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 Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 39  |  Issue : 3  |  Page : 171-176

Clinically significant red blood cell antibodies in multitransfused Egyptian thalassemic patients


1 Department of Clinical Pathology, Faculty of Medicine, Ain Shams University Hospital, Cairo, Egypt
2 Department of Pediatrics, Faculty of Medicine, Ain Shams University Hospital, Cairo, Egypt

Date of Submission15-Oct-2014
Date of Acceptance22-Oct-2014
Date of Web Publication31-Dec-2014

Correspondence Address:
Heba Tallah N El Sayed
Department of Clinical Pathology, Faculty of Medicine, Ain Shams University Hospital, Cairo 11556
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-1067.148253

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  Abstract 

Background Red blood cell (RBC) alloimmunization is a major challenge to repeated transfusions in β-thalassemia-major patients. The aim of this study was to evaluate the magnitude of RBC alloimmunization and autoimmunization in regularly transfused Egyptian patients with β-thalassemia major and analyze factors that may be responsible for the development of antibodies.
Patients and methods This study was conducted on 200 Egyptian β-thalassemia-major patients with age less than 16 years, who routinely visited the Pediatric Hematology Clinic of Ain Shams University Hospital for regular transfusions. All the patients underwent antibody screening. Patients with a positive antibody screen were further tested for antibody identification. The data were analyzed to find out the frequency of alloimmunization, and the patients' records were revised to analyze the factors influencing it. In case of pan-positivity of the antibody-screening cells and the autocontrol, adsorption by autogenic RBCs was performed to uncover the presence of alloantibodies.
Results RBC alloantibodies were found in 21 (10.5%) patients. The most frequent alloantibodies encountered were anti-Kell (52.4%) and anti-E (19%). Autoantibodies were encountered in only one patient. They were the warm autoantibody type, and adsorbtion using autogenic RBCs was succesful in eliminating them. The frequency of blood transfusion, the transfusion index, serum ferritin, and the age at first transfusion showed a statistically significant correlation with alloimmunization ( P < 0.05). In contrast, there was no statistically significant association between the patients' sex, age, the ABO, Rh blood groups, and the spleen state and alloimmunization (P > 0.05).
Conclusion We conclude that alloimmunization to RBC antigens is a relatively frequent finding among Egyptian transfusion-dependent thalassemic patients. The most frequent antibodies detected were against the Kell and Rh blood groups, mainly anti-Kell and anti-E. The majority of alloantibodies detected in this study were clinically significant.

Keywords: adsorption, alloimmunization, autoimmunization


How to cite this article:
El Sewefy DA, Al Feky MA, Abdel Fatah MF, El Sakhawy YN, Ragab IA, El Sayed HN. Clinically significant red blood cell antibodies in multitransfused Egyptian thalassemic patients. Egypt J Haematol 2014;39:171-6

How to cite this URL:
El Sewefy DA, Al Feky MA, Abdel Fatah MF, El Sakhawy YN, Ragab IA, El Sayed HN. Clinically significant red blood cell antibodies in multitransfused Egyptian thalassemic patients. Egypt J Haematol [serial online] 2014 [cited 2019 Dec 15];39:171-6. Available from: http://www.ehj.eg.net/text.asp?2014/39/3/171/148253


  Introduction Top


β-Thalassemia major is a congenital hemolytic anemia caused by defects in the β-globin chain synthesis. The α-globin chain that is produced in excess is responsible for the ineffective erythropoiesis and shortened red blood cell (RBC) survival. Blood transfusion is the mainstay of care in individuals with thalassemia major. The purpose of transfusion is to improve the anemia and to suppress ineffective erythropoiesis. Repeated transfusions prevent most of the serious growth, skeletal, and neurological complications of thalassemia major [1] .

However, repeated transfusions do pose a number of clinical problems. The beneficial effect of each unit transfused is accompanied by the possibility of some adverse effects. The adverse effects more commonly seen in these patients due to repeated transfusions include iron overload, an increased rate of transfusion-transmitted infections, and alloimmunization to RBC antigens [2] .

RBC alloimmunization remains a major challenge to repeated transfusions in these patients. Alloimmunization occurs when an incompatible antigen is introduced in an immune-competent host. The development of such antibodies can significantly complicate transfusion therapy due to the occurrence of acute or delayed types of hemolytic transfusion reactions, difficulty in finding compatible blood units, and increase in the risk of hemolytic disease of the newborn when a female patient becomes pregnant. It can also cause difficulty in hematopoetic bone marrow/stem cell transplantation and increase the chances of graft rejection in patients who are candidates for such therapy in future [3] . Because blood transfusion in these patients is necessary for life, early detection of these alloantibodies is very important. Screening for antibodies followed by their identification can help in the prevention of alloimmunization side effects [4] .

