CN1276526A - Mcv分析中的稀释剂以及确保其具有不随时间变化的一致性的方法 - Google Patents
Mcv分析中的稀释剂以及确保其具有不随时间变化的一致性的方法 Download PDFInfo
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Abstract
一种MCV分析方法,它包括如下步骤:血样采集后立即用抗凝结剂处理;用一种含有预定渗透浓度的预定表面活性剂的试剂稀释采集后经抗凝结剂处理并且经过至少72小时时间的血样,以强制血样中的红血球发生形态变化;并使用颗粒分析仪分析该血样。该试剂包括一种非离子表面活性剂,一种用来调节渗透压的盐,和水,该试剂的渗透压(π)大约是150—400mOsm/kg,pH值是6.0—8.5。表面活性剂的浓度是0.0005%到0.5%,并具有10—20的亲水/亲油平衡(HLB)。
Description
本发明涉及分析血样中的平均红细胞容积(MCV),特别是涉及一种用颗粒分析仪在进行MCV分析的过程中使用的试剂和方法。
现在,临床检查领域广泛使用了自动血液分析仪。作为医疗诊断过程中对病人作检查的装置,它们被设计为可对血液成分进行快速分析。这些装置能够进行全部血球数(CBC)的多种分析,包括诸如红血球(RBC)数、白血球(WBC)数、白血球分类、血红蛋白浓度(Hb)、血细胞比容(Ht)和血小板数(PLT)这些项目。
MCV是从这些细胞分析值中计算得到重要项目中的一项。MCV可以由血细胞比容被红细胞数所除,就是Ht/RBC值而得到,血细胞比容就是一个单位容积的全血中红血球的百分含量。那么MCV就是单个红血球的平均容积,相应的其单位是fL、飞升、或10-15L。
上述血液分析仪采用流式血细胞计数器。在流式血细胞计数器中,稀释后的血液以高速通过一个非常细小的喷嘴导入流动槽,并且,喷嘴导出的液流被包覆液体(层流)的圆柱形流所包围。通过使该层流变窄,可以由此将样品流聚集至如下程度,即该样品流基本上呈一个细胞接一个细胞的线性序列。从而这些样品流中细胞的通过被该层流控制,通过采用激光照射液流,用光学方法检测该细胞,以及通过测量电阻和电导率,用电学方法检测该细胞。
为给上述的血液分析仪中使用的流式血细胞计数器分析制备全血样品,样品一般必须使用抗凝剂如EDTA盐处理,并用生理等渗溶液稀释。
作为用来稀释整个血液的稀释剂,一般有如下溶液例如生理盐水、林格式溶液(Ringer’s solution)、洛克式溶液(Lock’s solution)和台罗德式溶液(Tyrode’s solution)。在前述血液分析器的分析过程中也可以使用上述稀释剂。然而通常,每台分析仪选用最佳稀释剂。
在制备MCV分析的全血样品过程中,目前技术上存在的主要问题是实际的MCV会随血液采集后经历的时间而改变。临床诊断中MCV值是必要而又重要的,它应在血样采集后立即获得,也就是,该值应当与原始血液的MCV值相一致。
实践中,作为CBC一部分的MCV值直到采集血样72小时后才会被测量。而此时这样的血样再也不能产生适宜的MCV分析值了。血液采集后,样品中的RBC膨胀,使得经过采集后的经历时间所测得的MCV值渐渐大于原始RBC的平均容积。原始MCV才是诊断所需的真正测量。
日本专利公告8-33388(1996),日本专利公告7-82010(1995)和日本专利申请公开8-122327(1996)公开了各种含有非离子型表面活性剂的水溶液。然而,不管这些溶液中的哪一种用作流式血细胞计数器的包覆液,都需要在流动腔内加入非离子型表面活性剂以消除气泡。
另外,日本专利公告1-33780(1989)公开了一种稀释剂,在其中加入一种非离子型表面活性剂以抵销防腐剂对红细胞的影响,最终目的是在所测的MCV中抑制由于防腐剂而产生的稀释后的变化。然而,日本专利公告1-33780没有公开在所测的MCV中抑制因为血样采集后的经历时间而产生的变化的方法。
