Clinical Diagnostic for the Quantitative Analysis of Leukocyte Oxidative Burst in Human Whole Bloods
临床诊断人体全血白细胞氧化破裂的定量检测

· Functional test ex vivo

来自体内的功能测试

· Evaluation of single cells to detect heterogenous populations

评价检测单个细胞异质种群

· Whole blood assay: No isolation procedures and optimal culture medium

全血检测：没有隔离程序和最佳培养基

· Physiological stimulans for phagocytes: Bacteria and fMLP

吞噬细胞生理兴奋劲：细菌和fMLP

· Dose response: Low and high stimulant

剂量反应:低和高的兴奋剂

· No electrostatic artifacts in polystyrol tubes compared to latex beads

没有静电工件比较乳胶珠子聚苯乙烯管内

· Standardized test procedure

标准化测试程序

· Exclusion of aggregation artifacts by DNA staining

通过DNA染色排除聚合工件

· Compatible with whole blood of mice and rats

兼容的小鼠和大鼠全血

· Fast assay: Whole assay time is 1.5 hours

快速测定:整个试验时间是1.5小时

100 analyses. Clinical Diagnostic for the Quantitative Analysis of Leukocyte Oxidative Burst in Human Whole Blood. Evaluation by flow cytometry.

100次分析。临床诊断人体全血白细胞氧化破裂的定量检测。通过流式细胞术进行评估。

Clinical Diagnostic for the Quantitative Analysis of Leukocyte Oxidative Burst in Human Whole Bloods

SUMMARY and EXPLANATION

BURSTTEST (PHAGOBURST) allows the quantitative determination of leukocyte oxidative burst in heparinized whole blood. It contains unlabeled opsonized bacteria (E.coli), phorbol 12-myristate 13-acetate (PMA) and the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP) as stimulants, Dihydrorhodamine (DHR) 123 as a fluorogenic substrate and necessary reagents. It determines the percentage of phagocytic cells which produce reactive oxidants (conversion of DHR 123 to R 123) and their enzymatic activity (amount of R 123 per cell).

The evaluation of these bioactivities should be performed by flow cytometry.

BURSTTEST(PHAGOBURST)允许定量测定白细胞氧化闯入肝素化全血。 它包含未标记的促进调理作用的细菌(大肠杆菌),佛波醇12十四烷酸乙酸13(PMA)和趋化作用的多肽n甲酰遇见亮氨酸板式换热器(fMLP)作为兴奋剂,二氢若丹明(DHR)123作为一个荧光衬底和必要的试剂。它决定了吞噬细胞产生活性氧化剂的比例(转换DHR 123 到 R 1233)及其酶活性(每个细胞R 123的数量)。

这些生物活性的测定应该有流式细胞术完成。

APPLICATIONS

The diagnostic kit is intended to investigate the altered oxidative burst activity found in various disorders and to evaluate the effects of drugs.

Reduced or missing burst activity is observed in inborne defects like the chronic granulomatous disease (CGD). CGD is a heterogenous group of inherited disorders that usually manifests itself during the first two years of life (3, 4). The disease is characterized clinically by repeated and life-threatening infections caused by bacterial and fungal organisms. These infections typically consist of pneumonia, lymphadenitis, or abscesses that involve lymph nodes, lungs, and liver. The NADPH oxidase is the enzyme system responsible for producing superoxide anion, which is quickly converted to hydrogen peroxide and hydroxyl radicals. Abnormalities in the constituent peptides of the NADPH oxidase enzyme system lead to the dysfunctions characteristic of CGD. Neutrophils from CGD patients fail to produce a significant oxidative burst following stimulation. Different forms of CGD are described (classical X-linked CGD and autosomal recessive patterns). BURSTTEST (PHAGOBURST?) is a rapid and sensitive method for the diagnosis of CGD and for the detection of X-linked carriers.

The oxidative burst of granulocytes is impaired in transplant patients and patients with AIDS (6). The spontaneous and fMLP-induced neutrophil respiratory burst was shown to be increased in neonates with laboratory signs of infection (7). Various immunomodulators (e.g., cytokines (GM-CSF, G-CSF, TNF) or drugs) seem to have effects on the oxidative burst. By using fMLP as a low stimulant one can investigate additive or priming effects (8) of test substances.

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

诊断测试组旨在研究改变了的氧化破裂活动中发现各种障碍和评估药物的影响。

观察细胞的减少或缺失的破裂活动像慢性肉芽肿性疾病(CGD)

慢性肉芽肿性疾病是一个异构群遗传疾病,通常体现在生命的最初两年期间(3、4)。这种疾病的临床特征是由于细菌和真菌重复和危机生命的感染。这些感染通常包括肺炎、淋巴腺炎或脓肿涉及到淋巴结、肺和肝。NADPH氧化酶的酶系统是负责生产超氧化物阴离子,迅速转化为过氧化氢和羟基自由基。NADPH氧化酶系统在组成多肽中的畸形导致慢性肉芽肿性疾病功能障碍性特点。CGD患者的中性粒细胞刺激后无法产生显著的氧化破裂。BURSTTEST (PHAGOBURST?)是一种快速有效诊断慢性肉芽肿性疾病和检测X连锁隐性遗传疾病携带者的方法。

氧化破裂的粒细胞在需要移植的病人和艾滋病患者中受损。无意识和诱发性fMLP嗜中性粒细胞破裂被证明会增加新生儿实验室感染的迹象。各种免疫调节剂（如细胞激素(GM-CSF, G-CSF, TNF)或药品）似乎会对氧化破裂产生影响。通过使用fMLP作为兴奋剂可以研究添加剂或激发效应(8)的测试物质。

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。

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 (oxidative burst) and oxygen-independent mechanisms (1, 2).

BURSTTEST (PHAGOBURST?) allows the quantitative determination of leukocyte oxidative burst. The BURSTEST kit contains unlabelled opsonized E.coli bacteria as particulate stimulus, the protein kinase C ligand phorbol 12-myristate 13-acetate (PMA) as high stimulus and the chemotactic peptide N-formyl-MetLeuPhe (fMLP) as low physiological stimulus, Dihydrorhodamine (DHR) 123 as a fluorogenic substrate (5) and necessary reagents. Heparinized whole blood is incubated with the various stimuli at 37°C, a sample without stimulus serves as negative background control. Upon stimulation, granulocytes and monocytes produce reactive oxygen metabolites (superoxide anion, hydrogen peroxide, hypochlorous acid) which destroy bacteria inside the phagosome. Formation of the reactive oxidants during the oxidative burst can be monitored by the addition and oxidation of DHR 123. The reaction is stopped by addition of LYSING SOLUTION, which removes erythrocytes and results in a partial fixation of leukocytes. After one washing step with WASHING SOLUTION, DNA STAINING SOLUTION is added to exclude aggregation artifacts of bacteria or cells. The percentage of cells having produced reactive oxygen radicals are then analyzed as well as their mean fluorescence intensity (enzymatic activity).