Int J Hematol
DOI 10.1007/s12185-016-2056-5
ORIGINAL ARTICLE
A monocentric retrospective study comparing pulse
cyclophosphamide therapy versus low dose rituximab in the
treatment of refractory autoimmune hemolytic anemia in adults
Rong Fu1 · Siyang Yan1 · Xiaoming Wang1 · Guojin Wang1 · Wen Qu1 ·
Huaquan Wang1 · Yuhong Wu1 · Hong Liu1 · Jia Song1 · Jin Guan1 · Limin Xing1 ·
Erbao Ruan1 · Lijuan Li1 · Hui Liu1 · Zonghong Shao1 
Received: 14 February 2016 / Revised: 24 June 2016 / Accepted: 26 June 2016
© The Japanese Society of Hematology 2016
Abstract This retrospective study aims at confirming
the efficacy and safety of low dose rituximab and pulse
cyclophosphamide in the treatment of refractory AIHA in
adults and making comparison of the two. Forty-nine adult
patients with refractory AIHA have been enrolled. Results
showed low dose rituximab combined with steroid therapy
(group B) got more CR (78.9 %, 15/19) compared to that
in intermittent intravenous cyclophosphamide combined
with steroid therapy (group A) (42.1 %, 8/19) (P  = 0.04)
at 6 months after treatment. The hemoglobin level in group
B was higher than group A at the time point of 1 month
(P  = 0.02) after treatments. The RFS in group A was
87.9 % at 6 months and 82.7 % at 12 months, which were
no significant difference with group B (91.1 % at 6 months
and 86.0 % at 12 months) (P = 0.81). Both the two thera-
pies were well tolerated with pulmonary infections as the
most common side effects. In conclusion, low dose rituxi-
mab combined with steroid therapy presents to be a better
choice in the treatment of refractory AIHA in adults com-
paring with pulse cyclophosphamide therapy.
Keywords  Refractory autoimmune hemolytic
anemia · Low dose rituximab · Intermittent intravenous
cyclophosphamide
R. Fu and S. Yan contributed equally to this manuscript
* Rong Fu
florai@sina.com
* Zonghong Shao
shaozonghong@sina.com
1
Department of Hematology, Tianjin Medical University
General Hospital, 154 Anshan Street, Heping District,
Tianjin 300052, People’s Republic of China
Introduction
Autoimmune hemolytic anemia (AIHA) is a relatively
uncommon autoimmune disease in which red blood cells
are destructed by autoantibodies, thus leads to hemoly-
sis [1]. According to the thermal range of the reactivity of
autoantibody, classifications of AIHA include warm, cold
or mixed type of AIHA [2, 3]. Corticosteroids, splenectomy
and conventional immunosuppressive drugs are traditional
treatment of AIHA [4]. The general agreement is that cor-
ticosteroids represent the first-line treatment for patients
with warm antibody type AIHA while splenectomy and
rituximab are the second-line treatments [5]. However, the
treatment for refractory AIHA patients is still a challenge
for doctors.
Rituximab, a monoclonal antibody directed against the
CD20 antigen expressed on B cells, has been reported to be
effective for autoimmune diseases such as systemic lupus
erythematosus, multiple sclerosis, thrombotic thrombocy-
topenic purpura and acquired hemophilia [6]. Its efficacy in
autoimmune hematological disorders is probably mediated
not only through elimination of pathogenic autoantibod-
ies, but also by disturbing the potency of B-lymphocytes as
antigen-presenting and cytokine-producing cells [7].
Intermittent intravenous cyclophosphamide (pulse
therapy) was reported to be effective for a number of
autoimmune diseases [8]. And before the introduction of
rituximab in the therapy of AIHA, cyclophosphamide was
thought to be highly effective and was often used as sec-
ond-line treatment [9, 10]. The mechanism for pulse cyclo-
phosphamide in the treatment of autoimmune disorders
seems to be suppression of both T and B lymphocyte num-
bers and function, thus results in diminished autoantibody
production [11]. Lymphocytes are highly sensitive to cyclo-
phosphamide while primitive hematopoietic progenitors
13
 R. Fu et al.

