WO2024050856A1 - 一种辅助血细胞分析仪判定异常血小板聚集的方法和系统 - Google Patents

一种辅助血细胞分析仪判定异常血小板聚集的方法和系统 Download PDF

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WO2024050856A1
WO2024050856A1 PCT/CN2022/118419 CN2022118419W WO2024050856A1 WO 2024050856 A1 WO2024050856 A1 WO 2024050856A1 CN 2022118419 W CN2022118419 W CN 2022118419W WO 2024050856 A1 WO2024050856 A1 WO 2024050856A1
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platelet aggregation
blood cell
cell analyzer
threshold
sample
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PCT/CN2022/118419
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高天翼
梅燕萍
何帮顺
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南京市第一医院
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/483—Physical analysis of biological material
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    • G01N33/49—Blood

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  • the present invention relates to the field of in vitro diagnostic technology, particularly a method and system for assisting a blood cell analyzer in determining abnormal platelet aggregation.
  • Platelets are an important component of blood cells and have the functions of adhesion, aggregation, release, procoagulation, and contraction of blood clots. They can also maintain the integrity of the vascular endothelium to achieve anticoagulant effects.
  • the number of platelets directly affects the patient's hemostatic function. Platelet count is the basis for the reliability of platelet parameters, and the accuracy of its test results is crucial. There are many factors that affect the accuracy of platelet count. Platelet aggregation is one of the more common interference factors, which can lead to false platelet count (PTCP). PTCP can occur in healthy or diseased individuals of different ages and genders.
  • Existing automatic hematology analyzers often have high sensitivity indicators but relatively low specificity indicators.
  • the automatic blood cell analyzer prompts an alarm for abnormal platelet aggregation
  • the inspector often contacts the patient and re-draws blood using a different anticoagulant blood collection tube to correct the impact of platelet aggregation on the platelet count.
  • the test result was negative after re-collecting blood. This results in a waste of test reagents and consumables, and also greatly delays the time for patients to obtain reports and seek medical treatment in a timely manner.
  • the object of the present invention is to provide a method and system for assisting a blood cell analyzer in determining abnormal platelet aggregation, so as to improve the problem of low specificity indicators of existing automatic blood cell analyzers.
  • a method for assisting a blood cell analyzer in determining abnormal platelet aggregation including the following steps:
  • the blood cell analyzer alarms abnormally and reads the platelet count of the instrument b per liter;
  • the threshold value of platelet aggregation index c is 21.36.
  • the number of fields of view under the microscope oil immersion field is n ⁇ 100.
  • the blood cell analyzer is an electrical impedance method blood cell analyzer.
  • the present invention also provides a system for using an auxiliary blood cell analyzer to determine abnormal platelet aggregation, including a blood cell analyzer module, a microscopy module and a threshold judgment module; the blood cell analyzer module is used to alarm and read abnormal platelet aggregation. Get the platelet count value a of the instrument; the microscopy module is used to microscopy the sample with abnormal platelet aggregation alarm, and read the microscopic platelet aggregation value a and the field of view number n under the microscope oil immersion field; the threshold judgment module is used to calculate platelets Aggregate the index c and determine the threshold.
  • the significant advantages of the present invention are: by calculating the platelet aggregation index c, the error rate of the abnormal platelet aggregation alarm of the blood cell analyzer can be significantly reduced, the test reagent consumables are saved, the test efficiency is improved, and it is an improvement over the current platelet aggregation alarm. A useful addition to counting methods.
  • Figure 1 is a schematic flow chart of the working method of the present invention.
  • Figure 2 is a distribution diagram of the relationship between platelet count and platelet aggregation in the present invention.
  • Figure 3 is a comparison chart of the corrected platelet aggregation index c of the present invention between the positive and negative control groups.
  • Figure 4 is a diagnostic curve chart for abnormal platelet aggregation.
  • Figure 5 is a schematic diagram of the platelet alarm of the blood cell analyzer:
  • Figure 5A is a schematic diagram of the platelet alarm of Sysmex XN;
  • Figure 5B is a schematic diagram of the platelet alarm of BC7500.
  • Example 1 collects 55 randomly selected platelet aggregation alarm samples from inpatients in Nanjing First Hospital within 190 days from February 2022 to early July 2022.
  • Example 2 collects 20 clinical Sysmex XN and Mindray BC7500 blood cell analyzer platelet aggregation alarm samples from inpatients in Nanjing First Hospital in early August 2022.
  • sample basic information and relevant blood routine data including patient age, gender, diagnosis, platelet count value in routine blood test, and corrected platelet count value after re-collection.
  • Use Mindray's blood cell analyzer BC-7500 or Sysmex's blood cell analyzer XN-2800 fully automatic blood cell analyzer for detection, and use the reagents and calibrators provided by the instrument.
  • Platelet detection is performed on the instrument after the hospital inspection LIS system number, directly put it into the test tube rack, and place it on Mindray's blood cell analyzer BC-7500 or Sysmex's blood cell analyzer XN-2800 fully automatic blood cell analysis Instrument detection. As shown in Figure 5, when the test detects platelet aggregation in the blood, the instrument automatically alarms for platelet aggregation (PLT Clumps).
  • the instrumented platelet count per liter b less than 100 ⁇ 10 9 is often used as an indicator of low platelet samples and clinical bleeding risk (less than 100 ⁇ 10 9 /L is a clinical intervention or patient diagnosis and treatment critical value), so the positive samples and negative samples per liter of instrument platelet count b are uniformly corrected to the platelet aggregation index c when the platelet count is 100 ⁇ 10 9.
  • the final formula is as follows:
  • the platelet aggregation index c required for final statistical verification was calculated according to the above formula. By comparing the positive sample with the negative control, it was found that the positive platelet aggregation index c was significantly higher than the negative control, as shown in Figure 3 (p ⁇ 0.05).
  • the platelet aggregation index c receiver operating characteristic curve (ROC) was drawn, as shown in Figure 4 , the c value is statistically significant, and the area under the receiver operating characteristic curve (AUC) is obtained.
  • YI (sensitivity + specificity) -1
  • the optimal cut off value is 21.36
  • the area under the curve is 99.4%.
  • the sensitivity is 92.3% and the specificity is 86.2%.
  • the effect of the present invention is verified using the platelet alarm specimen of the electrical impedance method blood cell analyzer.
  • the electrical impedance method blood cell analyzers used in this embodiment are Sysmex XN and Mindray BC7500 blood cell analyzers. All other manufacturers and models of blood cell analyzers can be judged by this method.
  • the blood cell analyzer alarms abnormally and reads the platelet count of the instrument b per liter;
  • the platelet values obtained in the first blood cell analysis instrument are all lower than 100 ⁇ 10 9 /L, which belongs to the clinical intervention value.
  • the results measured by the method adopted in the present invention are consistent with Existing clinical retesting standards are consistent.
  • the platelet value obtained in the first blood cell analysis instrument is higher than 300 ⁇ 10 9 /L, which belongs to the clinical intervention value.
  • the results measured by the method adopted in the present invention are also consistent with the existing clinical re-measurement standards. consistent.
  • the method for judging abnormal platelet aggregation through the auxiliary blood cell analyzer provided by the invention can judge the alarm of existing blood cell analysis instruments, has a wide trial range, is a useful supplement to the existing re-examination rules, saves reagent costs, and reduces the risk of Pain for patients and improve laboratory testing efficiency.

