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 Table of Contents  
Year : 2019  |  Volume : 4  |  Issue : 1  |  Page : 39-44

Spectrum of alloimmunization among multitransfused beta-thalassemia major patients

1 Safe Blood Transfusion Programme, Ministry of National Health Services, Government of Pakistan; Department of Pathology and Blood Bank, Pakistan Institute of Medical Sciences, Islamabad, Pakistan
2 Department of Bioinformatics and Biotechnology, International Islamic University, Islamabad, Pakistan
3 Department of Pathology and Transfusion Medicine, District Headquarters Hospital, Mandi Bahauddin, Punjab, Pakistan
4 Safe Blood Transfusion Programme, Ministry of National Health Services, Government of Pakistan, Islamabad, Pakistan
5 Department of Zoology, PMAS Arid Agriculture University, Rawalpindi, Pakistan

Date of Web Publication22-Apr-2019

Correspondence Address:
Dr. Usman Waheed
Safe Blood Transfusion Programme, Ministry of National Health Services, Government of Pakistan; Department of Pathology and Blood Bank, Pakistan Institute of Medical Sciences, Islamabad
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/GJTM.GJTM_52_18

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Introduction: The development of alloantibodies is a major problem among thalassemia major individuals due to periodic blood transfusions. The current study assessed the frequency and specificity of erythrocytes alloimmunization and also the variables affecting the extent of alloimmunization in repeatedly transfused thalassemia patients. Materials and Methods: This prospective, cross-sectional study was conducted at the Department of Blood Transfusion Services and Thalassemia Centre of Pakistan Institute of Medical Sciences from August 2017 to February 2018. Of the 475 thalassemia major patients, alloantibodies were screened and identified by 3-red cell antigen panel and afterward by an extended 11-cell antigen panel. The data analysis was done through SPSS version 20.0. Chi-square test was employed. Results: Alloantibodies were detected in 77 (16.2%) patients, 5 (6.5%) patients were found with double alloantibodies. Anti-D (31.1%) and Anti-E (29.9%) antibodies had the highest incidence and were found in 24 and 23 patients, independently. Anti-K antibody was observed in 11 (14.3%) and Anti-C in 3 (3.9%) patients. Anti-C (2.6%), Anti-E (2.6%), and Anti-Kpa (2.6%) all were found in two patients, individually. Similarly, Cw (1.3%), k (1.3%), Jka (1.3%), Anti-Fyb (1.3%), and Anti-s (1.3%) antibodies were detected in one patient each. Alloantibodies were common in males, splenectomized patients, in those who initiated their transfusions before 2 years of age, in patients receiving nonleukoreduced blood and in B and O blood group patients. Conclusion: The proper management and prudence are needed for thalassemia patients due to hemolytic nature of these alloantibodies. New techniques should be introduced to reduce the incidence of red blood cell alloimmunization.

Keywords: Alloimmunization, multiple transfusions, thalassemia major

How to cite this article:
Waheed U, Arshad M, Saeed M, Wazeer A, Farooq A, Arshad A, Zaheer HA. Spectrum of alloimmunization among multitransfused beta-thalassemia major patients. Glob J Transfus Med 2019;4:39-44

How to cite this URL:
Waheed U, Arshad M, Saeed M, Wazeer A, Farooq A, Arshad A, Zaheer HA. Spectrum of alloimmunization among multitransfused beta-thalassemia major patients. Glob J Transfus Med [serial online] 2019 [cited 2022 Jun 26];4:39-44. Available from: https://www.gjtmonline.com/text.asp?2019/4/1/39/256768

  Introduction Top

Beta-thalassemia major is the most prevailing autosomal recessive hemolytic disorder across the globe. It originates due to abnormal (absent/reduced/excessive) production of the β-globin chain. It causes an alarming situation initially in life; its victims suffer from severe anemia and fail to bloom properly.