Table 1  Baseline Characteristics Group A Group B P value

characteristics of the two groups
Number 27 22 0.31
Age (years) 48 (21–90) 40.5 (21–79)
median (range)
Sex ratio (male/female) 11/16 9/13 0.99
Lymphoproliferative disease 1 1 1
Autoimmune disease 5 6 0.70
Hemoglobin (g/L) 72.0 (39–106) 68.0 (33–107) 0.63
median (range)
Reticulocytes (%) 11.0 (1.3–35.2) 12.1 (0.2–26.5) 0.43
median (range)
Erythroid proliferative rate of ilium (%) 56.0 (18–89) 51.0 (1.5–76) 0.19
median (range)
Erythroid proliferative rate of sternum 59.0 (19.5–84) 50.3 (1–79) 0.18
median (range)
Unconjugated bilirubin (μmol/L) 20.0 (3.6–97) 15.9 (7.4–75) 0.43
median (range)
LDH (U/L) 383 (139–1072) 340 (179–3311) 0.72
median (range)
WAIHA 26 19 0.46
Mixed AIHA 1 3 0.46

are resistant to cyclophosphamide [12]. The reason is that
primitive hematopoietic progenitors have high levels of
aldehyde dehydrogenase, an enzyme that confers resistance
to cyclophosphamide [12]. So cyclophosphamide also has
efficiency in some refractory autoimmune diseases, such as
pemphigus vulgaris [13].
This retrospective study is aimed at evaluating and com-
paring the efficacy of low dose rituximab and pulse cyclo-
phosphamide in the treatment of severe refractory AIHA
which failed to two or more treatment modalities.
Materials and methods
Patients and treatments
Fifty-one adult patients with refractory AIHA in our hos-
pital were enrolled in this study during January 2010 to
October 2015, in which period there were 162 patients
diagnosed with AIHA in total. Among these refractory
AIHA patients, 43 patients were warm AIHA (WAIHA),
two patients were cold agglutinin disease (CAD) and six
patients were mixed-type AIHA. We excluded the two
patients of CAD and enrolled the other 49 patients in our
study. They all failed to respond to therapies including ster-
oid, intravenous immunoglobulin, oral cyclophosphamide
and some even splenectomy. The treatment for the patients
were based on their own will (intravenous cyclophospha-
mide or low dose rituximab), mainly according to eco-
nomic condition. Then the patients were divided into two
groups as following: Group A is steroid therapy combined
with intermittent intravenous cyclophosphamide (1 g as an
intravenous infusion on day 10, 20, 30 along); Group B is
steroid therapy combined with low dose rituximab (100 mg
as an intravenous infusion on days 7, 14, 21, 28 along).
Both the two groups also used intravenous immunoglobulin
(10 g as intravenous drop infusion, twice a week) simulta-
neously to help reduce the adverse reactions caused by the
possible impairment of immunity. Table 1 showed the char-
acteristics of the patients.
Among all the patients, 11 patients were secondary
to other autoimmune diseases as hypothyroidism (3/11),
hyperthyroidism (2/11), systemic lupus erythematosus
(4/11), autoimmune hepatitis (1/11), ankylosing spondyli-
tis (1/11) and connective tissue disease (1/11). There is one
patient who both had autoimmune hepatitis and hypothy-
roidism at the same time.
A search for detecting the serum level of complements
and autoimmune antibodies was performed in 28 patients,
and the results showed that all the patients did have some
abnormalities. For 25 patients (89.3 % of the total) had a
decrease in the serum level of either C3 or C4 or the both,
and for 22 patients (78.6 % of the total) showed a positive
result in the appearance of antinuclear antibodies (ANAs).
Besides, Ro-52 and anti-SSA antibody were positive in six
patients, dsDNA positive in nine patients. And for the com-
plements, our data showed that the level of serum Ig E, Ig
G and Ig M are more affected than the other two kinds.
As for the previous treatments, all patients had taken
steroid therapy (49/49) and two patients (one in group

