探究使用异种洗涤红细胞灌注液对人断肢进行离体机械灌注保存的实验流程及可能性。

收集来源于左下肢恶性肿瘤截肢患者断肢，肢体离断后置于4 ℃保温箱中转运至实验室。对断肢进行预处理，并将腘静脉、腘动脉插管后连接灌注系统，使用4 ℃肝素等渗NaCl溶液对断肢进行灌洗，直至从静脉端流出澄清的等渗NaCl溶液。膜式氧合灌注系统包括：膜式氧合器、蠕动泵、制氧机、储液装置和监护仪。灌注过程中对灌注液电解质和细胞损伤标记物进行监测，同时进行血常规和断肢末端经皮动脉血氧饱和度(SpO₂)检查；灌注结束后通过苏木精-伊红(HE)染色法进行组织学评价。

断肢热缺血和冷缺血时间分别为138和65 min。灌注开始后，断肢颜色恢复红润，远端表浅静脉充盈。灌注开始后前6 h断肢末端SpO₂维持在98%～100%，最终因断肢末端SpO₂下降，灌注终止，灌注时长7 h。灌注开始时白细胞和血小板均处于较低水平，白细胞在灌注开始后有较明显的下降，在3 h后出现上升，最终与灌注开始时数值接近；灌注开始后前4 h内血小板水平较为平稳，4 h后出现较大幅度的上升；灌注过程中红细胞和血红蛋白均呈波动趋势。灌注开始后前6 h内K^＋浓度较为稳定，之后有较大幅度上升；Na^＋浓度在整个灌注期间基本维持在生理状态；乳酸脱氢酶和肌酸激酶均呈上升趋势。HE染色示断肢肌肉细胞在灌注过程中体积逐渐恢复后又持续变小，肌间血管可见血栓形成。

初步验证利用基于异种红细胞对人断肢进行离体机械灌注的可行性，但其保存效果是否达到或接近生理状态并能维持功能，以及免疫排斥反应的程度仍需进一步论证。

To explore the experimental process and possibility of using external machine perfusion system based on xenogeneic washed red blood cell perfusate to preserve human amputated limb.

The amputated limb was collected from patients with left lower limb malignant tumor amputation, and the amputated limb was placed in an incubator at 4 ℃ and transported to the laboratory. The amputated limb was pretreated, and the popliteal vein and popliteal artery were intubated and connected to the perfusion system. The amputated limb was lavaged with heparin isotonic NaCl solution at 4 ℃ until the clarified isotonic NaCl solution flowed from the venous end. The membrane oxygenator perfusion system included membrane oxygenator, peristaltic pump, oxygen generator, liquid reservoir and monitor. The perfusion electrolyte and cell damage markers were monitored during perfusion, and the blood routine and percutaneous arterial oxygen saturation (SpO₂) of end limb were examined during perfusion, and histological evaluation was performed by hematoxylin-eosin (HE) staining after perfusion.

The time of warm and cold ischemia were 138 min and 65 min, respectively. After the beginning of perfusion, the amputated limb returned to ruddy color, and the distal superficial veins were filled. The SpO₂ of end limb remained at 98%-100% lasting 6 h, and finally the perfusion was terminated due to the decrease of SpO₂, which lasted 7 h. The levels of white blood cells and platelets were low at the beginning of perfusion. The white blood cells decreased significantly after the beginning of perfusion, and increased 3 h later, finally approaching the value at the beginning of perfusion. The platelet level was stable within 4 h, and increased significantly after 4 h. During the perfusion, both red blood cells and hemoglobin showed fluctuation trend. K⁺ concentration was stable within 6 h, and then increased significantly. Na⁺ concentration remained in physiological state during the whole perfusion period. Both lactate dehydrogenase and creatine kinase were on the rise. HE staining showed that the muscle cells of the amputated limb gradually recovered in volume during perfusion and then continued to shrink, and thrombi were observed in the intermuscular vessels.

The feasibility of external machine perfusion based on xenogeneic red blood cells to the amputated limb was preliminarily verified. However, whether the preservation effect reached or was close to the physiological state and the function could be maintained, as well as the degree of immune rejection still need to be further demonstrated.