OBJECTIVETo estimate whether the expression of glycosylphosphatidylinositol (GPI)-anchored protein in B lymphocytes could be an indicator for detecting paroxysmal nocturnal hemoglobinuria (PNH).

METHODSFlow cytometry and two-color McAbs were used to detect CD59 expression in B lymphocytes and granulocytes of 92 clinical samples. The feasibility of the indicators for diagnosis of PNH was assessed.

RESULTSB lymphocytes from healthy individuals mainly showed a single population of cells strongly positive for CD59, as the percentage of CD59 expression was (96.3 +/- 1.2)%. In PNH patients, B lymphocytes showed decreased expression of CD59, as well as negative cells and partly-positive cells were observed, as the percentage of CD59 expression was 10.5%-92.3%. In 26 of 28 non-PNH anemia patients, CD59 expression in B lymphocyte was normal, and in the other 2 patients, CD59 expression were decreased (< 95%), but no negative cell peak was observed. The precision CV of CD59 in B lymphocyte assay is less than 4.4%, stained samples could be kept stable for 24 h, the CD59 in B lymphocyte assay was 100% in accordance with routine assay (CD59 in granulocyte assay). The sensitivity of the indicators (CD59 in B lymphocytes and granulocytes) was 100%, the specificity was 97.4%, misdiagnosis rate was 2.6%, the rate of failing to diagnosis was 0%, the rate of positive forecast was 87.5%, the rate of negative forecast was 100%, the accuracy was 97.8%.

CONCLUSIONSThe test of CD59 expression in B lymphocytes by flow cytometry was simple, accurate and reproduceable. It could be a good marker for diagnosis of PNH because of high sensitivity and specificity.

B-Lymphocytes ; metabolism ; Biomarkers ; CD59 Antigens ; blood ; Female ; Flow Cytometry ; Glycosylphosphatidylinositols ; biosynthesis ; Granulocytes ; metabolism ; Hemoglobinuria, Paroxysmal ; diagnosis ; Humans ; Male ; Membrane Proteins ; metabolism ; Sensitivity and Specificity

In recent years, there have been lots of progresses in the studies on red cell diseases in China, especially bone marrow failure diseases including immuno-related pancytopenia, aplastic anemia, myelodysplastic syndrome, and paroxymal nocturnal hemoglobinuria. Numerous laboratory experiments as well as clinical researches have been carried out by Chinese hematologists, which brought about much clearer pathogenesis, more rational diagnosis methods and more effective therapies for red cell diseases.
Anemia, Aplastic ; diagnosis ; epidemiology ; etiology ; metabolism ; China ; Hematologic Diseases ; diagnosis ; epidemiology ; etiology ; metabolism ; Hemoglobinuria, Paroxysmal ; diagnosis ; epidemiology ; etiology ; metabolism ; Humans ; Myelodysplastic Syndromes ; diagnosis ; epidemiology ; etiology ; metabolism ; Pancytopenia ; diagnosis ; epidemiology ; etiology ; metabolism

BACKGROUND: Final diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) may take years demanding a quick diagnosis measure. We used the facts that PNH cells are damaged in acid, and reagents for measuring reticulocytes in Coulter DxH800 (Beckman Coulter, USA) are weakly acidic and hypotonic, to create a new PNH screening marker. METHODS: We analyzed 979 complete blood counts (CBC) data from 963 patients including 57 data from 44 PNH patients. Standard criteria for PNH assay for population selection were followed: flow cytometry for CD55 and CD59 on red blood cells (RBCs) to a detection level of 1%; and fluorescent aerolysin, CD24 and CD15 in granulocytes to 0.1%. Twenty-four PNH minor clone-positive samples (minor-PNH+) were taken, in which the clone population was <5% of RBCs and/or granulocytes. Excluding PNH and minor-PNH+ patients, the population was divided into anemia, malignancy, infection, and normal groups. Parameters exhibiting a distinct demarcation between PNH and non-PNH groups were identified, and each parameter cutoff value was sought that includes the maximum [minimum] number of PNH [non-PNH] patients. RESULTS: Cutoff values for 5 selected CBC parameters (MRV, RDWR, MSCV, MN-AL2-NRET, and IRF) were determined. Positive rates were: PNH (86.0%), minor-PNH+ (33.3%), others (5.0%), anemia (13.4%), malignancy (5.3%), infection (3.7%), normal (0.0%); within anemia group, aplastic anemia (40.0%), immune hemolytic anemia (11.1%), iron deficiency anemia (1.6%). Sensitivity (86.0%), specificity (95.0%), PPV (52.1%), and NPV (99.1%) were achieved in PNH screening. CONCLUSION: A new PNH screening marker is proposed with 95% specificity and 86% sensitivity. The flag identifies PNH patients, reducing time to final diagnosis by flow cytometry.
Antigens, CD15/metabolism ; Antigens, CD24/metabolism ; Antigens, CD55/metabolism ; Antigens, CD59/metabolism ; Biomarkers/metabolism ; Blood Cell Count ; Erythrocytes/cytology/metabolism ; Flow Cytometry ; Granulocytes/cytology/metabolism ; Hemoglobinuria, Paroxysmal/*diagnosis/metabolism ; Humans ; Sensitivity and Specificity

Paroxysmal nocturnal hemoglobinuria (PNH) is a progressive, systemic, life-threatening disease, characterized by chronic uncontrolled complement activation. A retrospective analysis of 301 Korean PNH patients who had not received eculizumab was performed to systematically identify the clinical symptoms and signs predictive of mortality. PNH patients with hemolysis (lactate dehydrogenase [LDH] > or = 1.5 x the upper limit of normal [ULN]) have a 4.8-fold higher mortality rate compared with the age- and sex-matched general population (P < 0.001). In contrast, patients with LDH < 1.5 x ULN have a similar mortality rate as the general population (P = 0.824). Thromboembolism (TE) (odds ratio [OR] 7.11; 95% confidence interval [CI] (3.052-16.562), renal impairment (OR, 2.953; 95% CI, 1.116-7.818) and PNH-cytopenia (OR, 2.547; 95% CI, 1.159-5.597) are independent risk factors for mortality, with mortality rates 14-fold (P < 0.001), 8-fold (P < 0.001), and 6.2-fold (P < 0.001) greater than that of the age- and sex-matched general population, respectively. The combination of hemolysis and 1 or more of the clinical symptoms such as abdominal pain, chest pain, or dyspnea, resulted in a much greater increased mortality rate when compared with patients with just the individual symptom alone or just hemolysis. Early identification of risk factors related to mortality is crucial for the management of PNH. This trial was registered at www.clinicaltrials.gov as NCT01224483.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antibodies, Monoclonal/therapeutic use ; Antibodies, Monoclonal, Humanized/therapeutic use ; Area Under Curve ; Child ; Dyspnea/etiology ; Female ; Hemoglobinuria, Paroxysmal/*diagnosis/drug therapy/mortality ; Hemolysis ; Humans ; Kaplan-Meier Estimate ; Kidney Diseases/complications/diagnosis ; L-Lactate Dehydrogenase/metabolism ; Male ; Middle Aged ; Odds Ratio ; ROC Curve ; Registries ; Republic of Korea ; Retrospective Studies ; Risk Factors ; Thromboembolism/complications/diagnosis ; Young Adult