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Nitroblue tetrazolium and Sudan Black B have been compared as pre-staining agents for human serum lipoproteins, using acrylamide gel gradients. Results from Nitroblue tetrazolium staining should be used with caution for low and very low density lipoproteins, and further work appears necessary on the reaction with alpha-lipoproteins.
Nitroblue tetrazolium (NBT) test was performed in forty-four diabetic patients of both sexes ranging in age from 15 to 64 years. Some of the patients were tested both before and after diabetes compensation. In cases of decompensated diabetes caused by dietary errors or by inadequate dosage of the antidiabetic drugs, NBT results were within normal range. Significantly higher values were observed in decompensation of diabetes caused by bacterial infection. This observation may be used to detect the real cause of diabetes decompensation.
The nitroblue tetrazolium (N.B.T.) test has been studied in 19 patients with moderate or severe acne vulgaris and 18 patients with subclinical or no acne. There was a significant elevation of the N.B.T. score in those patients with moderate or severe acne as compared with the control group. This is probably a result of the inflammatory reaction in and around the involved sebaceous gland follicles.
The reduction of nitroblue tetrazolium (NBT) by phagocytic leucocytes in peripheral blood has been examined in rabbits injected intratesticularly with Treponema pallidum, T. pallidum-free supernatant of orchitic testes, or normal rabbit testes supernatant. A significant (P less than 0.01) increase in NBT reduction was observed only in the infected animals.
In order to study the value of the tetrazolium nitroblue test (TNB) in the localisation of urinary tract infections, the authors performed this test in 33 patients with a urinary tract infection and in 20 control children suffering from no bacterial infection. The 33 infected children were divided into three groups on the basis of clinical and bacteriological criteria (dysuria, frequency, fever, abdominal pain, inflammatory syndrome, immunofluorescence of bacteriuria, serum antibody levels): -- group A (8 children): upper urinary infection -- group B (14 children): lower urinary infection -- group C (11 children): urinary infection of undetermined site. The number and percentage of TNB positive polynuclears was significantly higher in the children of group A than in the children of group B (p 0.001). By contrast there was no significant difference between the results obtained: in the non-infected control group and in group B -- in group A and group C. These results, confirming a previous study by Bjorksten and de Chateau, show the value of the TNB test in the localisation of urinary tract infection in the child.
Nitroblue tetrazolium test scores were obtained in 43 patients with psoriatic arthritis, 32 patients with rheumatoid arthritis, and 16 patients with osteoarthrosis. The mean score in all groups was similar and was higher than previously reported in patients awaiting heart surgery or with noninfective dermatoses. The test is not thought to be helpful in differentiating psoriatic arthritis from rheumatoid arthritis, and its usefulness in the diagnosis of septic arthritis superimposed on other forms of arthropathy is limited.
The effect of a mixture of nitroblue tetrazolium (NBT) and vitamin E on the metabolism and ultrastructure of polymorphonuclear leukocytes (PMN) from normal subjects or patients with chronic granulomatous disease (CGD) was determined in vitro. Increasing concentrations of NBT and vitamin E progressively decreased rates of oxygen consumption and 1-14C-glucose oxidation by normal PMN stimulated with particulates to a degree that exceeded either agent alone. NBT-vitamin-E also inhibited vacuole formation and the cytochemical release of myeloperoxidase-positive granules. The depressed oxidative metabolism and degranulation of NBT-vitamin-E-treated control PMN closely approximated the blunted responses of CGD PMN which were similar alone or in the presence of NBT-vitamin-E. In contrast to these effects, the highest concentration of NBT-vitamin-E used in the study did not damage, decrease rates of unstimulated oxidative metabolism of, or impair ingestion of particulates by control or CGD PMN. NBT and vitamin E impose a state on normal PMN which is remarkably similar to that observed in PMN from patients with CGD.
