| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
1
Institute of Laboratory Medicine and Pathobiochemistry and
2
Clinic of Pediatric Pneumology and Immunology, University Hospital, Charité, Campus Virchow-Klinikum of the Humboldt-University, Augustenburger Platz 1, 13353 Berlin, Germany
3
Department of Clinical Chemistry and Molecular Diagnostics, Clinic of the Philipps University Marburg, Baldingerstrasse, 35033 Marburg, Germany
a author for correspondence: fax 49-30-45069900, e-mail andreas.lun{at}charite.de
 |
Introduction |
|---|
 Top Introduction References |
|---|
The following clinical and laboratory findings indicate that assessment of granulocyte function is needed: increased susceptibility to bacterial infections, therapy-resistant infections, recurrent infections with nonpathogenic microorganisms, lymphadenitis, abscesses of liver or lung, osteomyelitis, recurrent stomatitis, or gingivitis. Granulocytopenia and defects of B cells or complement compartment must be excluded (17). Phagocytosis, adhesion molecules CD18 and CD11b for leukocyte adhesion defect I or CD15s for leukocyte adhesion defect II, and production of oxygen radicals upon stimulation for CGD can be tested by flow cytometric determinations. Disturbances such as Chédiak-Higashi syndrome, hyper IgE syndrome, or glycogenesis type Ib need other techniques.
One of the most common inherited granulocyte defects is CGD. The nitroblue tetrazolium dye reduction assay, the gold standard for diagnosis of CGD in the past (18)(19)(20), has been replaced to a large extent by flow cytometry-based procedures (18)(21)(22)(23). Commercially available flow cytometric assays have been used routinely in clinical laboratories. However, age-dependent reference values are not known or have been reported based on very small collectives (15)(24). Therefore, the aim of this study was to establish age-dependent reference values for phagocytosis and oxidative burst activities, both assessed by flow cytometry. In addition, the diagnostic efficiency of oxidative burst activities to detect CGD was examined in patients and heterozygous carriers of the disease.
The capacities of phagocytosis and oxidative burst were tested in 288 individuals. In all cases, an acquired or inherited immune deficiency syndrome had been excluded by history and/or extensive analysis of cellular and humoral immune functions. The age distribution was 1 month to 54 years.
To test the diagnostic efficiency, a group of seven patients with CGD (six males and one female) and six heterozygous carriers of CGD was evaluated. In all seven CGD patients investigated, the disease manifested during the first 2 years of life. The main clinical problems had been recurrent cervical purulent lymphadenitis with surgical interventions (n = 5) and liver granulomas (n = 4). Intrathoracic granulomas with abscesses (n = 2) and bihilar lymphadenopathy (n = 2) were also common. Two patients suffered from pulmonary tuberculosis, one from a long-lasting Bacille Calmette-Guérin inflammation after vaccination. In each case, bladder granulomas attributable to an Escherichia coli infection and diffuse small granulomas in the upper gastrointestinal tract could be observed. One boy presented with wound-healing disturbances after herniorrhaphy.
Oxidative burst and phagocytosis were measured quantitatively by fluorometric analysis using commercial methods (PHAGOBURST® and PHAGOTEST®; Orpegen Pharma) in heparinized whole blood (25). The analyses were performed within 4 h of sampling. Blood samples were transported and stored at room temperature.
In the oxidative burst activity experiments, upon stimulation with unlabeled opsonized bacteria (E. coli) as a particulate stimulus or the protein kinase C ligand phorbol 12-myristate 13-acetate (PMA) as a strong stimulus, granulocytes produced reactive oxygen metabolites. Radical formation was measured at 37 °C by conversion of dihydrorhodamine 123 to rhodamine 123 as a fluorogenic substrate (26)(27). A sample without stimulus served as negative background control. The reaction was stopped by addition of lysing solution, which removed erythrocytes and partially fixed leukocytes. After a washing step, DNA staining was performed to exclude aggregation artifacts from bacteria or cells. Cells were analyzed by flow cytometry (FACScan; Becton Dickinson), using Cell Quest software (Becton Dickinson) for data acquisition and analysis, and the results were expressed as mean fluorescence intensity (MFI) plotted as histograms. CalibriteTM beads (Becton Dickinson) were used to adjust instrument settings and set fluorescence compensation. Forward and sideways scatter were used to select granulocytes. The percentage of cells that did not produce oxygen radicals was between 1% and 15%. In all test series, blood samples taken from healthy adult donors were analyzed as an internal control.
