CN107976397B - 一种溶血剂及其制备方法 - Google Patents
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Abstract
一种用于白细胞分类计数的溶血剂，含有阳离子表面活性剂，能够有效溶解红细胞，含有非离子表面活性剂可以协作阳离子表面活性剂裂解红细胞，能迅速破坏红细胞膜，并完全溶解红细胞使红细胞碎片体积减小以至不干扰白细胞的分析；加入三氟甲磺酸可以降低膜的延展性，进而导致红细胞渗漏和溶血，能够增强血样中白细胞的稳定性及其分散性，同时能够增强表面活性剂的溶血作用，相辅相成，使白细胞分类计数数值更准确；加入磷酸盐缓冲液和乙二胺四乙酸二钠，能够正确区分嗜碱性粒细胞和其他白细胞；加入氯化钠保持白细胞在等渗溶液中状态的完整性。本发明提供的溶血剂无毒环保，溶血效果好且溶血后血样分散性佳，便于后续操作，且对仪器无损伤，无需对废液进行无害化处理。
Description
本发明涉及化学和医学检验技术领域，具体涉及一种溶血剂的制备及其应用。
背景技术
化验血可以检查出许多疾病。
血液由血浆和血细胞组成。
血浆相当于结缔组织的细胞间质，为浅黄色半透明液体，其中除含有大量水分以外，还有无机盐、纤维蛋白原、白蛋白、球蛋白、酶、激素、各种营养物质、代谢产物等，这些物质无一定的形态，但具有重要的生理功能。
血细胞又称“血球”，是存在于血液中的细胞，能随血液的流动遍及全身。血细胞分为三类：红细胞、白细胞、血小板。红细胞：成熟的红血球没有细胞核及细胞器，含有血红素，主要的功能是运送氧；白细胞：主要扮演免疫的角色，负责破坏及移除年老或异常的细胞及细胞残骸，及攻击病原体及外来物体。当病菌侵入人体时，白细胞能穿过毛细血管壁，集中到病菌入侵部位，将病菌包围后吞噬；血小板：负责凝血，把纤维蛋白原变成纤维蛋白，结成网状聚集红血球形成血栓，阻止更多血液流失，并帮助阻止细菌进入体内。
白细胞亦称白血球，为无色圆形细胞，比红细胞略大，有细胞核。可分为中性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞、淋巴细胞和单核细胞五种。在机体发生炎症或疾病时，白细胞数往往发生变化，故白细胞分析是血液检验中最常采用的检测项目之一。白细胞计数和分类不仅对疾病的诊断、鉴别、疗效观察和预后分析均有重要意义，而且对健康状态的评估也有一定的价值。
白细胞中的嗜碱性粒细胞大小约为10一15μm，呈圆形或椭圆形，细胞核与细胞质之比约为1∶2一1∶3，细胞核分叶状，常被颗粒覆盖，核染色质聚集，无核仁，细胞质含粗大、致密、深紫色或黑色颗粒，在健康人体血液中嗜碱性粒细胞大约占白细胞总数的0.5-1％，嗜碱性粒细胞增多见于慢性粒细胞白血病、何杰金氏病、癌转移、铅中毒等。
采用瑞氏试剂对细胞染色并进行人工镜检是白细胞五分类计数的“金标准”，但是随着人们对临床检测要求的不断提高，这种耗时、繁琐的标准方法越来越不适应如今的临床检测。随着医学血液学的发展，各种类型的血细胞分析仪配合专用试剂(如溶血剂等)的检测方法在国内得到了广泛的应用，这些全自动的仪器通过专用试剂处理红细胞，然后采用流式技术或电阻抗的计数原理，通过获取白细胞不同细胞器发射出的荧光信息和散射光信息将白细胞分成不同种类。
然而，由于不同类白细胞的体积可能差别不大，电阻抗原理只是将白细胞简单分类成淋巴细胞，单核细胞和粒细胞，并不能将嗜碱性粒细胞和嗜酸性粒细胞区分计数。也有的仪器使用的是光散射原理，将淋巴细胞、单核细胞，中性粒细胞，嗜酸性粒细胞区分和计数，但由于嗜碱性粒细胞数量较少，这种方法也不能很好的检测到嗜碱性粒细胞。
