ABSTRACT

Corresponding Author:
Dr. Seema Gupta,
#502, Westend Apartments,
Plot No–10, Sector–19A,
Nerul, Navi Mumbai–400706.
E-mail: seemaagupta@gmail.com

ABSTRACT

BACKGROUND

Thalassemia is the most common hereditary Anaemia. Lifelong and frequent red blood cell transfusions remain the main treatment for severe cases. Development of alloantibodies to red cell antigens is an important immune mediated delayed haemolytic transfusion reaction.

AIM

The objective of this study was to determine prevalence of alloimmunization among thalassemic patients of MGM Hospital, Kamothe.

SETTING AND DESIGN

Hospital based prospective study.

MATERIAL AND METHODS

This study was conducted in Department of Immunohaematology and Blood Transfusion of MGM Medical College and Hospital, Kamothe, Navi Mumbai, from January 2015–December 2015. Plasma of patients was screened for presence of any red cell alloantibodies on a commercial 3-cell panel by Column Agglutination Technique (CAT) (Diacell, Bio-Rad, Switzerland). Positive cases were subjected to antibody identification using 11-cell panel by CAT (Diacell, Bio-Rad, Switzerland).

STATISTICAL ANALYSIS

Statistical analysis of data was performed using the Statistical Package for the Social Sciences (SPSS) software Version 16.0. Discrete categorical data were presented as n (%). Continuous data were given as mean.

RESULT

Data from 171 thalassemic patients was analysed; 9 patients from among the 171 developed alloantibodies, the incidence of alloimmunization being 5.26%. Two out of these 9 patients developed multiple alloantibodies. Thus, a total of 11 alloantibodies were identified in the patient population. Investigations of the alloantibody specificity detected the Rh Group to be the most common (63.63%) followed by the MNS (18.18%) and Kell group (18.18%).

CONCLUSION

The alloimmunization rate was detected to be 5.26% among the multi-transfused thalassemic patients. After the diagnosis of thalassemia has been established, if the transfusions are started at an early age, then it will help prevent the development of alloantibodies in these patients.

KEYWORDS

Thalassemia, Allo-Immunization, Multiple Blood Transfusions, Allo-Antibodies, Auto-Antibodies. Other Blood Groups.

INTRODUCTION

Multiple transfusions are required in patients suffering from diseases like–thalassemia, sickle cell anaemia, patients on dialysis, cancer patients, etc. Alloimmunization means development of alloantibodies to red cell antigens due to disparity between donor and patient antigens.

Alloimmunization after exposure to red cell alloantigens depends on genetic and acquired patient-related factors, dose and the immunogenicity of the antigens.^[1,2] The exact kinetics of alloimmunization are not clear.^[3,4] This is an immune mediated delayed haemolytic transfusion reaction. According to published data, rates of alloimmunization in random patients vary from 0 to 3 percent.^[5] The factors for alloimmunization are complex and involve at least three main contributing elements: (1) The RBC antigenic difference between the blood donor and the recipient; (2) The recipient’s immune status; (3) The immunomodulatory effect of the allogeneic blood transfusions on the recipient’s immune system.^[4] The incidence of alloimmunization against RBC antigens depends on the demography of the population being studied.

Antibodies reacting at 37^oC in vitro and/or those reactive in the Indirect Antiglobulin Test (IAT) phase are considered to be clinically significant antibodies, which can lead to Haemolytic Transfusion Reactions (HTR) or Haemolytic Disease of Foetus and New-born (HDN). These are usually Immunoglobulin G (IgG) in nature. Since cellular assays and labelling studies are usually unavailable in routine laboratories, it is the historical data on the association of an antibody with HTRs and HDN, which is used to predict their clinical significance.^[6]

Finding a compatible unit for patients who have developed alloantibodies becomes a difficult task. Blood units negative for those antigens against which antibodies have been developed should be issued to such alloimmunized patients. Antigen typing of all the donor units is not routinely done in all the laboratories. Thus, alloimmunization especially in patients who are to receive multiple transfusions throughout their lives, complicates and limits the transfusion therapy. This contributes to technical complications along with morbidity and mortality. Blood units that have been antigen typed and matched for these minor antigens show expected in vivo survival rates and prevent adverse transfusion reactions in these patients.^[7]

Thalassemia is a congenital haemolytic disorder. It was first reported in literature in 1925, when Cooley and Lee described a form of severe anaemia, occurring in children which was associated with bone changes and splenomegaly. Although bone marrow transplantation is the only cure, regular blood transfusion is the available treatment for these patients.^[8] Alloimmunization becomes one of the major complications in multiple transfused thalassemic patients. The purpose of carrying out this study was to determine the prevalence of RBC alloantibodies and know the types of these antibodies among the thalassemic patients receiving multiple transfusions from our institute.

