AIHA with established associated diseases were considered as secondary AIHA. Forty-one (60.3% of total) cases had associated diseases, mainly infectious diseases (40 cases, 97.6%) such as respiratory infection (25 cases, 61.0%), enteritis (7 cases, 17.1%), Epstein-Barr virus/cytomegalovirus infection (7 cases, 17.1%), and soft tissue infection (1 case, 2.4%). During follow-up, 1 patient was diagnosed with ulcerative colitis, but the anemia recovered. Six patients presented with positive antinuclear antibody (ANA) at diagnosis and had presumed systemic lupus erythematosus (SLE). During follow-up, all had recovered from anemia and had no symptoms of SLE; repeated screening tests for SLE also showed insufficient evidence of SLE.
Of the patients enrolled, 64 cases (94.1%) were DAT positive with a total of 9 types of antibody combinations (Table 3), whereas 4 cases (5.9%) were DAT negative.
All patients’ Hb declined at diagnosis. Among all 68 cases, 7 cases (10.3%) had mild anemia (Hb>90 g/L); 24 cases (35.3%) had moderate anemia (Hb 60 to 90); 35 cases (51.5%) had severe anemia (Hb 30 to 60); 2 cases (2.9%) had very severe anemia (Hb<30). The average Hb was 61.0±21.2 g/L. Reticulocytosis as adequate response to AIHA was seen in 63 cases (92.6%), whereas normal reticulocyte level as inadequate response was seen in only 5 cases (7.4%). Leukocytosis was noted in 34 (50.0%) patients, leukopenia in 4 (5.9%) patients, and 30 (44.1%) cases had normal WBC levels. All ES patients had varied degrees of thrombocytopenia with the median platelet count decreased to 38.5×109/L (10 to 88×109/L).
Forty-five patients (66.2%) had an increased total bilirubin level with the median of 34.1 μmol/L. Forty-four (64.7%) cases had elevated indirect bilirubin and the median was 22.5 μmol/L. In AIHA-only patients, the median indirect bilirubin was higher than that in ES patients (P=0.03).
Of the 68 patients, the median LDH level was 596.4 U/L (229 to 5501) and there were 55 (80.9%) cases with LDH level exceeded the upper normal range of 382 U/L. There was no significant difference of average LDH between AIHA-only and ES groups (P=0.21).
Comparison of the Severity of Hemolysis
We stratified patients on the basis of age (1 y or below vs. above 1 y), initial presentations (AIHA-only vs. ES), antibody detected by DAT (single-antibody vs. combined-antibody), and presence of IgM (IgM-contained vs. non–IgM-contained) to compare the severity of hemolysis. The average Hb level was lower in the 1-year or below age group than in the above 1-year age group, combined-antibody group than single-antibody group, and IgM-contained group than non–IgM-contained group, respectively (P<0.05 for all). For free plasma Hb, there is no statistical difference between the comparison groups (P>0.05 for all) (Table 3).
All patients were treated with glucocorticoid as the first-line therapy with a starting dose of methylprednisolone 2 to 5 mg/kg/d based on the severity of the anemia. Once a response was seen, the methylprednisolone dose was tapered and changed to prednisone and stopped when the Hb and reticulocyte count normalized. Along with glucocorticoid, IVIG was given to 22 cases concurrently, with a dose of 0.4 to 1 g/kg for 1 to 3 times. All IVIGs were given after initial diagnosis, and no IVIG was used for relapsed cases. Of the 45 patients with complete follow-up data, 40 (88.9%) cases received first-line therapy only, 4 cases received second-line therapy such as 6-mercaptopurine due to poor response to glucocorticoid, and 1 patient received splenectomy after failure to respond to pharmacotherapy of glucocorticoid and cyclosporin A (Figure 2).
Of all 68 patients, 44 patients (64.7%) received washed red cell transfusion as supportive treatment due to severe anemia.
After treatment, all 45 cases achieved CR with the first-line therapy. At the cutoff point of follow-up, 29 cases remained in continuous CR (64.4%), whereas 16 cases relapsed (35.6%). Relapse occurred 0.5 to 36 months after initial diagnosis with a median time of 4 months. The treatment outcome is summarized in Figure 2.
On the basis of the follow-up data available of 45 cases, the median duration of therapy is 7.5 months (range, 1 to 83). Duration of therapy was longer in the ES group than in the AIHA-only group (P<0.05). However, the duration of therapy was not significantly impacted by age, IVIG, or types of serologic antibody groups. Relapse rates were also compared between all comparison groups, there was no significant difference (P>0.05 for all) (Table 4).
Of the 45 cases, 29 cases achieved continuous CR with glucocorticoid alone. The median duration of therapy was 5.5 months (range, 1 to 31) and the median follow-up time of 50 months (range, 17 to 79). Sixteen cases relapsed, and 9 cases achieved second CR with retreatment of glucocorticoid only and the median duration of therapy was 15 months (range, 7 to 80), and median follow-up time of 59 months (range, 16 to 98). There has been longer duration of glucocorticoid therapy in those patients who relapsed than in those who did not relapse (P<0.05).
