Effective centrifugation techniques for improving the frequency of metaphase spreads in lymphocytopenic patients

Cytogenetic biodosimetry is used for radiation dose assessment by evaluating chromosomal aberrations in peripheral blood lymphocytes. However, high-dose radiation may cause low absolute lymphocyte counts (ALCs), making it difficult to obtain sufficient metaphase spreads for analysis. This study aimed to optimize centrifugation settings to enrich metaphase spreads, particularly for lymphocytopenic patients.

Peripheral blood samples from four healthy donors and one lymphocytopenic patient were collected. Lymphocytes were harvested using one of four centrifugation settings. After an additional low-speed centrifugation step (200 × g for 1 minute) in each experiment, we evaluated slide quality using mitotic index (MI) and metaphase frequency (MF), which were calculated by the number of metaphases and blasts using Metafer 4 software.

We established an automatic measurement method for metaphases and blasts, adjusting the settings of Metafer 4. In four healthy donors, the strongest centrifugation (Exp. 4, 879 × g for 5 minutes) yielded the highest number of 'all cells' after harvest. Incorporating an additional low-speed centrifugation step significantly increased MF by 1.5- to 2-fold across all settings, with the greatest improvement observed in Exp. 4. This approach was applied to a lymphocytopenic patient, resulting in a 3.5-fold improvement of MF and the production of high-quality slides. MI was not significantly affected by centrifugation.

The combined high-speed and additional low-speed centrifugation method increased MF, improved slide quality by eliminating lower-density cells, and made it easier to analyze metaphase spreads. This method could be used for obtaining sufficient metaphase spreads in lymphocytopenic patients.