生物素标记红细胞脱落-再标记可能性的研究
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摘要
目的探讨利用生物素标记红细胞时测定到的红细胞存活率不随时间下降的原因。方法体外标记法:将1只C57BL/6供血小鼠用0.1 mL 10%水合氯醛麻醉后开胸从心脏穿刺取血0.8 mL,制备成25%红细胞悬液并用生物素在体外标记后平均分成6份分别输注到6只C57BL/6受血小鼠体内,受血的6只C57BL/6小鼠均分别于输注后1 h(基础值)及1、3、7、14、21、28 d尾静脉采血,流式细胞仪测定标记红细胞比例,计算输注到6只C57BL/6受血小鼠体内标记红细胞的存活率和寿命。体内标记法:另外取5只C57BL/6小鼠,直接将1 mg/300μL生物素过尾静脉分别输入这5只C57BL/6小鼠体内做体内标记红细胞,48 h后采集这5只C57BL/6小鼠全血并分别制备成25%标记红细胞悬液,共制备5份标本,从每份标本中取2等份(500μL/份)标记红细胞悬液分别与荧光染料链霉亲和素-R-藻红蛋白(SA-R-PE,终浓度10μg/mL)和结合荧光素的亲和素(FITC-AV,终浓度30μg/mL)反应,得到分别标记上SA-R-PE-生物素的红细胞(PE-RBC)和FITC-AV-生物素的红细胞(FITC-RBC),然后在体外将这2种单染红细胞混合、室温下共同孵育4 h后,流式细胞仪检测这5份混合孵育的标本中双染红细胞(PE-FITC-RBC)的比例,并在共聚焦显微镜下直接观察是否存在双染红细胞。结果输注到6只C57BL/6受血小鼠体内的标记红细胞的24 h存活率、半寿命和平均潜在寿命分别(98.9±15.6)%、(17.2±2.4) d和(34.2±4.3) d,标记红细胞的存活率以(2.7±0.4)%/d速率下降,但在83%(5/6)动物体内,标记红细胞的存活率超过基础值100%。5份标本内均出现双染红细胞(PE-FITC-RBC),比例为(0.8±0.5)%,并且共聚焦显微镜结果也证实了双染红细胞的存在。结论生物素标记红细胞输入动物体内后检测到的寿命在正常范围,但由于存在脱落-再标记现象,生物素标记后检测的红细胞存活率可能有波动。
Objective To discuss the reason that the red blood cell(RBC) survival rate measured by biotin labeling did not decrease with time. Methods Labeling in vitro: a C57 BL/6 donor mouse was anesthetized with 0.1 mL of 10% chloral hydrate, and then 0.8 mL blood was collected from the heart. RBCs resuspended to make 25% RBC suspension were labeled with biotin in vitro and divided into 6 parts, then infused respectively into six C57 BL/6 recipient mice. After transfusion, blood samples from six C57 BL/6 recipient mice were collected by tail vein on 1 h(basal value), 1 d, 3 d, 7 d, 14 d, 21 d, and 28 days respectively. The ratios of labeled RBCs were determined by flow cytometry. And further calculated the survival rates and lifespans of labeled RBCs infused to six C57 BL/6 recipient mice. Labeling in vivo: 1 mg/300 μL of biotin was, respectively, directly injected into another five C57 BL/6 mice for labeling RBCs in vivo.After 48 h, whole blood samples of the five C57 BL/6 mouse were collected and prepared into 25% labeled RBC suspensions, and total five samples were prepared.Two aliquots(500 μL/part) of labeled RBC suspension from each sample were combined with the fluorescent dye streptavidin-R-phycoerythrin(SA-R-PE, final concentration of 10 μg/mL) and Fluorescein-conjugated avidin(FITC-AV, final concentration of 30 μg/mL) and obtained RBCs labeled with SA-R-PE-biotin(PE-RBC) and FITC-AV-biotin RBCs(FITC-RBC). Then, the two single-stained RBCs were mixed in vitro and co-incubated for 4 hours at room temperature.The percentages of double-stained RBCs(PE-FITC-RBC)in the 5 mixed-incubated samples were detected by flow cytometry and the presence of double-stained RBCs was observed directly under confocal microscope. Results The 24-hour survival rate, half-life and mean potential life span of labeled RBCs infused into six C57 BL/6 recipient mice were(98.9±15.6)%,(17.2±2.4) d, and(34.2±4.3) d, respectively. The labeled RBCs decreased at a rate of(2.7±0.4) % daily. However, the percentages of labeled RBCs in 83%(5/6) animals were higher than the baseline values of 100%.After the two kinds of single-stained RBCs were mixed and co-incubated for 4 h in vitro,double-stained red blood cells(PE-FITC-RBC) were present in 5 specimens at a ratio of(0.8±0.5)%, and confocal microscopy results confirmed the presence of double-stained RBCs. Conclusion Labeled RBCs life spans are in the normal range after being infused into animals, but the RBC survival rate detected by biotin labeling method may fluctuate due to the phenomenon of shedding-relabeling.
