Patient characteristics
A total of 356 AIHA patients were identified, of which 274 cases (76.9%) were wAIHAs, 33 cases (9.3%) cAIHAs, and 49 cases (13.8%) mAIHAs. Table 1 showed the characteristics of all mAIHAs. In 49 cases with a median age of 45 years (range from 8 to 77) at initial diagnosis, 22 (44.9%) were males and 27 (55.1%) females. The majority of the 49 cases developed anemia-associated symptoms, such as fatigue (91.8%, 45 of 49), dizziness (32.7%, 16 of 49), chest tightness and exertional palpitations (20.4%, 10 of 49). Also, they presented hemolysis-associated symptoms with dark red urine (61.2%, 30 of 49), jaundice (71.4%, 35 of 49) and Raynaud’s phenomenon (22.4%, 11 of 49). The titer of cold agglutinins in patients with Raynaud’s phenomenon (median 1:1024) was extremely higher than those (median 1:96) without Raynaud’s phenomenon (P = 0.001).
About 35% patients presented initially with severe anemia (Hb < 60 g/L, mean 68.14 ± 23.20 g/L). The ratio of RET had a negative association with Hb levels (r = -0.426, P = 0.002), in addition, which appeared to be stronger positively correlated with LDH, TBIL and IBIL levels (r = 0.740; r = 0.747 ; r = 0.657; P < 0.001). However, the count of RET had no correlation with Hb levels (r = 0.125, P = 0.399). Markers indicating hemolysis including LDH, IBIL and TIBL levels were abnormally elevated in almost all cases. We found that TBIL and IBIL levels had negative association with Hb levels (r = -0.357, P = 0.012; r = -0.424, P = 0.002). The erythropoietin (EPO) levels, except for 3 cases with chronic kidney disease (CKD), showed a positive correlation with the Hb levels (r = -0.361, P = 0.015), which had no correlation with TBIL (P = 0.054), LDH (P = 0.806) levels and the ratio (P = 0.147) and count (P = 0.863) of RET. According to the abdominal ultrasonography results of 43 cases, 79.1% (34 of 43) patients developed splenomegaly, 16.3% (7 of 43) hepatomegaly, and 11.6% (5 of 43) hepatosplenomegaly, there was no statistical significance between spleen and liver sizes with clinical indicators, such as Hb levels, disease course, etiology, and antibody types (data not shown).
Characteristics of secondary mAIHAs
In all mAIHAs, 55.1% (27 of 49) were primary, and the others (44.9%, 22 of 49) secondary with undifferentiated connective tissue disease (UCTD), systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) and LPD being the most common etiologies. The laboratory tests showed that the group of secondary type had lower C3 levels (0.481 ± 0.182 g/L) than that (0.613 ± 0.210 g/L) in primary type (P = 0.03). Monoclonal immunoglobulin IgMκ were found in 6 of 43 patients, 3 of them were diagnosed as LPD, including 2 B-LPD and 1 Waldenström macroglobulinemia (WM) / lymphoplasmacytic lymphoma. The Ham test was positive in 12.2% (6 of 49) mAIHAs patients, most of them (5 of 6) were secondary cases.
Characteristics of mAIHAs patients with Evans syndrome
Eight cases (16.3%) were diagnosed as Evans syndrome in 49 mAIHAs compared to 12.4% (44 of 356) in AIHAs, in which 4 cases were secondary to CTDs. The characteristics of these patients are listed in Table 2. The IgM antibody was more easily detected in DAT in Evans syndrome patients than in the other 41 cases (P = 0.023). No Raynaud’s phenomenon was observed in these patients. There were no differences in Hb, the ratio of RET, C3, TBIL, IBIL, LDH, and EPO levels and relapse rate between Evans syndrome cases and the other 41 cases.
Treatments
At the end of follow-up, complete data for treatments were available for 45 patients, and 4 cases lost to follow-up. 71.1% (32 of 45) patients received second-line or even third-line treatments. The overall RR could be up to 93.3% (42.2% CR + 51.1% PR).
