Clin. exp. Immunol. (1975) 19, 309-318.
CELL DAMAGE AND DYE REDUCTION IN THE QUANTITATIVE NITROBLUE TETRAZOLIUM (NBT) TEST A. W. SEGAL AND A. J. LEVI
Northwick Park Hospital and Clinical Research Centre, Harrow, Middlesex (Received 24 July 1974)
SUMMARY
Nitroblue tetrazolium (NBT) is toxic to neutrophils; an effect which is greatly enhanced by endotoxin and latex particles. Cell damage, measured by the release of the cytoplasmic marker enzyme lactate dehydrogenase (LDH), was closely related to dye reduction. This suggests that, in this test, dye reduction occurs largely as a result of contact between intracellular reducing compounds and NBT following damage to the outer cell membrane. The expression of dye reduction as a function of LDH release should enhance the sensitivity of the quantitative NBT test by correcting for the observed intersubject variation in cell damage. The relationship between cell damage and dye reduction is a measure of the reducing capacity of the cell. This was normal in immature, bone marrow neutrophils, but diminished in neutrophils of patients with chronic granulomatous disease. INTRODUCTION Baehner & Nathan (1967) first observed that the redox dye nitroblue tetrazolium (NBT) was reduced by isolated neutrophils, that reduction was enhanced by phagocytosis and that neutrophils from patients with chronic granulomatous disease of childhood (CGDC) reduced the dye at an abnormally slow rate in the presence or absence of a phagocytic stimulus. The quantitative NBT test, in which an isolated leucocyte preparation is incubated together with NBT under standard conditions, and reduction of the dye quantitated spectrophotometrically after extraction into pyridine, is very useful in the diagnosis of CGDC (Baehner & Nathan, 1968). The metabolic basis of NBT reduction and the mechanism and site of contact between the reducing systems and NBT are unknown. This study was performed to investigate the effects of NBT, endotoxin and latex particles, separately and in various combinations, upon the cellular integrity of leucocytes and to examine the relationship between cell Correspondence: Dr A. W. Segal, Northwick Park Hospital, Harrow, Middlesex.
309
310
A. W. Segal and A. J. Levi
damage and dye reduction. The experimental system was similar to that employed in the quantitative NBT test, with the exception that KCN was excluded from the reaction mixture. Cell damage was determined by the release of lactate dehydrogenase (LDH), a cytoplasmic marker enzyme (Davies et al., 1973).
MATERIALS AND METHODS Separation of leucocytes Venous blood (15-100 ml) from eight normal subjects, taken into heparin (4 i.u./ml) was immediately mixed with an equal volume of cold (40C) separating solution, containing dextran in a balanced salt solution (Blackburn, Andrews & Watts, 1973) in a plastic measuring cylinder (Fig. 1). After sedimentation at 40C for 1 hr the erythrocyte-poor supernate was aspirated and centrifuged at 400 g at 40C for 10 min in a Sorval centrifuge. The supernate was discarded and erythrocytes haemolysed by the addition of 7 5 ml of cold (40C) sterile water to the cell pellet, which was resuspended by gentle pipetting up and down ten times. After 60 sec 2-5 ml of cold (40C) saline (0 54 M) was added to and mixed with the cell suspension to restore isotonicity. Twenty millilitres of cold (40C) Hanks's balanced salt solution (BSS, Oxoid) was added to and gently mixed with the cell suspension which was then centrifuged as above.
Incubation of cells with NBT and other materials The supernate was discarded and the cell pellet resuspended in BSS (2-10 ml depending upon the final volume required) by gentle pipetting up and down. A small sample of the cell suspension was removed to determine the neutrophil concentration (which ranged from 2 to 16 x 106/cmm). To each of two [ 0-ml samples of the cell suspension in disposable plastic test tubes was added 50 pil of either saline, endotoxin (300 pg/ml, E. coli 0127: B8, Difco) saline or dialysed latex particles (0-81 pm, Difco). The tubes were capped, mixed by inversion and incubated at 37°C for 10 min. A further 50 pl of the same additive was added to each tube. To one series of tubes was added 0 9 ml of phosphate (0 09 M) buffered saline (0 15 M) and to the other 0-9 ml of 0-1% NBT in phosphate-buffered saline. The suspensions were mixed by gentle shaking, incubated at 37°C for 30 min and centrifuged at 1000 g at 4°C for 20 min. One millilitre of the supernate from each tube was added to 10 ml of an aqueous solution containing bovine serum albumin (0 I1%, to stabilize the LDH) and Triton X-100 (0-1 Y, a detergent). This solution is henceforth referred to as the 'supernate fraction'.