如果能找到一种在血液采集72小时后测得的血细胞比容和红细胞数与血样原始值一致的方法,这将是本领域中的重要进步。
本发明使得采集后的血液经过一段时间后分析仍能产生一个与该样品原始值非常一致的MCV值,也就是与采集后立刻测量的MCV值相一致。
根据本发明的试剂是一种血样的水稀释溶液,它包括在有限的浓度范围内加入少量的预定表面活性剂。用合适的物质将该试剂的渗透压调至预定范围。
用于本发明的试剂是一种水溶液,它包括非离子型表面活性剂和用于将渗透压(π)调至大约150-400mOsm/Kg的一种盐或合适的物质。
该试剂优选的渗透压(π)大约是230-350mOsm/Kg;特别优选的是大约260-320mOsm/Kg的渗透压(π)。本发明实施例中的非离子型表面活性剂的例子可以是下面任何一个。
(1)
R-O(CH2CH2O)nH
(2)
R-COO(CH2CH2O)nH
(3)R-COO(CH2CH2O)nCO-R
在本发明的一个优选的实施例中,非离子型表面活性剂包括至少一个烷基链,一个烯基链和一个炔基链,分别有12-24个碳原子。
该非离子型表面活性剂较好包括一个聚氧乙烯链,该链上有5-70摩尔加成数的氧乙烯。
作为具体例子,下面所述的非离子型表面活性剂中的任何一种都可用于根据本发明的实施例中。a)吐温-80(加入20摩尔PEG的脱水山梨醇油酸酯),Croda公司“Crillet 4”。b)Steareth-20(十八醇+PEG 20),Croda公司“Volpo S-20”。c)PEG-60杏仁甘油酯(有60摩尔PEG的杏仁甘油单甘油酯和杏仁二甘油酯),Croda公司“Crovol A-70”。d)PEG-23油酸(加入PEG23酯的油酸),Croda公司“crodet O 23”。
更优选的非离子型表面活性剂是Oleth-20(聚氧乙烯[20]油醚)。这种表面活性剂含有一个聚氧乙烯油醚的混合物,其中每分子中氧乙烯单元的平均数大约是20。
将CTFA Name作为上述提到物质的总称。
应明确,在本发明的范围内,也可使用此处限定的非离子型表面活性剂的混合物。也应当明确,假如在其它方面符合此处给定的参量,在市场上买到的非离子型表面活性剂也适用于本发明。众所周知,市场上买到的非离子型表面活性剂不是化学纯材料。因此,在前述参数都可满足的条件下,根据本发明的试剂中采用相关非离子型结构所引入的伴生物和无关的材料,也在它们的范围之内。
特别重要的是这些表面活性剂或混合物在盐溶液中是透明的。该非离子型表面活性剂必需在水中完全可溶,并在最小的0.1%的重量浓度产生透明溶液。
根据渗透压和作为溶剂的盐溶液的成分能够合理确定试剂中含有的给定非离子型表面活性剂的浓度。然而,一般非离子型表面活性剂在约0.0005%~0.5%的低浓度范围内使用,优选的浓度范围是约0.001%~0.1%,最优选是约0.005%~0.05%的范围。
根据本发明,在盐溶液中使用非离子型表面活性剂,以此来为血液分析稀释和稳定血样。
该非离子型表面活性剂优选具有10-20的亲水/亲油平衡值。
根据本发明试剂的PH值优选是6.0~8.5。
本发明给出的试剂还包括一个用以稳定其PH值的缓冲溶液,一个用以调节试剂溶液渗透压的添加剂,一种防腐物质,或一种抗氧化剂。不特别限制合适的缓冲溶液;然而磷酸盐缓冲溶液,硼酸盐缓冲溶液,三(羟甲基)氨基甲烷盐酸(tris)缓冲溶液,咪唑缓冲溶液等都是优选的。
不特别限定用于调整试剂PH值的合适物质,但例如,它们可以是盐酸或氢氧化钠。
不特别限定用于调整渗透压的合适物质,例如,可以使用至少一种选自氯化钠或氯化钾的碱金属盐或碱土金属盐。或者,也可使用象蔗糖、葡萄糖等糖类或聚乙二醇。
根据本发明的方法包括:a)抗凝处理步骤,其中用抗凝剂处理血样;b)稀释步骤,其中用含有表面活性剂的试剂溶液稀释经抗凝剂处理的血样,确保样品中的红血球形态发生变化;c)分析步骤,其中,血样在取血后经过例如至少48小时的时间后接着稀释步骤用颗粒分析仪进行分析。
