Abstract
Severe hemolytic anemia in β-thalassemia major and β-thalassemias/HbE (β-TM) patients requires giving blood transfusions. Chronic blood transfusions lead to iron overload consequence with organs damage and risk of alloantibody-formation. This study evaluates the prevalence of red cell alloimmunization and estimates the risk of alloantibody-formation in chronic transfusion-dependent β-TM patients. This cross sectional study was conducted on 143 β-TM patients receiving regular transfusions. We tried to determine the frequency, types and factors influencing red cell alloimmunization in these transfusion-dependent β-TM patients. Median age of 25 (17.5 %) alloantibody-formation β-TM patients was 19.0 years (inter quartile 15.5–24.0 years). The alloantibodies were Anti-Rh (E) (13.1 %), Anti-Rh (D) (0.7 %). Thirty-four patients (23.8 %) of the sample had splenectomies of which 10 (29.4 %) had alloantibody-formation. The interval from first transfusion to antibody development varied from 1.5 to 14 years. Alloantibody-formation correlated with splenectomy and splenectomy correlated with number of transfusion (p < 0.005). In multiple logistic regression used to estimate the risk of alloantibodies formation with splenectomy; OR and 95 % CI were 2.88 (1.07–7.80), p = 0.037 after adjusting for other co-variates. The rate of red cell alloimmunization was 17.5 % and splenectomy associated with increased alloantibody-formation in these transfusion-dependent β-TM patients.
Keywords: β-Thalassemia, Alloimmunization, Alloantibody, Splenectomy, Transfusion
Introduction
Life-long red blood cell (RBC) transfusion remains the main treatment for severe β-thalassemia major and β°-mutant thalassemic Hb with HbE (β-TM) patients. However, the beneficial effect of the transfusion is accompanied with the possibility to cause adverse effects due to transfusion. The adverse affects in these β-TM patients due to chronic transfusions include iron overload, increased rate of transfusion transmitted infections and alloimmunization to red cell antigens. Alloimmunization is a response by the body’s immune system (alloantibody) against to donate RBC antigen. Though antibody formation can take place against any antigen present on the surface of red cells such as Rh and Kell blood group system [1]. The development of alloantibodies can significantly complicate transfusion therapy [2, 3], due to difficulty in getting compatible blood, delayed haemolytic transfusion reaction and life-threatening hyperhaemolysis syndrome [3–5].
Splenomegaly and hypersplenism are common clinical features of β-TM patients. Splenectomy is recommended intervention to reduce excessive blood consumption and consequent severe iron overload. Splenectomy is also ameliorates growth retardation or poor health, leukopenia, thrombocytopenia, increased transfusion demand, or symptomatic splenomegaly. However, all data of these β-TM patients included first diagnostic, time for first need blood transfusion and alloimmunization is very sketchy. In this study, we tried to determine the prevalence, types of alloantibody-formation and risk factors which may be responsible for the alloantibodies-formation in these β-TM patients receiving regular blood transfusions.
Materials and Methods
Subjects: From January to June 2013, a cross-sectional study was carried out in Nan Hospital, Nan Province, Northern Thailand. This study included 143 transfusion-dependent β-TM patients (68 males and 75 females, median aged = 16.0 (11.0–21.0) years) receiving regular blood transfusions from the Thalassemia Clinic Unit in Nan hospital. Diagnosis of these β-TM patients were established based on the age of onset of anemia, features of hemolysis on peripheral smear with increased fetal hemoglobin by Hb typing (EF, EFA, A2F and A2FA) with high performance liquid chromatography, hematological and clinical manifestation. Data from medical records were very sketchy, thus, the first age of received transfusion and estimated number of red blood cell packs for transfusions were uncertain. Clinical and transfusion records of all patients were analyzed, record variables include age, ferritin concentration, duration-time of transfusion, estimated number of red blood cell packs for transfusion and whether splenectomy was performed as patient variables. The parents of all study participants signed informed consent forms prior to enrollment. This study was approved by the Ethics Committees of Naresuan University and Nan Hospital.
