chronic granulomatous disease (CGD) was reported by Bass et al. in 1983 (1). ... (on a 4-decade logarithmic amplifier with channels from 1 to 10,000) obtained.
CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Mar. 1995, p. 227–232 1071-412X/95/$04.0010 Copyright q 1995, American Society for Microbiology
Vol. 2, No. 2
Rapid Whole-Blood Flow Cytometry Assay for Diagnosis of Chronic Granulomatous Disease MAURICE R. G. O’GORMAN*
AND
VIRGINIA CORROCHANO
Division of Immunology/Rheumatology, Department of Pediatrics, Northwestern University, Children’s Memorial Hospital, Chicago, Illinois 60614 Received 31 August 1994/Returned for modification 1 November 1994/Accepted 16 December 1994
Chronic granulomatous disease (CGD) is characterized by defective killing of intracellular microorganisms due to mutations in one of the four known components of the NADPH oxidase system. This system is responsible for the generation of superoxide and related antimicrobial oxidants. Diagnosis of CGD requires the demonstration of an abnormal oxidase system in the leukocytes of affected patients. Recently, several flow cytometry-based procedures which measure various reactive oxygen intermediates generated by the NADPH oxidase system have been developed. Most of the procedures developed to date require time-consuming granulocyte isolation, washing, and counting procedures, or they lack sensitivity. We have modified an existing procedure such that cell labelling and stimulation are performed directly in whole blood. Optimization of this procedure and its use in the diagnosis of patients with CGD or X-linked carriers are presented.
Our goal was to develop a sensitive procedure for the detection of reactive oxygen species (ROS) which could be performed rapidly and in a manner similar to whole-blood procedures for lymphocyte immunophenotyping or other granulocyte function assays. We have modified the procedure reported by Emmendo ¨rffer (2, 3, 12) to allow dye loading and granulocyte stimulation directly in whole blood. Following the prerequisite incubation periods, erythrocytes are lysed, the leukocytes are fixed, and the fluorescence in individual cells is measured by gating on the appropriate cell populations. This procedure is very sensitive and easy to perform. In this paper we discuss the optimization of this procedure for clinical use and present the results obtained with four families, each with at least one child with CGD.
The first flow cytometry-based assay for the diagnosis of chronic granulomatous disease (CGD) was reported by Bass et al. in 1983 (1). This procedure required the isolation, washing, and counting of granulocytes prior to loading the cells with the dye dichlorofluorescein diacetate (DCFH-DA). Most laboratories currently performing a flow cytometry-based CGDdiagnostic assay use this procedure or a slight modification of it. This assay requires relatively large volumes of blood, as polymorphonuclear cells (PMN) must be separated from whole blood prior to incubation of the cells with the dye. Additionally, once the PMN have been isolated they must be washed and counted, each step being relatively time-consuming. Whole-blood assays using DCFH-DA have been developed for the detection of reactive oxygen intermediates (4, 7, 8, 14). There are many variations of the procedure, including lysis of erythrocytes prior to labelling with DCFH-DA. Some of the whole-blood assays have been used in clinical settings; however, the sensitivity level of such assays may lead to problems in the identification of some X-linked carriers (4). The use of the uncharged nonfluorescent laser dye dihydrorhodamine 123 (DHR 123) to measure intracellular reactive oxygen metabolites was first reported by Rothe et al. (13). The dye readily permeates most membranes. In the presence of reactive oxygen intermediates generated during the respiratory burst, the dye is rapidly oxidized to produce the brightly fluorescent cationic compound rhodamine 123, which localizes in the mitochondria (5). DHR 123 is a more sensitive indicator of granulocyte respiratory-burst activity than DCFH-DA (12). Emmendo ¨rffer and colleagues have developed a clinical test utilizing DHR 123 (2, 3, 12). The test involves sedimentation, erythrocyte lysis, and washing and counting of granulocytes prior to dye loading and stimulation. The DHR 123 is added after granulocyte stimulation, as it was observed that DHR 123 inhibited the generation of reactive oxygen intermediates (2).
