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Nitroblue Tetrazolium: a Routine Test?
909
percentage of N.B.T.-positive neutrophils is raised, often greatly, in all systemic bacterial inftctions,
THE LANCET Nitroblue Tetrazolium: a Routine Test? NEUTROPHIL leucocytosis is a feature of many clinical situations and the cause is often self-evident. On occasion leucocytosis with or without fever may persist, and to establish the differential diagnosis between bacterial or other infections, leukaemia, carcinoma, or other conditions may be extremely difficult. The observation of toxic granulations in the neutrophil cytoplasm and the measurement of neutrophil alkaline phosphatase sometimes contribute to a diagnosis. The nitroblue-tetrazolium (N.B.T.) test may also yield valuable information. In 1967 BAEHNER and NATHAN1 showed that a small proportion of the neutrophils of a normal person could in vitro reduce the colourless soluble dye nitro blue tetrazolium to produce an insoluble blue-black formazan precipitate. The mechanism of this reduction of N.B.T. to form formazan in the cytoplasm of the phagocytic neutrophil remains unclear. Apparently the resting unstimulated neutrophil membrane is not permeable to N.B.T., so some membrane alteration has allowed entry of the dye. The actual reduction of N.B.T. seems to take place within the confines of the phagocytic vacuole and may be seen not only in neutrophils but also in eosinophils and monocytes. The reduction seems to depend on the activity of N.A.D.H. oxidase, an enzyme intimately involved in the oxygen consumption and bactericidal mechanism of the neutrophil. Against this background, PARK et aI.2 speculated that when neutrophils are concerned in phagocytosis in vivo during natural infection, a metabolic change takes place that leads to increased reduction of N.B.T. Such has indeed the to be case. proved The reduction of N.B.T. by neutrophils has now been studied by different workers 2-4 in a number of conditions, and a reproducible pattern has emerged. The absolute number and percentage of N.B.T.-
positive neutrophils is low in normal persons (less than 10%), in patients with fever and leucocytosis of non-bacterial origin (such as rheumatoid arthritis or disseminated lupus erythematosus), after surgical operations, and in uncomplicated viral infection. The test is also normal in bacterial infections that remain superficially localised. The number and 1. 2. 3. 4.
Baehner, R. L., Nathan, D.
G. Science, 1967, 155, 835. Park, B. H., Fikrig, S. M., Smithwick, E. M. Lancet, 1968, ii, 532. Feigin, R. D., Shackelford, P. G., Choi, S. C., Flake, K. K., Franklin, F. A., Eisenberg, C. S. Pediatrics, 1971, 78, 230. Matula, G., Paterson, P. Y. New Engl. J. Med. 1971, 285, 311.
including meningitis, endocarditis, osteomyelitis, and septic arthritis, empyema, and miliary tuberculosis. The test also seems to be positive in systemic fungal infection and in malaria 5-6 and other parasitic diseases.’ Normal newborn and premature infants seem, however, to have many positive neutrophils until the age of two months, so that the test has no diagnostic accuracy until this age. Conversely, the administration of corticosteroid drugs may be responsible for a false negative test in a bacterially infected patient. As might be expected, the leucocytosis of chronic granulocytic leukaemia seems to be unassociated with increased numbers of N.B.T.positive cells. Interestingly, in Hodgkin’s disease the test may be positive in the presence of fever and
neutrophil leucocytosis.9 Of considerable importance has been the finding that children with chronic granulomatous disease (c.G.D.), a condition characterised by increased susceptibility to infection with certain bacteria, have neutrophils that totally fail to reduce N.B.T. in vitro. No abnormality of immune function has been demonstrated in children with this disease. Although their neutrophils appear to phagocytose bacteria quite normally, they are unable to destroy engulfed bacteria of quite low virulence and the children die of chronic suppurative and granulomatous lesions. 10, 11 The basic defect is thought to lie in an enzyme system that contributes to normal lysosomal function. The lack of reduction of N.B.T. in vitro forms a very sensitive screening test for this disease. A positive N.B.T. test thus depends on two components. On the one hand, there must be sufficient bacteria or bacterial products present to impinge on the circulating neutrophils; on the other, the neutrophils must themselves be normal and have the capacity to reduce N.B.T. to formazan. Chronic granulomatous disease well exemplifies a defect in the second component. The N.B.T. test seems, therefore, to be clinically useful in three main situations. First, it may be very valuable in supporting a diagnosis of severe systemic infection with bacteria (or fungi) when other possibilities exist, and it may serve to suggest or exclude viral or non-infectious disease. Secondly, it may be a useful test with which to monitor patients and to detect early infection in those with increased susceptibility or in those in whom infection could be especially catastrophic, such as transplant recipients, patients undergoing immunosuppression for other reasons, or those with rheumatic valvular disease of the heart. 5. Andersen, B. R. Lancet, 1971, ii, 317. 6. Pujol-Moix, M. N. ibid. Oct. 16, 1971, p. 871. 7. Chretien, J. H., Garagusi, V. F. ibid. Sept. 4, 1971, p. 549. 8. Matula, G., Paterson, P. Y. ibid. 1971, i, 803. 9. Catovsky, D. Unpublished. 10. Holmes, B., Quie, P. G., Windhorst, D. B., Good, R. A.
