Transfusion and Apheresis Science 45 (2011) 167–170

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Transfusion and Apheresis Science

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Alloimmunization to red cells in thalassemics: Emerging problemand future strategies

Richa Gupta a,⇑, Deepak Kumar Singh b, Bharat Singh c, Usha Rusia b

a Department of Lab Medicine, Chacha Nehru Bal Chikitsalaya, Geeta Colony, Delhi, Indiab Department of Pathology, Guru Teg Bahadur Hospital, Delhi, Indiac Regional Blood Transfusion Centre, Guru Teg Bahadur Hospital, Delhi, India

a r t i c l e i n f o

Keywords:Transfusion

ThalassemiaAlloimmunizationAlloantibodyRed cells

1473-0502/$ - see front matter � 2011 Elsevier Ltddoi:10.1016/j.transci.2011.07.014

⇑ Corresponding author. Address: 132, Rajdhani NI.P. Extension, Patpadganj, Delhi 110092, India. Tel.: +9540951526.

E-mail address: richagupta0209@gmail.com (R. G

a b s t r a c t

Aims and objectives: To evaluate the magnitude of red cell alloimmunization in regularlytransfused patients with thalassemia major and analyse factors responsible for develop-ment of antibodies.Materials and methods: This cross sectional study was conducted on 116 thalassemics

receiving regular transfusions. All the patients underwent antibody screening. Patientswith positive antibody screen were further tested for antibody identification. The datawas analysed to find out the frequency, pattern and factors influencing red cell alloimmu-nization secondary to multiple transfusions.Results: Mean age of the patients was 14 years (range 1.5–27 years). Red cell alloantibodieswere found in 11 patients (9.48%). In four (36%) patients first transfusion was given before6 months of age and in seven (64%) patients, first transfusion was given after two years of age.The interval between consecutive transfusions varied from 18 to 35 days. The most commonantibody was Anti-E found in 4 (36.4%) patients, followed by Anti-K (three patients, 27.2%),Anti-Kpa (two patients, 18.2%) and Anti-Cw (two patients, 18.2%). The interval from first trans-fusion to antibody development varied from 1.5 to 14 years. None of the eight out of 116patients, who underwent splenectomy showed any antibody development.Conclusions: The rate of red cell alloimmunization was found to be 9.48% in thalassemicsreceiving regular transfusions. The incidence of alloantibody development was higher if firsttransfusion was received at more than 2 years of age. Early institution of red cell transfusionsand Rh and Kell phenotyping followed by provision of matched blood could preventalloimmunization.

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1. Introduction

Life long red cell transfusion is the main supportivetreatment for patients with thalassemia major. The use ofregular transfusions & chelation therapy has transformedthalassemia from a fatal disease in early childhood to achronic illness associated with prolonged survival [1].However, the beneficial effect of each unit transfused is

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accompanied with the possibility of some adverse effectsdue to transfusion. The adverse affects more commonlyseen in these patients due to repeated transfusions include– iron overload, increased rate of transfusion transmittedinfections and alloimmunization to red cell antigens. Theuse of Desferroxamine and recently, orally administerediron chelator ‘‘Deferasirox’’ has considerably reduced hepa-tic iron accumulation in these patients with minimal sideeffects [2]. With the advent of newer screening tests like4th generation ELISA and Nucleic Acid Amplification Tech-nique (NAT), the risk of transfusion transmitted infectionshas also been considerably reduced.

Fig. 1. Grading of reaction using gel technology.