The reduction of nitroblue tetrazolium (NBT) dye by neutrophils from 379 patients with infectious diseases and 268 controls has been examined. The mean NBT score was 29.8% (72.3% positive tests) in the 231 patients with non-tuberculous bacterial infections, 9.7% (28.1% positive tests) in the 135 patients with viral infections 5.3% (1.5% positive tests) in the controls. Positive tests were demonstrated in 1 of 7 patients with tuberculosis and in 4 of 6 with mycoplasma pneumonia. Patients with urinary tract infections or septicemia had the highest percentage of positive tests, particularly when the infections were caused by gram-negative bacteria. In acute bacterial infection, the 176 patients who had not received any antibacterial therapy prior to testing had a significantly higher mean NBT score and proportion (77.8%) of positive tests than the remaining 55 pretreated patients (54.5%). Recent antibiotic treatment seriously invalidates the NBT test results. In acute viral infection, 29 of the 38 positive tests were obtained from patients with acute hepatitis (mean score 20.0%) or infectious mononucleosis (mean score 9.3%). When evaluating the test results, special attention should be paid to patients with hepatitis. Endotoxin stimulated NBT tests disclosed normal enhancement of NBT reduction by neutrophils from the patients and the controls. Cautiously interpreted, the NBT reduction by neutrophils from the patients and the controls. Cautiously interpreted, the NBT test results may be useful as an adjunct in the differential diagnosis of major bacterial and viral infections.
The endotoxin--nitroblue tetrazolium (NBT) slide test was modified by precoating coverslips with phorbol myristate acetate instead of endotoxin. The percentage of control polymorphonuclear leukocytes reducing NBT was significantly (P less than .05) greater on phorbol myristate acetate-coated coverslips (99 +/- 0.21%) than on endotoxin-coated coverslips (96+/- 1.8%). Polymorphonuclear leukocytes from patients with chronic granulomatous disease did not reduce NBT on phorbol myristate acetate- or endotoxin-treated coverslips. NBT reduction by polymorphonuclear leukocytes from proven heterozygotic carriers of sex-linked chronic granulomatous disease was intermediate between NBT reductions by those from controls and patients. A statistically significant abnormality of NBT reduction was found in polymorphonuclear leukocytes from one carrier of chronic granulomatous disease with phorbol myristate acetate-treated, but not endotoxin-treated coverslips. The phorbol myristate acetate-NBT coverslip technic is a rapid, simple, reliable way to detect deficiencies in polymorphonuclear leukocytes from patients and carriers of chronic granulomatous disease.
A study was made of the test of restoration of the nitroblue tetrazolium (NBT) by the intact blood neutrophils and the neutrophils stimulated with various endotoxin doses of Gram-negative bacteria in 70 healthy persons. A high sensitivity of the test with the NBT for the quantitative assessment of the neutrophil response under conditions stimulating the phagocytic activity was demonstrated. A functional nonhomogeneity of the neutrophil population, the most distinct with the minimal stimulating action, was noted.
The general features of the reduction of nitroblue tetrazolium chloride (NBT) by NADH and phenazine methosulphate (PMS) have been studied under aerobic and anaerobic conditions. Under aerobic condition the reduction appears to be mediated through the intermediate formation of the superoxide anion radical O2-.; this reaction is strongly inhibited by superoxide dismutase and by a number of O2-. scavengers such as propyl gallate, (+)-catechin, manganous ions, reduced glutathione and benzoquinone. Cupric ions inhibited the overall reaction by reoxidising reduced PMS. Under anaerobic conditions, superoxide dismutase had only a small inhibitory action and, with the exception of cupric ions, the other substances mentioned above were ineffective as inhibitors. The data presented show that the use of NBT to detect the presence of O2-. is fraught with difficulties due to an equally rapid reduction of NBT by NADH and PMS under anaerobic conditions.
Peripheral-blood monocytes from normal individuals and from patients with malignant melanoma reduce nitroblue tetrazolium (NBT). A quantitative assay for dye reduction was applied to 25 healthy donors and 31 patients with malignant melanoma. NBT reduction expressed as dye reduction per monocyte was significantly impaired in patients with disseminated disease, and they responded poorly to a phagocytic stimulus. Monocytes from patients with micrometastatic disease, however, showed normal resting NBT reduction but, following exposure to a suspension of latex-polystyrene, showed significantly greater NBT reduction than those from normal individuals. Since NBT reduction is an indirect measure of intracellular hexose-monophosphate-shunt activity we conclude that the monocytes from patients with minimal disease are in some way activated.
