Study of Frequency and Characteristics of Red Blood Cell Alloimmunization in Thalassemic Patients: Multicenter Study from Palestine

Background. β-Thalassemia is a common inherited hemolytic disorder in Palestine. Red blood cell (RBC) transfusion is the principal treatment but it may cause RBC alloimmunization. This study was conducted to determine the prevalence and characteristics of RBC alloimmunization among thalassemic patients in northern governorates of Palestine. Methods. A prospective multicenter observational study was conducted in the thalassemia transfusion centers in the northern governorates of Palestine. The study included 215 thalassemia patients who received regular blood transfusions. Clinical and transfusion records of patients were examined. Antibody screening and identification was conducted using the microcolum gel technique. Results. Two hundred fifteen patients were included in the study. More than half (52.1%) of the patients were males. The median age of patients was 18 years (range: 12–24 years). The most frequent blood group was A (40.5%). Alloantibodies were detected in 12.6% of patients. Anti-D (33.3%), anti-K (25.9%) and anti-E (14.8%) were the most commonly isolated antibodies. There was no association between age, sex, starting age of transfusion, number of transfused units, history of splenectomy and alloimmunization. Conclusions. Anti-Rh and anti-K antibodies were common among this cohort of patients. Age, sex, starting age of transfusion, number of transfused units, and history of splenectomy could not predict the occurrence of alloimmunization.


Introduction
Beta thalassemia (β-thalassemia) is the most common inherited hemolytic disorder caused by partial or complete defect in globin chain [1,2]. Patients with β-thalassemia-major need regular blood transfusions throughout life. Blood is usually administered every 2-5 weeks in order to maintain the hemoglobin level between 9.5 and 10.5 g/dl [3,4].
Alloimmunization to red cell antigens is one of the important complications of chronic blood transfusion in addition to iron overload and transfusion-transmitted infections [5,6]. It can complicate transfusion therapy by causing delayed transfusion reactions and difficulties in finding compatible blood which may lead to increased morbidity and mortality in transfusion-dependent patients [7][8][9].
Alloimmunization rates among multiply-transfused thalassemia patients range from 2.5% to 42% in different regions of the world. e most common RBCs alloantibodies reported were ones against the Rhesus (Rh) and Kell antigens [10,11].
ere is only one previously published report about alloimmunization in multiply-transfused patients in Palestine. Samarah et al. reported the frequency of alloimmunization among sickle cell disease patients (7.76%) and the most frequent antibody was anti-K followed by anti-E antibodies [12]. e objectives of this study were to [1] assess alloimmunization among thalassemia patients in the northern governorates of Palestine, [2] determine the specificity of detected antibodies, and [3] to assess the possible association between previously reported risk factors and the development of alloimmunization.

Methods
A prospective multicenter observational study was conducted in all thalassemia transfusion centers in the northern governorates of Palestine (West Bank). Palestine consists of northern and southern governorates ( e West Bank and Gaza Strip, respectively).

Patients
e total number of transfusion-dependent thalassemic patients in the northern governorates is 500 (verbal communication from the TPFS-Palestine). In total, 215 thalassemic patients (β-thalassemia major and β-thalassemia intermedia) were conveniently selected and evaluated. Transfusiondependent patients who received at least 10 blood transfusions, of any age or gender were included. Multiply transfused patients with known connective tissue disease or any other autoimmune diseases were excluded.

Data Collection
Demographic and clinical information such as age, gender, status of the spleen, age of starting transfusion and number of transfused units were obtained directly from the participants (or their legal guardians) or from their medical files.

Laboratory Methods
Blood samples were collected from all patients. Plasma was screened for the presence of alloantibodies by using commercial three-cell panel (ID-Diacell I-II-II, Bio-Rad, Switzerland). e antibody specificity of all positive samples was determined using a commercial 11-cell identification panel (ID-DiaPanel, Bio-Rad, Switzerland).

Statistical Analysis
Statistical analysis was performed using the Statistical Package for the Social Science (SPSS) so ware (version 23.0; SPSS Inc., Chicago, IL, USA). Analysis included descriptive statistics, frequency distribution, mean, and standard deviation calculations. e relationship between alloimmunization and age, starting age of transfusion, and number of transfusions was assessed by Mann-Whitney U test. Chi-Square or Fisher's exact test was used to assess the association between gender and splenectomy and risk of alloimmunization. All reported values are two-sided, with a priori significance level of 0.05.

Ethical Considerations
Informed consent was obtained from patients or their legal guardians. e study was approved by Institutional Review Board committee at An-Najah National University, the research ethics committee at Al-Quds University and the Palestine Ministry of Health.

Assessment of Predictors for
Alloimmunization. Age of patients, starting age of transfusion, number of transfused blood units, gender, and splenectomy were not significantly different among alloimmunized and nonalloimmunized patients (Table 4).

Discussion
is study was conducted to determine the frequency and specificity of alloantibodies among transfusion-dependent thalassemia patients in the northern governorates. On the other hand, we assessed the association of previously reported risk factors and the development of alloimmunization in our cohort.
Predictors of alloimmunization among multiply transfused patients are still debatable. Our study showed no significant association with age and this is in agreement with Al-Mousawi et al. [27] and Elhence et al. [31]. On the other hand Al-Riyami et al. from Oman reported a significant association between age and alloimmunization [21].
Age at initiation of transfusion was not significantly different among alloimmunized and non-alloimmunized patients. is is in agreement with Karimi et al., Amin et al.,and Elhence et al. [18,26,31]. Other reports showed a significant association [24,27].
Contrary to other reports [21,23,26,29,32], we did not find a significant association between the numbers of transfused blood units the development of alloimmunization and this is in agreement with Al-Mousawi et al. [27].
Similar to our study, few reports did not find a significant relationship between alloimmunization and gender [21,23,27]. Other reports found female gender [24,33] or male [29] as a risk factor for alloimmunzion. 10. Limitations of the Study e incidence of thalassemia in Palestine is dwindling and this is due to the premarital screening program enforced by the Palestine Ministry of Health for beta thalassemia carriers and discouraging marriage between carriers. Clinical data were collected from a computerized system in the Palestinian Ministry of Health, but some data such as age of starting transfusion, transfusion frequency and number of transfused units was unavailable. erefore, age of starting transfusion and transfusion frequency were estimated by patients or guardians and the number of transfused units was estimated by calculations from the age of starting transfusion and transfusion frequency.

Conclusions
e high frequency of anti-D antibodies highlights the need to implement strict quality control programs in the local blood banks to test for weak D positive antigens. Red cell alloimmunization in transfusion dependent thalassemia patients can be minimized by phenotyping for Rh and Kell system.

Data Availability
Data supporting the findings will be provided upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.