HEMATOPATHOLOGY Original Article
A Rapid, Specific Assay for Superoxide Release from Phagocytes in Small Volumes of Whole Blood MICHAEL F. TOSI, M.D., AND AKBAR HAMEDANI, M.S.
performed with both soluble and particulate stimuli, and is sensitive enough to detect characteristic responses with as little as a single drop of blood. Patients with chronic granulomatous disease were readily identified using this assay. The ability to use very small blood volumes makes the assay suitable for use in prenatal diagnosis. The main disadvantage of this assay compared to the often-employed slide nitroblue tetrazolium test, with respect to testing for chronic granulomatous disease, is that carriers of x-linked chronic granulomatous disease are not readily detected with this chemiluminescence assay, as they are with the nitroblue tetrazolium test. (Key words: Chronic granulomatous disease; Lucigenin chemiluminescence; Superoxide) Am J Clin Pathol 1992; 97:566-573
A number of assays have been developed to measure the oxidative metabolic burst of phagocytic cells in response to a variety of stimuli. 1-4 Several of these have been used to evaluate the defect in NADPH-oxidase activity that characterizes the phagocytes of patients with chronic granulomatous disease of childhood (CGD).5"10 NADPHoxidase is a membrane-associated enzyme complex that requires the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) for the conversion of molecular oxygen to superoxide anion. 8 Most of these assays measure either the production of superoxide by these cells or products of reactions downstream, which, in turn, depend on superoxide production. 2 " For the most quantitative of such measurements, isolation of purified cell suspensions of neutrophils or monocytes usually is required so specific activity per cell can be determined. However, isolation of purified cell suspensions is time consuming, and can be expensive. In screening for CGD,
an ideal assay would be rapid, sensitive, and specific and at least semiquantitative. It would be of added practical value if very small volumes of blood could be used, so that along with the appropriate obstetrical expertise, it could be applied in prenatal diagnosis of this disorder.12 We developed an assay, based on the superoxide-specific chemiluminescence (CL) of lucigenin,13 that measures superoxide release by phagocytic cells in whole-blood samples. This assay does not require isolation of leukocytes from blood samples, can be performed using both soluble and particulate stimuli, and requires as little as a single drop of whole blood. MATERIALS AND METHODS
Subjects Forty healthy individuals ranging in age from 6 months to 39 years served as controls. Three children with CGD, ages 6 months, 11 months, and 2.5 years, were studied. One additional child, age 13 months, with cyclic neutropenia was included for comparison. From the Department of Pediatrics. Case Western Reserve University School of Medicine. Cleveland. Ohio. Preparation of Reagents Received May 29, 1991; received revised manuscript and accepted for Opsonized zymosan particles were prepared in batches publication August 19, 1991. by first boiling 250 mg zymosan particles (Sigma Chemical Address reprint requests to Dr. Tosi: Department of Pediatrics, Case Western Reserve University. 2101 Adelbert Road, Cleveland, Ohio 44106. Co., St. Louis, MO) in distilled water for 30 minutes, 566
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
A rapid, specific assay was developed to measure the release of superoxide anion from stimulated phagocytic cells in small volumes of whole blood. The assay is based on the chemiluminescence that results when lucigenin (bis-N-methyl acridinium nitrate) is reduced by superoxide. Heparinized whole blood, at volumes from 0.2 mL to a single drop from a 20-gauge needle, was mixed with lucigenin and either a soluble or particulate stimulus (phorbol myristate acetate or opsonized zymosan particles, respectively) in a standard volume of 1 mL. The chemiluminescence was measured at 3-minute intervals for a 30-minute period in a luminometer capable of automated operation. Characteristic plots of chemiluminescence versus time were obtained. This assay is rapid and simple, obviates the need to isolate leukocytes from whole blood, is specific for superoxide, can be
TOSI AND HAMEDANI Phagocyte Superoxide Assay with Small Volumes of Blood
Isolation of Peripheral Blood Leukocytes
Polymorphonuclear
