An Improved Nitroblue Tetrazolium Test Using Phorbol Myristate Acetate-coated Coverslips JOHN E. REPINE, M.D., BRAD RASMUSSEN, B.S., AND JAMES G. WHITE, M.D.
FOLLOWING PHAGOCYTOSIS, normal polymorphonuclear leukocytes develop a burst of oxidative metabolism and rapidly reduce nitroblue tetrazolium (NBT) to blue formazan.1B Polymorphonuclear leukocytes from patients who have chronic granulomatous disease fail to generate augmented biochemical activity or reduce N B T . 2 5 - 9 1 2 Polymorphonuclear leukocytes from heterozygotic carriers of chronic granulomatous disease usually show intermediate metabolic rates and NBT reduction because they have a mixed population of normal and abnormal polymorphonuclear leukocytes.'"•i4.i« T n e biochemical differences have formed the basis for tests of polymorphonuclear leukocyte function Received March 15, 1978; received revised manuscript and accepted for publication May 1, 1978. Supported in part by the Minnesota Medical Foundation, the Minnesota and American Heart Associations, the Basil O'Connor Starter Research Fund of the National Foundation of the March of Dimes, and NIH Grant HL-17132-1. Dr Repine is an Established Investigator of the American Heart Association. Brad Rasmussen is a medical student who was supported by a grant from the medical oncology section of the Department of Medicine. Address reprint requests to Dr. Repine: Webb-Waring Lung Institute, 4200 E. Ninth Avenue, Denver, Colorado 80262.
University of Minnesota Health Sciences Center, Minneapolis, Minnesota
designed to detect chronic granulomatous disease in patients and carriers. 2,5-9,11,12,14,16 While detection of the severe biochemical deficiency of polymorphonuclear leukocytes from patients who have chronic granulomatous disease is easily accomplished, identification of partial abnormalities of polymorphonuclear leukocytes from carriers is more difficult.4,7,10,11 In test systems that measure the function of a total population of polymorphonuclear leukocytes, a heterozygous carrier with only a few abnormal polymorphonuclear leukocytes, is often undetectable because normal polymorphonuclear leukocytes mask the presence of deficient polymorphonuclear leukocytes. 4,11 Various tests have been designed to evaluate the function of individual polymorphonuclear leukocytes in order to circumvent this problem. One such assay is the endotoxin-NBT slide technic of Ochs and Igo. 8 In this system polymorphonuclear leukocytes that reduce NBT after adhering to endotoxin-coated slides can be assessed individually. However, this test may not be optimal for detecting carriers of chronic granulomatous disease who have only a few defective polymorphonuclear leukocytes, since normal individuals often have some (approximately 10%) of NBT-negative polymorphonuclear leukocytes. We have modified the endotoxin-NBT slide test by precoating coverslips with phorbol myristate acetate, instead of endotoxin. Phorbol myristate acetate is a potent selective stimulus of neutrophilic oxidative metabolism and NBT reduction. 3,11-14 A greater percentage of control polymorphonuclear leukocytes were NBT-positive when stimulated by phorbol myristate acetate than when observed on endotoxin-coated coverslips. 8,11 The highly reproducible response of control polymorphonuclear leukocytes to phorbol myristate acetate stimulation made it possible to detect a statistically significant abnormality in polymorphonuclear leukocytes from a proven carrier of chronic granulomatous disease who had previously defied recognition by all tests, including the endotoxin-NBT slide test. 10,11
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Repine, John E., Rasmussen, Brad, and White, James G.: An improved nitroblue tetrazolium test using phorbol myristate acetate-coated coverslips. Am J Clin Pathol 71:582-585,1979. The endotoxin-nitroblue tetrazolium (NBT) slide test was modified by precoating coverslips with phorbol myristate acetate instead of endotoxin. The percentage of control polymorphonuclear leukocytes reducing NBT was significantly (P < .05) greater on phorbol myristate acetate-coated coverslips (99 ± 0.21%) than on endotoxin-coated coverslips (96 ± 1.8%). Polymorphonuclear leukocytes from patients with chronic granulomatous disease did not reduce NBT on phorbol myristate acetate- or endotoxin-treated coverslips. NBT reduction by polymorphonuclear leukocytes from proven heterozygotic carriers of sex-linked chronic granulomatous disease was intermediate between NBT reductions by those from controls and patients. A statistically significant abnormality of NBT reduction was found in polymorphonuclear leukocytes from one carrier of chronic granulomatous disease with phorbol myristate acetate-treated, but not endotoxin-treated coverslips. The phorbol myristate acetate-NBT coverslip technic is a rapid, simple, reliable way to detect deficiencies in polymorphonuclear leukocytes from patients and carriers of chronic granulomatous disease. (Key words: Neutrophils; Nitroblue tetrazolium [NBT]); Phorbol myristate acetate; Chronic granulomatous disease [CGD].)