Objective To discuss the reason that the red blood cell(RBC) survival rate measured by biotin labeling did not decrease with time. Methods Labeling in vitro: a C57 BL/6 donor mouse was anesthetized with 0.1 mL of 10% chloral hydrate, and then 0.8 mL blood was collected from the heart. RBCs resuspended to make 25% RBC suspension were labeled with biotin in vitro and divided into 6 parts, then infused respectively into six C57 BL/6 recipient mice. After transfusion, blood samples from six C57 BL/6 recipient mice were collected by tail vein on 1 h(basal value), 1 d, 3 d, 7 d, 14 d, 21 d, and 28 days respectively. The ratios of labeled RBCs were determined by flow cytometry. And further calculated the survival rates and lifespans of labeled RBCs infused to six C57 BL/6 recipient mice. Labeling in vivo: 1 mg/300 μL of biotin was, respectively, directly injected into another five C57 BL/6 mice for labeling RBCs in vivo.After 48 h, whole blood samples of the five C57 BL/6 mouse were collected and prepared into 25% labeled RBC suspensions, and total five samples were prepared.Two aliquots(500 μL/part) of labeled RBC suspension from each sample were combined with the fluorescent dye streptavidin-R-phycoerythrin(SA-R-PE, final concentration of 10 μg/mL) and Fluorescein-conjugated avidin(FITC-AV, final concentration of 30 μg/mL) and obtained RBCs labeled with SA-R-PE-biotin(PE-RBC) and FITC-AV-biotin RBCs(FITC-RBC). Then, the two single-stained RBCs were mixed in vitro and co-incubated for 4 hours at room temperature.The percentages of double-stained RBCs(PE-FITC-RBC)in the 5 mixed-incubated samples were detected by flow cytometry and the presence of double-stained RBCs was observed directly under confocal microscope. Results The 24-hour survival rate, half-life and mean potential life span of labeled RBCs infused into six C57 BL/6 recipient mice were(98.9±15.6)%,(17.2±2.4) d, and(34.2±4.3) d, respectively. The labeled RBCs decreased at a rate of(2.7±0.4) % daily. However, the percentages of labeled RBCs in 83%(5/6) animals were higher than the baseline values of 100%.After the two kinds of single-stained RBCs were mixed and co-incubated for 4 h in vitro,double-stained red blood cells(PE-FITC-RBC) were present in 5 specimens at a ratio of(0.8±0.5)%, and confocal microscopy results confirmed the presence of double-stained RBCs. Conclusion Labeled RBCs life spans are in the normal range after being infused into animals, but the RBC survival rate detected by biotin labeling method may fluctuate due to the phenomenon of shedding-relabeling.
引文
[1] Mock DM,Widness JA,Vengpedersen P,et al.Measurement of posttransfusion red cell survival with the biotin label.Transfus Med Rev,2014,28(3):114-125.
[2] Mock DM,tNalbant D,Kyosseva SV,et al.Development,validation,and potentialapplications of biotinylated red blood cells for posttransfusion kinetics and other physiological studies:evidenced-based analysis and recommendations.Transfusion,2018,58(8):2068-2081.
[3] Buchta C,Hanslik-Schnabel B,Weigl R,et al.Quality of drainageblood:Survival of red cells after re-transfusion and content of free hemoglobin and potassium.Int J Surg,2005,3(4):250-253.
