Acta haemat. 56: 73-77 (1976)
Response of Blood and Bone Marrow Neutrophils to the Nitroblue-Tetrazolium Test in Children C. C a ssim o s , S. S k la v u n u -Z u r u k z o g l u , D. C a triu and C. N anas Aristotle University of Thessaloniki, Thessaloniki
Key Words. Bone marrow neutrophils • Endotoxin ■ Neutrophils • Nitroblue-tetrazolium test Abstract. The ability of blood and bone marrow neutrophils to reduce nitrobluetetrazolium both before and after stimulation with Escherichia cctli endotoxin was investigated. The polymorphonuclear cells of the bone marrow are less able to re duce the dye than blood cells. This difference is maintained after stimulation by en dotoxin. Early forms of neutrophils (myelocytes and metamyelocytes) have some re ducing ability but this is more marked after stimulation with endotoxin.
In recent years numerous observations have emphasized the value of the nitroblue-tetrazolium (NBT) reduction test in the differential diagno sis of bacterial infections. In normal subjects, a positive reaction occurs in 4-14°/o of blood neutrophils. During bacterial infection, there is an in creased number of NBT-positive neutrophils [2, 5-8, 10], The reduction of the dye seems to occur more readily when the cell oxidative metabo lism is stimulated by bacterial endotoxin [11], As very little is known about the NBT reduction capacity of the bone marrow phagocytes [3] we have decided to apply this test to blood neu trophils and to bone marrow granulocytic cells simultaneously in order to compare their capacity to reduce NBT.
Samples of peripheral blood and bone marrow from 19 children with various dis eases were studied. These diseases were: acute lymphoblastic leukemia in remission
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Materials and Methods
74
C assimos/S klavunu-Z urukzoglu /C atriu /N anas
Fig. 1. Myelocytes reducing NBT in bone marrow.
2, infectious mononucleosis 1, various viral infections 4, nephrotic syndrome 1, sep ticemia 1, aplastic anemia 1, sickle cell anemia 1, infection of the urinary tract 3, Mediterranean fever 1, rheumatoid arthritis 2, Wilms’ tumor 1, and herpetic stoma titis 1. The NBT test was performed according to the method of P ark el at. [10], The test was also carried out after the phagocytes had been stimulated in vitro with Esch erichia coli endotoxin [11], At least 1,000 cells were counted from each sample and the result was expressed as a percentage.
Results
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In table I, the distribution of NBT scores are shown. The mean per centage of NBT-positive blood neutrophils was 9.3°/o with a range of 0-48% before stimulation, and 25.5% with a range of 2-64% after stimu lation. For the assessment of the results of the NBT test on the bone marrow cells, the reaction of the immature neutrophils (myelocytes and metamye locytes) has been considered separately from that of the mature neutro phils (band forms and segmented neutrophils). The mean percentage of the NBT-positive immature neutrophils was 0.09% with a range of 0-1% before stimulation, and 2.24% with a range of 0-14% after stimulation. The mean percentage of NBT-positive mature bone marrow neutrophils was 2% with a range of 0-15% before stimulation, and 8.7% with a range of 0-36% after stimulation.