Laboratory Determination
Every time, when these β-TM patients came to receive transfusion, a 5 mL blood sample was collected in each tube. Serum sample was collected for blood grouping, cross matching and ferritin concentration testing. Plasma sample was collected and divided into two aliquots with the identification code of the patient. One plasma sample was used for antibody screening, and the other was kept at −30 °C in deep freezer for antibody identification in future in case of a positive antibody screen. Screening for alloantibodies was done by using gel card technology [6] using screening cell from The Thai Red Cross Society, Thailand. The antibody screen of each sample was done by a medical technologist or blood bank transfusion technologist without knowledge of the corresponding patient details. Screening cells were used as reagent cells for screening irregular antibodies, which was undertaken by column gel agglutination (CGA) technique at 37 °C and room temperature (23 °C) using LISS Coombs and NaCI gel cards (DiaMed) respectively. They were also subjected to Direct Coombs test by CGA technique. Specimens found positive for irregular antibody were subjected to alloantibody identification using screening cells panel by CGA. The screening cell panel covered antigens of all known blood groups including Rh (D, C, E, c, e, Cw), Kell (K, k, Kpa, Kpb, Jsa, Jsb), Kidd (Jka, Jkb), Duffy (Fya, Fyb), P1, Lewis (Lea, Leb), MNS (M, N, S, s), Lutheran (Lua, Lub) and Xga. Grading and scoring of the agglutination in the gel cards was done by two medical technologist or blood bank technologist without knowledge of the clinical and laboratory data of the patient. The grading and scoring was done according to the distribution of agglutination throughout the gel matrix.
Statistical Analysis
Statistical analysis was performed by using SPSS (v 13.0 SPSS Inc, Chicago, USA). All variables were given as median and interquartile. Comparisons between groups were conducted by using Mann–Whitney test. The correlation between all variables was analyzed by Spearman’s correlation. For the analysis of risk factors associated with red cell alloimmunization, all β-TM patients from the Thalassemia Clinic Unit in Nan hospital already known with and without red cell antibodies were added for multivariate testing. Variables suspected including splenectomy, ferritin concentration, duration-time of transfusion and estimated number of red blood cell packs for transfusion to be associated with alloantibody-formation were entered into logistic regression models. All p-values <0.05 were considered as significant.
Results
A total of 143 β-TM patients were included in this study. The median age of the β-TM patients was 16.0 (11.0–21.0) years. There were 68 males and 75 females. All of them had received regular transfusion with leukodepleted red cell during a period ranging from 2 to 26 years. The time interval between transfusions in these patients varied from 18 to 35 days. Twenty-five (17.5 %) β-TM patients developed alloantibody. We compared some clinical characteristics and risk factors of both β-TM patients with alloantibody-formation and Non alloantibody-formation as shown in Table 1. Types of alloantibodies were identified in β-TM patients in the present study as shown in Table 2. Twenty-five β-TM patients with alloantibody, there were ten males and fifteen females. Eleven of these 25 β-TM patients who developed alloantibodies underwent a splenectomy. We also found fourteen none-splenectomised β-TM patients had developed alloantibodies. The most common antibody was Anti E (present in 13 cases) followed by Anti c, M, Lea, Mia which developed in four cases. There were four β-TM patients who developed two antibodies (Anti D and Anti c; Anti E and Anti Mia; Anti E and Anti Jka; Anti c and Anti E) and two β-TM patients who developed three antibodies (Anti C, Anti e and Anti Jka; Anti c, Anti E and Anti Mia) as shown in Table 2. For Spearman’s correlation analysis, we found that the patient’s age was significantly correlated with alloantibody-formation. While alloantibody-formation was significantly correlated with splenectomy. Splenectomy was also significantly correlated with estimated number of red blood cell packs for transfusion as shown in Table 3. We used multiple logistic regression analysis to examine effects of variables in the association of these variables with alloantibody-formation in β-TM patients. Alloantibody-formation showed strong association with splenectomy, which remained highly significant after adjusting laboratory co-variables; ORs and 95 % CIs were 2.88 (1.07, 7.80), statistics are listed in Table 4.
Table 1.
