In VitroLabeling of Solid Tissues with Tritiated Thymidine for Autoradiographic Detection of S-Phase Nuclei

Abstract

In vitro measurement of the thymidine labeling index (TLI) of solid tissues requires hyperbaric oxygenation and is potentiated by blockade of thymidylate synthetase by 5-fluoro-2'-deoxyuridine (FUdR) to favor uptake of tritiated thymidine (3H-TdR). Hyperbaric oxygenation can be achieved in a simple system through injection of oxygen into rubber-stoppered test tubes. Incubations are carried out in Hanks' balanced salt solution in a shaker bath at 37 C for 2 hours; an FUdR concentration of approximately 1 micron is optimal. Autoradiographic exposure for 1 week or less is sufficient for TLI measurements on human tissues. With 3 to 4 atmospheres oxygen tension, incorporation of 3H-TdR is sufficient for TLI measurement throughout slices of tissue cut 1 mm thick or less. Mincing of tissue is not necessary, and the anatomic continuity seen in ordinary histological preparations is preserved. A gradient of labeling intensity is present from the surface to the interior of the tissue, but sufficient intensity of labeling for detection of DNA synthesis can be achieved in the interior of the section. The gradient can be reduced only slightly by prior incubation in 3H-TdR with hyperbaric oxygen at 0 C. The method permits TLI measurements on the same specimens, including needle biopsies, that are used for pathologic diagnosis.

Cell kinetics of histologic variants ofin situ breast carcinoma

“…The purpose of the FUdR was to lower intracellular thymidine phosphate pools by blockade of thymidylate synthetase. Details of the labeling procedure have been published (14).…”

In vitro thymidine labelling of human pulmonary neoplasms

“…Fabrikant et al (1969) claimed that maximal depth of labelling could be achieved using an oxygen pressure of 3 atmospheres; no improvement on labelling beyond 500,pm into the tissue could be obtained by raising the oxygen pressure above 3 atmospheres. Meyer & Connor (1977) Secondly, the choice of which areas to consider for counting TLI often seems quite arbitrary. Meyer & Connor (1977), Chavaudra et al (1979 and others have commented that much of the current data available on in vitro human tumour [3H]dT labelling may underestimate tumour cell TLIs since areas deep in the fragments with suboptimal labelling are being counted.…”

“…Meyer & Connor (1977) Secondly, the choice of which areas to consider for counting TLI often seems quite arbitrary. Meyer & Connor (1977), Chavaudra et al (1979 and others have commented that much of the current data available on in vitro human tumour [3H]dT labelling may underestimate tumour cell TLIs since areas deep in the fragments with suboptimal labelling are being counted. Hainau et al (1977) would score tumour cells in a field where no labelled tumour cells were present if labelled stroma was seen, this being taken as evidence of adequate conditions for tumour cell labelling.…”

Comparison of monoclonal antibody Ki-67 reactivity with grade and DNA flow cytometry of breast carcinomas

“…Although information can be gained lFrom mitotic counting of histological sections, this is a tedious procedure, and measurement of the thymidine labelling index (TLI) has been used more extensively for asessing proliferation. The technique employs tritiated thymidine incorporation, with subsequent autoradiography (Mayer & Connor, 1977), which may restrict the number of laboratories undertaking this as a routine prognostic marker. The introduction of DNA flow cytometry, with its applicability to fixed, paraffin embedded tissue (Hedley et al, 1983) has proved to be of value in determining S-phase content of breast carcinomas (Walker & Camplejohn, 1986) but again there is a restriction in its availability.…”