Nitroblue-Tetrazolium Test
75
Table I. Percentage of neutrophil cells reducing NBT in blood and bone marrow in various diseases Case Diagnosis No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Acute lymphoblastic leukemia Acute lymphoblastic leukemia Infectious mono nucleosis Viral infection Viral infection Viral infection Viral infection Nephrotic syndrome Septicemia Sickle cell anemia Aplastic anemia Urinary tract infection Urinary tract infection Urinary tract infection Mediterranean fever Rheumatoid arthritis Rheumatoid arthritis Wilms’ tumor Herpetic stomatitis
Mean percentage
Blood before stimulation
Bone marrow after stimulation
mature neutrophils immature phils before after before stimula- stimula- stimulation tion tion
neutroafter stimulation
21
0
1
0
0
14
21
0
1
0
0.1
0 9 8 2.5 3 3 48 2 7 10.5 3 19 7 24 5.5 0.5 6
48 52 22 3.5 13 14 64 8 11.5 40 8 26 16 64 24 2 18
0 0 0 0 0 5 15 1 6 3 0 2 2 4 0
15 0 I 2 2 11 35 3 8 8 1 10 22 36 2 6 2
9.3
25.5
2
4.5
0
0
8.7
0 0 0.2 0 0.5 0 0 0 0 0 0 1 0 0 0 0 0 0.09
4.5 0 0 0 0 0.5 10 2 0 0.5 0 4 14 7 0 0 0
2.24
The pool of mature nonproliferating bone marrow cells represents ap proximately 60% of the total phagocytic capacity of the bone marrow cells. Myeloblasts and promyelocytes are not able to phagocytize [12]. Phagocytic activity is attained at the myelocyte to metamyelocyte stage. The polymorphonuclear neutrophils (PMN) have the maximal phagocytic efficiency. Our results show that the NBT reducing capacity of the imma ture neutrophils is significantly lower than that of mature marrow PMN.
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Discussion
76
C assimos/S klavunu-Z urukzoglu /C atriu /N anas
It is, however, noteworthy that in most cases the reduction of NBT imma ture marrow PMN was observed after stimulation with endotoxin, imply ing that under appropriate stimulatory conditions an apparently function ally immature cell can become functionally mature. M c C all et al. [9] made the observation that immature myelocytes and promyelocytes do not reduce NBT (perhaps the difference in our results is due to the fact that we used bone marrow cells instead of peripheral cells of patients with acute myelogenous leukemia). Furthermore, we have been able to show that these cells can be stimulated with endotoxin. C a tovsky and G o lto n [3] also applied the NBT reduction test to immature blood and bone marrow cells in an effort to differentiate acute myelocytic from monocytic leukemia. The great difference between the percentage of positive peripheral and marrow neutrophils was unexpected. This difference was statistically sig nificant (p
Response of Blood and Bone Marrow Neutrophils to the Nitroblue-Tetrazolium Test in Children C. C a ssim o s , S. S k la v u n u -Z u r u k z o g l u , D. C a triu and C. N anas Aristotle University of Thessaloniki, Thessaloniki
Key Words. Bone marrow neutrophils • Endotoxin ■ Neutrophils • Nitroblue-tetrazolium test Abstract. The ability of blood and bone marrow neutrophils to reduce nitrobluetetrazolium both before and after stimulation with Escherichia cctli endotoxin was investigated. The polymorphonuclear cells of the bone marrow are less able to re duce the dye than blood cells. This difference is maintained after stimulation by en dotoxin. Early forms of neutrophils (myelocytes and metamyelocytes) have some re ducing ability but this is more marked after stimulation with endotoxin.
In recent years numerous observations have emphasized the value of the nitroblue-tetrazolium (NBT) reduction test in the differential diagno sis of bacterial infections. In normal subjects, a positive reaction occurs in 4-14°/o of blood neutrophils. During bacterial infection, there is an in creased number of NBT-positive neutrophils [2, 5-8, 10], The reduction of the dye seems to occur more readily when the cell oxidative metabo lism is stimulated by bacterial endotoxin [11], As very little is known about the NBT reduction capacity of the bone marrow phagocytes [3] we have decided to apply this test to blood neu trophils and to bone marrow granulocytic cells simultaneously in order to compare their capacity to reduce NBT.
Samples of peripheral blood and bone marrow from 19 children with various dis eases were studied. These diseases were: acute lymphoblastic leukemia in remission
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/29/2019 1:03:06 PM
Materials and Methods
74
C assimos/S klavunu-Z urukzoglu /C atriu /N anas
Fig. 1. Myelocytes reducing NBT in bone marrow.