In the phagocytosis experiments, bacteria were opsonized with antibodies and complement of pooled serum samples and subsequently bound by leukocytes. Because phagocytosis is temperature-dependent, control experiments were performed on ice. After 10 min of incubation at 37 °C, phagocytosis was interrupted by placing the samples on ice and adding ice-cold quenching solution; uningested bacteria were excluded from analysis. After several washing steps, red blood cells were lysed, followed by incubation with DNA staining solution to exclude aggregation artifacts from bacteria or cells. Data were acquired as in the oxidative burst test.
To test the analytic precision for phagocytosis and oxidative burst
activities, 20 samples were analyzed in duplicate, and the CV
was calculated using the following formula: CV (%) = (SD/mean
value) x 100, where
 |
The CVs for the burst test were 14% and 11% for stimulation by E. coli and PMA, respectively, with MFI values of 498 arbitrary units (AU) and 610 AU. For phagocytosis, the CV was 15%, with a MFI of 610 AU. In addition, 20 stored measuring protocols were gated and evaluated twice using Cell Quest, and the CVs were calculated from duplicates, using the formula described above, to estimate the impact of gating of granulocytes on the imprecision of the methods. The CV for gating was 15%.
Statistical analyses were carried out using SPSS for Windows.
Phagocytosis reference limits were calculated for all age groups
together; the 2.5th and 97.5th percentiles for the MFI were 496 and
2738 AU, respectively (Fig. 1
A, left panel). The distributions of values between the
different age groups did not differ significantly; the mean values were
between 1000 and 1250 AU.
|
In the oxidative burst experiments, without stimulation neutrophils did
not produce relevant amounts of oxygen radicals as indicated by a
MFI of 33 AU (97.5th percentile). For stimulation of granulocytes by
opsonized E. coli, a reference interval of 138–964
AU (2.5th to 97.5th percentiles) was calculated (Fig. 1A
, middle
panel). There were no significant differences between age groups. A
slightly different pattern was found after stimulation with PMA
(Fig. 1A
, right panel). Overall, the values were higher than those for
stimulation with E. coli.
To test the diagnostic efficiency of the test system for oxidative
burst, a group of seven patients with known CGD and six heterozygous
carriers of CGD was tested. As depicted in Fig. 1A
, in all CGD patients
the oxygen burst activity, expressed as fluorescence intensity after
stimulation with E. coli or PMA, was below the 1st
percentile of the reference collective. Heterozygous individuals
presented with values between the 1st and 50th percentiles of the
reference interval. However, carriers can be easily identified by their
unique pattern of fluorescence activity. Carriers possessed two
distinct subsets of neutrophils; one subset revealed normal function,
whereas the other revealed the defect in oxygen radical formation. Fig. 1B
illustrates this phenomenon in a family with affected and unaffected
siblings. Whereas the patient revealed the expected deficiency in
oxygen radical formation, the father showed normal values for
stimulation with both E. coli and PMA. The mother and sister
as carriers revealed the typical "split population".
In this study, we aimed to establish age-dependent reference values for fluorometric assays of granulocyte functions as tested by flow cytometry. Although there was high interindividual variation in all age groups, significant differences between age groups were not detected. Therefore, reference values can be provided independent of age. The diagnostic efficiency was then tested in patients and carriers of CGD, and the data clearly indicated that the reference values allowed for correct identification of this disease in all patients. Reference values have also been presented by other authors, but they differ from our data (15)(23)(24)(28). There are several reasons that preanalytical and analytical issues (25) may account for such differences. Important aspects include the equipment and reagents used for lysing of red blood cells (29). The above-mentioned factors have an effect on the test result. However, in our study all of these aspects have been taken into consideration and allowed us to define reference values that are at least useful to identify patients with CGD, the most common hereditary leukocyte defect. In daily routine, it is important to include internal controls, which may consist of a sample from a healthy control donor.
For testing of granulocyte functions, flow cytometric tests have advantages compared with conventional assay systems: they allow exact quantification of functions, and results are obtainable within a short period of time, usually within the same day.
|
References |
|---|
|
TopIntroductionReferences |
|---|
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  A. Lun, J. Roesler, and H. Renz Unusual Late Onset of X-linked Chronic Granulomatous Disease in an Adult Woman after Unsuspicious Childhood Clin. Chem., May 1, 2002; 48(5): 780 - 781. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
and 
), and heterozygous carriers (
and •). The lower and upper margins of the
black box represent the 1st and 2.5th percentiles, the
line inside the gray box is the 50th percentile, and the
upper margin of the gray box represents the 97.5th
percentile of the reference collective. (B), oxidative
burst in a family with CGD. A, father; B,
mother; C–E, children. Lower panels,
histograms of oxidative burst in family members: the CGD patient
(C), the healthy father (A), and the
heterozygous mother and sister (B and D,
respectively). The histogram for E is not presented
because it was similar to B and D.