We performed Kaplan-Meier survival analysis for time to CR for the patient cohort who entered remission (n=38) as well as the patient cohort with time to second remission after relapse (n=9). The median time is 5.8 months versus 10 months, and there is no statistical difference between these 2 groups (P=0.14) (Supplemental Figure 1, Supplemental Digital Content 1, http://links.lww.com/JPHO/A119).
Side effects of glucocorticoid occurred during therapy including moon face (26 cases) and hypertension (2 cases). No deep vein thrombosis, pulmonary emboli, hyperglycemia, or gastrointestinal hemorrhage were observed during the duration of follow-up.
Among 20 cases of ES, 13 cases had long-term follow-up data (as discussed in the Results section). Only 1 patient was still on low-dose glucocorticoid 83 months into therapy with platelet count at 60×109/L after initial disease relapse. One patient failed frontline glucocorticoid and responded well to splenectomy in combination with cyclosporine without further therapy. The median duration of therapy for the case cohort was 15 (3 to 83) months. The relapse rate is 38.5% (5/13). In one of the relapsed cases, with only thrombocytopenia without the findings of recurrent AIHA, glucocorticoid was given.
AIHA is an uncommon disease that can affect patients at any age. The incidence in adults is around 1 to 3 cases per 100,000 per year,15,16 whereas it is estimated that the annual incidence in children is around 3 per million.3,17 In 4 previous reports of childhood AIHA, the median age at diagnosis was 10.5 months, 3.8 years, 6.5 years, and 11 years, respectively.3,5–7 In our study, the median age of onset was 16 months (Table 1), and 63.2% cases were 3 years of age or younger. The age distribution varies significantly in published literature. We do not have the information for the patient selection in other publications and, noticeably, the median age of onset was 10.5 months in the Brazil study. In our study, between 2006 and 2013, consecutive cases that fulfilled the diagnosis criteria were enrolled. Furthermore, we observed that patients less than 1-year old tend to present with a lower Hb level at diagnosis as shown in Table 3. It might be partly due to physiological anemia, and should be taken into consideration prior making a diagnosis and management of AIHA.
Positive DAT is the diagnostic hallmark of AIHA; however, it may be absent in some cases (Table 1). Some studies showed that usually, DAT positivity needed a certain number of antibody molecules sensitizing all red blood cells,18–21 therefore AIHA with a lower quantity of autoantibodies may present with negative DAT, which can be a diagnostic pitfall. Four cases in our study were diagnosed as AIHA with negative DAT, yet all other diagnostic measures met the criteria, and the efficacy of glucocorticoid therapy validated the accuracy of the diagnosis.
There are a variety of antibody combination types in DAT. Earlier studies indicated that IgG/IgG+C3 was associated with severe hemolysis and relatively poor treatment outcomes, whereas C3 correlated with lower relapse rates3,22; another report showed that compared with IgG alone, IgG combined with IgM or IgA can lead to more severe hemolysis.23 Consistent with published data, our study showed that patients with combined-antibody or IgM-contained serologic profiles tended to have more severe anemia.
In some prior reports, ES accounted for 13.6% to 64.3% of all cases of AIHA and as reported had a chronic, refractory nature, variable treatment response, and high relapse rate.9–11,24,25 In our study, the degree of anemia was similar between ES group and AIHA-only group; however, the duration of therapy in ES patients was longer than that of isolated AIHA. Therefore, clinically the glucocorticoid therapy should be prolonged in ES and tapered off with more consideration.
The mechanism of glucocorticoid to treat AIHA is probably downregulation of Fc receptors on phagocytes and reduced IL-2 production.26 A prior study showed that in adult AIHA, the response rate of glucocorticoid was 68.6% in the acute phase and 35% in the prolonged treatment.27 In our study, all cases responded to glucocorticoid in acute phase. Even after relapse, 56.3% cases achieved CR with glucocorticoid treatment only. These findings confirmed that glucocorticoid is the mainstay in childhood AIHA treatment.
Second-line therapy includes immunosuppressors such as 6-mercaptopurine, cyclosporine A, and cyclophosphamide.28–30 Recently, rituximab has been proven effective in refractory AIHA.13,14 Splenectomy as third-line therapy would be considered when these medicines failed. However, as the glucocorticoid treatment outcome of childhood AIHA is better than that of adults, these treatment approaches are less common in children, especially the use of rituximab in undeveloped regions due to its high price (Table 1).
AIHA may be primary or secondary to other diseases, including infections, autoimmune disorders, and malignancies. Data published before showed that in adults the main cause of AIHA was systemic autoimmune diseases such as SLE.31 The presence of ANA in the normal population has been reported without evidence of hemolytic anemia or autoimmune disease. In the Chinese population there have been 2 publications to our best knowledge that showed that 2.2% to 5.6% of healthy children were tested positive for ANA.32,33 In our study of childhood population, secondary AIHA was mainly associated with infectious disease, whereas systemic autoimmune disease was rare. Perhaps longer follow-up may reveal more underlying systemic autoimmune diseases.
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autoimmune hemolytic anemia (AIHA); Evans syndrome (ES); children; glucocorticoid
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