Importance of screening and identification of alloantibodies in multi-transfused patients of thalassemia major

Introduction: Thalassemia is a form of inherited autosomal recessive blood disorders characterized by abnormal formation of hemoglobin. These patientsneed blood transfusion on regular basis to maintain the hemoglobin level in the body.The frequent transfusions received by thalassemia major patients, expose them to the risk of contracting infectious diseases, and development of complication such as iron overload and alloimmunization. The production of antibodies against such alloimmunization induces further hemolysis. Subject and methodology: The main objective of the study was to find out clinically significant antibodies in multi-transfused thalassemiamajor patients to prevent hemolysis and to reduce frequency of blood transfusion there by reducing morbidity and mortality. A prospective and observational study comprising of total 205 thalassemic patients were included in the study (females 99 and males 106) in the age ranging from3to 43 years who had received more than 10 units of blood within one year. Majority of them were β thalassemia major followed by Eβ and sickle cell disease.Apart from ABO and Rh grouping and issuing of blood by proper crossmatching the alloantibodies were detected by using 3 cell and 11 cell panel by gel technique.Alloantibodies against Rh phenotypes were more than 90%. Discussion and conclusion:Findingof unexpected antibodies must be a part of all pretransfusion testing procedure which will help to accomplish more effective and uneventful blood transfusionof multi-transfused thalassemia patient. Production of alloantibodiesinmulti-transfused thalassemia patients can be prevented by screening for minor blood groups from beginning in addition to ABO and Rh grouping.


Introduction:
Thalassemia isa congenital hemolytic disorder caused by partial or complete absence of alpha or beta globin chains and a common genetic disorder distributed throughout the world but more prevalent in south east Asia and Mediterranean countries and India 1 . Approximately 400,000 babies are born with serious hemoglobinopathies worldwide with about 270 millioncarriers 2 . It has been seen that approximately 3% of world population carry beta thalassemia gene. In Europe it was most prevalent in Italy and Greece. Comparatively frequencies between 2.5% and 14.9% have been reported in India 3 . It was observed that 1000/1.5 million per year live births have β-thalassemia 4 .In India, nearly 8000-10000 new thalassemia (homozygous) were born every year and more common in Punjabis, Sindhis, Bengalis, and Gujaratis 5 . The standard protocol for management of β-thalassemia major is regular blood transfusion every 3to 4 weeks and needs blood transfusion throughout life. In country like Indiablood transfusion remains only affordable mean for most of the patients. It is the common practice in most of blood banks to issue the blood units in the Eastern part of India by preforming major cross-matching between the recipient's serum and donor red cells by tube method and by tube AHG method. Usually in most blood banks blood is issued ABO matched compatible blood by tube methods (saline technique). On repeated blood transfusion red cell alloimmunization occurs because of antigenicdifferences between donor and recipient RBCs of the minor blood groups which are not usually tested during cross matching 6 .With the development of alloantibodies following repeated transfusion process leads complications of the patents which needs more transfusion due to hemolytic reaction and significantly complicate transfusion therapy 7 .

Aims and Objectives:
The study was conducted with the aims 1. To find out the antibodies developed due to dissimilarities of Donors and recipients' minor groups. 2. Hemolysis of such patients can be prevented by properly matched blood transfusion. 3. Repeated transfusion in short periods can be prevented.

Materials and Methods:
The study was conducted from June 2016to October 2018 in the department of Pathology and Blood Bank, B.S. Medical College, Bankura West Bengal, India after taking permission from Institutional ethical committee.Prospective and observational study was performed. Total 205 transfusion dependent patients between age group of 3 to 43 years (106 males & 99 females) who received more than 10 units of blood both whole blood and PRBCwere included in this study. Transfusion dependent thalassemia patient having HIV, HCV and HBV positivity were excluded from the study. Clinical diagnosis, numbers of blood units received with interval of transfusion and history of splenectomy noted. Transfusion protocol of hemoglobin level maintained around 9gm/dl. All patients were instructed to estimate hemoglobin on regular interval. As per protocol in our institute we start blood transfusion at 7.0 gm /dl and only PRBC were supplied to the patients. Major and minor coombs crossmatch done by gel technique. The results were noted accordingly.The antibodies (alloantibodies) of all participants were detected by using commercial three-cell panel More than 99% of clinically significant antibodies were detected by using three vials set of screening cells.If there was agglutination in the 3 set panel, a set panel of 11 screening cells was required to identify the specific antigen.All results were tabulated, and statistical analysis was performed through SPSS software (version 17.0).

