|Year : 2017 | Volume
| Issue : 2 | Page : 58-63
Risk for red cell immunization among thalassemic patients
Eman R Saifeldeen MD 1, Mohamed A Awad1, Youssef A El-Tonbary2, Doaa A Aladle1, Doaa M Elghannam1
1 Department of Clinical Pathology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
2 Department of Pediatrics, Faculty of Medicine, Mansoura University, Mansoura, Egypt
|Date of Submission||10-Mar-2016|
|Date of Acceptance||13-Apr-2016|
|Date of Web Publication||6-Oct-2017|
Eman R Saifeldeen
Department of Clinical Pathology, Faculty of Medicine, Mansoura University, Mansoura
Source of Support: None, Conflict of Interest: None
Background Thalassemia major is a common health problem in Egypt. However, limited data are available on the frequency of red blood cell (RBC) alloimmunization and autoimmunization in transfusion-dependent thalassemia patients.
Objectives The primary aim of the current study was to determine the prevalence of alloimmunization and autoimmunization in multitransfused thalassemic patients and to assess potential clinical factors associated with RBC antibody formation.
Patients and methods Sixty-five multitransfused thalassemic patients (33 male and 32 female; mean age, 9 years; range 2–37 years) who attended Mansoura University Hospital for Children were included in our study. Antibody screening and identification were carried out with the ID card microtyping system and three-cell and 11-cell panels were used (Serascan Diana 3 and Identisera Diana), respectively.
Statistical analysis Statistical analysis was performed using excel program (Microsoft Office, 2010) and statistical package for social science program (SPSS Inc., Chicago, Illinois, USA), version 20. Qualitative data were presented as frequency and percentage. The χ2 and Fisher exact tests were used to compare groups. Quantitative data were presented as mean and SD or median and range. The Kolmogrov–Smirnov test was used for testing normality. For comparison between two groups, Student’s t-test or the Mann–Whitney (for nonparametric) were used. Logistic regression analysis was applied for the prediction of development of antibodies (P is significant if ≤0.05 at confidence interval 95%).
Results Of 65 patients, 15 cases (23.1%) had been alloimmunized. The majority of alloantibodies were directed against the Kell (12.3%) and Rh systems (6.2%). RBC autoantibodies developed in 9.2% of patients. Only 6.2% of patients had simultaneous alloimmunization and autoimmunization. We found a significant association between RBC immunization and age at first transfusion (P=0.02), splenectomy (P<0.001), and presence of alloimmunization and autoimmunization (P=0.022).
Conclusion Red cell alloimmunization is an important risk in thalassemia patients. Most alloantibodies were of the anti-K and the anti-Rh type. Extended phenotype matched blood transfusion for Rh and Kell antigens needs to be explored for preventing alloimmunization in thalassemia patients.
Keywords: alloimmunization, autoimmunization, thalassemia
|How to cite this article:|
Saifeldeen ER, Awad MA, El-Tonbary YA, Aladle DA, Elghannam DM. Risk for red cell immunization among thalassemic patients. Egypt J Haematol 2017;42:58-63
|How to cite this URL:|
Saifeldeen ER, Awad MA, El-Tonbary YA, Aladle DA, Elghannam DM. Risk for red cell immunization among thalassemic patients. Egypt J Haematol [serial online] 2017 [cited 2017 Oct 20];42:58-63. Available from: http://www.ehj.eg.net/text.asp?2017/42/2/58/216113
| Introduction|| |
Thalassemia is the most common hereditary anemia in Egypt. Trait carrier rates range from 6 to 10%. Ten thousand cases are registered, whereas more than 20 000 are not registered . Regular transfusions are essential for patients with thalassemia major to maintain growth and development during childhood and to sustain good quality of life during adulthood . Repeated blood transfusion can stimulate the patient’s immune system and results in the formation of antierythrocyte antibodies. Although autoantibodies appear with less frequency, they can result in clinical hemolysis and cross-matching problems. Alloimmunization against red blood cells (RBCs) will increase the need for blood transfusions in patients with thalassemia. Some alloantibodies are clinically significant that are known to cause hemolytic transfusion reactions and hemolytic disease of the newborn and limit the availability of further safe transfusion but others are clinically insignificant . 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 homogenous populations ,,,.
