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Malar J. 2019; 18：262.
在线发布2019年7月31日。doi：10.1186/s12936-019-2890-z
Infected RBC flag/parameter provided by Mindray BC-6800 haematology analyzer aid the diagnosis of malaria
迈瑞BC-6800血液分析仪提供的感染红细胞标志/参数辅助疟疾诊断
1孙毅， # 1 向代军， # 1 陈晨， 1 尚和， 1 桓琦， 2 王成斌
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Associated Data 关联数据
Abstract 摘要
Background 背景
The Mindray BC-6800 haematology analyzer (BC-6800) provides a dedicated flag ‘Infected RBC’ (InR) and the number of InR (InR#)/the permillage of InR (InR‰) in routine blood testing as a screening tool for malaria in endemic areas. This study sought to evaluate the effectiveness of the BC-6800 flag parameter for aiding the diagnosis of malaria.
迈瑞BC-6800血液分析仪（BC-6800）在常规血液检测中提供了一个专用的标记“感染红细胞”（InR）和InR数（InR#）/InR千分之一（InR‰），作为疟疾流行地区的筛查工具。本研究旨在评估BC-6800标记参数辅助诊断疟疾的有效性。
Methods 方法
A total of 181 samples were tested using the Mindray BC-6800 haematology analyzer, including 117 malaria-infected samples collected from Yunnan, China, and 64 samples from healthy controls. Microscopy examination was conducted as reference when stained thick blood film revealed the presence of malaria parasites identified as Plasmodium vivax and Plasmodium falciparum. The receiver operating characteristic (ROC) curve analysis was developed using Analyse-it v4.92.3. The Kappa value was determined to evaluate the agreement between BC-6800 and light microscopy.
使用迈瑞BC-6800血液分析仪共检测了181份样本，其中包括117份来自中国云南的疟疾感染样本和64份来自健康对照的样本。当染色的厚血膜显示存在鉴定为间日疟原虫和恶性疟原虫的疟原虫时，进行显微镜检查作为参考。使用Analyse-it v4.92.3开发受试者工作特征（ROC）曲线分析。测定Kappa值以评价BC-6800与光学显微镜之间的一致性。
Results 结果
The sensitivity of InR‰ generated by BC-6800 for P. vivax and P. falciparum was 88.3 and 24.1%, respectively; specificity of InR‰ for malaria parasites was 84.3 and 84.3%, respectively; positive predictive value and negative predictive value was 89.4 and 82.7% for P. vivax, and 52.8 and 60.3% for P. falciparum. There was a strong correlation between ΔWBC and InR‰ (R2 = 0.9731 for P. vivax and R2 = 0.9757 for P. falciparum). There was also a significant correlation between parasitaemia and InR# in P. vivax-infected samples (R2 = 0.734). InR# was evaluated using ROC curve analysis, the area under the ROC curve is 0.95 with a 95% confidence interval of 0.926 to 0.974, and the cut-off value is 0.01 × 109/L for P. vivax. However, the ring stage and the early trophozoite stage of Plasmodium cannot be detected easily on BC-6800, possibly because of the small size and low nucleic acid content of these stages.
BC-6800计算的InR ‰对间日疟原虫和恶性疟原虫的敏感性分别为88.3和24.1%，特异性分别为84.3和84.3%，阳性预测值和阴性预测值分别为89.4和82.7%，恶性疟原虫分别为52.8和60.3%。ΔWBC与InR‰呈强相关（间日疟原虫R 2 = 0.9731，恶性疟原虫R 2 = 0.9757）。在间日疟原虫感染的样品中，寄生虫血症与InR#之间也存在显著相关性（R 2 = 0.734）。用ROC曲线分析法计算InR#，ROC曲线下面积为0.95，95%可信区间为0.926 ~ 0.974，间日疟原虫的临界值为0.01 × 10 ~（#3）#/L。然而，疟原虫的环状期和早期滋养体期在BC-6800上不容易检测到，可能是因为这些阶段的体积小且核酸含量低。
Conclusions 结论
The findings suggest that the flag ‘InR’ and the parameters ‘InR#/InR‰’ provided by the BC-6800 haematology analyzer could be used to screen for malaria in a clinical setting.
研究结果表明，BC-6800血液分析仪提供的“InR”标记和参数“InR#/InR‰”可用于在临床环境中筛查疟疾。
关键词：疟疾，寄生虫病，自动报警，感染红细胞，光学显微镜，BC-6800分析仪
Background 背景
Malaria is a vector-borne infectious disease that continues to have high morbidity and mortality globally [1]. The primary clinical presentation of malaria is fever or flu-like symptoms or a history of fever and flu-like symptoms. Diagnosis based only on clinical symptoms has very low specificity [2] as there is no combination of symptoms that reliably distinguishes malaria from other causes of fever or flu.
疟疾是一种病媒传播的传染病，在全球范围内仍然具有高发病率和死亡率[ 1]。疟疾的主要临床表现是发热或流感样症状，或有发热和流感样症状史。仅基于临床症状的诊断具有非常低的特异性[ 2]，因为没有可靠地将疟疾与其他发热或流感原因区分开来的症状组合。
Light microscopy, malaria nucleic acid amplification (PCR) test and malaria rapid diagnostic tests (RDTs) are used for parasitological diagnosis of malaria. Malaria PCR is not commonly used due to its high cost; RDTs are now more common, but not yet the regular test in non-endemic areas; microscopic examination of stained blood films remains the standard and most commonly used diagnosis method. Although this is the standard method, it is labour intensive and requires a high level of expertise to scan thick blood films for the presence of malaria parasites and thin blood films to determine the type of malaria [3, 4].
