INDIAN JOURNAL OF PEDIATRICS Vol. 42
May, 1975
PRACTICAL USE OF NITROBLUE TETRAZOLIUM DISORDERS OF CHILvREN*
No. 328
T E S T IN F E B R I L E
K . D . AJWANI, H. SAXENA, N. SmOHAL AND K . VISHNOI
Ifanpur Pyrexla is a very common feature of many diseases in children. It may be accompanied b~' polymorphonuclear leucocytosis. A etiologically, three possible diagnoses are entertained, viz., bacterial infection, viral infection and n~n-infective conditions. T h e increased reduction of nltroblue tetrazolium (NBT) dye by tile neutrophils of patients with systemic bacterial infection has been fairly well established (Park et al. 1968, Felgin et al. 1971, M a t u l a and Paterson 1971). A positive test favours the diagnosis of bacterial infection while a negative test would support a diagnosis of viral or non-infective lesion provided the neutrophils themselves are normal. I n the present communication we report our results on tile applicability of the NBI' test in the differential diagnosis o f pyrexia in p a e d i a t r i c patients. Patients and Methods T w e n t y healthy children of both sexes were chosen as controls. T h e i r ages varied from a few weeks to ten years. Fifty-six children with pyrexia were taken as the test material.
* From the Department of Pathology. G.S V.M" Medical College, Kanpur, U.P. Received on ~'ebruary 14, 1975.
A slight modification of" the original m e t h o d of Park et al. (1968) was employed. Blood was collected in paraffinised small ivals containing 75-100 units of heparin per ml. of blood and the vlal was gently shaken. T w o drops of the heparinised blood were added to a mixture of equal volumes of 0.2~'o N B T solution and 0.15 M phosphate buffered saline ( p H 7 . 2 ) . After incubation for 15 minutes in a water bath at37~ 15 minutes at room temperature, one well mixed drop was smeared on a glass slide. T h e smear was air dried and fixed in methanol. Staining o f the smear was done by 0.5% safranin for one minute. T h e slide was then washed in running tap water for two minutes, air dried and screened under the oil-immerslon lens for N B T positive neutrophils. N B T positive cells were scored in a count of IC0 neutrophils a n d were expressed as a percentage. This modification of the m e t h o d obviates the use o f disposable plastic syringes and tubes during collection of the blood and the use of siliconised glassware in the rest of the test. F u r t h e r , staining by safranin, in place o f Wright's ~tain as originally used by Park et al. (1968), gives a sharp contrast to N B T positive ceils.
120
VOL. 42, No. 328
INDIAN JOURNAL OP PEDIATRICS
T h e soluble nitrolue tetrazolium dye is precipitated as insoluble formazan which appears as one large black deposit in the neutrophil cytoplasm (Fig. 1). These are counted as N B T positive cells. Some o f the N B T negative neutrophils may show small black particles. Occasionally monocyles may show this deposit in the form of small particles. It is not seen in eosinophils and lymphocytes. Extracellular formazan m a y be seen near platelet clumps and neutrophils.
R esul ts In healthy children, the percentage o f N B T positive neutrophils ranged from 1 to 13, with a mean o f 6.5. Scores above 13%, therefore, w e r e treated as positive N B T tests~
Table 1.
O f the 56 consecutive children with pyrexia, only 30 gave posilive NBT scores ( T a b l e 1). In 34 children a positive pathogen was isolated in smear or culture, while 5 others had trophozoits of Plasmo~lium vivax in the peripheral blood smear. The N B F test was singularly nega, ive in viral fevers; fevers associated ~:ith tuberculosis and rheumatic fever with the single exception of one case of tubercular meningitis. T h e N B T score was highest in 0yogenic meningitis and bacterial respiratory infections.
