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.     

Indicators of spontaneous and stimulated nitroblue tetrazole test of polymorphous-nuclear leucocytes in acute dysentery patients

140 healthy individuals and 93 sick with acute dysentery were subjected to an examination by spontaneous and by bacterial preparations stimulated reaction with nitroblue tetrazole (NBT test). Indicators in healthy persons were normal in the spontaneous, and increased in the NBT test, stimulated by bacterial preparations. Indicators of the spontaneous NBT test in patients with acute dysentery were raised with a maximum in the period of early convalescence. Stimulation by a live shigella culture--the dysentery vaccine--revealed by means of Sonne diagnostic high, and when endotoxin from Serratia marcescens and dysenterin was used as an inductor, mild indicators of NBT test activity. When a polyvalent agglutinating dysentery serum was used as a stimulator, the activity increased considerably, and a simultaneous use of serum and vaccine had an inhibiting effect on the indicators of the stimulated NBT test. The obtained results testify the sufficient high reserve possibilities of leucocytes towards complete phagocytosis and the efficiency of the NBT test, stimulated by bacterial preparations for the study of functional and metabolic activity of leucocytes in the process of acute bacterial dysentery.     

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.     

Biomimetic nanoparticles for siRNA delivery in the treatment of leukaemia

Leukaemia is a bone marrow cancer occurring in acute and chronic subtypes. Acute leukaemia is a rapidly fatal cancer potentially causing death within a few weeks, if untreated. Leukaemia arises as a result of disruption to haematopoietic precursors, caused either by acquired gene fusions, gene mutations or inappropriate expression of the relevant oncogenes. Current treatment options have made significant progress, but the 5 year survival for acute leukaemia remains under 10% in elderly patients, and less than 50% for some types of acute leukaemia in younger adults. For chronic leukaemias longer survival is generally expected and for chronic myeloid leukaemia patients on tyrosine kinase inhibitors the median survival is not yet reached and is expected to exceed 10 years. Chemotherapy and haematopoietic stem cell transplantation (HSCT) for acute leukaemia provide the mainstay of therapy for patients under 65 and both carry significant morbidity and mortality. Alternative and superior therapeutic strategies for acute leukaemias are urgently required. Recent molecular-based knowledge of recurring chromosome rearrangements, in particular translocations and inversions, has resulted in significant advances in understanding the molecular pathogenesis of leukaemia. Identification of a number of unique fusion genes has facilitated the development of highly specific small interfering RNAs (siRNA). Although delivery of siRNA using multifunctional nanoparticles has been investigated to treat solid cancers, the application of this approach to blood cancers is at an early stage. This review describes current treatments for leukaemia and highlights the potential of leukaemic fusion genes as therapeutic targets for RNA interference (RNAi). In addition, the design of biomimetic nanoparticles which are capable of responding to the physiological environment of leukaemia and their potential to advance RNAi therapeutics to the clinic will be critically evaluated.     

Active immunotherapy in treatment of acute leukaemia

Active non-specific immunotherapy has been used to prolong remissions in acute lymphoblastic leukaemia. The series reported here used Bordetella pertussis vaccine in a controlle trial after intensive chemotherapy. Possibly immunotherapy delayed the onset of relapse in the treated patients, but no long-term remissions were obtained. Further work is needed to establish the role of immunotherapy in general, and the use of B. pertussis vaccine in particular, in the treatment of acute leukaemia.     

Bacterial glutaminase in treatment of acute leukaemia.

A glutaminase-asparaginase enzyme from Achromobacter sp has antitumour activity in vitro and in animals. Glutaminase was administered in doses of 3500-20 000 IU/m2 body surface area/day to six patients with acute lymphoblastic leukaemia (ALL) and three patients with acute myeloid leukaemia (AML). The enzyme had a blood half life of 80 minutes but depletion of blood glutamine persisted for 12 hours after single doses. Seven patients, including four (two with AML and two with ALL) resistant to asparaginase, received repeated doses of glutaminase. Antileukaemic effects were observed in all seven; one elderly patient developed metabolic acidosis. Study of this new antileukaemic agent in patients with acute leukaemia at an earlier stage of their disease is now justified.     