MATERIALS AND METHODS Basic method. The procedure was developed to be performed in a manner similar to the standard whole-blood lysis methods used in many laboratories for routine immunophenotyping. One hundred microliters of whole blood was added to the appropriate tubes, diluted 1:10 with calcium- and magnesium-free phosphate-buffered saline (PBS) (Northwestern University Cancer Center, Chicago, Ill.), and incubated in a shaking water bath at 378C with 25 ml of DHR 123 (Molecular Probes, Eugene, Oreg.) at a final concentration of 2.5 mg/ml. DHR 123 was stored at a stock concentration of 5 mg/ml in N,N-dimethylformamide (Sigma, St. Louis, Mo.) at 2708C. (Note: N,N-dimethylformamide will dissolve some plastics.) After a 15-min incubation period, various stimuli (discussed
TABLE 1. Titration of DHR 123 in a whole-blood flow cytometry assay for the detection of ROS Fluorescence obtained with DHR 123 concn (mg/ml) of a: Sample
DHR 123 only DHR 123 1 PMA
1.5
3.0
l7.3 6 0.8
12.4 6 4.2
4.5
21.8 6 13.9
6.0
22.9 6 9.8
508.9 6 119 812.8 6 110.1 1,128.3 6 275.3 1,365.6 6 188
a DHR 123 concentrations are final concentrations added. Results are expressed as means 6 standard deviations of the mean logarithmic fluorescence (on a 4-decade logarithmic amplifier with channels from 1 to 10,000) obtained with a stimulus of 60 ng of PMA per ml in the standard assay performed with two different lots of DHR 123.
* Corresponding author. Mailing address: The Children’s Memorial Hospital, 2300 Children’s Plaza, Chicago, IL 60614. Phone: (312) 8803070. Fax: (312) 880-3739. Electronic mail address: mogorman@ anima.nums.nwu.edu. 227
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FIG. 1. Mean DHR 123 fluorescence of granulocytes with different stimuli. Different concentrations of PMA, fMLP and opsonized zymosan were added to whole blood preincubated with DHR 123. Results are expressed as the mean channel of fluorescence on a 4-decade logarithmic amplifier.
below) were added and the blood was incubated for an additional 15 min at 378C in a shaking water bath. Following the last incubation the samples were centrifuged at 400 3 g for 5 min, and the supernatant was discarded. The pellet was resuspended in 2 ml of erythrocyte lysing solution (Ortho-mune lysing reagent; Ortho Diagnostic Systems, Raritan, N.J.) and allowed to stand at room temperature for 10 min. The cells were then washed in a solution containing 2.5% sodium azide (Sigma) and 10% fetal bovine serum (GIBCO, Grand Island, N.Y.) in PBS and resuspended in 0.7 ml of a 1% paraformaldehyde solution (Electron Microscopy Sciences, Washington, Pa.). The samples were then run on the flow cytometer, and the levels of fluorescence in the appropriate populations were measured. Flow cytometry analysis. Following fixation, the samples were acquired on a FACScan flow cytometer (Becton Dickinson, Mountain View, Calif.). The ma-
chine is equipped with an argon ion air-cooled laser which emits a peak line of fluorescence of 488 nm at 15 mW. At least 10,000 events were acquired by using LYSYS II software (Becton Dickinson). Both forward-angle and right-angle light scatter signals were adjusted for optimum results for the detection of the three major leukocyte populations. The forward-scatter threshold signal was adjusted to exclude debris and unlysed erythrocytes. All parameters were optimized on nonstimulated, lysed whole blood which had not been loaded with DHR 123. Fluorescence of this sample was acquired on the photomultiplier tube used routinely to collect fluorescein isothiocyanate emissions through a 525-nm band pass filter. Logarithmically amplified signals were adjusted so that the peak fluorescence was in the first decade and a clear rise and fall of this peak were observed. Background fluorescence was determined by measuring fluorescence in the cells which were loaded with dye but not stimulated and comparing it with
TABLE 2. Results of standard CGD flow cytometry assaya Population 1 (CGD-affected PMN) Family member % of total PMN
c
MFC
Population 2 (normal PMN) d
NOI
% of total PMN
MFC
NOI
Result of NBT test (% normal)b
Healthy controle Mother XXJe CGD son AIXJ 1e CGD son ANXJ 2e
0 17 100 100
NA 38.5 15.4 13.7
NA 2.9 1.2 1.1
100 83 0 0
568 484 NA NA
45 36.4 NA NA
96 67 0 0
Healthy control Mother XXN Brother EZXN CGD son ELXN 1 CGD son RXN 2
0 82 0 100 100
NA 12.6 NA 5.7 9.5
NA 1.8 NA 0.9 1.0
100 18 100 0 0
1,457 1,355 1,665 NA NA
217 199.3 146 NA NA
98 40 90 3 0
Healthy control Father MXH Mother TXM CGD son RXM
0 0 24 100
NA NA 31.6 5.6
NA NA 3.2 0.9
100 100 76 0
1,296 626.4 550.3 NA
125.2 60.8 56.7 NA
95 94 90 0
