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[Rapid diagnosis of paroxysmal nocturnal hemoglobinuria by gel test agglutination]
J M Navenot1, D Bernard, Y Petit-Frioux
1Centre Régional de Transfusion Sanguine, Nantes.
Abstract:
Murine monoclonal antibodies (MoAbs) directed against DAF (Decay Accelerating Factor, CD55 antigen) and MIRL (Membrane Inhibitor of Reactive Lysis, CD59 antigen) were used to identify the affected red cells (CD55-/CD59-) of PNH patients. MoAbs NaM16-4D3 (CD55, IgG2a) and NaM77-1E5 (CD59, IgG3) weakly agglutinate red cells and represent powerful tools to quantitate normal (PNHI) and abnormal (PNHII and PNHIII) cells from PNH patients by indirect flow cytometry. MoAbs NaM125-7H10 (CD55) and NaM123-6G12 (CD59), both IgM, were selected for their agglutinating properties and used for the separation of PNHI from PNHII and PNHIII red cells by the gel test technology. From analysis of artificial mixtures of DAF+ and DAF- cells, a direct relationship was established between fluorescent cells detected by flow cytometry, and erythrocytes agglutinated in microtyping cards. The method was further confirmed by analysis of ten blood samples from PHN patients and represent an alternative to classical hemolysis tests. On the basis of our experience we propose the following for the diagnosis of PNH: 1) agglutination test with NaCl microtyping cards using IgM CD55 and CD59; 2) flow cytometry analysis for accurate quantitation of CD55-/CD59- red cells.
Insights
Monoclonal antibodies targeting Decay Accelerating Factor (CD55) and Membrane Inhibitor of Reactive Lysis (CD59) aid in diagnosing paroxysmal nocturnal hemoglobinuria (PNH). These antibodies enable accurate identification and quantification of affected red blood cells.
Area of Science:
- Hematology
- Immunology
- Cell Biology
Background:
- Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by a deficiency of specific complement regulatory proteins on red blood cells.
- Decay Accelerating Factor (DAF, CD55) and Membrane Inhibitor of Reactive Lysis (MIRL, CD59) are crucial for preventing complement-mediated hemolysis.
- Identifying PNH patients relies on detecting red blood cells lacking these protective antigens.
Purpose of the Study:
- To develop and validate novel monoclonal antibody-based methods for PNH diagnosis.
- To utilize antibodies against CD55 and CD59 for accurate identification and quantification of PNH red blood cells.
- To establish an alternative diagnostic approach to traditional hemolysis tests for PNH.
Main Methods:
- Murine monoclonal antibodies (MoAbs) targeting CD55 and CD59 were employed.
- Indirect flow cytometry was used with weakly agglutinating MoAbs (IgG2a, IgG3) for cell quantitation.
- Gel test technology utilized strongly agglutinating IgM MoAbs for separating PNH cell populations.
- Artificial mixtures of DAF+ and DAF- cells were analyzed to correlate flow cytometry and agglutination results.
Main Results:
- A direct relationship was established between flow cytometry detection of fluorescent cells and erythrocyte agglutination in microtyping cards.
- The developed methods were validated using blood samples from ten PNH patients.
- The agglutination and flow cytometry techniques demonstrated effectiveness in identifying and quantifying CD55-/CD59- red cells.
Conclusions:
- Monoclonal antibodies against CD55 and CD59 offer a reliable alternative to classical hemolysis tests for PNH diagnosis.
- The proposed diagnostic strategy includes agglutination tests using IgM CD55 and CD59 antibodies and flow cytometry for precise quantitation.
- These antibody-based methods provide accurate identification and quantification of PNH red blood cells, aiding in PNH diagnosis and management.