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Abstract

一种辅助血细胞分析仪判定异常血小板聚集的方法和系统。该方法包括如下步骤：S1、取血液样本放置入血细胞分析仪进行血小板计数；S2、血细胞分析仪异常报警，读取仪器血小板计数的每升值b；S3、取报警样本制作血涂片并进行镜检，读取镜检血小板聚集数值a和显微镜油镜视野下的视野数n；S4、根据如下公式计算血小板聚集指数c：c=( a×10 14) /(b×n)；S5、设定c的阈值，当c大于阈值时，判定样本为阳性；当c值≤阈值时，判定样本为阴性。该方法与现有技术相比，其显著优点是：通过计算血小板聚集指数c，可以显著降低血细胞分析仪异常血小板聚集报警的出错率，节省了检验试剂耗材，提高了检验效率，是对当前血小板计数方法的有益补充。

Description

一种辅助血细胞分析仪判定异常血小板聚集的方法和系统 技术领域
本发明涉及体外诊断技术领域，特别是一种辅助血细胞分析仪判断异常血小板聚集的方法和系统。
背景技术
血小板是血细胞的重要组成部分，具有粘附、聚集、释放、促凝和使血块收缩的功能，并且可维持血管内皮的完整性，从而达到止凝血的功效。血小板的数量直接影响患者的止凝血功能。血小板计数是血小板参数可靠性的基础，其检测结果准确性至关重要。血小板计数准确性的影响因素很多，血小板聚集是较常见的干扰因素之一，会导致血小板的假性减少(PTCP)。PTCP可以发生在不同年龄和性别的健康或患病个体中。现有的自动血细胞分析仪往往敏感度指标较高，但特异性指标相对较低。
文献1：孟令章.Sysmex NX-1000血细胞分析仪血小板聚集报警信息分析.医学研究与教育.2021年4月第38卷第2期.血小板聚集报警的敏感性为98.15％，特异性为63.5％。
当自动血细胞分析仪提示血小板聚集异常报警时，检验员常采取联系患者使用不同抗凝剂采血管重新采血，以纠正血小板聚集对血小板计数的影响。但因现有的血细胞分析仪特异性比较低，因此，采用重新采血后，其检测结果为阴性。从而造成检验试剂耗材的浪费，同时也极大地延迟了患者获取报告及时就医的时间。
因此，临床上亟需建立一套自动血细胞分析仪辅助判断异常血小板聚集的方法。
发明内容
本发明的目的在于一种辅助血细胞分析仪判断异常血小板聚集的方法和系统，以提高现有自动血细胞分析仪特异性指标较低的问题。
实现本发明目的的技术解决方案如下：
一种辅助血细胞分析仪判断异常血小板聚集的方法，包括如下步骤：
S1、取血液样本放置入血细胞分析仪进行血小板计数；
S2、血细胞分析仪异常报警，读取仪器血小板计数的每升值b；
S3、取报警样本制作血涂片并进行镜检，读取镜检血小板聚集数值a和显微镜油镜视野下的视野数n；
S4、根据如下公式计算血小板聚集指数c：
Figure PCTCN2022118419-appb-000001
S5、设定c的阈值，当c大于阈值时，判定样本为阳性；当c值≤阈值时，判定样本为阴性。
进一步的，c阈值通过如下方法得到：通过血小板聚集指数c受试者工作特征曲线得到受试者工作曲线下面积，通过计算约登指数＝(敏感度+特异度)-1，找到约登指数最大点，以此点计算c的阈值。优选的，血小板聚集指数c的阈值为21.36。
进一步的，显微镜油镜视野下的视野数n≥100。
进一步的，血细胞分析仪为电阻抗法血细胞分析仪。
相应的，本发明还提供一种应用辅助血细胞分析仪判断异常血小板聚集的方法的系统，包括血细胞分析仪模块、镜检模块和阈值判断模块；血细胞分析仪模块用于对血小板异常聚集报警和读取仪器血小板计数数值a；镜检模块用于对血小板异常聚集报警的样本进行镜检，并读取镜检血小板聚集数值a和显微镜油镜视野下的视野数n；阈值判断模块用于计算血小板聚集指数c并对阈值进行判定。