2, infectious mononucleosis 1, various viral infections 4, nephrotic syndrome 1, sep ticemia 1, aplastic anemia 1, sickle cell anemia 1, infection of the urinary tract 3, Mediterranean fever 1, rheumatoid arthritis 2, Wilms’ tumor 1, and herpetic stoma titis 1. The NBT test was performed according to the method of P ark el at. [10], The test was also carried out after the phagocytes had been stimulated in vitro with Esch erichia coli endotoxin [11], At least 1,000 cells were counted from each sample and the result was expressed as a percentage.
Results
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In table I, the distribution of NBT scores are shown. The mean per centage of NBT-positive blood neutrophils was 9.3°/o with a range of 0-48% before stimulation, and 25.5% with a range of 2-64% after stimu lation. For the assessment of the results of the NBT test on the bone marrow cells, the reaction of the immature neutrophils (myelocytes and metamye locytes) has been considered separately from that of the mature neutro phils (band forms and segmented neutrophils). The mean percentage of the NBT-positive immature neutrophils was 0.09% with a range of 0-1% before stimulation, and 2.24% with a range of 0-14% after stimulation. The mean percentage of NBT-positive mature bone marrow neutrophils was 2% with a range of 0-15% before stimulation, and 8.7% with a range of 0-36% after stimulation.
Nitroblue-Tetrazolium Test
75
Table I. Percentage of neutrophil cells reducing NBT in blood and bone marrow in various diseases Case Diagnosis No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Acute lymphoblastic leukemia Acute lymphoblastic leukemia Infectious mono nucleosis Viral infection Viral infection Viral infection Viral infection Nephrotic syndrome Septicemia Sickle cell anemia Aplastic anemia Urinary tract infection Urinary tract infection Urinary tract infection Mediterranean fever Rheumatoid arthritis Rheumatoid arthritis Wilms’ tumor Herpetic stomatitis
Mean percentage
Blood before stimulation
Bone marrow after stimulation
mature neutrophils immature phils before after before stimula- stimula- stimulation tion tion
neutroafter stimulation
21
0
1
0
0
14
21
0
1
0
0.1
0 9 8 2.5 3 3 48 2 7 10.5 3 19 7 24 5.5 0.5 6
48 52 22 3.5 13 14 64 8 11.5 40 8 26 16 64 24 2 18
0 0 0 0 0 5 15 1 6 3 0 2 2 4 0
15 0 I 2 2 11 35 3 8 8 1 10 22 36 2 6 2
9.3
25.5
2
4.5
0
0
8.7
0 0 0.2 0 0.5 0 0 0 0 0 0 1 0 0 0 0 0 0.09
4.5 0 0 0 0 0.5 10 2 0 0.5 0 4 14 7 0 0 0
2.24
The pool of mature nonproliferating bone marrow cells represents ap proximately 60% of the total phagocytic capacity of the bone marrow cells. Myeloblasts and promyelocytes are not able to phagocytize [12]. Phagocytic activity is attained at the myelocyte to metamyelocyte stage. The polymorphonuclear neutrophils (PMN) have the maximal phagocytic efficiency. Our results show that the NBT reducing capacity of the imma ture neutrophils is significantly lower than that of mature marrow PMN.
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/29/2019 1:03:06 PM
Discussion
76
C assimos/S klavunu-Z urukzoglu /C atriu /N anas
It is, however, noteworthy that in most cases the reduction of NBT imma ture marrow PMN was observed after stimulation with endotoxin, imply ing that under appropriate stimulatory conditions an apparently function ally immature cell can become functionally mature. M c C all et al. [9] made the observation that immature myelocytes and promyelocytes do not reduce NBT (perhaps the difference in our results is due to the fact that we used bone marrow cells instead of peripheral cells of patients with acute myelogenous leukemia). Furthermore, we have been able to show that these cells can be stimulated with endotoxin. C a tovsky and G o lto n [3] also applied the NBT reduction test to immature blood and bone marrow cells in an effort to differentiate acute myelocytic from monocytic leukemia. The great difference between the percentage of positive peripheral and marrow neutrophils was unexpected. This difference was statistically sig nificant (p