1966, i, 1225. 11. Windhorst, D. B., Holmes, B., Good, R.
A. ibid.
1967, i,
Lancet,
737.
910
it may help to identify patients with congenital defects of neutrophil function such as c.G.D. Its introduction into the repertoire of the routine
Thirdly,
hospital laboratory
seems
justified.
Measles in Vaccinated Communities IN the United States measles vaccine has been widely applied in an attempt to eradicate the disease, and the epidemic pattern has changed. The national campaign started in 1966 and there was at first a striking decline in measles incidence. Later, epidemics appeared among immunised communities and the total incidence of the disease rose, although the total number of cases is much lower than before.l In the United Kingdom also, the epidemic expected in 1969 was prevented, but it eventually arrived out of season in 1970. What are the reasons for the relative failure of these two extensive vaccination campaigns Trials of measles vaccine to eliminate measles ? have shown that it produced antibody in 95% of those vaccinated, and in the United Kingdom the Medical Research Council conducted a protection trial using the Schwartz strain, either alone or preceded by killed vaccine, which showed that the living vaccine was over 90% effective2 SPENCER et awl.3 discussed the epidemiological basis for the U.S. eradication hopes. Since the vaccine is so highly effective, and because lack of susceptibles and their subsequent recruitment by birth is the accepted explanation for the waves of epidemic disease, it seemed reasonable to suppose that vaccination of about 75 % or more of the children at one year of age would control the disease. This figure was based on the work of HEDRICK,4 who estimated the susceptible population in Baltimore from 1900 to 1931 by recording notifications on the one hand and recruitment by birth on the other. He found that epidemics in Baltimore stopped before all susceptibles were infected. At Willowbrook State School routine vaccination of all admissions has completely controlled the disease for nearly fifteen years in that nstitution. Similarly, in Rhode Islandmeasles incidence has fallen more than tenfold after a widespread and intensive vaccination campaign started in 1966; and the infection now appears mainly in children of military personnel or of Portuguese immigrants, who were out of the State during the campaign and were not vaccinated. The explanation for the failure of the campaign in Britain is that too few children have been vaccinated. This poor uptake of vaccine was largely attributable to a lack of conviction among Summary. U.S. Morbidity and Mortality Weekly Report, 1971, 20, 293. Practitioner, 1971, 206, 458. Spencer, D. J., Hull, H. B., Langmuir, A. D. Publ. Hlth Reps, Wash. 1967, 82, 253. Hedrick, A. W. Am. J. Hyg. 1933, 17, 613. Scott, D. Am. J. Epidem. 1971, 94, 37.