Healthy control Father SXD Mother SXD CGD daughter AXD CGD daughter MXD
0 0 0 100 100
NA NA NA 12.2 13.9
NA NA NA 1.4 2.0
100 100 73 0 0
2,536 2,763 2,640 NA NA
329 368 325.9 NA NA
NA NA NA NA NA
Results for four families are shown. The standard assay was performed with 60 ng of PMA per ml and 2.5 mg of DHR 123 per ml. NBT dye reduction slide assay performed according to standard techniques (15). A value of $85% positive for dye reduction is considered normal. Note that mother TXM would have been considered normal by the NBT slide assay. c MFC from PMA-stimulated cells. d Calculated as MFC of PMA-stimulated cells/MFC of nonstimulated cells. e Samples were held overnight prior to processing and analysis. a b
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FIG. 2. Kinetics of PMA-induced DHR fluorescence in the three major leukocyte populations. (A) The mean fluorescence was measured in granulocytes, monocytes, and lymphocytes by gating on each population. The diagram shows forward-angle light scatter versus right-angle light scatter. (B) Cells were preincubated with 2.5 mg of DHR 123 per ml and stimulated with 60 ng of PMA per ml. Time (in minutes) is indicated on the x axis; the MFC is indicated on the y axis. Results are representative of one experiment.
fluorescence in cells loaded with dye and stimulated. Following data acquisition, the data were analyzed by LYSYS II and recorded as the mean fluorescence. According to the formula described by Epling et al. (4), we determined the neutrophil oxidative index (NOI) by calculating the ratio of the mean fluorescence of the stimulated cells to the mean fluorescence observed in the background control cells. In cases in which two distinct fluorescent populations were observed (i.e., X-linked carriers), the mean fluorescence of each population was obtained and the NOI for each population was calculated. Response curves for PMA, opsonized zymosan, and fMLP. Various concentrations of phorbol 12-myristate 13-acetate (PMA), opsonized zymosan, and f-Met-Leu-Phe (fMLP) were assessed for their abilities to generate the production of ROS. PMA (Sigma) was diluted in dimethyl sulfoxide (Sigma) at a concentration of 5 mg/ml and stored at 2708C. Working dilutions were then made in PBS. Concentrations of 15 to 400 ng of PMA per ml were tested. The zymosan dilution (Sigma) was made up to 0.1 g in 10 ml of sterile PBS, boiled for 10 min, washed twice, and resuspended in 10 ml of PBS. One hundred milligrams of zymosan was then opsonized by incubation in 1 ml of pooled normal human serum in a shaking water bath at 378C for 1 h. Once opsonized, the zymosan was washed and resuspended to the desired concentration in PBS. Concentrations of 0.1 to 0.5 mg/ml were tested. A 1022 M solution of fMLP (Sigma) was prepared in dimethyl sulfoxide and stored as the stock solution. Tenfold serial dilutions were then made in PBS. Concentrations of 1024 to 1029 M were tested.
Detection of ROS after various holding times prior to processing and/or analysis. Blood samples obtained from each individual were treated with EDTA or with heparin and analyzed for their applicability in the generation of ROS. Additionally, some of the samples were prepared fresh (within 6 h of blood draw) or after being held at room temperature for 24 h. The samples were analyzed immediately or after being held overnight at 48C. Detection of ROS in granulocytes, monocytes, and lymphocytes. Recently it has been reported that ROS are detectable in lymphocytes (8). By the whole blood method described herein, list mode data from each of the three major leukocyte populations (i.e., monocytes, lymphocytes, and granulocytes) detectable by integrated analysis of the forward- and right-angle light scatter signals were analyzed. At various time points after stimulation we measured the fluorescence in the three populations. Study, subjects, controls, and X-linked carriers. Peripheral blood was obtained from 33 healthy donors, and the mean fluorescence channel (MFC) of the stimulated and nonstimulated granulocytes was determined. For each subject the NOI was calculated, and the lowest value detected was used as the cutoff for a normal oxidative burst (i.e., a burst not consistent with a diagnosis of CGD). Peripheral blood was obtained from four CGD-affected families, including five affected male children (X-linked CGD), one affected female child (autosomal recessive-p67-phox deficiency), one nonaffected male child, three obligate Xlinked carrier mothers, one unaffected father, and the parents of the child with autosomal recessive CGD. The MFC of each population was determined, and the NOI of each population was calculated.