A sensitive method is presented for measurement of changes in the permeability of mitchondria in cultured cells. Rat heart endothelioid cells were used to determine the penetration rate of nitroblue tetrazolium (NitroBT) or other reactants into mitochondria in situ. Nitroblue formazan, produced as a consequence of succinate dehydrogenase activity in the mitochondria, was eluted and measured with a spectrophotometer. Prior injury of cells with hypo-osmolar solutions increased the rate of formazan production. Several methods are described or suggested for the statistical analysis of the data.
The ability of bacteria to reduce nitroblue tetrazolium (NBT) to formazan, and the inhibition of this process by antibiotics, have been utilized in a method to determine antibiotic concentrations in serum. Escherichia coli, growing in the presence of increasing but subinhibitory concentrations of antibiotics, produces diminished amounts of formazan. This is photometrically quantified and the optical density obtained with the sample is compared with those obtained with know concentrations. Levels of aminoglycosides below 1 microgram/ml can be measured in the presence of other antibiotics also, and the assay can be carried out in three hours. Good correlation was obtained when 49 serum samples containing varying concentrations of sisomicin were tested simultaneously by the NBT assay and a disc diffusion method.
Identification of mouse bone marrow colony growth in soft agar cultures has been realized by layering of the plates with nitroblue tetrazolium at the concentrations of 1 mg/ml. Granulocyte-macrophage colonies appear dark-blue coloured on the yellow surrounding medium and can be fixed, stored at 4 degrees C and counted few days later. This method allows the cultures to be assessed macroscopically and makes a better enumeration of small clusters even if spread and consisting of only 30-40 cells.
The hearts from 81 cases of suspected myocardial infarction were stained with the nitroblue tetrazolium (NBT) test to show damaged heart muscle in the gross at necropsy. Thirty-seven cases were of stated clinical age less than 12 h and 27 of these were less than 5 h. Seven out of 17 cases under 1 h were negative with NBT, but all other cases showed either focal diminution of staining with the dark blue diformazan or patchy red staining with the monogormazan of NBT. Thus the method may be of diagnostic value at necropsy from 1 h onwards after the time of apparent infarction (stated clinical onset). Satisfactory results were obtained up to 3 days at ambient temperature after death and for longer when the corpse was stored at 4 degrees C.
The influence of pH, dye concentration and sample storage on the histochemical nitroblue tetrazolium (NBT) test in 45 patients with acute bacterial infections and 51 healthy individuals has been examined. Even minor deviations in pH and NBT dye concentration markedly influenced the reduction of NBT to formazan by patient and control neutrophils, and the differentiation between results in these groups was optimal at pH 7.2 and a concentration of 0.05% NBT in the final blood-NBT mixture. Storage of heparinized blood samples for more than 2 hours at 21 degrees C and for more than 6 hours at 4 degrees C is not recommended due to overlap between results in patients and controls. The error involved in counting of NBT-positive neutrophils accounted for the major discrepancies tests in patients and controls and in tests performed on different days. The reproducibility of the endotoxin stimulated test was inferior to that of the unstimulated test.
It was originally our intention to study the suitability of the nitroblue-tetrazolium (NBT) test for differentiating the causes of granulocytic pleocytosis of the cerebrospinal fluid (CSF). However, several methodological problems were encountered and required extensive preliminary studies. The test was initially performed on 51 CSF samples from 37 patients using the method described by Park et al. in 1968. Although bacterial meningitis was demonstrated in 19 patients, the NBT test resulted in more than 11% NBT positive granulocytes in only four cases. No NBT positive cells were found in 24 of the samples tested. These rather poor results are not surprising if one considers that NBT is only taken up by granulocytes as macrocomplex containing heparin or fibrinogen. CSF does not contain measurable quantities of fibrinogen--either under normal conditions or in cases of viral or bacterial meningitis. A comparative study of the NBT test with and without the addition of heparin was performed using CSF samples from six patients with proven bacterial meningitis. Without heparin there were less than 11% NBT positive granulocytes in five cases, while all samples demonstrated more than 11% positive cells after addition of heparin. Best results were achieved with 70 IU of heparin in each sample. Preparation and evaluation were facilitated by use of 0.1% NBT solution and concentration of the CSF cells in a sedimentation chamber.