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2019; 18: 262.
Malar J. 2019; 18:262.
Published online 2019 Jul 31. doi: 10.1186/s12936-019-2890-z
在线发布2019年7月31日。doi:10.1186/s12936-019-2890-z
PMCID: PMC6668162 PMCID:PMC6668162
PMID: 31366365 PMID:31366365

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

Yi Sun,#1 Daijun Xiang,#1 Chen Chen,1 Shang He,1 Huan Qi,2 and Chengbin Wangcorresponding author1
孙毅, # 1 向代军, # 1 陈晨, 1 尚和, 1 桓琦, 2 王成斌 corresponding author 8#

Associated Data 关联数据

Data Availability Statement
数据可用性声明

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。

Keywords: Malaria, Parasitaemia, Automatic alert, Infected RBC, Light microscopy, BC-6800 analyzer
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 []. 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 [] 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 [, ].
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 []. 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 []. 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 [] and determine the developmental stage of cultured Plasmodium falciparum []. 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 []. 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 []. 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 [, ]. 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 []. 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).