Abstract
BACKGROUND: ABO-incompatibility between donor and recipient is not a barrier for successful allogeneic hematopoietic stem cell transplantation even though it is well established that major ABO incompatibility may lead to prolonged destruction of donor-derived erythrocytes and prolonged transfusion requirements.
OBJECTIVE: To explore the effect of ABO-incompatible on clinical characteristics in allogeneic-hematopoietic stem cell transplantation.
DESIGN: A retrospective observation.
SETTING: Department of Hematology, the Affiliated Drum Tower Hospital of Nanjing University Medical School.
PARTICIPANTS: Fourteen patients (11 males and 3 females, aged 15-60 years old) with malignant hematologic diseases who received ABO-incompatible allogeneic hematopoietic stem cell transplantation in the Affiliated Drum Tower Hospital of Nanjing University Medical School from May 2002 to September 2007 were recruited for this study. Of the 14 patients, 7 were human leukocyte antigen (HLA)-matched, and the other 7 were HLA-half-matched. Controls were 11 patients who received ABO-compatibility bone marrow transplantation during the same period. Written informed consents for receiving allogeneic hematopoietic stem cell transplantation were obtained from each recipient. The donors were sibling sister, sibling brother, son and mother, and they all agreed to provide marrow for transplantation. This experiment was given an approval by the Ethics Committees of the hospital.
METHODS: Regimen conditioning: HLA-matched transplantation regimen conditioning consisted of busulfan (Bu) and cyclophosphamide (Cy). HLA-half-matched transplantation regimen conditioning adopted GIAC program from Beijing People's Hospital. The GIAC program consisted of 4 parts: G: granulocyte colony-stimulating factors used for donors; I: stronger immunosuppressive regimen conditioning used for recipients; A: antihuman thymocyte globulin added; C: combined transplantation of bone marrow and peripheral blood; Perfusion of hematopoietic stem cells: The marrow from ABO- incompatible donor depleted erythrocytes by hydroxyethyl starch sedimentation.
MAIN OUTCOME MEASURES: Adverse reaction, complication and hematologic recovery after ABO-incompatibility stem cell transplantation.
RESULTS: One out of fourteen recipients developed pure red cell aplasia (PRCA) and dropped out of final analysis. Hematologic recovery: The median time of erythrocyte recovery after ABO-incompatible stem cell transplantation was delayed compared with ABO compatible stem cell transplantation (t=2.352, P < 0.05). There were no significant differences in the recovery of neutrophils and platelets between ABO-incompatible group and ABO-compatible group (P > 0.05). The median time of recovery of the erythrocyte and the blood type switching was delayed in HLA-mis-matched allogeneic hematopoietic stem cell transplantation compared with HLA-matched allogeneic hematopoietic stem cell transplantation, but without significant difference (P > 0.05). Complications: During the stem cell transfusion following transplantation, none of 14 patients had hemolytic complications or delayed haemolysis.
CONCLUSION: There was no evidence of ABO-incompatibility between donor and recipient is a barrier for successful allogeneic hematopoietic stem cell transplantation.

INTRODUCTION

With improvements in transplant technology, more transplant recipients now survive free of the disease for which they were transplanted. ABO-incompatibility between donor and recipient is not a barrier for successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) [1-4], but conflicting data still exist concerning its influence on transplant outcome, graft-versus-host diseases (GVHD), relapse incidence, and overall survival [5-8]. Therefore, we performed a retrospective analysis of the recovery of erythrocytic system in 14 patients who underwent ABO-incompatible allo-HSCT in order to investigate the therapeutic effects and complications of ABO-incompatible allo-HSCT.