General characteristics of healthy control, β-thalassemia major and β-thalassemia/HbE patients (β-TM patients)
| Variables | Non-AAb formation in β-TM patients | AAb formation in β-TM patients | p-value |
|---|---|---|---|
| (n = 118) | (n = 25) | ||
| Age (years) | 15.5 (11.0–21.0)a | 19.0 (15.5–24.0)a | 0.009 |
| BMI (kg/m2) | 16.9 (14.6–19.6) | 16.7 (14.6–18.6) | 0.142 |
| Hb (g/L) | 70.0 (57.0–84.0) | 68.0 (57.0–82.0) | 0.328 |
| Hct (%) | 21.8 (18.1–27.2) | 21.4 (18.4–26.2) | 0.177 |
| WBC × 109 (cells/L) | 10.53 (7.69–30.16) | 11.89 (7.69–30.16) | 0.418 |
| Platelet × 109 (cells/L) | 271.0 (205.0–530.0) | 274.0 (206.0–540.0) | 0.248 |
| Ferritin (pmol/L) | 3478.9 (2485.9–7379.9) | 5000.0 (2223.0–8680.3) | <0.001 |
| Splenectomy (%) | 23 (67.6 %) | 11 (32.4 %) | <0.001 |
| dTT (year) | 12.0 (8.0–15.0) | 12 (11.0–15.0) | 0.107 |
| eNT | 1248 (832–1560) | 1248 (1144–1560) | 0.107 |
BMI body mass index, Hb hemoglobin, Hct hematocrit, WBC white blood cell count, AAb-formation alloantibodies-formation, eNT estimated number of red blood cell-packs for transfusion, dTT duration-time of transfusion
aMedian and (interquartile)
Table 2.
Prevalence of red cell alloimmunization and type of alloantibodies-formation were found in 25 β-TM patients
| 25 β-TM patients | Alloantibodies | Splenectomy | ||||
|---|---|---|---|---|---|---|
| No | Sex | Age | ABO gr | Rh type | ||
| 1 | M | 19 | B | positive | C, e, Jka | Yes |
| 2 | M | 22 | B | positive | c, E, Mia | No |
| 3 | F | 23 | O | positive | E, Jka | Yes |
| 4 | F | 25 | O | positive | c, E | Yes |
| 5 | M | 21 | B | positive | Le, a | Yes |
| 6 | F | 11 | O | positive | c, E | Yes |
| 7 | F | 22 | O | positive | E, Mia | Yes |
| 8 | F | 17 | O | positive | D, c | No |
| 9 | F | 21 | B | positive | E | Yes |
| 10 | F | 14 | O | positive | E | Yes |
| 11 | M | 32 | O | positive | E | Yes |
| 12 | F | 33 | O | positive | c | Yes |
| 13 | M | 17 | O | positive | E | Yes |
| 14 | M | 14 | AB | positive | E | No |
| 15 | F | 17 | B | positive | E | No |
| 16 | F | 17 | A | positive | E | No |
| 17 | M | 17 | B | positive | E | No |
| 18 | M | 19 | B | positive | E | No |
| 19 | F | 25 | O | positive | E | No |
| 20 | M | 30 | O | positive | E | No |
| 21 | F | 15 | O | positive | E | No |
| 22 | F | 8 | B | positive | E | No |
| 23 | F | 28 | A | positive | M | No |
| 24 | F | 16 | AB | positive | Mia | No |
| 25 | M | 15 | O | positive | Auto Ab | No |
| 118 β-TM patients | Negative | 23 | ||||
Table 3.
Correlation of variables among age, ferritin concentration, splenectomy and alloantibodies formation in β-TM patients
| Correlation between parameters | Correlation coefficient | |
|---|---|---|
| r | p-value | |
| Age | ||
| AAb-formation | 0.221 | 0.008 |
| Splenectomy | 0.244 | 0.003 |
| eNT | 0.932 | <0.001 |
| AAb-formation | ||
| Splenectomy | 0.219 | 0.009 |
| Splenectomy | ||
| eNT | 0.221 | 0.008 |
AAb-formation alloantibodies-formation, eNT estimated number of red blood cell-packs for transfusion
Table 4.