现有技术中公开了多种用于白细胞分类的试剂，如乐家新【乐家新，一种用于血液白细胞五分类计数的溶血剂及其用途[P]，中国专利： CN201110074470.9，申请日：2011-03-28】涉及一种用于血液白细胞五分类计数的溶血剂及其用途。所述的溶血剂由溶血剂A与溶血剂B组成。所述的溶血剂A含有聚氧乙烯型非离子表面活性剂、有机酸、季胺盐阳离子表面活性剂助溶剂、稳定剂和渗透压调节剂；所述的溶血剂B含有碱性无机盐和渗透压调节剂。该溶血剂能够将白细胞分为淋巴细胞、单核细胞、嗜酸性粒细胞、嗜中性粒细胞和嗜碱性粒细胞，获得各类白细胞数量。虽然上述专利记载的技术方案可以有效区分各类白细胞，但是对于仅需要区分嗜碱性粒细胞和其他白细胞来说，其所采用的溶血剂配方较为复杂繁琐，组分较多，而且使用上也需要按照一定的操作步骤来，简便性较差。
溶血剂是血液细胞分析仪最常用的试剂之一，也是白细胞及各分群计数、含量测定的关键试剂。被稀释的血液加入溶血剂后，红细胞溶解，释放血红蛋白，后者与溶血剂中相关成分结合形成血红蛋白衍生物，进入血红蛋白检测系统，在特定波长(一般在530-550nm)下比色，吸光度的变化与液体中血红蛋白含量成正比，仪器便可显示其浓度同时，不同类型的白细胞淋巴细胞、单核细胞、中性粒细胞等经溶血剂作用后形成明显的差异，根据这些差异，仪器可以将血液样本中的白细胞分成几个类群，并对白细胞总数及各类群的数量进行统计。
冯剑军等公开了一种溶血剂及其应用和白细胞分类计数方法。这种溶血剂相比当下组分较为复杂的溶血剂产品使用更加方便快捷，溶血时间快，无需特殊温度要求即可配合电阻抗法测量仪器对嗜碱性粒细胞和其他白细胞的分类计数，但是分类计数数值精度较差。【冯剑军、朱漩、蒋玉林，一种溶血剂及其应用和白细胞分类计数方法，申请号：201410620796.0，申请日：2014-11-05】
对于临床上应用的溶血剂，现有技术有的溶血剂只适用于白细胞三分类，即把白细胞分为：淋巴细胞、单核细胞和中性粒细胞。无法区分嗜碱性粒细胞和嗜酸性粒细胞；有的溶血剂可以把白细胞分为五类：即淋巴细胞、单核细胞、中性粒细胞、嗜碱性粒细胞和嗜酸性粒细胞。但是这种溶血剂往往含有氰化物，对人体、环境都有毒害，排出的废液严重污染环境；有的溶血剂不含氰化物，但是白细胞五分类数值精度较差；国外进口原装溶血剂，效果较好，但是价格昂贵。能研究适用于我国，原材料立足于国产，效果与原装进口相当，或者优于进口试剂，对于白细胞五分类数值准确，白细胞总数结果满意的溶血剂是目前临床医学检验技术的当务之急。
发明内容
本发明的目的在于克服上述已有技术不足，提供一种用于白细胞分类计数的溶血剂，溶血速度快、使用方便，能够正确区分嗜碱性粒细胞和其他白细胞。溶血剂能迅速破坏红细胞膜，并完全溶解红细胞使红细胞碎片体积减小以至不干扰白细胞的分析。
为了实现上述目的，本发明提供如下技术方案：
一种溶血剂包含：以两种阳离子表面活性剂为主，它可以溶解红细胞，加入一种非离子表面活性剂协作阳离子表面活性剂溶解红细胞，对红细胞的溶解彻底，作用迅速，对白细胞的检测无干扰；加入三氟甲磺酸保持白细胞的完整性，使白细胞分类计数数值更准确；加入磷酸盐缓冲液，使白细胞的形状完好，有利于白细胞分类；加入乙二胺四乙酸二钠，更好的区分嗜碱性粒细胞和嗜酸性粒细胞；加入氯化钠保持白细胞在等渗溶液中状态的完整性。
一种溶血剂的制备，质量百分比是：
上述所有组分之和等于100％。
各原料组分的加入顺序及操作方法为：
A：取水加入十八烷基三甲基氯化铵、月桂酰基三甲基溴化铵、聚乙二醇辛基苄基醚，搅拌使完全溶解。
B：取水加入磷酸氢二钾、磷酸二氢钠、氯化钠、乙二胺四乙酸二钠，搅拌使完全溶解。
C：把B倒入A中，三氟甲磺酸调pH＝7.35-7.45，0.45μm滤膜过滤。
实施例一
上述所有组分之和等于100。
配制方法：
A：取水量的二分之一加入十八烷基三甲基氯化铵、月桂酰基三甲基溴化铵、聚乙二醇辛基苄基醚，搅拌使完全溶解。
B：取剩余的水加入磷酸氢二钾、磷酸二氢钠、氯化钠、乙二胺四乙酸二钠，搅拌使完全溶解。
C：把B倒入A中，三氟甲磺酸调pH＝7.35，0.45μm滤膜过滤。测电导率＝12.11 Sm/cm；
实施例二
上述所有组分之和等于100。
配制方法：
A：取水量的二分之一加入十八烷基三甲基氯化铵、月桂酰基三甲基溴化铵、聚乙二醇辛基苄基醚，搅拌使完全溶解。
B：取剩余的水加入磷酸氢二钾、磷酸二氢钠、氯化钠、乙二胺四乙酸二钠，搅拌使完全溶解。