本发明与现有技术相比，其显著优点是：通过计算血小板聚集指数c,可以显著降低血细胞分析仪异常血小板聚集报警的出错率，节省了检验试剂耗材，提高了检验效率，是对当前血小板计数方法的有益补充。
附图说明
图1是本发明的工作方法流程示意图。
图2是本发明的血小板计数与血小板聚集关系分布图。
图3是本发明的校正后血小板聚集指数c在阳性与阴性对照组比较图。
图4是血小板异常聚集诊断曲线图。
图5是血细胞分析仪的血小板报警示意图：
其中图5A是Sysmex XN的血小板报警示意图；图5B是BC7500的血小板报警示意图。
具体实施方式
以下结合附图对本发明的优选实施例进行说明，应当理解，此处所描述的优选实施例仅用于说明和解释本发明，并不用于限定本发明。
1、对象
实施例1以收集2022年2月至2022年7月初随机选的190天内在南京市第一医院住院患者血常规检测血小板聚集报警样本55例。
实施例2以收集2022年8月初在南京市第一医院住院患者临床Sysmex XN以及迈瑞BC7500血细胞分析仪血小板聚集报警样本20例。
采集样本基本信息和相关血常规数据，包括患者年龄、性别、诊断、血常规检测血小板计数值以及重新采集纠正后血小板计数值。
2、仪器与试剂
采用迈瑞公司血细胞分析仪BC-7500或希森美康公司血细胞分析仪XN-2800全自动血细胞分析仪检测，使用仪器配套的试剂与校准品。
显微镜型号OLMPUS CX21，日本奥林巴斯株式会社公司
美国BDEDTA-K2真空抗凝管。
瑞氏吉姆萨染液(中国珠海贝索生物技术公司))。
3、检测方法
(1)血小板检测在仪器上操作程序医院检验LIS系统编号后，直接放入试管架后，置于迈瑞公司血细胞分析仪BC-7500或希森美康公司血细胞分析仪XN-2800全自动血细胞分析仪检测。如图5所示，当检测发现血内有血小板聚集时，仪器自动报警血小板聚集(PLT Clumps)。
(2)血涂片及瑞士染色的步骤研究参照人工镜检遵循《全国临床检验操作规程》(第 四版)，具体步骤如下：
①充分混匀疑似血小板聚集标本，在玻片近一端1/3处，加一滴血样(约40-50ul)；
②呈30°至45°角使血滴沿推片迅速散开，快速、平稳地推动推片至载玻片另一端；
③待血片晾干后，按照1：1加入数滴瑞士染液以及PH6.8磷酸盐缓冲液，以覆盖整个血膜为宜；
④待5分钟后，流水冲去染液，晾干后显微镜下镜检计数血小板聚集数。
4、阳性与阴性判断标准：
如图5所示，血小板聚集或血小板直方图异常报警标本，采用其他抗凝剂(如枸橼酸钠)采血管采血后相同仪器重新检测，血小板计数显著改变(>20％)判定为血常规血小板异常聚集样本，阳性。反之，如采血后血小板计数与报警时计数值≤20％，则判定为血常规血小板正常生理聚集计数样本，阴性。
实施例1：公式推理
将20例血常规血小板正常生理聚集计数样本进行人工镜检。在人工镜检时计数时，须按照从左向右，由上至下的顺序，城墙式计数至少100个视野，以确保结果的准确可靠。否则观察视野不够，可能不能准确计数血小板聚集数，导致结果偏差。同时在人工镜检时，将n个油镜(物镜×100)视野下血小板聚集数a统一校正为1000个视野下血小板聚集数n’。以镜检血小板聚集数值a为纵坐标，以仪器血小板计数的每升值b为横坐标，进行20例样本位置标示，最终得到图2结果，a与b呈正相关。
根据现有的标准，仪器血小板计数的每升值b小于100×10 9常被作为低血小板样本以及提出临床出血风险的指标(小于100×10 9/L是临床需要干预或者对患者进行诊疗的一个临界值)，因此将仪器血小板计数的每升值b的阳性样本与阴性统一校正为血小板计数为100×10 9时血小板聚集指数c，最终公式如下：
