WO2022100234A1 - 一种用于荧光强度基底计算的方法 - Google Patents

一种用于荧光强度基底计算的方法 Download PDF

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WO2022100234A1
WO2022100234A1 PCT/CN2021/116361 CN2021116361W WO2022100234A1 WO 2022100234 A1 WO2022100234 A1 WO 2022100234A1 CN 2021116361 W CN2021116361 W CN 2021116361W WO 2022100234 A1 WO2022100234 A1 WO 2022100234A1
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peak
fluorescence intensity
value
output data
base
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张胜军
汤四媛
罗继全
李昆鹏
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三诺生物传感股份有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • 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
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • G—PHYSICS
    • 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
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

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  • the present invention relates to the technical field of fluorescence immunodetection, and more particularly, to a method for calculating the basis of fluorescence intensity.
  • the base signal is the noise base, also known as the interference signal, which depends on factors such as the autofluorescence of the target detection material in the system, the dark current of the photodetector and the offset voltage of the op amp.
  • the base signal generated during the test may also be affected by the test strip tomography, etc., resulting in unevenness in the base signal curve. Therefore, in order to improve the accuracy of the measurement, the base signal needs to be removed.
  • the prior art adopts the traditional method of subtracting the fixed base data, which will lead to large errors in the calculated T peak area, peak value, and C peak area and peak value, and even when detecting samples with lower concentrations, there is a situation where the instrument cannot measure the value. .
  • the purpose of the present invention is to provide a method for calculating the fluorescence intensity base in order to make up for the deficiencies of the prior art.
  • a method for fluorescence intensity base calculation comprising the following steps:
  • the determination of the bases corresponding to the T peaks and the C peaks includes the following steps: combining the peak starting position points (X S , Y S ) and the peaks obtained in step (2) Connect the end point (X E , Y E ) to obtain the linear function f(X) corresponding to the line, and use the linear function f(X) as the calculation function of the base B; the base B is each output data
  • the peak position points (X C , Y C ) of the T peak and the C peak in step (2) are determined by the maximum value or the average amplitude method.
  • the maximum or average amplitude method is in the prior art and will not be described in detail here.
  • the determination of the peak starting position point and the end position point corresponding to the T peak and the C peak described in step (3) all include the following steps: the abscissa X C of the peak position point is respectively subtracted and added to the peak.
  • the peak width W is a preset value.
  • the technician Before the instrument leaves the factory, the technician will determine a plurality of peak width values corresponding to the detection items one-to-one according to the types of items that the instrument needs to detect. In the actual detection process, the corresponding peak width value is called according to the different detection items.
  • technicians determine the peak width for a certain test item, they use an empirical method to determine it. Generally, the following methods are used:
  • an initial peak width value perform multiple detections (such as 10 times) for a sample of a certain concentration, and collect multiple groups of original fluorescence intensity values (such as 10 groups).
  • a certain original fluorescence intensity value corresponding to the concentration is calculated as the final fluorescence intensity detection value, so that multiple final fluorescence intensity detection values (such as 10) can be obtained;
  • the initial peak width value will be used; calculate the coefficient of variation CV of these multiple (such as 10) final fluorescence intensity detection values, when the coefficient of variation CV is within the acceptable range (such as less than 3%, or less than 1%, etc.), the initial peak width value set previously is used as the final peak width value; otherwise, another different initial peak width value is reset, and the above calculation method is used to verify again until a suitable peak width value is found. .
  • the initial peak width value is determined by the experience of the technician, and the verification process is only to determine a more accurate peak width value from an approximate range.
  • the following methods can be used:
  • n is the number of statistical samples, that is, the number of final fluorescence intensity detection values (that is, the number of detections); x i is the i-th final fluorescence intensity detection value; is the average value of the nth final fluorescence intensity detection value.
  • the determination of the peak position points (X C , Y C ) of the T peak and the C peak described in step (2) includes the following steps: calculating the slope value K i of the fluorescence intensity point within the preset retrieval range of the peak , sort the slope value with its corresponding output data serial number X from small to large as the arrangement order to obtain the corresponding slope array; the slope value K i is calculated according to the following formula:
  • x is the serial number of the output data
  • y is the fluorescence intensity value corresponding to the serial number of the output data
  • m is a preset known integer, m ⁇ 1
  • i is the serial number of the output data; traverse the obtained slope array to find the maximum value of the slope value and the minimum value, find the corresponding output data serial numbers according to the maximum value and the minimum value of the slope value, denoted as X max , X min , by finding the output data corresponding to the value with the smallest absolute value of the slope value between X max and X min Serial number, the fluorescence intensity point corresponding to the output data serial number is the peak position point (X C , Y C ).
