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目的 探讨不同温度热处理及IgG抗体诱导对红细胞衰亡的影响。方法 设3组分别用50、56及60℃3个温度加热处理2%RhD+红细胞悬液5、10、20 min;另外设设1组,以RhDIgG抗体75μL与2%RhD+红细胞悬液150μL混合,同时设对照组(以PBS代替RhD),37℃30 g振荡(离心),诱导时间分别为0、16、24、40、48及72 h。用Alexa Fluor 488 Annexin V/Dead Cell试剂盒检测红细胞的衰亡比例。结果 流式检衰亡细胞比例(%):50℃/5 min、56℃/5 min、60℃/5 min组分别为1.09±0.32 vs 5.44±0.51 vs 16.35±0.53,50℃/10 min、56℃/10 min、60℃/10 min组分别为1.03±0.17 vs 9.27±0.25 vs 29.42±1.21,50℃/20 min、56℃/20 min、60℃/20 min分别为1.66±0.21 vs17.74±0.81 vs 61.38±4.34(P<0.01);除了50℃/5 min和50℃/10 min 2个组外,同一温度下,各组随着加温时间的延长,衰亡细胞比例增大,不同温度加热20 min时,死亡细胞比例(AV-PI+及AV+PI+红细胞之和)较5 min和10min有所升高,尤以60℃组最为明显,但红细胞破碎严重,检测到的细胞总数较少,死亡细胞比率(%):60℃/5 min、60℃/10 min、60℃/20 min组分别为0.51±0.59 vs 0.46±0.11 vs 6.20±1.70(P<0.01)。抗体诱导试验:诱导时间0、16、24、40、48、72 h的衰亡和死亡细胞的比例(%)各自分别为0.76±0.20、0.07±0.01,6.44±0.22、3.78±0.51,8.29±0.23、6.36±0.24,13.34±0.96、9.27±0.51,15.01±2.07、10.21±2.05,31.20±10.20、10.34±2.06(P<0.01)。结论 加热及抗体处理诱导均会引起红细胞衰亡;提示血清学试验中以热处理温度≤56℃、热处理时间<10 min为宜。
Objective To investigate the effects of heat treatment at different temperatures and the induction of IgG on the decline of erythrocytes. Methods 3 groups were treated with 50, 56 and 60 ℃ 3 temperature heat treatment of 2% RhD + red cell suspension for 5,10,20 min; another set of 1 group, with RhDIgG antibody 75μL and 2% RhD + 150μL erythrocyte suspension mixed, At the same time set the control group (instead of RhD PBS), 37 ℃ 30 g oscillation (centrifugation), induction time were 0,16,24,40,48 and 72 h. The rate of erythrocyte death was measured using Alexa Fluor 488 Annexin V / Dead Cell kit. Results The percentage of apoptotic cells in flow cytometry was 1.09 ± 0.32 vs 5.44 ± 0.51 vs 16.35 ± 0.53 at 50 ℃ / 5 min, 56 ℃ / 5 min and 60 ℃ / 5 min, respectively The results were as follows: 1.03 ± 0.17 vs 9.27 ± 0.25 vs 29.42 ± 1.21, 50 ° C / 20 min, 60 ° C / 20 min and 60 ° C / 10 min respectively, and 1.66 ± 0.21 vs 17.74 ± 0.81 vs 61.38 ± 4.34 (P <0.01). Except for 50 ℃ / 5 min and 50 ℃ / 10 min, the percentage of apoptotic cells increased with the increase of heating time at the same temperature The percentage of dead cells (sum of AV-PI + and AV + PI + erythrocytes) increased at 5 min and 10 min after 20 min heating, especially at 60 ℃, but the number of red blood cells was severely broken. The percentage of dead cells (%) was 0.51 ± 0.59 vs 0.46 ± 0.11 vs 6.20 ± 1.70 (P <0.01) at 60 ℃ / 5 min, 60 ℃ / 10 min and 60 ℃ / 20 min respectively. Antibody induction test: The percentage of apoptotic and dead cells at the induction time of 0, 16, 24, 40, 48 and 72 h were 0.76 ± 0.20,0.07 ± 0.01,6.44 ± 0.22,3.78 ± 0.51,8.29 ± 0.23 , 6.36 ± 0.24, 13.34 ± 0.96, 9.27 ± 0.51, 15.01 ± 2.07, 10.21 ± 2.05, 31.20 ± 10.20, 10.34 ± 2.06 (P <0.01). Conclusion Heating and antibody treatment induced erythrocyte cell death. It is suggested that the heat treatment temperature≤56 ℃ and the heat treatment time <10 min in serological test are appropriate.