Arseniev L, Goudeva L, Kadar J G, Südmeier I, Battmer K, Matheja S, Mitschulat H, Stangel W, Link H
Abteilung Hämatologie und Onkologie, Medizinische Hochschule Hannover, Germany.
Infusionsther Transfusionsmed. 1995 Jun;22(3):152-8. doi: 10.1159/000223115.
Cryopreservation is the only available method for the long-time maintenance of blood cells. The present study was designed to prove: (i) the reliability of multiparameter flow cytometry (MFC) for estimation of CD34+ cells in frozen-thawed cell suspensions and (ii) the acceptability of a new teflon container for the cryopreservation of hematopoietic progenitor cells.
Each of 15 ABO-compatible buffy coats (BC) were pooled, and mononuclear cells (MNC) were then separated with the Fresenius AS 104 device (n = 10). MNC harvested by apheresis were then divided into 2 portions and transferred pairwise into either the new Fresenius or into Gambro cryopreservation containers. Paired samples were frozen at controlled rates (9% DMSO final concentration) and stored at -196 degrees C in liquid nitrogen for 2 weeks. Leukocyte, MNC and differential blood counts and proportions of CD3+, CD4+, CD8+, CD14+ and CD34+ cells were assessed from the pooled BC, the apheresis products, and the frozen-thawed samples. Methyl cellulose culture assays as well as trypan blue viability staining were also carried out.
The mean content of the divided apheresis products was 4.9 x 10(9) leukocytes with 86% MNC, 6.89 x 10(6) CD34+ cells, 2.1 x 10(5) granulocyte-macrophage colony-forming units (CFU-GM) and 7.1 x 10(5) erythroid burst-forming units (BFU-E). As expected, there were virtually no granulocytes after freezing in both types of container. The corresponding mean cell content was as follows: 6.3 x 10(6) CD34+ cells, 2.5 x 10(5) CFU-GM, and 8.1 x 10(5) BFU-E in Fresenius containers, and 6.1 x 10(6) CD34+ cells, 1.3 x 10(5) CFU-GM, and 7.7 x 10(5) BFU-E in Gambro containers. The mean MNC viability of the samples frozen in Fresenius was 81.5% and 82.7% in the Gambro containers. MFC was found to compare with stained smear differentials. CD34+ cell counts correlated with CFU-GM (0.69, p = 0.03) and BFU-E (0.63, p = 0.02) colony formation.
The study reported here revealed no significant differences between the 2 types of storage containers. The new Fresenius teflon container could thus be recommended for cryopreservation of hematopoietic progenitor cells. MFC provided reliable data on CD34+ cell content and leukocyte subset composition of the frozen-thawed cell suspension.
冷冻保存是长期维持血细胞的唯一可用方法。本研究旨在证明：（i）多参数流式细胞术（MFC）用于评估冻融细胞悬液中CD34+细胞的可靠性，以及（ii）一种新型聚四氟乙烯容器用于造血祖细胞冷冻保存的可接受性。
将15份ABO血型相容的 Buffy 层（BC）合并，然后用费森尤斯AS 104设备分离单个核细胞（MNC）（n = 10）。通过单采术采集的MNC随后分为2份，并成对转移到新的费森尤斯或 Gambro 冷冻保存容器中。配对样本以可控速率冷冻（最终二甲基亚砜浓度为9%），并在液氮中-196℃保存2周。从合并的BC、单采术产品和冻融样本中评估白细胞、MNC以及CD3+、CD4+、CD8+、CD14+和CD34+细胞的分类计数和比例。还进行了甲基纤维素培养测定以及台盼蓝活力染色。
分离的单采术产品的平均含量为4.9×10⁹个白细胞，其中86%为MNC，6.89×10⁶个CD34+细胞，2.1×10⁵个粒细胞-巨噬细胞集落形成单位（CFU-GM）和7.1×10⁵个红系爆式集落形成单位（BFU-E）。如预期的那样，在两种类型的容器中冷冻后几乎没有粒细胞。相应的平均细胞含量如下：费森尤斯容器中为6.3×10⁶个CD34+细胞，2.5×10⁵个CFU-GM和8.1×10⁵个BFU-E；Gambro容器中为6.1×10⁶个CD34+细胞，1.3×10⁵个CFU-GM和7.7×10⁵个BFU-E。在费森尤斯中冷冻的样本的平均MNC活力在Gambro容器中为81.5%和82.7%。发现MFC与染色涂片分类计数结果相符。CD34+细胞计数与CFU-GM（0.69，p = 0.03）和BFU-E（0.63，p = 0.02）集落形成相关。
本研究报告显示两种类型的储存容器之间无显著差异。因此，新型费森尤斯聚四氟乙烯容器可推荐用于造血祖细胞的冷冻保存。MFC提供了关于冻融细胞悬液中CD34+细胞含量和白细胞亚群组成的可靠数据。
