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Chronic lymphocytic leukemia (CLL) is the most common leukemia in the United States. Metaphase-based cytogenetic tests, such as G-Band karyotyping, are among the most effective to detect CLL and provide significant prognostic information. However, the use of metaphase cytogenetics is currently problematic due to the low mitotic index of most CLL cells in vitro cultures. Even when metaphases can be generated in the presence of traditional B-cell mitogen LPS, the quality is often poor and aberrations escape detection.We hypothesized that immuno-stimulatory interleukin-2(IL-2) plus cytosine-phosphodiester-guanine oligodeoxynucleotide (CpG ODN) can work as a novel B-cell mitogen to stimulate bone marrow cultures which result in a higher mitotic index than regular standard bone marrow cultures stimulated with LPS. This will increase the clonal chromosomal aberration detection rate in patients with CLL.Bone marrow samples from CLL patients were divided and parallel cultures were set up using LPS and CpG Oligonucleotide/ IL-2 (IL-2/CpG) as mitogens, respectively. Mitotic index was read under the microscope blindly by three different readers (SQ, LV, RM). G-banding, and Spectral Karyotyping (SKY) were performed to confirm and compare abnormalities.The readings showed that mitotic index in IL-2/CpG stimulated bone marrow cultures was seven times higher than that of standard LPS bone marrow cultures with an average standard deviation of "0.92'" and CI of 95%, p less than 0.05. G-Banding and Spectral Karyotyping (SKY) showed the same abnormalities in IL-2/CpG found in LPS Bone marrow cultures.According to the results, IL-2/CpG cultures should be used in the cytogenetic lab for chromosomal analysis instead of LPS due to the higher mitotic index that helps in reducing false negative results. Further research should be done in order to lower false negative CLL detection results.