Assessment of human leukocyte viability in vitro cell culture conditions at the university teaching hospital of Butare (CHUB)

Abstract

Cytogenetic is the study of chromosomal number and structural observable under a microscopy. It is a valuable tool to diagnose genetic disorders caused by chromosomal abnormalities, as well as acquired conditions such as leukemia. One common cytogenetic technique, karyotyping, involves culturing blood leukocytes in special culture media, usually Roswell Park Memorial Institute (RPMI) 1640, which contains hormones and growth factors that stimulate cell division and proliferation. After three to four days, cells are harvested at the metaphase stage, when chromosomes are most condensed and readily visualized under microscope.The success of karyotyping depends on cell viability and proliferation. Factors such as nuclear alteration in dying cells, low mitotic index, and poorly defined chromosomal pattern can hinder accurate analysis. Moreover, since cytogenetic laboratories are usually located in major facilities, delays in sample processing can impair cell viability. A successful karyotyping is achieved when blood samples are cultured within 24 hours of collection; aging samples greatly reduce mitotic activity, sometimes resulting in complete absence of mitosis. Despite this limitation, there is lack of studies on cell viability during cell culturing for karyotyping at CHUB. This study aimed to assess leucocytes viability prior to culturing and to evaluate the viability of cells cultured on RPMI by using Trypan Blue exclusion method among patients attending the genetic service at CHUB. A cross-sectional experimental study wa conducted on 50 heparinized blood samples from adult males and females. Leukocyte count was done by using the Sysmex XN-3500.Cell viability test was assessed at three time points: after 24 hours at room temperature, 72 hours of refrigeration and finally at 72 hours of culturing on RPMI. Data were analyzed using SPSS Version 25 .RPMI 1640 significantly preserved leucocytes viability after 72 hours of culture :13,34% higher than 24 hrours at room temperature after blood collection. Prolonged Refrigeration impacted leukocyte viability: 4.2% reduction of cell viability . The leucocyte count from fresh samples at 24 hours did not reliably predict the cell viability of leukocytes isolated after RPMI 1640 culture