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Introduction

Acute lymphoblastic leukemia (ALL) mainly occurs in children aged 3–7 years. The abnormal proliferation of B- or T-type mature lymphocytes can inhibit normal hematopoietic function, resulting in fever, leukocytosis, decrease in levels of platelet and neutrophil, eventually leading to anemia, then primitive cells invaded into extramedullary tissue, causing hepatosplenomegaly and a series of lesions (1,2). With the continuous improvement of diagnosis method and chemotherapy, the 5-year survival rate of ALL children has been increased to 80%. ALL has become one of the malignant tumors with the highest cure rate. But effective treatments are still lacking for special subtypes and high risk cases. In addition, the application of chemotherapy is challenged by the serious complications and high recurrence rate caused by toxicity and side-effects (3–5). However, chemotherapy is still the preferred treatment, especially for child patients.

Allogeneic blood transfusion has now become an important radical treatment for ALL. It will lead to a series of complications, and even death if graft vs. host disease occurs (6). Blood from donor with blood relationship is preferred, but blood from donor without blood relationship is also allowed to be used in allogeneic blood transfusion (7). However, studies have shown that allogeneic blood transfusion can inhibit recipient's immune function, which in turn increase postoperative infection, and promote tumor growth, metastasis and recurrence (8). Studies have shown that, after allogeneic blood transfusion, the number of CD4+ T cells would be reduced, and IL-2 level would be decreased. IL-2 is an essential factor for the activation of B, CD8+ T and NK cells, and the decrease in the level of IL-2 can lead to immunosuppression (9). Interleukin-6 (IL-6) is also known as B-cell differentiation factor. IL-6 is secreted by monocytes and activated Th cells, and its role is to maintain the growth and differentiation of B cells, stimulate the production of immunoglobulin, and induce IL-2 expression, so as to participate in multiple processes of inflammatory response (10,11). Activated T cells and other monocytes can secrete soluble interleukin-2 receptor (sIL-2R), a low-affinity receptor for IL-2 that is involved in the regulation of IL-2-mediated lymphocyte activation (12). sIL-2R is recognized as a marker of multiple lymphomas and blood tumors. Level of sIL-2R in blood is closely related to the prognosis of patients with hematological malignancies (13). It has been reported that high levels of sIL-2R in the blood of patients with lymphoma suggest poor prognosis. The possible explanation is that sIL-2R may bind to serum-free IL-2 to reduce the function of IL-2 in regulating cellular immunity (14,15).

In this study, ALL children received allogeneic blood transfusion therapy, and serum levels of IL-6 and sIL-2R were detected before and after treatment to analyze the immune status of children after transfusion and to predict the prognosis. Our study provided references for follow-up studies.

Materials and methods

General information

A total of 91 children with ALL admitted to Nanfang Hospital of Southern Medical University (Guangzhou, China) from June 2014 to January 2017 were selected as the study group. Inclusion criteria: i) with ALL confirmed by bone marrow cell morphology, cytochemistry, cytogenetics and immunophenotyping; ⅱ) children aged 3–12 years; and ⅲ) parents signed informed consent. Exclusion criteria: i) combined with lung, kidney, liver and other functional abnormalities; and ⅱ) with severe mental dysfunction. Among 91 patients, there were 43 males and 48 females, and the age ranged from 3 to 12 years with an average age of 6.98±1.45 years. Patients were randomly divided into allogeneic blood transfusion group (n=38) and non-transfusion group (n=53). Allogeneic blood transfusion group included 17 males and 21 females, the age ranged from 3 to 12 years with an average age of 6.98±1.45 years. The non-transfusion group included 26 males and 27 females, and the age ranged from 3 to 12 years with an average age of 7.09±1.42 years. No significant differences in general information were found between the two groups. In addition, a total of 64 healthy children who attend physical examination in the hospital were also selected from June 2014 to January 2017 as the control group. None of the children had history of recent infection. The control group included 30 males and 34 females, and the age ranged from 3 to 12 years with an average age of 6.71±1.39 years. No significant differences in general information were found between the study and control groups. The study was approved by the Ethics Committee of Nanfang Hospital and informed consents were signed by the guardians of the patients.

