Analysis of clinical characteristics of 92 patients with paroxysmal nocturnal hemoglobinuria: A single institution experience in China

Abstract Objectives We performed a retrospective analysis to investigate the clinical characteristics and therapeutic strategies of Chinese paroxysmal nocturnal hemoglobinuria (PNH) patients, and assessed the efficacy and safety of glucocorticoid in PNH patients. Methods The clinical data of 92 PNH cases in our hospital were analyzed, including clinical manifestation, laboratory examination, treatment efficacy, and survival. Results The main clinical manifestations of these patients included hemoglobinuria, anemia, fatigue, dyspnea, headache, abdominal pain, and erectile dysfunction. Glucocorticoid is still the first‐line treatment for PNH patients to control hemolytic attack, and the short‐term remission rate (12 months) is 79.01% (64/81). Meanwhile, the overall survival (OS) of 10 years after diagnosis was estimated as 70.77% (46/65). Moreover, Cox proportional risk model for multivariate analysis showed that the increase in LDH multiple, thrombosis complications, and complicated with bone marrow failure were the independent adverse prognostic factors affecting the survival of PNH patients. Conclusion Paroxysmal nocturnal hemoglobinuria patients in mainland China have various clinical features, while lower incidences of thrombosis and renal damage. Thrombosis and bone marrow failure are two complications with worse prognosis.

anchors leads to absence of all proteins that utilize GPI to attach to the plasma membrane.
The diagnosis of PNH is based on the detection of GPI-anchored proteins (such as CD59, CD55) or the GPI-anchor (flaer) by flow cytometry, which is more sensitive and replaced the Ham test and sucrose lysis tests. 3 Significant advances have been reported in the detection of the GPI-AP-deficient cells, as well as in the pathophysiology of the disease. In treatment aspect, recombinant human anticomplement C5 monoclonal antibodies, new complement inhibitors, and allogeneic hematopoietic stem cell transplantation have made great progress in the field of PNH therapy. [4][5][6][7][8] China as a developing country, eculizumab is not yet available in China, and we have no experience with eculizumab, so the traditional treatment of PNH is aimed at protecting PNH clone, reducing complement attack and destruction, and alleviating hemolysis. Glucocorticoid is still the pre-

| Patients
Ninety-two patients diagnosed as PNH from January 2005 to December 2015 in Tianjin Medical University General Hospital were enrolled in this retrospective study. It is worth mentioning that the 92 PNH patients enrolled in our study were all outpatients. Among them, patients with severe acute hemolytic episodes are treated in the income ward. For patients with mild illness, patients who did not have hemolysis were outpatient visits (including oral glucocorticoid therapy, PNH clone monitoring), and the income wards continued to be treated as soon as acute hemolysis or aggravation occurred.
The clinical data of the patients were collected including gender, age, clinical classification, clinical manifestations, and laboratory findings. Paroxysmal nocturnal hemoglobinuria is classified into three different clinical forms: classic-PNH, PNH in the setting of another bone marrow failure syndrome (PNH/AA, PNH/MDS), and subclinical PNH (non-hemolytic PNH). 4,9 The PNH diagnostic criteria refer to Chinese expert consensus on the diagnosis and treatment of PNH (2014), 10 and we also synthetically analyzed the results of the international PNH Working Group on PNH treatment. 11,12 The chronic kidney disease (CKD) classification was divided into five stages according to eGFR(mL/min/1.73 m 2 ), which were as follows: grade 1 (eGFR > 90), grade 2 (eGFR60-89), grade 3 (eGFR30-59), grade 4 (eGFR15-29), and grade 5 (eGFR < 15).
The research was in compliance of the declaration of Helsinki, and the protocol was approved by the ethical committee of Tianjin Medical University General Hospital. Consent for research and publication was obtained from participants and/or their immediate family if certain participants had passed away. In order to avoid losing clinical features, we collect the symptoms and signs of patients through the quality of life assessment questionnaire (FACIT Weakness Scale and EORTC QLQ-C30 Questionnaire divided into questionnaires, general health status, and working status).

| Treatment and therapeutic index
All patients received glucocorticoid treatment after diagnosis. The initial treatment included methylprednisolone intravenous infusion of 1 mg/kg/d and vitamin E (300 mg/day), and the dose of methylprednisolone may increase as appropriate if hemolytic crisis. Patients who had granulocytopenia (<1.5 × 10 9 ) at admission were treated with granulocyte stimulating factor (2-5 ug to anticoagulation. Except for two patients with bleeding tendency during the treatment, the other nine patients were treated with anticoagulation for 2 weeks. 13,14 Chinese hematology experts have formulated the criteria for the efficacy of glucocorticoids in the treatment of PNH, 10

| Statistical analysis
All data were analyzed by SPSS 21.0 statistical software. The classification variables were compared by Mann-Whitney U test (two groups) or Kruskal-Wallis test (three groups and above). Chi-square test or Fisher exact test was used to test the difference. Kaplan-Meier method was used to describe the distribution of survival status. Log-rank test was used to analyze the prognosis of single factor, and Cox proportional regression model was used for multivariate analysis. P < .05 showed significant difference.

