One hundred and three PNH clone cases referred to a tertiary medical center in Brazil from December 1999 through December 2011 were retrospectively reviewed. A total of 398 patients were screened for the PNH clone using a FCM assay.5,14 The diagnosis of PNH was established by detecting a GPI-AP deficient clone greater than 0.1%, with at least two cell lineages showing GPI deficient populations. The study included 103 patients who had demonstrated the presence of a PNH clone and had available clinical data. The date of PNH diagnosis was based on the first positive FCM analysis. Patients with co-morbid AA were subclassified as severe or non-severe according to published criteria.17,18

The diagnosis of PNH was established by the detection of an unequivocal positive PNH clone by multicolor FCM assay5,11 using a FACSCalibur® cytometer (BD Biosciences, San Jose, USA) and CellQuest Pro software (BD Biosciences, San Jose, USA). The proteins studied were CD55 and CD59 on red cells, neutrophils and monocytes; CD16, CD24 and CD66b on neutrophils and CD14 on monocytes. PNH clones were defined by the presence of GPI-AP deficient cells at a frequency greater than 0.1% of neutrophils, monocytes and red cells, and the proportion of GPI-AP deficient cells (clone size) was defined by the highest level of these cells lacking GPI-anchored proteins.

Debris was thresholded out, and at least 50,000 events in leukocyte tubes and 20,000 events in red cell tubes were collected and analyzed using Paint-a-gate® (BD Biosciences, San Jose, USA) or Infinicyt™ (Cytognos, Salamanca, Spain) software (for samples tested after January 2009). Red cells and granulocytes were identified based on forward and side scatter, and by staining with CD41a FITC and CD45 PercP, respectively. The gates used to define GPI negative populations were established by using normal red cells and granulocytes as controls.

Patients were assigned to one of the three subcategories based on the recently proposed PNH working clinical classification1:

• (I)

  The Classic PNH subcategory included patients with clinical and laboratory evidence of intravascular hemolysis (such as hemoglobinuria, hemoglobinemia, and elevated LDH and bilirubin) but no evidence of bone marrow failure;

• (II)

  Patients in the PNH/AA subcategory were defined by the presence or a history of bone marrow failure in conjunction with clinical and laboratory evidence of intravascular hemolysis;

• (III)

  Patients in the subclinical PNH (PNH-sc/AA) subcategory were identified by the presence of bone marrow failure but without clinical or laboratory evidence of hemolysis.

The criteria for bone marrow failure included bone marrow hypoplasia (cellularity<50%) and at least two of the following three laboratory abnormalities: hemoglobin level <12g/dL, absolute neutrophil count <1.50×109/L, and platelet count (PLT) <100×109/L. Pancytopenia was considered when the three hematopoietic lineages were affected. Haptoglobin values were not used in this study.

Overall survival (OS) was calculated from the date of diagnosis to the date of death or the date of last follow-up. Survival analysis was performed using the Kaplan–Meier method. The distributions of the presentation of characteristics were compared between the three subcategories and between Classic PNH and PNH-sc/AA by the chi-squared or Fisher's exact test as necessary for categorical variables, and by the Kruskal–Wallis (three subcategories) or Mann–Whitney (two groups) test for continuous variables. The percentages of GPI-AP deficient red blood cells and granulocytes were compared using Student's t-test. All statistical analyses were performed using the Statistica v.8 program. A p-value <0.05 was considered statistically significant.

The Ethics Committee of the Hospital de Clinicas, Universidade Federal do Paraná, Brazil approved the study.

Of the 398 patients tested, 125 (31.4%) had a detectable PNH population. Of these, 103 (51 male and 52 female), who had clinical data available, were studied. The median age at presentation was 24.1 years (range: 5.5–62.0 years). There were 24 patients below the age of 18 years (range: 5.5–17.9 years; median: 14.7). All these young patients were in the aplastic groups (11 HPNsc/AA and 13 HPN/AA).

Hemoglobinuria was identified during monitoring in 43 (41.7%) patients, while it was the initial symptomatic manifestation in only 19 (18.4%) patients. The frequencies of infections and bleeding were 46.6% and 47.1%, respectively. Ninety-eight patients (95.1%) presented with asthenia, 33 (32.0%) with abdominal pain, and 14 (13.6%) with renal insufficiency; 17 (16.5%) developed thrombosis during monitoring and 49 patients (47.6%) had documented hemolysis. Twenty-six patients (25.2%) with aplasia at diagnosis developed the PNH clone and hemolysis in a median of 2.35 years after diagnosis. Of these patients, eleven (10.7%) developed hemolysis five years after the diagnosis of aplasia.

Peripheral blood abnormalities were present in 101 patients: 83 (80.6%) had pancytopenia, 12 (11.6%) anemia and thrombocytopenia, and four (3.9%) had anemia and leukopenia. The median hemoglobin level was 8.8g/dL (range: 3.8–14.5g/dL), the median absolute neutrophil count was 0.94×109/L (range: 0.26–1.45×109/L) and the median platelet count was 25×109/L (range: 2–294×109/L). The median LDH concentration was 328U/L (range: 30–7970U/L; normal range 190–240U/L). Ninety patients (87.4%) had hypocellular bone marrow.

Patients included in this study were divided into the subcategories of Classic PNH (10 patients), PNH/AA (39 patients), and PNH-sc/AA (54 patients) based on the proposed PNH working clinical classification.1 The initial characteristics of the subgroups are summarized in Table 1.

