Maria Nowakowska Anna, Borek-Dorosz Aleksandra, Leszczenko Patrycja, Adamczyk Adriana, Pieczara Anna, Jakubowska Justyna, Pastorczak Agata, Ostrowska Kinga, Marzec Katarzyna Maria, Majzner Katarzyna
Jagiellonian University in Kraków, Faculty of Chemistry, Department of Chemical Physics, 2 Gronostajowa St., Krakow, Poland.
Jagiellonian University in Kraków, Faculty of Chemistry, Department of Chemical Physics, 2 Gronostajowa St., Krakow, Poland; Jagiellonian University in Kraków, Doctoral School of Exact and Natural Sciences, 11 Lojasiewicza St., Krakow, Poland.
Spectrochim Acta A Mol Biomol Spectrosc. 2023 May 5;292:122408. doi: 10.1016/j.saa.2023.122408. Epub 2023 Jan 25.
Leukemias are a remarkably diverse group of malignancies originating from abnormal progenitor cells in the bone marrow. Leukemia subtypes are classified according to the cell type that has undergone neoplastic transformation using demanding and time-consuming methods. Alternative is Raman imaging that can be used both for living and fixed cells. However, considering the diversity of leukemic cell types and normal leukocytes, and the availability of different sample preparation protocols, the main objective of this work was to verify them for leukemia and normal blood cell samples for Raman imaging. The effect of glutaraldehyde (GA) fixation in a concentration gradient (0.1 %, 0.5 %, and 2.5 % GA) on the molecular structure of T-cell acute lymphoblastic leukemia (T-ALL) and peripheral blood mononuclear cells (PBMCs) was verified. Changes in the secondary structure of proteins within cells were indicated as the main effect of fixation, as shown by an increase in band intensity at 1041 cm, characteristic for in-plane δ(CH) deformation in phenylalanine (Phe). Different sensitivity of mononuclear and leukemic cells to fixation was observed. While the 0.1 % concentration of GA was too low to preserve the cell structure for an extended period of time, a GA concentration of 0.5 % seemed optimal for both normal and malignant cells. Chemical changes in PBMCs samples stored for 11 days were also investigated, which manifested in numerous modifications in the secondary structure of proteins and the content of nucleic acids. The impact of cell preculturing for 72 h after unbanking was verified, and there was no significant effect on the molecular structure of cells fixed with 0.5 % GA. In summary, the developed protocol for the preparation of samples for Raman imaging allows for the effective differentiation of fixed normal leukocytes from malignant T lymphoblasts.
白血病是一组起源于骨髓中异常祖细胞的极为多样的恶性肿瘤。白血病亚型是根据使用耗时且要求严格的方法发生肿瘤转化的细胞类型来分类的。另一种方法是拉曼成像，它可用于活细胞和固定细胞。然而，考虑到白血病细胞类型和正常白细胞的多样性，以及不同样品制备方案的可用性，这项工作的主要目的是针对白血病和正常血细胞样本验证这些方案用于拉曼成像的效果。验证了戊二醛（GA）浓度梯度（0.1%、0.5%和2.5% GA）对T细胞急性淋巴细胞白血病（T-ALL）和外周血单个核细胞（PBMC）分子结构的影响。细胞内蛋白质二级结构的变化被表明是固定的主要作用，如1041 cm处谱带强度增加所示，这是苯丙氨酸（Phe）面内δ(CH)变形的特征。观察到单核细胞和白血病细胞对固定的敏感性不同。虽然0.1%浓度的GA过低，无法长时间保存细胞结构，但0.5%的GA浓度似乎对正常细胞和恶性细胞都是最佳的。还研究了储存11天的PBMC样本的化学变化，这些变化表现为蛋白质二级结构和核酸含量的大量改变。验证了细胞解冻后预培养72小时的影响，对用0.5% GA固定的细胞分子结构没有显著影响。总之，所开发的用于拉曼成像样本制备的方案能够有效区分固定的正常白细胞和恶性T淋巴母细胞。
