title: A novel flow cytometry-based method of analyzing Heinz bodies.
abstract: BACKGROUND
  Heinz bodies are important to diagnosing and managing patients. However, microscopic examination of Heinz bodies has several disadvantages, demonstrating the need for a better method. We explored the potential use of flow cytometry to examine Heinz bodies.
  
  
  METHODS
  Whole-blood samples were collected from patients deficient in G6PD and healthy volunteers. Acetylphenylhydrazine was used to induce formation of Heinz bodies in red blood cells (RBCs). Then, RBCs positive for Heinz bodies were examined using a FACSCanto II cytometer.
  
  
  RESULTS
  RBCs treated with acetylphenylhydrazine formed Heinz bodies and emitted a broad spectrum of fluorescence that could be detected by flow cytometry. The maximum emission of fluorescence was observed at 45 min after the incubation with acetylphenylhydrazine. In addition, the fluorescence emitted was stable for at least 72 h. The flow cytometer could detect the RBCs positive for Heinz bodies even if they made up as little as 0.1% of the total RBC population. Furthermore, the percentage and number, respectively, of RBCs positive for Heinz bodies in G6PD-deficient patients and normal donors exhibited a mean ± standard deviation (SD) of 68.9 ± 27.5 vs. 50.9 ± 28.6 and 96 014 ±35 732 cells/μL vs. 74 688 ± 36 514 cells/μL.
  
  
  CONCLUSIONS
  Heinz bodies induced by acetylphenylhydrazine emit fluorescence, and this fluorescence could be examined using flow cytometry. Our study suggests the potential use of the developed method to investigate the formation of Heinz bodies in clinical samples.
authors.affiliation: Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand.
authors.forename: D
authors.initials: D
authors.lastname: Palasuwan
authors.affiliation: Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand.
authors.forename: A
authors.identifier: http://orcid.org/0000-0002-1903-0512
authors.initials: A
authors.lastname: Palasuwan
authors.affiliation: Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand.
authors.forename: A
authors.initials: A
authors.lastname: Charoensappakit
authors.affiliation: Research Division, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: E
authors.initials: E
authors.lastname: Noulsri
mesh_terms: D004912:Erythrocytes / Q000378:metabolism* / Q000473:pathology; D005260:Female; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005955:Glucosephosphate Dehydrogenase Deficiency / Q000097:blood* / Q000473:pathology; D006366:Heinz Bodies / Q000378:metabolism*; D006801:Humans; D008297:Male
keywords: Heinz body; acetylphenylhydrazine; flow cytometry; red blood cell