Whole blood from healthy adult volunteers was mixed with dextran at 6 g/L (MW = 400,000, Sigma Chemical Co.), and red blood cells were allowed to sediment for 30 to 40 minutes. The resulting leukocyte-rich plasma was removed and centrifuged for 10 minutes at 600g. Cells from the resulting pellet were resuspended in PBS, layered over a Ficoll-Hypaque cushion (5.4% Ficoll, Sigma Chemical; 9% sodium Hypaque, Winthrop-Laboratories, New York, NY), and centrifuged for 20 minutes at 400g. The cell pellet was recovered, residual red cells were eliminated by hypotonic lysis, and the remaining neutrophils (PMN) were resuspended in cold PBS at 107 cells/mL. The purity and viability of these PMN suspensions were uniformly more than 95%.16 Measurement of PMN Superoxide Production by Cytochrome C Reduction A modification of a previously described method2 was used. Neutrophils (2.5 X 106) were preincubated for 5 minutes at 37 °C in the presence of SOD at concentrations ranging from 0 to 100 jtg/mL. Horse heart ferricytochrome c (0.08 mmol/L, Sigma Chemical Co.) and a stimulus, either 1 fig PMA or 1 mg opsonized zymosan, were added in a final volume of 1.0 mL in PBS and the mixtures were tumbled gently at 37 °C for 10 minutes.
Resting control cells were incubated with buffer in place of stimuli. The reactions were terminated by placing the incubation tubes in an ice-water bath. Cells were pelleted by centrifugation and supernatants were analyzed by spectrophotometry at 550 nm. Superoxide release, measured by cytochrome C reduced, was expressed in nanomoles 10 minutes/10 6 PMN. Chemiluminescence
Assay
Varying volumes of heparinized whole blood, ranging from 0.2 mL to a single drop from a 20-gauge needle (approximately 12 fih), were added in duplicate to cuvettes in a final volume of 1 mL PBS that contained 10~5 mol/ L lucigenin and a stimulus, either 1 mg of opsonized zymosan particles prepared as described above or 1 ng PMA. Resting controls were performed in the absence of stimuli. In experiments to confirm that lucigenin CL was similar to cytochrome reduction in its specificity for superoxide, 106 isolated PMN were used instead of whole blood, with 1 ng PMA as the stimulus. Mixtures were incubated at 37 °C in an LKB 1251 automated luminometer that was equipped for continuous stirring during measurements (LKB-Wallac, Turku, Finland). This instrument is capable of processing up to 25 samples in a single run. Using an IBM PC-compatible phagocytosis program (LKB-Wallac) with an AT&T PC 6300 computer (AT & T, Dayton, OH), the instrument was set to measure the intensity of light emission from each sample at 3minute intervals during an entire assay period of 30 minutes (45 minutes for the assays with isolated PMN). At the end of the assay the same program was used to obtain a printed plot of CL (output in mV from the sensing photomultiplier tube) versus time, as well as the peak CL value for each sample during the assay. As many as five samples could be plotted on a single graph. Plots were scaled automatically to accommodate the highest value among any samples being plotted. For direct comparisons of the results of different individuals or different assay conditions, scales were set manually to accommodate the highest value in any group of compared samples within the upper 20% of the scale. In assays to confirm the specificity of the whole-blood lucigenin CL assay for superoxide, paired samples were assayed in the presence or absence of 100 Mg/mL SOD. RESULTS Specificity of Lucigenin Chemiluminescence for Superoxide: Comparison with Cytochrome Reduction in Assays with Isolated PMN As shown in Figure 1, the addition of SOD at increasing concentrations resulted in progressively greater inhibition
Vol. 97 • No. 4
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
washing three times in Dulbecco's phosphate-buffered saline (PBS), pH 7.4 (GIBCO Laboratories, Grand Island, NY), then pelleting at 1,500^ for 10 minutes. Fresh human serum was prepared by allowing whole blood to clot in glass tubes for 30 minutes at 25 °C, separating the clots, and centrifuging the tubes at l,000g for 10 minutes. The serum was collected and immediately placed in a tube on ice. The pellet of 250 mg boiled, washed zymosan was resuspended in 25 mL of the fresh human serum and incubated at 37 °C with gentle agitation for 60 minutes. The opsonized zymosan was washed three times, resuspended in PBS at 10 mg/mL, and stored in aliquots at - 8 0 °C. 1415 Phorbol myristate acetate (PMA; Sigma Chemical Co.) was dissolved in a minimal volume of dimethyl sulfoxide, brought to a stock concentration of 100 j^g/mL in PBS, and stored at —80 °C. Lucigenin (bis-N-methyl acridinium nitrate; Sigma Chemical Co.) was dissolved in a minimal volume of dimethyl sulfoxide, brought to a stock concentration of 10 -4 mol/L in PBS, and stored at 4 °C. Recombinant human superoxide dismutase (SOD), a gift of Biotechnology General, New York, was dissolved in PBS at 2 mg/mL and stored in 100-/uL aliquots at - 2 0 °C.