血小板聚集指数
Figure PCTCN2022118419-appb-000002
(n:显微镜油镜视野(物镜×100)下视野计数(n≥100)；a:血小板聚集数；b:仪器血小板计数。)
根据上述公式计算得到最终统计验证所需血小板聚集指数c，通过比较阳性样本与阴性对照，发现阳性较阴性血小板聚集指数c显著增高如图3所示(p<0.05)。
以29例(包含上述20例血常规血小板正常生理聚集计数样本)作为阴性组和26例阳性标本绘制血小板聚集指数c受试者工作特征曲线(receiver operating characteristic curve，ROC)，如图4所示，c值具有统计学意义，得到受试者工作曲线下面积(area under the receiver operating characteristic curve，AUC)，通过计算约登指数(Youden’sindex，YI)，YI＝(敏感度+特异度)-1，找到YI最大点，以此点计算临界(cut-off)值。通过测算，最佳cut off值为21.36，曲线下面积为99.4％。如图4所示，采用21.36作为血小板聚集指数c阈值时，灵敏度为92.3％，特异性为86.2％。
实施例2：方法实施与效果验证
以电阻抗法血细胞分析仪血小板报警的标本进行本发明的效果验证。本实施例所采用的电阻抗法血细胞分析仪为Sysmex XN以及迈瑞BC7500血细胞分析仪。其余厂家和型号血细胞分析仪均可以用此方法进行判断。
如图1所示方法步骤，具体如下：
S1、取血液样本放置入血细胞分析仪进行血小板计数；
S2、血细胞分析仪异常报警，读取仪器血小板计数的每升值b；
S3、取报警样本制作血涂片并进行镜检，读取镜检血小板聚集数值a和显微镜油镜视野下的视野数n(镜检时按照从左向右，由上至下的顺序，城墙式计数至少100个视野)；
S4、根据如下公式计算血小板聚集指数c：
Figure PCTCN2022118419-appb-000003
S5、设定c的阈值(21.36)，当c大于阈值时，判定样本为阳性；当c值≤阈值时， 判定样本为阴性。
具体结果如下：
Figure PCTCN2022118419-appb-000004
Figure PCTCN2022118419-appb-000005
同时将此20例报警样本，采用现有技术方法进行检测，以枸橼酸钠为抗凝剂，重新采集患者血液，并用同型号Sysmex XN或者迈瑞BC7500全自动血细胞分析仪分析患者血小板计数变化情况，最终其实验结果与本发明所采用的判断方法结果一致。具体如下：
Figure PCTCN2022118419-appb-000006
Figure PCTCN2022118419-appb-000007
以样本编号为1,3,5,9,12为例，首次血细胞分析仪器中得到的血小板数值均低于100×10 9/L属于临床干预值，通过本发明采用的方法测定得到的结果与现有的临床重新测定的标准一致。
以样本编号为17为例，首次血细胞分析仪器中得到的血小板数值高于300×10 9/L属于临床干预值，通过本发明采用的方法测定得到的结果与现有的临床重新测定的标准也一致。
这说明本发明所提供的方法不仅适用于临床计数值在正常范围内的(100-300)×10 9/L仪器报警检测，也适用于临床数值需要干预的仪器报警检测。
通过本发明所提供的辅助血细胞分析仪判断异常血小板聚集的方法可以对现有的血细胞分析仪器报警的进行判断，试用范围广，是对现有复检规则进行有益补充，节省了试剂成本，减轻患者痛苦，提高实验室检验效率。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。

