April 1978
638
TheJournalofPEDIATRICS
Nitroblue tetrazolium test in neonates
Barry D. Chandler, M.D., Neena Kapoor, M.D., Barbara E. Barker, Ph.D., Robert J. Boyle, M.D., and William Oh, M.D.,* Providence, R. L
THE NITROBLUE TETRAZOLIUM test was introduced by Parks et al 1 in 1968 as an aid in the diagnosis of acute bacterial infection. Its use is based on the observation that neutrophil reduction of NBT to an intracellular formazan precipitate is increased under conditions of bacterial infection. The test has had limited use in the diagnosis of neonatal sepsis because of spuriously high values found in normal infants. ~ Barker and Farnes 3 observed that falsely high and widely varied NBT values were obtained in adults if high concentrations of heparin were used. Using a micromethod, they further showed that limiting heparin concentration to 1 unit/ml gave normal NBT values of less than 5% in adults and older children. ~ We hypothesized that if the heparin concentration in the NBT solution were similarly controlled, normal values may be defined in newborn subjects, making the test available for future clinical testing in such situations as neonatal sepsis.
MATERIALS AND M E T H O D S lnitiaUy, 24 clinically well normal term newborn infants were NBT tested (103 determinations) at the time of screening for phenylketonuria (age two to four days). Heel puncture blood (44.7h) was collected in a nonheparinized capillary tube and immediately added to equal volumes of 0.2% NBT solution in a 0.1M phosphate buffer (pH = 7.0) containing varying concentrations of heparin (1 to 70 units/ml NBT-blood solution). The mixtures were From the Department of Pediatrics, Women and Infants Hospital of Rhode Island, The Department of Pathology, Rhode Island Hospital, and Brown University, Section of Reproductive and Developmental Medicine. *Reprint address: Department of Pediatrics, Women and Infants Hoapital of Rhode Island, 50 Maude St., Providence, RI 02908.
Vol. 92, No. 4, pp. 638-640
incubated in a 37~ water bath for 25 minutes. Smears were made from samples of each solution, and were Wright stained in the usual manner, Duplicate slides were made on each infant; 100 neutrophils were counted on each slide and an average of NBT-positive cells was obtained from combining both cell counts. Endotoxinstimulated specimens (Salmonella abortus equi, 50 m g / m l NBT-blood solution) were collected, and these confirmed that each infant had neutrophil opsonization capability. I
Abbreviation used. NBT: nitroblue tetrazolium
In the second part of the study, 58 infants of varying gestational ages (range 25 to 43 weeks) and birth weights (range 755 to 5,240 gin) admitted to the Women & Infants Hospital Special Care Nursery were NBT and NBTendotoxin tested from heel puncture blood obtained between one and five days of age. These infants were admitted to the Special Care Nursery for various clinical conditions other than sepsis and respiratory distress, and were clinically stable when the NBT tests were performed. The methods for the NBT test was similar to that used in the first study, using one unit of heparin per milliliter of NBT-blood solution. RESULTS Fig. 1 presents the results from the initial study of normal newborn infants and shows that the percentage of NBT positivity was variable but that at 1 to 25 units of heparin concentration in the NBT-blood solution, the values were cgnsistently within the range of 0 to 35%. At a higher heparin concentratioh, spuriously high values were observed (M +_ SD of 34 _+ 12.6, 40 __+ 17.6, and 33 + 18.7% at 40, 50, and 70% heparin Concentrations, respectively). Fig. 2 illustrates the NBT values obtained from the group of 58 infants studied with 1 unit/ml of
0022-3476/78/0492-0638500.30/0 9 1978 The C. V. Mosby Co.
Volume 92 Number 4
N B T test in n e o n a t e s
63 9
NBT (% POSITIVITY) 70
60-
(3)
(Io)
-MSSD 50-
( )=n
(7)
t
40; 30.
(16)
123)
20
t
10
I
0
o
I
I0
I 30
20
I 40
I 50
1 60
I 70
Units of Heporin/ml NBT-Blood Solution Fig. 1. Relationship between concentration of heparin in the NBT-blood solution and percent nitroblue tetrazolium positivity (nonendotoxin stimulated).
NBT
% POSITIVITY)
.30
0 0
20
0
(3O
0 0
0 0
0 0
0
0 O0 (3O
0 0 CO
0 0 0
I0
0
(3O
0 0 0 0 0
0 0 0 0
0
0
0
0
I 26
I 28
I 30
,, 32
o
GESTATION
,, 34
0
1 36
I.
38
0 CO CO 0 O(2O O0 0 OO 0 0 0 OOaO 0
np,
40-43
(Weeks)
Fig. 2. Nitroblue tetrazolium positivity of neutrophils in infants (gestational age 25 to 40 weeks) with no evidence of sepsis or respiratory distress (with 1 unit heparin/ml NBT-blood solution).
