Wong Linda, Hill Beth L, Hunsberger Benjamin C, Bagwell C Bruce, Curtis Adam D, Davis Bruce H
Trillium Diagnostics, LLC, Brewer, Maine.
Verity Software House, Topsham, Maine.
Cytometry B Clin Cytom. 2015 Jul-Aug;88(4):227-35. doi: 10.1002/cyto.b.21217. Epub 2015 Feb 6.
Leuko64™ (Trillium Diagnostics) is a flow cytometric assay that measures neutrophil CD64 expression and serves as an in vitro indicator of infection/sepsis or the presence of a systemic acute inflammatory response. Leuko64 assay currently utilizes QuantiCALC, a semiautomated software that employs cluster algorithms to define cell populations. The software reduces subjective gating decisions, resulting in interanalyst variability of <5%. We evaluated a completely automated approach to measuring neutrophil CD64 expression using GemStone™ (Verity Software House) and probability state modeling (PSM).
Four hundred and fifty-seven human blood samples were processed using the Leuko64 assay. Samples were analyzed on four different flow cytometer models: BD FACSCanto II, BD FACScan, BC Gallios/Navios, and BC FC500. A probability state model was designed to identify calibration beads and three leukocyte subpopulations based on differences in intensity levels of several parameters. PSM automatically calculates CD64 index values for each cell population using equations programmed into the model. GemStone software uses PSM that requires no operator intervention, thus totally automating data analysis and internal quality control flagging. Expert analysis with the predicate method (QuantiCALC) was performed. Interanalyst precision was evaluated for both methods of data analysis.
PSM with GemStone correlates well with the expert manual analysis, r(2) = 0.99675 for the neutrophil CD64 index values with no intermethod bias detected. The average interanalyst imprecision for the QuantiCALC method was 1.06% (range 0.00-7.94%), which was reduced to 0.00% with the GemStone PSM. The operator-to-operator agreement in GemStone was a perfect correlation, r(2) = 1.000.
Automated quantification of CD64 index values produced results that strongly correlate with expert analysis using a standard gate-based data analysis method. PSM successfully evaluated flow cytometric data generated by multiple instruments across multiple lots of the Leuko64 kit in all 457 cases. The probability-based method provides greater objectivity, higher data analysis speed, and allows for greater precision for in vitro diagnostic flow cytometric assays.
Leuko64™（Trillium诊断公司）是一种流式细胞术检测方法，用于测量中性粒细胞CD64表达，可作为感染/脓毒症或全身性急性炎症反应存在的体外指标。Leuko64检测目前使用QuantiCALC，这是一种半自动化软件，采用聚类算法来定义细胞群体。该软件减少了主观设门决策，使得不同分析人员之间的变异性<5%。我们评估了一种使用GemStone™（Verity软件公司）和概率状态建模（PSM）来测量中性粒细胞CD64表达的完全自动化方法。
使用Leuko64检测方法处理了457份人类血液样本。样本在四种不同的流式细胞仪型号上进行分析：BD FACSCanto II、BD FACScan、BC Gallios/Navios和BC FC500。设计了一种概率状态模型，基于几个参数强度水平的差异来识别校准微珠和三个白细胞亚群。PSM使用编入模型的方程自动计算每个细胞群体的CD64指数值。GemStone软件使用PSM，无需操作员干预，从而完全自动化数据分析和内部质量控制标记。采用谓词法（QuantiCALC）进行专家分析。对两种数据分析方法评估了不同分析人员之间的精密度。
GemStone的PSM与专家手动分析相关性良好，中性粒细胞CD64指数值的r(2)=0.99675，未检测到方法间偏差。QuantiCALC方法的不同分析人员间平均不精密度为1.06%（范围0.00 - 7.94%），使用GemStone PSM时降至0.00%。GemStone中不同操作员之间的一致性为完美相关，r(2)=1.000。
CD64指数值的自动定量产生的结果与使用基于标准设门的数据分析方法的专家分析密切相关。PSM成功评估了所有457例中来自多个批次Leuko64试剂盒的多种仪器生成的流式细胞术数据。基于概率的方法提供了更高的客观性、更高的数据分析速度，并允许体外诊断流式细胞术检测具有更高的精密度。