Recombinant human colony stimulating factor-granulocyte/macrophage and -granulocyte, but not macrophage induce the development of a respiratory burst in primary human myeloid leukemic cells in vitro

Respiratory burst develops in myeloid blast cells if they differentiate functionally along the monocytic or granulocytic lineage. Using the nitroblue tetrazolium (NBT) assay we studied the effects of recombinant human granulocyte/macrophage colony stimulating factor (rhuGM-CSF), rhuG-CSF and rhuM-CSF on development of respiratory burst activity in primary blast cells from patients with myeloid leukemia. Assessing suspension cultures containing cells from patients with acute myeloid leukemia (AML, n = 13) or myeloid-blast crisis (myBC) of chronic myeloid leukemia (CML, n = 5) it was found that the percentage of NBT positive cells was increased by at least 20% as compared to control cultures by rhuGM-CSF in 6/17 cases, by rhuG-CSF in 7/17 cases and by rhuM-CSF in 0/16 cases, representing in 'responders' a mean increase of 267% and 270% in the absolute number of NBT positive cells by rhuGM-CSF and rhuG-CSF, respectively. Morphological examination of cultured cells from 'responders', as compared to controls, showed decreased blast cell content but generally no evidence of terminal differentiation. The demonstration of Auer rods in NBT positive cells indicates that respiratory burst developed in a leukemic clone. These findings may be of physiological, pathophysiological and clinical relevance.     

Src kinase signaling in leukaemia

Role of Src kinases in acute lymphoblastic leukaemia has been recently demonstrated in leukaemia mouse model. Retained activation of Src kinases by the BCR-ABL oncoprotein in leukaemic cells following inhibition of BCR-ABL kinase activity by imatinib indicates that Src activation by BCR-ABL is independent of BCR-ABL kinase activity and provides an explanation for reduced effectiveness of the BCR-ABL kinase activity inhibitors in Philadelphia chromosome-positive acute lymphoblastic leukaemia. Simultaneous inhibition of kinase activity of both BCR-ABL and Src kinases results in long-term survival of mice with acute lymphoblastic leukaemia. Leukaemic stem cells exist in acute lymphoblastic leukaemia, and complete eradication of this group of cells would provide a curative therapy for this disease.     

Distribution of Acute Leukaemia in Time and Space. Studies in New Zealand

The distribution of leukaemia in time and space was examined by tracing the date of onset and the place of residence in all cases of childhood leukaemia and of acute leukaemia in adults, totalling 1,003 cases, which occurred in New Zealand during the 12 years from 1953 to 1964.Significant seasonal variations in the onset were found in adults but not in children. Time-space clustering did not occur in adults, but a significant tendency towards it was found in children aged less than 6 years. Since a similar tendency has previously been found by two other groups of investigators it is suggested that leukaemia in young children may have a different aetiology from that occurring at other ages.     

Plasma transcobalamins in haematological disorders

Plasma UBBC-B12 and transcobalamins were measured in 112 patients suffering from different haematological disorders. The data showed different patterns of changes in plasma transcobalamin profile in different haematological disorders. Plasma UBBC-B12 and transcobalamins were significantly higher than normal in untreated chronic myeloid leukaemia, acute promyelocytic leukaemia, nutritional megaloblastic anaemia and in refractory anaemias with hypercellular marrow. Normal levels of these proteins were noted in chronic lymphatic leukaemias, in primary and secondary hypereosinophilic states and in multiple myeloma. Subnormal levels of these proteins were observed in hypoplastic anaemia and acute lymphoblastic leukaemia. Chronic myeloid leukaemia patients during blast crisis and acute myeloid leukaemia patients except those suffering from acute promyelocytic leukaemia showed varying pattern of plasma transcobalamins depending on type of blast crisis or FAB subtype of AML. The significance of these changes in plasma transcobalamins have been discussed along with the experience of other workers in this field.     