13
A monocentric retrospective study comparing pulse cyclophosphamide therapy versus low dose…

Table 2  Responses to the Group A (N = 27) Group B (N = 22) P value

treatments
Response rate CR OR CR OR CR OR
After 1 month 10/27 25/27 11/22 22/22 0.36 0.50
(N = 27) (N = 22)
After 3 months 12/25 24/25 14/21 20/21 0.20 1
(N = 25) (N = 21)
After 6 months 8/19 17/19 15/19 17/19 0.04 1
(N = 19) (N = 19)
After 12 months 5/9 8/9 12/14 14/14 0.16 0.39
(N = 9) (N = 14)
After 24 months 2/5 5/5 6/8 7/8 0.29 1
(N = 5) (N = 8)

OR complete response + partial response, CR complete response (Hb ≥ 12 g/dL), PR partial response

(Hb > 10 g/dL or at least 2 g/dL increase in Hb
Group A steroid therapy + pulse cyclophosphamide intravenously
Group B steroid therapy + low dose rituximab

A and one in group B) had received splenectomy (2/49),
one patient in group A had taken splenic embolization.
Plasma exchange had been performed on two patients in
group A.
This study was approved by the Ethical Committee of
the Tianjin Medical University. Written informed consent
was obtained from the patients for the publication of this
report and any accompanying images.
Evaluation
Complete clinical examination, blood counts, and hemo-
lytic markers were performed at enrollment and at month
3, 6, 12 and 24. Overall response (OR) was divided in
complete response (CR), defined as Hb > 12 g/dL without
recent transfusion and normalization of hemolytic mark-
ers, and partial response (PR), defined as Hb ≥ 10 g/dL or
at least 2 g/dL increase in Hb from baseline, and without
recent transfusion [14].
Statistical analysis
Descriptive statistics included mean ± SD or median
(range) as appropriate for continuous variables and fre-
quency (percentage) for categorical variables. Univariate
analysis involved the Chi square test or Fisher exact test as
appropriate to compare categorical variables and student’s
t test with stterthwaite method were used to analyze con-
tinuous variables. Kaplan–Meier curve analysis was used to
describe relapse-free survival (RFS, %) and log-rank test to
compare both curves. Relapse-free survival was calculated
as the time from corticosteroid treatment onset to relapse,
failure, or death, whichever occurred first. All P values
were two-sided, and those below 0.05 were considered
statistically significant. All the analyses were performed
with the software SPSS, version 13.
Results
Response to treatment
Table  2 showed responses of the two groups at different
times. After a complete course, the patients achieved com-
plete reaction as 37.0 % in group A and 50 % in group B
(P  = 0.36). Both the two groups show a good response
after a complete course of therapy. The data also showed
that at 6 months after treatment, group B got significantly
higher CR compared to group A (P = 0.04), which means
low dose rituximab may have a greater and faster effect on
refractory AIHA in adults than pulse cyclophosphamide. At
all the four observe points of time, over 90 % patients of
two groups had OR response, showing highly effective for
both treatments.
Table  3 shows hematological parameters in patients
of two groups at different time after the treatments. The
hemoglobin level in group B was higher than that in group
A at the time point of 1 month (P = 0.02) after treatments.
At the later time points, CR in the two groups had no sig-
nificant difference any more. Comparing to group A, the
patients in group B appeared to have a quicker and greater
recovery.
There were 17 patients in group A and 10 patients in
group B were red blood cell transfusion-dependent at their
enrollment. After the treatments, 25 of them became eryth-
rocyte transfusion-independent except two patients in group
A who failed to respond to the treatment. The two failed
patients were one male aged 65-year-old with confirmed

13
 R. Fu et al.

lymphoma and one male aged 31-year-old with 2 years his-

P value
Unconjugated bilirubin (μmol/L) tory of AIHA. After treatment, the two patients still kept

0.07
0.43
0.70
0.42
0.11
low serum level of Hb and need red cell transfusion.
7.1 ± 3.6
8.1 ± 6.5
8.0 ± 4.9
9.1 ± 3.7
9.5 ± 3.6
Relapse

In group A, one patient relapsed after 3 months of therapy;

B

two relapsed after 6 months and one after 9 months. In

9.8 ± 6.2
9.4 ± 3.8
7.5 ± 3.2
7.6 ± 4.7
5.5 ± 3.4
group B, there were two patients relapsed after 3 months,
two after 6 months, one after 15 months and one after
A