Formation of autoantibodies against RBCs, in association with RBC alloimmunization, has been documented in previous studies [5] . Autoantibodies are directed against the individual's own RBCs, which can result in clinical hemolysis and difficulty in the cross-matching procedure. Patients with autoantibodies may have a higher transfusion rate and often require immunosuppressive drugs, splenectomy, or alternative treatments [6] .

Pretransfusion testing should always include three stages: first, blood grouping for determining the ABO blood group and the Rh type of both the recipient and the donor specimens. Second, the recipient's plasma should be screened for the presence of unexpected red blood cell antibodies. Third, after selection of a donor unit of the appropriate group and type, cross-matching should be performed to determine whether the donor cells are compatible with the recipient's plasma [7] .


  Aims Top


The aims of this study were to detect the presence of alloantibodies and autoantibodies in a group of regularly transfused β-thalassemic Egyptian patients, to identify their types and frequencies, and to evaluate the different possible factors influencing the development of alloantibodies.


  Patients and methods Top


This study included 200 Egyptian β-thalassemic patients with age 16 years or younger, who routinely visited the Pediatric Hematology Clinic of Ain Shams University Hospital for regular blood transfusion. The clinical and transfusion records of all patients were evaluated for the age of the patient, the age at which blood transfusion was started, the frequency and the amount of blood transfusion, splenectomy, and the blood transfusion index of the previous year, which was determined by dividing the total amount of packed RBC transfusion per year divided by the patient's weight.

The following laboratory investigations were performed using standard blood bank techniques in the central blood bank of Ain Shams University:

  1. ABO and D blood grouping (by the gel card method using the Diamed system ABO-Rh/Reverse Grouping; Diamed, Cressier sur Morat, Switzerland).
  2. Antibody screening to detect alloantibodies against foreign RBC antigens [the indirect antiglobulin test using an antibody-screening panel (DiaCell I+II+III; Diamed)].
  3. Antibody identification, in cases of positive screening, to identify the antibody type [the indirect antiglobulin test using an antibody identification panel (DiaPanel; Diamed)].
  4. RBC phenotyping, for the antigen of which the antibody type was identified [using Diamed rare antisera (e.g. anti-Kell and anti-C)].


Autocontrol column was added to identify the autoantibodies. In case of pan-positivity of all the columns rescreening using the immediate spin technique was carried out to separate autoantibodies from the alloantibodies that could be present. If the immediate spin was negative (i.e. warm autoantibodies were present), adsorption by autogenic RBCs was carried out using Warm Autoantibody Removal Medium (WARM) reagent (Immucor/Gamma, Canada).

Statistical analysis

Data were analyzed using SPSS (version 15.0.1, 2001; SPSS Inc., Chicago, Illinois, USA) software. Comparison between groups with regard to qualitative data was carried out using χ2 -test. The Fisher exact test was used instead of the χ2 -test when the table contained values less than 5. Comparison between two groups regarding quantitative nonparametric data was carried out using Mann-Whitney U-test (Z value).

The P value was calculated for all parameters and was evaluated as follows: P value of 0.05 or more was considered nonsignificant (NS), P value of less than 0.05 was considered significant (S), and P value less than 0.01 was considered highly significant (HS).


  Results Top


Of the 200 patients, 109 (54.5%) were males and 91 (45.5%) were females with a male to female ratio of 1.2 : 1. Their ages ranged from 1.5 to 16 years with a mean of age 8.7 ± 4 years. The ABO blood groups of the 200 patients were as follows: 72 (36%) patients had blood group A, 57 (28.5%) had blood group B, 53 (26.5%) had blood group O, 18 (9%) had blood group AB, 187 (93.5%) were Rh positive and 13 (6.5%) were Rh negative. With regard to the spleen state 61 (30.5%) patients were splenectomized, whereas 139 (69.5%) were not ([Table 1]).
Table 1 Distribution of the studied patients as regard general data

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The mean age at which transfusion started for the patients was 16 months. As for the frequency of blood transfusion, the mean number was 17 U/year, 59 (29.5%) patients were transfused with more than 17 U/year and 141 (70.5%) were transfused with less than 17 U/year.

