Glasser L, Fiederlein R L
Department of Pathology, College of Medicine, University of Arizona, Tucson.
Am J Clin Pathol. 1990 May;93(5):662-9. doi: 10.1093/ajcp/93.5.662.
Neutrophils are considered fragile cells, easily damaged by improper handling. Assays of neutrophil function frequently require preliminary isolation procedures that may be potentially harmful. The authors did an extensive investigation of the effects of currently used isolation procedures on a broad spectrum of functional assays that included: 1) phagocytosis; 2) bacterial killing by a culture technique and tritiated thymidine labeling; 3) candidacidal activity by methylene blue dye exclusion and differential Giemsa staining; 4) quantitative unstimulated and histochemical stimulated nitroblue tetrazolium dye reduction; 5) chemiluminescence; 6) random locomotion; and 7) chemotaxis by the agarose method. In addition, cells were examined morphologically by electron microscopy, and the granule density was quantitated by morphometric analysis. Isolation techniques included erythrocyte sedimentation, hypotonic lysis, gradient density separation using Ficoll-Hypaque, and counterflow centrifugal elutriation. The purest neutrophil suspensions were obtained by density gradient separation and counterflow centrifugal elutriation with mean neutrophil percentages of greater than or equal to 94%. Regardless of the isolation procedure, neutrophils were similar in all groups (P greater than .05) in the following studies: phagocytosis, nitroblue tetrazolium dye reduction, bacterial killing, candidal killing, inhibition of candidal germ tube formation, and cytoplasmic granulation. Differences were noted in assays of neutrophil migration and chemiluminescence. Neutrophil suspensions isolated by counterflow centrifugal elutriation and Ficoll-Hypaque had the highest scores for random migration and chemotaxis. These differences can be related to the purity of the neutrophil suspension rather than the harmful effects of the isolation procedures. Erythrocyte contamination affected both the slope and the time to peak response in the chemiluminescence assay. Exposure of neutrophils to cold temperatures (0-4 degrees C) for 1.5 hours impaired both random locomotion and chemotaxis. Current recommendations for the storage, transport, and preparation of leukocytes for neutrophil function studies need to be reassessed.
中性粒细胞被认为是脆弱的细胞，容易因处理不当而受损。中性粒细胞功能检测常常需要进行初步的分离程序，而这些程序可能具有潜在危害。作者对当前使用的分离程序对一系列功能检测的影响进行了广泛研究，这些检测包括：1）吞噬作用；2）通过培养技术和氚标记胸腺嘧啶核苷标记进行细菌杀伤；3）通过亚甲蓝染料排斥和吉姆萨染色鉴别进行念珠菌杀伤活性检测；4）定量未刺激和组织化学刺激下的硝基蓝四氮唑染料还原；5）化学发光；6）随机运动；7）通过琼脂糖法进行趋化性检测。此外，通过电子显微镜对细胞进行形态学检查，并通过形态计量分析对颗粒密度进行定量。分离技术包括红细胞沉降、低渗裂解、使用聚蔗糖-泛影葡胺进行梯度密度分离以及逆流离心淘析。通过密度梯度分离和逆流离心淘析获得了最纯的中性粒细胞悬液，中性粒细胞平均百分比大于或等于94%。在以下研究中，无论采用何种分离程序，各实验组的中性粒细胞均相似（P大于0.05）：吞噬作用、硝基蓝四氮唑染料还原、细菌杀伤、念珠菌杀伤、念珠菌芽管形成抑制以及细胞质颗粒化。在中性粒细胞迁移和化学发光检测中发现了差异。通过逆流离心淘析和聚蔗糖-泛影葡胺分离得到的中性粒细胞悬液在随机迁移和趋化性方面得分最高。这些差异可能与中性粒细胞悬液的纯度有关，而非分离程序的有害影响。红细胞污染影响了化学发光检测中的反应斜率和达到峰值的时间。将中性粒细胞暴露于低温（0 - 4摄氏度）1.5小时会损害随机运动和趋化性。中性粒细胞功能研究中白细胞储存、运输和制备的当前建议需要重新评估。