C：把B倒入A中，三氟甲磺酸调pH＝7.40，0.45μm滤膜过滤。测电导率＝12.21 Sm/cm；
实施例三
上述所有组分之和等于100。
配制方法：
A：取水量的二分之一加入十八烷基三甲基氯化铵、月桂酰基三甲基溴化铵、聚乙二醇辛基苄基醚，搅拌使完全溶解。
B：取剩余的水加入磷酸氢二钾、磷酸二氢钠、氯化钠、乙二胺四乙酸二钠，搅拌使完全溶解。
C：把B倒入A中，三氟甲磺酸调pH＝7.38，0.45μm滤膜过滤。测电导率＝12.09 Sm/cm；
实施例四
上述所有组分之和等于100。
配制方法：
A：取水量的二分之一加入十八烷基三甲基氯化铵、月桂酰基三甲基溴化铵、聚乙二醇辛基苄基醚，搅拌使完全溶解。
B：取剩余的水加入磷酸氢二钾、磷酸二氢钠、氯化钠、乙二胺四乙酸二钠，搅拌使完全溶解。
C：把B倒入A中，三氟甲磺酸调pH＝7.42，0.45μm滤膜过滤。测电导率＝12.11 Sm/cm；
实施例五
上述所有组分之和等于100。
配制方法：
A：取水量的二分之一加入十八烷基三甲基氯化铵、月桂酰基三甲基溴化铵、聚乙二醇辛基苄基醚，搅拌使完全溶解。
B：取剩余的水加入磷酸氢二钾、磷酸二氢钠、氯化钠、乙二胺四乙酸二钠，搅拌使完全溶解。
C：把B倒入A中，三氟甲磺酸调pH＝7.45，0.45μm滤膜过滤。测电导率＝12.03 Sm/cm；
实施例六
上述所有组分之和等于100。
配制方法：
A：取水量的二分之一加入十八烷基三甲基氯化铵、月桂酰基三甲基溴化铵、聚乙二醇辛基苄基醚，搅拌使完全溶解。
B：取剩余的水加入磷酸氢二钾、磷酸二氢钠、氯化钠、乙二胺四乙酸二钠，搅拌使完全溶解。
C：把B倒入A中，三氟甲磺酸调pH＝7.35，0.45μm滤膜过滤。测电导率＝12.34 Sm/cm；
本发明中，通过利用阳离子表面活性剂，使用量少但对红细胞的裂解能力好，降低膜的延展性，进而导致细胞渗漏和溶血。本发明采用三氟甲磺酸作为溶液酸碱调节剂，能够增强溶血后血样的稳定性及其分散性，同时能够增强表面活性剂的溶血作用，相辅相成，减少操作时间。本发明提供的溶血剂无毒环保，溶血效果好且溶血后血样分散性佳，便于后续操作，且对仪器无损伤，无需对废液进行无害化处理。
表1 本发明与进口原装溶血剂的对比试验
从上表可以看出，本发明的溶血剂与进口原装溶血剂在白细胞总数，以及白细胞五分类的数值测定结果与进口试剂比较无显著性差异，完全可以替代进口试剂。
附：
白细胞分类计数正常值参考范围
化验结果临床意义
(1)中性粒细胞：增多见于急性和化脓性感染、各种中毒(酸中毒、尿毒症、铅中毒等)，组织损伤、恶性肿瘤、急性大出血、急性溶血等。减少见于伤寒、副伤寒、麻疹、流感等传染病；化疗、放疗。再生障碍性贫血、自身免疫性疾病等。
(2)嗜酸粒细胞：增多见于过敏性疾病、皮肤病、寄生虫病、某些血液病，射线照射后、脾切除术后、传染病恢复期等。减少见于伤寒、应用糖皮质激素、促肾上腺皮质激素等。
(3)淋巴细胞：增多见于某些传染病(百日咳、水痘、麻疹、风疹、流行性腮腺炎、病毒性肝炎、淋巴细胞性白血病和淋巴瘤等)。减少见于多种传染病的急性期，免疫缺陷病等。
(4)单核细胞：增多见于结核病、伤寒，感染性心内膜炎、疟疾、单核细胞白血病、黑热病及传染病的恢复期等。
(5)嗜碱粒细胞：多见于慢性粒细胞性白血病、嗜碱粒细胞白血病、霍奇金病、脾切除术后等。
Claims (2)
1.一种溶血剂，其特征在于：
溶血剂的组分为：
上述所有组分之和等于100％。
2.根据权利要求1所述溶血剂的制备方法，其特征在于：各原料组分的加入顺序及操作方法为：
A：取水加入十八烷基三甲基氯化铵、月桂酰基三甲基溴化铵、聚乙二醇辛基苄基醚，搅拌使完全溶解；
B：取水加入磷酸氢二钾、磷酸二氢钠、氯化钠、乙二胺四乙酸二钠，搅拌使完全溶解；
C：把B倒入A中，三氟甲磺酸调pH＝7.35-7.45，0.45μm滤膜过滤。
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