567
HEMATOPATHOLOGY Original Article
568
of both cytochrome reduction and lucigenin CL by isolated peripheral blood PMN. In this representative set of experiments, SOD at 100 Mg/mL inhibited both reactions by more than 90%. On inspection, the inhibition curves suggest that lucigenin CL may be slightly more sensitive than cytochrome reduction to SOD, but this could be explained by the fact that there are 2.5 times as many PMN per milliliter in the cytochrome reduction assay. Catalase or heat-inactivated SOD at 100 ^g/mL had no effect on the results for either assay (data not shown). These results confirmed the specificity of lucigenin CL for superoxide anion in this assay system.
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Figure 2 shows the CL curves during a 30-minute period for 8 of the 40 healthy individuals studied. The results shown are for assays performed with 0.2 mL of whole blood. In preliminary experiments it was determined that 0.2 mL of whole blood in a final volume of 1 mL PBS (20% vol/vol) was the concentration of blood that gave
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A Rapid, Specific Assay for Superoxide Release from Phagocytes in Small Volumes of Whole Blood MICHAEL F. TOSI, M.D., AND AKBAR HAMEDANI, M.S.
performed with both soluble and particulate stimuli, and is sensitive enough to detect characteristic responses with as little as a single drop of blood. Patients with chronic granulomatous disease were readily identified using this assay. The ability to use very small blood volumes makes the assay suitable for use in prenatal diagnosis. The main disadvantage of this assay compared to the often-employed slide nitroblue tetrazolium test, with respect to testing for chronic granulomatous disease, is that carriers of x-linked chronic granulomatous disease are not readily detected with this chemiluminescence assay, as they are with the nitroblue tetrazolium test. (Key words: Chronic granulomatous disease; Lucigenin chemiluminescence; Superoxide) Am J Clin Pathol 1992; 97:566-573
A number of assays have been developed to measure the oxidative metabolic burst of phagocytic cells in response to a variety of stimuli. 1-4 Several of these have been used to evaluate the defect in NADPH-oxidase activity that characterizes the phagocytes of patients with chronic granulomatous disease of childhood (CGD).5"10 NADPHoxidase is a membrane-associated enzyme complex that requires the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) for the conversion of molecular oxygen to superoxide anion. 8 Most of these assays measure either the production of superoxide by these cells or products of reactions downstream, which, in turn, depend on superoxide production. 2 " For the most quantitative of such measurements, isolation of purified cell suspensions of neutrophils or monocytes usually is required so specific activity per cell can be determined. However, isolation of purified cell suspensions is time consuming, and can be expensive. In screening for CGD,
an ideal assay would be rapid, sensitive, and specific and at least semiquantitative. It would be of added practical value if very small volumes of blood could be used, so that along with the appropriate obstetrical expertise, it could be applied in prenatal diagnosis of this disorder.12 We developed an assay, based on the superoxide-specific chemiluminescence (CL) of lucigenin,13 that measures superoxide release by phagocytic cells in whole-blood samples. This assay does not require isolation of leukocytes from blood samples, can be performed using both soluble and particulate stimuli, and requires as little as a single drop of whole blood. MATERIALS AND METHODS