疟疾的寄生虫学诊断采用光学显微镜、疟疾核酸扩增试验和疟疾快速诊断试验。疟疾PCR由于成本高而不常用; RDT现在更常见，但在非流行地区尚未成为常规检测;染色血膜的显微镜检查仍然是标准和最常用的诊断方法。虽然这是标准方法，但它是劳动密集型的，需要高水平的专业知识来扫描厚血膜以确定疟疾寄生虫的存在和薄血膜以确定疟疾的类型[ 3，4]。
Malaria is prevalent in parts of Southeast Asia, South Asia, Africa and South America and, therefore, there is a demand for malaria screening in these areas [5–7]. Haematology analysis technology has improved considerably in the last 70 years, particularly with the introduction of automated haematology analyzers. Modern analyzers are capable of processing hundreds of samples per hour by using flow cytometry, and such techniques could aid in the diagnosis of malaria [8–11]. A recent study introduced a novel analyzer, which was able to detect infected red blood cells (iRBCs) in blood samples from mice infected with rodent malarial parasites [12] and determine the developmental stage of cultured Plasmodium falciparum [13]. Although this research is promising, most analyzers using numeric and graphical data to detect malaria fail to move beyond the research phase and are not clinically applicable due to the careful monitoring required to detect malaria parasites. A more reliable and accessible detection method for malaria is a dedicated alert message/flag that is incorporated into routine complete blood count (CBC) analysis when a positive case is detected. This could support earlier detection and potentially reduce adverse outcomes related to malaria infection.
疟疾在东南亚、南亚、非洲和南美洲的部分地区流行，因此，这些地区需要进行疟疾筛查[ 5- 7]。在过去的70年里，血液学分析技术有了很大的进步，特别是随着自动血液学分析仪的引入。现代分析仪能够通过使用流式细胞术每小时处理数百个样本，并且这种技术可以帮助诊断疟疾[ 8- 11]。最近的一项研究介绍了一种新型分析仪，该分析仪能够检测感染啮齿动物疟疾寄生虫的小鼠血液样本中受感染的红细胞（iRBC）[ 12]，并确定培养的恶性疟原虫的发育阶段[ 13]。虽然这项研究是有希望的，但大多数使用数字和图形数据来检测疟疾的分析仪未能超越研究阶段，并且由于检测疟疾寄生虫所需的仔细监测而不适用于临床。 一种更可靠和更容易获得的疟疾检测方法是在检测到阳性病例时纳入常规全血细胞计数（CBC）分析的专用警报信息/标志。这可以支持早期检测，并有可能减少与疟疾感染有关的不良后果。
For clinicians without extensive technical experience or expertise, the BC-6800 automated haematology analyzer (Mindray, Shenzhen, China) could be used to detect malaria parasites and to calculate parasitaemia in blood samples through cytometry analysis. The BC-6800 analyzer provides CBC parameters, reticulocyte and its fractions, and nucleated red blood cells (NRBC) value. It also provide a dedicated flag for ‘InR’ and ‘InR#/InR‰’ parameters. These are research use only parameters roughly equivalent to the number of InR (InR#)/the permillage of InR (InR‰) for the malaria iRBCs in a sample [14]. BC-6800 light scatter and fluorescence three-dimensional analysis technology (SF Cube) detects ‘iRBCs’ using signals generated by side-scattered light (SC, representing the internal cell structure and its contents), forward-scattered light (FS, indicating iRBC size), and side fluorescent light (FL, corresponding to DNA content). The flag is generated without the use of any special reagents.
对于没有广泛技术经验或专门知识的临床医生，BC-6800自动血液分析仪（迈瑞，中国深圳）可用于检测疟疾寄生虫，并通过细胞计数分析计算血液样本中的寄生虫血症。BC-6800分析仪提供CBC参数、网织红细胞及其组分和有核红细胞（NRBC）值。它还为“InR”和“InR#/InR‰”参数提供专用标志。这些研究仅使用大致相当于样本中疟疾iRBC的InR数量（InR#）/InR千分率（InR‰）的参数[ 14]。BC-6800光散射和荧光三维分析技术（SF Cube）利用侧向散射光（SC，代表细胞内部结构及其内容物）、前向散射光（FS，代表iRBC大小）和侧向荧光（FL，对应DNA含量）产生的信号检测“iRBC”。该标记是在不使用任何特殊试剂的情况下生成的。
The present study aimed to evaluate the utility of the infected RBC flag and parameters InR#/InR‰ in routine blood testing as a malaria screening tool in endemic areas. Furthermore, a comparison study was carried out for the flag information of CBC parameters between control group and the malaria group with P. vivax and P. falciparum.
本研究的目的是评估感染红细胞标志和参数InR#/InR‰在疟疾流行区常规血检中作为疟疾筛查工具的实用性。并对间日疟和恶性疟患者全血细胞计数指标的标志信息进行了比较研究。
Methods 方法
Samples and technical principles
样品和技术原理
EDTA anti-coagulated blood samples were collected from Tengchong, Yunnan Province (an endemic area in the border region of China and Myanmar) between May and August in 2016 according to physicians’ request. Some 117 malaria-infected samples were analysed, including 96 from P. vivax-infected patients (aged 3–69 years; 80 men and 16 women) and 21 from P. falciparum-infected patients (aged 6–60 years; 14 men and 7 women). Sixty-four healthy subjects from a non-endemic area in China were collected as the control group (aged 8–70 years; 47 men and 17 women). Samples were classified as malaria positive only when microscopy examination of stained thick blood film revealed the presence of at least one of the four malaria parasite forms: ring form, trophozoite, schizont and/or gametocytes. All blood samples were examined using both light microscopy and BC-6800 haematology analyzer using the manufacturer recommended reagents, calibrator and controls. Analyzer performance was monitored daily using three levels of quality control material.
