from the mantle which has a low proliferation rate. Some follicles may lack the typical pattern, so multiple follicles should be assessed: this is the case if the section is purely through the light zone.

Mixed pattern hyperplasia

Follicles show a high level of staining and interfollicular areas a low to moderate level

Progressive transformation of germinal centres

There are large follicles within which there are small aggregates of proliferating cells: the non-proliferating cells are T-cells or mantel zone B-cells. The pattern overlaps with that of the floral variant of follicular lymphoma. The few large follicles showing PTGC are present alongside obviously benign reactive follicles.

Castleman's disease

A nodular pattern of staining of low intensity, similar to follicular lymphoma, but the nodules are smaller and more widely spaced.

Follicular lymphoma

Follicles show a lower level of staining than in follicular hyperplasia and polarity is lacking. Margins are ill-defined. Grade 3 lymphomas may have a proliferation rate approaching that of benign follicles but are distinguished by the uniformity of the pattern among many follicles.

Floral variant of follicular lymphoma

There are large follicles within which there are small aggregates of proliferating cells: the non-proliferating cells are centrocytes. The pattern overlaps with that of progressive transformation of germinal centres. In addition to the floral type follicles, there are typical neoplastic follicles.

Marginal zone lymphoma

Diffuse low level of (interfollicular) staining, with or without residual follicles. Colonisation of follicles gives a pattern similar to that of follicular lymphoma.

Small lymphocytic lymphoma

Ki-67 accentuates the proliferation centres, which may resemble the pattern of neoplastic follicles.

Burkitt and Burkitt-like lymphoma

proliferation index approaching 100%

Transformation of indolent lymphomas

May be suggested by an anomalously high proliferation rate

T-cell rich B-cell lymphoma


The neoplastic cells are highlighted

Hodgkin lymphoma

Angioimmunoblastic T-cell lymphoma


Diagnostic utility

The rate of cell proliferation as assessed by Ki-67 immunoreactivity has been studied as a prognostic indicator in numerous malignant neoplasms and shown to correlate with tumour grade and clinical course. This includes:

It has also been used to distinguish benign from malignant neoplasms.


1 Lindboe, C.F. and Torp, S.H. Comparison of Ki-67 equivalent antibodies. J Clin Pathol 2002;55:467-71.

2 Torp, S.H. Proliferative activity in human glioblastomas: evaluation of different Ki67 equivalent antibodies. Mol Pathol 1997;50:198-200.

3 Hall, P.A., Richards, M.A., Gregory, W.M., d'Ardenne, A.J., Lister, T.A. and Stansfeld, A.G. The prognostic value of Ki67 immunostaining in non-Hodgkin's lymphoma. J Pathol 1988;154:223-35.

4 Zuber, P., Hamou, M.F. and de Tribolet, N. Identification of proliferating cells in human gliomas using the monoclonal antibody Ki-67. Neurosurgery 1988;22:364-8.

5 Ueda, T., Aozasa, K., Tsujimoto, M., Ohsawa, M., Uchida, A., Aoki, Y., Ono, K. and Matsumoto, K. Prognostic significance of Ki-67 reactivity in soft tissue sarcomas. Cancer 1989;63:1607-11.

6 Veronese, S.M., Gambacorta, M., Gottardi, O., Scanzi, F., Ferrari, M. and Lampertico, P. Proliferation index as a prognostic marker in breast cancer. Cancer 1993;71:3926-31.

7 Bryant RJ, Banks PM,O'Malley D P. Ki67 staining pattern as a diagnostic tool in the evaluation of lymphoproliferative disorders. Histopathology 2006; 48:505-15

8 Wang SA, Wang L, Hochberg EP, et al. Low histologic grade follicular lymphoma with high proliferation index: morphologic and clinical features. Am J Surg Pathol 2005; 29:1490-6

9 Jalava P, Kuopio T, Juntti-Patinen L, et al. Ki67 immunohistochemistry: a valuable marker in prognostication but with a risk of misclassification: proliferation subgroups formed based on Ki67 immunoreactivity and standardized mitotic index. Histopathology 2006; 48:674-82

10 Gerdes J, Li L, Schlueter C, et al. Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67. Am J Pathol 1991; 138:867-73

11 Palestro G, Ponti R, Chiusa L, et al. Cell proliferation, bcl-2, c-myc, p53 and apoptosis as indicators of different aggressiveness in small lymphocytic lymphoma (SLL). Eur J Haematol 1997; 59:148-54


This page last revised 3.11.2006.

©SMUHT/PW Bishop