NBT-blood solution. There was no difference between NBT values obtained from infants of varying gestati0nal
COMMENT The use of high concentrations of heparin in the NBT
ages or birth weights (graph for birth weight not shown), and the values ranged from 1 to 33%.
test has been shown to cause increased dye reduction in adult neutrophils2 Our data indicate that a similar
640
Chandler et al.
phenomenon is observed in neonates. Furthermore, the spuriously high value is accentuated by the higher NBT values in the neonate compared to that in adults, even with uniform heparin concentration of 1 unit/ml NBTblood solution ( < 5% in adult 4 and up to 33% in newborn infants). The precise reason for the higher NBT values in the newborn infant is not known. However, Park et al r have shown that neutrophils from normal neonates have slightly increased oxygen consumption and carbon dioxide production, indicating increased hexose monophosphate shunt activity in these cells as compared to that in normal adult neutrophils. This could explain our mean normal NBT value of 13.5%, compared to normal adult values of less than 5%. The reason for the elevated NBT values with high heparin concentration is not increased neutrophilic hexose monophosphate shunt activity. ~,~ Furthermore, electron microscopy of neutrophils in the presence of heparin has not demonstrated any discernible membrane damage6; these investigators also postulated that increased heparin concentration may cause an increase in cell membrane permeability, thereby leading to greater dye reduction and higher NBT values.
The Journal o f Pediatrics April 1978
In summary, we have defined the normal NBT in the neonates by using a fixed dose of heparin preparation of NBT solution. Its potential usage cfinical diagnosis of bacterial sepsis remains explored.
values in the in the to be
REFERENCES
1. Park BH, Fikrig SM, and Smithwick GM: Infection and nitroblue tetrazolium reduction by neutrophils, Lancet 2:532, 1968. 2. Humbert JR, Jurtz M, and Hathaway WE: Increased reduction of nitroblue tetrazolium by neutrophils of newborn infants, Pediatrics 45:125, 1970. 3. Barker BE, and Farnes P: Nitroblue tetrazolium (NBT) test--A micromethod, Clin Res 20:524, 1972. 4. Farnes P, Barker BE, and Forman EN: The nitrobluetetrazolium (NBT) test in clinical medicine-some current views, RIMed J 56:109, 1973. 5. Vieira J, Herd PA, and Barker BE: Effectof heparin on nitroblue tetrazolium (NBT Test) and carbohydrate metabolism in human neutrophils, Clin Res 21:977, 1973. 6. Barker BE: Personal communication. 7. Park BH, Holmes B, and Good RA: Metabolic activities in leukocytes of newborn infants, J PEDIATa76:237, 1970. 8. Hohn DC, and Lehmer RI: Mechanism of the heparin effect on the nitroblue tetrazolium slide test, Infect Immun 10:772, 1974.
638
TheJournalofPEDIATRICS
Nitroblue tetrazolium test in neonates
Barry D. Chandler, M.D., Neena Kapoor, M.D., Barbara E. Barker, Ph.D., Robert J. Boyle, M.D., and William Oh, M.D.,* Providence, R. L
THE NITROBLUE TETRAZOLIUM test was introduced by Parks et al 1 in 1968 as an aid in the diagnosis of acute bacterial infection. Its use is based on the observation that neutrophil reduction of NBT to an intracellular formazan precipitate is increased under conditions of bacterial infection. The test has had limited use in the diagnosis of neonatal sepsis because of spuriously high values found in normal infants. ~ Barker and Farnes 3 observed that falsely high and widely varied NBT values were obtained in adults if high concentrations of heparin were used. Using a micromethod, they further showed that limiting heparin concentration to 1 unit/ml gave normal NBT values of less than 5% in adults and older children. ~ We hypothesized that if the heparin concentration in the NBT solution were similarly controlled, normal values may be defined in newborn subjects, making the test available for future clinical testing in such situations as neonatal sepsis.
MATERIALS AND M E T H O D S lnitiaUy, 24 clinically well normal term newborn infants were NBT tested (103 determinations) at the time of screening for phenylketonuria (age two to four days). Heel puncture blood (44.7h) was collected in a nonheparinized capillary tube and immediately added to equal volumes of 0.2% NBT solution in a 0.1M phosphate buffer (pH = 7.0) containing varying concentrations of heparin (1 to 70 units/ml NBT-blood solution). The mixtures were From the Department of Pediatrics, Women and Infants Hospital of Rhode Island, The Department of Pathology, Rhode Island Hospital, and Brown University, Section of Reproductive and Developmental Medicine. *Reprint address: Department of Pediatrics, Women and Infants Hoapital of Rhode Island, 50 Maude St., Providence, RI 02908.