[4] Liao XY,Zuo SS,Meng WT,et al.Intraoperative blood salvage may shorten the lifespan of red blood cells within 3 days postoperatively:a pilot study[J/OL].Medicine,2017,96(39).2017-09-29[2018-09-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626293/pdf/medi-96-e8143.pdf
[5] 杨健萍,叶铁真.红细胞寿命检测方法及其应用的研究进展.国际输血及血液学杂志,2016,39(5):428-433.
[6] Ma YJ,Zhang HD,Ji YQ,et al.A modified carbon monoxide breath test for measuring erythrocyte lifespan in small animals[J/OL].Biomed Res Int,2016,2016:7173156.2016-05-12[2018-09-02]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4880698/pdf/BMRI2016-7173156.pdf
[7] Nalbant D,Cancelas JA,Mock DM,et al.In premature infants there is no decrease in 24-hour posttransfusion allogeneic red blood cell recovery after 42 days of storage.Transfusion,2018,58(2):352-358.
[8] Kuruvilla DJ,Widness JA,Nalbant D,et al.Estimation of adult and neonatal RBC lifespans in anemic neonates using RBCs labeled at several discrete biotin densities.Pediatr Res,2017,81(6):905-910.
[9] International Committee for Standardization in Haematology.Recommended method for radioisotope red-cell survival studies.Br J Haematol,1980,45(4):659-666.
[10] Hoffmann-Fezer G,Mysliwietz J,M?rtlbauer W,et al.Biotin labeling as an alternative nonradioactive approach to determination of red cell survival.Ann Hematol,1993,67(2):81-87.
[11] Donald M.Mock MD PhD,Widness JA,Strauss R G,et al.Posttransfusion red blood cell (RBC) survival determined using biotin-labeled RBCs has distinct advantages over labeling with 51 Cr.Transfusion,2012,52(7):1596-1598.
[12] Suzuki T,Dale G L.Biotinylated erythrocytes:in vivo survival and in vitro recovery.Blood,1987,70(3):791-795.
[13] Mock DM,Lankford GL,Widness J A,et al.Measurement of circulating red cell volume using biotin-labeled red cells:validation against 51Cr-labeled red cells.Transfusion,2010,39(2):156-162.
[14] Dholakia U,Bandyopadhyay S,Hod E A,et al.Determination of RBC survival in C57BL/6 and C57BL/6-Tg(UBC-GFP) mice.Comp Med,2015.65(3):196-201.
[15] Widness JA,Kuruvilla DJ,Mock DM,et al.Autologous infant and allogeneic adult red cells demonstrate similar concurrent post-transfusion survival in very low birth weight neonates.J Pediatr,2015,167(5):1001-1006.
[16] An G,Widness JA,Mock DM,et al.A Novel physiology-based mathematical model to estimate red blood cell lifespan in different Human Age Groups.AAPS J,2016,18(5):1182-1191.
[17] Mock DM,Matthews NI,Zhu S,et al.Comparison of red blood cell survival in sheep determined using red blood cells labeled with either biotin at multiple densities or [14C]cyanate:validation of a model to study human physiology and disease.Transfusion,2012.52(5):963-973.
[18] 张逸伟,曾汉维.生物素合成的进展.华南理工大学学报(自然科学版),2001,29(2).58-65
[19] 徐常龙,严平,王琳,等.N-羟基琥珀酰亚胺生物素酯的合成与应用.化工中间体,2007(1):15-16.
[20] Schmidt RL,Mock DM,Franco RS,et al.Antibodies to biotinylated red blood cells in adults and infants:improved detection,partial characterization,and dependence on red blood cell-biotin dose.Transfusion,2017,57(6):1488-1496.
[21] Bogusiewicz A,Mock NI,Mock DM.Release of biotin from biotinylated proteins occurs enzymatically and nonenzymatically in human plasma.Anal Chem ,2004,331(2):260-266.
[2] Mock DM,tNalbant D,Kyosseva SV,et al.Development,validation,and potentialapplications of biotinylated red blood cells for posttransfusion kinetics and other physiological studies:evidenced-based analysis and recommendations.Transfusion,2018,58(8):2068-2081.
