ABSTRACT
Southeast Asian ovalocytosis (SAO) is characterized by the misfolding of band 3 protein in red blood cells (RBC). The abnormal structure of the band 3 protein results in dysmorphic RBC and related functions. Previous data showed that in vitro storage under hypothermic conditions alters band 3 protein structure and function. Microvesiculation includes shedding of RBC membranes, called RBC-derived microparticles/extracellular vesicles (RMP/EVs), and storage lesions. Unfortunately, there is no evidence of RBC microvesiculation under in vitro storage conditions in heterozygous SAO individuals. This study determined the generation of REVs and procoagulant activity during the storage of SAO blood samples in southern Thailand. Venous blood was collected from eight SAO and seven healthy individuals, preserved in citrate phosphate dextrose-adenine 1 (CPDA-1) at 4 °C for 35 days. The absolute numbers of REVs and PS-expressing RBCs were analyzed using flow cytometry. The procoagulant activity of the produced extracellular vesicles was determined by a clotting time assay. The results showed a significant increase in the number of REVs and PS-expressing RBCs in the SAO blood samples. Significantly correlated PS externalization and procoagulant activity were observed in the SAO blood samples. These lines of evidence indicate that the abnormality of the Band 3 protein is possibly involved in aberrant microvesiculation, exerting procoagulant activity in vitro. Increased pools of REV production and abnormal storage lesions in SAO blood samples should be a concern. Notably, the mechanisms underlying membrane vesiculation depend on the extent of blood cell storage under hypothermic conditions.
ABSTRACT
INTRODUCTION: 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. CONCLUSION: 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.