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FIG. I. Light micrograph of control polymorphonuclear leukocytes on a phorbol myristate acetate-coated coverslip. The control polymorphonuclear leukocytes become large blue NBT-positive cells. x800.
• • * * *
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FIG. 2. Light micrograph of polymorphonuclear leukocytes from an individual hemizygotic for chronic granulomatous disease on a phorbol myristate acetate-coated coverslip. The patient's polymorphonuclear leukocytes, which are small and red, maintain a wellpreserved nuclear and cytoplasmic appearance, are considered NBT-negative cells. x800. FIG. 3. Light micrograph polymorphonuclear leukocytes from a carrier of chronic granulomatous disease on a phorbol myristate acetatecoated coverslip. The carrier polymorphonuclear leukocytes are a mixture of NBTpositive and NBT-negative cells, x 1,200.
Materials and Methods Investigation was approved by the Human Volunteers Committee of the University of Minnesota. Blood was donated by three persons hemizygotic for chronic granulomatous disease,512 three obligate maternal carriers of chronic granulomatous disease,111416 and eight control subjects. The individuals were free of infection and not taking drugs at the time of the study. NBT reduction was measured on glass coverslips by use of the technic of Ochs and Igo.8 Briefly, 0.1 ml endotoxin (lipopolysaccharide B, Escherichia coli 0.26:B6)* or 0.1 fj.g phorbol myristate acetatet/ml was * Difco, Detroit, Michigan. t Midland Consolidated Corp., Midland, New York.
allowed to air-dry on coverslips. Phorbol myristate acetate and endotoxin solutions were prepared and stored as previously described. 8111314 Drops of whole blood obtained by finger-stick or venipuncture were then placed on uncoated, endotoxin-coated, and phorbol myristate acetate-coated coverslips, which were incubated at 37 C for 45 min in a moist chamber. The clot that formed on each coverslip was removed by washing, leaving granulocytes and monocytes adherent to the coverslip. Coverslips were then placed on a drop of NBT-serum solution on a glass slide and reincubated for 20 min. Subsequently, coverslips were removed, washed, fixed in methanol, counterstained in safranin, and air-dried. Four hundred polymorphonuclear leukocytes on coverslips were examined in
A.J.C.P. • May 1979
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Table 1. Nitroblue Tetrazolium (NBT) Reduction by Polymorphonuclear Leukocytes from Controls and Patients or Carriers of Chronic Granulomatous Disease (CGD) NBT Reduction (% Cells Reducing NBT) (Mean ± SE) Slide Treatment None
CGD Carriers (n = 3)
Control Subjects (n = 8)*
CGD Patients (n = 3)
59 ± 2.4 (17 determinations)
0(5)*
50 (3 determinations)
0(5)*
72 (3 determinations)*
0 (5)*
69 (3 determinations)*
.001 1
1
Phorbol myristate acetate-coated
99 ± 0.21 (14 determinations)
Endotoxin-coated
96 ± 1.8 (12 determinations)
1 .05 1
1
* Significantly different from control values. P < .01.
