EBV was detected in B lymphocytes from all 29 sufferers tested (Physique 1; Table 1), except for the 4 patients with T-cell chronic active EBV (CAEBV) from Japan. copy number per cell. Although we detected CD21, the EBV B-cell receptor, on EBV-infected B cells, we could not detect it on virus-infected T cells. These findings expand the range of cell types infected in the blood. Determining the number of EBV genomes per cell and the type of cells infected in patients with high EBV loads may provide additional prognostic information for the development of EBV lymphoproliferative diseases. Introduction Epstein-Barr computer virus (EBV) infects more than 90% of the human population.1 In immunocompetent hosts, the computer virus is latent in B cells of the peripheral blood and is not associated with disease.2C4 However, in immunocompromised patients, immune surveillance to the computer virus is often impaired, a larger number of B cells are infected with EBV, and the computer virus can contribute to lymphoproliferative disease. Approximately 1%-20% of transplant recipients can develop posttransplantation lymphoproliferative disease GO6983 (PTLD) during the first 12 months after transplantation, and approximately 90% of these cases are EBV positive.5 Persons with AIDS have a 60-fold increased risk of developing lymphoma, compared with the general population, and virtually all Hodgkin and non-Hodgkin lymphomas that occur in the late stages of HIV infection are EBV positive.6 Although EBV establishes a latent infection in peripheral blood B cells of healthy people, less is known about the phenotype of virus-infected cells in the blood of immunocompromised persons with high EBV DNA loads. Most studies have focused on the phenotype of virus-infected B cells in transplant recipients.2,7C11 However, EBV can infect cells other than B cells, including T cells, natural killer (NK) cells, monocytes, and pre-Langerhans cells.12C16 Several techniques have been developed to detect EBV in cells. In situ hybridization using a probe that detects the EBV-encoded RNAs (EBERs) is considered the best test for localizing latent EBV in tissue samples.17 Combined staining for EBERs and antibodies to cell-surface markers for tissues on microscope slides, or for peripheral blood by flow cytometry,18 has been used to determine the phenotype of the EBV-infected cells. Although detection of EBERs indicates that cells are infected with EBV, this test cannot provide an estimate of the number of EBV genomes present per cell. We describe a new technique (Immuno-FISH) that combines immunofluorescent staining for surface proteins (using antibodies directly conjugated to fluorochromes) and fluorescent in situ hybridization for EBV DNA. This technique allows the simultaneous determination of the cell type infected by EBV and quantification of EBV copy GO6983 GO6983 number in the infected cell. We show that EBV is present not only in B cells, but also in a large percentage of DNM3 other cell types in the peripheral blood of patients with high EBV DNA loads. In addition, we correlate the number of EBV genomes per cell with the phenotype of the infected cells. Methods Study participants Patients had blood drawn after informed consent was obtained in accordance with the Declaration of Helsinki under protocols approved by the Institutional Review Boards of the National Institute of Allergy and Infectious Diseases, the National Malignancy Institute, the National Heart, Lung, and Blood Institute (patients 1-23), Nagoya University Hospital (patients 24-27), or the University of Maryland and the National Institute of Allergy and Infectious Diseases (patients 28-29). For patients from the United States, we selected those whose EBV DNA loads GO6983 were more than 5000 copies per million cells (normal is usually 200 copies/million cells) and, for patients from Japan, more than 50 000 copies per g of DNA. Measurement of EBV DNA in blood For patients 1-23 and 28-29, the EBV DNA load data were reported as the number of EBV genomes per 106 cells. Peripheral blood mononuclear cells (PBMCs) were lysed and EBV quantitative real-time polymerase chain reaction (qPCR) was performed as previously described19 (see supplemental data, available on the Web site; see the Supplemental Materials link at the top of the online article). Immuno-FISH procedure Cryopreserved PBMCs were thawed at 37C, washed once in media and once in phosphate-buffered saline (PBS), and then resuspended in a solution of 0.2N acetic acid, 0.02N HCl, in Tris HCl 0.1M (adjusted to pH 3.7) for 15 minutes at room heat. The cells were washed twice in PBS and incubated with fluorochrome-conjugated monoclonal antibodies.