OBJECTIVETo determine whether affected reticulocytes could be a reliable marker for the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH), we analyzed CD59-antigen expression on the membranes of reticulocytes and erythrocytes.

METHODSWe studied 10 PNH patients and 5 healthy volunteers by two-color flow cytometry with a membrane permeable fluorescent dye, thiazole orange (TO), and anti-CD59 monoclonal antibodies (MoAb). TO was introduced to gate reticulocytes and anti-CD59 MoAb were used to identify glycosylphosphatidylinositol (GPI)-deficient cells.

RESULTSCells from healthy individuals were only CD59 positive. However, in all PNH patients, CD59-antigen expression could be divided into 3 types: type I cells (CD59 normally positive), type II cells (CD59 partly positive) and type III cells (CD59 negative). The majority of reticulocytes belonged to type III cells, GPI-deficient cells (61.0%). In addition, the percentage of affected reticulocytes was higher than that of erythrocytes.

CONCLUSIONSAnalyzing PNH reticulocytes was important, because most patients had elevated numbers of reticulocytes, which represent more closely the recent erythroid output of BM. However, circulating mature erythrocytes were subject to complement-mediated intravascular lysis. Therefore, the percentage of abnormal erythrocytes may not accurately reflect the proliferation rate of normal and abnormal erythroid progenitor cells. Thus, affected reticulocytes could be a more reliable indicator for the diagnosis of PNH than mature erythrocytes.

Adult ; CD59 Antigens ; analysis ; Erythrocyte Count ; Erythrocytes ; cytology ; immunology ; Female ; Flow Cytometry ; Hemoglobinuria, Paroxysmal ; immunology ; Humans ; Immunophenotyping ; Male ; Middle Aged ; Reticulocytes ; cytology ; immunology

OBJECTIVETo establish a flow cytometric internal standard method for counting platelet-derived microparticles (PMPs) and to study its clinical significance.

METHODSPMPs suspension (platelet poor plasma, PPP) was extracted by gradual centrifugation. According to the size of PMPs, 3 microm and 0.8 microm latex beads were used as internal standards for the quantitation. PMPs were counted by adjusting flow cytometric discrimination and voltage of forward scatter and side scatter.

RESULTSIn 30 healthy donors, the average concentration of resting PMPs was (1.2 x 10(5) +/- 5.7 x 10(4))/ml and that of activated PMPs was (1.6 x 10(6) +/- 9.1 x 10(5))/ml. Compared with healthy donors, PMPs mean value was significantly higher (P < 0.001) in 18 patients with coronary artery disease, 12 with acute cerebral infraction and 23 with chronic renal failure [the average PMPs concentration, (6.1 x 10(5) +/- 2.5 x 10(5))/ml, (6.8 x 10(5) +/- 3.4 x 10(5))/ml and (5.9 x 10(5) +/- 3.1 x 10(5))/ml respectively]. However, no significant difference in PMPs concentration was observed in 25 patients with acute leukemia and severe thrombocytopenia during the aplastic phase after chemotherapy [(1.3 x 10(5) +/- 6.1 x 10(4))/ml, (P > 0.05)].

CONCLUSIONSPMPs is a useful indicator in monitoring platelet activation, and plays an important role in thrombotic disease. By flow cytometric internal standard method, PMPs can be counted rapidly and accurately, which may be very helpful in interlaboratory comparative studies.

Adolescent ; Adult ; Aged ; Blood Platelets ; chemistry ; ultrastructure ; Cell Membrane ; chemistry ; ultrastructure ; Child ; Child, Preschool ; Coronary Disease ; blood ; Flow Cytometry ; methods ; Humans ; Kidney Failure, Chronic ; blood ; Middle Aged ; Particle Size ; Platelet Activation ; Platelet Count ; Platelet Membrane Glycoproteins ; analysis ; Reference Standards