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FIG. 3. DHR fluorescence in PMA-stimulated granulocytes. The graph illustrates the dose response effects of increased PMA concentrations on the MFCs observed for the peripheral blood obtained from seven healthy donors. (See Materials and Methods for description of the basic assay.)
RESULTS Response curves for different stimuli. The optimal concentration of dye used to load the granulocytes prior to stimulation with 60 ng of PMA per ml was between 2.5 and 5 mg/ml (Table 1). The concentration of dye giving the most significant increase in fluorescence after a 15-min incubation period, while maintaining low background fluorescence, was determined for each new lot number. The results obtained with the different stimuli are summarized in Fig. 1. Zymosan was an effective mediator for the generation of the oxidative burst, with optimal results occurring at a concentration of 0.5 mg/ml. Higher levels of opsonized zymosan in the samples interfered with the light scatter signals. Because of the complexities of preparing opsonized zymosan, it is not used routinely for the clinical procedures. The fMLP generated a very weak response in this assay, resulting in a slight increase in fluorescence at the concentrations used. The most potent stimulus was PMA at a concentration of 240 ng/ml. This level of PMA was, however, toxic to the cells and resulted in decreased recovery of granulocytes (as determined by decreased cell counts and a degenerating light scatter pattern) (data not shown). PMA at a concentration of 60 ng/ml induced a consistent increase in fluorescence (i.e., the generation of ROS) (see Fig. 3) while maintaining the characteristic light scatter pattern of the granulocyte cluster (see Fig. 2A). Anticoagulant and holding of samples prior to preparation and analysis. The results of our assays were comparable and did not differ significantly when either EDTA or heparin was used as the anticoagulant. The background fluorescence expressed as the MFC in cells with dye but no stimulus was 11.9 6 5.7 in the sample containing EDTA, compared with an MFC of 12.4 6 5.3 for heparinized samples (n 5 4). The MFC following stimulation with 60 ng of PMA per ml was 1,496 6 655 in the sample containing EDTA, compared with an MFC of 1,536 6 537 for the samples (n 5 4) containing heparin. Processing the samples fresh and then analyzing them after 24 h resulted in a 34% 6 16% (mean 6 standard deviation, n 5 4) reduction in the MFC compared with the mean fluorescence obtained when the samples were analyzed immediately. Holding the sample for 24 h prior to processing and then
analyzing it immediately also resulted in a significant decrease in the mean fluorescence (56% 6 30%, n 5 4). Detection of ROS in granulocytes, monocytes, and lymphocytes. ROS were detectable in granulocytes and monocytes after a 15-min incubation with PMA as the stimulus. After approximately 30 min, ROS were detectable in the lymphocyte population as a very minimal increase in fluorescence. Even after 60 min, the increased fluorescence in the lymphocyte population was minimal (Fig. 2). Assay for ROS in normal controls, CGD patients, and Xlinked carriers. The lower limit for normal ROS production (calculated as the NOI) was established by studying 33 donors over a 6-month period. With a stimulus of 60 ng of PMA per ml and a DHR 123 loading concentration of 2.5 mg/ml, the MFC in the stimulated granulocytes ranged from 503 to 2,248 (mean 6 standard deviation 5 1,400 6 568). The mean background fluorescence in this population (i.e., the population preincubated with DHR 123 but not PMA stimulated) was 13 6 7 channels. Figure 3 depicts the dose response effects of PMA in whole blood obtained from seven different healthy controls. The NOIs in this population of adult individuals ranged from 32 to more than 300, with an average NOI of 149. An NOI greater than 30 is considered normal, i.e., not consistent with a diagnosis of CGD. The average NOI for the five patients with CGD was 1.0 6 0.1. The average NOI for the population of normal granulocytes in the three X-linked carriers was 192, and the average NOI for the population of granulocytes expressing X chromosomes with the CGD mutation was 2.4. Figure 4 illustrates the results obtained from a CGD family with the X-linked form of the disease. The patient’s PMN fail to generate ROS as indicated by the absence of an increase in fluorescence following stimulation with PMA, i.e., an NOI of 0.9. The patient’s mother’s results indicate the presence of both normal (NOI 5 57) and abnormal populations of PMN; i.e., cells with the affected X chromosome do not elaborate ROS (NOI 5 3). The healthy control’s NOI was 125. Finally, the father’s stimulated PMN were all brightly fluorescent and had an NOI consistent with normal ROS production (NOI 5 61; data not shown in Fig. 4). Table 2 is a summary of the results obtained from four families, each with at least one child
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FIG. 4. Flow cytometry results obtained by using the standard assay for one CGD family. First column, positive control (father); second column, affected son; third column, affected mother. The first row shows the autofluorescent signal generated by untreated cells, the second row shows the background fluorescence in cells incubated with dye but not stimulated, and the third row shows the results of the PMA-stimulated samples preincubated with dye. All results represent the fluorescence in the electronically gated granulocyte population.
