The Introduction and Evaluation of Ki-67 as a read-out method of in vitro lymphocyte proliferation and its application in the investigation of immunodeficiency.

  • Elizabeth Walker

Student thesis: Unknown

Abstract

Abstract In the United Kingdom, a significant minority of people, have a primary or secondary immune deficiency, leaving them vulnerable to severe, recurrent, persistent or unusual bacterial, viral and fungal infections. As part of investigating these patients, an in vitro lymphocyte proliferation assay is used to measure cell meditated immunity, where patient lymphocytes are stimulated with mitogens and/or antigens in order to measure the functional response of a patients’ T cells. Lymphocyte proliferation assays are also used to monitor immune suppressive therapy and assess immune function after bone marrow transplantation. The method commonly used to measure lymphocyte proliferation is tritiated thymidine incorporation (3[H] thymidine incorporation). However, this assay has issues such as a high Coefficient of Variation (CV), the inability to distinguish different lymphocyte populations and the use of radioactivity. Alternative assays for the measurement of lymphocyte proliferation are welcome. The intra-cellular protein, Ki-67, is widely used as a proliferation marker in vitro, mainly within research. It is expressed in proliferating cells but not in G0 resting cells. A method was developed for utilising Ki-67 to measure lymphocyte proliferation in vitro. It was optimised as a whole blood assay, for use in healthy controls, by designing a suitable antibody panel, demonstrating the ability to detect Ki-67 in proliferating cells but not unstimulated cells and determining the optimal conditions for red blood cell (RBC) lysing. Eighteen healthy controls and 25 patients being investigated for immune deficiency were tested by Ki-67. Inter- and intra-assay variation were found to be less than 20%. Costings and the practicalities of introducing the Ki-67 assay for routine use were also considered. Normal ranges were devised on the healthy controls and an approach using the mean and 3SDs was adopted. Twenty five patients and 8 Healthy Controls (HC) were tested by Ki-67 assay and 3[H] thymidine incorporation and results compared. Concordance was good although there were several discrepancies which were further investigated. The use of the Ki-67 assay in the measurement of antigen-specific T cell responses to VZV antigen was also examined. Different methods were tried to identify Ki-67 positive T cells responding to VZV antigen, however, no significant increase in Ki-67 positive T cells could be identified. Stimulation with Covid peptides and Tetanus toxoid did produce a significant increase in Ki-67 positive T cells, demonstrating the Ki-67 method is suitable for identifying antigen-specific T cells. The Ki-67 assay for lymphocyte proliferation is suitable for use as a routine test for the investigation of immune deficiency and the use of the assay could be extended to other areas of the clinical immunology laboratory.
Date of Award31 Dec 2023
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorJohn Grainger (Supervisor)

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