SUBJECTS AND METHODS

Subjects
Fourteen patients [11 males and 3 females, averaged 34 years of age (range 15-60 years old), with a mean disease course of 7 months] with malignant hematologic diseases who received ABO-incompatible allo-HSCT in the Affiliated Drum Tower Hospital of Nanjing University Medical School from May 2002 to September 2007 were recruited for this study. Five patients had received transplantation for acute myeloid leukemia, seven patients for chronic myelogenous leukemia (two patients for accelerated phase), one patient for aplastic anemia, one patient for stage Ⅳ non-Hodgkin's lymphoma. We reported the ABO-incompatibility transplantation in 14 patients, including 4 ABO minor mismatched, 6 major mismatched and 4 bi-directionally mismatched pairs. In which, 6 patients had received human leukocyte antigen (HLA)-mis-matched transplantation. All the patients had no active diseases and had normal functions of heart and lung. Controls were 11 patients who received ABO-compatibility bone marrow transplantation during the same period. Written informed consents

for receiving allo-HSCT were obtained from each recipient. This experiment was approved by the Ethics Committees of the hospital. The concrete general data of included patients are in Table 1.

Methods
Grafts
The CD34 content of allografts was significantly higher in the patients receiving HLA-mismatch transplantation compared with those receiving HLA-match transplantation: 5.6(range,4.5-10.5) versus 3.2 (range 2.3-4.5) ×106 CD34+ cells per kilogram, respectively. The MNC content of allografts was also higher for patients receiving HLA-mismatch transplantation compared with those receiving HLA-match transplantation: 7.8(range 5.7-12.8) ×108 versus 3.1 (range 2.8-3.7) ×108 CD3+ cells per kilogram, respectively.

Regimen conditioning
HLA-matched transplantation regimen conditioning consisted of busulfan (Bu) and cyclophosphamide (Cy). HLA-mis-matched transplantation regimen conditioning adopted GIAC program from Beijing People's Hospital (G: granulocyte colony-stimulating factors used for donors; I: stronger immunosuppressive regimen conditioning used for recipients; A: antihuman thymocyte globulin added; C: combined transplantation of bone marrow and peripheral blood.

Processing of bone marrow from donor
The marrow from major ABO- incompatible donor depleted erythrocytes by hydroxyethyl starch (0.06 volume fraction) sedimentation and that from the minor ABO-incompatible hematopoietic stem cell grafts depleted the plasm before perfusion. Prior to and following marrow perfusion, recipients were hydrated with half physiological saline and diuresed with intravenous furosemide to maintain a urine flow greater than 100 mL/h. Thirty minutes prior to perfusion, all patients were premedicated intravenously with 10 mg of dexamethasone.

Prevention and treatment of complications following transplantation
Prevention of GVHD was performed with cyclosporin A (CSA) and short-range methotrexate(MTX); Support therapy included using antibiotics and G-CSF prophylacticly. According to the blood type of donor-recipient, we performed red cell and platelet transfusion when the platelets of patients were less than 20×109 L-1 or hemoglobin less than < 60 g/L. All the blood products must be irradiated before transfusion.

Statistical analysis
Statistical analysis was performed using SPSS 6.0 For Windows software. t test was used for comparing the difference in measurement data.

RESULTS

Quantitative analysis of participants
Fourteen patients who received ABO-incompatible allo-HSCT and eleven who received ABO-compatible allo-HSCT were recruited for this study. Among the 14 patients, 1 suffered from pure red cell aplasia and dropped out of final analysis.

Reconstruction of hematologic function
In 13 of 14 patients undergoing ABO-incompatibility stem cell transplantation, the median time of recovery of erythrocytes (haemoglobin at least 80 g/L), neutrophils　 (neutrophil count at least 0.5×109 L-1) and platelets (platelet count at least 20×109 L-1) were 43.2 (range 13-79) days, 16.0 (range 11-20) days and 22.0 (range,12-26) days, respectively. Meanwhile, in the 11 patients undergoing ABO-compatibility stem cell transplantation, the median time of recovery of erythrocytes, neutrophils and platelets were 30.6(range14-48) days, 17.8(range 14-30) days and 20.4(range11-34) days, respectively. The median time of erythrocyte recovery after ABO-incompatible stem cell transplantation was delayed compared with ABO compatible stem cell transplantation. There were no significant differences in the median time of recovery of neutrophils and platelets between the ABO-incompatibility group and the ABO-compatibility group (P > 0.05) (Table 2).