Results Morphology The morphologic features of polymorphonuclear leukocytes studied on phorbol myristate acetate-coated slides are shown in Figure 1. Nearly all cells from control subjects became large blue NBT-positive cells on phorbol myristate acetate-treated coverslips (Fig. 1), in striking contrast to polymorphonuclear leukocytes from patients with chronic granulomatous disease, which were small, red, and had a well-preserved nuclear and cytoplasmic appearance. NBT-positive cells were not found on coverslips from patients with chronic granulomatous disease (Fig. 2). Mixed populations of NBT-positive and NBT-negative cells were apparent on coverslips made from blood samples from heterozygotic carriers of chronic granulomatous disease (Fig. 3). NBT Reduction The percentage of polymorphonuclear leukocytes from control subjects that reduced NBT was significantly (P < .01) greater on phorbol myristate acetatecoated coverslips (99 + 0.21*) than on endotoxincoated (96+1.8$) or blank (59 ± 2.4$) coverslips t Mean ± SE.
(Table 1). Cells from patients hemizygotic for chronic granulomatous disease did not reduce NBT on blank, endotoxin-coated, or phorbol myristate acetate-treated coverslips. Specimens from obligate carriers of chronic granulomatous disease had NBT-positive and NBTnegative cells (Table 1). On slides coated with phorbol myristate acetate or endotoxin, the mean percentages of NBT-positive polymorphonuclear leukocytes from heterozygotic carriers were intermediate between the means for controls and patients who had chronic granulomatous disease (Table 1). An abnormality in NBT reduction by polymorphonuclear leukocytes from an obligate carrier of chronic granulomatous disease whose defect had not been detectable by other methods, including the endotoxinNBT slide test,1011 was demonstrated on slides coated with phorbol myristate acetate. When tested on three occasions by the use of blank or endotoxin-coated coverslips, the mean percentages of NBT-positive polymorphonuclear leukocytes from this carrier (60 and 92%, respectively) were within 2 SD of the mean for polymorphonuclear leukocytes from control subjects (59 ± 14$ or 96 ± 8,t respectively). Repeat testing on three occasions, using slides coated with phorbol myristate acetate, revealed that 92% of the polymorphonuclear leukocytes from this carrier reduced NBT. This mean value was outside of the mean of NBT-positive cells from control subjects (99 ± 0.9*). Thus, it was possible to consider this individual abnormal at a 95% level of confidence using the phorbol myristate acetatecoated, but not the endotoxin-coated, NBT slide test. Discussion In the present investigation the endotoxin-NBT slide test was modified by precoating coverslips with phorbol myristate acetate instead of endotoxin. Phorbol myristate acetate is a chemical that stimulates alterations in normal polymorphonuclear leukocytes that closely resemble changes developing in cells after phag-
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
random sequence with the light microscope, and the numbers of large, NBT-positive (blue) and small, NBTnegative (red) cells were quantitated. The cells were examined in a double-blind fashion by three independent observers. The results obtained by the three observers were averaged and used to calculate the percentage of polymorphonuclear leukocytes reducing NBT. The numbers of cells that adhered to phorbol myristate acetate- and endotoxin-coated coverslips were comparable.
