Valuation Method for Milk Somatic Cell Count Reference Materials and the Uncertainty Evaluation
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摘要: 建立了基于显微成像流式细胞技术对牛奶体细胞数量标准物质中的牛奶体细胞浓度进行测量的方法。该方法通过细胞核荧光染料溴化乙锭(Ethidium Bromide)对牛奶体细胞进行标记,显微成像流式细胞技术提供的单细胞多参数显微图像可有效辅助流式细胞分析方法中对目标细胞的圈门识别,从而提高圈门策略的正确性和重复性。通过多份不同体细胞浓度的牛奶体细胞标准物质样本的多次测量分析证明,该方法具有良好的室内重复性和日间重复性。建立了基于显微成像流式技术的牛奶体细胞数量标准物质中牛奶体细胞浓度测量方法的数学模型,分析确定了该测量方法不确定度主要来源于测量重复性、样本前处理、试剂加样、圈门操作和仪器测量体积偏差等。综合评定,针对研究制备的牛奶体细胞数量标准物质候选物V级(~1.0×106 /mL),基于显微成像流式细胞技术的牛奶体细胞浓度测量方法的测量相对不确定度为4.84%。
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关键词:
- 计量学 /
- 牛奶体细胞 /
- 细胞浓度 /
- 不确定度 /
- 显微成像流式细胞技术
Abstract: A method based on microscopic imaging flow cytometry was established to measure the concentration of milk somatic cells in the standard substance for milk somatic cell count. This method labels milk somatic cells using the fluorescent dye Ethium Bromide, and the single cell multi parameter microscopic images provided by flow cytometry can effectively assist in identifying target cells in flow cytometry analysis, thereby providing the correctness and repeatability of the gate strategy. Through multiple measurements and analysis of milk somatic cell standard substance samples with different concentrations of somatic cells, it has been proven that this method has good indoor and daytime repeatability. A mathematical model for measuring the concentration of milk somatic cells in a standard substance for milk somatic cell count based on microscopic imaging flow cytometry technology was established. The analysis determined that the uncertainty of this measurement method mainly comes from measurement repeatability, sample pretreatment, reagent addition, gate operation, and measurement volume. Comprehensive evaluation, targeting the candidate V-grade (~1.0×106/mL) of the standard substance for milk somatic cell count prepared in the study, the relative uncertainty of the measurement method for milk somatic cell concentration based on microscopic imaging flow cytometry is 4.84%.-
Key words:
- metrology /
- milk somatic cells /
- cell concentration /
- uncertainty /
- microscopic imaging flow cytometry
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表 1 测量方法室内重复性统计
Table 1. Indoor repeatability statistics of measurement methods
样本编号 1 2 3 4 5 6 7 I 细胞浓度(×106/m)L) 0.11 0.13 0.12 0.10 0.09 0.13 0.11 RSD(%) 3.6 3.1% 3.3% 2.9% 3.4% 3.7 3.7% II 细胞浓度(×106/m)L) 0.23 0.21 0.20 0.19 0.18 0.20 0.21 RSD(%) 2.7% 3.3% 3.0% 2.5% 3.1% 2.9% 2.7% III 细胞浓度(×106/m)L) 0.44 0.44 0.39 0.41 0.45 0.44 0.44 RSD(%) 2.9% 1.7% 2.0% 1.9% 2.6% 2.1% 2.6% IV 细胞浓度(×106/m)L) 0.90 0.88 0.80 0.85 0.89 0.83 0.88 RSD(%) 1.4% 1.7% 2.3% 2.3% 1.5% 1.9% 1.4% V 细胞浓度(×106/m)L) 1.06 1.05 1.08 1.05 0.99 0.98 1.01 RSD(%) 1.5% 1.2% 1.0% 1.4% 1.2% 1.4% 2.0% 表 2 测量方法日间重复性统计
Table 2. Measurement method daytime repeatability statistics
样本编号
时间(天)1 2 3 F P I 1 0.13±0.004 0.13±0.004 0.12±0.003 2.23 0.099 2 0.13±0.004 0.13±0.005 0.13±0.005 0.99 0.193 3 0.11±0.004 0.10±0.003 0.10±0.003 1.05 0.153 II 1 0.23±0.006 0.21±0.004 0.20±0.006 4.10 0.078 2 0.21±0.005 0.21±0.006 0.24±0.006 2.08 0.166 3 0.19±0.005 0.18±0.005 0.21±0.005 3.85 0.093 III 1 0.44±0.008 0.42±0.007 0.42±0.007 2.78 0.126 2 0.41±0.008 0.42±0.008 0.41±0.008 1.01 0.313 3 0.39±0.008 0.39±0.008 0.39±0.009 0.75 0.400 IV 1 0.90±0.013 0.90±0.014 0.90±0.013 0.95 0.359 2 0.88±0.015 0.89±0.015 0.89±0.014 1.89 0.0963 3 0.88±0.012 0.88±0.016 0.90±0.015 2.89 0.128 V 1 1.08±0.011 1.11±0.014 1.10±0.012 4.25 0.095 2 1.05±0.014 1.05±0.016 1.04±0.016 1.07 0.177 3 0.99±0.012 1.00±0.015 1.01±0.015 2.75 0.333 表 3 前处理方法细胞回收率数据分析
Table 3. Analysis of Cell Recovery Data for Pre processing Methods
样本编号 处理前(×106/mL) 处理后(×106/mL) 回收率(%) 1 1.02 0.99 96.6 2 1.10 1.03 93.6 3 0.96 0.91 94.5 4 0.99 0.94 95.0 5 1.07 0.99 92.3 6 1.12 1.02 90.8 7 1.07 0.98 91.6 8 0.92 0.86 93.3 9 0.99 0.95 95.6 10 1.13 1.07 94.6 平均回收率 93.8% RSD 1.95% 表 4 测量不确定度分量汇总表
Table 4. Summary of Measurement Uncertainty Components
不确定度来源 量值 标准不确定度u 相对不确定度urel(%) 测量重复性u1 10000 123.5 1.24 前处理细胞回收u2 93.8% 1.8% 1.95 试剂加样u3 0.83 0.024 0.015 圈门操作u4 10000events 50events 0.50 仪器测量体积偏差u5 100μL 0.5μL 0.50 测量相对不确定度 2.42% 测量扩展不确定度 4.84% -
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