Results:
Among 205 transfusions dependent thalassemia patients were included in the study for detection alloantibodies of which 99 were females and 106 were males. The clinical data of the patients are given below (Table 1 and Table 2).  159 transfusion dependent patient who did not show any alloantibody excluded from the study with advice to complete subgrouping of C, c,E,e and k in addition to ABO and RhDgrouping. Further study was done with II cell panel of 46 patients who had developed alloantibodies and were detected specific antibodies. After testing the 46 patients with II cell panel it was observed that alloantibodies against c was highest (34.78%) and lowest was against k antigen (6.52%). 93.48 % of patient showed antibodies against Rh phenotype (Table 3).  Development of alloantibodies were more in female than male and about 100% alloantibody were against Rh and K blood grouping.

Discussion and conclusion:
Thalassemia is a congenital hemolyticanemia of which thalassemia major patients need lifelong blood transfusion from early childhood to raised hemoglobin level and reduces the skeletal deformities associated with excessive erythropoiesis 8 . Although blood transfusion is life saver for thalassemia it may introduce infection which can be prevented by proper transfusion transmitted infection (TTI) testing of blood. Multiple transfusion introduces a multitude of alloantigenof which red blood cell related antigens are most important red blood cellspecific antigens which stimulate the immune system and produce alloantibodies 9.10 . Although due to improved techniques TTI cancontrol but red cell alloimmunization is still an important factor for reducing hemoglobin spite of extensive research and improved techniques of blood transfusion the red blood cell alloimmunization remain a major problem in transfusion dependent thalassemic patients specially patients from eastern part of India, as pretransfusion antibody screening is not routinely performed 11 .
In the present study we try to evaluate screening and identification of alloantibodies in multitransfused patients of thalassemia (including Hemoglobinopathy), in Thalassemia unit of B.S. Medical college & Hospital, Bankura. There were several studies in India and different parts of world, but most studies were done in urban areas without any documentation of such studies in rural areas.In a report of Subhra Dutta et al.reportedRed blood cell (RBC) alloimmunization in 5.6% patients and maximum alloimmunization occurred in the age group of 21-40 years 11 .AzitaAzarkeivan et al. in a study (2014) observed 11.3% alloimmunization of which74% patients were with single alloantibody &36% patients developed more than one antibodyand the most common alloantibodies were anti-Rh antibodies 12 .The present study observed 22.43%alloimmunization out of which 80.43% patient showed single antibody and 19.57% patients developed more than one antibody.About 93.48 % of patient showed antibodies against Rh blood group and occurred mostly in the age group of 10-15 years.Reisner et al observed alloimmunization are more with female patients than male 13 .
Incidence and type of alloantibody observer by several workers weredifferent and Rh related alloantibodies were most common alloantibodies. The incident of development of alloantibodies by different workers are shown in Table 5. Anti-c being the most common specificity Alloimmunization to red cell antigens is due to immune response usually stimulated by the transfusion of blood products and is one of the complications of RBC transfusions. Developments of anti-red blood cell antibodies still a major problem in thalassemia major patients.
In present study showed frequency of red blood cell (RBC) alloimmunization among thalassemia patients with regular transfusions were Rh blood group system antibodies and accounted 93.48% of alloantibodies.
Usually the minor antigens play the role of alloimmunization. Our data shows that the frequency of alloimmunization to minor RBC antigens is high in our multi-transfused thalassemia patients. The high incidence may be due tolack of homogeneity of RBC antigens between the blood donors and recipients and not matching withminor Rh Antigen. We concluded that for multi-transfused thalassemia patient identification of alloantibodies must be done fortransfusion of well-matched donor (lacking specific antigen against which the antibody developed) for desire rise of hemoglobin level and thereby reduces the transfusion frequencies as well as increase transfusion interval.Blood transfusion is life saving for thalassemia patients and it is the principal method of management of these patients in spite of that it may be associated with complication like RBC alloimmunization. Detectionfor unexpected antibodies should be a part of all pretransfusion testing procedure which will help to accomplish more effective and uneventful blood transfusion.

Conflict of Interest:
We declare no conflict of interest among authors. Ethical Approval: We conducted the study after obtaining the ethical approval from Ethical Committee of B.S. Medical College (written permission obtained). Funding statement: None. Individual Authors Contribution:All authors participated in all sections of the study and contributed in all sections.