Alloimmunisation in sickle cell patients of western Odisha: A tertiary care centre study

RBC carries numerous protein and carbohydrate antigens on their surface. Out of 347 red cell antigens recognized by international society of blood Transfusion, 308 antigens are clustered in 36 blood Group systems. Except naturally occurring anti-A and anti-B antibodies all others are unexpected. Out of these some like Duffy, Kell, Kidd, MNS, P and certain Rh types are considered clinically signiﬁcant. Only few studies for prevalence of irregular red cell alloantibody have been done. Those studies were done either in general population or in thalassemia patients. Few studies were done on sickle cell disease patients but all are outside India and those are signiﬁcant. But no studies have been done till now on prevalence of alloantibody in sickle cell disease patients in India. Again the western part of Odisha is with high patient load of sickle cell disease. This study is very useful for this part of Odisha as complication due to the alloantibody can be managed properly. Both the patients and the clinician will be beneﬁted by this study.


Introduction
Sickle cell disease is a hereditary haemoglobinopathy, characterised by chronic anaemia, recurrent painful episodes and irreversible organ damage. Transfusion of red cells is a common intervention to treat and prevent the complication. Patients with sickle cell disease have high risk of alloantibody formation. Alloantibody may cause haemolytic transfusion reaction (acute or delayed) or decrease in the survival of transfused RBCs.
Red blood cell alloimmunization results from the genetic red blood cell antigen disparity between donor and recipient or from mother and fetus. The first reports on alloimmunization date from the 17th century describing hydropic stillborns. This disease, today known as hemolytic disease of the fetus or newborn (HDFN), is caused by immune IgG antibodies from the mother directed against the red blood cells of the fetus A red blood cell unit contain red cells that express an array of multiple alloantigens, each of which can potentially induce an antibody response. It is therefore surprising that humoral alloimmunisation to red cell is rare. Indeed, when ABO COMPATIBLE, D matched red blood cells are used, only approximately 3% of transfused patients become alloimmunized, even following multiple red blood cell transfusion.
The alloimmunisation frequency varies with both the blood group antigen and the underlying genetics and pathophysiology of the recipient. Alloimmunisation rate are substantially higher in sickle cell anemia. 1 the reasons for this are 2. Alteration in immunobiology due to sickle cell disease. 3. Lickage disequilibrium with immunoregulatory genes close to the globin gene. 2 The red cell alloantibodies are not equally distributed among transfused patients. Rather patients who have made an alloantibody against one blood group antigen are more likely to make additional antibody often subsequent transfusion. Those not responding to antigen in initial transfusion are unlikely to develop the antibody. The two groups are responder and non-responder. These observations are practical ramification for management of patients requiring chronic transfusion therapy as sickle cell disease.
Matching blood for multiple antigens (e.g. kell, kidd &duffy) is both costly and time consuming. So many places matching for ABO & D is done during initial treatment and later on once the patient makes one red cell alloantibody, extensively matched blood is provided for subsequent transfusion, patient who does not make alloantibody continue to receive red blood cell matched only for ABO & D.
This saves the resources but results in development of at least one red cell alloantibody in responder, which would have been avoided.

Potential mechanisms
1. The recipient genetically negative for the antigens. 2. The transfused donor red cell carries the antigen. 3. The recipient MHC class-II molecules are capable of presenting a red cell allogenic peptide containing a variant amino acid found in the donor but not in the recipient.

Additional factors
1. Genetic determinants other than red cell antigen and MHC class-II. 2. Environmental factors affecting the donor limit. 3. Environmental factor s affecting the transfusion recipient.

Other genetic determinants
Rs660 polymorphism in the Ro52 gene is associated with kinetics of alloimmunisation in sickle cell disease patients, 2 although the function of Ro52 (also called SSA1 and TRIM21) is only practically characterised. It appears to be immunoregulatory gene products. Thus, a role for Ro52 in regulating alloimmunisation is logical; however correctly the association is only correlative and causal role is yet to be tested. The risk of alloimmunisation can be reduced by choosing blood matched for Rh and Kell groups in SCD. 3 Patients who are already alloimmunised should undergo extended red cell phenotypic matching (C, c, D, E, e, K, k, Jka, Jkb, Fya, Fyb, Kpa, Kpb, MNS, Lewis) with some centres also employing red cell genotyping to increase the accuracy of Rh typing, and in locating compatible units.

