Klinkmann Gerd, Doss Fanny, Doss Sandra, Schwarz Antje, Reichert Susanne, Reuter Daniel A, Selleng Kathleen, Thiele Thomas, Mitzner Steffen, Altrichter Jens
Department of Anesthesiology, Intensive Care Medicine and Pain Therapy, University Medical Center, Rostock, Germany.
Department of Extracorporeal Immunomodulation, Fraunhofer Institute for Cell Therapy and Immunology, Rostock, Germany.
Transfus Med Hemother. 2024 Apr 12;51(6):383-392. doi: 10.1159/000537698. eCollection 2024 Dec.
Granulocyte concentrates (GCs) are usually prepared by single-donor apheresis after G-CSF pretreatment and have to be transfused within 24 h after cell collection because of the rapid decrease in pH and cell survival due to high lactate production by red blood cell contamination. GCs pooled from buffy coats of whole blood donations could improve the availability of these products. Methods to reduce red blood cell and platelet contamination may improve storability. We developed a manufacturing process for pooled GCs and investigated cell viability and functionality over time.
Six ABO blood group-identical buffy coats were pooled. Subsequently, the red blood cells spontaneously sedimented after the addition of hydroxyethyl starch. The resulting leukocyte-enriched supernatant was washed twice with saline to reduce platelets and was resuspended in ABO-identical donor plasma. The leukocyte concentrate was transferred to a platelet storage bag and stored up to 72 h at 20-24°C w/o agitation. Cell count and viability, pH, blood gases, phagocytosis, and oxidative burst activity were monitored.
The number of red blood cells and platelets was reduced to 0.4% and 6.1% of the baseline levels. About 50% of the original present leukocytes could be extracted ( = 76). In the course of 72 h of storage, there were no significant changes in white blood cell counts ( = 0.12). The viability exceeded 98% during the entire period. The rate of granulocytes performing phagocytosis and oxidative burst remained above 95% anytime.
GCs prepared from pooled buffy coats provide a precious alternative to granulocytes obtained from apheresis. Reduction of red blood cells and platelets by more than 90% extends the maximum shelf life of GCs from 24 h to 72 h. For a therapeutic dose of at least 1 × 10 granulocytes, 15-20 buffy coats are required.
粒细胞浓缩物（GCs）通常在粒细胞集落刺激因子（G-CSF）预处理后通过单采供血者的血液成分单采制备，由于红细胞污染导致乳酸大量产生，pH值迅速下降且细胞存活率降低，因此必须在细胞采集后24小时内输注。从全血捐献的 Buffy 层中汇集的GCs可以提高这些产品的可用性。减少红细胞和血小板污染的方法可能会提高其储存性。我们开发了一种汇集GCs的制造工艺，并研究了随时间变化的细胞活力和功能。
汇集6个ABO血型相同的Buffy层。随后，加入羟乙基淀粉后红细胞自然沉降。将得到的富含白细胞的上清液用生理盐水洗涤两次以减少血小板，并重悬于ABO血型相同的供体血浆中。将白细胞浓缩物转移至血小板储存袋中，在20-24°C下不搅拌储存长达72小时。监测细胞计数和活力、pH值、血气、吞噬作用和氧化爆发活性。
红细胞和血小板数量分别降至基线水平的0.4%和6.1%。约50%的原始白细胞可以被提取（n = 76）。在储存72小时的过程中，白细胞计数没有显著变化（P = 0.12）。在整个期间，活力超过98%。进行吞噬作用和氧化爆发的粒细胞比例在任何时候都保持在95%以上。
由汇集的Buffy层制备的GCs为通过血液成分单采获得的粒细胞提供了一种宝贵的替代方案。将红细胞和血小板减少90%以上可将GCs的最长保质期从24小时延长至72小时。对于至少1×10⁹个粒细胞的治疗剂量，需要15-20个Buffy层。