本发明优选的一个方面是,在颗粒分析仪中基于电阻原理进行血液分析;层流电阻装置是更优选的。
该分析过程可以是这样一个过程,在经过至少72小时的时间后接着稀释步骤用颗粒分析仪分析血液。
分析前,全血需用抗凝剂进行处理。
一个标准的抗凝处理是,血液被采集后立即将适量例如EDTA盐的抗凝剂加到全血样品中去。
以下给出一个抗凝剂例子的分子式:
优选使用真空血液采集管获取样品,并优选是在血液采集时就用抗凝结剂处理该样品。
本发明的稀释溶液强迫血样红血球中的形态变化,并且与血样作用可高达至少72小时,因此能产生与那些样品采集后立即测定所取得的数据相一致的MCV结果。
本发明的作用机理还没有被证明。但已经发现使用调整到预定渗透压的溶液稀释血样并含有预表面活性剂,能迫使血样的红血球发生形态变化,就保持了所分析的MCV的一致性。该形态变化达到一定程度使得采集后经过一段时间后,测得的红血球的MCV值能与血样采集后立即测得的MCV值在诊断可接受的范围内相等。所以,本发明可提供贯穿血样整个诊断过程中一致的MCV值。
根据本发明,通过将新采集的血样和已经过72小时的血样悬浮到稀释溶液试剂中,并分别用显微镜检测样品,可证明该观点。在这两个样品中都发现了类似程度的裂口红细胞。
通过使用一种根据本发明的试剂,可以限制血样的MCV随时间的变化。在样品采集和其后的至少72小时之内,不同时间所测得的MCV分析的差异能被控制在大约±4(fL)内,在诊断可接受的范围内这是很好的。
本发明前述和其它的目的、特征、方面和优点从下面的详细描述中将更为清楚。实施例1
用EDTA-3K抗凝结剂处理血样。然后分别在血样刚采集后和采集后72小时使用Sysmex公司的自动血液分析仪SE-9500进行MCV分析。血样应在25℃下储存。试剂A(传统型)15mM磷酸盐缓冲溶液
用氯化钠将该试剂的渗透压调至大约250mOsm/Kg,和试剂B 15mM磷酸盐缓冲溶液;
0.015%的聚氧乙烯(20)油醚
用氯化钠将该试剂的渗透压调至大约320mOsm/Kg,用氢氧化钠将它的PH值调到大约7.8。
实施例2
样品1 平均细胞飞升容积(fL) | ||||
试剂 | 血液采集后的时间 | 变化 | 百分比 | |
立时 | 72小时 | |||
试剂A | 83.8(fL) | 97.1(fL) | +13.3(fL) | +15.9(%) |
试剂B | 88.2(fL) | 90.4(fL) | +2.2(fL) | +2.5(%) |
样品2 平均细胞飞升容积(fL) | ||||
试剂 | 血液采集后的时间 | 变化 | 百分比 | |
立时 | 72小时 | |||
试剂A | 89.7(fL) | 103.9(fL) | +14.2(fL) | +15.8(%) |
试剂B | 93.1(fL) | 96.0(fL) | +2.9(fL) | +3.1(%) |
样品3 平均细胞飞升容积(fL) | ||||
试剂 | 血液采集后的时间 | 变化 | 百分比 | |
立时 | 72小时 | |||
试剂A | 89.6(fL) | 103.9(fL) | +14.3(fL) | +16.0(%) |
试剂B | 93.9(fL) | 96.0(fL) | +2.1(fL) | +2.2(%) |
用EDTA-3K抗凝结剂处理血样。然后分别在血样刚采集后和采集后48小时使用Sysmex公司的自动血液分析仪SE-9500进行MCV分析。血样应在25℃下储存。试剂A 15mM磷酸盐缓冲溶液用氯化钠将该试剂的渗透压调至大约250mOsm/Kg,用氢氧化钠将它的PH值大约调到7.8。试剂C 15mM磷酸盐缓冲溶液;用氯化钠将该试剂的渗透压调至大约285mOsm/Kg,用氢氧化钠将它的PH值大约调到7.8。试剂D 15mM磷酸盐缓冲溶液;0.015%的聚氧乙烯(20)油醚