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LABORATORY SUGGESTIONS
for evaluating polymorphonuclear leukocytes from patients with other diseases is currently under investigation. References 1. Baehner RL, Boxer LA, Davis J: The biochemical basis of nitroblue tetrazolium reduction in normal human and chronic granulomatous disease. Blood 48:309-313. 1976 2. Baehner RL, Nathan DG: Quantitative nitro blue tetrazolium test in chronic granulomatous disease. N Engl J Med 278: 971-976, 1968 3. DeChatelet LR, Shirley PS, Johnston RB Jr: Effect of phorbol myristate acetate on the oxidative metabolism of human polymorphonuclear leukocytes. Blood 47:545-554, 1976 4. Dupree E, Smith CS, MacDougall NLT, et al: Undetected carrier state in chronic granulomatous disease. J Pediatr 81:770-778, 1972 5. Holmes B, Page AR, Good RA: Studies of the metabolic activity of leukocytes from patients with a genetic abnormality of phagocytic function. J Clin Invest 46:1422-1432, 1967 6. Nathan DG: NBT reduction by human phagocytes. N Engl J Med 290:280-281, 1974 7. Nathan DG, Baehner RL, Weaver DK: Failure of nitro blue tetrazolium reduction in the phagocytic vacuoles of leukocytes in chronic granulomatous disease. J Clin Invest 48: 1895-1904, 1969. 8. Ochs HD, Igo RP: The NBT slide test: A simple screening method for detecting chronic granulomatous disease and female carriers. J Pediatr 83:77-82, 1973 9. Park BH, Holmes BM, Rodey GE, et al: Nitroblue-tetrazolium test in children with fatal granulomatous disease and newborn infants. Lancet 1:157-160, 1969 10. Repine JE, Clawson CC: Quantitative measurement of the bactericidal capacity of neutrophils from patients and carriers of chronic granulomatous disease. J Lab Clin Med 90:522528, 1977 11. Repine JE, Clawson CC, White JG, et al: Spectrum of function of neutrophils from carriers of sex-linked chronic granulomatous disease. J Pediatr 87:901-907, 1975 12. Repine JE, White JG, Clawson CC, et al: Effects of phorbol myristate acetate on the metabolism and ultrastructure of neutrophils in chronic granulomatous disease. J Clin Invest 54:83-90, 1974 13. Repine JE, White JG, Clawson CC, et al: The influence of phorbol myristate acetate on oxygen consumption by polymorphonuclear leukocytes. J Lab Clin Med 83:911-920, 1974 14. Repine JE, White JG, Clawson CC, et al: The influence of phorbol myristate acetate on the metabolism of neutrophils from carriers of sex-linked chronic granulomatous disease. J Lab Clin Med 85:82-86, 1975 15. Strauss RG, Mauer AM, Asbrock T. et al: Stimulation of neutrophil oxidative metabolism by the alternate pathway of complement activation: A mechanism for the spontaneous NBT test. Blood 45:843-849. 1975 16. Windhorst DB, Holmes B. Good RA: A newly defined X-linked trait in man with demonstration of the Lyon effect in carrier females. Lancet 1:737-739, 1967
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
ocytosis of bacteria. Phorbol myristate acetate is a potent stimulator of oxygen consumption, superoxide production, glucose [1-14C] oxidation, and NBT reduction. 3 " -14 In contrast to endotoxin, phorbol myristate acetate does not require serum or complement. This may prove to be a major advantage for the phorbol myristate acetate-NBT slide test, because activation of the alternate pathway of complement activation may in itself produce NBT reduction by polymorphonuclear leukocytes.15 Phorbol myristate acetate does not significantly increase oxidative metabolic activity or NBT reduction of polymorphonuclear leukocytes from patients who have chronic granulomatous disease.112 Therefore, it may prove to be an ideal agent for application to the coverslip technic. Phorbol myristate acetate stimulated NBT reduction by nearly every control cell, with a high degree of consistency. This reliability made the phorbol myristate acetate-NBT slide test very useful for detecting partial abnormalities of polymorphonuclear leukocyte function in predominantly normal populations of cells. The detection of an abnormality in polymorphonuclear leukocytes from a previously undefined obligate carrier of chronic granulomatous disease supports the value of the new approach described in this report. A test that reliably measures the function of individual cells is important, since it is necessary for optimal identification of the carrier state for sex-linked chronic granulomatous disease. According to the Lyon hypothesis of permanent inactivation of an X chromosome, the phenotypic expression of heterozygotes will range from normal to abnormal levels. Thus, in some carriers the number of abnormal polymorphonuclear leukocytes may be very small. Moreover, it is generally accepted that an individual must have test results that are consistently 2 SD beyond the mean before being considered abnormal. Thus, the phorbol myristate acetate-treated coverslip test offers a useful assay for reliable detection of statistically significant abnormalities in carriers with small numbers of abnormal polymorphonuclear leukocytes. The phorbol myristate acetate-NBT coverslip test is a simple, rapid, reproducible method that can be performed using only a single drop of venous blood. Its effectiveness as an assay
