临床诊断人体全血白细胞吞噬的定量检测

 

Whole blood assay:人体全血检测

 

· No isolation procedures and optimal culture medium

 

   没有隔离程序和最佳培养基

 

· Suppressible fluorescence (quenching)

 

    可隐藏的荧光(淬火)

 

· Physiological target

 

    生理目标

 

· Standardized fluorescence intensity

 

    标准化的荧光强度

 

· Compatible for simultaneous immunofluorescence

 

       兼容,同时免疫荧光

 

· pH dependent FITC fluorescence

 

       pH值依赖的牛血清白蛋白荧光

 

· No electrostatic charging or absorption artifacts like latex

 

    没有静电充电或吸收构件例如乳胶

 

· Favourable size range of bacteria

 

    有利的细菌尺寸范围

 

· Exclusion of aggregation artifacts by DNA staining

 

       通过DNA染色排除聚合工件

 

 

Clinical Diagnostic for the Quantitative Analysis of Leukocyte Phagocytosis in Human Whole Blood

 

SUMMARY and EXPLANATION

 

PHAGOTEST® is a complete diagnostic kit for the investigation of the phagocytic function of granulocytes and monocytes [1, 2] in whole blood. This test kit allows the quantitative determination of leukocyte phagocytosis in heparinized whole blood. It contains fluorescein (FITC) labelled opsonized bacteria (E. coli-FITC) and necessary reagents. It measures the overall percentage of monocytes and granulocytes showing phagocytosis in general (ingestion of one or more bacteria per cell) and the individual cellular phagocytic activity (number of bacteria per cell). The evaluation of the results can be done either by flow cytometry or by fluorescence microscopy.

 

摘要及说明

 

PHAGOTEST是一个完整的诊断试剂盒用于研究单核细胞吞噬功能的全血粒细胞[1,2]。这个测试套件在全血的肝素化允许定量检测白细胞吞噬功能。它包含荧光素(牛血清白蛋白)标记过的使(真菌)易受调理素作用细菌(大肠杆菌牛血清白蛋白)和必要的试剂。它能测量单核细胞的总百分比和一般情况下粒细胞单核细胞吞噬(每个细胞摄入的一个或多个细菌)和个体细胞的吞噬活性(每个细胞的细菌数量)。测试的结果可以通过流式细胞术或荧光显微镜显示。

 

APPLICATIONS

 

PHAGOTEST® is intended to investigate the phagocytic activity found in various disorders and to evaluate the effects of drugs. Abnormal phagocytosis can occur with a variety of clincal disorders (3, 4). The defects can be associated with the neutrophil itself or with an immunoglobulin or complement defect. Known inborne defects are actin dysfunction, tuftsin deficiency and complement receptor C3bi deficiency. These deficiencies can result in increased susceptibility to infection due to defective neutrophil phagocytosis.

 

Acquired defects associated with altered phagocytic activity can be observed in trauma, diabetes, renal failure, and infection. Reduced phagocytosis was found in patients with recurrent bacterial skin and sinopulmonary infections (5), in patients with wound infections from burns (6), in patients with AIDS (7), in neonates or in elderly people.

 

Various immunomodulators (cytokines such as interleukin-2 or interferon-, lactic acid bacteria and plant extracts such as from Echinaceae Purpureae herba) can increase the phagocytic activity of neutrophils and monocytes. These effects can be investigated in vitro or ex vivo (8, 9).

 

PHAGOTEST® also allows the study of the phagocytic activity of HL-60 promyelocytic leukemia cells or of isolated monocytes or macrophages.

 

The diagnostic kit is also applicable on blood of mice, rats, rabbits, dogs, cattle and other species.

 

应用：

 

PHAGOTEST®旨在研究在各种不同中发现吞噬活性以及评价药物的作用。异常吞噬作用会产生于各种临床疾病(3, 4)。缺陷可以与中性粒细胞本身，免疫球蛋白或者不提缺陷相关联。已知的缺陷是肌动蛋白功能障碍,tuftsin缺乏症和补体受体C3bi缺乏症。由于有缺陷的嗜中性粒细胞吞噬作用这些缺陷可以导致感染的易感性增加而导致感染。

 

获得的缺陷与改变了的吞噬活性可以被观察到与外伤、糖尿病、肾功能衰竭、感染相关联。减少吞噬作用在患者复发性细菌性皮肤和窦肺感染(5),在伤口感染患者从伯恩斯(6),导致艾滋病患者(7),在新生儿或老年人被发现。

 

 

各种免疫调节剂(细胞因子如白细胞介素-2或干扰素-,乳酸菌和植物提取物如Echinaceae Purpureae草)可以增加中性粒细胞和单核细胞的吞噬活性。这些影响可以研究体外或间接体内疗法(8、9)。

 

PHAGOTEST®还允许研究的噬菌作用的活动早幼粒细胞白血病细胞hl 60的或孤立的单核细胞或巨噬细胞。

 

TEST PRINCIPLES

 

Phagocytosis by polymorphonuclear neutrophils and monocytes constitutes an essential arm of host defense against bacterial or fungal infections. The phagocytic process can be separated into several major stages: chemotaxis (migration of phagocytes to inflammatory sites), attachment of particles to the cell surface of phagocytes, ingestion (phagocytosis) and intracellular killing by oxygen-dependent and oxygen-independent mechanisms (1, 2).