用氯化钠将该试剂的渗透压调至大约285mOsm/Kg,用氢氧化钠将它的PH值大约调到7.8。
平均细胞飞升容积(fL) | ||||
试剂 | 血液采集后的时间 | 变化 | 百分比 | |
12小时 | 48小时 | |||
试剂A | 93.5(fL) | 102.6(fL) | +9.1(fL) | +9.7(%) |
试剂C | 93.4(fL) | 103.7(fL) | +10.3(fL) | +11.0(%) |
试剂D | 92.7(fL) | 95.4(fL) | +2.7(fL) | +2.9(%) |
结果表明稀释剂渗透压的增加设有对采样后经过一段时间的血样MCV值变化产生控制。然而,稀释剂中非离子表面活性剂的加入对控制MCV的变化中被证明有很好的效果。实施例3
在实施例3中,采用通过改变试剂D的渗透压制得的试剂测量MCV值的变化。试剂渗透压可通过改变氯化钠的加入量来调节。血样应在25℃下储存。使用Sysmex公司的自动血液分析仪SE-9500分析样品以测定MCV值。试剂D: π=285mOsm/Kg(PH7.8)试剂E: π=250mOsm/Kg(PH7.8)试剂F: π=268mOsm/Kg(PH7.8)试剂G: π=300mOsm/Kg(PH7.8)试剂H: π=320mOsm/Kg(PH7.8)
实施例3-1 平均细胞飞升容积(fL) | ||||
试剂 | 血液采集后的时间 | 变化 | 百分比 | |
12小时 | 48小时 | |||
试剂A | 94.5(fL) | 103.7(fL) | +9.2(fL) | +9.7(%) |
试剂E(π250) | 93.4(fL) | 98.3(fL) | +4.9(fL) | +5.2(%) |
试剂D(π285) | 92.3(fL) | 94.2(fL) | +1.8(fL) | +2.0(%) |
实施例3-2 平均细胞飞升容积(fL)
试剂 | 血液采集后的时间 | 变化 | 百分比 | |
12小时 | 48小时 | |||
试剂A | 97.4(fL) | 106.7(fL) | +9.3(fL) | +9.5(%) |
试剂F(π268) | 95.4(fL) | 99.0(fL) | +3.6(fL) | +3.8(%) |
试剂D(π285) | 94.1(fL) | 94.3(fL) | +0.2(fL) | +0.2(%) |
实施例3-3 平均细胞飞升容积(fL) | ||||
试剂 | 血液采集后的时间 | 变化 | 百分比 | |
12小时 | 48小时 | |||
试剂A | 94.5(fL) | 103.4(fL) | +8.9(fL) | +9.4(%) |
试剂D(π285) | 92.3(fL) | 95.9(fL) | +3.6(fL) | +3.9(%) |
试剂G(π300) | 91.6(fL) | 95.0(fL) | +3.4(fL) | +3.7(%) |
试剂H(π320) | 91.6(fL) | 93.5(fL) | +1.9(fL) | +2.1(%) |
结果表明,通过将试剂的渗透压保持在260-320mOsm/kg的范围之内,MCV的变化可以控制在±4(fL)之内。在该渗透压范围之内,较高的渗透压对MCV变化有更强的控制力。实施例4
在实施例4中,将不同浓度的非离子表面活性剂聚氧乙烯(20)油醚加到试剂A中,通过采用由此制得的试剂测量MCV值的变化。血样应在25℃下储存。使用Sysmex公司的自动血液分析仪SE-9500分析样品以测定MCV值。试剂A: 0.000%非离子表面活性剂试剂E: 0.015%非离子表面活性剂试剂I: 0.150%非离子表面活性剂试剂J: 0.300%非离子表面活性剂
平均细胞飞升容积(fL) | ||||
