Rapid whole-blood flow cytometry assay for diagnosis of chronic granulomatous disease

Abstract

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.

Resistance ofNeisseria gonorrhoeaeto non-oxidative killing by adherent human polymorphonuclear leucocytes

“…The genetic deficiency in CGD donor #2, also male, has not been defined. A whole‐blood flow cytometry assay was used to demonstrate that PMNs from donor #2 produced no respiratory burst (O'Gorman and Corrochano, 1995).…”

M1/70 attenuates blood-borne neutrophil oxidants, activation, and myofiber damage following stretch injury

“…Samples were collected again 24-h postinjury immediately before tissue harvest. Sodium-citrate-treated whole blood aliquots for flow cytometry were kept at room temperature, and the procedure of cell preparation was started within 30 min of collection (43). EDTAtreated aliquots for IL-8 ELISA were maintained on ice and immediately centrifuged at 2,750 rpm for 10 min at 4°C to avoid cytokine synthesis or degradation in vitro.…”

Correlation between flow cytometry and molecular findings in autosomal recessive chronic granulomatous disease: A cohort study from Oman

“…8,9 The test utilizes dihydrorhodamine 123 (DHR 123) dye to measure intracellular ROS generated during the respiratory burst upon phorbol 12-myristate 13-acetate (PMA) stimulation. 10,11 Flow cytometry is sensitive for CGD diagnosis and in predicting carrier status, and has been suggested for predicting underlying genotypes. 9,11 While patients with XL-CGD have been found to have no oxidative burst activity on flow cytometry, maternal carriers are characterized by a bimodal flow cytometry-generated fluorescent pattern.…”

“…10,11 Flow cytometry is sensitive for CGD diagnosis and in predicting carrier status, and has been suggested for predicting underlying genotypes. 9,11 While patients with XL-CGD have been found to have no oxidative burst activity on flow cytometry, maternal carriers are characterized by a bimodal flow cytometry-generated fluorescent pattern. 9,11 However, a smaller proportion of these patients have been described as having a modest oxidative burst activity with broad peaks, which is more consistent with AR-CGD caused by p47 phox deficiency.…”

“…9,11 While patients with XL-CGD have been found to have no oxidative burst activity on flow cytometry, maternal carriers are characterized by a bimodal flow cytometry-generated fluorescent pattern. 9,11 However, a smaller proportion of these patients have been described as having a modest oxidative burst activity with broad peaks, which is more consistent with AR-CGD caused by p47 phox deficiency. 9 Screening carriers by flow cytometry can fail in detecting the mosaic pattern of neutrophil activation in carriers that have skewed X-chromosome inactivation, which leads to a normal flow cytometry result.…”