Subjects Forty healthy individuals ranging in age from 6 months to 39 years served as controls. Three children with CGD, ages 6 months, 11 months, and 2.5 years, were studied. One additional child, age 13 months, with cyclic neutropenia was included for comparison. From the Department of Pediatrics. Case Western Reserve University School of Medicine. Cleveland. Ohio. Preparation of Reagents Received May 29, 1991; received revised manuscript and accepted for Opsonized zymosan particles were prepared in batches publication August 19, 1991. by first boiling 250 mg zymosan particles (Sigma Chemical Address reprint requests to Dr. Tosi: Department of Pediatrics, Case Western Reserve University. 2101 Adelbert Road, Cleveland, Ohio 44106. Co., St. Louis, MO) in distilled water for 30 minutes, 566
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
A rapid, specific assay was developed to measure the release of superoxide anion from stimulated phagocytic cells in small volumes of whole blood. The assay is based on the chemiluminescence that results when lucigenin (bis-N-methyl acridinium nitrate) is reduced by superoxide. Heparinized whole blood, at volumes from 0.2 mL to a single drop from a 20-gauge needle, was mixed with lucigenin and either a soluble or particulate stimulus (phorbol myristate acetate or opsonized zymosan particles, respectively) in a standard volume of 1 mL. The chemiluminescence was measured at 3-minute intervals for a 30-minute period in a luminometer capable of automated operation. Characteristic plots of chemiluminescence versus time were obtained. This assay is rapid and simple, obviates the need to isolate leukocytes from whole blood, is specific for superoxide, can be
TOSI AND HAMEDANI Phagocyte Superoxide Assay with Small Volumes of Blood
Isolation of Peripheral Blood Leukocytes
Polymorphonuclear
Whole blood from healthy adult volunteers was mixed with dextran at 6 g/L (MW = 400,000, Sigma Chemical Co.), and red blood cells were allowed to sediment for 30 to 40 minutes. The resulting leukocyte-rich plasma was removed and centrifuged for 10 minutes at 600g. Cells from the resulting pellet were resuspended in PBS, layered over a Ficoll-Hypaque cushion (5.4% Ficoll, Sigma Chemical; 9% sodium Hypaque, Winthrop-Laboratories, New York, NY), and centrifuged for 20 minutes at 400g. The cell pellet was recovered, residual red cells were eliminated by hypotonic lysis, and the remaining neutrophils (PMN) were resuspended in cold PBS at 107 cells/mL. The purity and viability of these PMN suspensions were uniformly more than 95%.16 Measurement of PMN Superoxide Production by Cytochrome C Reduction A modification of a previously described method2 was used. Neutrophils (2.5 X 106) were preincubated for 5 minutes at 37 °C in the presence of SOD at concentrations ranging from 0 to 100 jtg/mL. Horse heart ferricytochrome c (0.08 mmol/L, Sigma Chemical Co.) and a stimulus, either 1 fig PMA or 1 mg opsonized zymosan, were added in a final volume of 1.0 mL in PBS and the mixtures were tumbled gently at 37 °C for 10 minutes.
