Detection of Erythrocytes Deficient of Glycosylphosphatidyl-inositol Anchored Membrane Proteins in Patients with Paroxysmal Nocturnal Hemoglobinuria by the Toxin HEC Secreted by Aeromonas hydrophila J-1

Abstract

Objective: To study the feasibility of diagnosing paroxysmal nocturnal hemoglobinuria (PNH) with toxin HEC, the abbreviation of hemolytic, entreotoxigenicity and cytotoxity secreted by Aeromonas hydrophila J-1.

Methods: The crude toxin HEC was extracted from the culture medium of Aeromonas hydrophila J-1 by precipitating with saturated (NH₄)₂SO₄ and then purified through DEAE52. Purified toxin HEC is different from Aerolysin in molecular weight and necessity of activation. Crude toxin is prepared possessed same effect as purified ones. This crude toxin was used to act on red blood cells (RBCs) from patients with PNH, non-PNH anemia, and normal persons. Absorbance at 630 nm was measured to quantitate the extent of hemolysis. Toxin HEC treated and untreated RBCs were both stained with anti-CD59 monoclonal antibody and FITC labeled goat-anti-mouse IgG. The percentage of CD59⁺ cells was detected by flow cytometry (FCM).

Results: After toxin HEC treatment, RBCs from PNH patients showed resistance to the toxin hemolysis, which was negatively related to the percentage of CD59⁺ cells, while RBCs from normal persons and non-PNH anemic patient were nearly totally lysed.

Conclusion: Detection of RBCs resistance to toxin HEC can be used for the diagnosis of PNH.

Keywords:

• Paroxysmal nocturnal hemoglobinuria
• Toxin HEC
• CD59
• Glycosylphosphatidylinositol-anchored protein
• Aeromonas hydrophila