The median ages of PNH-sc/AA, PNH/AA and Classic PNH patients were 25.1, 21.5 and 34.7 years, respectively (p-value=0.016). The clinical symptoms of hemolysis, hemoglobinuria and thrombosis were higher in the hemolytic forms of PNH (PNH/AA and Classic PNH) than in PNH-sc/AA (p-value<0.001), whereas infection and fever were higher in the PNH-sc/AA group (p-value<0.001). The median numbers of neutrophils and platelets were significantly lower in the aplastic groups than in the Classic PNH group (p-value for both <0.001). On the other hand, the Classic PNH group had higher hemolytic markers, such as LDH (p-value=0.001), reticulocyte count (p-value <0.001) and bilirubin levels (p-value <0.001 – Table 2).

FCM data showed that larger PNH clones were associated with classical PNH and thrombotic events, while smaller PNH clones were associated with bone marrow aplasia. At diagnosis PNH clone sizes in red cells, granulocytes and monocytes were significantly different between the three clinical subcategories (p-value <0.001) for all the studied monoclonal antibodies (CD55, CD59, CD14, CD16, CD24 and CD66b). The median PNH clone percentages in red cells were 0.04% (range: 0–18.0%), 15.8% (range: 0–99.7%) and 82.2% (range: 0–99.85%) in PNH-sc/AA, PNH/AA and Classic PNH, respectively (p-value <0.001). Moreover, the median PNH clone percentages in granulocytes were 7.3% (range: 0.3–68.7%), 63.0% (range: 1.7–99.8%) and 98.0% (range: 81.3–100.0%) in PNH-sc/AA, PNH/AA and Classic PNH, respectively (p-value <0.001 – Figures 1–3).

Figure 1.

Median percentages of paroxysmal nocturnal hemoglobinuria clone in erythrocyte and neutrophil clones.

(0.16MB).

Figure 2.

Number of patients with predominantly small erythrocyte clones in paroxysmal nocturnal hemoglobinuria with subclinical features and aplastic anemia, intermediate clones in paroxysmal nocturnal hemoglobinuria with aplastic anemia and large clones in classic paroxysmal nocturnal hemoglobinuria.

(0.11MB).

Figure 3.

Number of patients with predominantly small granulocyte clones in paroxysmal nocturnal hemoglobinuria with subclinical features and aplastic anemia, intermediate in paroxysmal nocturnal hemoglobinuria with aplastic anemia and large in classic paroxysmal nocturnal hemoglobinuria.

(0.11MB).

Seventeen patients presented with thrombotic events: six in the Classic PNH group, nine in the PNH/AA group and two in the PNH-sc/AA group. There were five cases of deep vein thrombosis (DVT), five abdominal thromboses, five arterial thromboses with ischemic stroke, one sinus venous thrombosis and one renal vein thrombosis. Two PNHsc/AA patients had DVT despite the absence of hemolytic symptoms, and died. Four of the PNH/AA patients died due to thrombotic events. Regarding the PNH clone, clones larger than 50% were seen in neutrophils of 13 (76.5%) patients (p-value <0.001), and in erythrocytes of six (35.3%) patients (p-value=0.005 – Table 3).

The differences between patients with or without thrombotic events are shown in Table 4. There were statistically significant differences in the number of platelets (p-value=0.001), LDH (p-value=0.002) and in median percentages of the PNH clone in neutrophils (p-value <0.001) and erythrocytes (p-value=0.008). The median neutrophil PNH clone in thrombotic patients was 92.7% (range: 3.4–100%) vs. 21.8% (range: 0.25–99.9%) in non-thrombotic patients. The median erythrocyte PNH clone was 31.8% (range: 0.0–97.1%) vs. 1.2% (range: 0.0–92.2%) in thrombotic and non-thrombotic patients, respectively. The bone marrow cellularity, hemoglobin concentration, leukocyte count and absolute reticulocyte count were similar between the two groups. There were six deaths among patients with thrombotic events (35.3%), and eight (9.3%) among the non-thrombotic group.

As a retrospective study, the cohort of patients received different treatments depending on the physician and time criteria. The majority of patients (81.9%) of aplastic groups (PNH-sc/AA and PNH/AA) received cyclosporine A-based immunosuppressive therapy with or without corticosteroids some time during the follow-up, and 12 patients (12.7%) received anti-human thymocyte immunoglobulin immunosuppressive therapy. Hematopoietic stem cell transplantation (HSCT) was performed in 27 patients (28.7%), the majority of whom were in the PNH-sc/AA group. Nine PNH/AA and two Classic PNH patients received HSCT due to hemolytic symptoms. Eculizumab was introduced in 2010, and five patients received this drug until the conclusion of the study.

The median follow-up period for all patients was 49.2 months (range: 2.4–310 months), with an OS of 81.7% at ten years after diagnosis. There was a slight difference in survival between PNH-sc/AA and the other two groups (76.5% vs. 87.9%; p-value=0.112). In total 14 patients died, nine in the PNH-sc/AA group and five in the PNH/AA group. The causes of death were sepsis (six patients), pulmonary thromboembolism (four patients), mesenteric thrombosis (two patients), severe graft-versus-host disease (GVHD) after HSCT (one patient) and sudden death (one patient). No death was reported in the classical PNH group by the end of the follow-up period.