Materials and Methods
All the tests are done by gel card method. Liss/coombs card (Matrix), Card centrifuge (85g), Incubator (37 0 C), Workstation, Pipettes (10, 25 & 50µl), Screening cell pane, Isotonic saline solution (LISS), Bottle top dispenser Reagents: -The matrix AHG (Coombs) Test Card contains six microtubes, prefilled with a gel in a suitable buffer containing Anti-Human IgG and Monoclonal Anti- The matrix AHG (Coombs) Test Card is suitable for Direct Coombs test, Indirect Coombs test including compatibility testing, antibody screening and antibody identification.

Methods
The study was conducted in The Department of transfusion medicine and Department of Pathology VSSIMSAR, BURLA from November 2017 to August 2019 and the study was Prospective and observational study. The study was conducted on the Sickle cell disease patients coming for red cell transfusion to Blood bank, VSSIMSAR, Burla.
Principle:-As the Matrix gel card containing red blood cells is centrifuged under specific conditions, the red blood cells sensitized with antibody will agglutinate in presence of the Anti-Human Globulin reagent in the gel matrix and will be trapped in the gel column. The red blood cells, which do not get trapped in the gel matrix, are palleted at the bottom of the column. The reaction is then read and graded according to their reactivity pattern.

Results
Out of total 110 patients screened for alloantibody 15 patients were found to be positive for alloantibody. That is 13.64% of patients developed alloantibodies. Which is significant and requires immediate attention.
1. Among the 15 patients 7 were males and 8 were females. So the alloantibody distribution shows little female predominance.

Discussion
Till now there are no published data on incidence of alloimmunisation among sickle cell disease patients in western part of Odisha and India. Thus, study was aimed to investigate the frequency of alloimmunisation among these patients.
The rate of alloimmunisation observed in present study is 13.64% which is comparable to study done by L.A.M. Bashawri, Damman, Soudi Arbia, 4 who found it to be 13.7%. Study done by J Sin et al, amsterdom 5 and Wendell F. Rosse et al, Chicago 6 found it total 22% and 18.6% respectively, which is little higher than the present study. Another study done by Fekri Samarah et al, Palestine 7 found 7.76%, which is much lower than the present study. Except the last study all other study found higher frequency of alloantibody formation in sickle cell disease as mentioned in different literatures and books.
These differences in the rate of RBC alloimmunization among SCD patients support the importance of ethnic/genetic differences between patients and donors. Although the cost of antigen matching is high, further studies are needed to investigate the influence of this factor on the rate of alloimmunization. Another factor that could contribute to the relatively low rate in the last study is that SCD patients are not checked for RBC alloantibodies after each transfusion which may lead to missing the detection of transitory alloantibodies. 7 Mean age of alloantibody production in present study is 22.2yrs. Which is 28.8yrs in study by L.A.M. Bashawri  Two patients out of 15 patients in present study were of paediatric age groups. This is quite similar to most of the other studies. It is reported that SCD children who were first transfused at the age of 10 years and older had a higher rate of alloantibodies compared to those who were transfused before that age. 9 Eight out of 15 patients are female in present study, showing slight female predominance. This is similar to the study by L.A.M. Bashawri and most of other studies. Only the study by F. Samarah found no difference in gender. It has been suggested in many literatures that the rate of alloimmunisation was greater for women than for men. The reason for this is more need of red cell transfusion and complications related to pregnancy. 10 The predominant blood group in present study is O+ve followed by B+ve, A+ve and AB+ve. The study shows result similar to the study done by L.A.M. Bashawri and most of the other studies available. 11,12 Comparison of different studies with present study