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Evaluation of nitroblue-tetrazolium test in low-birth-weight infants
March 1974
The Journal of P E D I A T R I C S
441
Evaluation of nitroblue-tetrazolium test & low-birth-weight infants The nitroblue-tetrazofium (NBT) test has been modified and compared with the conventional method of Park. Sign([icant increases of NBT-positive cells werefound in infected low-birth-weight infants. In infants with respiratory distress the values of NBT-positive cells are regularly high. Both absolute number and percentage of NBT-positive cells facilitate clinical separation of bacterial and nonbacterial infections and are useful in monitoring infectious diseases and antibacterial treatment in low-birth-weight infants.
Pitsa K. Kalpaktsoglou, M.D.,* Constantine P. Padiatellis, M.D., John
A. Sofatzis, M.D., and Calliope B. Metaxas, M.D., Athens, Greece
THE CONVENTIONAL method of performing the nitroblue-tetrazolium (NBT) test has not proved useful in distinguishing bacterial infection in low-birth-weight infants. 1-3While working with the NBT test in small infants we discovered that reproducible results could be obtained by using 20 to 30 h of blood obtained from a plantar heel prick. Using a procedure of direct analysis with supravital staining of cells, dilution in a standard WBC pipette, and direct counting of cells in a calibrated counting chamber, the variations observed with the original method were reduced. MATERIAL
AND METHODS
In an initial study of the NBT test a comparison was made with blood obtained by venipuncture and blood obtained by heel prick from 32 infants. The data show clearly no significant differences in the results obtained in the two types of blood sampling in these infants. NBT test. Capillary blood, 20 to 30 h, obtained from a
From the Department of Neonatal lmmunobiology and Department of Pediatrics, "Marika Eliadi"Maternity Hospital. Supported by WeUcome Trust, London, Grant BECH/ EM/P. 3011, and by World Health Organization, Geneva, Grants R100346 and N3/181/43. *Reprint address: Dept. of Neonatal Imrnunobiology "Marika Eliadi" Maternity Hospital, 2 Helena VenizelosSquare, Athens 601, Greece.
freely flowing plantar heel prick was drawn halfway into a heparinized microhematocrit tube, repeatedly inverted to avoid coagulation, and discharged into a small well made by pressing the rounded end of a small test tube into a 2 • 2 cm. piece of parafilm " M " (American Can Co., Neenah, Wis.). Then the conventional method of Park and associates4 was applied. After incubation, the test was read in two ways: (1) on cover slip smears as described by Park and associates 4 and, (2) by making a 1:10 dilution of the blood-NBT dye mixture with 3 per cent acetic acid in a nonsiliconized standard WBC pipette and by direct counting of the NBT-negative and NBT-positive cells (the latter with black cytoplasmic deposit) in a Neubauer chamber, in the same manner as a standard white blood cell count. The percentage of NBT-positive neutrophils was determined by calculation from the number of NBT-negative and NBT-positive cells obtained from the counting chamber and from the standard differential count. Patients. Four hundred (400) NBT tests were performed on capillary blood by the two methods and were compared. We tested all infants entering the intensive care nursery and the premature nursery at the "Marika Eliadi" Maternity Hospital during the first two weeks of the months August, September, October, November, and December in 1972. The infants were studied immediately and then twice a week for the first two weeks a n d once each week thereafter for a period of one to
Vol. 84, No. 3, pp. 441-443
442
Kalpaktsoglou et al.
The Journal of Pediatrics March 1974
Table I. Absolute number and percentage of NBT-positive cells in normal and infected low-birth-weight infants
Low-birth-weight infants
Number of patients
Absolute number
Percentage
Mean • S.E.M.
Mean +_ S.E.M.
X2
Normal
91
783 + 36
14.8 _+ 0.8]
Infected
106
2,105 • 103
46.6 • 1.9
l
44.0
Percentile
<
99.95
S.E.M. = Standard error of the mean. Table II. Absolute number and percentage of NBT-positive cellsin low-birth-weight newborn infants
Low-birth-weight newborn infants
Number of patients
Absolute number
Percentage
Mean • S.E.M.
Mean +_ S.E.M.
