Karapetyan Knarik, Gizhlaryan Mane, Kalinovskaia Olga, Hovhannisyan Anna, Tadevosyan Gohar, Matinyan Lilit, Tamamyan Gevorg, Ghazaryan Narine
Department of Molecular Biology, Hematology Center After Prof. R.H.Yeolyan, Yerevan, Armenia.
Pediatric Cancer and Blood Disorders Center of Armenia, Hematology Center After Prof. R.H: Yeolyan, Yerevan, Armenia.
Mol Cytogenet. 2023 Jul 27;16(1):17. doi: 10.1186/s13039-023-00649-x.
A precise diagnosis of central nervous system involvement in acute lymphoblastic leukemia (ALL) requires comprehensive knowledge of morphological analysis, with a focus on the quantity and quality of cells being examined. Some research has utilized techniques such as immunocytochemistry, flow cytometry, polymerase chain reaction (PCR), and interphase fluorescence in situ hybridization (iFISH) on cerebrospinal fluid (CSF) cytospin samples to detect any remaining leukemic cells in the CSF. To obtain reliable results using immunocytochemistry and flow cytometry, it is essential to use freshly collected specimens within a limited timeframe. At the same time, PCR requires a sufficient number of cells for DNA extraction. On the other hand, the iFISH procedure on CSF cytospin samples can be challenging and requires practice. Therefore, there is a need for a fast, easy method that will be affordable and marketable in laboratories where the above methods are not available, or the sample is insufficient to use those methods.
The samples were prepared by centrifugation of 1 mL aliquots of CSF collected into EDTA tubes. The CSF sample was centrifuged at 3000 rpm for 3 min, the supernatant was removed, and the pellet was placed in KCl hypotonic solution for 5 min at 37 °C. Other steps (fixation, hybridization, wash steps, and analysis) were the same as in the standard protocol for blood samples. The BCR-ABL1 rearrangements were performed and evaluated in 200 interphase cells.
90% of Ph(+) cells were found in CSF.
We propose a significantly streamlined iFISH method for detecting blast/residual leukemic cells in acute lymphoblastic leukemia using CSF as a complementary test option.
准确诊断急性淋巴细胞白血病（ALL）中枢神经系统受累情况需要全面掌握形态学分析知识，重点关注所检查细胞的数量和质量。一些研究已利用免疫细胞化学、流式细胞术、聚合酶链反应（PCR）以及脑脊液（CSF）细胞离心涂片样本的间期荧光原位杂交（iFISH）等技术来检测脑脊液中残留的白血病细胞。为通过免疫细胞化学和流式细胞术获得可靠结果，在有限时间内使用新鲜采集的标本至关重要。同时，PCR需要足够数量的细胞用于DNA提取。另一方面，脑脊液细胞离心涂片样本的iFISH操作可能具有挑战性且需要实践经验。因此，需要一种快速、简便的方法，在无法使用上述方法或样本不足以采用这些方法的实验室中既经济又适用。
将收集到EDTA管中的1 mL脑脊液等份进行离心制备样本。脑脊液样本以3000 rpm离心3分钟，去除上清液，将沉淀置于37℃的氯化钾低渗溶液中5分钟。其他步骤（固定、杂交、洗涤步骤和分析）与血液样本的标准方案相同。对200个间期细胞进行BCR-ABL1重排检测和评估。
在脑脊液中发现90%的Ph(+)细胞。
我们提出一种显著简化的iFISH方法，用于检测急性淋巴细胞白血病中的原始/残留白血病细胞，将脑脊液作为一种补充检测选项。