  • the slope value K i is used to represent the data change trend between the fluorescence intensity point corresponding to the output numerical number i and the two adjacent fluorescence intensity points before and after; There are other methods of technology to perform the calculation.
  • any positive integer value of m can be selected according to actual needs.
  • the theoretically calculated slope The value should be more accurate; however, it is sufficient to select 1 or 2 for m in actual operation, so m is preferably 1 or 2 in this case.
  • the determination of the peak starting position point and the end position point corresponding to the T peak and the C peak described in step (3) all include the following steps: find the corresponding fluorescence intensity point according to the obtained X max and X min ,
  • the fluorescence intensity points are the starting position points (X S , Y S ) and the ending position points (X E , Y E ) of the peak.
  • the fluorescence immunoassay analyzer in step (1) adopts ADUCM360 as the main chip for data acquisition, the set test frequency f is 500Hz, and the test time t is 3s.
  • the method for calculating the fluorescence intensity base by first determining the peak positions of the T peak and the C peak in the fluorescence intensity scattergram, as well as the starting position and the ending position of the T peak and the C peak, Then connect the corresponding peak starting point and the peak ending point to obtain the linear function corresponding to the output data serial number of the line, that is, the T peak and the C peak respectively obtain their corresponding basis functions;
  • the method enables to calculate the peak area of T peak, C peak or the peak area of T peak and C peak when calculating the concentration, first subtract its corresponding fixed base or subtract its corresponding base function for correction, which is compared with the existing fixed base.
  • the calculation method is more conducive to reducing the error of concentration detection, and effectively solves the technical problem that the instrument cannot measure the value when detecting samples of lower concentration.
  • FIG. 1 is a scattergram of fluorescence intensity obtained in Example 1 of the present invention.
  • This embodiment takes the detection of a sample with a low concentration as an example to describe the method for calculating the fluorescence intensity base described in this embodiment, and the operation of calculating the concentration by applying the method.
  • a method for fluorescence intensity base calculation comprising the following steps:
  • the fluorescent immune analyzer adopts ADUCM360 as the main chip for data acquisition, the set test frequency f is 500Hz, and the test time t is 3s, so that the output data serial number X is the abscissa and the fluorescence intensity value. 1500 fluorescence intensity points are obtained on the coordinate system where Y is the ordinate, and the corresponding fluorescence intensity scattergram is obtained; the specific scattergram is shown in Figure 1;
  • Different detection items may have low C peak data or low T peak data.
  • the left peak may be the C peak
  • the right peak may be the T peak
  • the right peak may be the C peak
  • the left peak may be the T peak.
  • the left wave peak is the C peak
  • the right wave peak is the T peak.
  • the determination of the bases corresponding to the T peaks and the C peaks includes the following steps: combining the peak starting position points (X S , Y S ) and the peaks obtained in step (2) Connect the end point (X E , Y E ) to obtain the linear function f(X) corresponding to the line, and use the linear function f(X) as the calculation function of the base B; the base B is each output data
  • the determination of the peak position points (X C , Y C ) of the T peak and the C peak described in step (2) includes the following steps: calculating the slope value Ki of the fluorescence intensity point within the preset retrieval range of the peak, The slope value is sorted by its corresponding output data serial number X from small to large as the arrangement order to obtain the corresponding slope array; the slope value Ki is calculated according to the following formula:
  • x is the serial number of the output data
  • y is the fluorescence intensity value corresponding to the serial number of the output data
  • i is the serial number of the output data; traverse the obtained slope array to find the maximum value of the slope value and the minimum value, find the corresponding output data serial numbers according to the maximum value and the minimum value of the slope value, denoted as X max , X min , by finding the output data corresponding to the value with the smallest absolute value of the slope value between X max and X max Serial number, the fluorescence intensity point corresponding to the output data serial number is the peak position point (X C , Y C ).