根据医生要求，于2016年5月至8月在云南省腾冲市（中缅边境地区的流行区）采集EDTA抗凝血样本。分析了约117份疟疾感染样本，其中96份来自间日疟原虫感染患者（年龄3-69岁; 80名男性和16名女性），21份来自恶性疟原虫感染患者（年龄6-60岁; 14名男性和7名女性）。选择非流行区健康体检者64例作为对照组，年龄8-70岁，男47例，女17例。只有当染色的厚血膜的显微镜检查显示存在四种疟原虫形式中的至少一种时，样品才被归类为疟疾阳性：环状、滋养体、鞭毛体和/或配子体。使用光学显微镜和BC-6800血液分析仪，使用制造商推荐的试剂、校准品和质控品，对所有血液样本进行检查。 每天使用三种水平的质控材料监测分析仪性能。
The BC-6800 haematology analyzer used sheath flow impedance, laser scatter and SF Cube analysis technology. The SF Cube analysis technology is three-dimensional using information from laser light scatter at two angles and fluorescent signals for cell differentiation and counting [15]. In the BC-6800 differentiating (DIFF) channel, the fluorescent staining technology was adopted after the sample was mixed with DIFF lyse. For samples infected with malaria, RBC and white blood cell (WBC) sub-populations were differentiated by their size and complexity using lysing. Due to the different content of nucleic acid in WBC sub-populations, the volume of fluorescent dye staining the nucleic acid substances was different: the low-angle light scatter reflects cell size, the high-angle light scatter reflects intracellular granularity, and the intensity of fluorescent signal reflects the degree to which the cell is stained. Since multiple proteins play a role in generating increased rigidity of Plasmodium-infected erythrocytes, that would result in resistance to lysis [16, 17]. Through three-dimensional signal analysis of the cells processed with lyse, the DIFF channel differentiates the sub-populations, including lymphocytes, monocytes, neutrophils, Plasmodium-infected RBC, and eosinophils, as well as identifies and flags abnormal cells such as immature granulocytes, abnormal lymphocytes and blast cells.
BC-6800血液分析仪采用鞘流阻抗、激光散射和SF Cube分析技术。SF Cube分析技术是三维的，使用两个角度的激光散射信息和荧光信号进行细胞分化和计数[ 15]。在BC-6800分化（DIFF）通道中，样品与DIFF裂解液混合后采用荧光染色技术。对于感染疟疾的样品，RBC和白色血细胞（WBC）亚群通过它们的大小和复杂性使用裂解来区分。由于白细胞亚群中核酸含量不同，荧光染料染色核酸物质的体积也不同：低角度光散射反映细胞大小，高角度光散射反映细胞内粒度，荧光信号的强度反映细胞被染色的程度。 由于多种蛋白质在产生疟原虫感染的红细胞的刚性增加中起作用，这将导致对裂解的抗性[ 16，17]。通过对用裂解物处理的细胞进行三维信号分析，DIFF通道区分亚群，包括淋巴细胞、单核细胞、中性粒细胞、疟原虫感染的RBC和嗜酸性粒细胞，以及识别和标记异常细胞，如未成熟粒细胞、异常淋巴细胞和母细胞。
Comparison with light microscopy and quality control
与光学显微镜和质量控制的比较
Thin and thick blood films were prepared for staining and parasite investigation. Blood films were sent to Tengchong Laboratory in a standard slide box for staining with 3% Giemsa and microscopic examination [18]. Parasitaemia was determined from thick blood films by counting the number of parasites per 200 WBCs. Thick blood films were classified as positive if one or more malaria parasites were observed and negative if no parasites were observed after examining at least 100 ‘oil-lens’ fields (i.e. at a magnification of ×1000). Thin smears were further examined after parasites were seen in the thick smears in order to measure parasitaemia and identify the species of malaria parasites. All the blood smears were examined using a CX21 light microscope (Olympus, Tokyo, Japan).
制备薄的和厚的血膜用于染色和寄生虫调查。将血片置于标准载玻片盒中送至腾冲实验室，进行3%吉姆萨染色和显微镜检查[ 18]。通过计数每200个WBC的寄生虫数量，从厚血膜确定寄生虫血症。如果在检查至少100个“油镜”视野（即放大1000倍）后观察到一个或多个疟原虫，则厚血膜被归类为阳性，如果没有观察到疟原虫，则为阴性。在厚涂片中观察到寄生虫后，进一步检查薄涂片，以测量寄生虫血症并识别疟疾寄生虫的种类。使用CX 21光学显微镜（Olympus，Tokyo，Japan）检查所有血液涂片。
The malaria microscopist was blinded to BC-6800 results. Similarly, healthy controls were blinded to microscopy results. In order to assure malaria form quality of the microscopic examinations, the entire positive and 10% of the negative slides were sent to a senior malaria microscopist and re-examined at the Tengchong Laboratory. An experienced reader was assigned to re-examine the discrepant slides.
疟疾显微镜专家对BC-6800结果不知情。同样，健康对照对显微镜检查结果不知情。为了保证显微镜检查的疟疾形态质量，将全部阳性和10%的阴性切片送至高级疟疾显微镜师，并在腾冲实验室重新检查。指定一名经验丰富的阅片员重新检查不一致的玻片。
Ethical consideration 伦理思考
This study obtained ethical approval from the ethics committee of the General Hospital of the People’s Liberation Army. Individual informed consent was obtained from adults and from the parents or guardians of children under 18 years old when they presented in hospital. In addition, verbal assents were given to minors.