The aim of this study was to explore the frequency of red cell alloantibodies and autoantibodies and to determine risk factors that might influence alloimmunization with the hope to minimize some of the transfusion-associated risks in those patients.
| Patients|| |
A total of 65 transfusion-dependent thalassemia major patients who attended the Hematology Unit of Mansoura University Children Hospital were included in the study. There were 33 male and 32 female patients, with a mean age of 9 years (range 2–37 years). They received regular blood transfusions matched for ABO-Rh(D). At first, the patient’s age, sex, age at first blood transfusion, ABO and Rh blood group, history of splenectomy, and ethnic background were recorded in questionnaires after receiving written consent from each patient, before transfusion. Patients with connective tissue disease, autoimmune diseases, or those receiving immunosuppressive drugs were excluded from study. This study was approved by the hospital’s ethical committee.
| Methods|| |
A volume of 10 ml of venous blood was collected in two separate tubes: a K2-EDTA tube for autocontrol and a tube without anticoagulant for antibody screening and antibody identification. Initially, antibody screening was carried out with a three-cell panel (Serascan Diana 3; Diagnostic Grifols, Barcelona, Spain) according to standard blood bank methods. Two parts of serum and one part of RBCs were mixed and evaluated in three phases (room temperature, 37°C, and AHG phases). All data were entered in predefined tables. In case of a positive screen, antibody identification was performed in the same phases as antibody screening, using a 11-cell panel (Identisera Diana; Diagnostic Grifols). Finally, according to the presented antigram pattern of each panel, the type of specific antibody against each antigen was determined. An autocontrol was also put simultaneously to determine the presence of autoantibody.
| Results|| |
Characteristics of thalassemic patients are summarized in [Table 1]. A total of 65 multitransfused thalassemia major patients who received regular blood transfusion at an interval of 0.5–4 months were enrolled in the study. The blood group of patients was as follows: ‘O’ in 29.2% of cases; ‘A’ in 18.5% of cases; ‘B’ in 36.9% of cases; and ‘AB’ in 15.4% of cases. Rh was positive in 89.2% of cases and negative in 10.8% of cases. Age at first transfusion ranged from 0.5 to 5 years, with a mean of 0.5 years. The duration for receiving transfusion ranged from 1.5 to 36.5 years, with a median of 8 years. Alloimmunization was present in 15 cases (23.1%) and autoimmunization in six cases (9.2%), with a significant difference between alloimmunization and autoimmunization (P=0.022) ([Table 2]).
[Table 3] demonstrates the distribution of alloantibodies in all studied patients. Fifteen patients developed 18 types of alloantibodies. The most common alloantibodies were anti-Kell (12.3%) and anti-Rh (6.2%) (anti-D, anti-c, and anti-E). Anti-K was seen in 6.2% of cases, anti-k in 3.1% of cases, anti-D in 3.1% of cases, anti-c in 1.5% of cases, anti-fy-b in 1.5% of cases, anti-lu-a in 1.5% of cases, and anti-Jkb in 1.5% of cases. Three cases presented with multiple antibodies: anti-E+anti-S (1.5%), anti-K+anti-S (1.5%), and anti-K+anti-N (1.5%). Patients with ages over 5 years at first transfusion had significantly higher risk of having alloantibodies (P=0.020), as shown in [Table 4]. Moreover, the total number of transfused bags/year, intervals between transfusions, and duration of transfusion did not predict the presence of alloantibodies. However, risk for autoantibody formation was not predicted by the mentioned variables. Alloimmunized patients showed significantly older age at first transfusion when compared with autoimmunized patients (P=0.021) ([Table 4]).
|Table 4 Relative risk of transfusion history on red blood cell immunization in all studied patients|
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[Table 5] demonstrates the effect of splenectomy status over RBC immunization. Patients with splenectomy showed a significantly higher incidence of alloimmunization. However, patients with splenomegaly showed a significantly lower incidence of the presence of alloimmunization compared with nonalloimmunized patients (P<0.001 for both).
|Table 5 Distribution of antibodies according to spleen status in all studied patients|
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| Discussion|| |
The objective of this study was to explore the frequency of red cell alloantibodies and autoantibodies among β-thalassemia patients who received regular transfusions and to determine risk factors that might influence RBC immunization.
