Nitroblue tetrazolium (NBT) test: criteria for false positivity and its use in practice.

The criteria for false positivity of the NBT test were described including absence of classical clinical signs of bacterial infection, negative blood (and, if necessary, other) cultures, and lack of response of antibacterial treatment as the basis for appreciation of positive NBT test result as false-positive. A case of acute lymphoblastic leukaemia with all the criteria being fulfilled was described.     

Flow cytometric evaluation of nitro blue tetrazolium (NBT) reduction in human polymorphonuclear leukocytes

Oxidative metabolic burst of activated human polymorphonuclear leukocytes (PMN) is most commonly investigated in clinical practice by evaluating nitroblue tetrazolium (NBT) reduction at the single cell level. Reduced NBT precipitates where the redox reaction has taken place and can be visualized as PMN-associated dark blue granules of formazan in light microscopy. Although widely used and not technically demanding, this method remains subjective and labor intensive, especially when large numbers of samples need to be investigated. We developed a new flow cytometry technique in which PMN membrane was rendered fluorescent by a short incubation with fluorescein-conjugated Concanavalin A. PMN were then incubated with NBT and increasing doses of a suitable stimulus, such as phorbol myristate acetate (PMA). Formazan has a distinct peak of absorption at 520 nm that represents the peak of emission of fluorescein. As a consequence, formazan quenches the PMN-associated fluorescence. Data show that a dose-dependent reduction of fluorescence can be obtained using graded amounts of PMA in normal PMN cultures. PMN-associated fluorescence remains unchanged in control patients with chronic granulomatous (CGD) disease, a disorder characterized by a selective impairment of PMN oxidative metabolism. Electronic cell size increases upon PMA incubation in normal PMN, irrespective of the presence of NBT. Conversely, forward light scatter intensity decreases in the presence, but not in the absence, of NBT indicating that the phenomenon is due to the capacity of formazan to absorb/scatter the incident light. The present method for easily detecting NBT reducing activity at single cell level by flow cytometry makes use of commonly available, inexpensive reagents and standard instrumentation. It could become a useful test for clinical purposes.     

Nitroblue tetrazolium test in patients with breast cancer undergoing mastectomy and radiotherapy

The reduction of nitrotetrazolium blue with stimulated and unstimulated granulocytes was performed in 40 patients with breast cancer. Blood was collected from peripheral vein before surgical intervention, during surgery from vein draining of tumor, two weeks after surgery and three months after surgery when radiotherapy was finished. In parallel experiments the NBT test was made in the peripheral blood of 20 healthy individuals. We have observed decreased reduction of nitrotetrazolium blue in stimulated granulocytes, which may indicate that granulocytes from these patients are unable for efficient stimulation. No evident effect concerning the advancement of disease and applied therapy could be found. The importance of determining the NBT test in cancer patients is briefly discussed.     

Effect of interferon on the increased reduction of nitroblue tetrazolium (NBT)]

Human leukocyte interferon (HL-IF) enhanced the NBT reduction of human peripheral neutrophil in vitro. Dose relation between IF activity and the NBT reduction was recognized. Heat-inactivated HL-IF, HL-IF neutralized by anti-IF serum or heterologous IF could not increase the NBT reduction.     

Differentiation of HL-60 promyelocytic leukemia cells monitored by flow cytometric measurement of nitro blue tetrazolium (NBT) reduction

Reduction of nitro blue tetrazolium (NBT) to insoluble blue formazan granules occurs during the stimulus-induced respiratory burst of mature granulocytes and is routinely used as an indicator of the extent of granulocytic differentiation of HL-60 acute promyelocytic leukemia cells. In the present study, the differentiation of HL-60 leukemia cells induced by dimethylsulfoxide (DMSO) or retinoic acid was monitored by flow cytometric (FCM) measurement of forward and 90 degree light scatter of NBT treated cells. Two-parameter correlated analysis permitted a distinction between cells with increased forward and decreased 90 degree light scatter (NBT-), and cells with decreased forward and increased 90 degree light scatter (NBT+). Fixation of NBT treated cells with 1% paraformaldehyde facilitated flow cytometric analysis, and allowed differences in NBT reduction to be quantitated. DMSO-induced cells expressed an all-or-none reduction of NBT to formazan, compared with retinoic acid treated cells that exhibited a graded response. Three parameter flow cytometric analysis of HL-60 leukemia cells stained with propidium iodide in combination with NBT allowed the determination of the cell cycle distribution of NBT-treated cells.     

Nitroblue tetrazolium reduction by human blood neutrophils. II. The influence of sodium and potassium ions

Activity of blood neutrophils of 18 healthy volunteers (4 men and 14 women aged from 20 to 32 years) was investigated in the Nitroblue Tetrazolium (NBT) reduction reaction in Na- and K-phosphate buffers (0.1 M, pH 7.3) after a preliminary incubation of heparinized blood (37 degrees C, 0-7 h). It was found that the percentage of NBT-positive neutrophils in the presence of Na+ is higher than in the presence of K+. The dependence of percentage of NBT-positive neutrophils on the reaction time is described by a kinetic equation with constants k1 and k2, characterizing accordingly, speeds of NBT absorption and reduction by neutrophils. It has been shown that k1, k2 and k1/k2 values differ in potential phagocytes and in neutrophils, which are not capable of adherence. The kinetics constants of the latter cells do not depend on both cations and incubation time, while the percentage of NBT-positive potential phagocytes, and their k1 and k2 in the presence of Na+ are higher than in the presence of K+, and the values of described parameters are changed during incubation. During incubation the percentage of NBT-positive neutrophils increases to result in a partial aggregation of thrombocytes, as a consequence of excretion in the blood of products involved in this process.     