  • step (3) the determination of the peak starting position point and the end position point corresponding to the T peak and the C peak all include the following steps: find the corresponding fluorescence intensity point according to the obtained X max and X min ,
  • the fluorescence intensity points are the starting position points (X S , Y S ) and the ending position points (X E , Y E ) of the peak.
  • step (4) The specific operation process of above-mentioned step (4) in this embodiment is:
  • the method for calculating the fluorescence intensity base includes the following steps: calculating the T/C peak ratio or the T/C peak area ratio; and obtaining the corresponding concentration according to the obtained ratio.
  • the method for obtaining the corresponding concentration of the obtained ratio adopts the prior art, and will not be described in detail here.
  • the calculation of the T/C peak area ratio includes the following steps: subtracting the coordinates of each fluorescence intensity value from the peak starting position (250, 3869.22) to the ending position (500, 4040.16) of the T peak respectively.
  • step (1) is the same as that of Embodiment 1, except that the base calculation is performed by taking the fluorescence intensity value corresponding to the middle point of the T peak position point and the C peak position point (the corresponding horizontal The coordinates are 653.5) as the fixed base.
  • the ordinate of the T peak position and the ordinate of the C peak position are respectively subtracted from the fixed base to obtain the corrected T peak-to-peak value and C peak-to-peak value, which are calculated according to the T/C peak ratio or T/C peak.
  • the process of calculating the concentration of the area ratio is the same as that in Example 1.
  • Example 1 Combining Example 1 and Comparative Example 1, it can be seen that the method for calculating the fluorescence intensity base of the present invention can still produce values when detecting samples with a low concentration so as to detect a more accurate concentration, which is compared with the existing fixed base calculation method.