| The characteristics of PNH patients
The clinical features and laboratory examinations of 92 PNH patients were showed in Table
The total course of glucocorticoid treatment was 7.5

| Adverse effects of glucocorticoid therapy
The most common adverse events during glucocorticoids were

| Complications
In

| D ISCUSS I ON
PNH is a rare, life-threatening, and debilitating clonal blood disorder caused by an acquired mutation in the phosphatidylinositol glycan (PIG)-A gene. Clinically, PNH is characterized by a classical triad of acquired Coombs-negative intravascular hemolytic anemia, thrombophilia, and various degrees of bone marrow failure. [15][16][17] The resulting chronic intravascular hemolysis is the underlying cause of PNH morbidities and mortality. Our study showed that both sexes can be affected, and the ratio of male to female is 1.7:1. The median age of PNH patients at diagnosis was 37.4 years old, the male is slightly higher, the age span is very significant, and our case-series study and previous reports revealed a slight preponderance of male in PNH.
The clinical manifestations included hemoglobinuria (54%), anemia (78%), hemorrhage (13%), pancytopenia (20.99%), and F I G U R E 1 Glucocorticoid treatment efficacy in PNH patients. After 2-4 wk treatment, the level of Hb increased significantly, while the levels of Ret, LDH, and TBIL were reduced, and the above hemolysis indicators were significantly different before and after treatment (A). The proportion of CD59-negative neutrophils is positively related to the level of LDH in 81 PNH patients (r = .351, P = .0013) (B). The proportion of CD59negative neutrophils in 11 PNH patients with combined thrombosis is significantly higher than those without thrombosis (C) (P = .0028, ** represents P < .01, *** represents P < .001) thrombus (12%). Though PNH was usually described as a intravascular hemolysis, PNH-associated cytopenias were common in our study. Bone marrow failure is the PNH common clinical manifestations, occurs to some degree in all PNH patients, and, in its most extreme form, presents as immune-mediated severe aplastic anemia. Although many patients showed a two-line more blood cell reduce, but confirmed by bone marrow aspiration and biopsy, bad hyperplasia only 6.17%. We also evaluated the common symptoms of PNH patients and compared the differences between physician evaluation and patient self-assessment. The results showed a significant difference in abdominal pain, headache, dysphagia, and erectile dysfunction. These differences remind us that we need to pay much attention to the common symptoms of PNH patients when they are interrogation, because patients often do not mention these symptoms voluntarily. Clinicians should ask patients voluntarily, so that they do not miss the data of these common symptoms. And we analyzed the correlation between the level of LDH and the ratio of CD59 − NEU, and the results showed that the size of PNH clones was positively correlated with hemolysis degree. The detection of PNH cloning provides the gold standard for diagnosis on the one hand and helps to predict clinical symptoms and complications on the other hand. 18 The complications in the patients consist of recurrent infections, thrombotic events, hepatosplenomegaly, renal function damage, and abnormal coagulation function. In our study, recurrent infection was the main problem, which may be due to a significant proportion of the patients with bone marrow failure.
Later-stage renal dysfunction or failure is a major cause of morbidity and mortality in PNH. Studies have reported that the five-year mortality rate for PNH patients is about 35% and median survival time is about 10-15 years, and kidney failure contributes to 8%-18% of PNH-related deaths. 19 Clark 20 reported about 32% of PNH patients with renal failure (CKD3-5). Previous reports showed that the mechanism of renal damage is considered to be persistent intravascular hemolysis, plasma-free hemoglobin, and/or micro-thrombus repeatedly acting on renal tissue. The large amount of free hemoglobin produced by hemolysis seriously consumes nitric oxide, which leads to the increase in renal artery resistance, the decrease in renal blood flow, and the impairment of renal function. 21,22 In our study of the 81 patients, one patient had been identified as specifically having chronic renal impairment in a review of their medical histories with later-stage CKD (Stages 3-5). Twelve of these 81 patients had been identified to have one or more major clinical kidney (MCK) events. And there is a significant difference in LDH between patients with normal renal function and CKD patients; therefore, it is speculated that the mechanism of PNH combined with renal damage may be closely related to intravascular hemolysis, and the specific mechanism of PNH combined with renal dysfunction needs further study in large samples.
The estimated OS of 10 years after diagnosis was 70.09% in our study, which was similar with 71% reported by Fujioka, 23 and 75% by de Latour, 9 but higher than 50% by Dacie and Lewis. 24 We compared the OS of 10 years after diagnosis from the following five aspects: the degree of LDH elevation, the degree of PNH clone increase, whether it was associated with thrombosis or bone marrow failure and the hemolytic attack frequency, the results showed that   34 There is also research and development of oral C5 inhibitors. 35 These two formulations improve the inconvenience caused by intravenous injection of eculizumab. It is hoped that in the near future, eculizumab can be listed in China and benefit from PNH patients in China.

| CON CLUS ION
Paroxysmal nocturnal hemoglobinuria is a life-threatening blood disorder in which patients are at risk for end-organ damage and organ failure because of chronic hemolysis. Renal dysfunction or damage is a common and progressive medical complication in patients with PNH that also contributes to mortality in these patients. Our retrospective review of 92 PNH patients over a 10-year period provided us a useful information, including the clinical manifestation, laboratory examination, treatment, complications, and prognostic factors influencing survival, which would help us to understand the pathogenesis of PNH.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.