本研究获得了中国人民解放军总医院伦理委员会的伦理批准。从成人和18岁以下儿童的父母或监护人处获得个人知情同意书。此外，对未成年人给予口头同意。
Data analysis 数据分析
Data entry was performed using Microsoft Office Excel. Statistical analysis was performed using SPSS 22.0 (SPSS Inc, Chicago, IL, USA) analyzer Microsoft tool box. It was compared on the blood cell parameters and suspect flags between control group and iRBC group in BC-6800 analyzer. Pearson’s correlation or Spearman’s rank correlation was also used to determine the association between control group and malaria group (P. vivax group and P. falciparum group). Sensitivity, specificity and predictive values for the detection of different Plasmodium species were calculated and compared the P. vivax group and P. falciparum group. InR# representing the number of ‘iRBC’ was evaluated using ROC curve analysis, reporting the area under the curve (AUC) and its confidence interval (CI). ROC analysis was completed using Analyse-it v4.92.3. The Kappa value was calculated to evaluate the agreement between BC-6800 and light microscopy. A P value less than 0.05 was considered statistically significant.
使用Microsoft Office Excel进行数据录入。使用SPSS 22.0（SPSS Inc，芝加哥，IL，USA）分析仪Microsoft工具箱进行统计分析。在BC-6800全自动血细胞分析仪上比较对照组和iRBC组的血细胞参数和可疑标志。皮尔森相关或斯皮尔曼等级相关也用于确定对照组与疟疾组（间日疟组和恶性疟组）之间的关联。计算检测不同疟原虫种属的敏感性、特异性和预测值，并对间日疟原虫组和恶性疟原虫组进行比较。使用ROC曲线分析评价代表“iRBC”数量的InR#，报告曲线下面积（AUC）及其置信区间（CI）。使用Analyse-it v4.92.3完成ROC分析。计算Kappa值以评价BC-6800与光学显微镜之间的一致性。P值小于0.05被认为具有统计学显著性。
Results 结果
A special cluster showed in BC-6800 scattergram with malaria-infected samples
BC-6800散点图显示疟疾感染样本的一个特殊聚类
BC-6800 WBC DIFF plot displayed a distinct cluster for malaria parasites (Fig. 1a, yellow spots); SF Cube scatter plot permits clearer view of infected cell cluster (Fig. 1b, yellow spots). These findings suggested that samples with malaria parasites, especially schizont and/or gametocytes, show a distinct cluster and unique location on SF Cube.
BC-6800 WBC DIFF图显示疟疾寄生虫的明显聚类（图1a，黄色斑点）; SF Cube散点图可以更清楚地观察感染细胞聚类（图1b，黄色斑点）。这些发现表明，疟疾寄生虫的样品，特别是疟原虫和/或配子体，显示出独特的簇和独特的位置在SF立方体。
White blood cell scattergrams generated by the Mindray BC-6800 haematology analyzer. a An example of the gates lymphocyte, monocyte, neutrophil, infected RBC (yellow spots) and eosinophil in a representative patient with malaria; b the distinct cluster of malaria parasites (yellow spots) in three-dimensional analysis scattergrams
迈瑞BC-6800血液分析仪生成的白色血细胞散点图。a代表性疟疾患者的门淋巴细胞、单核细胞、中性粒细胞、受感染的RBC（黄色斑点）和嗜酸性粒细胞的示例; b三维分析散点图中疟疾寄生虫（黄色斑点）的独特集群
Performance of InR by BC-6800 in comparison to the reference light microscopy
BC-6800与参考光学显微镜相比测定InR的性能
The sensitivity and specificity of light microscopy for P. vivax and P. falciparum infection were compared with that of InR‰ by BC-6800. The sensitivity of the InR‰ by BC-6800 for P. vivax and P. falciparum was 88.3 and 24.1%, respectively. The specificity of the InR‰ by BC-6800 for P. vivax and P. falciparum was 84.3 and 84.3%, respectively. Details of these results can be found in Table 1.
并与BC-6800测定InR‰的敏感性和特异性进行比较。BC-6800测定间日疟原虫和恶性疟原虫的InR‰敏感性分别为88.3%和24.1%。BC-6800测定间日疟原虫和恶性疟原虫的InR‰的特异性分别为84.3%和84.3%。这些结果的详细信息见表1。
Table 1 表1
Performance of InR‰ by BC-6800 identified in comparison to the reference light microscopy
与参考光学显微镜相比，BC-6800鉴别InR‰的性能
| P. vivax (95% CI) P. vivax（95%CI） | P. falciparum (95% CI) p。恶性疟原虫（95%CI） | |
|---|---|---|
| Sensitivity | 88.3% (83.73–93.77%) 88.3%（83.73-93.77%） | 24.1% (19.43–40.42%) 24.1%（19.43-40.42%） |
| Specificity | 84.3 (76–90.55%) 84.3（76-90.55%） | 84.3% (76–90.55%) 84.3%（76-90.55%） |
| Positive likelihood ratio 阳性似然比 | 5.69 (3.66–8.82) 5.69（3.66-8.82） | 1.85 (1.06–3.22) 1.85（1.06-3.22） |
| Negative likelihood ratio 阴性似然比 | 0.12 (0.08–0.20) 0.12（0.08-0.20） | 0.84 (0.71–0.99) 0.84（0.71-0.99） |
| Disease prevalence 疾病流行 | 60% (53.89–65.89%) 60%（53.89-65.89%） | 42.25 (35.07–49.67%) 42.25（35.07-49.67%） |
| Positive predicitve value 正预测值 | 89.4% (83.73–93.77%) 89.4%（83.73-93.77%） | 52.8 (40.89–72.96%) 52.8（40.89-72.96%） |
| Negative predicitve value 负预测值 | 82.7% (76–90.55) 82.7%（76-90.55） | 60.3 (53.54–69.78%) 60.3（53.54-69.78%） |
| Total consistent rate 总一致率 | 86.7% | 58.9% |
| Kappa value | 0.738 | 0.143 |
InR# was evaluated using ROC curve analysis. In the P. vivax group, AUC is 0.95, with a 95% CI of 0.926 to 0.974 and Youden indices of 0.726. The best cut-off is 0.01 × 109/L. However, in the P. falciparum group, area under ROC curve is only 0.55, meaning it is not amenable to screen P. falciparum. The ROC curves are shown in Fig. 2.