Resting control cells were incubated with buffer in place of stimuli. The reactions were terminated by placing the incubation tubes in an ice-water bath. Cells were pelleted by centrifugation and supernatants were analyzed by spectrophotometry at 550 nm. Superoxide release, measured by cytochrome C reduced, was expressed in nanomoles 10 minutes/10 6 PMN. Chemiluminescence
Assay
Varying volumes of heparinized whole blood, ranging from 0.2 mL to a single drop from a 20-gauge needle (approximately 12 fih), were added in duplicate to cuvettes in a final volume of 1 mL PBS that contained 10~5 mol/ L lucigenin and a stimulus, either 1 mg of opsonized zymosan particles prepared as described above or 1 ng PMA. Resting controls were performed in the absence of stimuli. In experiments to confirm that lucigenin CL was similar to cytochrome reduction in its specificity for superoxide, 106 isolated PMN were used instead of whole blood, with 1 ng PMA as the stimulus. Mixtures were incubated at 37 °C in an LKB 1251 automated luminometer that was equipped for continuous stirring during measurements (LKB-Wallac, Turku, Finland). This instrument is capable of processing up to 25 samples in a single run. Using an IBM PC-compatible phagocytosis program (LKB-Wallac) with an AT&T PC 6300 computer (AT & T, Dayton, OH), the instrument was set to measure the intensity of light emission from each sample at 3minute intervals during an entire assay period of 30 minutes (45 minutes for the assays with isolated PMN). At the end of the assay the same program was used to obtain a printed plot of CL (output in mV from the sensing photomultiplier tube) versus time, as well as the peak CL value for each sample during the assay. As many as five samples could be plotted on a single graph. Plots were scaled automatically to accommodate the highest value among any samples being plotted. For direct comparisons of the results of different individuals or different assay conditions, scales were set manually to accommodate the highest value in any group of compared samples within the upper 20% of the scale. In assays to confirm the specificity of the whole-blood lucigenin CL assay for superoxide, paired samples were assayed in the presence or absence of 100 Mg/mL SOD. RESULTS Specificity of Lucigenin Chemiluminescence for Superoxide: Comparison with Cytochrome Reduction in Assays with Isolated PMN As shown in Figure 1, the addition of SOD at increasing concentrations resulted in progressively greater inhibition
Vol. 97 • No. 4
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
washing three times in Dulbecco's phosphate-buffered saline (PBS), pH 7.4 (GIBCO Laboratories, Grand Island, NY), then pelleting at 1,500^ for 10 minutes. Fresh human serum was prepared by allowing whole blood to clot in glass tubes for 30 minutes at 25 °C, separating the clots, and centrifuging the tubes at l,000g for 10 minutes. The serum was collected and immediately placed in a tube on ice. The pellet of 250 mg boiled, washed zymosan was resuspended in 25 mL of the fresh human serum and incubated at 37 °C with gentle agitation for 60 minutes. The opsonized zymosan was washed three times, resuspended in PBS at 10 mg/mL, and stored in aliquots at - 8 0 °C. 1415 Phorbol myristate acetate (PMA; Sigma Chemical Co.) was dissolved in a minimal volume of dimethyl sulfoxide, brought to a stock concentration of 100 j^g/mL in PBS, and stored at —80 °C. Lucigenin (bis-N-methyl acridinium nitrate; Sigma Chemical Co.) was dissolved in a minimal volume of dimethyl sulfoxide, brought to a stock concentration of 10 -4 mol/L in PBS, and stored at 4 °C. Recombinant human superoxide dismutase (SOD), a gift of Biotechnology General, New York, was dissolved in PBS at 2 mg/mL and stored in 100-/uL aliquots at - 2 0 °C.
567
HEMATOPATHOLOGY Original Article
568
of both cytochrome reduction and lucigenin CL by isolated peripheral blood PMN. In this representative set of experiments, SOD at 100 Mg/mL inhibited both reactions by more than 90%. On inspection, the inhibition curves suggest that lucigenin CL may be slightly more sensitive than cytochrome reduction to SOD, but this could be explained by the fact that there are 2.5 times as many PMN per milliliter in the cytochrome reduction assay. Catalase or heat-inactivated SOD at 100 ^g/mL had no effect on the results for either assay (data not shown). These results confirmed the specificity of lucigenin CL for superoxide anion in this assay system.
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Figure 2 shows the CL curves during a 30-minute period for 8 of the 40 healthy individuals studied. The results shown are for assays performed with 0.2 mL of whole blood. In preliminary experiments it was determined that 0.2 mL of whole blood in a final volume of 1 mL PBS (20% vol/vol) was the concentration of blood that gave
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