168 R. Gupta et al. / Transfusion and Apheresis Science 45 (2011) 167–170

Red cell alloimmunization remains a major challenge torepeat transfusions in these patients. Alloimmunizationoccurs when an incompatible antigen is introduced in animmunocompetent host. Formation of alloantibodies tored cell antigens may be stimulated by either transfusionof red cells or fetomaternal bleed. Though such antibodiesare infrequently observed in patients receiving occasionaltransfusions, their development is much more commonin patients receiving repeat red cell concentrates. Thedevelopment of such antibodies can significantly compli-cate transfusion therapy due to the occurrence of acuteand delayed type of haemolytic transfusion reactions, diffi-culty in finding compatible blood units and increased riskof haemolytic disease of the newborn when such a patientbecomes pregnant [3]. Also, it can cause difficulty in hae-matopoetic bonemarrow/stem cell transplantation and in-crease the chances of graft rejection in patients who arecandidates for such therapy in future.

Though antibody formation can take place against anyantigen present on the surface of red cells, such antibodieshave been more frequently observed against antigensbelonging to the Rh and Kell blood group system [4].

The aims of the present study were (1) to determine thefrequency of red cell alloantibodies in patients of thalasse-mia major receiving regular blood transfusions; (2) to ana-lyse factors which may be responsible for the developmentof these antibodies; (3) to find out the type of antibody andextent to which provision of Rh and Kell matched bloodcould prevent alloimmunization in these patients.

2. Materials and methods

This study included 110 patients of thalassemia majorreceiving regular blood transfusions in the hospital. Clini-cal and transfusion records of all the patients were ana-lysed to record parameters like age of the patient at thetime of diagnosis, age at which first transfusion was given,intervals between transfusions and whether splenectomywas performed as a part of management. Patient parame-ters including haemoglobin, haematocrit, reticulocytecount, total and indirect bilirubin, results of haemoglobinelectrophoresis, HbA, Hb A2 and Hb F levels were alsorecorded.

Every time a patient came to receive transfusion, a 5 mlblood sample was collected in anticoagulant EDTA for anti-body screening and identification. A sample without anti-coagulant was also collected for blood grouping and crossmatching. Sample for antibody screening was collected inan EDTA tube since plasma is preferable to serum whencarrying out antibody screening by gel card method [5].Plasma was separated by centrifugation at 1000 rpm for2 min (Microcentrifuge, model-ID Centrifuge 24-S, By ID-Diamed, Switzerland). The separated plasma was dividedinto two aliquots and both were labelled with identifica-tion code of the patient.

Antibody screening was performed on one plasma sam-ple and the other was kept at �30 �C in deep freezer forantibody identification in future in case of a positive anti-body screen. Screening for alloantibody was done by gelcard technology using three cell panel (ID-Diacell I-II-III,

Diamed, Switzerland). The antibody screen of each samplewas done by a trained transfusion technologist withoutknowledge of the corresponding patient details. Thescreening cell panel covered antigens of all known bloodgroups including Rh, Kell, Kidd, Duffy, P, Lewis, MNS,Lutheran and Xg. The reaction was performed in both Coo-mbs and Enzyme phase to increase the sensitivity of theprocedure. For enzyme phase of reaction papainized cells(ID-Diacell I-II-III-P, Diamed, Switzerland) available ready-made with the manufacturer were used. Grading and scor-ing of the agglutination in the gel cards was done bydoctors without knowledge of the clinical and laboratorydata of the patient. The grading and scoring was doneaccording to the distribution of agglutination throughoutthe gel matrix as shown in Fig. 1.

In case of a positive result on initial screen, antibodyidentification was done using the 11 cell identification pa-nel (ID DiaPanel, Diamed, Switzerland). An autocontrol wasalso put simultaneously to exclude the presence ofautoantibody.

3. Results

A total of 116 patients of thalassemia major were in-cluded in this study. The age of the patients ranged from1.5 to 27 years with a mean age of 14.25 years. There weresixty males and fifty-six females (M:F ratio of 1.07:1). All ofthem received regular transfusion with leukodepleted redcell concentrates during a period ranging from 1 to26.6 years.