The highest burden of thalassemia is observed in malaria-prevalent countries. It occurs with high prevalence in a broad belt extending from Mediterranean, Middle East, Indian subcontinent, South China, and Southeast Asian regions. Carrier frequency of thalassemia is about 5.6% in Pakistan with an estimated 5,000 new cases of thalassemia major born annually, and the total number may exceed 50,000 children.[1],[2]

The proposed management for beta-thalassemia major is regular blood transfusions, spleen removal, folate intake, excess iron expulsion with deferoxamine, and stem cell transplantation.[3] Stem cell transplantation has remained very successful in the treatment of these patients, but its limitation lies in affordability for our low- and middle-class society.[4] Hence, regular blood transfusion every 3–4 weeks is the only convenient way of treatment available in these patients to maintain the Hb level from 9 to 11.5 g/dl.[5] However, in long-term, regular blood transfusion schedule is confronted with several difficulties such as alloimmunization, organ damage due to excessive iron accumulation, and bloodborne diseases.

Erythrocyte alloimmunization is the production of antibodies due to foreign red cell antigens inherited from a heterogeneous individual of the similar species.[6] Effective factors for alloimmunization are erythrocyte antigenic variation between provider and recipient, the immune condition of the recipient and the immunomodulatory impact of isogenic blood transfusion on recipient's defense system. It acts as an impediment to safe blood transfusion supply, causes infantile hemolytic afflictions and varied hemolytic transfusion reactions.[7] Thalassemia patients are more prone toward the development of alloantibodies due to periodic blood transfusions, and studies reveal that immunoglobulin G antibodies exist in the serum of multitransfused victims.[8]

In majority of the blood centers across Pakistan, blood grouping is performed only for ABO and Rh antigens followed by a major crossmatch. The antibody screening and identification are not routinely performed and only a few centers have this facility. Published literature suggests the most frequently occurring erythrocyte alloantibodies are against the Rh and Kell antigens, followed by antigens of Kidd, Duffy, MNS, and other minor blood group types.[9] However, in our settings, limited data are available regarding these blood types and alloimmunization associated with them.

Due to lack of awareness with respect to minor blood group antigens, resulting complications, and their potentially fatal consequences, this study was conducted in multitransfused beta-thalassemia major patients to assess the rate and specificity of erythrocyte alloantibodies and to find the variables affecting the extent of alloimmunization in repeatedly transfused thalassemia major patients.

  Materials and Methods Top

The study protocol was approved from the Institutional Ethical Review Board. A prospective, cross-sectional analysis of 475 thalassemia major patients who had already received (>10) transfusions was conducted at the Department of Blood Transfusion Services and Thalassemia Centre of Pakistan Institute of Medical Sciences. The hospital is a 1300-bedded tertiary care facility in the federal capital. The study was carried out from August 2017 to February 2018. Frequently transfused patients with other hemoglobin disorders were excluded from the study. An informed consent was obtained in all the cases from the parents of thalassemic children.

According to the standard WHO phlebotomy protocols, about 5 cc blood sample was collected; 2 cc in ethylenediaminetetraacetic acid tube while and 3 cc in serum tube. All participants name, age, and blood group were noted. A questionnaire was designed to collect clinical data regarding age at the time of diagnosis and initiation of blood transfusion, transfusion span, Hb level at each transfusion, hepatomegaly, and spleen status from positive patients through interview method.

After blood clotting, serum was separated by centrifugation at 1,500 g for 10 min. Employing routine blood bank procedures serum was analyzed before each transfusion for detection of new antibodies to red blood cell (RBC) antigens. A commercially prepared DiaCell I + II + III (DiaMed, Switzerland) was used for antibody screening by adding 100 μl of patient's serum and 50 μl of commercially prepared washed RBCs in two phases, instantaneous NaCl phase, and 37°C LISS-anti human globulin phase. After centrifugation tubes were examined for agglutination and hemolysis. Coombs-control IgG was used to check the negative reactions. After antibody screening, antibody specificity against minor blood group antigens was determined in positive samples by DiaPanel and afterward was confirmed through ID-DiaPanel (DiaMed, Switzerland). DiaPanel has the similar procedure as employed for DiaCell I + II + III. The data analysis was done through SPSS statistical software version 20.0 (IBM Corp., Armonk, NY, USA). Chi-square test was used to assess statistical significance between clinical data.