Methods

Hepatitis virus, cytomegalovirus and herpes virus and other infections were not detected in the donors, and the donors had the blood type O. Cross-matching was performed before transfusion to ensure the compatibility between the recipient and donor. Lymphocytes were separated from blood using the Baxter CS-3000 Plus Blood Cell Separator (Baxter, Deerfield, IL, USA) to obtain erythrocyte suspension and platelets. Patients in the allogeneic blood transfusion group were transfused with allogeneic erythrocyte suspension and platelet (3.5 U), as previously reported (16). Blood transfusion was not performed in the non-transfusion group. Peripheral venous blood (3 ml) was extracted from each participant before and at 4, 8 and 12 weeks after blood transfusion. Blood samples were centrifuged (2,010 × g) for 8–12 min to separate serum. Serum was stored at −20°C before use. Levels of IL-6 and sIL-2R were detected by enzyme-linked immunosorbent assay (ELISA) using kits (human IL-6 ELISA kit, cat. no. BMS213-2TEN; human sIL-2R kit, cat. no. BMS212-2; Thermo Fisher Scientific, Inc., Waltham, MA, USA). OD values as 450 nm were measured using a microplate reader (Bio Rad Laboratories, Inc., Hercules, CA, USA).

Observational measurements

i) To observe the changes of serum IL-6 and sIL-2R levels in the study and control group; ⅱ) to observe the changes of serum IL-6 level in the allogeneic blood transfusion and non-transfusion group before and at 4, 8 and 12 weeks after blood transfusion group; ⅲ) to observe the changes of serum sIL-2R level in the allogeneic blood transfusion and non-transfusion group before and at 4, 8 and 12 weeks after blood transfusion group.

Statistical analysis

SPSS 19.0 software (IBM Corp., Armonk, NY, USA) was used. Measurement data are expressed as the mean ± standard deviation, and comparisons between the groups were performed by t-test. Enumeration data were expressed as cases, and comparisons between the groups were performed by Chi-square test. Comparisons within the groups were performed by analysis of variance to repeated measurements and the post hoc test was Dunnetts test. P<0.05 was considered to be statistically significant.

Results

Changes in serum levels of IL-6 and sIL-2R in the study and control group

Before transfusion, levels of serum IL-6 and sIL-2R in the study group were significantly lower than those in control group (p<0.05) (Table I).

Table I. Serum levels of IL-6 and sIL-2R in the study and control group (mean ± standard deviation).

Table I.

Serum levels of IL-6 and sIL-2R in the study and control group (mean ± standard deviation).

Groups	Cases	IL-6 (U/ml)	sIL-2R (pg/ml)
Study	91	237.49±68.42	1.28±0.34
Control	64	474.23±121.65	7.32±2.19
t-test		101.05	22.14
P-value		<0.05	<0.05

[i] IL-6, interleukin-6; sIL-2R, soluble interleukin-2 receptor.

Changes of serum IL-6 levels in the allogeneic blood transfusion and non-transfusion group

There was no significant difference in serum IL-6 levels between the two groups before transfusion (p>0.05). Serum IL-6 level was stable in non-transfusion group for 12 weeks. Serum IL-6 level significantly increased at 4 weeks after transfusion in the allogeneic blood transfusion group, and gradually decreased at 8 and 12 weeks after transfusion, but was still significantly higher than the level before transfusion (p<0.05). Serum levels of IL-6 were significantly higher in the allogeneic blood transfusion group than in non-transfusion group during 12 weeks after transfusion (p<0.01) (Table II).

Table II. Changes of serum IL-6 levels in the allogeneic blood transfusion and non-transfusion group (U/ml).

Table II.

Changes of serum IL-6 levels in the allogeneic blood transfusion and non-transfusion group (U/ml).