many doctors that measles in Britain is worth pre-
venting, despite the Public Health Laboratory Service report on the complications of natural measles 6; and doubts were strengthened by the withdrawal of the Beckenham 31 strain. This strain is associated with a higher incidence of fever and rash, and might therefore produce more encephalitis, than the Schwartz strain. It is, however, quite clear that both strains of attenuated virus give rise to less reactions than the original Enders B vaccine, widely used in the U.S.A. without untoward incident.’ A third reason for lack of enthusiasm for the vaccine has been the fear that immunity may not be lifelong and that the effect may be to delay measles until a more vulnerable age, The second common cause of disappointment in vaccine performance is a considerable number of infections among vaccinated individuals. Such a situation causes much disquiet, and always merits thorough investigation. It is, however, completely high vaccine effectiveness. WYLL compatible with 8 and WITTE found 32 cases in unvaccinated pupils and 15 in vaccinated pupils at a particular school, and a study of the whole outbreak and calculation of attack-rates led to the conclusion that the vaccine was over 90 % effective. This figure agrees well with the M.R.C. trial and with serological studies of vaccine performance. Vaccine performance can, however, be less than satisfactory. For example, in another outbreak9 the vaccine almost certainly failed because it had been given to children under the age of 9 months, when maternal antibody often prevents adequate multiplication of vaccine virus. A similar cause of vaccine failure in the U.S.A. concerned Enders B vaccine, which had to be used with y-globulin to cut down untoward reactions. On this schedule, the dose of y-globulin had to be carefully calibrated so that the vaccine virus multiplied sufficiently to produce an immunising infection, but not enough to cause reactions; and sometimes too much y-globulin was
given. A third source of vaccine failure is the lability of the living attenuated virus. LERMAN and GOLD10 found in one school 17 cases of measles-11 unvaccinated and 6 vaccinated. 5 of the children who had measles despite vaccine had been vaccinated by one doctor, who stored vaccine in a refrigerator which, on busy days when it was constantly open, reached a temperature of 18°C. Another possible reason for failure may be waning of immunity, especially with more attenuated strains like Schwartz. Again, the full effectiveness of less attenuated strains may be reduced by simultaneous administration of y-globulin or prior administration
1. Measles Surveillance
2. 3. 4. 5.
6. Miller, D. L. Br. med. J. 1964, ii, 75. 7. U.S. Measles Surveillance 1969. Communicable Diseases Center report no. 7. 8. Wyll, S. A., Witte, J. J. J. Am. med. Ass. 1971, 216, 1306. 9. Baretta, R. O. Pediatrics, 1970, 46, 397. 10. Lerman, S. J., Gold, E. J. Am. med. Ass. 1971, 216, 1311.
percentage of N.B.T.-positive neutrophils is raised, often greatly, in all systemic bacterial inftctions,
THE LANCET Nitroblue Tetrazolium: a Routine Test? NEUTROPHIL leucocytosis is a feature of many clinical situations and the cause is often self-evident. On occasion leucocytosis with or without fever may persist, and to establish the differential diagnosis between bacterial or other infections, leukaemia, carcinoma, or other conditions may be extremely difficult. The observation of toxic granulations in the neutrophil cytoplasm and the measurement of neutrophil alkaline phosphatase sometimes contribute to a diagnosis. The nitroblue-tetrazolium (N.B.T.) test may also yield valuable information. In 1967 BAEHNER and NATHAN1 showed that a small proportion of the neutrophils of a normal person could in vitro reduce the colourless soluble dye nitro blue tetrazolium to produce an insoluble blue-black formazan precipitate. The mechanism of this reduction of N.B.T. to form formazan in the cytoplasm of the phagocytic neutrophil remains unclear. Apparently the resting unstimulated neutrophil membrane is not permeable to N.B.T., so some membrane alteration has allowed entry of the dye. The actual reduction of N.B.T. seems to take place within the confines of the phagocytic vacuole and may be seen not only in neutrophils but also in eosinophils and monocytes. The reduction seems to depend on the activity of N.A.D.H. oxidase, an enzyme intimately involved in the oxygen consumption and bactericidal mechanism of the neutrophil. Against this background, PARK et aI.2 speculated that when neutrophils are concerned in phagocytosis in vivo during natural infection, a metabolic change takes place that leads to increased reduction of N.B.T. Such has indeed the to be case. proved The reduction of N.B.T. by neutrophils has now been studied by different workers 2-4 in a number of conditions, and a reproducible pattern has emerged. The absolute number and percentage of N.B.T.-
positive neutrophils is low in normal persons (less than 10%), in patients with fever and leucocytosis of non-bacterial origin (such as rheumatoid arthritis or disseminated lupus erythematosus), after surgical operations, and in uncomplicated viral infection. The test is also normal in bacterial infections that remain superficially localised. The number and 1. 2. 3. 4.