 

PHAGOTEST® allows the quantitative determination of leukocyte phagocytosis (ingestion of bacteria). It measures the percentage of phagocytes which have ingested bacteria and their activity (number of bacteria per cell). The phagocytosis test kit contains fluorescein-labelled opsonized Escherichia coli bacteria and other necessary reagents. Heparinzed whole blood is incubated with the fluorescein-labelled E.coli bacteria at 37°C, a negative control sample remains on ice. The phagocytosis is stopped by placing the samples on ice and adding QUENCHING SOLUTION. This solution allows the discrimination between attachment and internalization of bacteria by quenching the FITC fluorescence of surface bound bacteria leaving the fluorescence of internalized particles unaltered. After two washing steps with WASHING SOLUTION erythrocytes are then removed by addition of LYSING SOLUTION. The DNA STAINING SOLUTION, which is added just prior flow cytometric analysis, excludes aggregation artifacts of bacteria or cells. The percentage of cells having performed phagocytosis are then analyzed as well as their mean fluorescence intensity (number of ingested bacteria).

 

The E.coli bacteria are opsonized with immunoglobulin and complement of pooled sera. Cells of the phagocytic system (monocytes, polymorphonuclear neutrophils) have receptors for a complement component (C3b) and for the constant part of the immunoglobulin molecule (Fc) mediating the adhesion of the bacteria to the cell surface. By utilizing both opsonized and nonopsonized bacteria, which are also available, both opsonic capacity and phagocytosis can be measured at the same time. Thus, it can be determined whether abnormal phagocytosis is due to a failure in the opsonization process or to a defect in the ingestion capability of the phagocyte.

 

测试原理

 

多形核中性粒细胞和单核细胞的吞噬作用是构成防御细菌或真菌感染一个主要防御基本臂。吞噬的过程可以分为几个主要阶段:趋化性(吞噬细胞迁移到炎症),附件的颗粒的吞噬细胞到细胞表面,摄入(吞噬作用)和胞内杀害氧依赖性和氧气独立机制(1、2)。

 

PHAGOTEST®允许定量测定白细胞吞噬作用(摄入细菌)。它衡量吞噬细胞的百分比包括它们摄入的细菌和它们的活性(每个细胞的细菌数量)。吞噬功能测试套件包括荧光素标记促进大肠杆菌的调理作用的细菌和其他必要的试剂。Heparinzed全血是与荧光标记的大肠杆菌在37°C培育。阴性的控制样品在冰上存活。停止吞噬功能需将样品放置在冰上并加入淬火溶液。这种溶液通过淬火的牛血清白蛋白的荧光细菌表面束缚不变的内化粒子能辨别出细菌附着物和内化物。经过红细胞冲洗液两次冲洗过程并移除添加溶液剂，DNA染色溶液加入前流流式细胞分析,排除了聚合工件的细菌或细胞。细胞的百分比进行了吞噬作用然后分析以及他们的平均荧光强度(摄入细菌的数量)。

 

REAGENTS PROVIDED

 

提供的试剂

 

· Fluorescein-labelled opsonized Escherichia coli bacteria

 

荧光素标记促进大肠杆菌的调理作用的细菌

 

· QUENCHING SOLUTION

 

猝火溶液

 

· DNA STAINING SOLUTION

 

DNA染色溶液

 

· LYSING SOLUTION

 

溶血剂

 

· WASHING SOLUTION

 

利用流式细胞技术及Phagotest试剂盒可以方便准确地定量检测人全血白细胞吞噬功能，方便全血分析，不必分离或培养细胞，在样品处理的过程中用溶解液将红细胞裂解，细胞与细菌根据他们不同的DNA含量得以区分，中性粒细胞与单核细胞也能以其不同的大小密度区分。准确在于试剂盒中有FITC荧光标记的经调理的的大肠杆菌，细胞吞噬后就有荧光，并且吞入的数量与荧光强度成正比，而附着在细胞表面未被吞噬的细菌因加入的淬灭液而失去荧光，因此可以定量测得发生吞噬作用细胞占总细胞的百分率和单个细胞的吞噬活性。

 

 

诊断试剂盒也适用于小老鼠、大老鼠、兔子、狗、牛和其他物种的血液。