For each patient, the annual blood requirement as volume of transfused blood per kg of body weight was calculated and expressed as transfusion index. The mean value of the transfusion index was 139.6. Serum ferritin and blood hemoglobin values were also collected from the patients' sheets to correlate with the alloimmunization.

Using antibody-screening tests and autocontrol, alloantibodies were detected in 21 (10.5%) patients. One of the patients had multiple alloantibodies and autoantibodies, whereas the rest of the patients were free of clinically significant antibodies. The encountered alloantibodies were anti-Kell in 11 (52.4%) cases, anti-E in four (19%), and anti-C in three (14.2%). The least prevalent alloantibodies were anti-D, anti-C, and anti-Fyb, which were detected in one case each. As a confirmatory step for all alloimmunized cases, phenotyping for the patients' RBCs was carried out to ensure that the patient was negative for the antigen of which the antibody identification revealed. In all the positive cases (100%), the RBCs phenotyping was negative for the relevant antibody ([Table 2]).
Table 2 Percentage of alloantibodies and autoantibodies in alloimmunized patients

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Of 109 male and 91 female patients, 15 and six patients were alloimmunized, respectively. The sex of the patients and alloimmunization showed a nonsignificant association (P = 0.142). As for the spleen state and the age of the patients the P values were more than 0.05 (P = 0.138 and 0.202, respectively). The ABO system showed a nonsignificant association with alloimmunization (P > 0.05); however, on comparing each blood group as a separate entity, patients with blood group B were seen to have a lower frequency and patients with blood group O to have a higher frequency of alloimmunization (P = 0.014 and 0.048, respectively), in contrast to the A and AB blood groups, in which the association was nonsignificant (P = 0.92 and 0.39) ([Table 3]).
Table 3 Comparing qualitative data as regards alloimmunization

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The age at first transfusion showed a significant correlation (P = 0.027) with alloimmunization. The risk of alloimmunization appeared to be greater in patients who started transfusion therapy after the first few months of life ([Table 5]).

With regard to the frequency of blood transfusion in patients, the mean number of transfused units per year was taken as a cutoff (17 U/year). A statistically highly significant correlation was found between the development of alloimmunization and the frequency of blood transfusions, as the 21 patients with alloantibodies (100% of the alloimmunized cases) were receiving more than 17 U/year (P < 0.01). The mean number of transfused units per year was 38 in positive cases compared with 15 U/year in negative cases ([Table 4]).
Table 4 Comparing the frequency of transfusion in positive and negative alloimmunized patients

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Table 5 Correlation of alloimmunization with quantitative data

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Also, a highly significant correlation was found between the transfusion index and alloimmunization (P < 0.01). With regard to the serum ferritin a highly significant correlation was observed (P < 0.01) ([Table 5]).

Among the positive cases, autoantibodies were detected in only one (4.8%) thalassemic splenectomized male patient with blood group O positive. The immediate spin was negative (warm antibodies). After removal of the warm autoantibodies by adsorption, the antibody identification revealed two different alloantibody formations (anti-Kell and anti-C). Data from his sheet showed that he started blood transfusion at the age of 1 year and had higher frequency of blood transfusion (>17 U/year) and elevated serum ferritin and transfusion index.


  Discussion Top


This study was carried out on 200 previously diagnosed Egyptian β-thalassemic major patients whose samples were referred to the central blood bank of Ain Shams University Hospital for routine blood transfusion. These samples were examined for the presence of RBC alloantibodies and autoantibodies.

Twenty-one (10.5%) of the 200 patients were found to have alloantibodies to RBCs. These data were in accordance with those reported by previous works [8],[9] .In contrast, another Egyptian study recorded that the rate of alloimmunization was 28.4% [10] . The lower rate of alloimmunization in our study compared with this study may be because of the fact that all thalassemic patients in Ain Shams University Hospital are routinely injected with steroid therapy before each transfusion as a prophylaxis against allergy and transfusion reactions.

Also, lower rates of alloimmunization were recorded in most of the western countries because the patients are routinely transfused with phenotyped blood, at least for the Rh-Kell systems. In contrast, blood transfusion in most hospitals in Egypt is matched according to the ABO and Rh (D) antigens only [11] .