Vincristine and Prednisone for the Induction of Remissions in Acute Childhood Leukaemia

A total of 65 children with acute lymphoblastic leukaemia and seven with other types of acute leukaemia received treatment with a combination of vincristine and prednisone. In all 122 courses of treatment were given. Of 22 patients with acute lymphoblastic leukaemia who received this as their first treatment, all achieved complete remission. The complete remission rates were 82% for patients with acute lymphoblastic leukaemia in their first relapse, 63% in the second relapse, and much lower in subsequent relapses and in the patients with other types of acute leukaemia. Alopecia and gastrointestinal and neuromuscular toxicity occurred respectively in 51%, 29%, and 21% of instances, only the last of these side-effects of vincristine being dose-related. Most of the complete remissions were obtained with a total dose of vincristine which carried only a low risk of neurotoxicity.     

Decreasedin vitro chemosensitivity of tumour cells in patients suffering from malignant diseases with a poor prognosis

The aim of the study was to assess the predictive value of MTTin vitro assay for evaluation of tumour cell resistance/sensitivity to cytotoxic drugs. We analyzed 105 samples of malignant cells of different origin. The study included patients with a diagnosis of acute and chronic lymphatic leukaemia, acute and chronic myeloid leukaemia, non-Hodgkin lymphoma, carcinoma of the lung, stomach and liver, rhabdomyosarcoma and breast carcinoma. The results demonstrate outstanding chemosensitivity in the majority of childhood acute lymphoblastic leukaemias, medium chemosensitivity of adult haematopoietic malignant diseases and chemoresistance of solid tumour cells. Our preliminary data suggest a good correlation betweenin vitro MTT assay and clinical curability of individual malignant diseases.Abbreviations ALL acute lymphoid leukaemia - AML acute myeloid leukaemia - CML chronic myeloid leukaemia - LCS₅₀ 50% leukaemic cell survival     

Multidrug resistance (MDR) genes in haematological malignancies

The emergence of drug resistant cells is one of the main obstacles for successful chemotherapeutic treatment of haematological malignancies. Most patients initially respond to chemotherapy at the time of first clinical admission, but often relapse and become refractory to further treatment not only to the drugs used in the first treatment but also to a variety of other drugs. Laboratory investigations have now provided a cellular basis for this clinical observation of multidrug resistance (MDR). Expression of a glycoprotein (referred to as P-glycoprotein) in the membrane of cells made resistantin vitro to naturally occurring anticancer agents like anthracyclines, Vinca alkaloids and epipodophyllotoxins, has been shown to be responsible for the so-called classical MDR phenotype. P-glycoprotein functions as an ATP-dependent, unidirectional drug efflux pump with a broad substrate specificity, that effectively maintains the intracellular cytotoxic drug concentrations under a non-cytotoxic threshold value. Extensive clinical studies have shown that P-glycoprotein is expressed on virtually all types of haematological malignancies, including acute and chronic leukaemias, multiple myelomas and malignant lymphomas. Since in model systems for P-glycoprotein-mediated MDR, drug resistance may be circumvented by the addition of non-cytotoxic agents that can inhibit the outward drug pump, clinical trials have been initiated to determine if such an approach will be feasible in a clinical situation. Preliminary results suggest that some haematological malignancies, among which are acute myelocytic leukaemia, multiple myeloma and non-Hodgkin's lymphoma, might benefit from the simultaneous administration of cytotoxic drugs and P-glycoprotein inhibitors. However, randomised clinical trials are needed to evaluate the use of such resistance modifiers in the clinic.Abbreviations ALL acute lymphocytic leukaemia - AML acute myelocytic leukaemia - BM bone marrow - CAT chloramphenicol acetyltransferase - CLL chronic lymphocytic leukaemia - CML chronic myelocytic leukaemia - CR complete remission - HCL hairy cell leukaemia - MDR multidrug resistance - MDS myelodysplastic syndrome - MM multiple myeloma - MoAb monoclonal antibody - NHL non-Hodgkin's lymphoma - PB peripheral blood - PCR polymerase chain reaction - PLL prolymphocytic leukaemia - RMA resistance modifying agent - VAD vincristine, doxorubicin, dexamethasone     

