CN107238524B - 一种改良型特殊染色诊断液 - Google Patents
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Abstract
本发明提供了一种改良型特殊染色诊断液，旨在提供一种检测灵敏度高，检测结果可靠的用于宫颈癌筛查的检测试剂。检测时染色液中的醋酸可以使病变部位出现醋酸白化反应区，即可在醋白反应区域行阴道镜下活组织检查。该种方法利用亚甲蓝显色和醋酸白化反应双重定位及指示，提高宫颈/阴道/外阴癌前病变的检出率。本发明染色液接触空气时稳定性高，可避免快速氧化造成假阳性结果，充分保证检测结果准确性。该改良型特殊染色液，由下述重量百分比的组分制成：30％～70％医药级丙二醇、3％～7％冰醋酸、0.1％～5％维生素C、0.05％～5％叶酸、0.05％～5％乙酸钠、0.03％～0.5％亚甲基蓝、1％～10％果糖、1％～20％葡萄糖、0.1％～0.5％蔗糖脂肪酸酯、纯化水定容。
Description
技术领域
本发明涉及一种检测试剂，具体地说，属于特殊染色及酶组织化学染色诊断(液)，是一种活细胞染色剂；本发明还涉及该检测试剂的组成及制备方法。
背景技术
目前，宫颈癌筛查临床大量依赖实验室多项检查，如：液基细胞学检查(TCT)、HPV基因分型检测、阴道镜检查等、而上述检查之前，医生对患者的拟诊存在依据不清晰的问题，同时出报告时间长，检查费用较高，病人容易流失。用于上皮组织进行细胞特殊染色的方法主要有醋酸白化反应、碘试验、以及亚甲基蓝染色等。特殊染色液体外诊断疾病有很长的历史，是一种侵入式体外染色诊断方法，广泛用于宫颈上皮组织等常规病变的初筛。
有研究认为调节染色液采用的是乙酸，我们研究发现，单独调节乙酸的量难以被解决极易氧化的问题，并且要实现醋酸白化反应，必须保证3％～5％冰醋酸，否则不能实现棉签染色与组织醋酸白化反应区的双重定位。
3％～5％的醋酸溶液是很容易挥发的液体，在妇女病普查中一般先配制好放在大的密封良好的容器中，用时再分次少量倒入小的容器中，尽可能保持醋醋溶液的浓度，以防因挥发后浓度降低而影响肉眼观察的效果。同时因其在操作出可引起局部微微的烧灼感，所以在检查时被检查者不容易接受，引起不适。
液基细胞学检查出现假阴性的原因，与宫颈肥大、病变范围局限或较靠近穹隆处、取材者的经验、取材细胞量多少及取材时出血量多等情况有关。而改良型特殊染色诊断液试验则是一种相对简单、较少依赖操作仪器的检查方法，较为直观，用肉眼直接观察即可得出结果，阳性者可做阴道镜下活检进一步明确诊断，而且，其费用低廉，经济性和可行性较好，可应用于妇科门诊的每一位患者，因此，具有实用价值和推广前景。
目前市场该类特殊染色剂多以二甲基亚砜(DMSO)为主要溶剂，但研究表明，DMSO存在一定的毒性作用，与蛋白质疏水基团发生作用，导致蛋白质变性，具有血管毒性和肝肾毒性，能够灼伤皮肤并使皮肤有刺痛感，如同所见的皮疹及水泡一样，因该类染色剂需涂抹于人体组织，因此二甲基亚砜存在安全风险。
目前市场该类特殊染色剂氧化作用强，因此稳定性都难以解决，在临床使用需现配现用，其稳定性只有十几分钟，非常不方便，也不便于大范围筛查及门诊量大的临床检查。
发明内容
本发明主要解决的技术问题是提供一种改良型特殊染色诊断液，旨在提供一种检测灵敏度高，检测结果可靠的用于宫颈癌筛查的检测试剂。检测时染色诊断液中的醋酸可以使病变部位出现醋酸白化反应区，即可在醋白反应区域行阴道镜下活组织检查。该种方法利用亚甲蓝显色和醋酸白化反应双重定位及指示，提高宫颈/阴道/外阴癌前病变的检出率。
为解决上述技术问题，本发明提供了一种改良型特殊染色诊断液，所述染色诊断液技术方案通过下列实行。