X2
Normal
31
967 _+ 96
14.2 + 1.8]
With respiratory distress
29
3,615 + 339
35.6 + 2.1.1
I
37.5
Percentile
<
99.95
S.E.M. = Standard error of the mean. three months. The tests were performed and recorded without knowledge of the infants' condition by those reading the test. The groups of the infants were defined and established after the data had been recorded. The patients were divided into normal and infected lowbirth-weight infants and into normal low-birth-weight newborn infants and low-birth-weight newborn infants with respiratory distress. The infants were initially divided into subgroups on the basis of their gestational and postnatal age, but since no significant difference attributed to the above two criteria were observed, the results obtained from the subgroups were combined for further analysis. RESULTS The values obtained by the conventional method of NBT test, as shown by Park 3 and others, 1,2 did not distinguish clearly between the infected and the healthy low-birth-weight infants. By contrast, with the new technique distinct and significant difference in the absolute number and percentage of NBT-positive cells was recorded. As shown in Table I, the mean values of NBTpositive cells in the nonlnfected infants regularly fell below 1,300 positive cells per cubic millimeter and below 20 per cent. In infected low-birth-weight infants the absolute number of NBT-positive cells was greater than 1,300 and the percentage greater than 20. The difference
of the NBT values obtained from the above two groups is statistically significant. In infants with respiratory distress, either associated with or not obviously associated with bacterial infection, the NBT-positive cells regularly exceeded the absolute number of 3,000 cells per cubic millimeter and the percentage of 30. Table II lists the values of NBT-positive cells obtained in normal lowbirth-weight newborn infants and in low-birth-weight newborn infants with respiratory distress. Serial studies in the same patients comparing the absolute number and the percentage of NBT-positive cells show that the increased percentage appears to last longer than the absolute number of positive cells. The latter seems to reflect immediately the effects of antibiotic therapy and bacterial infection. On Figs. 1 and 2 serial data are recorded from representative cases of infected low-birth-weight infants. DISCUSSION We have developed a new technique for performing and evaluating the NBT supravital staining which seems to be applicable to recognition and evaluation of bacterial infection in low-birth-weight newborn infants and lowbirth-weight infants. Both absolute number of NBT-positive cells greater than 1,300 cells per cubic millimeter and NBT-positive neutrophils greater than 20 per cent can be taken as a
Volume 84 Number 3
Evaluation o f nitroblue-tetrazolium
body
443
weight
NBT ~o NBT
%
test
absol.
NH r
N~ --__
N I~ r
. t , . ,, s
N "
o v~
k,.
80 5000
5o
o
70O0
3000_
3000
3---q
J
. _ ~
5000
/ /
3000 40
200
~
~
3000 ooo
2000_
1o 2O
o
o
lOO 0
10 AGE
20
30 /N
40
50
60
I 70
DAYS
Fig. 1. Absolute number and percentage of NBT-positive cells in a low-birth-weight infant with cytomegalovirus and bacterial infection. Treatment: ampicillin; . . . . . . . colistin sulfate; sodium dimethoxyphenyl penicillin; and ~ gentamicin sulfate.
strong warning of the presence of a clinically significant bacterial infection in term and low-birth-weight infants. Unlike Cocchi and associates) we did not find low values in v e r y small low-birth-weight infants, nor did we find the absolute n u m b e r or percentage of NBT-positive cells to relate to gestational age, size, or postnatal age of the infants. Spuriously low levels were, h o w e v e r , sometimes obtained in infants immediately prior to death, e v e n w h e n death could be attributed to bacterial sepsis. A surprising result was the observation that infants with severe respiratory distress s e e m to h a v e inordinately high values of NBT-positive cells. Thus the N B T test could not be useful in the diagnosis o f infection in infants with hyaline m e m b r a n e disease.
__ 0
JO00_ I
0
0
f
10 20
I
I
I
~
i
I
I
30
40
50
60
70
80
90
AGE
IN
DAYS
Fig. 2. Absolute number and percentage of NBT-positive cells in a low-birth-weight infant with bacterial infection. Treatment: ampicillin; gentamicin sulfate.
We thank Mr. Demetrius Kipiotis and Miss Susan Paschalidis for their technical assistance.
REFERENCES
1. Cocchi, P., Mori, S., and Becattini, A.: NBT test in premature infants, Lancet 2: 1426, 1969. 2. Humbert, J. R., Kurtz, M., and Hathaway, W. E.: Increased reduction of nitroblue tetrazolium by neutrophils of newborn infants, Pediatrics 45: 125, 1970. 3. Park, B. H.: The use and limitations of the nitroblue tetrazolium test as a diagnostic aid, J. PED1ATK.78" 376, 1971. 4. Park, B. H., Fikrig, S. M., and Smithwick, E. M.: Infection and nitroblue-tetrazoluim reduction by neutrophils, a diagnostic aid, Lancet 2: 532, 1968.
