The nitroblue tetrazolium (NBT) test in renal transplantation

The nitroblue tetrazolium (NBT) test has been evaluated as a means of differentiating episodes of infection and rejection in 10 cadaver kidney transplant recipients. Normal NBT values were observed during all 11 episodes of acute rejection. A significant elevation of the NBT score was encountered in six of eight episodes of infection after transplantation. A positive NBT result is useful additional evidence in favour of infection in patients in whom the differentiation of infection and rejection is proving difficult.     

Routine application of the nitroblue tetrazolium test in the clinical laboratory

A modified nitroblue tetrazolium test (NBT) is described which is suitable for routine application in the haematology or bacteriology laboratory and which provides a rapid aid to the diagnosis of bacterial infection.Hitherto published methods have recommended the use of heparinized whole blood samples for the performance of the NBT test. However, we have demonstrated that the use of the sucrose polymer Ficoll permits the test to be carried out on buffy coats prepared from venous blood anticoagulated in sequestrene (EDTA).The mean percentage of NBT-positive neutrophils in 60 healthy control subjects was 6.1. The mean percentage of NBT-positive neutrophils in 56 patients with confirmed or presumed bacterial infection was 34.2. Scores within the normal range were observed in 13 patients in this group. Forty-eight individuals with viral infection yielded a mean percentage of NBT-positive neutrophils of 8.5. Six patients in this group gave scores above the upper limit of normal.Both pathological groups are described in detail and applications of this test are suggested.     

An improved nitroblue tetrazolium test using phorbol myristate acetate-coated coverslips.

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.     

Comparative study of the endotoxin-stimulated nitroblue tetrazolium test in disease and health.

Endotoxin-stimulated nitroblue tetrazolium (NBT) reduction was evaluated in 853 individuals: 270 healthy controls, 334 with various non-neoplastic conditions, 220 with solid non-lymphomatous tumours, and 29 with lymphoma. Each of the above groups was divided into three age subgroups: less than 60, 60-69, and greater than or equal to 70 years. In the controls and in patients with nonmalignant diseases, significantly lower values were recorded for elderly subjects (greater than or equal to 60 years) compared with younger subjects of the same group, whereas in cancer patients the results were independent of age. Under the age of 60, stimulated values for both patient groups were significantly lower than the control values, and in patients with solid non-lymphomatous tumours significantly lower values were attained than in the other patients. NBT reduction in lymphoma patients was comparable to that of the controls. In the elderly (age greater than or equal to 70) no significant differences were noted between patients and controls. It is suggested that stimulated NBT reduction declines with advancing age. While this test clearly demonstrates some leucocyte dysfunction in solid cancer, its value in investigating neutrophil behaviour in elderly subjects is questioned.     

Heterogeneity in chronic granulomatous disease detected with an improved nitroblue tetrazolium slide test

The microscopic nitroblue tetrazolium (NBT) slide test, used to score the ability of individual phagocytic leukocytes to produce superoxide, was improved according to the following procedure. Purified granulocyte suspensions are incubated with NBT and fixed in suspension, thereafter centrifuged on microscope slides and stained with nuclear fast red. This method precludes stimulation and selection of cells by adherence and washing. The number of formazan grains per cell can be judged in a semiquantitative way. In parallel incubations, the cells are stained with May-Grünwald/Giemsa, allowing identification of formazan-positive and -negative cells. The test discriminates well between cells from normal individuals, cells from patients with chronic granulomatous disease (CGD), and cells from heterozygotes for the X-linked form of CGD. Several patients and heterozygotes with an autosomal or variant form of CGD were detected with decreased NBT-reducing activity in their neutrophils and/or eosinophils. The relation between NBT-reductase activity of the phagocytes and the clinical situation is discussed.     

Cell damage and dye reduction in the quantitative nitroblue tetrazolium (NBT) test.

Nitroblue tetrazolium (NBT) is toxic to neutrophils; an effect which is greatly enhanced by endotoxin and latex particles. Cell damage, measured by the release of the cytoplasmic marker enzyme lactate dehydrogenase (LDH), was closely related to dye reduction. This suggests that, in this test, dye reduction occurs largely as a result of contact between intracellular reducing compounds and NBT following damage of the outer cell membrane. The expression of dye reduction as a function of LDH release should enhance the sensitivity of the quantitative NBT test by correcting for the observed intersubject variation in cell damage. The relationship between cell damage and dye reduction is a measure of the reducing capacity of the cell. This was normal in immature, bone marrow neutrophils, but diminished in neutrophils of patients with chronic granulomatous disease.     

Serial nitroblue tetrazolium tests in the management of infection

Two cases are described in which extensive use was made of the nitroblue tetrazolium (NBT) test. In the first case the advantages of using this technique to diagnose and control infection is shown; in the second the considerable advantage of the speed of the technique. In both of these cases the test made a material contribution to the management of the patient, and it is concluded that the test brings bacteriological control of the patient within the immediate clinical area, thus overcoming one of the principal disadvantages of the classical bacteriological methods.     