An important finding of our study was that all the antibodies detected are known to be clinically significant, that is, capable of causing hemolytic transfusion reactions and hemolytic disease of the newborn. Although hemolytic transfusion reaction was not reported in any of the patients, the increased rate of hemolysis and shortened survival of transfused RBC s were evident by the increased frequency of transfusions in all patients [12] .

With regard to the prevalence of alloantibodies detected in this study, the majority of detected antibodies against antigens were of the Rh-Kell system, with anti-Kell being 52.4% and anti-E being 19%. The finding that these two antibodies accounted for the predominance of cases of alloimmunization agrees with the data reported by many workers [11],[12],[13],[14] . In the current study, anti-D was detected in only one patient, whereas a study performed in Iran reported that anti-D was detected in 22 of 101 alloimmunized patients who were negative for the D antigen and had contact RBCs with positive Rh or took D u -positive blood bags [13] .

In this study, only one (0.5%) patient developed autoantibodies together with anti-Kell and anti-C. However, many previous studies observed higher results exceeding 25% [11],[15] . The pathogenesis of erythrocyte autoantibody formation after transfusion is not well understood. However, clinical evidence of autoimmune hemolytic anemia has been seen with high amounts of RBC-associated IgG. It was also suggested that alloantibody binding to the RBCs could lead to conformational changes of the antigenic epitope that ultimately stimulates production of autoantibodies. Patients with autoantibodies may have a higher transfusion rate and often require immunosuppressive drugs, splenectomy, or alternative treatments [16] .

As regard the factors influencing alloimmunization, many factors had been studied by many researchers, trying to minimize the rate of alloimmunization especially in the chronically transfused patient. One of the most important risk factors was the age at first transfusion. In the present study, there was a significant relation between alloimmunization and the age of the first transfusion. It has been thought that transfusion at an early age may offer some protection against RBC alloimmunization because of immune tolerance in young children [17] .In contrast, there were no statistical significant difference between patients' age and sex with regard to the immunization rate (P > 0.05).

In the current study, there was no statistically significant difference between splenectomized and nonsplenectomized patients as regards the alloimmunization rate (47.6 and 52.4%, respectively; P > 0.05). However, other studies have reported a high incidence of RBC alloimmunization and autoimmunization among patients who underwent splenectomy [15] . They stated that the absence of a spleen may further enhance the immune response to the infused foreign antigens, which were not effectively filtered.

Most studies stated that the relationship between the number of units transfused and alloimmunization was unknown in thalassemia. Moreover, it was reported that the interval between transfusions did not appear to play a significant role in antibody development as similar interval was observed between all the patients. However, the interval shortened after the development of the antibodies due to decreased survival of foreign RBCs [6] .

In our study, using the mean number of transfused blood units as a cutoff, it was found that the frequency of blood transfusion was more than 17 U/year in all alloimmunized patients (P < 0.001). We also found that the frequency of transfusion could be better judged by the transfusion index wherein the annual blood requirement as volume of transfused blood per kg of body weight was calculated [18] . The transfusion index showed high statistical significance with regard to alloimmunization (P < 0.01).

Although blood hemoglobin and serum ferritin concentrations were not mentioned in other studies, their values were collected from the patients' sheets to correlate with the clinical significance of alloimmunization. For serum ferritin, the correlation was highly significance (P < 0.01). The increase in the blood transfusion index and frequency in transfusion can explain the increase in the ferritin levels. For blood hemoglobin, the correlation with the alloimmunization was nonsignificant (P > 0.05). Also, the increased frequency of transfusion can explain the higher than expected hemoglobin levels in alloimmunized patients.

We conclude that alloimmunization to RBC antigens is a relatively frequent finding among Egyptian transfusion-dependent thalassemic patients. The most frequent antibodies detected were against the Kell and Rh blood groups; mainly anti-Kell and anti-E. The majority of alloantibodies detected in this study were clinically significant. The most important factors that had contributed to this problem were the multiple allogenic blood exposure with the majority of patients being transfused with blood matched for ABO and D antigens only, and the delay in the age at which transfusion started. In our study, alloimmunized patients had increased transfusion frequency, increased transfusion index, and increased serum ferritin, all of which could complicate the general condition of these patients.

We recommend for the blood transfusion-dependent thalassemic patients to have antibody-screening test performed before each transfusion. Also, blood transfusion-dependent patients must be phenotyped (Rh-Kell) once diagnosed and before their first transfusion to receive Rh-Kell phenotyped blood, thus preventing later complications.


  Acknowledgements Top


Conflicts of interest

There are no conflicts of interest.

 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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