  • the method is more conducive to reducing the error of concentration detection, and effectively solves the technical problem that the instrument cannot measure the value when detecting samples of lower concentration.

Abstract

本发明涉及荧光免疫检测技术领域，更具体地说，涉及一种用于荧光强度基底计算的方法。本发明所述用于荧光强度基底计算的方法，通过先确定所述荧光强度散点图中T峰、C峰的峰值位置点，以及T峰、C峰的起始位置点、终止位置点，然后将对应峰起始点和峰终止点连线得到该线关于输出数据序号对应的线性函数，即T峰、C峰分别得到其对应的基底函数；采用本发明所述用于荧光强度基底计算的方法，使得在计算浓度时计算T峰、C峰的峰值或T峰、C峰的峰面积先减去其对应的固定基底或减去其对应的基底函数进行校正，其相较现有固定基底计算方法更有利于降低浓度检测的误差，并有效解决在检测较低浓度样本时仪器测不出值的技术问题。

Description

一种用于荧光强度基底计算的方法 技术领域
本发明涉及荧光免疫检测技术领域，更具体地说，涉及一种用于荧光强度基底计算的方法。
背景技术
在采用荧光免疫分析仪进行荧光免疫检测的过程中，系统所产生的基底信号被叠加至检测到的荧光强度数据中，会对测量精度有较大影响。基底信号即噪声基底，又叫干扰信号，其取决于系统中目标检测物质的自发荧光、光电探测器的暗电流和运放失调电压等因素。此外，测试过程中产生的基底信号还可能受试条层析等影响，从而出现基底信号曲线出现凹凸不平的现象，因此为了提高测量的准确性，需要去除基底信号。
现有技术采用传统减去固定基底数据的方法，会导致计算的T峰面积、峰值以及C峰面积、峰值存在较大误差，甚至在检测较低浓度样本时，存在仪器测不出值的情况。
故，现有技术具有较大的改进空间。
发明内容
本发明的目的是为了弥补现有技术的不足，提出一种用于荧光强度基底计算的方法。
为了达到上述目的，本发明通过以下技术方案实现：
一种用于荧光强度基底计算的方法，包括以下步骤：
(1)采用荧光免疫分析仪以设定的测试频率f、测试时间t对试剂卡进行测试，按序输出荧光强度数据，从而在以输出数据序号X为横坐标、荧光强度值Y为纵坐标的坐标系上得到(f×t)个荧光强度点，得到对应的荧光强度散点图；
(2)确定所述荧光强度散点图中T峰、C峰的峰值位置点(X C，Y C)；
(3)确定所述T峰、C峰的起始位置点(X S，Y S)、终止位置点(X E，Y E)；
(4)分别确定T峰、C峰的基底；所述T峰、C峰对应的基底的确定均包括以下步骤：将步骤(2)所得峰起始位置点(X S，Y S)和峰终止位置点(X E，Y E)连线，得到该线对应的线性函数f(X)，将所述线性函数f(X)作为基底B的计算函数；所述基底B为每个输出数据序号X对应于所述线性函数f(X)的函数值，即B＝f(X)，从而使得所述荧光散点图中波峰范围内每个荧光强度点均有对应的基底。
根据以上方案，步骤(4)还包括分别将T峰、C峰的峰值位置点的横坐标X C代入线性函数f(X)，得到T峰、C峰对应的基底B C＝f(X C)，以B C＝f(X C)作为波峰范围内的每个荧光强度点的基底。
根据以上方案，步骤(2)中所述T峰、C峰的峰值位置点(X C，Y C)采用最大值或平均幅值法进行确定。所述最大值或平均幅值法为现有技术，在此不再详述。
根据以上方案，步骤(3)中所述T峰、C峰对应的峰起始位置点、终止位置点的确定均包括以下步骤：将峰值位置点的横坐标X C分别减去、加上峰宽W的一半，计算出峰起始位置点的横坐标X S和峰终止位置点的横坐标X E，即X S＝X C-0.5W、X E＝X C+0.5W，再根据X S、X E在所述荧光强度散点图中找到其对应的纵坐标，从而确定到峰起始位置点(X S，Y S)和峰终止位置点(X E，Y E)。
其中，峰宽W为一个预设值，在仪器出厂前，技术人员会根据仪器需要检测的项目种类，确定多个与检测项目一一对应的峰宽值。在实际检测过程中，根据检测项目的不同调用对应的峰宽值。技术人员在针对某一个检测项目来确定峰宽时，采用经验方式来确定，一般会采用如下方法进行：