Ferrous iron mediated oxidation of arachidonic acid: studies employing nitroblue tetrazolium (NBT)

The oxidation of arachidonic acid by ferrous sulfate provides a useful model to study the role of iron in lipid oxidation reactions. We have employed nitroblue tetrazolium (NBT) in the present investigation to evaluate the mechanism of this reaction. In the presence of arachidonic acid, Fe⁺⁺, and O₂, the yellow dye NBT was rapidly reduced to the blue form, NBTH₂. The molar ratio of arachidonic acid to Fe⁺⁺ in this rapid reaction was 1:1, showing an interaction of one fatty acid molecule per iron molecule. Approximately one molecule of NBT was reduced per four molecules of arachidonic acid and Fe⁺⁺. Reduction of NBT was accompanied by oxidation of Fe⁺⁺ to Fe⁺⁺⁺, suggesting the transfer of four electrons from the Fe⁺⁺ to NBT to reduce the dye. Arachidonic acid was found to be unchanged when extracted at the end of the reaction, indicating formation of a complex that could dissociate leaving intact arachidonic acid. Evidence for the presence of such a complex which slowly dissociates during the reaction was obtained after longer incubations with small amounts of arachidonic acid. NBT reduction was not inhibited by agents which hydrolyze superoxide, by catalase or by agents which trap hydroxy radicals. We, therefore, propose a model in which NBT traps a radical generated on the arachidonic acid molecule. The proposed model suggests mechanisms for other fatty acid oxidation reactions such as prostaglandin and hydroperoxy fatty acid synthesis.     

Serum immunoglobulins in workers exposed to silicogenic risk with tuberculin-negative test and hyperergia

Changes in serum proteins and immunoglobulins IgG, IgA, and IgM were analysed comparatively in workers exposed to silicogenic particles in dependence on the hyperergic test or negative tuberculin test. Hypoalbuminemia with an increase in alpha-2 globulins was found in both groups. In addition, increase in gamma globulins was noticed in the group of hyperergic subjects. The electrophoretic picture in the hyperergic patients resembled that reported in human pulmonary tuberculosis. In the hyperergic group, a significant increase in IgG and occasionally in IgA was found. In the tuberculin-negative subjects, a less definite immunologic picture was found, with a slight but significant decrease in IgG while IgA was normal. In both groups, IgM were significantly decreased.     

Enhancement of nitroblue tetrazolium dye reduction by leucocytes exposed to a factor released from sensitised lymphocytes.

Leucocytes from purified protein derivative (PPD) skin test positive individuals, after exposure to PPD in vitro, show a significant increase in nitroblue tetrazolium (NBT) dye reduction when compared to leucocytes from PPD negative donors. The active principle was shown to be soluble, and was released from lymphocytes after a rapid, immunologically specific interaction with the sensitising antigen.     

Urinary homovanillic acid (HVA) and vanillymandelic acid (VMA) in workers exposed to manganese dust

The neurotoxicity of manganese (Mn) is well known, however, the neurochemical effect caused by this metal is less well investigated. In this study, urinary homovanillic acid (HVA) and vanillymandelic acid (VMA), two end products of catecholamine metabolism, were measured in 39 workers chronically exposed to Mn in a manganese smelting plant. The average duration of Mn exposure was 17.4 yr. Nineteen nonexposed workers were also studied. Concentrations of Mn in serum (MnS) and in urine (MnU) were measured by Zeeman graphite furnace atomic absorption spectrophotometry (ZAAS), and HVA and VMA determined by high performance liquid chromatography (HPLC). For Mn-exposed workers, the concentration of MnS was nearly 2.8 times (1.61 ± 0.16 mg/L vs 0.56 ± 0.16 mg/L) and MnU about 4.5 times higher (7.62 ± 0.17 mg/L vs 1.69 ± 0.16 mg/L) than the nonexposed. Although the geometric mean concentration of HVA in exposed workers was similar to that of the nonexposed (3.09 ± 1.39 mg/g ere. vs 2.99 ± 1.40 mg/g cre.), the VMA concentration was significantly higher (3.02 ± 1.43 mg/g cre. vs 2.49 ± 1.58 mg/g cre.,p = 0.033). Multiple regression analysis showed that although there were no correlations between any of these parameters with the duration of exposure to Mn, both HVA and VMA showed significant correlations with increase in MnS and MnU. These data provide evidence that exposure to Mn was associated with measurable increase in catecholamine metabolites. This finding is compatible with recent observations in laboratory animals that Mn interferes with neurochemical metabolism.     

Gamma-aminobutyric and glutamic acid levels in the brain of rats exposed to noise and vibration on ships

The influence of different levels of noise and vibration on the content of GABA and glutamic acid in the brain and on behavioural characteristics of rats have been studied on 60 white rats under voyage conditions. A correlation is determined between biochemical and physiological reactions of the central nervous system in animals and duration and level of the influence of the studied factors.