Glucocorticoids. At the end of follow-up, 13 primary patients (28.9%) received glucocorticoids only, of which 46.2% got CR and 53.8% PR, however, only 4 (30.8%) patients reached successful drug withdrawal. Furthermore, we investigated the side effects of glucocorticoids in total of 45 cases, 10 cases (22.2%) presented glucocorticoids-induced hyperglycemia, and 35 cases (77.8%) showed decrease of bone mineral density, of which 2 cases presented osteoporotic fracture.
Low-dose rituximab. Eight primary patients received low-dose rituximab as second-line treatment with the continuing use of glucocorticoids, up to 87.5 % (7 of 8) patients responded effectively with a median time of 4 weeks. The reasons for receiving second-line option were recurrence after the use of glucocorticoids (6 cases) and poor response of glucocorticoids (2 cases). Only 2 of 6 cases relapsed again with a median follow-up time of 30 months (range from 6 to 81). And the response rate of patients with the use of low-dose rituximab plus glucocorticoids was 75%.
Splenectomy. Three recurrent patients underwent splenectomy, one died of lung infection and one intracerebral hemorrhage at the 8 and 15 months after splenectomy respectively. The other one got PR and still live well.
Immunosuppressive drugs. Three primary patients got glucocorticoids combined with cyclosporine of which one got CR, one PR and one NR.
Treatments targeting to secondary mAIHAs. Two patients secondary to B-LPD received chemotherapy and hemolysis was controlled afterwards. One patient secondary to WM incompletely responded to chemotherapy and still needs steroids to control hemolysis. For 10 cases secondary to SLE, 9 were applied with glucocorticoids plus hydroxychloroquine, and 6 cases got CR. Notably, none of them researched drug discontinuation, and the other one received steroids plus mycophenolate and got PR.
Complications
Up to 35.6% (16 of 45) patients developed infections, including 2 lung fungal infections, 13 lung bacterial infections and 1 sepsis of Escherichia coli. Thrombotic events were recorded in 8.9% (4 of 45) patients. 2 patients were accompanied with acute cerebral thrombo-infarction. 2 cases developed deep venous thrombosis (DVT).
Relapse
The relapse rate was as high as 60% (27 of 45) in total 45 patients. We found 10 of 27 (37.0%) relapse happened during upper respiratory tract infection, 8 of 27 (29.6%) during steroids tapering and 9 of 27 (33.3%) during steroids withdraw. Besides, multivariate analysis indicated that Hb levels at onset (P = 0.044) and etiology (P < 0.001) of primary or secondary were significantly and positively correlated with relapse. By means of a binary logistic regression model (χ2 = 13.82, P = 0.008), lower initial Hb levels and primary etiology instead of age (P = 0.236) and gender (P = 0.156) were found as two independent risk predictors for recurrence. As a continuous variable, 1 g/L lower in Hb levels led to a 3.6% higher risk of recurrence outcome (P = 0.029, 95% CI 1.004 - 1.070). In addition, the risk of recurrence in the primary group was 6.3 times higher than in the secondary group (P = 0.019, 95% CI 0.034 - 0.736).
Survival
The 5-year overall survival (OS) rate was 93.6%. At the time of analysis, 5 patients died, 4 of whom were due to lung infections and 1 accompanied with reduced activity of coagulation factors (factor IX, XI, XII) mainly because of intracerebral hemorrhage. The clinical characteristics and therapy of these patients were shown in Table 3. To determine the prognostic factors for survival, KM survival analysis of univariate factors supported the results that lung infection (χ2 = 6.109, P = 0.013) and age ≥ 60 years old (χ2 = 8.501, P = 0.004) were responsible for death in Figure 1. Moreover, by a multivariate Cox regression model, only age ≥ 60 years old represented an independent prognostic risk factor for mAIHA survival (P = 0.032, HR = 11.060, 95% CI 1.225 - 99.872).