LABORATORY DIAGNOSTIC TECHNIQUE FOR PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA Russian patent published in 2016 - IPC G01N33/533 

FIELD: medicine.

SUBSTANCE: examined blood sample is stained with anti-CD235a (FITC)/ CD59(PE)/CD71 (APC) monoclonal antibodies. The presence of PNH clone in erythrocytes and reticulocytes is stated by the absence of CD59 defensive protein on a reticulocyte membrane recovered by a gate of erythrocytic marker CD235a and transferrin receptor CD71. Assessing the PNH clone in reticulocytes in the gate CD71+ requires collecting at least 20000 events, the gate CD71+ is used to plot FSC(log) vs SSC(log) diagram, whereon reticulocytes are recovered by means of the supplementary gate CD71str, thereby clearing a reticulocyte population from debris and duplexes by sequential gating. If observing 100% CD59-positive reticulocytes, the absence of the PNH clone is stated, and the absence of paroxysmal nocturnal haemoglobinuria is diagnosed. If the patient with clinical symptoms is found to have CD59-negative reticulocytes with the derived values of more than 1%, the presence of the PNH clone is diagnostically stated; the additional study observing an international standard protocol is suggested to prove the diagnosis of paroxysmal nocturnal haemoglobinuria. If observing CD59-negative reticulocytes with the derived values of 0.1-1%, the minor PNH clone is stated and recommended to re-measure 6 months later for a value increase and development of paroxysmal nocturnal haemoglobinuria; if this size of the clone is proved, the subclinical form of paroxysmal nocturnal haemoglobinuria free from clinical signs is diagnosed.

EFFECT: using the multiparameter gating of CD71+ cells makes it possible to eliminate debris, duplexes and non-specifically bound monoclonal antibodies from analysis.

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