MATERIAL AND METHODS

The study was conducted from January 2015 to December 2015 at the Department of Immunohaematology and Blood Transfusion, Kamothe, Navi Mumbai. All thalassemia patients (α or β, major or intermedia) registered with the thalassemia clinic at our institute were included in this study. Clearance from the Ethical Committee of the Institute was obtained before starting the study. Patient’s history was elicited and the following data was noted–name of patient, age, sex, blood group, diagnosis, age at diagnosis and initiation of transfusion therapy, transfusion interval, splenectomy status, any other special interventions.

Thalassemia patients are transfused to keep target Hb levels between 9–11.5 g/dL according to the institutional transfusion policy. Each time a blood requisition was received for thalassemia patients, a fresh sample of about 3 mL was collected in ethylene diamine tetra acetate containing tube. The sample was centrifuged at 3000 rpm for 3 mins. and the plasma obtained was used to perform alloantibody screening by using commercially available 3–cell panel (Diacell, Bio-Rad, Switzerland). Blood samples of patients having a positive screening test were then tested for alloantibody identification using commercially available 11 cell panel (Dia-cell, Bio-Rad, Switzerland). Autoantibodies were detected by incubating patient’s own plasma with patient’s own RBCs at 37^oC for 10 min. on gel card containing poly-specific antihuman globulin (anti IgG + C3d).

Ethics

This study was approved by the Ethical Review Committee of the Institute. Participants were briefed about the study and their consent was obtained.

Statistics

Statistical analysis of data was performed using the Statistical Package for the Social Sciences (SPSS) software Version 16.0. Discrete categorical data were presented as n (%). Continuous data were given as mean. This study was approved by the Ethical Review Committee of the Institute. Informed consent of the patients or their guardians was obtained.

RESULT

Data from 171 thalassemic patients was analysed. The mean age 9.61 years, ranging from 6 months to 62 years. Among the total patients, 47.4% were males and 52.6% were females; 9 patients from among the 171 developed alloantibodies, the incidence of alloimmunization being 5.26%. Two out of these 9 patients developed multiple alloantibodies. Thus, a total of 11 alloantibodies were identified in the patient population.

Investigations of the alloantibody specificity detected the Rh Group to be the most common (63.63%), followed by the MNS (18.18%) and Kell group (18.18%). Among the Rh group the incidence of antibodies was as follows–Anti-D = 27. 27%, Anti-C (18.18%), Anti-E (9.09%) and Anti-C (9.09%); 77.8% of the patients developed a single alloantibody, while 22.2% developed multiple alloantibodies [Table 1]. All patients who developed dual antibodies were having at least one antibody belonging to the Rh blood group system.

Difference between mean age among alloimmunized and non-alloimmunized was not statistically significant. Age at which first blood transfusion was received was significantly higher in alloimmunized (28.7 months) than in non-alloimmunized patients (15.2 months). The development of alloantibodies did not co-relate with the total number of transfusions received by the patients. Higher frequency of alloimmunization were observed among the male patients [Table 2].

The distribution of ABO and Rh blood group among the thalassemic patients is shown in Table 3. On group-wise analysis, it was noted that most of the patients who developed alloimmunization belonged to the B group (44.44%). Higher frequency of alloimmunization was detected among the patients having Rh negative blood group (77.7%).

Auto-antibodies were detected in 3 patients (3.5%). Mean age of developing auto-antibodies was found to be higher (16.3 years) than patients not developing auto-antibodies (11.8 years). No statistical gender difference was found for the development of allo and auto-antibodies. Development of auto-antibodies co-related with increasing number of blood transfusions received by the patients [Table 2].