浓度(%) | 血液采集后的时间 | 变化 | 百分比 | |
12小时 | 48小时 | |||
试剂A(0.000%) | 95.8(fL) | 105.9(fL) | +10.1(fL) | +10.5(%) |
试剂E(0.015%) | 95.4(fL) | 100.0(fL) | +5.6(fL) | +5.9(%) |
试剂I(0.150%) | 96.6(fL) | 100.9(fL) | +4.3(fL) | +4.5(%) |
试剂J(0.300%) | 96.2(fL) | 100.4(fL) | +4.2(fL) | +4.4(%) |
该结果表明宽浓度范围内的非离子表面活性剂对控制MCV值的变化比较有效。实施例5
通过在试剂E中加入大约0.015%的聚氧乙烯(20)油醚制备实施例5中的试剂,然后用下列物质将该稀释剂的渗透压调至285mOsm/Kg。
试剂D: 氯化钠
试剂K: 蔗糖
试剂L: 葡萄糖
试剂M: 聚乙二醇(分子量400)
在实施例5中测定用来调节试剂渗透压的上述物质对MCV变化的影响。血样应在25℃下储存。使用Sysmex公司的自动血液分析仪SE-9500分析样品以测定MCV值。
实施例5-1 平均细胞飞升容积(fL) | ||||
试剂 | 血液采集后的时间 | 变化 | 百分比 | |
12小时 | 48小时 | |||
试剂A | 94.7(fL) | 105.1(fL) | +10.4(fL) | +11.0(%) |
试剂D | 92.5(fL) | 94.8(fL) | +2.3(fL) | +2.5(%) |
试剂K | 91.6(fL) | 94.9(fL) | +3.3(fL) | +3.6(%) |
试剂L | 93.0(fL) | 95.8(fL) | +2.7(fL) | +2.9(%) |
实施例5-2 平均细胞飞升容积(fL) | ||||
试剂 | 血液采集后的时间 | 变化 | 百分比 | |
12小时 | 48小时 | |||
试剂A | 94.4(fL) | 104.7(fL) | +10.3(fL) | +10.9(%) |
试剂D | 92.8(fL) | 95.6(fL) | +2.8(fL) | +3.0(%) |
试剂M | 92.9(fL) | 93.6(fL) | +0.7(fL) | +0.8(%) |
一般用来调节渗透压的物质并不仅限于实施例5-1和实施例5-2中所给出的。然而,该结果表明盐、糖和聚氧乙烯油醚是比较好的。
选择这几个实施例仅是为了解释本发明,很明显,本领域的技术人员可以在该公开文本的基础上作出变化和修改,而不脱离由本发明附加权利要求和它们的等同物所限定的范围。
Claims (17)
1.一种用于MCV分析的血样稀释剂,所述的试剂是水溶液,它包括:
至少一种非离子表面活性剂;
和一种用于将该水溶液渗透压调至大约150~400mOsm/Kg的物质;其中
选择所述非离子表面活性剂和用于调节渗透压的物质,以使所述的稀释剂能确保采集后经过一段时间后的血样的MCV测定值与血样采集后立即测定所得的MCV值在诊断可接受的范围一致。
2.如权利要求1所述的血样稀释剂,其中渗透压调至大约230~350mOsm/Kg。
3.如权利要求2所述的血样稀释剂,其中渗透压调至大约260~320mOsm/Kg。
5.如权利要求5所述的血样稀释剂,其中所述的非离子表面活性剂是聚氧乙烯(20)油醚。
6.如权利要求1所述的血样稀释剂,其中所述的非离子表面活性剂具有10-20的亲水/亲油平衡值。
7.如权利要求1所述的血样稀释剂,其中所述的非离子表面活性剂在水溶液中的浓度是5.0×10-4%到5.0×10-1%。
8.如权利要求1所述的血样稀释剂,其中所述的稀释剂的PH值是6.0-8.5。