Discussion T h e usefulness of the N B T dye reduction in the support o f bacterial infection is clearly established ill our study and the findings are in general agreement
N B T scores. N B T scores m per cent
No. of cases
Mean
Range
H e a l t h y children
20
6.5
1-13
Bacterial pneumonia
11
30.0
15-46
Gastroenteritls ( E. coti)
8
19.0
14-30
Pyogenic meningitis
5
42.0
18-52
R h e u m a t i c fever
5
5.5
3-12
Primary tuberculosis
7
8.0
2-13
Viral encephalitis
5
6.3
2-12
I0
8.0
2-18
5
35.0
30-39
T u b e r c u l a r meningitis M a l a r i a (P. vivax)
AIWANI ET A L . ~ N I T R O B L U E TETRAZOL1UM TEST IN FEBRILE DISORDERS
with earlier series (Park et al. 1968, Feigin 1971, Park 1971, Hawkins 1973, Gordon et al. 1973).
et al.
T h e exact mechanism o f increased reduction of nitroblue tetrazolium within the neulrophils in bacterial infection is still not very clear. Park et af. (1968) were o f the opinion that neutrophils undergo in vivo metabolic changes on phagocytosis of bacteria, provided the neutrophils themselves are normal. False negative NBT tests are observed in transient malfunction of the neutrophils as reported by Rubinstein and Pelet (1973) and in chronic granulomatous di,ease (Baehner and N a t h a n 1968). Park and Good (1970) described a stimulated N B T test to differentiate transient neutrophil dysfunction from the congenital defect of chronic granulomatous disease. G o r d o n et al. (1973) emphasized two important criteria to be fulfilled before results of the N B T test are v a l i d : one, all components of phagocytosis are operating; and two, bacterial infection is systemic. Allen (1973) stated that increase in scavenger like activity of the neutrophils leads to an increased N B T activity, reacting probably through the hexosemonophosphate shunt. Segal et al. (1973) stated that keeping in view the considerable variability of conditions in which the NBT test is positive, the test reflects the presence of an acute stressful situation rather than its underlying cause. French and MacFarlane (1970) consider that the NBT score seems to reflect removal of fibrin by phagocytosis and release of fibrinolytic enzymes. This would explain why the N B I test may be positive in recent myocardial infarction.
121
T h e N B T test is consistently negative in primary tuberculosis and tubercular meningitis (Park et al. 1c68, M a t u l a and Paterson 1971). In one of our cases o f tubercular meningitis, the N B T score was 18 per cent but the possibility of another systemic infection could not be ruled out as the child died soon after admission. It clearly indicates that the neutrophil has little role to play in immune reactions against tubercle bacilli. N B T is reportedly negative in viral infections (Park e t a l . 1968, Matula and Paterson 1971, Feigin et al. 1971, G o r d o n et al. 1973). However, recently H e l i u m and Solberg (1973) reported repeatedly positive N B T test in 14 cases o f acute viral hepatitis. T h e N B F response reverted to normal during the recovery phase. It is surprising that N B T is positive in parasitic diseases (Chretien and Garagusi 1971), especially malaria (Pujol Moix 1971, Matula and Paterson 1971, Anderson 1971). Since it is understood that parasites excite eosinophilic response both in tissues and blood, the positive N B T test could be explained on the basis o f release of toxins or necrotic debris o f the parasites bringing about an increased metabolic activity within the neutrophils.
Summary Fifty-slx children with pyrexia were subjected to the N B T spontaneous qualitative test. T h e test was positive in pyrexia due to systemic bacterial infection with the exception o f tuberculosis, and in malaria. T h e test was negative in pyrexia associated with viral fevers and non-infective fevers. T h e oractical use of the N B T test in the differential diagnosis o f febrile disorders in children is confirmed. T h e technical m e t h o d of performing the N B T test was
INDIAN JOURNAL OF PED[ATRICS
PLATE
Fig 1. T h e cell shows distinct f o r m a z a n deposits w i t h i n a n e u t r o p h i l on t h e lcft. T h e r~egative cell is seen at t h e right side (arrow). T h e red blood cells a p p e a r ghost-like in t h e b a c k g r o u n d S a f r a n i n x 700. AJWANI ET A L . - - P R A C T I C A L USE OF NITROBLUE TETRAZOLIUM TEST IN FEBRILE CHILDREN
Fig. 2. A single t r u n c u s arising from t h e left superior angle o f t h e left ventricle.
Fig, 3.
T b o r a c l c a n d a b d o m i n a l viscera in position of" s i t u / z inversus.