其组成和配比由下述重量百分比组分制成：30％～70％医药级丙二醇、3％～5％冰醋酸、0.1％～5％维生素C、0.05％～5％叶酸、0.05％～5％乙酸钠、0.03％～0.5％亚甲基蓝、1％～10％果糖、1％～20％葡萄糖、0.3％～2.5％蔗糖脂肪酸酯、纯化水定容。
所述的医药级丙二醇粘性和吸湿性好，并且无毒；30％～70％医药级丙二醇和20％～80％纯化水的混合液可作为药物经皮肤或粘膜吸收的渗透促进剂，能延缓维生素C的水解，增加其稳定性。
所述的蔗糖脂肪酸酯具有表面活性，能降低表面张力，同时有良好的乳化、分散增溶、润滑、渗透、起泡、黏度调节、防止老化、抗菌等性能。
所述的染色诊断液PH值为5.0-5.5。
所述的制备方法依次包括下述步骤：
按权利要求1所述的重量百分数称取各个组分；
将步骤(1)称取的叶酸、蔗糖脂肪酸酯用少量纯化水溶解后加入医药级丙二醇中，搅拌溶解；
将维生素C加入步骤(2)溶液中，搅拌混匀；
将果糖、葡萄糖用适量纯化水溶解后加入步骤(3)溶液中，搅拌混匀；
将亚甲基蓝加入至步骤(4)溶液中，搅拌溶解后继续搅，20～30分钟；
将冰醋酸和乙酸钠混合溶液按加入至步骤(5)溶液中搅拌均匀，用纯化水定容即得。
所述的染色诊断液为淡黄棕色；当染色诊断液经棉签涂抹于宫颈组织后，如果有癌变细胞存在，叶酸复合物就会经过这些细胞表面的叶酸受体，迅速进入细胞浆，同时还原型的亚甲基蓝也被带入细胞浆内，乙酸促使细胞内环境为PH5.0-5.5，叶酸复合物、还原型亚甲基蓝与叶酸受体分离。游离的叶酸被还原成四氢叶酸，参与一碳单位的代谢过程中；还原型无色亚甲基蓝则被细胞内广泛存在的活性氧(ROS)氧化，变为氧化型亚甲基蓝，由于这些生物大分子的进入，使得细胞内的渗透压升高，致使氧化型亚甲基蓝逸出细胞外，则可在棉签上显现出颜色，如棉签无变色，则表明无病变细胞；如果棉签变色(紫黑色或蓝黑色)，则提示该部位细胞表面的叶酸受体数量异常增高，细胞内的活性氧增加；同时，染色剂中的冰醋酸也可以使病变部位出现醋酸白化反应区，即可在醋白反应区域行阴道镜下活组织检查；本染色液诊断利用亚甲基蓝显色和醋酸白化反应双重定位及指示，提高宫颈/阴道/外阴癌前病变的检出率。
所述的染色诊断液稳定性好，打开密封包装，24小时内不氧化变色，适合大范围筛查使用；优于市场其他同类染色剂。
我们通过361例临床样本验证，改良型特殊染色诊断液临床检出率与TCT、PCR等方法学具备等效性，改良型特殊染色诊断液使用更加方便，不需制片及上机检测前处理等复杂程序，临床值得推广使用。
具体实施方式
改良型特殊染色诊断液是叶酸受体介导的肿瘤筛查和早期诊断新技术，本发明解决了其稳定性差、易氧化、不便于使用的问题。下面结合具体实施方式对本发明的权利要求作进一步的详细说明，但本发明并不限于此，任何人在本发明权利要求范围内所做的有限次的修改仍在本发明权利要求保护范围之内。
实施例1
医药级丙二醇3.0g、冰醋酸0，3g、维生素C0.1g、叶酸0.1、乙酸钠0.1g、亚甲基蓝0.01g、果糖0.1、葡萄糖0，1、蔗糖脂肪酸酯0.03g、纯化水定容至1000ml(g)。
实施例2
医药级丙二醇4.0g、冰醋酸0.35g、维生素C0.2g、叶酸0.2、乙酸钠0.2g、亚甲基蓝0.02g、果糖0.2g、葡萄糖0.2g、蔗糖脂肪酸酯0.05g、纯化水定容至1000ml(g)。
实施例3
医药级丙二醇5.0g、冰醋酸0.4g、维生素C0.3g、叶酸0.3、乙酸钠0.3g、亚甲基蓝0.03g、果糖0.3g、葡萄糖0.3g、蔗糖脂肪酸酯0.06g、纯化水定容至1000ml(g)。
实施例4
医药级丙二醇6.0g、冰醋酸0.45g、维生素C0.4g、叶酸0.4g、乙酸钠0.4g、亚甲基蓝0.04g、果糖0.5g、葡萄糖0.5g、蔗糖脂肪酸酯0.1g、纯化水定容至1000ml(g)。
实施例5
