Bashiri Dezfouli Ali, Pourfathollah Ali Akbar, Nikougoftar-Zarif Mahin, Khosravi Mohammad, Tajrishi Mona, Ezzati Nasim, Kashani Khatib Zahra, Abbasi Sourki Parvaneh, Valizadeh Maryam
Tarbiat Modares University, Faculty of Medical Science, Tehran, Iran; Iranian Blood Transfusion Organization, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.
Tarbiat Modares University, Faculty of Medical Science, Tehran, Iran; Iranian Blood Transfusion Organization, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.
Hematol Transfus Cell Ther. 2022 Apr-Jun;44(2):197-205. doi: 10.1016/j.htct.2020.09.155. Epub 2020 Dec 21.
The isolation of captured peripheral blood mononuclear cells (PBMNCs) from leukoreduction filters (LRFs) can be of great importance in terms of bringing the lost cells back into use.
The aim of this study was to evaluate various methods based on their potential to recover the peripheral blood cells from LRFs with a focus on mononuclear cells (MNCs).
For cell isolation from LRFs, three distinct methods (back-flushing, direct and vacuum pump) were compared through the calculation of the yield of isolated MNCs. The viability of extracted cells was determined by the flow cytometry technique. Moreover, the recovered MNCs were characterized regarding the presence of blood stem cell purification. The cell culture, microscopic observation, and immunophenotyping were employed to characterize the blood stem cells (hematopoietic, mesenchymal and progenitor endothelial stem cells).
The yield of isolation obtained in the back-flushing, direct and vacuum pump methods were 17.7 ± 1.28, 17.3 ± 0.96 and 21.2 ± 0.90 percent, respectively. Although the highest potential for total blood cell recovery belonged to the vacuum pump method, the lowest cell viability (85.73 ± 4.84%) was observed in this method. However, the isolation process of the back-flushing and direct methods had less effect on cell viability. The characterization of the isolated MNCs displayed that the dominant positive phenotype was for CD34/CD45, indicating hematopoietic stem cells. In addition, the endothelial stem/progenitor cells were significantly detected as CD31/CD133 positive cells.
According to our results and considering the safety and efficiency potential of each of the applied methods, the back-flushing in comparison with the other methods can be considered a suitable procedure for MNC isolation from LRFs.
从白细胞滤除器（LRF）中分离捕获的外周血单核细胞（PBMNC）对于使丢失的细胞重新得到利用具有重要意义。
本研究旨在评估各种从LRF中回收外周血细胞的方法，重点是单核细胞（MNC）。
为了从LRF中分离细胞，通过计算分离出的MNC的产量，比较了三种不同的方法（反冲法、直接法和真空泵法）。采用流式细胞术技术测定提取细胞的活力。此外，对回收的MNC进行血液干细胞纯化情况的表征。采用细胞培养、显微镜观察和免疫表型分析来表征血液干细胞（造血干细胞、间充质干细胞和祖细胞内皮干细胞）。
反冲法、直接法和真空泵法的分离产量分别为17.7±1.28%、17.3±0.96%和21.2±0.90%。虽然全血细胞回收潜力最高的是真空泵法，但该方法观察到的细胞活力最低（85.73±4.84%）。然而，反冲法和直接法的分离过程对细胞活力的影响较小。分离出的MNC的表征显示，主要的阳性表型是CD34/CD45，表明是造血干细胞。此外，内皮祖细胞被显著检测为CD31/CD133阳性细胞。
根据我们的结果并考虑到每种应用方法的安全性和效率潜力，与其他方法相比，反冲法可被视为从LRF中分离MNC的合适方法。
