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A monoclonal antibody against bromodeoxyuridine (BrdUrd) was produced, and a rapid slide technique (RPMB technique) was developed for the estimation of S-phase cells in a population using this antibody. Bone marrow cells from patients with acute nonlymphocytic leukemia (ANLL) were studied by both the RPMB technique and tritiated thymidine (3HdThd) labeling index studies. The percentage of S-phase cells obtained by each method was compared in 50 samples, and the correlation coefficient was r = 0.89.A "double label" method is also described in which cells were simultaneously incubated with either BrdUrd and 3HdThd or BrdUrd and tritiated cytosine arabinoside (3HAra-C).The samples were first processed by the Key terms: Anti-BrdUrd antibody, double labeling, acute nonlymphocytic leukemia, cell cycle characteristics, cytosine arabinoside Certain halogenated pyrimidines such as 5-bromo-2'deoxyuridine (BrdUrd) and 5-iodo-2'-deoxyuridine (IUdR) can be incorporated into DNA in place of thymidine, and consequently they have been used for studies of DNA replication for a number of years (1,11,17). Both polyclonal (4,16) and monoclonal (3,12) antibodies have been developed against these compounds and have been successfully utilized for quantitating the proportion of S-phase cells in a population (5) and for studying effects of drugs on DNA synthesis of cells (2) and the frequency of sister chromatid exchanges (9).We developed a rapid slide technique (RPMB technique) for determination of S-phase cells in a population using a monoclonal anti-BrdUrd antibody (RPMB I). In this immunofluorescent assay, every S-phase cell demonstrated bright green fluorescence. Additionally, we were able to quantitate fluorescence per cell by using an automated microphotometer system attached to the fluorescence microscope, and we showed that the degree of fluorescence was directly proportional to the amount of DNA synthesis in individual cells.A "double-label" technique was then established by incubating freshly obtained human leukemic cells with BrdUrd and tritiated thymidine (3HdThd) simultaneously. The samples were first processed by RPMB technique, so that every S-phase cell revealed bright green fluorescence. Following the application of photographic emulsion and development by autoradiography (ARG),we were able to demonstrate black grains of 3HTdR overlying the nucleus of every fluorescent cell. This double label technique not only validated the RPMB technique but allowed us to introduce two probes simultaneously into the DNA.

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A rapid and convenient method for estimating S-phase cells in a population was developed which detects bromodeoxyuridine (BrdU) incorporation into DNA by means of monoclonal anti-BrdU antibodies. This immunofluorescence technique (RPMB technique) was compared to autoradiographic (ARG) detection of tritiated thymidine (3HTdr) grains incorporated into the DNA. Using incubation periods for BrdU and 3HTdr ranging from one minute to one hour and detecting their incorporation by ARG and RPMB techniques, it became apparent that the RPMB technique was far more sensitive than ARG in addition to being extremely easy to perform. Some possible utilities of the RPMB technique are discussed.