医药级丙二醇7.0g、冰醋酸0.5g、维生素C0.5g、叶酸0.5g、乙酸钠0.5g、亚甲基蓝0.05g、果糖1.0g、葡萄糖1.5g、蔗糖脂肪酸酯0.2g、纯化水定容至1000ml(g)。
实施例1至5中任意一种改良型特殊染色诊断液制备方法，依次包括下述步骤：
按实施例1至5中要求所述的重量百分数称取各个组分；
将步骤(1)称取的叶酸、蔗糖脂肪酸酯用少量纯化水溶解后加入医药级丙二醇中，搅拌溶解；
将维生素C加入步骤(2)溶液中，搅拌混匀；
将果糖、葡萄糖用适量纯化水溶解后加入步骤(3)溶液中，搅拌混匀；
将亚甲基蓝加入至步骤(4)溶液中，搅拌溶解后继续搅，20～30分钟；
将冰醋酸和乙酸钠混合溶液按加入至步骤(5)溶液中搅拌均匀，用纯化水定容即得。
为了更好的说明本发明的优点，我们通过361例临床样本验证，改良型特殊染色诊断液临床检出率与TCT、PCR等方法学具备等效性，改良型特殊染色诊断液使用更加方便，不需制片及上机检测前处理等复杂程序，临床值得推广使用。
表1改良型特殊染色诊断液与TCT法、PCR法检查结果比较(％)
表1
表2宫颈检查阳性与阴道镜下宫颈活检符合率比较
表2
表3改良型染色诊断液与TCT法、PCR法检查结果比较(％)
表3
上述统计学按t检验方法，差异均无统计学意义(P＞0.05)。
改良型特殊染色诊断液使用方法：(1)用棉签蘸取溶液，然后用力在宫颈附近上皮组织表面涂抹3-5圈，最后在宫颈口按压3～5秒钟；(2)立即取出棉签，观察棉签的颜色变化(请务必在30秒内观察，切忌长时间反复观察)；(3)存留于上皮组织的多余液体，用干棉签擦干即可。
改良型特殊染色诊断液使用时对颜色的判读：(1)棉签无颜色变化，或者棉签变为淡绿色，提示上皮组织无异常病变；(2)棉签变为墨绿色或蓝色、黑色，提示上皮组织有异常病变，建议进一步体检；(3)宫颈隐性出血者，表现在棉签上是小黑点，此黑色不做判断依据。
本发明制备方法简单，无三废污染产生；使用中不产生需特殊处理的医疗垃圾。
Claims (5)
1.一种改良型特殊染色诊断液，其特征在于，其组成和配比由下述重量百分比组分制成：30％～70％医药级丙二醇、3％～5％冰醋酸、0.1％～5％维生素C、0.05％～5％叶酸、0.05％～5％乙酸钠、0.03％～0.5％亚甲基蓝、1％～10％果糖、1％～20％葡萄糖、0.3％～2.5％蔗糖脂肪酸酯、纯化水定容。
2.根据权利要求1所述的一种改良型特殊染色诊断液，其特征在于：所述的30％～70％医药级丙二醇和20％～80％纯化水的混合液可作为药物经皮肤或粘膜吸收的渗透促进剂，能延缓维生素C的水解，增加其染色诊断液稳定性。
3.根据权利要求1所述的一种改良型特殊染色诊断液，其特征在于：所述染色诊断液中0.3％～2.5％蔗糖脂肪酸酯具有表面活性，能降低表面张力，使染色诊断液同时有良好的乳化、分散增溶、润滑、渗透、起泡、黏度调节、防止老化、抗菌性能。
4.根据权利要求1所述的一种改良型特殊染色诊断液，其特征在于：所述的染色诊断液PH值为5.0-5.5。
5.根据权利要求1所述的一种改良型特殊染色诊断液，其特征在于，制备方法依次包括下述步骤：
(1)按权利要求1所述的重量百分数称取各个组分；
(2)将步骤(1)称取的叶酸、蔗糖脂肪酸酯用少量纯化水溶解后加入医药级丙二醇中，搅拌溶解；
(3)将维生素C加入步骤(2)溶液中，搅拌混匀；
(4)将果糖、葡萄糖用适量纯化水溶解后加入步骤(3)溶液中，搅拌混匀；
(5)将亚甲基蓝加入至步骤(4)溶液中，搅拌溶解后继续搅，20～30分钟；
(6)将冰醋酸和乙酸钠混合溶液按加入至步骤(5)溶液中搅拌均匀，用纯化水定容即得。
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