The Journal of P E D I A T R I C S
441
Evaluation of nitroblue-tetrazolium test & low-birth-weight infants The nitroblue-tetrazofium (NBT) test has been modified and compared with the conventional method of Park. Sign([icant increases of NBT-positive cells werefound in infected low-birth-weight infants. In infants with respiratory distress the values of NBT-positive cells are regularly high. Both absolute number and percentage of NBT-positive cells facilitate clinical separation of bacterial and nonbacterial infections and are useful in monitoring infectious diseases and antibacterial treatment in low-birth-weight infants.
Pitsa K. Kalpaktsoglou, M.D.,* Constantine P. Padiatellis, M.D., John
A. Sofatzis, M.D., and Calliope B. Metaxas, M.D., Athens, Greece
THE CONVENTIONAL method of performing the nitroblue-tetrazolium (NBT) test has not proved useful in distinguishing bacterial infection in low-birth-weight infants. 1-3While working with the NBT test in small infants we discovered that reproducible results could be obtained by using 20 to 30 h of blood obtained from a plantar heel prick. Using a procedure of direct analysis with supravital staining of cells, dilution in a standard WBC pipette, and direct counting of cells in a calibrated counting chamber, the variations observed with the original method were reduced. MATERIAL
AND METHODS
In an initial study of the NBT test a comparison was made with blood obtained by venipuncture and blood obtained by heel prick from 32 infants. The data show clearly no significant differences in the results obtained in the two types of blood sampling in these infants. NBT test. Capillary blood, 20 to 30 h, obtained from a
From the Department of Neonatal lmmunobiology and Department of Pediatrics, "Marika Eliadi"Maternity Hospital. Supported by WeUcome Trust, London, Grant BECH/ EM/P. 3011, and by World Health Organization, Geneva, Grants R100346 and N3/181/43. *Reprint address: Dept. of Neonatal Imrnunobiology "Marika Eliadi" Maternity Hospital, 2 Helena VenizelosSquare, Athens 601, Greece.
freely flowing plantar heel prick was drawn halfway into a heparinized microhematocrit tube, repeatedly inverted to avoid coagulation, and discharged into a small well made by pressing the rounded end of a small test tube into a 2 • 2 cm. piece of parafilm " M " (American Can Co., Neenah, Wis.). Then the conventional method of Park and associates4 was applied. After incubation, the test was read in two ways: (1) on cover slip smears as described by Park and associates 4 and, (2) by making a 1:10 dilution of the blood-NBT dye mixture with 3 per cent acetic acid in a nonsiliconized standard WBC pipette and by direct counting of the NBT-negative and NBT-positive cells (the latter with black cytoplasmic deposit) in a Neubauer chamber, in the same manner as a standard white blood cell count. The percentage of NBT-positive neutrophils was determined by calculation from the number of NBT-negative and NBT-positive cells obtained from the counting chamber and from the standard differential count. Patients. Four hundred (400) NBT tests were performed on capillary blood by the two methods and were compared. We tested all infants entering the intensive care nursery and the premature nursery at the "Marika Eliadi" Maternity Hospital during the first two weeks of the months August, September, October, November, and December in 1972. The infants were studied immediately and then twice a week for the first two weeks a n d once each week thereafter for a period of one to
Vol. 84, No. 3, pp. 441-443
442
Kalpaktsoglou et al.
The Journal of Pediatrics March 1974
Table I. Absolute number and percentage of NBT-positive cells in normal and infected low-birth-weight infants
Low-birth-weight infants
Number of patients
Absolute number
Percentage
Mean • S.E.M.
Mean +_ S.E.M.
X2
Normal
91
783 + 36
14.8 _+ 0.8]
Infected
106
2,105 • 103
46.6 • 1.9
l
44.0
Percentile
<
99.95
S.E.M. = Standard error of the mean. Table II. Absolute number and percentage of NBT-positive cellsin low-birth-weight newborn infants
Low-birth-weight newborn infants
Number of patients
Absolute number
Percentage
Mean • S.E.M.
Mean +_ S.E.M.