In our study, we demonstrated an alloimmunization incidence rate of 23.1% (15 of 65) in transfusion-dependent thalassemia patients. Autoimmunization was found in six patients (9.2%). There were no available data about the time from the start of transfusion to antibody formation ([Table 2]).
The rate of alloimmunization in the present study is similar to that found in the study by Hussein et al. , who found an alloimmunization rate of 22.8% among Egyptian thalassemic patients. The high rate of alloimmunization demonstrated in their study is probably caused by the antigenic difference between donors’ RBCs and the recipient’s blood. Similar results were obtained by Singer et al. , who found 22% alloimmunization rate among Asian thalassemic patients. The high incidence reported in the study by Singer and colleagues was attributed to RBC antigenic difference in blood donors being white and most of the recipients being Asians. Ameen and colleagues reported 30% alloimmunization rate among Arab thalassemic patients. This high incidence of alloimmunization can be attributed to the great heterogeneity of the population in Kuwait .
Several factors might have contributed to the high alloimmunization rate observed in this study, including lack of better-matched donors for those patients and the use of nonleukodepleted blood. The immunomodulatory role of white blood cells in transfused blood could have contributed to the high incidence of alloantibodies observed in our group of patients. Leukocytes in blood components may cause an enhanced B-cell function that may result in increased alloimmunization to RBC antigens .
Singer et al. , Ameen et al. , Pahuja et al. , and Hussein et al.  demonstrated a significant effect of leucoreduction in multitransfused patients, showing lower alloantibody incidence rates. However, others demonstrated no significant association between leucoreduction and low alloantibody formation ,. A recent survey of transfusion policies suggests that RBC filtration has become a standard practice in many institutions that are likely to care for thalassemia patients in the USA and Canada .
In our study, six patients developed autoantibody formation (9.2%), of whom four had combined alloantibodies ([Table 2]). Autoimmunization accompanying alloimmunization was also found in a study conducted in Kuwait by Ameen et al. , who found that 11% of their transfusion-dependent thalassemia patients developed RBC autoantibodies. Similarly, Vichinsky et al.  reported that 6.5% of their patients developed autoantibodies associated with alloimmunization. In an Asian study by Singer et al. , they reported that 25% of their patients developed autoantibody formation. However, a study in Malaysia found that only one patient of 63 transfusion-dependent thalassemia patients developed RBC autoantibodies .
Possible theories to explain the formation of autoantibodies together with alloantibodies include failure to regulate alloantibody-induced lymphoproliferation, as well as altered processing and presentation of alloantigens to T cells . Alloantibodies binding to RBCs could lead to conformational changes of the antigenic epitopes that ultimately stimulate the production of autoantibodies . It is possible that certain people are genetic responders who have an increased tendency to develop RBC autoantibodies, and the tendency toward autoantibody formation could reflect an overall dysfunction of the immune system .
Autoantibodies may result from an imbalance between pathogenic and regulatory arms of the immune response because antigen-specific IL-10-secreting regulatory T cells can be detected during periods of remission . However, Th1 responses are present during active disease .
Our results demonstrated 18 alloantibodies found in 15 patients. Of that, the most frequent alloantibodies found in our study were KEL-related (12.3%) followed by Rh-related (6.1%) ([Table 3]). This is in accordance with the findings of Thompson et al. , who found that, in most western countries, the most common alloantibodies in thalassemia patients are directed against Rh (C and E) and Kell antigens.
This predominance is expected, given the strong immunogenicity of these particular antigens and antigenic discrepancy between donors and recipients, which is mostly noted for Rh and Kell antigens. Therefore, for the prevention of alloimmunization, transfusion of blood should be matched for Rh and K antigens . However, the potential to form RBC alloantibodies to unmatched antigens still exists .
In the current study, 50 patients involved did not produce RBC alloantibodies despite regular RBC transfusion early in life. Twelve alloimmune thalassemic patients developed only single alloantibody despite regular transfusion with antigen-positive RBCs. Three alloimmune patients developed multiple RBC alloantibodies ([Table 2]). It is possible that certain people are genetic responders who have an increased tendency to develop RBC alloantibodies and others are not. The tendency toward alloantibody formation could reflect an overall dysfunction of the immune system .