设定一个初始峰宽值，针对某一浓度的样本进行多次检测(比如10次)，分别可以采集到多组原始荧光强度值(比如10组)，根据现有荧光强度值数据处理方式，针对这多组原始荧光强度值，计算出与浓度对应的某个原始荧光强度值作为最终荧光强度检测值，这样可以获得多个最终荧光强度检测值(比如10个)；在现有的最终荧光强度检测值计算过程中，会使用到初始峰宽值；计算这多个(比如10个)最终荧光强度检测值的变异系数CV，当变异系数CV在可接受范围内时(比如小于3％，或者小于1％等)，则将前面设定的初始峰宽值作为最终峰宽值；否则重新设定另外一个不同的初始峰宽值，按上述计算方式再次验证，直到找到合适的峰宽值。
初始峰宽值由技术人员的经验确定，验证的过程只是从一个大概范围内确定一个更准确的峰宽数值。变异系数CV的计算，可采用如下方法：
Figure PCTCN2021116361-appb-000001
其中，n为统计的样本数量，即最终荧光强度检测值的个数(也即检测的次数)；x i为第i个最终荧光强度检测值；
Figure PCTCN2021116361-appb-000002
为第n个最终荧光强度检测值的平均值。
根据以上方案，步骤(2)中所述T峰、C峰的峰值位置点(X C，Y C)的确定，均包括以下步骤：计算峰预设检索范围内荧光强度点的斜率值K i，将斜率值以其对应的输出数据序号X 从小到大作为排列顺序进行排序得到对应的斜率数组；所述斜率值K i按以下公式计算：
Figure PCTCN2021116361-appb-000003
其中，x为输出数据序号；y为输出数据序号对应的荧光强度值；m为预设的已知整数，m≥1；i为输出数据序号；遍历所得的斜率数组找出斜率值的最大值和最小值，根据斜率值的最大值、最小值找到其对应的输出数据序号记为X max、X min，通过在X max与X min之间找到斜率值绝对值最小的值所对应的输出数据序号，该输出数据序号对应的荧光强度点即为峰值位置点(X C，Y C)。
所述斜率值K i用于表示输出数值序号i对应的荧光强度点与前后相邻的两个荧光强度点之间的数据变化趋势；所述斜率值除了采用上述公式计算外，也可采用现有技术的其他方法进行计算。
根据以上方案，所述m可根据实际需要选取任意正整数值，其取值越大，在计算斜率值公式时，选取的连续数据点越多，计算工作量也越大，理论上计算的斜率值应该越准确；然而在现实操作时所述m选取1或2已足够，因此本案所述m优选为1或2。
根据以上方案，步骤(3)中所述T峰、C峰对应的峰起始位置点、终止位置点的确定均包括以下步骤：根据所得的X max、X min找到其对应的荧光强度点，所述荧光强度点即为峰的起始位置点(X S，Y S)、终止位置点(X E，Y E)。
根据以上方案，步骤(1)中所述荧光免疫分析仪采用ADUCM360作为数据采集主芯片，所述设定的测试频率f为500Hz，所述测试时间t为3s。
本发明的有益效果在于：
本发明所述用于荧光强度基底计算的方法，通过先确定所述荧光强度散点图中T峰、C峰的峰值位置点，以及T峰、C峰的起始位置点、终止位置点，然后将对应峰起始点和峰终止点连线得到该线关于输出数据序号对应的线性函数，即T峰、C峰分别得到其对应的基底函数；采用本发明所述用于荧光强度基底计算的方法，使得在计算浓度时计算T峰、C峰的峰值或T峰、C峰的峰面积先减去其对应的固定基底或减去其对应的基底函数进行校正，其相较现有固定基底计算方法更有利于降低浓度检测的误差，并有效解决在检测较低浓度样本时仪器测不出值的技术问题。
附图说明
图1为本发明实施例1所得的荧光强度散点图。
具体实施方式
为了更好地理解本发明，下面结合实施例进一步阐明本发明的内容，但本发明不仅仅局限于下面的实施例。
实施例1
本实施例以检测一个浓度偏低的样本为例，说明本实施例所述用于荧光强度基底计算的方法，及应用其进行计算浓度的操作。
一种用于荧光强度基底计算的方法，包括以下步骤：
(1)采用荧光免疫分析仪以设定的测试频率f、测试时间t对试剂卡进行测试，按序输出荧光强度数据，从而在以输出数据序号X为横坐标、荧光强度值Y为纵坐标的坐标系上得到(f×t)个荧光强度点，得到对应的荧光强度散点图；
进一步地，所述荧光免疫分析仪采用ADUCM360作为数据采集主芯片，所述设定的测试频率f为500Hz，所述测试时间t为3s，从而在以输出数据序号X为横坐标、荧光强度值Y为纵坐标的坐标系上得到1500个荧光强度点，得到对应的荧光强度散点图；具体的散点图见图1；
不同的检测项目可能C峰数据偏低，也可能T峰数据偏低。如图1所示，可能左侧波峰为C峰、右侧波峰为T峰，也可能右侧波峰为C峰、左侧波峰为T峰。此处按左侧波峰为C峰、右侧波峰为T峰来计算。
(2)确定所述荧光强度散点图中T峰、C峰的峰值位置点(X C，Y C)；
(3)确定所述T峰、C峰的起始位置点(X S，Y S)、终止位置点(X E，Y E)；