ZINGDE ET A L . ~ T R U E
ISOLATED LAEVOCARDIA IN A NEONATE
DISORDERS OF
May, 1975
PRACTICAL USE OF NITROBLUE TETRAZOLIUM DISORDERS OF CHILvREN*
No. 328
T E S T IN F E B R I L E
K . D . AJWANI, H. SAXENA, N. SmOHAL AND K . VISHNOI
Ifanpur Pyrexla is a very common feature of many diseases in children. It may be accompanied b~' polymorphonuclear leucocytosis. A etiologically, three possible diagnoses are entertained, viz., bacterial infection, viral infection and n~n-infective conditions. T h e increased reduction of nltroblue tetrazolium (NBT) dye by tile neutrophils of patients with systemic bacterial infection has been fairly well established (Park et al. 1968, Felgin et al. 1971, M a t u l a and Paterson 1971). A positive test favours the diagnosis of bacterial infection while a negative test would support a diagnosis of viral or non-infective lesion provided the neutrophils themselves are normal. I n the present communication we report our results on tile applicability of the NBI' test in the differential diagnosis o f pyrexia in p a e d i a t r i c patients. Patients and Methods T w e n t y healthy children of both sexes were chosen as controls. T h e i r ages varied from a few weeks to ten years. Fifty-six children with pyrexia were taken as the test material.
* From the Department of Pathology. G.S V.M" Medical College, Kanpur, U.P. Received on ~'ebruary 14, 1975.
A slight modification of" the original m e t h o d of Park et al. (1968) was employed. Blood was collected in paraffinised small ivals containing 75-100 units of heparin per ml. of blood and the vlal was gently shaken. T w o drops of the heparinised blood were added to a mixture of equal volumes of 0.2~'o N B T solution and 0.15 M phosphate buffered saline ( p H 7 . 2 ) . After incubation for 15 minutes in a water bath at37~ 15 minutes at room temperature, one well mixed drop was smeared on a glass slide. T h e smear was air dried and fixed in methanol. Staining o f the smear was done by 0.5% safranin for one minute. T h e slide was then washed in running tap water for two minutes, air dried and screened under the oil-immerslon lens for N B T positive neutrophils. N B T positive cells were scored in a count of IC0 neutrophils a n d were expressed as a percentage. This modification of the m e t h o d obviates the use o f disposable plastic syringes and tubes during collection of the blood and the use of siliconised glassware in the rest of the test. F u r t h e r , staining by safranin, in place o f Wright's ~tain as originally used by Park et al. (1968), gives a sharp contrast to N B T positive ceils.
120
VOL. 42, No. 328
INDIAN JOURNAL OP PEDIATRICS
T h e soluble nitrolue tetrazolium dye is precipitated as insoluble formazan which appears as one large black deposit in the neutrophil cytoplasm (Fig. 1). These are counted as N B T positive cells. Some o f the N B T negative neutrophils may show small black particles. Occasionally monocyles may show this deposit in the form of small particles. It is not seen in eosinophils and lymphocytes. Extracellular formazan m a y be seen near platelet clumps and neutrophils.
R esul ts In healthy children, the percentage o f N B T positive neutrophils ranged from 1 to 13, with a mean o f 6.5. Scores above 13%, therefore, w e r e treated as positive N B T tests~
Table 1.
O f the 56 consecutive children with pyrexia, only 30 gave posilive NBT scores ( T a b l e 1). In 34 children a positive pathogen was isolated in smear or culture, while 5 others had trophozoits of Plasmo~lium vivax in the peripheral blood smear. The N B F test was singularly nega, ive in viral fevers; fevers associated ~:ith tuberculosis and rheumatic fever with the single exception of one case of tubercular meningitis. T h e N B T score was highest in 0yogenic meningitis and bacterial respiratory infections.
Discussion T h e usefulness of the N B T dye reduction in the support o f bacterial infection is clearly established ill our study and the findings are in general agreement
N B T scores. N B T scores m per cent
No. of cases
Mean
Range
H e a l t h y children
20
6.5
1-13
Bacterial pneumonia
11
30.0
15-46
Gastroenteritls ( E. coti)
8
19.0
14-30
Pyogenic meningitis
5
42.0
18-52
R h e u m a t i c fever
5
5.5
3-12
Primary tuberculosis
7
8.0
2-13
Viral encephalitis
5
6.3
2-12
I0
8.0
2-18
5
35.0
30-39
T u b e r c u l a r meningitis M a l a r i a (P. vivax)
AIWANI ET A L . ~ N I T R O B L U E TETRAZOL1UM TEST IN FEBRILE DISORDERS
with earlier series (Park et al. 1968, Feigin 1971, Park 1971, Hawkins 1973, Gordon et al. 1973).
et al.