We have found a significant association between RBC immunization and the age at first transfusion (before or after 5 years) (P=0.020) ([Table 4]). The full capacity to produce immune antibodies develops slowly after birth. The immunization rate − that is, immunological tolerance − in preterm infants is very low to negligible because of functionally immature B cells . Transfusion at an early age (<1–3 years old) may offer some immune tolerance and protection against alloimmunization in thalassemia patients despite greater transfusion burden ,. However, Karimi et al. , Ahmed et al. , and Hussein et al.  found that the incidence of alloimmunization was not influenced by the age at which transfusions were started.
In our study, we found no significant association between number of transfused blood bags and alloantibody or autoantibody formation. This is in accordance with the findings of van de Watering et al. , Bhatti et al. , and Ahmed et al. . In contrast, Singer et al. , Zalpuri et al.  and Vichinsky et al.  found a relation between the number of blood bags transfused and antibody formation in thalassemic patients who received multiple transfusions.
We found no significant difference in RBC immunization as regards sex. This is in accordance with the findings of Ameen et al. , El Danasoury et al. , and Thompson et al. , who found similar results. However, in a recent review of the literature, Verduin et al.  observed a higher RBC alloimmunization rate in transfused female patients with sickle cell disease only and not in other diseases that require multiple blood transfusions. Zalpuri et al.  found that female sex is associated with increased risk for alloimmunization, as women are more susceptible to exposure of alloantigens during pregnancy, miscarriages, abortions, and childbirth. Moreover, they found that there is well-documented evidence that certain human leukocyte antigen types are associated with enhanced response to some RBC antigens such as Kell, Duffy, and Kidd .
In the present study, patients who underwent splenectomy had a higher alloimmunization rate. There was a significant association between splenectomy and presence of RBC immunization (P<0.001) ([Table 5]). Our findings are in agreement with those of Singer et al. , who observed a higher rate of alloimmunization in patients who underwent splenectomy compared with patients who did not undergo splenectomy (P=0.06) .
After splenectomy, there are conformational changes in the RBCs due to erythrocyte fragmentation, membrane deformation, or alloantibody binding. These findings are consistent with senescent erythrocytes and may expose new antigens and promote or enhance an immune reaction, as known to occur in aging and impaired RBCs. It is likely that the absence of an efficient filtering system for removal of damaged erythrocytes enhances the process . However, in an Iranian study, there was no significant difference in the frequency of splenectomy between alloimmunized and nonalloimmunized patients .
With splenectomy, a marked absolute lymphocytosis, mostly due to an increase in B lymphocytes, was reported with the use of both nonleukoreduced and leukoreduced blood .The lymphocytosis is accompanied by an increase in serum immunoglobulins, immune complexes, and cells bearing surface immunoglobulins, which are the result of the immunomodulatory effect of blood elements, absence of spleen, and recipient’s immune status ,.
| Conclusion and recommendations|| |
To minimize the hazards of the immunological reactions following blood transfusion, pretransfusion matching of blood for ABO and Rh D antigens is important as a routine work, which helps in determining the true blood groups and assists in the identification of suspected alloantibodies and selection of antigen-negative RBCs for transfusion. Extended phenotype matched blood transfusion for Rh (other than D) and Kell antigens needs to be explored in preventing alloimmunization in thalassemia patients.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Hussein E, Desooky N, Rihan A, Kamal A. Predictors of red cell alloimmunization in multitransfused Egyptian patients with β-thalassemia. Arch Pathol Lab Med
Weatherall D, Clegg J. Diagnosis and management of thalassaemia. The Thalassaemia syndromes
. 4th ed. Oxford, UK: Blackwell Publishing; 2001.