[3] Buchta C,Hanslik-Schnabel B,Weigl R,et al.Quality of drainageblood:Survival of red cells after re-transfusion and content of free hemoglobin and potassium.Int J Surg,2005,3(4):250-253.
[4] Liao XY,Zuo SS,Meng WT,et al.Intraoperative blood salvage may shorten the lifespan of red blood cells within 3 days postoperatively:a pilot study[J/OL].Medicine,2017,96(39).2017-09-29[2018-09-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626293/pdf/medi-96-e8143.pdf
[5] 杨健萍,叶铁真.红细胞寿命检测方法及其应用的研究进展.国际输血及血液学杂志,2016,39(5):428-433.
[6] Ma YJ,Zhang HD,Ji YQ,et al.A modified carbon monoxide breath test for measuring erythrocyte lifespan in small animals[J/OL].Biomed Res Int,2016,2016:7173156.2016-05-12[2018-09-02]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4880698/pdf/BMRI2016-7173156.pdf
[7] Nalbant D,Cancelas JA,Mock DM,et al.In premature infants there is no decrease in 24-hour posttransfusion allogeneic red blood cell recovery after 42 days of storage.Transfusion,2018,58(2):352-358.
[8] Kuruvilla DJ,Widness JA,Nalbant D,et al.Estimation of adult and neonatal RBC lifespans in anemic neonates using RBCs labeled at several discrete biotin densities.Pediatr Res,2017,81(6):905-910.
[9] International Committee for Standardization in Haematology.Recommended method for radioisotope red-cell survival studies.Br J Haematol,1980,45(4):659-666.
[10] Hoffmann-Fezer G,Mysliwietz J,M?rtlbauer W,et al.Biotin labeling as an alternative nonradioactive approach to determination of red cell survival.Ann Hematol,1993,67(2):81-87.
[11] Donald M.Mock MD PhD,Widness JA,Strauss R G,et al.Posttransfusion red blood cell (RBC) survival determined using biotin-labeled RBCs has distinct advantages over labeling with 51 Cr.Transfusion,2012,52(7):1596-1598.
[12] Suzuki T,Dale G L.Biotinylated erythrocytes:in vivo survival and in vitro recovery.Blood,1987,70(3):791-795.
[13] Mock DM,Lankford GL,Widness J A,et al.Measurement of circulating red cell volume using biotin-labeled red cells:validation against 51Cr-labeled red cells.Transfusion,2010,39(2):156-162.
[14] Dholakia U,Bandyopadhyay S,Hod E A,et al.Determination of RBC survival in C57BL/6 and C57BL/6-Tg(UBC-GFP) mice.Comp Med,2015.65(3):196-201.
[15] Widness JA,Kuruvilla DJ,Mock DM,et al.Autologous infant and allogeneic adult red cells demonstrate similar concurrent post-transfusion survival in very low birth weight neonates.J Pediatr,2015,167(5):1001-1006.
[16] An G,Widness JA,Mock DM,et al.A Novel physiology-based mathematical model to estimate red blood cell lifespan in different Human Age Groups.AAPS J,2016,18(5):1182-1191.
[17] Mock DM,Matthews NI,Zhu S,et al.Comparison of red blood cell survival in sheep determined using red blood cells labeled with either biotin at multiple densities or [14C]cyanate:validation of a model to study human physiology and disease.Transfusion,2012.52(5):963-973.
[18] 张逸伟,曾汉维.生物素合成的进展.华南理工大学学报(自然科学版),2001,29(2).58-65
[19] 徐常龙,严平,王琳,等.N-羟基琥珀酰亚胺生物素酯的合成与应用.化工中间体,2007(1):15-16.
[20] Schmidt RL,Mock DM,Franco RS,et al.Antibodies to biotinylated red blood cells in adults and infants:improved detection,partial characterization,and dependence on red blood cell-biotin dose.Transfusion,2017,57(6):1488-1496.
[21] Bogusiewicz A,Mock NI,Mock DM.Release of biotin from biotinylated proteins occurs enzymatically and nonenzymatically in human plasma.Anal Chem ,2004,331(2):260-266.