585
FOLLOWING PHAGOCYTOSIS, normal polymorphonuclear leukocytes develop a burst of oxidative metabolism and rapidly reduce nitroblue tetrazolium (NBT) to blue formazan.1B Polymorphonuclear leukocytes from patients who have chronic granulomatous disease fail to generate augmented biochemical activity or reduce N B T . 2 5 - 9 1 2 Polymorphonuclear leukocytes from heterozygotic carriers of chronic granulomatous disease usually show intermediate metabolic rates and NBT reduction because they have a mixed population of normal and abnormal polymorphonuclear leukocytes.'"•i4.i« T n e biochemical differences have formed the basis for tests of polymorphonuclear leukocyte function Received March 15, 1978; received revised manuscript and accepted for publication May 1, 1978. Supported in part by the Minnesota Medical Foundation, the Minnesota and American Heart Associations, the Basil O'Connor Starter Research Fund of the National Foundation of the March of Dimes, and NIH Grant HL-17132-1. Dr Repine is an Established Investigator of the American Heart Association. Brad Rasmussen is a medical student who was supported by a grant from the medical oncology section of the Department of Medicine. Address reprint requests to Dr. Repine: Webb-Waring Lung Institute, 4200 E. Ninth Avenue, Denver, Colorado 80262.
University of Minnesota Health Sciences Center, Minneapolis, Minnesota
designed to detect chronic granulomatous disease in patients and carriers. 2,5-9,11,12,14,16 While detection of the severe biochemical deficiency of polymorphonuclear leukocytes from patients who have chronic granulomatous disease is easily accomplished, identification of partial abnormalities of polymorphonuclear leukocytes from carriers is more difficult.4,7,10,11 In test systems that measure the function of a total population of polymorphonuclear leukocytes, a heterozygous carrier with only a few abnormal polymorphonuclear leukocytes, is often undetectable because normal polymorphonuclear leukocytes mask the presence of deficient polymorphonuclear leukocytes. 4,11 Various tests have been designed to evaluate the function of individual polymorphonuclear leukocytes in order to circumvent this problem. One such assay is the endotoxin-NBT slide technic of Ochs and Igo. 8 In this system polymorphonuclear leukocytes that reduce NBT after adhering to endotoxin-coated slides can be assessed individually. However, this test may not be optimal for detecting carriers of chronic granulomatous disease who have only a few defective polymorphonuclear leukocytes, since normal individuals often have some (approximately 10%) of NBT-negative polymorphonuclear leukocytes. We have modified the endotoxin-NBT slide test by precoating coverslips with phorbol myristate acetate, instead of endotoxin. Phorbol myristate acetate is a potent selective stimulus of neutrophilic oxidative metabolism and NBT reduction. 3,11-14 A greater percentage of control polymorphonuclear leukocytes were NBT-positive when stimulated by phorbol myristate acetate than when observed on endotoxin-coated coverslips. 8,11 The highly reproducible response of control polymorphonuclear leukocytes to phorbol myristate acetate stimulation made it possible to detect a statistically significant abnormality in polymorphonuclear leukocytes from a proven carrier of chronic granulomatous disease who had previously defied recognition by all tests, including the endotoxin-NBT slide test. 10,11
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Repine, John E., Rasmussen, Brad, and White, James G.: An improved nitroblue tetrazolium test using phorbol myristate acetate-coated coverslips. Am J Clin Pathol 71:582-585,1979. The endotoxin-nitroblue tetrazolium (NBT) slide test was modified by precoating coverslips with phorbol myristate acetate instead of endotoxin. The percentage of control polymorphonuclear leukocytes reducing NBT was significantly (P < .05) greater on phorbol myristate acetate-coated coverslips (99 ± 0.21%) than on endotoxin-coated coverslips (96 ± 1.8%). Polymorphonuclear leukocytes from patients with chronic granulomatous disease did not reduce NBT on phorbol myristate acetate- or endotoxin-treated coverslips. NBT reduction by polymorphonuclear leukocytes from proven heterozygotic carriers of sex-linked chronic granulomatous disease was intermediate between NBT reductions by those from controls and patients. A statistically significant abnormality of NBT reduction was found in polymorphonuclear leukocytes from one carrier of chronic granulomatous disease with phorbol myristate acetate-treated, but not endotoxin-treated coverslips. The phorbol myristate acetate-NBT coverslip technic is a rapid, simple, reliable way to detect deficiencies in polymorphonuclear leukocytes from patients and carriers of chronic granulomatous disease. (Key words: Neutrophils; Nitroblue tetrazolium [NBT]); Phorbol myristate acetate; Chronic granulomatous disease [CGD].)