(4)分别确定T峰、C峰的基底；所述T峰、C峰对应的基底的确定均包括以下步骤：将步骤(2)所得峰起始位置点(X S，Y S)和峰终止位置点(X E，Y E)连线，得到该线对应的线性函数f(X)，将所述线性函数f(X)作为基底B的计算函数；所述基底B为每个输出数据序号X对应于所述线性函数f(X)的函数值，即B＝f(X)，从而使得所述荧光散点图中波峰范围内每个荧光强度点均有对应的基底。
进一步地，步骤(4)还包括分别将T峰、C峰的峰值位置点的横坐标X C代入线性函数f(X)，得到T峰、C峰对应的基底B C＝f(X C)，以B C＝f(X C)作为波峰范围内的每个荧光强度点的基底。
进一步地，步骤(2)中所述T峰、C峰的峰值位置点(X C，Y C)的确定，均包括以下步骤：计算峰预设检索范围内荧光强度点的斜率值Ki，将斜率值以其对应的输出数据序号X从小到大作为排列顺序进行排序得到对应的斜率数组；所述斜率值Ki按以下公式计算：
Figure PCTCN2021116361-appb-000004
其中，x为输出数据序号；y为输出数据序号对应的荧光强度值；m为预设的已知整数，m＝1；i为输出数据序号；遍历所得的斜率数组找出斜率值的最大值和最小值，根据斜率值的最大值、最小值找到其对应的输出数据序号记为X max、X min，通过在X max与X max之间找到斜率值绝对值最小的值所对应的输出数据序号，该输出数据序号对应的荧光强度点即为峰值位置点(X C，Y C)。
进一步地，步骤(3)中所述T峰、C峰对应的峰起始位置点、终止位置点的确定均包括以下步骤：根据所得的X max、X min找到其对应的荧光强度点，所述荧光强度点即为峰的起始位置点(X S，Y S)、终止位置点(X E，Y E)。
步骤(2)、(3)的计算结果见下表1：
表1
Figure PCTCN2021116361-appb-000005
本实施例设定m＝1进行斜率值计算，本领域技术人员可根据实际需要设定不同正整数值，其计算结果与本实施例相差不大，因此本案不再累述。
上述步骤(4)在本实施例中的具体操作过程为：
1、将T峰的峰起始位置点(250，3869.22)和其终止位置点(500，4040.16)连线，该线对应的线性函数f(X)＝0.68376X+3698.28，即为T峰的基底B的计算函数B＝f(X)＝0.68376X+3698.28；同样地，将C峰的峰起始位置点(807，4891.56)和其终止位置 点(1057，5319.68)连线，该线对应的线性函数f(X)＝1.71248X+3509.589，即为C峰的基底B的计算函数B＝f(X)＝1.71248X+3509.589，从而使得所述荧光散点图中波峰范围内每个荧光强度点均有对应的基底。
2、分别将T峰、C峰的峰值位置点的横坐标X C代入线性函数f(X)，得到T峰、C峰对应的基底B C＝f(X C)，即将T峰的峰值位置点的横坐标X C＝375代入线性函数f(X)＝0.68376X+3698.28，得到T峰对应的基底B C＝f(X C)＝0.68376×375+3698.28＝3984.69；将C峰的峰值位置点的横坐标X C＝932代入线性函数f(X)＝1.71248X+3509.589，得到C峰对应的基底B C＝f(X C)＝1.71248×932+3509.589＝5105.62，以B C＝f(X C)作为波峰范围内的每个荧光强度点的基底。
所述用于荧光强度基底计算的方法用于计算浓度时，包括以下步骤：计算T/C峰值比值或T/C峰面积比值；根据所得比值得到对应的浓度。将所得比值得到对应浓度的方法采用的是现有技术，在此不再详述。
其中，所述T/C峰值比值的计算，包括以下步骤：将T峰的位置点对应的纵坐标Y C＝4341.27减去其对应的基底B C＝3984.69,得到校正后的T峰荧光强度值为4341.27-3954.69＝386.58；将C峰的位置点对应的纵坐标Y C＝9318.05减去其对应的基底B C＝5105.62,得到校正后的C峰荧光强度值为9318.05-5105.62＝4212.43；校正后的T峰荧光强度值除以校正后的C峰荧光强度值得到T/C峰值比值为386.58/4212.43＝0.092。
其中，所述T/C峰面积比值的计算，包括以下步骤：将T峰的峰起始位置点(250，3869.22)至终止位置点(500，4040.16)的各荧光强度值的坐标分别减去其对应的固定基底B＝f(X)＝0.68376X+3698.28，得到校正后的荧光强度值，计算校正后的荧光强度值的总和得到校正后的T峰的峰面积；将C峰的峰起始位置点(807，4891.56)至终止位置点(1057，5319.68)的各荧光强度值的坐标分别减去其对应的固定基底B＝f(X)＝1.71248X+3509.589，得到校正后的荧光强度值，计算校正后的荧光强度值的总和得到校正后的T峰的峰面积；校正后的T峰的峰面积除以校正后的C峰的峰面积值得到T/C峰面积比值。