Out of total 116 patients, 11 (9.48%) patients developedalloantibodies. Profile of patients with alloantibodies isshown in Table 1. Out of 11 patients with alloantibodiesthere were seven males and four females. In seven patients(63.6%) transfusion was initiated after two years of age.The time interval between transfusions in these patientsvaried from 18 to 35 days. None of these 11 patients whodeveloped alloantibodies underwent a splenectomy. Sixpatients out of 116 had undergone splenectomy due to in-crease in the requirement of blood but none of the splenec-tomised patient developed alloantibodies.

The antibody characteristics of all the 11 patients werealso studied (Table 2). The most common antibody wasfound to be Anti E (present in 4 cases) followed by Anti Kwhich developed in 3 cases. Anti Cw and Anti Kpa devel-oped in two patients each. There was one patient whodeveloped multiple antibodies (Anti E and Anti Kpa).Majority (7/12) of the antibodies belonged to the Rh bloodgroup system and the rest (5/12) belonged to the Kell

Table 1Profile of patients with Alloantibodies.

Case No. Sex Current age in years Age at 1st transfusion Interval B/W trans. (days) Splenectomy

1 F 8 6 25 No2 M 11 6 30 No3 F 17 3 18 No4 M 12 <1 35 No5 F 12 2 20 No6 M 10.5 <1 20 No7 M 5 <1 33 No8 M 19 2.5 23 No9 M 10 3 28 No

10 M 8 2 20 No11 F 11 <1 20 No

Table 2Antibody characterstics in the eleven cases.

Case No. Allo Ab(s) Bld. Gp. Reactivity Clinical significance

NaCl Coombs Enzyme

1 Anti k Kell � + + HTR, HDN2 Anti E, Rh +, � + + HTR, HDN

Anti kpa Kell � +, �3 Anti Cw Rh +, � + + HTR, HDN4 Anti Cw Rh +, � + + HTR, HDN5 Anti K Kell � + + HTR, HDN6 Anti E Rh +, � + + HTR, HDN7 Anti D Rh � + + HTR, HDN8 Anti E Rh +, � + + HTR, HDN9 Anti Kpa Kell � + +, � HTR, HDN10 Anti E Rh +, � + + HTR, HDN11 Anti K Kell � + + HTR, HDN

R. Gupta et al. / Transfusion and Apheresis Science 45 (2011) 167–170 169

blood group system. Testing for antibodies in saline phasewas insufficient to detect all the antibodies but all of theantibodies could be detected by carrying out the reactionin the enzyme and Coombs phase.

4. Discussion

The present study assessed the rate of alloimmuniza-tion in 116 patients of thalassemia major and the risk fac-tors associated with antibody development. Leucodepletedblood was routinely used for transfusion. The rate ofalloimmunization in the patients was found to be 9.8%with 11 out of 116 patients developing alloantibodies. Pre-vious studies have reported rates of alloimmunization inpatients of thalassemia major from 5–30% (Table 3). Sincethalassemia is more prevalent in Mediterranean countries

Table 3Alloimmunization rates in Thalassemics.

Authors Year Place Rate (mean) (%)

Spanos et al. 1990 Greece 22.6Sirchia et al. 1991 Italy 3.1Singer et al. 2000 USA 22Ameen et al. 2003 Iran 30Bhatti et al. [13] 2004 Pakistan 6.84Noor Haslina et al. [14] 2006 Malaysia 8.6Wang et al. 2006 Taiwan 37Ansari et al. 2007 Iran 3.75

and Southeast Asia, most of the studies on red cell alloim-munization are from these countries. Highest rate (30%)was reported by Ameen et al. [6] in patients of Iranian des-cent in 2000, while much lower rates were reported byother authors [4,7]. Only two previous studies reportingrate of alloantibody formation have been done in Indianpatients with thalassemia major (Table 4). One such studywas conducted by Pradhan et al. in 2001 in Bombay [8].They found an alloimmunization rate of eight percentamongst 100 patients of thalassemia major. Pahuja et al.in 2009 estimated the rate of alloantibody formation in pa-tients of thalassemia major as 2.7% which is very low ascompared to studies from other countries [9]. Moreover,none of these studies analysed factors which may beresponsible for alloantibody formation in these patients.