Cell lysis One millilitre of the aqueous solution of Triton X-100 and albumin was added to the remaining supernate and cell pellet, mixed by vigorous manual shaking and centrifuged at 2000 g at 4°C for 20 min. 0-2 ml of the supernate, henceforth referred to as the 'subnate lysate' was transferred to fresh plastic tubes and stored at 4°C. Extraction offormazan The tubes to which NBT had been added, containing the remainder of the subnate lysate and cell debris or cell debris and formazan, were washed three times with 2-0 ml of 1 N HCl, the washings from each tube were pooled in 15 ml glass centrifuge tubes and centrifuged at 2000 g at room temperature for 20 min. The supernate was discarded, 9 0 ml of pyridine
Cell damage and dye reduction in NBT test
311
7]hr 40C
BSS + Dextran
0
DextranL400g.x
in
Haemolytic shock
Neutrophil Cel l
count
min
suspension additions
Cells in BSS
37'Cx 10,30 min Then IOOOgx2O V1
U+H
+N BT
AlbuminTriton X-100 solution
LDH measurement Wash/ three time
> LC
With
\DIVSO
ml ~~~~~I
x12000min~ /Soniate
OD515nm 20Og 0
X
It~i
FIG. 1. Diagramatic representation of the methods used for cell separation, incubation of leucocytes with various additives, in the presence and absence of NBT, and the measurement of LDH release from and NBT reduction by these cells.
was added to each tube which was then sonicated in a MSE 150 W ultrasonic disintegrator at an interpeak amplitude of 10 gm for 15 sec, with the 3 0 mm titanium probe tip positioned immediately above the cell button; incubated at 370C for 1 hr and then centrifuged at 2000 g at room temperature for 10 min. Residual formazan was extracted from the plastic tubes by the addition of 1 0 ml of dimethyl sulphoxide (DMSO) which was incubated in the tube at 37°C for 1 hr and then added to and mixed with the corresponding extract of formazan in pyridine. The optical density was read at 515 nm on a Gifford spectrophotometer against a blank containing a mixture of 9 0 ml of pyridine and 1 0 ml of DMSO. H
A. W. Segal and A. J. Levi
312
Measurement of LDH The lactate dehydrogenase (LDH) was assayed in the supernate fraction and subnate lysate by determining the rate of oxidation of NADH at 340 nm following exactly the instructions issued with Biochemica Test Combination LDH UV-test number 15948 TLAC. Assays were always completed within a few hours of obtaining the samples. The release of LDH into the supernate fraction was expressed as a percentage of the total LDH concentration assayed in the subnate lysate. Duplicate studies were performed by the same methods on neutrophils from two patients with CGDC. Studies were also performed on neutrophils from the bone marrow of two normal subjects. Two millilitres of sternal marrow aspirate was mixed with 10 0 ml of autologous plasma containing heparin (4 i.u./ml) as anticoagulant. Differential cell counts were performed on the isolated marrow cells. Random samples of suspensions of normal neutrophils were examined by light microscopy after incubation with NBT.
RESULTS The e.ftct of endotoxin and latex on the release of LDH from leucocytes, in the presence and absence of NBT LDH release from cells was increased when NBT was present in the incubation mixture. Latex and endotoxin markedly enhanced LDH release in the presence but not in the absence of NBT (Tables 1 and 2). TABLE 1. The effect of endotoxin and latex, in the presence and absence of NBT, on the release of LDH from cells
Additive to cell suspension Saline Endotoxin Latex NBT
NBT+endotoxin NBT+Latex
Percentage of LDH activity in supernate compared with control (saline only)* 100
103-2 (8 2) 86-3 (5*7) 1322 (11-2) 222-8 (25-7) 268-9 (40 6)
Number of studies 8 3 3 8 8 8
* LDH release (as a percentage of the total LDH released after cell lysis) is expressed as a percentage of LDH release from control cells (exposed to saline only). Mean values (s.e.) are shown.