Baehner, R. L., Nathan, D.
G. Science, 1967, 155, 835. Park, B. H., Fikrig, S. M., Smithwick, E. M. Lancet, 1968, ii, 532. Feigin, R. D., Shackelford, P. G., Choi, S. C., Flake, K. K., Franklin, F. A., Eisenberg, C. S. Pediatrics, 1971, 78, 230. Matula, G., Paterson, P. Y. New Engl. J. Med. 1971, 285, 311.
including meningitis, endocarditis, osteomyelitis, and septic arthritis, empyema, and miliary tuberculosis. The test also seems to be positive in systemic fungal infection and in malaria 5-6 and other parasitic diseases.’ Normal newborn and premature infants seem, however, to have many positive neutrophils until the age of two months, so that the test has no diagnostic accuracy until this age. Conversely, the administration of corticosteroid drugs may be responsible for a false negative test in a bacterially infected patient. As might be expected, the leucocytosis of chronic granulocytic leukaemia seems to be unassociated with increased numbers of N.B.T.positive cells. Interestingly, in Hodgkin’s disease the test may be positive in the presence of fever and
neutrophil leucocytosis.9 Of considerable importance has been the finding that children with chronic granulomatous disease (c.G.D.), a condition characterised by increased susceptibility to infection with certain bacteria, have neutrophils that totally fail to reduce N.B.T. in vitro. No abnormality of immune function has been demonstrated in children with this disease. Although their neutrophils appear to phagocytose bacteria quite normally, they are unable to destroy engulfed bacteria of quite low virulence and the children die of chronic suppurative and granulomatous lesions. 10, 11 The basic defect is thought to lie in an enzyme system that contributes to normal lysosomal function. The lack of reduction of N.B.T. in vitro forms a very sensitive screening test for this disease. A positive N.B.T. test thus depends on two components. On the one hand, there must be sufficient bacteria or bacterial products present to impinge on the circulating neutrophils; on the other, the neutrophils must themselves be normal and have the capacity to reduce N.B.T. to formazan. Chronic granulomatous disease well exemplifies a defect in the second component. The N.B.T. test seems, therefore, to be clinically useful in three main situations. First, it may be very valuable in supporting a diagnosis of severe systemic infection with bacteria (or fungi) when other possibilities exist, and it may serve to suggest or exclude viral or non-infectious disease. Secondly, it may be a useful test with which to monitor patients and to detect early infection in those with increased susceptibility or in those in whom infection could be especially catastrophic, such as transplant recipients, patients undergoing immunosuppression for other reasons, or those with rheumatic valvular disease of the heart. 5. Andersen, B. R. Lancet, 1971, ii, 317. 6. Pujol-Moix, M. N. ibid. Oct. 16, 1971, p. 871. 7. Chretien, J. H., Garagusi, V. F. ibid. Sept. 4, 1971, p. 549. 8. Matula, G., Paterson, P. Y. ibid. 1971, i, 803. 9. Catovsky, D. Unpublished. 10. Holmes, B., Quie, P. G., Windhorst, D. B., Good, R. A.
1966, i, 1225. 11. Windhorst, D. B., Holmes, B., Good, R.
A. ibid.
1967, i,
Lancet,
737.
910
it may help to identify patients with congenital defects of neutrophil function such as c.G.D. Its introduction into the repertoire of the routine
Thirdly,
hospital laboratory
seems
justified.