Effectiveness of the nitroblue tetrazolium test in demonstrating reduced bactericidal activity of polymorphonuclear neutrophils in schistosomal hepatosplenomegaly and ascites.

The nitroblue tetrazolium (NBT) test was carried out on blood from ten normal individuals and 20 schistosomal patients, ten of whom had hepatosplenomegaly and ten hepatosplenomegaly and ascites. The test revealed defective neutrophils in patients who had hepatosplenomegaly accompained by ascites. The phagocytic and bactericidal effects from five of each of the two groups of patients; results substantiated tose obtained by the NBT screening test. The NBT screening test thus proved useful in detecting acquired defective function of neutrophils in a helminthic parasitic disease that is accompanied by frequent bacterial infections.     

Leucocyte migration and nitroblue tetrazolium assay in Nigerian children with bacteremia and malaria parasitemia

The prevalence of malaria parasitemia, bacteremia, certain hematological parameters, leucocyte migration index and nitroblue tetrazolium dye reduction were determined in 147 Nigerian children (4.24+/-2.88 years of age). Sixty (40.8%), 28(19.1%) and 26(17.7%) had malaria parasitemia only, bacteremia only and both malaria parasitemia and bacteremia, respectively. Four genera of bacteria, i.e E. coli, Proteus, Staphylococcus and Salmonella, were detected in subjects with both malaria parasitemia and bacteremia. The 4 bacterial genera and Klebsiella were detected in subjects with bacterial infection only. P. falciparum (68%), P. malariae (25%) and P. ovale (7%) were the species of malaria parasites identified in our subjects. Bacteremia was most prevalent in subjects with hemoglobin AA (HbAA) (60.7%) followed by HbAC (21.45%). Packed cell volume (PCV) and Hb concentration were similar in all groups but mean counts of red blood cells (RBC) and white blood cells (WBC) were statistically significantly lower in subjects with malaria parasites only compared to the controls. Leucocyte migration was significantly reduced in children with bacteremia only or both malaria parasitemia and bacteremia compared to controls, while the nitroblue tetrazolium assay was significantly reduced in children with bacteremia only. It may be concluded that malaria parasitemia significantly affects both leucocyte migration and nitroblue tetrazolium assay.     

A quantitative nitroblue tetrazolium assay for determining intracellular superoxide anion production in phagocytic cells

Conventionally, a semi-quantitative microscopic nitroblue tetrazolium (NBT) assay is used to determine the production of superoxide anion (O2(-)) in various phagocytic cells. This microscopic assay is conducted by counting the cells containing blue NBT formazan deposits, which are formed by reduction of the membrane permeable, water-soluble, yellow-colored, nitroblue tetrazolium (Y-NBT) by O2(-). However, this assay is semi-quantitative and is prone to observer bias. In the present study, we modified the NBT assay by dissolving the blue formazan particles using 2M potassium hydroxide and dimethylsulfoxide and then measured its absorbance using a microplate reader at 620nm. The absorbance of dissolved NBT increased in proportion to cell number (r = 0.9907), incubation time, and stimulus concentration. To test the usefulness of this modified assay, we compared the abilities of a number of types of phagocytic cells to produce O2(-). The cells examined included murine macrophage cell lines (RAW 264.7 and J774), freshly prepared murine peritoneal macrophages and neutrophils, a human myeloid cell line (PLB-985), and freshly prepared human peripheral blood neutrophils. In addition, we demonstrate that nitric oxide produced by RAW 264.7 cells does not interfere with the modified colorimetric NBT assay. Taken together, our results indicate that the modified colorimetric NBT assay is simple, sensitive, and quantitative, and that it can be used to determine the amounts of intracellular O2(-) produced by phagocytic cells. Thus, this assay is sensitive enough to measure, quantitatively, even the small amounts of O2(-) produced in monocytes and macrophages that are not detectable by the conventional microscopic NBT assay.     

Study of opsonic factors by the nitroblue tetrazolium reduction reaction by human neutrophils

A method of determining nonspecific opsonins in the system of human neutrophilic phagocytosis is suggested. The method is based on assessment of the intensity of metabolic stimulation of neutrophils by killedSerratia marcescens cells in the presence of opsonizing substrate. Serum opsonic activity levels were determined in 44 healthy blood donors.Department of Bacterial Allergies, Institute of Epidemiology and Microbiology, Kazan. (Pressented by Academician of the Academy of Medical Sciences of the USSR A. D. Ado.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 89, No. 2, pp. 214–215, February, 1980.     

Polymorphonuclear leukocytes stimulation by immune complexes. Assessment by nitroblue tetrazolium dye reduction

Insoluble immune complexes and immune aggregates were found to stimulate the activity of the hexose monophosphate shunt as measured by nitroblue tetrazolium reduction in human polymorphonuclear leukocytes. This phenomenon depends more on the size of the complex than on its ability to activate complement.