C 1992 Wiley-Liss, Inc.

Cytometry 13:615-620 (1992)

Neutrophil Function Screening in Patients With Chronic Granulomatous Disease by a Flow Cytometric Method C.L. Epling, D.P. Stites, T.M. McHugh, H.O. Chong, L.L. Blackwood, and D.W. Wara Departments of Laboratory Medicine and Pediatrics, University of California, San Francisco, San Francisco, California Received for publication September 20, 1991; accepted December 19, 1991

Neutrophils from patients with chronic (NBT) test. For the DCF test, the ratio of granulomatous disease (CGD) fail to pro- mean fluorescence intensity of stimuduce a significant oxidative burst follow- lated to non-stimulated neutrophils was ing stimulation. We have evaluated the (5 for CGD patients and from 16 to >50 use of flow cytometry and the dye 2’,7’- for healthy individuals. With the DCF dichlorofluorescein diacetate (DCF) for test, two populations of neutrophils routine screening for deficiencies of neu- could be identified in samples from four trophil oxidative burst. A range for DCF carriers of X-linked CGD, although two fluorescence for phorbol myristate ace- carriers of AR CGD had NBT and DCF tate stimulated and non-stimulated neu- results in the normal range. Our data trophils was established based on data suggest the DCF test is a sensitive and from 52 healthy adults. Samples from convenient method for detecting CGD. three patients with suspected neutrophil 0 1992 Wiley-Liss, Inc. dysfunction, three patients with X-linked CGD, and one patient with autosomal re- Key terms: Flow cytometry, oxidative cessive (AR) CGD were evaluated with burst, dichlorofluorescein diacetate both the DCF assay and the quantitative (DCF), nitroblue tetrazolium dye reducnitroblue tetrazolium dye reduction tion test (NBT)

An oxidative burst in neutrophils was discovered in 1933 when it was observed that canine neutrophils increased their oxygen uptake while ingesting bacteria (4). Neutrophils can be activated by either particulate agents or soluble factors such as phorbol myristate acetate (PMA). Activation generates a n oxidative burst that includes a n increase in oxygen uptake, the stimulation of the hexose monophosphate shunt (HMPS), and the production of hydrogen peroxide (16,211. In 1957, Berendes et al. (7) described a new granulomatous disease characterized by sex-linked inheritance, hypergammaglobulinemia, lymphadenopathy, granulomatous infiltrations of organs, and recurrent infections. Invitro studies of neutrophils from patients with this syndrome demonstrated that Staphylococcus aureus was ingested by these cells but not killed (15). Chronic granulomatous disease (CGD) was soon recognized a s resulting from a n absent respiratory burst related to defects in the nicotinamideadenine dinucleotide phosphate (NADPH) oxidase enzyme system (1,10,24). Two types of inheritance patterns, X-linked

(20,22,23) and autosomal recessive (AR) (9,12,25), have been well characterized. Many in vitro assays have been developed to detect deficient oxidative burst in neutrophils. One of the first tests used for the diagnosis of CGD was the nitroblue tetrazolium dye reduction (NBT) test (2). In this test, neutrophils are incubated with NBT solution and a neutrophil stimulant. The dye, initially clear and yellow, enters the cell and is reduced to microscopically visible blue formazan in the presence of products of oxidative burst. The slide NBT test has been modified to quantitatively determine the amount of NBT dye reduced by stimulated neutrophils by extracting the dye and measuring its absorbance spectrophotometrically (3). Although these results are less subjective

Address reprint requests to C.L. Epling, Department of Laboratory Medicine, Medical Center a t the University of California, 505 Parnassus Avenue, Box 0100, San Francisco, CA 94143-0100.

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EPLING ET AJ,.

Table 1 Clinical Histories of Patients and Carriers Patient

x1

Status CGD

Sex M

Age 2

NBT” AB

x2 x3

CGD CGD

M M

530 nm. Prior to performing each assay, the fluorescein channel was monitored using glutaraldehyde fixed chicken red blood cells (Becton Dickinson, San Jose, CA). The data were collected using a logarithmic scale for side scatter and fluorescein amplification. Forward and side scatter plots were used to identify the neutrophil population. The mean fluorescence intensity (MFI) from neutrophils exposed only to PBS was considered resting or background DCF fluorescence. Neutrophils stimulated by PMA had a n increased MFI which was attributed to oxidation of DCF by hydrogen peroxide. The ratio of the MFI from stimulated neutrophils and resting neutrophils (SiR) is the neutrophil oxidative index (NOI). The difference between the MFI of stimulated neutrophils and resting neutrophils (S-R) is the net fluorescence (NF). Two experiments were performed to test the specificity of the DCF assay for oxidative burst. To measure the ability of neutrophils in a lysed whole blood preparation to absorb DCF and fluoresce in the presence of hydrogen peroxide, non-stimulated cells were incubated with 200 pM hydrogen peroxide (J.T. Baker, Phillipsburg, NJ) for 30 min a t 37"C, followed by flow cytometric analysis. In a second set of experiments, the effect of 10 mM of D-mannose (Sigma Chemicals, St. Louis, MO), which reduces oxygen consumption and superoxide production (19) was evaluated. D-glucose and D-galactose (10 mM, Sigma Chemicals, St. Louis, MO) were included in the experiments a s controls. The carbohydrates were added prior to stimulation with PMA and their effect on DCF fluorescence measured by flow cytometry. To evaluate the ability of catalase to improve the fluorescent channel separation between abnormal CGD neutrophils and normal neutrophils, catalase a t 1,000 Units/ml (Sigma Chemicals, St. Louis, MO) was added in place of KCN to 1) a cell suspension from a normal individual, prior to incubation with either hydrogen peroxide or PMA, and 2) mixtures of blood from a CGD patient and a normal individual, prior to the 30 min incubation with PBS or PMA.

RESULTS Specificity of the DCF Assay When a solution containing DCF and non-stimulated neutrophils was incubated with 200 pM hydrogen peroxide (n = 4), the MFI of the neutrophils increased from 66 (232) to 1,787 (t1,037), a 27-fold increase in fluorescence (25). Mannose, added prior to activation with PMA, reduced the MFI of stimulated neutrophils by

Table 2 Effect of Carbohydrates on DCF Fluorescence" Percent DCF MFI change 3,572 2 695 1,508 ? 314 -58b 2 4 2,955 ? 503 -19' 2 8 2.929 997 -23d 5 10 "DCF MFI, Dichlorofluorescein mean fluorescence intensity; PMA, phorbol myristate acetate. b n = 4 , P < 0.005 when compared to samples with PMA only. "n =3, P < 0.10 when compared to samples with PMA only. dn= 3, P < 0.05 when compared t o samples with PMA only. Additive PMA PMA + 10 mM Mannose PMA + 10 mM Glucose PMA + 10 mM Galactose

*

more than 50%;less effect was observed with glucose or galactose (Table 2).

Samples From Normal Individuals and Patients The results of the DCF tests from the 3 patients with suspected neutrophil dysfunction (with normal NBT test results), 4 patients with CGD, 6 carriers of CGD, and the data from 52 healthy individuals are listed in Table 3. Values for NO1 and NF from normal individuals were ranked and a lower normal limit was determined which excluded 5% of the data collected. These values were >19 for NO1 and >1,000 for NF. There was no overlap between the lowest values from healthy individuals (16for NO1 and 657 for NF) and those of CGD patients (

Neutrophil function screening in patients with chronic granulomatous disease by a flow cytometric method.

Neutrophils from patients with chronic granulomatous disease (CGD) fail to produce a significant oxidative burst following stimulation. We have evalua...
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