The relationship between NBT reduction by and LDH release from neutrophils There was a close correlation between NBT reduction and LDH release by cells exposed to NBT alone or in the presence of endotoxin or latex particles (Fig. 2). Three such comparisons between LDH release and NBT reduction in six normal subjects resulted in a correlation coefficient of 0-89 which is highly statistically significant (P
CELL DAMAGE AND DYE REDUCTION IN THE QUANTITATIVE NITROBLUE TETRAZOLIUM (NBT) TEST A. W. SEGAL AND A. J. LEVI
Northwick Park Hospital and Clinical Research Centre, Harrow, Middlesex (Received 24 July 1974)
SUMMARY
Nitroblue tetrazolium (NBT) is toxic to neutrophils; an effect which is greatly enhanced by endotoxin and latex particles. Cell damage, measured by the release of the cytoplasmic marker enzyme lactate dehydrogenase (LDH), was closely related to dye reduction. This suggests that, in this test, dye reduction occurs largely as a result of contact between intracellular reducing compounds and NBT following damage to the outer cell membrane. The expression of dye reduction as a function of LDH release should enhance the sensitivity of the quantitative NBT test by correcting for the observed intersubject variation in cell damage. The relationship between cell damage and dye reduction is a measure of the reducing capacity of the cell. This was normal in immature, bone marrow neutrophils, but diminished in neutrophils of patients with chronic granulomatous disease. INTRODUCTION Baehner & Nathan (1967) first observed that the redox dye nitroblue tetrazolium (NBT) was reduced by isolated neutrophils, that reduction was enhanced by phagocytosis and that neutrophils from patients with chronic granulomatous disease of childhood (CGDC) reduced the dye at an abnormally slow rate in the presence or absence of a phagocytic stimulus. The quantitative NBT test, in which an isolated leucocyte preparation is incubated together with NBT under standard conditions, and reduction of the dye quantitated spectrophotometrically after extraction into pyridine, is very useful in the diagnosis of CGDC (Baehner & Nathan, 1968). The metabolic basis of NBT reduction and the mechanism and site of contact between the reducing systems and NBT are unknown. This study was performed to investigate the effects of NBT, endotoxin and latex particles, separately and in various combinations, upon the cellular integrity of leucocytes and to examine the relationship between cell Correspondence: Dr A. W. Segal, Northwick Park Hospital, Harrow, Middlesex.
309
310
A. W. Segal and A. J. Levi
damage and dye reduction. The experimental system was similar to that employed in the quantitative NBT test, with the exception that KCN was excluded from the reaction mixture. Cell damage was determined by the release of lactate dehydrogenase (LDH), a cytoplasmic marker enzyme (Davies et al., 1973).
MATERIALS AND METHODS Separation of leucocytes Venous blood (15-100 ml) from eight normal subjects, taken into heparin (4 i.u./ml) was immediately mixed with an equal volume of cold (40C) separating solution, containing dextran in a balanced salt solution (Blackburn, Andrews & Watts, 1973) in a plastic measuring cylinder (Fig. 1). After sedimentation at 40C for 1 hr the erythrocyte-poor supernate was aspirated and centrifuged at 400 g at 40C for 10 min in a Sorval centrifuge. The supernate was discarded and erythrocytes haemolysed by the addition of 7 5 ml of cold (40C) sterile water to the cell pellet, which was resuspended by gentle pipetting up and down ten times. After 60 sec 2-5 ml of cold (40C) saline (0 54 M) was added to and mixed with the cell suspension to restore isotonicity. Twenty millilitres of cold (40C) Hanks's balanced salt solution (BSS, Oxoid) was added to and gently mixed with the cell suspension which was then centrifuged as above.
Incubation of cells with NBT and other materials The supernate was discarded and the cell pellet resuspended in BSS (2-10 ml depending upon the final volume required) by gentle pipetting up and down. A small sample of the cell suspension was removed to determine the neutrophil concentration (which ranged from 2 to 16 x 106/cmm). To each of two [ 0-ml samples of the cell suspension in disposable plastic test tubes was added 50 pil of either saline, endotoxin (300 pg/ml, E. coli 0127: B8, Difco) saline or dialysed latex particles (0-81 pm, Difco). The tubes were capped, mixed by inversion and incubated at 37°C for 10 min. A further 50 pl of the same additive was added to each tube. To one series of tubes was added 0 9 ml of phosphate (0 09 M) buffered saline (0 15 M) and to the other 0-9 ml of 0-1% NBT in phosphate-buffered saline. The suspensions were mixed by gentle shaking, incubated at 37°C for 30 min and centrifuged at 1000 g at 4°C for 20 min. One millilitre of the supernate from each tube was added to 10 ml of an aqueous solution containing bovine serum albumin (0 I1%, to stabilize the LDH) and Triton X-100 (0-1 Y, a detergent). This solution is henceforth referred to as the 'supernate fraction'.