  Results Top

Of 475 thalassemia major patients, males and females were 54.53% and 45.47%, respectively, (P < 0.7999) with a male-to-female ratio of 1.20:1. The mean age was 8.5 years, about 72.63% patients were from 2 to 10 years age group. The average age at the time of diagnosis and the first transfusion was six months and transfusion span was one month (range 1–3 m). About 10.1% of the study participants were splenectomized. All patients were receiving leukodepleted RBCs. The mean Hb level at the time of transfusion was 7.0 g/dl and very high serum ferritin level was observed (880–13,790 μg/l). Blood group “B” was most prevalent 41.5% [Table 1]. Among clinical features, hepatomegaly was seen in all patients. Different sizes such as 2 cm, 2–5 cm, and >5 cm were observed in 23 (29.87%), 24 (31.17%), and 30 (38.96%) alloimmunized patients, respectively. Background diseases included hepatitis-B (20%), hepatitis-C (30%), HIV (0.63%), heart diseases (10%), and diabetes (1%–2%).
Table 1: Clinical features of beta-thalassemia major patients

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Alloimmunization was detected in 16.2% (n = 77) patients. Blood group B was most prevalent 41.55% (n = 32) and AB was least prevalent 10.38% (n = 8) in alloimmunized patients. The rate of alloimmunization among both Rh-positive (P = 0.9022) and Rh-negative (P = 0.489) patients was nonsignificant, respectively.

After recurrent transfusions over the years, transfusion requirement increased and transfusion interval reduced to 1–2 weeks in these patients. Increased rate of immunization 64.94% (50/77) was observed among 11–28 years of age groups. Prevalence of alloimmunization was found to be highly significant among these age groups. The frequency of immunization was more (14.29%) in splenectomized patients compared to nonsplenectomized patients (11.69%) (P < 0.1738). Higher rate of immunization was observed in patients who received transfusion before the age of two years (P < 0.0000). Of 16.2% (n = 77), double alloantibodies were detected only in 6.5% (n = 5) patients. Most frequently occurring antibodies were of Rh blood group system followed by Kell system. Anti-D and Anti-E had the highest incidence and were found in 24 and 23 patients independently. Anti-K antibody was observed in 11 and Anti-c in 3 patients. Anti (-C,-e, and,-Kpa) all were found in two patients, individually. Similarly, Cw, k, Jka, Anti-Fyb, and Anti-s antibodies were detected in one patient each [Table 2].
Table 2: The frequency of alloimmunization in thalassemia patients (n=77)

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  Discussion Top

This study was conducted to evaluate the rate of alloimmunization in multitransfused beta-thalassemia major patients. Alloimmunization was detected in 16.2% (n = 77) patients (male – 43 and female – 34). Our results are consistent with the previous studies which reported 3%–37% RBC immunization rate.[7],[9],[10],[11],[12],[13],[14]

Among 77 immunized patients, the present study showed that double alloantibodies were detected only in 6.5% (n = 5) patients. Most frequently occurring antibodies were of Rh blood group system followed by the Kell system. Anti-D and Anti-E had the highest incidence and were found in 24 and 23 patients, independently. Anti-K antibody was observed in 11 and Anti-c in 3 patients. Anti (-C, -e, and, -Kpa) all were found in two patients, individually. Similarly, Cw, k, Jka, Anti-Fyb, and Anti-s antibodies were detected in one patient each.

A similar study from Karachi, Pakistan, reported similar RBC alloantibodies ratio, that is. 10.75% (n = 40). Frequency of single and multiple antibodies was 32 (82%) and 8 (20%), respectively, whereas E and K antibodies were more common 32.5% and 27.5%, respectively. Among the alloimmunization patients, the mean age was almost similar to the present study, that is, 10.63 years (standard deviation ± 5.587). Splenomegaly and hepatomegaly were reported in 77.5% and 45% patients, whereas splenectomy was found in one case.[7]

Another similar study conducted by Franchini et al. reported 11.4% RBC alloimmunization with higher rate against Rh and Kell antigens 52.4% and 25.6%, respectively, overall alloantibodies for Rh and Kell antigens accounted for 78% of the cases.[15]

In another study, Zaidi et al. analyzed 162 thalassemia patients and reflected the relatively low rate of immunization as compared to the present study, 4 (8·6%) patients with Anti-E,-K,-e, and -D as most prevalent antibodies.[16] The high rate of alloimmunization in the present study may be attributed to divergence of red cell antigens among providers and thalassemia patients as explained by the prior studies conducted on Asian population, in the USA, and on Arab thalassemia patients.[9],[14] As donors from different ethnic origins come to our department from all over Pakistan, they may represent diversity regarding their minor blood type antigens.