24 months. Overall the ratios of relapse for both two groups

seem to be acceptable.
P value

Meanwhile, there were four patients with mixed type of

0.28
0.98
0.46
0.84
0.89

AIHA. One in group A, got PR after one course, relapsed

after 3 months and after another course reached CR for
286.1 ± 106.2

14 months; Three patients in group B, one reached PR and

228.0 ± 105.9
288.7 ± 70.8

225.9 ± 72.7
208.6 ± 44.4

two reached CR. The relapse risks seem raise with time
after treatment and reached peak to some degree (Fig. 1).
The RFS in group A was 87.9 % at 6 months, 82.7 %
B

at 12 months. And in group B the RFS was 91.1 % at

6 months and 86.0 % at 12 months, which was a bit higher
324.6 ± 141.9
286.9 ± 114.5
251.8 ± 132.7
204.0 ± 55.0
218.3 ± 43.9

than that in group A (Fig. 2). Group B appeared to show a

LDH (U/L)

better response both in the time and effective duration com-

paring with group A, though there was no obvious signifi-
A

cant significance (P = 0.81).

P value

Safety
1.00
0.24
0.49
0.31
0.88

Treatments were well tolerated, no infusion side reac-

2.7 ± 2.2
1.9 ± 0.9
1.8 ± 0.6
1.9 ± 0.5
2.0 ± 1.2

tion was observed. However, we did observe that many

patients in the two groups had infections during the treat-
B

ments (Table 4). There were 10 patients in group A and

12 in group B had pulmonary infections. Two patients in
2.7 ± 2.0
2.3 ± 1.3
2.0 ± 0.7
1.7 ± 0.3
1.9 ± 0.9

group A developed central nervous system infection at 5

Ret (%)

and 8 months after treatment, respectively. Another patient

A

Group A steroid therapy + pulse cyclophosphamide intravenously

in group A was affected as skin soft tissue infection after

P value

0.02
0.06
0.27
0.10
0.27
122.8 ± 16.8
127.2 ± 12.5
125.1 ± 17.2
131.8 ± 18.2
127.3 ± 19.2
Table 3  Hematological parameters in patients