Autologous responses to human leukaemic cells in mixed leucocyte culture

Summary Cryopreserved leukaemic blasts and remission non-T cells from 22 patients with acute leukaemia (15 lymphocytic, 7 non-lymphocytic) were tested as stimulators of autologous remission T cells and normal allogeneic T cells in primary and secondary MLC. In most cases the autologous response elicited by leukaemic cells was less than or equal to that elicited by remission non-T cells. However, T cells from 2 patients in long-standing first remission from ANLL displayed greater proliferation in response to leukaemic blasts than to remission non-T cells in both primary and secondary MLC. The results are suggestive of sensitization of these 2 patients to leukaemia-specific antigens, but other possible explanations are discussed. Abbreviations used: MLC, mixed leucocyte culture; ANLL, acute non-lymphocytic leukaemia; ALL, acute lymphoblastic leukaemia; AMLR, autologous mixed lymphocyte reaction; NK cells, natural killer cells; MNC, mononuclear cells     

Leukocyte acid phosphatase and metabolic efficiency of phagocytes in the first lactation trimester of cows from a leukaemic herd

In Black-and-White cattle, polymorphism of acid phosphatase (AcP) of blood leukocytes is determined by a pair of autosomal alleles. The aim of the study was to determine the relationship between AcP polymorphism and the metabolic efficiency of phagocytes in the first months after calving of cows naturally infected with the bovine leukaemia virus. The studied population consisted of 91 Black-and-White cows aged 3-6 years, from one herd. Enzootic bovine leukaemia (EBL) was diagnosed with the immuno-enzymatic ELISA method and a PCR molecular test. Additionally, agarose gel electrophoresis and the cytochemical method were used to determine the AcP polymorphism and activity in leukocytes. The metabolic activity of phagocytes was determined by the nitroblue tetrazolium (NBT) reduction test. Significant differences in metabolic efficiency of granulocytes were observed between cows representing different AcP phenotypes. No significant differences in levels of the analysed indices were observed between the EBL-positive and EBL-negative cows and between the three subsequent months after calving.     

Risk of acute childhood leukaemia in Sweden after the Chernobyl reactor accident. Swedish Child Leukaemia Group.

OBJECTIVE--To evaluate the risk of acute childhood leukaemia in areas of Sweden contaminated after the Chernobyl reactor accident in April 1986. DESIGN--Population based study of childhood leukaemia diagnosed during 1980-92. SETTING--Coordinates for places of residence of all 1.6 million children aged 0-15 years; aerial mapped areas of Sweden heavily contaminated after the Chernobyl accident. SUBJECTS--888 children aged 0-15 years with acute leukaemia diagnosed in Sweden during 1980-92, identified with place of birth and residence at diagnosis. MAIN OUTCOME MEASURES--Risk of leukaemia in areas contaminated after the Chernobyl accident compared with the rest of Sweden and in the same areas before the accident. RESULTS--During six and a half years of follow up after the accident the odds ratio for acute leukaemia was 0.9 (95% confidence interval 0.6 to 1.4) in highly contaminated areas (> or = 10 kBq/m2) compared with the same areas before the accident. For the subgroup acute lymphoblastic leukaemia in children aged under 5 years at diagnosis the odds ratio was 1.5 (0.8 to 2.6). For all cases diagnosed after May 1986 in highly contaminated areas compared with areas of low contamination the odds ratio was 0.9 (0.7 to 1.3). For acute lymphoblastic leukaemia in children aged under 5 years at diagnosis the odds ratio was 1.2 (0.8 to 1.9) in highly contaminated areas compared with areas of low contamination. Dose-response analysis showed no correlation between the degree of contamination and the incidence of childhood leukaemia. CONCLUSION--There has been no significant increase in the incidence of acute childhood leukaemia in areas of Sweden contaminated after the Chernobyl reactor accident.     