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FIG. I. Light micrograph of control polymorphonuclear leukocytes on a phorbol myristate acetate-coated coverslip. The control polymorphonuclear leukocytes become large blue NBT-positive cells. x800.
• • * * *
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FIG. 2. Light micrograph of polymorphonuclear leukocytes from an individual hemizygotic for chronic granulomatous disease on a phorbol myristate acetate-coated coverslip. The patient's polymorphonuclear leukocytes, which are small and red, maintain a wellpreserved nuclear and cytoplasmic appearance, are considered NBT-negative cells. x800. FIG. 3. Light micrograph polymorphonuclear leukocytes from a carrier of chronic granulomatous disease on a phorbol myristate acetatecoated coverslip. The carrier polymorphonuclear leukocytes are a mixture of NBTpositive and NBT-negative cells, x 1,200.
Materials and Methods Investigation was approved by the Human Volunteers Committee of the University of Minnesota. Blood was donated by three persons hemizygotic for chronic granulomatous disease,512 three obligate maternal carriers of chronic granulomatous disease,111416 and eight control subjects. The individuals were free of infection and not taking drugs at the time of the study. NBT reduction was measured on glass coverslips by use of the technic of Ochs and Igo.8 Briefly, 0.1 ml endotoxin (lipopolysaccharide B, Escherichia coli 0.26:B6)* or 0.1 fj.g phorbol myristate acetatet/ml was * Difco, Detroit, Michigan. t Midland Consolidated Corp., Midland, New York.
allowed to air-dry on coverslips. Phorbol myristate acetate and endotoxin solutions were prepared and stored as previously described. 8111314 Drops of whole blood obtained by finger-stick or venipuncture were then placed on uncoated, endotoxin-coated, and phorbol myristate acetate-coated coverslips, which were incubated at 37 C for 45 min in a moist chamber. The clot that formed on each coverslip was removed by washing, leaving granulocytes and monocytes adherent to the coverslip. Coverslips were then placed on a drop of NBT-serum solution on a glass slide and reincubated for 20 min. Subsequently, coverslips were removed, washed, fixed in methanol, counterstained in safranin, and air-dried. Four hundred polymorphonuclear leukocytes on coverslips were examined in
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Table 1. Nitroblue Tetrazolium (NBT) Reduction by Polymorphonuclear Leukocytes from Controls and Patients or Carriers of Chronic Granulomatous Disease (CGD) NBT Reduction (% Cells Reducing NBT) (Mean ± SE) Slide Treatment None
CGD Carriers (n = 3)
Control Subjects (n = 8)*
CGD Patients (n = 3)
59 ± 2.4 (17 determinations)
0(5)*
50 (3 determinations)
0(5)*
72 (3 determinations)*
0 (5)*
69 (3 determinations)*
.001 1
1
Phorbol myristate acetate-coated
99 ± 0.21 (14 determinations)
Endotoxin-coated
96 ± 1.8 (12 determinations)
1 .05 1
1
* Significantly different from control values. P < .01.