X2
Normal
31
967 _+ 96
14.2 + 1.8]
With respiratory distress
29
3,615 + 339
35.6 + 2.1.1
I
37.5
Percentile
<
99.95
S.E.M. = Standard error of the mean. three months. The tests were performed and recorded without knowledge of the infants' condition by those reading the test. The groups of the infants were defined and established after the data had been recorded. The patients were divided into normal and infected lowbirth-weight infants and into normal low-birth-weight newborn infants and low-birth-weight newborn infants with respiratory distress. The infants were initially divided into subgroups on the basis of their gestational and postnatal age, but since no significant difference attributed to the above two criteria were observed, the results obtained from the subgroups were combined for further analysis. RESULTS The values obtained by the conventional method of NBT test, as shown by Park 3 and others, 1,2 did not distinguish clearly between the infected and the healthy low-birth-weight infants. By contrast, with the new technique distinct and significant difference in the absolute number and percentage of NBT-positive cells was recorded. As shown in Table I, the mean values of NBTpositive cells in the nonlnfected infants regularly fell below 1,300 positive cells per cubic millimeter and below 20 per cent. In infected low-birth-weight infants the absolute number of NBT-positive cells was greater than 1,300 and the percentage greater than 20. The difference
of the NBT values obtained from the above two groups is statistically significant. In infants with respiratory distress, either associated with or not obviously associated with bacterial infection, the NBT-positive cells regularly exceeded the absolute number of 3,000 cells per cubic millimeter and the percentage of 30. Table II lists the values of NBT-positive cells obtained in normal lowbirth-weight newborn infants and in low-birth-weight newborn infants with respiratory distress. Serial studies in the same patients comparing the absolute number and the percentage of NBT-positive cells show that the increased percentage appears to last longer than the absolute number of positive cells. The latter seems to reflect immediately the effects of antibiotic therapy and bacterial infection. On Figs. 1 and 2 serial data are recorded from representative cases of infected low-birth-weight infants. DISCUSSION We have developed a new technique for performing and evaluating the NBT supravital staining which seems to be applicable to recognition and evaluation of bacterial infection in low-birth-weight newborn infants and lowbirth-weight infants. Both absolute number of NBT-positive cells greater than 1,300 cells per cubic millimeter and NBT-positive neutrophils greater than 20 per cent can be taken as a
Volume 84 Number 3
Evaluation o f nitroblue-tetrazolium
body
443
weight
NBT ~o NBT
%
test
absol.
NH r
N~ --__
N I~ r
. t , . ,, s
N "
o v~
k,.
80 5000
5o
o
70O0
3000_
3000
3---q
J
. _ ~
5000
/ /
3000 40
200
~
~
3000 ooo
2000_
1o 2O
o
o
lOO 0
10 AGE
20
30 /N
40
50
60
I 70
DAYS
Fig. 1. Absolute number and percentage of NBT-positive cells in a low-birth-weight infant with cytomegalovirus and bacterial infection. Treatment: ampicillin; . . . . . . . colistin sulfate; sodium dimethoxyphenyl penicillin; and ~ gentamicin sulfate.
strong warning of the presence of a clinically significant bacterial infection in term and low-birth-weight infants. Unlike Cocchi and associates) we did not find low values in v e r y small low-birth-weight infants, nor did we find the absolute n u m b e r or percentage of NBT-positive cells to relate to gestational age, size, or postnatal age of the infants. Spuriously low levels were, h o w e v e r , sometimes obtained in infants immediately prior to death, e v e n w h e n death could be attributed to bacterial sepsis. A surprising result was the observation that infants with severe respiratory distress s e e m to h a v e inordinately high values of NBT-positive cells. Thus the N B T test could not be useful in the diagnosis o f infection in infants with hyaline m e m b r a n e disease.
__ 0
JO00_ I
0
0
f
10 20
I
I
I
~
i
I
I
30
40
50
60
70
80
90
AGE
IN
DAYS
Fig. 2. Absolute number and percentage of NBT-positive cells in a low-birth-weight infant with bacterial infection. Treatment: ampicillin; gentamicin sulfate.
We thank Mr. Demetrius Kipiotis and Miss Susan Paschalidis for their technical assistance.
REFERENCES
1. Cocchi, P., Mori, S., and Becattini, A.: NBT test in premature infants, Lancet 2: 1426, 1969. 2. Humbert, J. R., Kurtz, M., and Hathaway, W. E.: Increased reduction of nitroblue tetrazolium by neutrophils of newborn infants, Pediatrics 45: 125, 1970. 3. Park, B. H.: The use and limitations of the nitroblue tetrazolium test as a diagnostic aid, J. PED1ATK.78" 376, 1971. 4. Park, B. H., Fikrig, S. M., and Smithwick, E. M.: Infection and nitroblue-tetrazoluim reduction by neutrophils, a diagnostic aid, Lancet 2: 532, 1968.