采用ROC曲线分析法评价InR#。在间日疟原虫组中，AUC为0.95，95% CI为0.926 - 0.974，约登指数为0.726。最佳临界值为0.01 × 10 9 /L。而恶性疟原虫组的ROC曲线下面积仅为0.55，不适于筛查恶性疟原虫。ROC曲线如图2所示。
The difference of InR and InR‰ in various infection density groups
不同感染密度组间InR及InR‰的差异
As shown in Table 2, the results presented that the infection densities in microscopy (χ2 = 16.230, P < 0.001) in various InR groups were unequal, and the difference was statistically significant. In addition, these results displayed that the infection density in InR group II and group III were higher than that in group I.
由表2可知，不同InR组显微镜下感染密度不等（χ 2 = 16.230，P < 0.001），差异有统计学意义。此外，这些结果显示，InR组II和组III的感染密度高于组I。
Table 2 表2
The difference of infection density in different groups (Plasmodium vivax) [M (P25, P75)]
不同组别间日疟原虫感染密度的差异[M（P25，P75）]
| The number of InR InR的数量 | Infection density in microscope (109/L) 显微镜下感染密度（10 9 /L） | χ 2 | P |
|---|---|---|---|
| Group I | 1388 (932, 2040) | 16.23 | < 0.001 |
| Group II | 2575 (1217, 4485)* | ||
| Group III | 2957 (2105, 5201)* |
I: The range of InR# was [0, 0.1); II: The range of InR# was [0.1, 2); III: The range of InR# was ≥ 2
I：InR#范围为[0，0.1）; II：InR#范围为[0.1，2）; III：InR#范围≥ 2
* Compared to group I, P < 0.05
* 与I组相比，P < 0.05
Blood cell parameters and suspect flags between control group and malaria groups in BC-6800 analyzer
BC-6800血细胞分析仪中对照组与疟疾组的血细胞参数及可疑标志
WBC parameters and suspect flags between control group and malaria group
对照组和疟疾组之间的WBC参数和可疑标志
Automated WBC counts and suspect flags from BC-6800 haematology analyzer are shown in Table 3. The correlation between the count of InR and cell blood count was analysed in the P. vivax-infected patient group and in the P. falciparum-infected group. The result showed that there was a clear relationship between ΔWBC (WBCDIFF–WBCBASO) and InR‰ (P. vivax group R2 = 0.973). (WBCDIFF is the number of WBC counting in the DIFF channel with mild lyse, and WBCBASO is the number of WBC counting in the BASO channel with severe membrane destruction in the Mindray BC-6800 haematology analyzer). A better relationship was present between ΔWBC and InR‰ in the P. falciparum group, however, the InR flags were only flagged in 5 of all 21 P. falciparum patients by the BC-6800 analyzer (Fig. 3). The correlation between the number of InR (‰) and P. vivax was R2 = 0.734 (Fig. 4).
BC-6800血液分析仪的自动WBC计数和可疑标记见表3。对间日疟原虫感染组和恶性疟原虫感染组的InR计数与血细胞计数进行相关性分析。结果表明，ΔWBC（WBC DIFF -WBC BASO ）与InR‰（间日疟原虫组R 2 = 0.973）有明显的相关性。(WBC DIFF 为迈瑞BC-6800血液分析仪中DIFF通道中轻度溶解的WBC计数数，WBC BASO 为BASO通道中膜严重破坏的WBC计数数）。恶性疟原虫组中ΔWBC与InR‰之间存在更好的相关性，然而，BC-6800分析仪仅在所有21例恶性疟原虫患者中的5例中标记出InR标记（图3）。InR（‰）与间日疟原虫的相关性为R 2 = 0.734。
Table 3 表3
Blood cell parameters and suspect flags between control group and malaria groups in BC-6800 analyzer
BC-6800血细胞分析仪中对照组与疟疾组的血细胞参数及可疑标志
| Parameters | Control group (n = 64) 对照组（n = 64） | Malaria group (n = 117) 疟疾组（n = 117） | p 1 | p 2 | |
|---|---|---|---|---|---|
| P. vivax (n = 96) P.间日疟（n = 96） | P. falciparum (n = 21) P.恶性疟原虫（n = 21） | ||||
| WBC [DIFF] count (× 109/L) WBC [DIFF]计数（× 10 9 /L） | 6.94 (2.75 to 22.65) 6.94（2.75至22.65） | 6.46 (2.13 to 21.0) 6.46（2.13至21.0） | 6.14 (2.55 to 12.44) 6.14（2.55至12.44） | 0.04 | 0.03 |
| WBC [BASO] count (× 109/L) WBC [BASO]计数（× 10 9 /L） | 6.90 (2.74 to 22.84) 6.90（2.74至22.84） | 5.42 (1.06 to 11.31) 5.42（1.06至11.31） | 5.70 (2.29 to 9.76) 5.70（2.29至9.76） | 0.02 | 0.03 |
| WBC [DIFF]–WBC [BASO] (× 109/L) WBC [DIFF]-WBC [BASO]（× 10 9 /L） | 0.04 (− 5.8 to 16.12) 0.04（-5.8至16.12） | 1.75 (− 0.37 to 1.6) 1.75（-0.37至1.6） | 0.5 (− 0.67 to 2.06) 0.5（-0.67至2.06） | 0.01 | 0.01 |
| RBC (1012/L) | 4.93 (3.13 to 7.06) 4.93（3.13至7.06） | 4.66 (1.64 to 6.82) 4.66（1.64至6.82） | 4.54 (2.49 to 6.53) 4.54（2.49至6.53） | 0.72 | 0.68 |
| HGB (g/L) | 136.78 (97 to 208) 136.78（97至208） | 128.54 (52 to 209) 128.54（52至209） | 125.15 (62 to 165) 125.15（62至165） | 0.83 | 0.81 |