Results Morphology The morphologic features of polymorphonuclear leukocytes studied on phorbol myristate acetate-coated slides are shown in Figure 1. Nearly all cells from control subjects became large blue NBT-positive cells on phorbol myristate acetate-treated coverslips (Fig. 1), in striking contrast to polymorphonuclear leukocytes from patients with chronic granulomatous disease, which were small, red, and had a well-preserved nuclear and cytoplasmic appearance. NBT-positive cells were not found on coverslips from patients with chronic granulomatous disease (Fig. 2). Mixed populations of NBT-positive and NBT-negative cells were apparent on coverslips made from blood samples from heterozygotic carriers of chronic granulomatous disease (Fig. 3). NBT Reduction The percentage of polymorphonuclear leukocytes from control subjects that reduced NBT was significantly (P < .01) greater on phorbol myristate acetatecoated coverslips (99 + 0.21*) than on endotoxincoated (96+1.8$) or blank (59 ± 2.4$) coverslips t Mean ± SE.
(Table 1). Cells from patients hemizygotic for chronic granulomatous disease did not reduce NBT on blank, endotoxin-coated, or phorbol myristate acetate-treated coverslips. Specimens from obligate carriers of chronic granulomatous disease had NBT-positive and NBTnegative cells (Table 1). On slides coated with phorbol myristate acetate or endotoxin, the mean percentages of NBT-positive polymorphonuclear leukocytes from heterozygotic carriers were intermediate between the means for controls and patients who had chronic granulomatous disease (Table 1). An abnormality in NBT reduction by polymorphonuclear leukocytes from an obligate carrier of chronic granulomatous disease whose defect had not been detectable by other methods, including the endotoxinNBT slide test,1011 was demonstrated on slides coated with phorbol myristate acetate. When tested on three occasions by the use of blank or endotoxin-coated coverslips, the mean percentages of NBT-positive polymorphonuclear leukocytes from this carrier (60 and 92%, respectively) were within 2 SD of the mean for polymorphonuclear leukocytes from control subjects (59 ± 14$ or 96 ± 8,t respectively). Repeat testing on three occasions, using slides coated with phorbol myristate acetate, revealed that 92% of the polymorphonuclear leukocytes from this carrier reduced NBT. This mean value was outside of the mean of NBT-positive cells from control subjects (99 ± 0.9*). Thus, it was possible to consider this individual abnormal at a 95% level of confidence using the phorbol myristate acetatecoated, but not the endotoxin-coated, NBT slide test. Discussion In the present investigation the endotoxin-NBT slide test was modified by precoating coverslips with phorbol myristate acetate instead of endotoxin. Phorbol myristate acetate is a chemical that stimulates alterations in normal polymorphonuclear leukocytes that closely resemble changes developing in cells after phag-
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
random sequence with the light microscope, and the numbers of large, NBT-positive (blue) and small, NBTnegative (red) cells were quantitated. The cells were examined in a double-blind fashion by three independent observers. The results obtained by the three observers were averaged and used to calculate the percentage of polymorphonuclear leukocytes reducing NBT. The numbers of cells that adhered to phorbol myristate acetate- and endotoxin-coated coverslips were comparable.