A study from neighbor country reported 16.32% rate of alloimmunization with clinically important alloantibodies of Kell and Rh blood group systems among 8 of 49 thalassemia patients.[17] Koçyiǧit et al. revealed alloantibodies from Rhesus, Kell, and Kidd systems in 9 (6.4%) out of 139 transfusion-seeking thalassemia major and intermedia patients.[18]

Hussein et al., discovered 123 (22.8%) alloantibodies in 62 repeatedly transfused thalassemia patients. Clinically significant alloantibodies were Anti (-E,-D,-C, and -c) succeeded by Anti-Kell, -MNS, -Kidd, -Duffy, -Le, -Lu, and -P1.[19] Ameen et al. investigated Anti-K and -E as frequent alloantibodies in 57 (30%) patients among a total of 190 Thalassemia major carriers.[14] Thompson et al. reported the prevalence of alloimmunization to be 16.5% in 115 of 697 thalassemia patients and identified alloantibodies mainly belonged to Rh, Kell, Duffy, Kidd, Lewis, and MNS blood group systems.[20]

In this study, the prevalence of alloimmunization was comparatively higher in males (55.84%) compared to females (44.16%). This shows concurrence with the results of the past studies conducted in Pakistan [21],[22] and Egypt.[19] Contrary to this, many studies have reported a high rate of alloimmunization in females as compared to males. In our study, 11 (22.9%) of 48 splenectomized patients were detected with red cell alloantibodies. This can be strengthened by the previous studies, which reported that patients with lost spleen showed a higher frequency of alloimmunization compared to nonsplenectomized patients.[9],[19] While according to Pahuja et al. for the eliciting immunization, splenectomy is not good criteria.[23] The antigens present on RBC and other immunogens are very different like anatomical compartmentalization. While antigens from infectious pathogens and solid organ transplants drain into local lymph nodes, Moreover, during the blood circulation RBCs remain segregated and become the target of splenic and hepatic antigen-presenting cells. Therefore, the spleen plays a key role in alloimmunization against transfused red cells.

In the present study, 47 (61.04%) of alloimmunized patients started the transfusion before the age of 6 months and remaining 30 (38.96%) received their first transfusion at the age of 2.5 years. This is in contrast with a previous study which showed a low rate of alloimmunization in patients who started blood transfusions (<3 years of age).[24] While studies by Bhatti et al. and Gader et al. found no correlation between age at the 22th time of initial transfusion and development of alloantibodies.[25] In our study, transfusion span was 30 days in nonalloimmunized patients but 10–15 days in alloimmunized patients. This gives the evidence that immunization against foreign antigens is the crucial factor for increasing the transfusion requisite in thalassemia patients.

To avoid the alloimmunization consequences in thalassemia patients, cross matching should be done at least for Rh and Kell systems as the majority of antigens reported in our study and previous studies conducted in Pakistan are of Rhesus and Kell blood group system. Regular antibody screening and identification will alleviate the problem of alloimmunization to much extent. This will not only ameliorate the patient safety but will also lessen the requirement for blood transfusion, which will prove more cost economical for a health-care center with meager reserves.

  Conclusion Top

A significant ratio of alloantibodies observed in this study necessitates the regular monitoring of erythrocyte alloimmunization in thalassemia patients, particularly which are suffering from varied hemolytic transfusion reactions. Limited data are available regarding detection of alloantibodies and provision of leukodepleted blood from the Pakistani population. Large-scale national studies are required to confirm and generalize these findings.


This work was supported by the Department of Blood Transfusion Services, Pakistan Institute of Medical Sciences, Islamabad. The authors are grateful to Khawaja Junaid Shokat, Product Manager, Sind Medical Stores, for his technical assistance in the conduction of the study.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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Saqib MA, Ansar R. Burden of thalassemia; time to act. Pak J Med Res 2017;56:66-7.  Back to cited text no. 2
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Qidwai A, Mansoor N, Syeda A, Maheen H, Mohammad I, Malik F. Trends of red cell alloimmunization in β thalassemia major patients: A single center retrospective study in Karachi. J Blood Disord Treat 2018;1:3-5.  Back to cited text no. 7
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Franchini M, Forni GL, Marano G, Cruciani M, Mengoli C, Pinto V, et al. Red blood cell alloimmunisation in transfusion-dependent thalassaemia: A systematic review. Blood Transfus 2019;17:4-15.  Back to cited text no. 15
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