流式细胞术快速测定红细胞渗透脆性的应用

doi:10.3969/j.issn.1673-8640.2016.010.017

摘要/Abstract

摘要：

目的建立流式细胞术快速检测红细胞渗透脆性的方法,评估流式细胞仪测定红细胞渗透脆性的可能性。方法收集30例抗凝外周血,每例标本均一分为二,1份在采集2 h内检测（正常对照组）,另1份经37 ℃孵育24 h（阳性对照组）;另收集3例临床确诊的遗传性球形红细胞增多症患者作为疾病组。分别使用流式细胞术检测各组红细胞渗透脆性。通过流式细胞术测定红细胞残留百分比确定生理盐水和无菌水最佳体积比,确定乙二胺四乙酸（EDTA）和肝素2种抗凝剂对其的影响。另收集14例肝素抗凝标本,同时采用改良Sanford法和流式细胞术测定红细胞残留百分比,对比评价流式细胞术和改良Sanford法测定红细胞渗透脆性的特点。结果采用流式细胞术检测正常对照组的红细胞残留百分比为72.03% ± 5.68%,明显高于阳性对照组（46.47% ± 15.83%,P<0.05）;疾病组红细胞残留百分比为5.76% ± 3.54%,明显低于正常对照组（P<0.001）。当生理盐水和无菌水体积比为1.2∶0.8时,正常对照组和阳性对照组红细胞残留百分比差异最大（P<0.05）;与EDTA抗凝剂比较,肝素抗凝的外周血更适合于红细胞渗透脆性检测。流式细胞术与改良Sanford法测定红细胞渗透脆性有较好的一致性,但流式细胞术敏感性更高。结论流式细胞术测定红细胞渗透脆性具有操作简便、敏感性更好等特点,有实际应用的可行性。

关键词: 红细胞渗透脆性, 流式细胞术, Sanford法

Abstract:

Objective To establish a rapid determination of erythrocyte osmotic fragility by flow cytometry,and to evaluate the feasibility of determining erythrocyte osmotic fragility by flow cytometry. Methods A total of 30 anticoagulation peripheral blood specimens were collected,and each specimen was divided into 2 parts,one group collected within 2 h as normal control group and another group incubated at 37 ℃ for 24 h as positive control group. A total of 3 patients diagnosed as hereditary spherocytosis were enrolled as disease group. The erythrocyte osmotic fragility was determined by flow cytometry. The residue percentage of red blood cells was analyzed by flow cytometry. The optimal volume ratio of normal saline and sterile water was identified. The influence of ethylene diamine tetraacetic acid（EDTA） and heparin anticoagulants was evaluated. A total of 14 cases of heparin anticoagulation specimens were collected,and the red blood cell residue percentage was determined by improved Sanford and flow cytometry simultaneously. The characterization of determining erythrocyte osmotic fragility by flow cytometry and improved Sanford was evaluated.Results By flow cytometry,red blood cell residue percentages were 72.03% ± 5.68% in normal control group and 46.47% ± 15.83% in positive control group（P<0.05）. The disease group showed a significant reduction in red blood cell residue percentage（5.76% ± 3.54%） compared to that in normal control group（P<0.001）. When the volume ratio of normal saline to sterile water was 1.2∶0.8,there was significant difference of red blood cell residue percentage between the 2 groups（P<0.05）. Compared with EDTA anticoagulant,heparin anticoagulant was more suitable for the determination of erythrocyte osmotic fragility. There was a good consistency between flow cytometry and improved Sanford in the determination of erythrocyte osmotic fragility,but flow cytometry had better sensitivity than improved Sanford. Conclusions Flow cytometry has the characteristics of operation simplicity and sensitivity,and has a practical application feasibility in the determination of erythrocyte osmotic fragility.

Key words: Erythrocyte osmotic fragility, Flow cytometry, Sanford

中图分类号:

R446.1

王维维, 袁向亮, 费奇力, 沈立松. 流式细胞术快速测定红细胞渗透脆性的应用[J]. 检验医学, 2016, 31(10): 911-916.

WANG Weiwei, YUAN Xiangliang, FEI Qili, SHEN Lisong. Application of flow cytometry for the rapid determination of erythrocyte osmotic fragility[J]. Laboratory Medicine, 2016, 31(10): 911-916.

