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Many, but not all, infants born to mothers infected with the human immunodeficiency virus (EIV) are infected in utero. We have now shown that mothers who have

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Two-color whole blood lysis is the assay of choice for lymphocyte immunophenotyping because of the additional information it provides. Recently, artifactual double-staining of some specimens has been observed with this assay. In these cases, the samples appear to be uncompensated for spectral overlap or to inappropriately coexpress two antigens simultaneously. This artifact can result in the apparent coexpression of CD4 and CD8 (observed in lymphoblastic processes) or of CD5 and CD20 (characteristic of chronic lymphocytic leukemia) in normal persons, leading to an erroneous diagnosis. Using plasma, serum, or immunoglobulin preparations from donors who exhibit this artifact we sought to determine 1) the source of the artifact and 2) ways to overcome it. This staining is apparently due to an immunoglobulin in the donors' serum and plasma which does not have specific reactivity with mouse immunoglobulin. Washing whole blood samples or blocking with mouse immunoglobulin is a convenient way of avoiding this artifact. 0 1994 Wiley-Liss, Inc.Key terms: Flow cytometry, immunophenotyping, monoclonal antibodies, fluorescein, phycoerythrin Lymphocyte immunophenotyping of specimens from human immunodeficiency virus (H1V)-infected persons or lcukemia patients has become a routine procedure in many clinical laboratories. To minimize any artifacts that may result from density gradient centrifugation, a whole blood assay has been recommended for immunophenotyping lymphocytes (1,2). In this assay, whole blood is incubated with monoclonal antibodies that are labeled with fluorochromes (direct immunofluorescence). The red blood cells are lysed, and the remaining cells of interest (leukocytes) are analyzed o n a flow cytometer. The flow cytomcter processes light scatter and fluorescence data from the specimens. Currently, two-color immunofluorescence is the method of choice ( 1,2) because it allows better identification of cell populations that may possess more than one antigen of interest and provides more information than single-color immunofluorescence.Recently, we as well as others ( 3 ) have observed aberrant labeling in some specimens which appears to be uncompensated fluorescence or inappropriate double-laheling. In some cases, erroneous results were reported 0 1994 Wiley-Liss, Inc.when CD4 and CD8 cells were artifactually double-labeled and interpreted to be a population of immature T cells or when artifactual double expression of CD5 and CD20 was interpreted as a lymphocytic malignancy (Marti, personal communication).We report the data from four different laboratories concerning this artifactual two-color staining in specimens, including the frequency of occurrence and the conditions under which it is observed. This artifact is idiosyncratic and is apparently due to an immunoglobulin found in plasma and serum of random donors which is not antimouse immunoglobulin antibodies. The immunoglobulin appears to crosslink mouse monoclonal antibodies, possibly through the Fc portion of the antibodies.

There is a close relationship between the response of mononuclear phagocytes to bacterial infections and their capacity to inhibit tumor growth. Macrophages obtained from mice infected with Listeria monocytogenes or with Bacillus Calmette-Gu~rin show enhanced bactericidal activity; in addition, these cells are reported to inhibit the growth of tumors in vivo (1, 2) and destroy tumor cells in vitro (3-5). These findings suggest that mononuclear phagocytes have the capacity to play a pivotal role in defending the host against neoplasms.To date, however, most of the systems in which macrophage cytotoxicity has been examined have involved incubation of the effector cells with the tumor cell targets in vitro. Cytotoxicity has generally been assessed by release of a radioactive label previously incorporated into the target cell or by a measurement of the number of viable tumor cells remaining at the end of the assay. However, there is no quantitative standard by which cytotoxic effects in vitro can be correlated with inhibition of tumor growth in vivo. Numerous studies have described the destruction in vitro of 60-70% of an inoculum of tumor cells by suitably activated macrophages (6, 7). Yet, if activated macrophages are to make a meaningful contribution to the eradication of neoplasms, the macrophages must be capable of destroying the clonogenic tumor cells within a tumor bed in the tumor-bearing host.To evaluate the efficacy of mononuclear phagocytes in tumor resistance, we have examined the capacity of these cells to inhibit the formation of tumors by B559 cells, a subclone of the B 16 melanoma (8, 9), in its syngeneic host, the C57BL/6 mouse. We have chosen this system for several reasons. The B16 melanoma arose spontaneously. Like most spontaneously arising tumors it is not immunogenic (as assayed in challengeprotection experiments) and does not elicit concomitant immunity (Results). The tumor has been extensively studied, grows rapidly to form lethal tumors in vivo, and is easy to propagate and to clone in vitro. The experiments described below examine the tumorigenic potential of B559 cells when these cells are injected into syngeneic mice together with various putative cytotoxic cells.