L. half of patients with EBV+ T/NK-LPDs had genetic defects associated with immunodeficiency. However, hemophagocytic lymphohistiocytosis, and not genetic defects, was the only parameter correlated with poor prognosis of EBV+ T/NK-LPDs. Conclusions Determination of EBV-infected cell types among PBMCs is a valuable tool for the differential diagnosis of EBV+ hematological diseases. In this study, determination of Epstein-Barr virus-infected cell types in peripheral blood mononuclear cells of 291 patients with high Epstein-Barr virus loads were retrospectively investigated, which indicate it is a valuable tool for Epstein-Barr virus-associated hematological diseases. value <.05 in the univariate analysis were further included in a multivariate analysis, with 2 and Fisher exact tests used for categorical variables, and the Mann-Whitney test for quantitative variables. The EBV DNA levels were log-transformed before the correlation and regression analyses. The Pearson test was used for correlations. Differences were considered Thiomyristoyl statistically significant at <. 05 (2 sided). RESULTS Determination of EBV-Infected Cell Types To determine EBV-infected cell Thiomyristoyl types, PBMCs isolated from patients were fractionated into B, T, and NK cells using MACS, purities of which were confirmed by flow cytometry to be 97%C99% for B and T cells and 91%C95% for NK cells (Figure 1A). The resulting cells were then analyzed with real-time PCR to amplify the genomic as a surrogate marker for EBV and with FISH assay with probes against EBER (Figure 1B and 1C). Twenty-six patients were examined with both real-time PCR and FISH, and the EBV-infected cell types identified by SARP1 real-time PCR were highly consistent with FISH results. Furthermore, the EBV DNA copy number determined by real-time PCR was correlated with the number of EBER-positive cells by FISH (Figure 1D). Therefore, in the subsequent Thiomyristoyl studies, we used real-time PCR to determine the EBV-infected cell types for better clinical feasibility. Thiomyristoyl Open in a separate window Figure 1. Validation of magnetic-activated cell sorting (MACS) and real-time polymerase chain reaction (PCR) for determining Epstein-Barr virus (EBV)Cinfected lymphocyte cell types. Patient peripheral blood mononuclear cells (PBMCs) were fractionated into B, T, and natural killer (NK) cells using MACS. The purities of the postsorting B, T, and NK cells were confirmed with flow cytometry and subsequent fluorescent in situ hybridization (FISH) analysis, using EBV-encoded small nuclear RNA (EBER) like a probe ((Standard FISH images from 3 individuals are shown. White colored arrows show EBER-positive cells, and figures represent the percentage of EBER-positive cells. Related results of real-time PCR analysis from your same 3 individuals are demonstrated. Dotted lines show EBV DNA levels recognized in unfractionated PBMCs. Correlation between percentage of EBER-positive cells and EBV DNA level in 26 individuals with EBV-positive PBMCs. Abbreviations: FSC, ahead scatter; EBER, EBV-encoded small nuclear RNA; NK, natural killer. Dominant EBV-Infected Cell Types in Different EBV Disease Entities A total of 291 individuals were ultimately enrolled in this study. In the immunodeficiency group, 45 individuals were identified as having dominant B-cell-type illness, including posttransplantation lymphoproliferative disorder (PTLD) (n = 2), posttransplantation status (n = 13), the use of immunosuppressant medicines (n = 23), or the presence of an autoimmune disease (n = 7), and 1 patient with PTLD after renal transplantation was identified as having NK-cell-type illness. The additional 245 individuals in the immunocompetent group exhibited numerous EBV disease entities, summarized in Table 1. Table 1. Summary of Epstein-Barr.