with CGD. Our laboratory has routinely used the nitroblue tetrazolium (NBT) dye reduction slide test for the diagnosis of CGD and for the identification of X-linked carriers (15). The diagnosis of CGD obtained with the NBT test correlated 100% with the results obtained in the CGD diagnosis by whole-blood assay (Table 2). Interestingly, mother TXM, clearly identified as a carrier by the flow cytometry-based assay, would have been considered normal on the basis of the NBT slide assay (90% of the cells reduced the dye). DISCUSSION We have developed a rapid and sensitive flow cytometry whole-blood assay for the detection of abnormal granulocytes in patients with CGD and in carriers with the X-linked form of the disease. The assay is optimal with a sample which is processed within 8 h, and the results did not depend on whether heparin or EDTA was used. Specimens can be held overnight prior to processing, or the samples can be processed and then held overnight. Both of these holding procedures resulted in reduced fluorescence compared with processing and analyzing within 8 h; however, the NOI remained greater than 30, i.e.,
not consistent with a diagnosis of CGD. We recommended that each laboratory establish its own ranges using the appropriately processed samples. For routine clinical testing, three samples are processed: (i) a sample with dye only as the background control, (ii) a PMA-stimulated sample as the positive control, and (iii) an fMLP-stimulated sample as a low-positive control. We have previously shown that fMLP generates a rapid increase in the level of expression of b2 leukocyte integrins on the surface of granulocytes (9). Interestingly, fMLP stimulated a rapid increase in the surface expression of adhesion molecules without a concomitant generation of a strong oxidative burst. We have developed this procedure to allow for the dye loading and stimulation of cells directly in whole blood. Unlike Emmendo ¨rffer and colleagues (2), we did not observe an inhibitory effect of DHR 123 on granulocyte stimulation. One explanation for this discrepancy may be that both the dye loading and the granulocyte stimulation occur directly in whole blood. The carrier status of mothers with male children suffering from CGD was easily and objectively measured. In three of the families studied, the results obtained with the mothers’ blood
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were consistent with their being carriers for CGD. On the basis of the results obtained with the NBT slide test, mother TXM would have been considered normal, as 90% of her cells were reported to have reduced the dye. When tested with our wholeblood assay the mother clearly showed a population of abnormal granulocytes (approximately 25%), consistent with an Xlinked-carrier status. Interestingly, random X-chromosome inactivation was observed to be not so random. None of the CGD carrier mothers had an even proportion of normal and abnormal PMN. It is unlikely that the nonrandom X chromosome inactivation observed was due to a selective advantage in those cells with normal X chromosomes, since one of the mothers had less than 20% normal PMN. It remains controversial (6, 10) whether lymphocytes are able to generate ROS. Recent publications (10, 11) indicated that after 45 min ROS could be detected in lymphocytes by flow cytometry. The authors suggest that some elaborate form of complementation is occurring. Although this is possible, similar to what is suggested by Emmendo ¨rffer (3), we also suggest that the increased fluorescence observed in the lymphocyte population may simply be the passive diffusion of oxidized fluorescent dye into these cells. In an experiment in which we mixed normal and CGD granulocytes together, we observed a positive shift in fluorescence in the CGD granulocytes (data not shown). This may be a form of complementation, although this could be due to the diffusion of dye into the CGD granulocytes. Additionally, in the CGD carrier mothers, the abnormal or CGD granulocytes expressed higher levels of fluorescence than the granulocytes from the CGD-affected sons. This has been observed by others (3). In the mother with fewer normal granulocytes (,20%), this increase in fluorescence in the abnormal granulocytes did not occur. We feel that the increases in fluorescence in the lymphocytes observed by our group and by Rabesandratana’s group (11) may be the results of diffusion of oxidized dye from the granulocytes into the lymphocytes. Formal complementation studies may have to be performed to resolve this issue. In summary, we feel that we have developed an assay for the diagnosis of CGD and for the detection of X-linked carriers of the CGD mutation which can be performed rapidly and routinely with less than 500 ml of whole blood. This technique should be particularly useful for the diagnosis of CGD in very young infants. ACKNOWLEDGMENTS This work was supported by grant 2430 from the Children’s Memorial Institute for Education and Research.