Groups	Cases	Before transfusion	4 weeks	8 weeks	12 weeks
Allogeneic blood transfusion	38	238.42±49.21	97.42±38.31a	62.91±25.25a, b	134.12±53.21a, c
Non-transfusion	53	236.98±67.39	229.38±58.13	241.38±73.76	225.09±64.07
t-test		0.37	32.86	50.19	13.03
P-value		>0.05	0.001	<0.001	0.002

a P<0.05, compared with the level before transfusion within a group

b p=0.042, compared with 4 weeks after transfusion

c p=0.037, compared with 4 weeks after transfusion. IL-6, interleukin-6.

Changes of serum sIL-2R levels in the allogeneic blood transfusion and non-transfusion group

There was no significant difference in serum sIL-2R levels between the two groups before transfusion (p>0.05). Serum sIL-2R level was stable in non-transfusion group for 12 weeks. Serum sIL-2R level significantly increased at 4 weeks after transfusion in the allogeneic blood transfusion group, and gradually decreased after that, but was still significantly higher than the level before transfusion (p<0.01). Serum levels of sIL-2R were significantly higher in the allogeneic blood transfusion group than in non-transfusion group during 12 weeks after transfusion (p<0.05) (Table III).

Table III. Changes of serum sIL-2R levels in the allogeneic blood transfusion and non-transfusion group (pg/ml).

Table III.

Changes of serum sIL-2R levels in the allogeneic blood transfusion and non-transfusion group (pg/ml).

Groups	Cases	Before transfusion	4 weeks	8 weeks	12 weeks
Allogeneic blood transfusion	38	1.30±0.35	3.91±1.27a	3.46±1.14a, b	2.75±0.87a, c
Non-transfusion	53	1.26±0.32	1.29±0.37	1.23±0.29	1.27±0.32
t-test		0.57	3.69	8.64	4.43
P-value		>0.05	0.037	0.008	0.005

a P<0.05, compared with the level before transfusion within a group

b p=0.089, compared with 4 weeks after transfusion

c p=0.043 compared with 4 weeks after transfusion. sIL-2R, soluble interleukin-2 receptor.

Discussion

Childhood ALL is a malignant hematologic disease that seriously affects children's physical and mental health and quality of life. Allogeneic blood transfusion has become another treatment option other than chemotherapy, and the combined use of these two treatments can inhibit the proliferation of lymphocytes, resulting in a better therapeutic effect (16). But allogeneic blood transfusion can suppress the immune function of the recipient (17). IL-6 is an important cytokine that maintains B-cell growth and differentiation and the production of antibodies. In this study, both the serum levels of IL-6 and sIL-2R were lower in the study group than in the control group, which was consistent with the fact that the children with ALL had decreased immunity. The results showed that IL-6 levels in serum were significantly increased after allogeneic blood transfusion. IL-6 levels were gradually decreased at 12 weeks after transfusion, but were still higher than pre-transfusion levels. It is consistent with previous studies which showed levels of IL-6 increased after transfusion (18), indicating that the ability immune system to produce antibodies was reduced. Therefore, infection should be avoided and antibiotics may be used (19). This study found that sIL-2R levels increased at 4 weeks after transfusion. Although decrease was observed after that, the levels were still higher than pre-transfusion levels. Studies reported that levels of IL-2 in serum of children were decreased after transfusion. IL-2 is one of the most important cytokines that regulates the cellular immune response, which could inhibit the children's cellular immune response (20). One reason for the decrease in IL-2 levels is the elevated sIL-2R levels in cells. sIL-2R can bind to serum IL-2, thereby reducing IL-2-mediated cellular immune responses (21). The above reports are consistent with the findings in our study.

In conclusion, our study showed that allogeneic blood transfusion could increase serum IL-6 and sIL-2R levels in ALL patients, so as to inhibit the children's cellular immune response and reduce the production of antibodies, resulting in low immunity in patients. Changes in the level of two cytokines suggest that attention should be paid on the immune status of children after transfusion, and preventive measures should be given if necessary.

Acknowledgements

Not applicable.

Funding

No funding was received.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

HZ and HYZ collected and analyzed the general data of patients. QC prepared the blood sample. CW, JB and PL were responsible for observational measurements. FZ helped with statistical analysis. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

The study was approved by the Ethics Committee of Nanfang Hospital of Southern Medical University (Guangzhou, China) and informed consents were signed by the guardians of the patients.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References