Cell lysis One millilitre of the aqueous solution of Triton X-100 and albumin was added to the remaining supernate and cell pellet, mixed by vigorous manual shaking and centrifuged at 2000 g at 4°C for 20 min. 0-2 ml of the supernate, henceforth referred to as the 'subnate lysate' was transferred to fresh plastic tubes and stored at 4°C. Extraction offormazan The tubes to which NBT had been added, containing the remainder of the subnate lysate and cell debris or cell debris and formazan, were washed three times with 2-0 ml of 1 N HCl, the washings from each tube were pooled in 15 ml glass centrifuge tubes and centrifuged at 2000 g at room temperature for 20 min. The supernate was discarded, 9 0 ml of pyridine
Cell damage and dye reduction in NBT test
311
7]hr 40C
BSS + Dextran
0
DextranL400g.x
in
Haemolytic shock
Neutrophil Cel l
count
min
suspension additions
Cells in BSS
37'Cx 10,30 min Then IOOOgx2O V1
U+H
+N BT
AlbuminTriton X-100 solution
LDH measurement Wash/ three time
> LC
With
\DIVSO
ml ~~~~~I
x12000min~ /Soniate
OD515nm 20Og 0
X
It~i
FIG. 1. Diagramatic representation of the methods used for cell separation, incubation of leucocytes with various additives, in the presence and absence of NBT, and the measurement of LDH release from and NBT reduction by these cells.
was added to each tube which was then sonicated in a MSE 150 W ultrasonic disintegrator at an interpeak amplitude of 10 gm for 15 sec, with the 3 0 mm titanium probe tip positioned immediately above the cell button; incubated at 370C for 1 hr and then centrifuged at 2000 g at room temperature for 10 min. Residual formazan was extracted from the plastic tubes by the addition of 1 0 ml of dimethyl sulphoxide (DMSO) which was incubated in the tube at 37°C for 1 hr and then added to and mixed with the corresponding extract of formazan in pyridine. The optical density was read at 515 nm on a Gifford spectrophotometer against a blank containing a mixture of 9 0 ml of pyridine and 1 0 ml of DMSO. H
A. W. Segal and A. J. Levi
312
Measurement of LDH The lactate dehydrogenase (LDH) was assayed in the supernate fraction and subnate lysate by determining the rate of oxidation of NADH at 340 nm following exactly the instructions issued with Biochemica Test Combination LDH UV-test number 15948 TLAC. Assays were always completed within a few hours of obtaining the samples. The release of LDH into the supernate fraction was expressed as a percentage of the total LDH concentration assayed in the subnate lysate. Duplicate studies were performed by the same methods on neutrophils from two patients with CGDC. Studies were also performed on neutrophils from the bone marrow of two normal subjects. Two millilitres of sternal marrow aspirate was mixed with 10 0 ml of autologous plasma containing heparin (4 i.u./ml) as anticoagulant. Differential cell counts were performed on the isolated marrow cells. Random samples of suspensions of normal neutrophils were examined by light microscopy after incubation with NBT.
RESULTS The e.ftct of endotoxin and latex on the release of LDH from leucocytes, in the presence and absence of NBT LDH release from cells was increased when NBT was present in the incubation mixture. Latex and endotoxin markedly enhanced LDH release in the presence but not in the absence of NBT (Tables 1 and 2). TABLE 1. The effect of endotoxin and latex, in the presence and absence of NBT, on the release of LDH from cells
Additive to cell suspension Saline Endotoxin Latex NBT
NBT+endotoxin NBT+Latex
Percentage of LDH activity in supernate compared with control (saline only)* 100
103-2 (8 2) 86-3 (5*7) 1322 (11-2) 222-8 (25-7) 268-9 (40 6)
Number of studies 8 3 3 8 8 8
* LDH release (as a percentage of the total LDH released after cell lysis) is expressed as a percentage of LDH release from control cells (exposed to saline only). Mean values (s.e.) are shown.
The relationship between NBT reduction by and LDH release from neutrophils There was a close correlation between NBT reduction and LDH release by cells exposed to NBT alone or in the presence of endotoxin or latex particles (Fig. 2). Three such comparisons between LDH release and NBT reduction in six normal subjects resulted in a correlation coefficient of 0-89 which is highly statistically significant (P