CLIN. DIAGN. LAB. IMMUNOL. We thank the families for their willing cooperation in this study and Gunter Valet for his helpful suggestions.
REFERENCES 1. Bass, D. A., P. Wallace, L. R. Dechatelet, P. Szejda, M. C. Seeds, and M. Thomas. 1983. Flow cytometric studies of oxidative product formation by neutrophils: a grade response to membrane stimulation. J. Immunol. 130: 1910–1917. 2. Emmendo ¨rffer, A., M. Hecht, M. L. Lohmann-Matthes, and J. Roesler. 1990. A fast and easy method to determine the production of reactive oxygen intermediates by human and murine phagocytes using dihydrorhodamine 123. J. Immunol. Methods 131:269–275. 3. Emmendo ¨rffer, A., M. Nakamura, G. Rothe, K. Spiekermann, M.-L. Lohmann-Matthes, and J. Roesler. 1994. Evaluation of flow cytometric methods for the diagnosis of chronic granulomatous disease variants under routine laboratory conditions. Cytometry 18:147–155. 4. Epling, C. L., D. P. Stites, T. M. McHugh, H. O. Chong, L. L. Blackwood, and D. W. Wara. 1992. Neutrophil function screening in patients with chronic granulomatous disease by a flow cytometric method. Cytometry 13:615–620. 5. Haugland, R. P. 1992. Probes for following endocytosis, p. 99–110. In K. D. Larison (ed.), Molecular probes: handbook of fluorescent probes and research chemicals. Molecular Probes Inc., Eugene, Oreg. 6. Himmelfarb, J., and K. A. Ault. 1993. Reply to Dr. Rabesandratana and Dr. Dornand. Cytometry 14:697. (Letter to the editor.) 7. Himmelfarb, J., R. M. Hakim, D. G. Holbrook, D. A. Leeber, and K. A. Ault. 1992. Detection of granulocyte reactive oxygen species formation in whole blood using flow cytometry. Cytometry 13:83–89. 8. Kamani, N., C. S. August, D. E. Campbell, N. F. Hassan, and S. D. Douglas. 1988. Marrow transplantation in chronic granulomatous disease: an update, with 6-year follow-up. J. Pediatr. 113:697–700. 9. O’Gorman, M. R. G., A. C. McNally, D. C. Anderson, and B. L. Myones. 1993. A rapid whole blood lysis technique for the diagnosis of moderate or severe leukocyte adhesion deficiency (LAD). Ann. N.Y. Acad. Sci. 677:427– 430. 10. Rabesandratana, H., and J. Dornand. 1993. Flow cytometric analysis of oxygen species formation in activated leukocytes. Cytometry 14:695–696. (Letter to the editor.) 11. Rabesandratana, H., A.-M. Fournier, M.-T. Chateau, A. Serre, and J. Dornand. 1992. Increased oxidative metabolism in PMA-activated lymphocytes: a flow cytometric study. Int. J. Immunopharmacol. 14:895–902. 12. Roesler, J., M. Hecht, J. Greihorst, M.-L. Lohmann-Matthes, and A. Emmendo¨rffer. 1991. Diagnosis of chronic granulomatous disease and of its mode of inheritance by dihydrorhodamine 123 and flow microcytofluorometry. Eur. J. Pediatr. 150:161–165. 13. Rothe, G., A. Oser, and G. Valet. 1988. Dihydrorhodamine 123: a new flow cytometric indicator for respiratory burst activity in neutrophil granulocytes. Naturwissenschaften 75:354–355. 14. Trinkle, L. S., S. R. Wellhausen, and K. R. McLeish. 1987. A simultaneous flow cytometric measurement of neutrophil phagocytosis and oxidative burst in whole blood. Diagn. Clin. Immunol. 5:62–68. 15. Windhorst, D. B., B. Holmes, and R. A. Good. 1967. Newly defined X-linked trait in man with demonstration of Lyon effect in carrier females. Lancet i:737–739.