Claims (6)

  1. 一种辅助血细胞分析仪判断异常血小板聚集的方法，其特征在于，所述方法包括如下步骤：
    S1、取血液样本放置入血细胞分析仪进行血小板计数；
    S2、血细胞分析仪异常报警，读取仪器血小板计数的每升值b；
    S3、取报警样本制作血涂片并进行镜检，读取镜检血小板聚集数值a和显微镜油镜视野下的视野数n；
    S4、根据如下公式计算血小板聚集指数c：
    Figure PCTCN2022118419-appb-100001
    S5、设定c的阈值，当c大于阈值时，判定样本为阳性；当c值≤阈值时，判定样本为阴性。
  2. 根据权利要求1所述的一种辅助血细胞分析仪判断异常血小板聚集的方法，其特征在于，血小板聚集指数c受试者工作特征曲线得到受试者工作曲线下面积，通过计算约登指数＝(敏感度+特异度)-1，找到约登指数最大点，以此点计算c的阈值。
  3. 根据权利要求2所述的一种辅助血细胞分析仪判断异常血小板聚集的方法，其特征在于，血小板聚集指数c的阈值为21.36。
  4. 根据权利要求1所述的一种辅助血细胞分析仪判断异常血小板聚集的方法，其特征在于，显微镜油镜视野下的视野数n≥100。
  5. 根据权利要求1所述的一种辅助血细胞分析仪判断异常血小板聚集的方法，其特征在于，步骤S1中所述血细胞分析仪为电阻抗法血细胞分析仪。
  6. 一种应用上述任意一项权利要求一种辅助血细胞分析仪判断异常血小板聚集的方法的系统，其特征在于，包括血细胞分析仪模块、镜检模块和阈值判断模块；
    所述血细胞分析仪模块用于对血小板异常聚集报警和读取仪器血小板计数数值a；
    所述镜检模块用于对血小板异常聚集报警的样本进行镜检，并读取镜检血小板聚集数值a和显微镜油镜视野下的视野数n；
    所述阈值判断模块用于计算血小板聚集指数c并对阈值进行判定。
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