Ameen R, Al-Shemmari S, Al-Humood S, Chowdhury RI, Al-Eyaadi O, Al-Bashir A. RBC alloimmunization and autoimmunization among transfusion-dependent Arab thalassemia patients. Transfusion
Spanos T, Karageorga M, Ladis V, Peristeri J, Hatziliami A, Kattamis C. Red cell alloantibodies in patients with thalassemia. Vox Sang
Singer ST, Wu V, Mignacca R, Kuypers FA, Morel P, Vichinsky EP. Alloimmunization and erythrocyte autoimmunization in transfusion-dependent thalassemia patients of predominantly Asian descent. Blood
Wang LY, Liang DC, Liu HC, Chang FC, Wang CL, Chan YS, Lin M. Alloimmunization among patients with transfusion-dependent thalassemia in Taiwan. Transfus Med
Pahuja S, Pujani M, Gupta SK, Chandra J, Jain M. Alloimmunization and red cell autoimmunization in multitransfused thalassemics of Indian origin. Hematology
Lannan KL, Sahler J, Spinelli SL, Phipps RP, Blumberg N. Transfusion immunomodulation − the case for leukoreduced and (perhaps) washed transfusions. Blood Cells Mol Dis
Schonewille H, van de Watering LM, Loomans DS, Brand A. Red blood cell alloantibodies after transfusion: factors influencing incidence and specificity. Transfusion
Thompson AA, Cunningham MJ, Singer ST, Neufeld EJ, Vichinsky E, Yamashita R et al.
Red cell alloimmunization in a diverse population of transfused patients with thalassaemia. Br J Haematol
Spinella PC, Dressler A, Tucci M, Carroll CL, Rosen RS, Hume H et al.
Survey of transfusion policies at US and Canadian children’s hospitals in 2008 and 2009. Transfusion
Vichinsky E, Neumayr L, Trimble S, Giardina PJ, Cohen AR, Coates T et al.
Transfusion complications in thalassemia patients: a report from the Centers for Disease Control and Prevention (CME). Transfusion
:972–981 quiz 971.
Noor Haslina MN, Ariffin N, Illuni Hayati I, Rosline H. Red cell autoantibodies among thalassaemia patients in Hospital Universiti Sains Malaysia. Singapore Med J
Barker RN, Shen CR, Elson CJ. T-cell specificity in murine autoimmune haemolytic anaemia induced by rat red blood cells. Clin Exp Immunol
Castellino SM, Combs MR, Zimmerman SA, Issitt PD, Ware RE. Erythrocyte autoantibodies in paediatric patients with sickle cell disease receiving transfusion therapy: frequency, characteristics and significance. Br J Haematol
Hall AM, Ward FJ, Vickers MA, Stott LM, Urbaniak SJ, Barker RN. Interleukin-10-mediated regulatory T-cell responses to epitopes on a human red blood cell autoantigen. Blood
Bashawri L, Ahmed M, AL-Qatary A, Ahmed M. Red cell alloimmunization in thalassaemia patients. Bahrain Med Bull
Ahmed AM, Hasan NS, Ragab SH, Habib SA, Emara NA, Aly AA. Red cell alloimmunization and autoantibodies in Egyptian transfusion-dependent thalassaemia patients. Arch Med Sci
Karimi M, Nikrooz P, Kashef S, Jamalian N, Davatolhagh Z. RBC alloimmunization in blood transfusion-dependent beta-thalassemia patients in southern Iran. Int J Lab Hematol
Van de Watering L, Hermans J, Witvliet M, Versteegh M, Brand A. HLA and RBC immunization after filtered and buffy coat-depleted blood transfusion in cardiac surgery: a randomized controlled trial. Transfusion
Bhatti FA, Salamat N, Nadeem A, Shabbir N. Red cell immunization in beta thalassaemia major. J Coll Physicians Surg Pak
Zalpuri S, Zwaginga JJ, van der Bom JG. Risk factors for alloimmunisation after red blood cell transfusions (r-FACT): a case cohort study. BMJ Open
El-Danasoury AS, Eissa DG, Abdo RM, Elalfy MS. Red blood cell alloimmunization in transfusion-dependent Egyptian patients with thalassemia in a limited donor exposure program. Transfusion
Verduin EP, Brand A, Schonewille H. Is female sex a risk factor for red blood cell alloimmunization after transfusion? A systematic review. Transfus Med Rev
Hodge G, Lloyd JV, Hodge S, Story C, Han P. Functional lymphocyte immunophenotypes observed in thalassaemia and haemophilia patients receiving current blood product preparations. Br J Haematol
Sibinga CTS. Immune effects of blood transfusion. Curr Opin Hematol
Marshall-Clarke S, Reen D, Tasker L, Hassan J. Neonatal immunity: how well has it grown up? Immunol Today
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]