Vol. 92, No. 4, pp. 638-640
incubated in a 37~ water bath for 25 minutes. Smears were made from samples of each solution, and were Wright stained in the usual manner, Duplicate slides were made on each infant; 100 neutrophils were counted on each slide and an average of NBT-positive cells was obtained from combining both cell counts. Endotoxinstimulated specimens (Salmonella abortus equi, 50 m g / m l NBT-blood solution) were collected, and these confirmed that each infant had neutrophil opsonization capability. I
Abbreviation used. NBT: nitroblue tetrazolium
In the second part of the study, 58 infants of varying gestational ages (range 25 to 43 weeks) and birth weights (range 755 to 5,240 gin) admitted to the Women & Infants Hospital Special Care Nursery were NBT and NBTendotoxin tested from heel puncture blood obtained between one and five days of age. These infants were admitted to the Special Care Nursery for various clinical conditions other than sepsis and respiratory distress, and were clinically stable when the NBT tests were performed. The methods for the NBT test was similar to that used in the first study, using one unit of heparin per milliliter of NBT-blood solution. RESULTS Fig. 1 presents the results from the initial study of normal newborn infants and shows that the percentage of NBT positivity was variable but that at 1 to 25 units of heparin concentration in the NBT-blood solution, the values were cgnsistently within the range of 0 to 35%. At a higher heparin concentratioh, spuriously high values were observed (M +_ SD of 34 _+ 12.6, 40 __+ 17.6, and 33 + 18.7% at 40, 50, and 70% heparin Concentrations, respectively). Fig. 2 illustrates the NBT values obtained from the group of 58 infants studied with 1 unit/ml of
0022-3476/78/0492-0638500.30/0 9 1978 The C. V. Mosby Co.
Volume 92 Number 4
N B T test in n e o n a t e s
63 9
NBT (% POSITIVITY) 70
60-
(3)
(Io)
-MSSD 50-
( )=n
(7)
t
40; 30.
(16)
123)
20
t
10
I
0
o
I
I0
I 30
20
I 40
I 50
1 60
I 70
Units of Heporin/ml NBT-Blood Solution Fig. 1. Relationship between concentration of heparin in the NBT-blood solution and percent nitroblue tetrazolium positivity (nonendotoxin stimulated).
NBT
% POSITIVITY)
.30
0 0
20
0
(3O
0 0
0 0
0 0
0
0 O0 (3O
0 0 CO
0 0 0
I0
0
(3O
0 0 0 0 0
0 0 0 0
0
0
0
0
I 26
I 28
I 30
,, 32
o
GESTATION
,, 34
0
1 36
I.
38
0 CO CO 0 O(2O O0 0 OO 0 0 0 OOaO 0
np,
40-43
(Weeks)
Fig. 2. Nitroblue tetrazolium positivity of neutrophils in infants (gestational age 25 to 40 weeks) with no evidence of sepsis or respiratory distress (with 1 unit heparin/ml NBT-blood solution).
NBT-blood solution. There was no difference between NBT values obtained from infants of varying gestati0nal
COMMENT The use of high concentrations of heparin in the NBT
ages or birth weights (graph for birth weight not shown), and the values ranged from 1 to 33%.
test has been shown to cause increased dye reduction in adult neutrophils2 Our data indicate that a similar
640
Chandler et al.
phenomenon is observed in neonates. Furthermore, the spuriously high value is accentuated by the higher NBT values in the neonate compared to that in adults, even with uniform heparin concentration of 1 unit/ml NBTblood solution ( < 5% in adult 4 and up to 33% in newborn infants). The precise reason for the higher NBT values in the newborn infant is not known. However, Park et al r have shown that neutrophils from normal neonates have slightly increased oxygen consumption and carbon dioxide production, indicating increased hexose monophosphate shunt activity in these cells as compared to that in normal adult neutrophils. This could explain our mean normal NBT value of 13.5%, compared to normal adult values of less than 5%. The reason for the elevated NBT values with high heparin concentration is not increased neutrophilic hexose monophosphate shunt activity. ~,~ Furthermore, electron microscopy of neutrophils in the presence of heparin has not demonstrated any discernible membrane damage6; these investigators also postulated that increased heparin concentration may cause an increase in cell membrane permeability, thereby leading to greater dye reduction and higher NBT values.
The Journal o f Pediatrics April 1978
In summary, we have defined the normal NBT in the neonates by using a fixed dose of heparin preparation of NBT solution. Its potential usage cfinical diagnosis of bacterial sepsis remains explored.
values in the in the to be
REFERENCES
1. Park BH, Fikrig SM, and Smithwick GM: Infection and nitroblue tetrazolium reduction by neutrophils, Lancet 2:532, 1968. 2. Humbert JR, Jurtz M, and Hathaway WE: Increased reduction of nitroblue tetrazolium by neutrophils of newborn infants, Pediatrics 45:125, 1970. 3. Barker BE, and Farnes P: Nitroblue tetrazolium (NBT) test--A micromethod, Clin Res 20:524, 1972. 4. Farnes P, Barker BE, and Forman EN: The nitrobluetetrazolium (NBT) test in clinical medicine-some current views, RIMed J 56:109, 1973. 5. Vieira J, Herd PA, and Barker BE: Effectof heparin on nitroblue tetrazolium (NBT Test) and carbohydrate metabolism in human neutrophils, Clin Res 21:977, 1973. 6. Barker BE: Personal communication. 7. Park BH, Holmes B, and Good RA: Metabolic activities in leukocytes of newborn infants, J PEDIATa76:237, 1970. 8. Hohn DC, and Lehmer RI: Mechanism of the heparin effect on the nitroblue tetrazolium slide test, Infect Immun 10:772, 1974.