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LABORATORY SUGGESTIONS
for evaluating polymorphonuclear leukocytes from patients with other diseases is currently under investigation. References 1. Baehner RL, Boxer LA, Davis J: The biochemical basis of nitroblue tetrazolium reduction in normal human and chronic granulomatous disease. Blood 48:309-313. 1976 2. Baehner RL, Nathan DG: Quantitative nitro blue tetrazolium test in chronic granulomatous disease. N Engl J Med 278: 971-976, 1968 3. DeChatelet LR, Shirley PS, Johnston RB Jr: Effect of phorbol myristate acetate on the oxidative metabolism of human polymorphonuclear leukocytes. Blood 47:545-554, 1976 4. Dupree E, Smith CS, MacDougall NLT, et al: Undetected carrier state in chronic granulomatous disease. J Pediatr 81:770-778, 1972 5. Holmes B, Page AR, Good RA: Studies of the metabolic activity of leukocytes from patients with a genetic abnormality of phagocytic function. J Clin Invest 46:1422-1432, 1967 6. Nathan DG: NBT reduction by human phagocytes. N Engl J Med 290:280-281, 1974 7. Nathan DG, Baehner RL, Weaver DK: Failure of nitro blue tetrazolium reduction in the phagocytic vacuoles of leukocytes in chronic granulomatous disease. J Clin Invest 48: 1895-1904, 1969. 8. Ochs HD, Igo RP: The NBT slide test: A simple screening method for detecting chronic granulomatous disease and female carriers. J Pediatr 83:77-82, 1973 9. Park BH, Holmes BM, Rodey GE, et al: Nitroblue-tetrazolium test in children with fatal granulomatous disease and newborn infants. Lancet 1:157-160, 1969 10. Repine JE, Clawson CC: Quantitative measurement of the bactericidal capacity of neutrophils from patients and carriers of chronic granulomatous disease. J Lab Clin Med 90:522528, 1977 11. Repine JE, Clawson CC, White JG, et al: Spectrum of function of neutrophils from carriers of sex-linked chronic granulomatous disease. J Pediatr 87:901-907, 1975 12. Repine JE, White JG, Clawson CC, et al: Effects of phorbol myristate acetate on the metabolism and ultrastructure of neutrophils in chronic granulomatous disease. J Clin Invest 54:83-90, 1974 13. Repine JE, White JG, Clawson CC, et al: The influence of phorbol myristate acetate on oxygen consumption by polymorphonuclear leukocytes. J Lab Clin Med 83:911-920, 1974 14. Repine JE, White JG, Clawson CC, et al: The influence of phorbol myristate acetate on the metabolism of neutrophils from carriers of sex-linked chronic granulomatous disease. J Lab Clin Med 85:82-86, 1975 15. Strauss RG, Mauer AM, Asbrock T. et al: Stimulation of neutrophil oxidative metabolism by the alternate pathway of complement activation: A mechanism for the spontaneous NBT test. Blood 45:843-849. 1975 16. Windhorst DB, Holmes B. Good RA: A newly defined X-linked trait in man with demonstration of the Lyon effect in carrier females. Lancet 1:737-739, 1967
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
ocytosis of bacteria. Phorbol myristate acetate is a potent stimulator of oxygen consumption, superoxide production, glucose [1-14C] oxidation, and NBT reduction. 3 " -14 In contrast to endotoxin, phorbol myristate acetate does not require serum or complement. This may prove to be a major advantage for the phorbol myristate acetate-NBT slide test, because activation of the alternate pathway of complement activation may in itself produce NBT reduction by polymorphonuclear leukocytes.15 Phorbol myristate acetate does not significantly increase oxidative metabolic activity or NBT reduction of polymorphonuclear leukocytes from patients who have chronic granulomatous disease.112 Therefore, it may prove to be an ideal agent for application to the coverslip technic. Phorbol myristate acetate stimulated NBT reduction by nearly every control cell, with a high degree of consistency. This reliability made the phorbol myristate acetate-NBT slide test very useful for detecting partial abnormalities of polymorphonuclear leukocyte function in predominantly normal populations of cells. The detection of an abnormality in polymorphonuclear leukocytes from a previously undefined obligate carrier of chronic granulomatous disease supports the value of the new approach described in this report. A test that reliably measures the function of individual cells is important, since it is necessary for optimal identification of the carrier state for sex-linked chronic granulomatous disease. According to the Lyon hypothesis of permanent inactivation of an X chromosome, the phenotypic expression of heterozygotes will range from normal to abnormal levels. Thus, in some carriers the number of abnormal polymorphonuclear leukocytes may be very small. Moreover, it is generally accepted that an individual must have test results that are consistently 2 SD beyond the mean before being considered abnormal. Thus, the phorbol myristate acetate-treated coverslip test offers a useful assay for reliable detection of statistically significant abnormalities in carriers with small numbers of abnormal polymorphonuclear leukocytes. The phorbol myristate acetate-NBT coverslip test is a simple, rapid, reproducible method that can be performed using only a single drop of venous blood. Its effectiveness as an assay
585