Group B steroid therapy + low dose rituximab

B

108.0 ± 23.3
118.2 ± 17.6
117.7 ± 22.4
122.0 ± 8.9
118.8 ± 4.9
Hb (g/L)
A

12 Months
24 Months
3 Months
6 Months
1 Month

Fig. 1  Relapse risk of two groups

13
A monocentric retrospective study comparing pulse cyclophosphamide therapy versus low dose…

Discussion

The treatment of refractory AIHA in adults has not reached

a worldwide agreement. Splenectomy was considered as
the usual options in the past days [13], however, it is lim-
ited for not highly effective enough and can result in a
higher complication rate of secondary AIHA [15]. Despite
the fact that nowadays rituximab has taken a new role [16],
the evidence for rituximab therapy is not so strong and
there is still no idea whether we should use standard dose
or low dose of it in our therapy. As for immune suppres-
sors, cyclophosphamide was popular as the second line
treatment before the appearance of rituximab [17]. Never-
theless, considering its efficacy and side effects are still in
Fig. 2  Proportion relapse-free of two groups doubt, cyclophosphamide has rarely been used in the treat-
ment of AIHA in western courntries nowadays [18]. How-
ever, in some developing countries, many patients still can-
Table 4  Infections of two groups not afford the use of rituximab because of its high price.
Group A Group B Therefore, cyclophosphamide maybe a much more practi-
(CYC + Pred) (RTX + Pred) cal treatment for the patients.
(N = 27) (N = 22) Our retrospective study aims at confirming the efficacy
Pulmonary infec- 10 12 and safety of low dose rituximab and pulse cyclophos-
tion phamide and making comparison of the two. In our study,
Skin soft tissue 1 1 the characteristics of patients have a bit differences. Our
infection patients appear to be younger and are more affected by
Intracranial infec- 2 0 autoimmune diseases comparing to the previous reports
tion [18, 19].
Sepsis 0 1 In the respect of response to the treatment, the OR
Total 13 14 P = 0.28 ratios in both groups reach as high as over 90 % and even
reach 100 % in group B after the patients had taken a com-
plete course of therapy. The decreases in the later time are
14 months of treatment. In group B, there was one patient mainly caused by the relapse. But the OR ratio is still high
infected with tuberculosis among those who got pulmonary in total (all over 90 %) and the two groups had almost no
infection. There was also a patient with skin soft tissue difference in the OR ratio at any time after the treatments
infection (1 month after treatment) and the pathogen was (Table  2). As for the CR ratios in two groups, they differ
confirmed as Norcardia. obviously to some degree. Group A has a lower ratio of
Among all the 49 patients there was one death. The 37.0 % in patients who could get a CR immediately after
female patient in group B got sepsis and died of respiratory they finished a complete course of therapy (1 month) com-
failure developed from severe pneumonia after 4 months pared to the 50 % in group B at the same time point. For
of the treatment. The other patients with infections of both the later time points as 3 months, 6 months after the treat-
groups all revived well after anti-infection treatment and ments, CR ratios in group A were never over 50 % while
lasted CR or PR response. never lower than 60 % that in group B. Six months after the
According to our data, there were five patients in group treatments is the time points which have static significance
A presented a decrease in WBC count after or during the for the two groups in the CR ratios (P = 0.04). If there are
treatment of cyclophosphamide. Two patients had the more patients in two groups, we may observe more differ-
decrease during the treatment, one patients decreased at ences. The same explanation also fits for the comparison
1 week after the treatment, and the other two decreased at of relapse risks of two groups. Besides, based on the data
1 month after the treatment. However, all these five patients of hematological parameters in patients of two groups at
just had a mild decrease, i.e., about 2.0–3.0 × 109/L and different time after the treatments, group B also appeared
the conditions soon got relived after the use of G-CSF. to perform better comparing to group A. The hemoglobin
Among them, two patients presented NR to the treatment level in group B was higher than that in group A at the time
and the other three got good response to the treatment. point of 1 month (P = 0.02) after the treatment (Table 3).