T h e exact mechanism o f increased reduction of nitroblue tetrazolium within the neulrophils in bacterial infection is still not very clear. Park et af. (1968) were o f the opinion that neutrophils undergo in vivo metabolic changes on phagocytosis of bacteria, provided the neutrophils themselves are normal. False negative NBT tests are observed in transient malfunction of the neutrophils as reported by Rubinstein and Pelet (1973) and in chronic granulomatous di,ease (Baehner and N a t h a n 1968). Park and Good (1970) described a stimulated N B T test to differentiate transient neutrophil dysfunction from the congenital defect of chronic granulomatous disease. G o r d o n et al. (1973) emphasized two important criteria to be fulfilled before results of the N B T test are v a l i d : one, all components of phagocytosis are operating; and two, bacterial infection is systemic. Allen (1973) stated that increase in scavenger like activity of the neutrophils leads to an increased N B T activity, reacting probably through the hexosemonophosphate shunt. Segal et al. (1973) stated that keeping in view the considerable variability of conditions in which the NBT test is positive, the test reflects the presence of an acute stressful situation rather than its underlying cause. French and MacFarlane (1970) consider that the NBT score seems to reflect removal of fibrin by phagocytosis and release of fibrinolytic enzymes. This would explain why the N B I test may be positive in recent myocardial infarction.
121
T h e N B T test is consistently negative in primary tuberculosis and tubercular meningitis (Park et al. 1c68, M a t u l a and Paterson 1971). In one of our cases o f tubercular meningitis, the N B T score was 18 per cent but the possibility of another systemic infection could not be ruled out as the child died soon after admission. It clearly indicates that the neutrophil has little role to play in immune reactions against tubercle bacilli. N B T is reportedly negative in viral infections (Park e t a l . 1968, Matula and Paterson 1971, Feigin et al. 1971, G o r d o n et al. 1973). However, recently H e l i u m and Solberg (1973) reported repeatedly positive N B T test in 14 cases o f acute viral hepatitis. T h e N B F response reverted to normal during the recovery phase. It is surprising that N B T is positive in parasitic diseases (Chretien and Garagusi 1971), especially malaria (Pujol Moix 1971, Matula and Paterson 1971, Anderson 1971). Since it is understood that parasites excite eosinophilic response both in tissues and blood, the positive N B T test could be explained on the basis o f release of toxins or necrotic debris o f the parasites bringing about an increased metabolic activity within the neutrophils.
Summary Fifty-slx children with pyrexia were subjected to the N B T spontaneous qualitative test. T h e test was positive in pyrexia due to systemic bacterial infection with the exception o f tuberculosis, and in malaria. T h e test was negative in pyrexia associated with viral fevers and non-infective fevers. T h e oractical use of the N B T test in the differential diagnosis o f febrile disorders in children is confirmed. T h e technical m e t h o d of performing the N B T test was
INDIAN JOURNAL OF PED[ATRICS
PLATE
Fig 1. T h e cell shows distinct f o r m a z a n deposits w i t h i n a n e u t r o p h i l on t h e lcft. T h e r~egative cell is seen at t h e right side (arrow). T h e red blood cells a p p e a r ghost-like in t h e b a c k g r o u n d S a f r a n i n x 700. AJWANI ET A L . - - P R A C T I C A L USE OF NITROBLUE TETRAZOLIUM TEST IN FEBRILE CHILDREN
Fig. 2. A single t r u n c u s arising from t h e left superior angle o f t h e left ventricle.
Fig, 3.
T b o r a c l c a n d a b d o m i n a l viscera in position of" s i t u / z inversus.
ZINGDE ET A L . ~ T R U E
ISOLATED LAEVOCARDIA IN A NEONATE
DISORDERS OF
