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Delayed Blood Processing Leads to Rapid Deterioration in the Measurement of the Neutrophil Respiratory Burst by the Dihydrorhodamine‐123 Reduction Assay
Author(s) -
Quach Alex,
Glowik Shan,
Putty Trishni,
Ferrante Antonio
Publication year - 2019
Publication title -
cytometry part b: clinical cytometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.646
H-Index - 61
eISSN - 1552-4957
pISSN - 1552-4949
DOI - 10.1002/cyto.b.21767
Subject(s) - respiratory burst , venipuncture , medicine , respiratory system , nadph oxidase , absolute neutrophil count , whole blood , granulocyte , neutrophile , rhodamine 123 , ex vivo , immunology , anesthesia , chemistry , biochemistry , neutropenia , oxidative stress , in vitro , toxicity , inflammation , multiple drug resistance , antibiotics
Background Neutrophils ex vivo in whole blood specimens are widely understood to decay rapidly when compared to other leukocytes, requiring assessment of neutrophil activity to be performed shortly after blood collection. There is a disparity in evidence for decay rates in measurements and recommended time‐frames for assaying neutrophil parameters in particular assays following blood collection. We, therefore, evaluated the decline in the neutrophil respiratory burst, typically screened for assessing congenital NADPH oxidase defects, over a shorter time‐course than previously published experiments. Methods The neutrophil respiratory burst was assessed by flow cytometric detection of DHR‐123 oxidation to rhodamine‐123 (Rho123), following stimulation of neutrophils by phorbol myristate acetate (PMA), in heparinized healthy donor blood specimens immediately following venipuncture, and then at 3 and 5 h later with ambient temperature or refrigerated specimen storage. Results A consistent time‐dependent decline in the Rho123 fluorescence of PMA‐stimulated neutrophils was detected in the healthy donor specimens, indicating a decay in respiratory burst activity. Neutrophil oxidative indexes calculated for half of the specimens at 3 and 5 h of age, fell below our normal laboratory lower limit. We also found that Rho123 histograms of PMA‐stimulated neutrophils from stored healthy donor specimens have a risk of misinterpretation due to mimicking the appearance of histograms from carriers of CGD and other NADPH oxidase defects. Refrigeration of specimens did not significantly minimize decay. Conclusions DHR assay of the neutrophil respiratory burst from blood specimens at 3 h post‐venipuncture and beyond can generate unreliable clinical measurements due to decay. © 2019 International Clinical Cytometry Society