| RDW-CV (%) | 13.45 (11.8 to 17.3) 13.45（11.8至17.3） | 14.55(11.2 to 32.4) 14.55（11.2至32.4） | 14.55 (12 to 18.2) 14.55（12至18.2） | 0.66 | 0.66 |
| RET (%) | 0.98 (0.33 to 2.67) 0.98（0.33至2.67） | 1.53 (0.43 to 12.47) 1.53（0.43至12.47） | 2.01 (0.52 to 7.58) 2.01（0.52至7.58） | 0.01 | 0.01 |
| IRF (%) | 4.44 (0 to 17.5) 4.44（0至17.5） | 9.90 (0.9 to 24.2) 9.90（0.9至24.2） | 6.80 (0.9 to 25.1) 6.80（0.9至25.1） | 0.01 | 0.02 |
| LFR (%) | 95.56 (82.5 to 100) 95.56（82.5至100） | 90.10 (56.1 to 99.1) 90.10（56.1至99.1） | 93.21 (74.9 to 99.1) 93.21（74.9至99.1） | 0.04 | 0.06 |
| MFR (%) | 4.28 (0 to 13.4) 4.28（0至13.4） | 8.11 (0.9 to 24.2) 8.11（0.9至24.2） | 6.23 (0.9 to 19.3) 6.23（0.9至19.3） | 0.03 | 0.04 |
| HFR (%) | 0.16 (0 to 4.2) | 1.79 (0 to 22.1) | 0.57 (0 to 5.8) | 0.01 | 0.02 |
| Anaemia (%) | 11 (17.18%) | 27 (28.13%) | 6 (28.57%) | 0.03 | 0.03 |
| PLT count (× 1010/L) | 193 (62 to 355) | 108 (8 to 289) | 149 (53 to 323) | 0.01 | 0.03 |
| Malaria flag/parameters | |||||
| InR flag | 0 | 85 (88.5%) | 5 (23.80%) | 0.01 | 0.01 |
| InR# | |||||
| InR‰ | |||||
P1, the P value compared P. vivax group to control group; P2, the P value compared P. falciparum group to control group; InR, parasite infected red blood cell; InR#, the number of infected red blood cell; InR‰, the permillage of infected red blood cell. RBC, red blood cell; HGB, haemoglobin; RDW, red blood cell volume distribution width; RET, reticulocyte; IRF, immature reticulocyte fraction; LFR, low fluorescence ratio; MFR, middle fluorescence ratio; HFR, high fluorescence ratio; PLT, platelet count; InR, parasite infected red blood cell; InR#, the number of infected red blood cell; InR‰, the permillage of infected red blood cell
P 1 为间日疟原虫组与对照组比较的P值; P 2 为恶性疟原虫组与对照组比较的P值; InR为疟原虫感染红细胞数; InR#为感染红细胞数; InR‰为感染红细胞千分率。RBC，红细胞; HGB，血红蛋白; RDW，红细胞体积分布宽度; RET，网织红细胞; IRF，未成熟网织红细胞分数; LFR，低荧光比; MFR，中荧光比; HFR，高荧光比; PLT，血小板计数; InR，寄生虫感染红细胞; InR#，感染红细胞数; InR‰，感染红细胞千分率
The correlation between the ‘Infected RBC’ (InR‰) and ΔWBC (WBCDIFF–WBCBASE). a The correlation between the InR‰ and ΔWBC in P. vivax; b the correlation between the InR‰ and ΔWBC in P. falciparum
“感染红细胞”（InR‰）与ΔWBC（WBC DIFF -WBC BASE ）之间的相关性。a间日疟原虫InR‰与ΔWBC的相关性; b恶性疟原虫InR‰与ΔWBC的相关性
The correlation between the ‘Infected RBC’ (InR‰) and P. vivax
感染红细胞与间日疟原虫的相关性
RBC parameters and suspect flags between control group and malaria group
对照组和疟疾组之间的RBC参数和可疑标志
Automated RBC counts and suspect flags from BC-6800 haematology analyzer are shown in Table 3. There was no significant difference in RBC, HGB and red blood cell volume distribution width (RDW) between the malaria group and control group (P > 0.05), but a significant difference in reticulocyte parameters was observed between P. vivax/P. falciparum patient group and the control group (P < 0.01).
BC-6800血液分析仪的自动RBC计数和可疑标记见表3。间日疟/恶性疟患者RBC、HGB、RDW与对照组比较差异无显著性（P > 0.05），网织红细胞参数与对照组比较差异有显著性（P < 0.01）。
PLT parameters and suspect flags between control group and malaria group
对照组和疟疾组之间的PLT参数和可疑标志
Automated platelets (PLT) counts and suspect flags from the BC-6800 haematology analyzer are shown in Table 3. There was significant difference in PLT count between P. vivax/P. falciparum patient groups and control group (P < 0.05).
BC-6800血液分析仪的自动血小板（PLT）计数和可疑标记见表3。间日疟/恶性疟患者组与对照组血小板计数比较差异有显著性（P < 0.05）。
Discussion 讨论
The BC-6800 haematology analyzer uses SF-Cube technology to achieve three-dimensional cell analysis for WBC, reticulocyte (RET) and NRBC with clinically relevant suspect flags [19, 20]. It provides a dedicated flag named ‘infected RBC?’ and parameters ‘InR#/InR‰’ to represent the possible presence of Plasmodium parasites and the causative agent of malaria infection. In this study, the parameter ‘infected RBC’ was used to analyse the diagnosis performance in patients with malaria, as well as routine parameters and suspect flags.