9.一种对保存高达至少72小时的血样进行MCV分析的方法,该方法包括:用抗凝剂处理所采集的血样的步骤;采用稀释剂稀释经抗凝剂处理的血样的步骤,目的在于确保血样中的红血球发生形态变化,而以便使采集后经过一段时间后的血样的MCV测定值与血样采集后立即测定所得的MCV值在诊断可接受的范围保持一致;和用颗粒分析仪分析血样获得MCV值的步骤。
10.如权利要求9所述的MCV分析方法,其中所述血样的分析步骤中,颗粒分析仪采用电阻原理。
11.如权利要求10所述的MCV分析方法,其中所述的颗粒分析仪是通过层流电阻原理分析的。
12.如权利要求9所述的MCV分析方法,其中稀释剂水溶液中含有至少一种非离子表面活性剂,以及一种用于将该水溶液的渗透压调至大约150-400mOsm/Kg的物质。
14.如权利要求13所述的MCV分析方法,其中非离子表面活性剂是聚氧乙烯(20)油醚。
15.如权利要求12所述的MCV分析方法,其中非离子表面活性剂具有10-20的亲水/亲油平衡值。
16.如权利要求12所述的MCV分析方法,其中非离子表面活性剂在水溶液中的浓度是5.0×10-4%到5.0×10-1%。
17.如权利要求12所述的MCV分析方法,其中稀释剂的PH值是6.0-8.5。
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-
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- 2000-05-30 AT AT00304578T patent/ATE315226T1/de not_active IP Right Cessation
- 2000-05-30 DE DE60025309T patent/DE60025309T2/de not_active Expired - Lifetime
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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CN101685050B (zh) * | 2008-09-26 | 2012-07-04 | 希森美康株式会社 | 血样稀释用试剂及用其测定平均红细胞容积的方法 |
CN101995455B (zh) * | 2009-08-14 | 2014-08-27 | 深圳迈瑞生物医疗电子股份有限公司 | 血细胞分析试剂、制备方法及稳定mcv的方法 |
CN104981691A (zh) * | 2013-03-12 | 2015-10-14 | 雅培实验室 | 用于分析红细胞和血小板的试剂、体系及方法 |
CN104981691B (zh) * | 2013-03-12 | 2020-03-31 | 雅培实验室 | 用于分析红细胞和血小板的试剂、体系及方法 |
Also Published As
Publication number | Publication date |
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EP1058114B1 (en) | 2006-01-04 |
DE60025309T2 (de) | 2006-08-24 |
EP1058114A3 (en) | 2001-12-12 |
ATE315226T1 (de) | 2006-02-15 |
EP1058114A2 (en) | 2000-12-06 |
JP2000356636A (ja) | 2000-12-26 |
US6225124B1 (en) | 2001-05-01 |
DE60025309D1 (de) | 2006-03-30 |
JP4086452B2 (ja) | 2008-05-14 |
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