对比例1
采用一种现有固定基底计算方法，其步骤(1)同实施例1相同，不同在于其基底计算是通过取T峰位置点和C峰位置点中间点对应的荧光强度值(其对应的横坐标为653.5)作为固定基底。计算浓度时，将T峰位置点的纵坐标、C峰位置点的纵坐标分别减去固定基座得到校正后的T峰峰值、C峰峰值，其根据T/C峰值比值或T/C峰面积比值计算浓度的过程同实施例1。
从图1可见，若按此方法确定的固定基底值要比T峰峰值大，那么计算后的T峰校正荧光强度值为负值，仪器将其重置为0,则最终浓度的测试结果为0。
结合实施例1与对比例1可见，本发明所述用于荧光强度基底计算的方法在检测浓度偏低的样本时仍能出值从而检测到较为准确的浓度，其相较现有固定基底计算方法更有利于降低浓度检测的误差，并有效解决在检测较低浓度样本时仪器测不出值的技术问题。
以上所述仅是本发明的较佳实施方式，故凡依本发明专利申请范围所述的构造、特征及原理所做的等效变化或修饰，均包括于本发明专利申请范围内。

Claims (8)

  1. 一种用于荧光强度基底计算的方法，其特征在于，包括以下步骤：
    (1)采用荧光免疫分析仪以设定的测试频率f、测试时间t对试剂卡进行测试，按序输出荧光强度数据，从而在以输出数据序号X为横坐标、荧光强度值Y为纵坐标的坐标系上得到(f×t)个荧光强度点，得到对应的荧光强度散点图；
    (2)确定所述荧光强度散点图中T峰、C峰的峰值位置点(X C，Y C)；
    (3)确定所述T峰、C峰的起始位置点(X S，Y S)、终止位置点(X E，Y E)；
    (4)分别确定T峰、C峰的基底；所述T峰、C峰对应的基底的确定均包括以下步骤：将步骤(2)所得峰起始位置点(X S，Y S)和峰终止位置点(X E，Y E)连线，得到该线对应的线性函数f(X)，将所述线性函数f(X)作为基底B的计算函数；所述基底B为每个输出数据序号X对应于所述线性函数f(X)的函数值，即B＝f(X)，从而使得所述荧光散点图中波峰范围内每个荧光强度点均有对应的基底。
  2. 根据权利要求要求1所述用于荧光强度基底计算的方法，其特征在于，步骤(4)还包括分别将T峰、C峰的峰值位置点的横坐标X C代入线性函数f(X)，得到T峰、C峰对应的基底B C＝f(X C)，以B C＝f(X C)作为波峰范围内的每个荧光强度点的基底。
  3. 根据权利要求要求1所述用于荧光强度基底计算的方法，其特征在于，步骤(2)中所述T峰、C峰的峰值位置点(X C，Y C)采用最大值或平均幅值法进行确定。
  4. 根据权利要求要求1所述用于荧光强度基底计算的方法，其特征在于，步骤(3)中所述T峰、C峰对应的峰起始位置点、终止位置点的确定均包括以下步骤：将峰值位置点的横坐标X C分别减去、加上峰宽W的一半，计算出峰起始位置点的横坐标X S和峰终止位置点的横坐标X E，再根据X S、X E在所述荧光强度散点图中找到其对应的纵坐标，从而确定到峰起始位置点(X S，Y S)和峰终止位置点(X E，Y E)。
  5. 根据权利要求要求1所述用于荧光强度基底计算的方法，其特征在于，步骤(2)中所述T峰、C峰的峰值位置点(X C，Y C)的确定，均包括以下步骤：计算峰预设检索范围内荧光强度点的斜率值K i，将斜率值以其对应的输出数据序号X从小到大作为排列顺序进行排序得到对应的斜率数组；所述斜率值K i按以下公式计算：
    Figure PCTCN2021116361-appb-100001
    其中，x为输出数据序号；y为输出数据序号对应的荧光强度值；m为预设的已知整数，m≥1；i为输出数据序号；遍历所得的斜率数组找出斜率值的最大值和最小值，根据斜率值的最大值、最小值找到其对应的输出数据序号记为X max、 X min，通过在X max与X min之间找到斜率值绝对值最小的值所对应的输出数据序号，该输出数据序号对应的荧光强度点即为峰值位置点(X C，Y C)。
  6. 根据权利要求要求5所述用于荧光强度基底计算的方法，其特征在于，所述m为1或2。
  7. 根据权利要求要求5所述用于荧光强度基底计算的方法，其特征在于，步骤(3)中所述T峰、C峰对应的峰起始位置点、终止位置点的确定均包括以下步骤：根据所得的X max、X min找到其对应的荧光强度点，所述荧光强度点即为峰的起始位置点(X S，Y S)、终止位置点(X E，Y E)。
  8. 根据权利要求要求1所述用于荧光强度基底计算的方法，其特征在于，步骤(1)中所述荧光免疫分析仪采用ADUCM360作为数据采集主芯片，所述设定的测试频率f为500Hz，所述测试时间t为3s。
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