Time of hematologic recovery and blood type switching in ABO-incompatible HLA half-matched and HLA-matched stem cell transplantation
The median time to the recovery of neutrophils (0.5× 109 L-1)and platelets (20×109 L-1) was on days 16.4 and 22 after hematopoietic stem cell transplantation, respectively in patients undergoing ABO-incompatibility HLA- mis-matched stem cell transplantation, and they were not significantly different as compared with patients undergoing ABO-incompatibility HLA-matched stem cell transplantation (P > 0.05). Additionally, the median time of the erythrocyte recovery and the blood type switching after ABO-incompatible HLA-mis-matched stem cell transplantation was delayed compared with HLA-matched allo-HSCT, but without significant difference (P > 0.05) (Table 3).

Complications
During the stem cell transfusion following transplantation, none of 14 patients had hemolytic complications or delayed haemolysis. Only one patient developed pure red cell plastic anemia. We treated the patient with erythropoietin, without success. Marrow examination showed that the erythroid in marrow was less than 5%, granulocytic and megakaryocytic accrementition developed well in the marrow on day 120. While the blood type switched to donors' type 8 months after transplantation. During the stem cell transfusion, none of 14 patients had hemolytic complications, such as chilly, shakes, fever, lumbodynia, dark brown urine and so on. After transfusion, urine routine test showed that results were all in the normal

DISCUSSION

In the current study, all the patients had full donor myeloid chimerism following ABO-incompatible allo-HSCT and there was no evidence ABO-incompatibility between donor and recipient is a barrier for successful allo-HSCT [1-4]. While we must pay attention to adopt strategies to minimize the possibility of acute hemolytic complications associated with perfusion of ABO-mismatched grafts[5-8]. The total amount of peripheral blood stem cells was about 200 mL. In general, it can be directly perfused after collected because of having few red cells. While the amount of marrow and the red cells and plasma in the marrow were large, we should treat the marrow or recipient before transfusion. During perfusion, we adopted fluid transfusion, diuresis, urine alkalization and use of corticosteroids.
In spite of regimen conditioning of HLA-mis-match stem cell transplantation adopted serious immunosuppressive agents and chemotherapy drugs, such as antithymocyte globulin (ATG), the neutrophil and platelet recovery were not delayed. We supposed it was related to mobilized marrow and peripheral blood stem cells including more mononuclear cells, CD34+ cells and colony-forming unit-granulocyte macrophage (CFU-GM)[9-10]. While we observed that the median time of the erythrocyte recovery and the blood-type switching after ABO-incompatible HLA-mis-matched stem cell transplantation was delayed compared with HLA-matched allo-HSCT, but without significant difference (P > 0.05). We should accumulate cases for further study. It's worth noting that 6 of 8 patients with HLA-matched allo-HSCT received perfusion of peripheral blood stem cells. Kanda et al [11] reported that the use of peripheral blood stem cells facilitated erythrocyte recovery, and most of recipients following peripheral blood stem cell transplantation had erythrocyte recovered before anti-host isohaemagglutin disappeared. So they considered even if existing anti-host isohaemagglutin, a great quantity of erythrocytic precursor cells in the peripheral blood stem cells facilitated erythrocyte recovery.
Most researchers considered no statistically significant difference was noted in hematologic recovery, complication and long-term survival following receiving ABO-incompatibility or ABO-compatibility allografts[12-13]. In the current study, the median time of erythrocyte recovery after ABO-incompatible stem cell transplantation was significantly delayed (P < 0.05). While pure red cell aphasia is an infrequent hematologic complication after allogeneic bone marrow transplantation [14-16]. We reported one out of 14 recipients developed pure red cell aplasia. In general, incidence of pure red cell aplasia increases in type O recipients following transplantation. It is believed to be due to a delayed fall in antidonor isohemagglutinin titer [4,17]. Whether the grafts depleted erythrocytes or T cells had no impact on the development of pure red cell aplasia. Although the mechanisms responsible for pure red cell aplasia developed following transplantation have not been fully elucidated, and pure red cell aplasia spontaneously relieves in some patients. Zhang et al reported that cyclosporine (CsA) withdrawal was necessary before resolution of pure red cell aplasia and commented that this may be related to its
inhibitory action on T- but not B-cell function. Some patients were resolved completely after treatment with ATG or ALG [14,18]. We report here a patient who developed post-BMT pure red cell aplasia. His sibling donor (brother) was HLA identical and ABO major mismatched, pure red cell aplasia did not improve after treatment with erythropoietin but the problem was resolved completely after treatment by plasma exchange. However, the aging patients should select ABO-compatible donor in order to avoid delayed donor red cell recovery and the development of pure red cell aplasia. If no way to avoid, it is suitable to select peripheral blood stem cell transplant and perfuse more mononuclearcells as possible.