The factors responsible for high rate of alloimmuniza-tion in thalassemia patients (preceded only by sickle cellanemia) are largely unknown. In our study, out of the 11patients who developed alloantibodies 63.6% were trans-fused at or after two years of age while 36.6% were trans-fused before one year of age. Various authors haveobserved a lower rate of alloimmunization in patients inwhom transfusion was initiated at an early age. Spanos

Table 4Alloimmunization rates in Thalassemics in India.

Authors Year Place Rate (mean) (%)

Pradhan et al. 2001 Bombay 8Pahuja et al. 2009 Delhi 2.37

170 R. Gupta et al. / Transfusion and Apheresis Science 45 (2011) 167–170

et al. and Michael-Merianou et al. [10,11] have shown thatalloimmunization rate was significantly lower in patientswho started transfusion therapy before three years of ageas compared to those who were started on transfusion la-ter. Ameen et al. [7] however reported a transfusion fre-quency of 30% in 190 thalassemics who all startedtransfusion therapy before one year of age.

Out of the total 116 subjects in our study, seven pa-tients underwent splenectomy but none of the sevendeveloped alloantibody. Of the eleven patients who did de-velop alloantibody, none was splenectomised. Someauthors have suggested that the rate of alloimmunizationis higher after splenectomy. In a study by Singer et.al.[12] in 2001, the incidence of red cell alloimmunizationin 64 Asian patients living in United states was 22% andsplenectomy was found to be a risk factor. They found36% rate of antibody formation in splenectomised subjectsas compared to 12.8% in non splenectomised subjects. Theauthors reasoned that removal of spleen led to nonfilteringof antigens from blood stream which was responsible forhigher rates in splenectomised subjects. Our results didnot substantiate findings of Singer et al.

The interval between transfusions does not appear toplay a significant role in antibody development since sim-ilar interval was observed between all the subjects. How-ever the interval shortened after the development of theantibody due to decreased survival of foreign red cells.

Another important finding was that all the antibodiesdetected are known to be clinically significant, that is,capable of causing haemolytic transfusion reactions andhaemolytic disease of the newborn. Though haemolytictransfusion reaction was not reported in any of the pa-tients, the increased rate of hemolysis and shortened sur-vival of transfused red cells was evident by the increasedfrequency of transfusions in all of these patients.

Therefore, every patient should be screened for unex-pected/irregular antibodies to prevent any untoward effectof transfusion. While antibody screening is included in thecompatibility testing protocol in the developed countries,it is not so in India and other developing countries wherecost and lack of awareness remains a major constraint.As a result very few centres in India carry out antibodyscreening, even in case of a multiply transfused patient.

To prevent alloimmunization to RBC antigens, it hasbeen recommended to develop programmes to provideantigen matched blood to all chronically transfusedpatients [15]. However the provision of antigen matchedblood would require formation of antigen pool for each pa-tient. The feasibility and cost effectiveness of such an ap-proach remains under question. However, as is evidentby this study and some previous studies, antibodies havebeen more frequently seen against Rh and Kell blood groupantigens [5]. Therefore, knowledge about the Rh and Kellstatus of the patients and selective transfusion of bloodmatched for Rh and Kell blood group antigens may be afeasible approach to prevent alloimmunization in most ofthe cases.

5. Conclusion

Due to the several hundreds of epitopes on red cells, anRBC transfusion is inevitably incompatible. The risk ofalloimmunization is much higher in patients transfusedrepeatedly with red cells such as patients of thalassemiamajor. Despite of lower rates reported by few studies con-ducted in this region, we found a 9.48% rate of alloantibodyformation in these patients. Therefore antibody screeningis strongly recommended at the time of transfusion forsuch patients. Morover it was found that all the antibodiesbelonged to the Rh and Kell blood group. Therefore provi-sion of Rh and Kell matched blood could prevent alloim-munization in majority of the patients thus preventinglater complications.

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