图/表 7

表1 改良Sanford法采用的NaCl溶液浓度梯度

项目	1	2	3	4	5	6	7	8	9	10	11	12	13
9 g/L NaCl（mL）	4.25	3.75	3.50	3.25	3.00	2.75	2.50	2.25	2.00	1.75	1.50	1.00	0.50
无菌水（mL）	0.75	1.25	1.50	1.75	2.00	2.25	2.50	2.75	3.00	3.25	3.50	4.00	4.50
NaCl（g/L）	8.50	7.50	7.00	6.50	6.00	5.50	5.00	4.50	4.00	3.50	3.00	2.00	1.00

图1 流式细胞术测定红细胞渗透脆性的设门方法

图2 流式细胞术检测肝素抗凝新鲜外周血标本和经37 ℃孵育24 h的标本的结果

图3 正常对照组、阳性对照组和疾病组红细胞残留百分比的比较

图4 流式细胞术测定红细胞渗透脆性的条件优化

图5 抗凝剂对红细胞残留百分比的影响红细胞残留百分比（%）

表2 Sanford 法与流式细胞术测定红细胞渗透脆性结果的比较

改良Sanford法	流式细胞术
改良Sanford法	+	-
+	7	0
-	4	3

参考文献 16

[1]	WARANG P,GUPTA M,KEDAR P,et al.Flow cytometric osmotic fragility-an effective screening approach for red cell membranopathies[J]. Cytometry B Clin Cytom,2011,80(3):186-190.
[2]	熊立凡. 临床检验基础[M]. 3版. 北京:人民卫生出版社,2005:8.
[3]	GOLAFSHAN HA,RANJBARAN R,KALANTARI T,et al.Evaluation of red cell membrance cytoskeletal disorders using a flow cytometric method in South Iran[J]. Turk J Hematol,2014,31(1):25-31.
[4]	CHESNEY A,GOOD D,REIS M.Clinical utility of flow cytometry in the study of erythropoiesis and nonclonal red cell disorders[J]. Methods Cell Biol,2011,103:311-332.
[5]	SANCHEZ-PEREZ A,BROWN G,MALIK R,et al.Rapid detection of haemotropic mycoplasma infection of feline erythocytes using a novel flow cytometric approach[J]. Parasit Vectors,2013,6:158.
[6]	LELLIOTT PM,LAMPKIN S,MCMORRAN BJ,et al.A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo[J]. Malar J,2014,13:100.
[7]	KING MJ,JEPSON MA,GUEST A,et al.Detection of hereditary pyropoikilocytosis by the eosin-5-maleimide(EMA)-binding test is attributable to a marked reduction in EMA-reactive transmembrane proteins[J]. Int J Lab Hematol,2011,33(2):205-211.
[8]	STOYA G,GRUHN B,VOGELSANG H,et al.Flow cytometry as a diagnostic tool for hereditary spherocytosis[J]. Acta Haematol,2006,116(3):186-191.
[9]	KING MJ,TELFER P,MACKINNON H,et al.Using the eosin-5-maleimide binding test in the differential diagnosis of hereditary spherocytosis and hereditary pyropoikilocytosis[J]. Cytometry B Clin Cytom, 2008,74(4):244-250.
[10]	TACHAVANICH K,TANPHAICHITR VS,UTTO W,et al.Rapid flow cytometric test using eosin-5-maleimide for diagnosis of red blood cell membrane disorders[J]. Southeast Asian J Trop Med Public Health,2009,40(3):570-575.
[11]	WON DI,SUH JS.Flow cytometric detection of erythrocyte osmotic fragility[J]. Cytometry B Clin Cytom,2009,76(2):135-141.
[12]	MOHANDAS N,GALLAGHER PG.Red cell membrane:past,present,and future[J]. Blood,2008,112(10):3939-3948.
[13]	BARCELLINI W,BIANCHI P,FERMO E,et al.Hereditary red cell membrane defects:diagnostic and clinical aspects[J]. Blood Transfus,2011,9(3):274-277.
[14]	张红军,徐酉华. 遗传性球形红细胞增多症并自身免疫性溶血性贫血1例[J]. 实用儿科临床杂志,2008,23(9):713.
[15]	CRISP RL,SOLARI L,VOTA D,et al.A prospective study to assess the predictive value for hereditary spherocytosis using five laboratory tests (cryohemolysis test, eosin-5'-maleimide flow cytometry,osmotic fragility test,autohemolysis test,and SDS-PAGE) on 50 hereditary spherocytosis families in Argentina[J]. Ann Hematol,2011,90(6):625-634.
[16]	TAO YF,DENG ZF,LIAO L,et al.Comparison and evaluation of three screening tests of hereditary spherocytosis in Chinese patients[J]. Ann Hematol,2015,94(5):747-751.