BC-6800血液分析仪使用SF-Cube技术实现WBC、网织红细胞（RET）和具有临床相关可疑标记的NRBC的三维细胞分析[ 19，20]。它提供了一个专用的标志，名为“感染的RBC？”参数“InR#/InR‰”表示可能存在疟原虫寄生虫和疟疾感染的病原体。在这项研究中，参数“感染的红细胞”被用来分析疟疾患者的诊断性能，以及常规参数和可疑标志。
The diagnostic performance of the InR flag for the diagnosis of Plasmodium was evaluated. Samples with malaria parasites show a distinct cluster and unique location on three-dimensional cell plots. The characteristics of the cluster are dependent on the species of Plasmodium as well as the size and number of the parasite present. The sensitivity and specificity for detecting P. vivax was not significant for P. falciparum. This may be a result of early trophozoites of P. falciparum that developed over 10 h in the peripheral blood concealed in the microvasculature, sinusoids or other slow blood flow before being developed into late trophozoites and schizonts [21, 22]. Trophozoites and schizonts of P. falciparum are rarely seen in the peripheral blood of infected patients. The ring and the gametocyte stage are the most commonly seen in a peripheral blood smear. However, the ring and the early trophozoites of Plasmodium cannot be detected easily on BC-6800, the main reason possibly being the small size and low nucleic acid content.
评价了InR标记用于诊断疟原虫的诊断性能。带有疟原虫的样本在三维细胞图上显示出独特的簇和独特的位置。簇的特征取决于疟原虫的种类以及存在的寄生虫的大小和数量。检测间日疟原虫的敏感性和特异性与恶性疟原虫差异不显著。这可能是恶性疟原虫早期滋养体在微血管、窦状隙或其他缓慢血流中隐藏的外周血中发育超过10小时，然后发育成晚期滋养体和滋养体的结果[ 21，22]。恶性疟原虫的滋养体和寄生虫很少出现在感染患者的外周血中。在外周血涂片中最常见的是环状和配子母细胞期。 但在BC-6800上不易检测到疟原虫的环状体和早期滋养体，主要原因可能是其体积小、核酸含量低。
InR flag being triggered by BC-6800 should meet two conditions. First, the malaria-infected specimens should be collected during the intra-erythrocytic phase Plasmodium. Second, the number of intra-erythrocytic phase Plasmodium should surpass a certain threshold [23]. Some P. vivax specimens were tested as false negatives because they did not meet these two conditions. This may be because the majority of samples among the P. vivax group are trophozoite and ring form, which are too small to be detected easily. Both microscopy and malaria RDT are very effective at identifying ring infections. In addition to its poor sensitivity for detecting P. falciparum, this study suggested the shortcoming should be improved in BC-6800 haematological analyzer. At present, this is the major limitation of broader use for P. falciparum in particular. The machine could be more effective for identifying P. falciparum in future.
BC-6800触发的InR标志应满足两个条件。首先，疟疾感染的标本应收集在红细胞内期疟原虫。其次，红细胞内期疟原虫的数量应超过一定的阈值[ 23]。一些间日疟原虫标本被检测为假阴性，因为它们不符合这两个条件。这可能是因为间日疟原虫组中的大多数样品是滋养体和环状的，它们太小而不易检测。显微镜和疟疾RDT在识别环感染方面都非常有效。BC-6800血细胞分析仪检测恶性疟原虫的灵敏度较低，提示其存在的不足之处有待改进。目前，这是恶性疟原虫广泛使用的主要限制。该机器在未来可能更有效地识别恶性疟原虫。
Several studies report that DIFF scattergram suspect flags can provide assistance in the diagnosis of malaria in P. vivax-infected patients [10, 24]. Results from the present study support this finding in the iRBC group. Research shows that high eosinophil count is found in the presence of P. vivax infection, and was observed in up to 39% P. vivax-infected patients [25]. In this study, results found that pseudo-eosinophilia was observed in only a small number of samples (P. vivax 6.25% and P. falciparum 14.29%). In P. vivax and P. falciparum-infected patients, results found that WBC count was higher in the DIFF channel (mild lyse) compared to the WBC/BASO channel (severe membrane destruction). A strong correlation was found between ∆WBC (WBCDIFF–WBCBASO) and the number of iRBC (R2 = 0.973) for P. vivax-infected patients (Fig. 3). A correlation was also found between parasitaemia and the number of iRBC for P. vivax-infected patients (R2 = 0.734; Fig. 4). This makes it possible to estimate the amount of P. vivax after samples are tested using the BC-6800.
几项研究报告，DIFF散点图可疑标记可以为间日疟原虫感染患者的疟疾诊断提供帮助[ 10，24]。本研究的结果支持iRBC组的这一发现。研究表明，在间日疟原虫感染的情况下发现高嗜酸性粒细胞计数，并在高达39%的间日疟原虫感染患者中观察到[ 25]。在这项研究中，结果发现仅在少数样本中观察到假性嗜酸性粒细胞增多（间日疟原虫6.25%和恶性疟原虫14.29%）。在间日疟原虫和恶性疟原虫感染的患者中，结果发现DIFF通道（轻度溶解）中的WBC计数高于WBC/BASO通道（严重膜破坏）。对于间日疟原虫感染的患者，发现iRBC（WBC DIFF -WBC BASO ）和iRBC数量（R 2 = 0.973）之间存在强相关性（图3）。还发现寄生虫血症与间日疟原虫感染患者的iRBC数量之间存在相关性（R 2 = 0.734;图4）。 这使得在使用BC-6800检测样品后可以估计间日疟原虫的数量。
Anaemia a common symptom of malaria and is associated with dyserythropoiesis and ineffective erythropoiesis [26]. The BC-6800 haematology analyzer provides additional parameters, including the immature platelet fraction (IPF) and immature reticulocyte fraction (IRF), both important in clinical markers for thrombocytopaenia and anaemia [27–29]. Results from the present study show P. vivax or P. falciparum parasitaemia are associated with abnormal reticulocyte parameters, such as RET scattergrams and IRF. Results also show platelet-associated flags, including PLT clump, IPF and PLT abnormal in malaria-positive samples were increased compared to the control group. These findings are in accordance with the study of Dubreuil et al. [30].