Associations between the two serum proteins haptoglobin and transferrin and leukaemia

Haptoglobin and transferrin (TF) types were determined for 134 patients with leukaemia of the four most common types: acute lymphocytic (ALL), chronic lymphocytic (CLL), acute myelocytic (AML) and chronic myelocytic leukaemia (CML). The phenotype HP1 was found to have an increased incidence in the total patient group due to an increased incidence in those with AML, ALL and CML compared with controls, but not in those with CLL. Although tests of association applied to each of the samples of the four common types of leukaemia produced no significant chi 2 values, they did indicate that the relative incidence (RI) was just under 2 for the groupings of the acute forms ALL and AML, the myelocytic forms AML and CML and for the combination of ALL, AML and CML, respectively. All these associations were statistically significant (p less than 0.05). Analysis of TF subtypes and leukaemia indicated a significantly increased frequency of TF C1C1 among leukaemia patients compared with controls (p less than 0.005). Analysis of the samples of each of the four common types suggested that while the RI was raised in all but ALL patients, the association was significant only in AML patients (p less than 0.05). However, when the two myelocytic types were combined the RI was 2.3 and the association was highly significant (p less than 0.005). No such association could be detected in the lymphocytic forms.     

The phagocytic ability of neutrophils and serum lysozyme activity in experimentally infected carp, Cyprinus carpio L.

The phagocytosis of neutrophils and serum lysozyme activity were investigated in carp experimentally infected with Pseudomonas alcaligenes and Aeromonas punctata. The total leucocyte numbers, relative leucocyte counts, nitroblue tetrazolium (NBT) reduction, NBT test rate, lysozyme activity and lysozyme index were examined on days 7, 14 and 21 after injection. On days 7 and 14 there was a significant increase in total leucocyte numbers and serum lysozyme activity, but a decrease in the NBT test rate and lysozyme index. NBT reduction was unchanged compared with the control group. On day 21 the total leucocyte numbers and lysozyme activity had declined and were less than control values, but there was a significant increase of the NBT test rate, NBT reduction and lysozyme index.     

Preliminary Clinical Assessment of I.C.R.F. 159 in Acute Leukaemia and Lymphosarcoma

I.C.R.F. 159, a new antitumour agent, has been assessed in six patients with acute leukaemia and three with lymphosarcoma. In all but two there was a considerable fall in circulating primitive cells, and in one there was bone-marrow evidence of a partial remission. Severe toxic effects were seen in only one case; they consisted of alopecia and gastroenteritis. It is suggested that I.C.R.F. 159 is worth further examination in all forms of acute leukaemia and lymphosarcoma.     

Comparison of the cytomorphological classification with the cytochemical differentiation of acute lymphatic leukemia in children]

In 100 children with acute lymphatic leukaemia the cytomorphological subclassification of the pathological cell type was made according to Mathé and the French-American-British Co-operative group (FAB). In addition, all cases of leukaemia were differentiated according to their cytochemical type. Lymphoblasts from 10 cases of leukaemia could be subclassified immunologically. From 71 children will ALL the survival rates of those cases of leukaemia subclassified cytomorphologically and the cytochemical reactions were compiled and partially compared. Microlymphoblastic leukaemia could be found to be the most frequent type of ALL at children's age. Prolymphocytic leukaemias were characterized by a favourable survival rate and the highest percentage of ALL with the PAS type. Macrolymphoblastic and microlymphoblastic cases of leukaemia revealed no essential differences of survival rate, but significant differences of cytochemical reactions.