DISCUSSION

The thalassemia syndromes are a heterogeneous group of inherited disorders caused by genetic lesions leading to decreased synthesis of one or more of the globin subunits.^[9] It is caused by a partial or complete defect in α or β globin chain synthesis. The globin chains that are produced in relative excess can damage the red cells or their precursors. As a result, there is an overall deficit of haemoglobin tetramers in the Red Blood Cells (RBC) and the Mean Corpuscular Volume (MCV) and Mean Corpuscular Haemoglobin (MCH) are reduced.^[10] Thalassemia is considered as the most common genetic disorder world-wide with a particularly high frequency in a broad belt, extending from the Mediterranean basin through the Middle East (Iran), India and Southeast Asia.^[11]

Early and regular blood transfusion therapy in patients with thalassemia decreases the complications of severe anaemia and prolongs survival. In the long term, however, the beneficial effects of transfusions are limited by complications such as chronic viral infections, haemosiderosis and alloimmunization against RBC.^[12]

The development of anti-RBC antibodies (Both allo- and auto-antibodies) remains a major problem. Repetition of transfusions for the treatment of thalassemia major provokes the patient’s immune system and produces anti-erythrocyte antibodies (Alloantibodies and/or autoantibodies). The development of alloantibodies and/or autoantibodies against RBC antigens complicate RBC cross-matching, shortens in vivo survival of transfused cells, delays provision of safe transfusions and may accelerate tissue iron loading.^[13,14] The literature reports various frequencies of alloimmunization depending on the homogeneity of the donor-recipient population, RBC phenotype matching policy and age at transfusion initiation. Reported alloimmunization rates ranged from 4% to 50% in thalassemia and were lower in more homogeneous populations.^[4,15-17] Some alloantibodies are haemolytic and may cause haemolytic transfusion reactions and limit the availability of further safe transfusion. Others are clinically insignificant.^[4,18]

The rates of alloimmunization in our study were found to be 5.26%, which were similar to results found in other studies conducted by Karimi et al^[19] (5.3%), Sirchia et al^[16] (5.2%) and Dhawan et al^[20] (5.64%).

Among the alloimmunized antibodies, the Rh blood group system was found to be the commonest in our study. Similar results were obtained in studies conducted by Dhawan et al^[20] (52.17%), Al-Joudi F et al^[21] (37.4%) and Sadeghian et al.^[11] We found alloimmunization to be associated more with male patients as found by Saied et al.^[22] The patients who received their first transfusion within 2 years of age were found to have a low rate of alloimmunization in our study. Similar findings were observed by Dhawan et al^[20] in the study conducted by them. Alloimmunization risk was significantly lower in haemoglobinopathy patients who started transfusion therapy at a very young age (<3 years) compared with those who started later in life, where an immature immune system and some form of the acquired immune tolerance to allogeneic RBC antigens is held responsible for the reduced alloimmunization risk.^{[13,23,24,25]}

In our study 3.5% patients developed auto-antibodies, which is very less as compared to the rates observed in the studies conducted by Dhawan et al^[20] (28.2%), Singer et al^[4] (25%), and Ammen et al^[26] (11%). The autoantibodies found among our patient pool did interfere with the compatibility testing and were not associated with any increased haemolysis. Although some reports found the existence of warm autoantibodies to be associated with clinically significant haemolysis.^[27] Others did not find this to be always the case.^[28]

Patients with autoantibodies may have a higher transfusion rate and often require immunosuppressive drugs, splenectomy or alternative treatments.^[29,30] Approaches for prevention of alloimmunization are under debate. They range from the provision of RBCs matched for all the major antigens associated with clinically significant antibodies to blood matched only for antibodies that have already been made. Reasons for controversy regarding following the best approach lay in the fact that many alloantibodies are not harmful and expensive prevention methods may therefore benefit only some patients.^[31,32,33] In addition, donor feasibility and the cost of RBC matching affects the approach of individual medical centres. There is limited data on the RBC phenotypes and the extent of alloimmunization among Asians.

CONCLUSION

The alloimmunization rate was detected to be 5.26% among the multi-transfused thalassemic patients. The blood group systems which were found to be commonest among the alloimmunized patients were Rh, MNS and Kell. The high frequency of Anti-D alloantibody implies that quality programmes for determination of weak D positive red blood cells should be improved and updated, so that the rate of alloantibody synthesis can be reduced. After the diagnosis of thalassemia has been established, if the transfusions are started at an early age, then it will help prevent the development of alloantibodies in these patients.

RBC antigen typing before the first transfusion and issue of antigen matched blood should be tried wherever possible. The patients who have already developed alloantibodies should be regularly checked for disappearance of old alloantibodies and appearance of new alloantibodies. All these measures would help the transfused RBC’s to survive in the patient for a longer period of time and prevent any haemolytic transfusion reactions.

ACKNOWLEDGEMENTS

We thank the Mahatma Gandhi Mission’s University for their permission for conducting this study and generous help with data collection.

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