贫血是疟疾的常见症状，与红细胞生成不良和无效红细胞生成有关[ 26]。BC-6800血液分析仪提供了其他参数，包括未成熟血小板分数（IPF）和未成熟网织红细胞分数（IRF），这两个参数在血小板减少症和贫血的临床标志物中都很重要[ 27- 29]。本研究的结果显示间日疟原虫或恶性疟原虫寄生虫血症与异常网织红细胞参数相关，如RET散点图和IRF。结果还显示，与对照组相比，疟疾阳性样本中的血小板相关标志，包括PLT团块、IPF和PLT异常增加。这些发现与Dubreuil等人的研究一致[ 30]。
Except for cost, malaria PCR and RDTs are obviously ahead of the BC-6800 in sensitivity. It is necessary to further improve the sensitivity, especially for the ring infections, in the apparatus. In this study, the associations were limited between the haematological parameters and the species of infection considering patient age, symptoms and prior treatment.
除成本外，疟疾PCR和RDTs在灵敏度上明显优于BC-6800。有必要进一步提高仪器的灵敏度，特别是对环感染的灵敏度。在本研究中，考虑到患者年龄、症状和既往治疗，血液学参数与感染种属之间的相关性有限。
Conclusions 结论
A dedicated ‘Infected RBC’ flag on a CBC result print-out provides an objective record and a trigger for further examination if malaria is suspected. Establishing a work protocol to further examine all flagged samples and use thick blood film microscopy and/or malaria RDTs to confirm or rule out malaria may reduce therapeutic intervention time and improve patient care outcomes. In malaria-endemic zones, ‘Infected RBC’ flag could serve as a rapid decision support tool when screening for malaria.
CBC结果打印件上的专用“感染RBC”标志提供了客观记录，并在怀疑疟疾时触发进一步检查。建立一个工作协议，进一步检查所有标记的样本，并使用厚血膜显微镜和/或疟疾RDT来确认或排除疟疾，可以减少治疗干预时间，改善患者护理结果。在疟疾流行区，“感染红细胞”标志可以作为筛查疟疾时的快速决策支持工具。
Acknowledgements 确认
The authors are grateful to the Department of Infectious Disease of Chinese People’s Liberation Army General Hospital for their advice of clinical research designing and article modification.
作者感谢中国人民解放军总医院感染科对临床研究设计和文章修改的建议。
Abbreviations 缩写
| iRBC | infected red blood cell 感染红细胞 |
| CBC | complete blood count 全血细胞计数 |
| NRBC | nucleated red blood cells 核红细胞 |
| InR | infected RBC |
| RBC | red blood cell 红细胞 |
| WBC | white blood cell 白色细胞 |
| ROC | receiver operating characteristic 受试者工作特征 |
| AUC | area under the curve 曲线下面积 |
| CI | confidence interval 置信区间 |
| RDW | red blood cell volume distribution width 红细胞体积分布宽度 |
| DIFF | differentiating |
| PLT | platelets |
| RET | reticulocyte |
| PPV | positive predictive value 阳性预测值 |
| NPV | negative predictive value 阴性预测值 |
| malaria RDT | malaria rapid diagnostic test 疟疾快速诊断试验 |
| SC | side scattered light 侧向散射光 |
| FS | forward scattered light 前向散射光 |
| FL | side fluorescent light 侧向荧光 |
Authors’ contributions 作者的贡献
SY designed the study, collected and tested the samples and wrote the paper. X-DJ was a major contributor in improving the study design and paper. CC analyzed the data. HS contributed to the improvement of data collection and analysis. QH operated training for the instrument. W-CB was director of the study. All authors read and approved the final manuscript.
SY设计了研究，收集和测试了样本，并撰写了论文。X-DJ是改进研究设计和论文的主要贡献者。CC分析了数据。HS为改进数据收集和分析作出了贡献。QH仪器操作培训。W-CB是该研究的负责人。所有作者均阅读并批准了最终手稿。
Funding 资金
This work is supported by the National Key Scientific Instrument and Equipment Development Project in China (No. 2012YQ18011708).
本工作得到了国家重点科学仪器设备研制项目（No.2012YQ18011708）的支持。
Availability of data and materials
数据和材料的可用性
The data used and/or analysed during the current study are available from the testing of blood samples collected from area mentioned above.
本研究期间使用和/或分析的数据可从上述区域采集的血液样本检测中获得。
Ethics approval and consent to participate
伦理批准和参与同意
This study obtained ethical approval from the ethics committee of the General Hospital of the People’s Liberation Army. Individual informed consent was obtained from adults and from the parents or guardians of children under 18 years old when they presented in hospital. In addition, verbal assents were given to minors.
本研究获得了中国人民解放军总医院伦理委员会的伦理批准。从成人和18岁以下儿童的父母或监护人处获得个人知情同意书。此外，对未成年人给予口头同意。
Competing interests 相互竞争的利益
The authors declare that they have no competing interests.
提交人声明，他们之间没有利益冲突。
Footnotes 脚注
Publisher's Note 出版商说明
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
施普林格自然在已出版地图和机构隶属关系中的管辖权主张方面保持中立。
Yi Sun and Daijun Xiang contributed equally to this work
孙毅和代军翔对这项工作作出了同样的贡献
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