Flow cytometric analysis of the granulocyte respiratory burst: a comparison study of fluorescent probes.

Chronic granulomatous disease (CGD) is a rare recessive disorder caused by defects in the NADPH oxidase enzyme complex of phagocytes (neutrophils, eosinophils and monocytes). CGD phagocytes fail to produce superoxide and other reactive oxygen species following cell activation (Malech, 1993). The products of oxidase activation can be measured in individual cells by flow cytometry using specific fluorescent probes that increase fluorescence upon oxidation (Trinkle et al., 1987). This approach can be used to confirm a diagnosis of CGD, and to detect the normal/abnormal phagocyte mixture that characterizes the X-linked CGD carrier state. Three fluorescent probes have been described as useful for this purpose: 2'7'-dichlorofluorescin diacetate (DCF) (Bass et al., 1983), 5,6-carboxy-2'7'-dichlorofluorescein diacetate, bis(acetoxymethyl) ester (C-DCF) (Hockenbery et al., 1993) and dihydrorhodamine 123 (DHR) (Rothe et al., 1988; Kinsey et al., 1987). A direct comparison between these three probes has not been reported. In this study we performed a direct comparison between these three probes, evaluating their ability in flow cytometric analysis to maximize fluorescent separation between activated CGD patient and normal granulocytes. Using a whole blood technique with phorbol myristate acetate (PMA) as an activator, it was found that DHR loaded normal granulocytes had a fluorescence intensity which, upon activation, was 48-fold higher than that of C-DCF loaded granulocytes and seven-fold higher than DCF loaded granulocytes (P < 0.001). Use of sodium azide to decrease the catabolism of H2O2 enhanced the fluorescence of DCF by 140%, C-DCF by 45% and DHR by 25%, suggesting that DCF is primarily sensitive to H2O2. DCF and DHR were then evaluated for sensitivity in the detection of small percentages of normal cells in a CGD/normal granulocyte mixture. Normal sub-populations as small as 0.1% could clearly be distinguished using DHR, while DCF was insensitive at this level. Based on these findings, we used DHR in an effort to detect normal granulocytes in a CGD patient following therapeutic granulocyte transfusion. We were able to detect normal granulocytes in the circulation for up to 18 h after transfusion. With these data we show that DHR is the most sensitive flow cytometric indicator for the detection of oxygen reactive species in activated granulocytes and is the best probe for evaluating CGD patients and carriers. In addition, our data suggest that DHR is a useful tool for monitoring circulating normal granulocytes in CGD patients following transfusion, and potentially will be a sensitive probe for assessing the success of such future technologies as gene therapy for CGD.

Genotype-dependent variability in flow cytometric evaluation of reduced nicotinamide adenine dinucleotide phosphate oxidase function in patients with chronic granulomatous disease.

We studied phagocyte reduced nicotinamide adenine dinucleotide phosphate function to evaluate production of reactive oxygen species in both X-linked and autosomal forms of chronic granulomatous disease. We found a consistent and significant difference between the activated granulocyte response of the X-linked (gp91-phagocyte oxidase) form of chronic granulomatous disease (n = 18) and that of the most common autosomal recessive (p47-phagocyte oxidase) form of the disease (n = 17). The data indicate that mutations in the p47-phagocyte oxidase component of the reduced nicotinamide adenine dinucleotide phosphate oxidase component do not completely prevent oxidation despite severe defects in superoxide generation.

Genetic correction of p67phox deficient chronic granulomatous disease using peripheral blood progenitor cells as a target for retrovirus mediated gene transfer.

Chronic granulomatous disease (CGD) can result from any of four single gene defects involving the components of the superoxide (O-2) generating phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We show that transduction of peripheral blood CD34+ hematopoietic progenitors from a p67phox deficient CGD patient with replication defective amphotropic retrovirus encoding p67phox (MFGS-p67phox) significantly corrected the CGD functional defect in phagocyte oxidase activity in vitro. Using a chemiluminescence assay of oxidase activity, we showed that transduced patient CD34+ progenitors differentiating to myeloid cells in culture produced 25% of the total superoxide produced by normal CD34+ progenitors differentiating in culture. A flow cytometric assay of oxidase activity used to assess the oxidase function of individual cells in the cultures indicated that up to 32% of maturing granulocytes derived from transduced CD34+ progenitors from the p67phox CGD patient were oxidase positive with the average level of correction per granulocyte of 85% of that seen with granulocytes in similar cultures of CD34+ progenitors from normal volunteers. Nitroblue tetrazolium dye reduction assays of colonies of transduced progenitors in soft agar indicated that in some studies restoration of oxidase activity occurred in myeloid cells within 44% of granulocyte-erythrocyte-monocyte colonies, and within 28% of the combined group of granulocyte colonies/monocyte colonies/granulocyte monocyte colonies. These high correction rates were achieved without any selective regimen to enrich for transduced cells. This study provides a basis for development of gene therapy for the p67phox deficient form of CGD.

Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease.

Little is known about the potential for engraftment of autologous hematopoietic stem cells in human adults not subjected to myeloablative conditioning regimens. Five adult patients with the p47(phox) deficiency form of chronic granulomatous disease received intravenous infusions of autologous CD34(+) peripheral blood stem cells (PBSCs) that had been transduced ex vivo with a recombinant retrovirus encoding normal p47(phox). Although marrow conditioning was not given, functionally corrected granulocytes were detectable in peripheral blood of all five patients. Peak correction occurred 3-6 weeks after infusion and ranged from 0.004 to 0.05% of total peripheral blood granulocytes. Corrected cells were detectable for as long as 6 months after infusion in some individuals. Thus, prolonged engraftment of autologous PBSCs and continued expression of the transduced gene can occur in adults without conditioning. This trial also piloted the use of animal protein-free medium and a blood-bank-compatible closed system of gas-permeable plastic containers for culture and transduction of the PBSCs. These features enhance the safety of PBSCs directed gene therapy.