REFERENCES

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ABO血型不合异基因造血干细胞移植
治疗恶性血液病★

邵晓雁，欧阳建，陈 兵，许景艳，杨永公，周 敏
南京大学医学院附属鼓楼医院血液科，江苏省南京市 210008
邵晓雁★，女，1977年生，江苏省苏州市人，汉族，2003年苏州大学毕业，硕士，主治医师，主要从事造血干细胞移植治疗恶性血液病的研究。
通讯作者：欧阳建，教授，主任医师，南京大学医学院附属鼓楼医院血液科，江苏省南京市 210008
摘要
背景：由于移植技术的提高,供受者ABO血型不合已不再是异基因造血干细胞移植的障碍，但是由于宿主血凝素抗体的持续存在,ABO血型不合异基因造血干细胞移植后常出现红细胞系统恢复的延迟。
目的：观察ABO血型不合异基因造血干细胞移植患者红细胞系统恢复情况,评价血型不合、人类白细胞抗原配型不相合对造血功能重建的影响。
设计：回顾性分析。　
单位：南京大学医学院附属鼓楼医院血液科。
对象：选择2002-05/2007-09在南京大学医学院附属鼓楼医院血液科行ABO血型不合异基因造血干细胞移植的恶性血液患者(受者)14例,男11例,女3例；年龄15~60岁。14例患者中7例供受者人类白细胞抗原配型完全相合,7例供受者人类白细胞抗原配型半相合。纳入同期ABO血型相合的造血干细胞移植患者11例为对照。受者在接受异基因造血干细胞移植前签署移植同意书，供者为同胞姊妹、胞弟、儿子、母亲，均同意提供用于移植的骨髓。实验经医院伦理委员会批准。
方法：①预处理方案：人类白细胞抗原配型全相合组采取马利兰和环磷酰胺为主的方案。人类白细胞抗原配型半不合组采用北京人民医院的GIAC方案。②造血干细胞输注：沉降供者骨髓,取上层有核细胞输给受者。
主要观察指标：观察ABO血型不合异基因造血干细胞移植的副反应、并发症及造血重建情况。
结果：14例ABO血型不合患者仅1例发生单纯红细胞再生障碍性贫血未进入结果分析。①造血功能重建情况：与对照组比较，ABO血型不合组血红蛋白恢复时间明显延迟(t=2.352, P < 0.05)，ABO血型相同与ABO血型不合组中性粒细胞和血小板恢复情况差异无显著性意义（P > 0.05）。ABO血型不合的人类白细胞抗原配型半不合造血干细胞组血红蛋白恢复和血型转换时间迟于全相合,但其差异无显著性意义（P > 0.05）。②并发症：14例ABO血型不合患者移植后成分输血过程未出现溶血反应,移植后也均未发生迟发性溶血反应。
结论：ABO血型不合不影响造血干细胞移植的效果，且较为安全。
关键词：造血干细胞移植；血型不合；疗效
中图分类号: R394.2 文献标识码: A 文章编号: 1673-8225(2008)12-02383-04
邵晓雁，欧阳建，陈兵，许景艳，杨永公，周敏.ABO血型不合异基因造血干细胞移植治疗恶性血液病[J].中国组织工程研究与临床康复，2008，12(12):2383-2386
[www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2383(ps).pdf]
（Edited by Song LP/Wang L）