Measles in Vaccinated Communities IN the United States measles vaccine has been widely applied in an attempt to eradicate the disease, and the epidemic pattern has changed. The national campaign started in 1966 and there was at first a striking decline in measles incidence. Later, epidemics appeared among immunised communities and the total incidence of the disease rose, although the total number of cases is much lower than before.l In the United Kingdom also, the epidemic expected in 1969 was prevented, but it eventually arrived out of season in 1970. What are the reasons for the relative failure of these two extensive vaccination campaigns Trials of measles vaccine to eliminate measles ? have shown that it produced antibody in 95% of those vaccinated, and in the United Kingdom the Medical Research Council conducted a protection trial using the Schwartz strain, either alone or preceded by killed vaccine, which showed that the living vaccine was over 90% effective2 SPENCER et awl.3 discussed the epidemiological basis for the U.S. eradication hopes. Since the vaccine is so highly effective, and because lack of susceptibles and their subsequent recruitment by birth is the accepted explanation for the waves of epidemic disease, it seemed reasonable to suppose that vaccination of about 75 % or more of the children at one year of age would control the disease. This figure was based on the work of HEDRICK,4 who estimated the susceptible population in Baltimore from 1900 to 1931 by recording notifications on the one hand and recruitment by birth on the other. He found that epidemics in Baltimore stopped before all susceptibles were infected. At Willowbrook State School routine vaccination of all admissions has completely controlled the disease for nearly fifteen years in that nstitution. Similarly, in Rhode Islandmeasles incidence has fallen more than tenfold after a widespread and intensive vaccination campaign started in 1966; and the infection now appears mainly in children of military personnel or of Portuguese immigrants, who were out of the State during the campaign and were not vaccinated. The explanation for the failure of the campaign in Britain is that too few children have been vaccinated. This poor uptake of vaccine was largely attributable to a lack of conviction among Summary. U.S. Morbidity and Mortality Weekly Report, 1971, 20, 293. Practitioner, 1971, 206, 458. Spencer, D. J., Hull, H. B., Langmuir, A. D. Publ. Hlth Reps, Wash. 1967, 82, 253. Hedrick, A. W. Am. J. Hyg. 1933, 17, 613. Scott, D. Am. J. Epidem. 1971, 94, 37.
many doctors that measles in Britain is worth pre-
venting, despite the Public Health Laboratory Service report on the complications of natural measles 6; and doubts were strengthened by the withdrawal of the Beckenham 31 strain. This strain is associated with a higher incidence of fever and rash, and might therefore produce more encephalitis, than the Schwartz strain. It is, however, quite clear that both strains of attenuated virus give rise to less reactions than the original Enders B vaccine, widely used in the U.S.A. without untoward incident.’ A third reason for lack of enthusiasm for the vaccine has been the fear that immunity may not be lifelong and that the effect may be to delay measles until a more vulnerable age, The second common cause of disappointment in vaccine performance is a considerable number of infections among vaccinated individuals. Such a situation causes much disquiet, and always merits thorough investigation. It is, however, completely high vaccine effectiveness. WYLL compatible with 8 and WITTE found 32 cases in unvaccinated pupils and 15 in vaccinated pupils at a particular school, and a study of the whole outbreak and calculation of attack-rates led to the conclusion that the vaccine was over 90 % effective. This figure agrees well with the M.R.C. trial and with serological studies of vaccine performance. Vaccine performance can, however, be less than satisfactory. For example, in another outbreak9 the vaccine almost certainly failed because it had been given to children under the age of 9 months, when maternal antibody often prevents adequate multiplication of vaccine virus. A similar cause of vaccine failure in the U.S.A. concerned Enders B vaccine, which had to be used with y-globulin to cut down untoward reactions. On this schedule, the dose of y-globulin had to be carefully calibrated so that the vaccine virus multiplied sufficiently to produce an immunising infection, but not enough to cause reactions; and sometimes too much y-globulin was
given. A third source of vaccine failure is the lability of the living attenuated virus. LERMAN and GOLD10 found in one school 17 cases of measles-11 unvaccinated and 6 vaccinated. 5 of the children who had measles despite vaccine had been vaccinated by one doctor, who stored vaccine in a refrigerator which, on busy days when it was constantly open, reached a temperature of 18°C. Another possible reason for failure may be waning of immunity, especially with more attenuated strains like Schwartz. Again, the full effectiveness of less attenuated strains may be reduced by simultaneous administration of y-globulin or prior administration
1. Measles Surveillance
2. 3. 4. 5.
6. Miller, D. L. Br. med. J. 1964, ii, 75. 7. U.S. Measles Surveillance 1969. Communicable Diseases Center report no. 7. 8. Wyll, S. A., Witte, J. J. J. Am. med. Ass. 1971, 216, 1306. 9. Baretta, R. O. Pediatrics, 1970, 46, 397. 10. Lerman, S. J., Gold, E. J. Am. med. Ass. 1971, 216, 1311.