13

At the later time points, level in the two groups did not
have significance any more. The results show that low
dose rituximab combined with steroid therapy can have a
quicker and greater effect on the refractory AIHA in adults
than intermittent intravenous cyclophosphamide combined
with steroid therapy.
According to our data, relapse risk in group A is higher
than that in group B as a whole, especially within the
period of 6 months to the 21 months after the treatments
(Figs. 1, 2), although the log rank test shows that P = 0.81.
Because our number of patients in both groups is not big
enough and many patients had been lost in the follow-up,
the tails of curves seem not so convincing as the other parts
and need further confirmation.
As for the safety aspect of the two treatments, our data
shows that infections after the treatment were common and
most of them were pneumonia (Table 4). They were cured
at most times except one patient died of infection. Intrac-
ranial infections also happened in a few patients while the
prognoses were all good. Skin soft tissue infections are also
been found in one patient in group A and another in group
B. This kind of infection has not been reported in the previ-
ous reports [14, 16, 17, 19–22]. In general, the side effects
that occurred did not differ between the two treatment
groups in our study (P = 0.28).
These data confirmed that pulse cyclophosphamide
intravenously and low dose rituximab are both highly effec-
tive in the treatment of refractory AIHA in adults. Fur-
thermore, low dose rituximab treatment seems to have a
quicker and greater effect the patients than that of cyclo-
phosphamide. Rituximab may also have lower relapse risk
than cyclophosphamide especially in the long term and
need further investigation in a large scale. The two kinds
of therapies are relatively safe but also need caution dur-
ing and after the usage. In conclusion, low dose rituxi-
mab combined with steroid therapy presents to be a better
choice than pulse cyclophosphamide therapy in the treat-
ment of refractory AIHA in adults.
Acknowledgments  This work was supported by the National Natu-
ral Science Foundation of China (Grant Nos. 81570106, 81570111,
814000888, 81400085), the Tianjin Municipal Natural Science Foun-
dation (Grant Nos. 14JCYBJC25400, 15JCYBJC24300).
Compliance with ethical standards  House; 2013.
Conflict of interest  All authors declare that they have no conflict of
interest.
References
1. Gehrs BC, Friedberg RC. Autoimmune hemolytic anemia[J]. Am
J Hematol. 2002;69(4):258–71.
13
R. Fu et al.
2. Michel M. Classification and therapeutic approaches in auto-
immune hemolytic anemia: an update. Expert Rev Hematol.
2011;4:607–18.
3. Packman CH. Hemolytic anemia due to warm autoandibodies.
Blood Rev. 2008;22:17–31.
4. King KE, Ness PM. Treatment of autoimmune hemolytic ane-
mia. Semin Hematol. 2005;42:131–6.
5. Zanella A, Barcellini W. Treatment of autoimmune hemolytic
anemias[J]. Haematologica. 2014;99(10):1547–54.
6. Garvey B. Rituximab in the treatment of autoimmune haemato-
logical disorders[J]. Br J Haematol. 2008;141(2):149–69.
7. Reff ME, Carner K, Chambers KS, et al. Depletion of B cells
in vivo by a chimeric mouse human monoclonal antibody to
CD20[J]. Blood. 1994;83(2):435–45.
8. McCune WJ, Golbus J, Zeldes W, et al. Clinical and immuno-
logic effects of monthly administration of intravenous cyclo-
phosphamide in severe systemic lupus erythematosus[J]. N Engl
J Med. 1988;318(22):1423–31.
9. Zupańska B, Sylwestrowicz T, Pawelski S. The results of pro-
longed treatment of autoimmune haemolytic anaemia[J]. Haema-
tologia. 1981;14(4):425–33.
10. Sakalová A, Hrubisko M. Cyclophosphamide in the treatment of
immune hemocytopenias[J]. Folia haematologica (Leipzig, Ger-
many: 1928). 1975;102(5):559.
11. Verlin M, Laros RK, Penner JA. Treatment of refractory throm-
bocytopenic purpura with cyclophosphamide[J]. Am J Hematol.
1976;1(1):97–104.
12. Jones RJ, Barber JP, Vala MS, et al. Assessment of aldehyde
dehydrogenase in viable cells[J]. Blood. 1995;85(10):2742–6.
13. Hayag MV, Cohen JA, Kerdel FA. Immunoablative high-
dose cyclophosphamide without stem cell rescue in a
patient with pemphigus vulgaris[J]. J Am Acad Dermatol.
2000;43(6):1065–9.
14. Barcellini W, Zaja F, Zaninoni A, et al. Sustained response
to low-dose rituximab in idiopathic autoimmune hemolytic
anemia[J]. Eur J Haematol. 2013;91(6):546–51.
15. Akpek G, McAneny D, Weintraub L. Comparative response
to splenectomy in coombs-positive autoimmune hemolytic
anemia with or without associated disease[J]. Am J Hematol.
1999;61(2):98–102.
16. Roumier M, Loustau V, Guillaud C, et al. Characteristics and
outcome of warm autoimmune hemolytic anemia in adults: new
insights based on a single-center experience with 60 patients[J].
Am J Hematol. 2014;89(9):E150–5.
17. Thabet AF, Faisal M. Pulse cyclophosphamide therapy in refrac-
tory warm autoimmune hemolytic anemia: a new perspective[J].
Indian J Hematol Blood Transfus. 2014;30(4):313–8.
18. Lechner K, Jäger U. How I treat autoimmune hemolytic anemias
in adults[J]. Blood. 2010;116(11):1831–8.
19. Birgens H, Frederiksen H, Hasselbalch HC, et al. A phase III
randomized trial comparing glucocorticoid monotherapy ver-
sus glucocorticoid and rituximab in patients with autoimmune
haemolytic anaemia[J]. Br J Haematol. 2013;163(3):393–9.
20. Zhang ZN. Hematology. Beijing: People’s Medical Publishing
21. Maung SW, Leahy M, O’Leary HM, et al. A multi-centre retro-
spective study of rituximab use in the treatment of relapsed or
resistant warm autoimmune haemolytic anaemia[J]. Br J Haema-
tol. 2013;163(1):118–22.
22. D’Arena G, Califano C, Annunziata M, et al. Rituximab for
warm-type idiopathic autoimmune hemolytic anemia: a ret-
rospective study of 11 adult patients[J]. Eur J Haematol.
2007;79(1):53–8.