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.
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].)
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
0002-9173/79/0500/0582 $00.70 © American Society of Clinical Pathologists
582
Vol. 71 . No. 5
LABORATORY SUGGESTIONS
583
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.
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
REPINE, RASMUSSEN, AND WHITE
584
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.
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.
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-
Vol. 71 • No. 5
LABORATORY SUGGESTIONS
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
for evaluating polymorphonuclear leukocytes from
patients with other diseases is currently under investigation.
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