Association of alloantibodies-formation with splenectomy after adjusting for their covariates in these β-TM patients
| Variables | Alloantibodies formation | ||
|---|---|---|---|
| OR | 95 % CI | p-value | |
| Splenectomya | 2.88 | 1.07–7.80 | 0.037 |
| Ferritin | 1.00 | 0.99–1.00 | 0.375 |
| eNT | 1.00 | 0.99–1.00 | 0.051 |
| Sex | 1.81 | 0.72–4.58 | 0.434 |
| Age | 1.81 | 0.72–4.58 | 0.008 |
aModel after adjusting for ferritin, eNT, sex and age
eNT estimated number of red blood cell-packs for transfusion
Discussion
All β-TM were received leucodepleted blood as routinely used for transfusion. The rate of alloimmunization in the patients was found to be 17.5 % with 25 out of 143 patients developing alloantibodies. Previous studies have reported rates of alloimmunization in patients of thalassemia from 4 to 50 % [7, 8]. The major of the antibodies belonged to the Rh and Kell blood group in our race. Therefore provision of Rh and Kell matched blood could prevent alloimmunization in majority of the β-TM patients thus preventing later complications. Since thalassemia is now world wild, albeit more prevalent in Southeast Asia and Mediterranean countries, most of the studies on red cell alloimmunization are from these countries. Ameen et al. [9] reported the highest rate (30 %) of red cell alloimmunization in β-TM patients of Iranian descent, while lower rates were reported by Sirchia et al. [10]. Pradhan et al. [11] found an alloimmunization rate of 8 % amongst 100 patients of thalassemia major in Bombay. Another was reported by Pahuja et al. [12] estimated the rate of alloantibody-formation in patients of thalassemia major as 2.7 % which is very low as compared to studies from other countries. However, none of these studies analyzed factors which may be responsible for alloantibody-formation in these patients. The factors responsible for high rate of alloimmunization in β-TM are largely unknown. In our study, 25 β-TM patients who developed alloantibodies were older, higher ferritin level and higher rate of splenectomy, while duration-time of transfusion and estimated numbers of red blood cell-pack for transfusion were not significantly different and the patient’s age at the start of transfusion was unknown. Our previous study [13] demonstrated that iron overload and oxidative stress were commonly occurred due to organ dysfunctions including splenomegaly in these β-TM patients. Splenectomy is recommended procedure to ameliorate growth retardation or poor health, reduce excessive blood consumption, severe iron overload, leukopenia, thrombocytopenia, increased transfusion demand, or symptomatic splenomegaly and associated not only to increased number of RBCs and nucleated red blood cells but also with higher frequency of complications [6, 8]. The risk of infection, most commonly sepsis from encapsulated micro-organisms (Streptococcus spp., Haemophilus spp. and Neisseria spp.), varies among studies, from more than 30-fold in comparison with normal population [14, 15]. Other negative effects include a raised risk of thrombotic complications and pulmonary hypertension, possibly due to the raised number of circulating platelets and immature red blood cells with alteration of the endothelial function, enhanced platelet activation, decreased levels of proteins C and S [16, 17] Then, a progressive increase of awareness of these negative effects may have contributed to lower the tendency to perform splenectomy in these β-TM patients. Many studies have suggested that the rate of alloimmunization is higher after splenectomy [18]. Singer et al. [8] demonstrated that splenectomy was the risk factor for red cell alloimmunization in Asian patients living in US. The authors reasoned that removal of spleen led to nonfiltering of antigens and damaged erythrocytes from blood stream which was responsible for higher rates of alloantibodies-formation in splenectomised subjects. Our result was also demonstrated that alloantibodies-formation was higher in splenectomised subjects and associated with splenectomy. Several studies have shown that splenectomy is associated with changes in both humoral and cell mediated immunity, including decreased serum IgM concentrations and raised serum IgG and IgA concentrations [19–22]. Thromboembolic events and hypercoagulable state have been reported in β-TM patients and more frequent in splenectomized patients [23]. These may cause from the procoagulant effect of anionic phospholipids on the surface of altered red blood cells and erythroblasts [10]. Kapadia et al. [24] also reported that β-TM patients have an immunological abnormality by lymphocytosis, alterations in lymphocyte function and increase in the absolute numbers of total peripheral blood lymphocytes and T and B cells. However, factors that might lead to the development of these abnormalities include iron overload, splenectomy [24], and the use of DFO, repeated exposure to allogeneic antigens in blood and as a potent inhibitor of T cell responsiveness to mitogens [25], and all possible significant factors contributing to immunological abnormalities were iron overload, deferoxamine use, and splenectomy. Iron overload has been implicated as a major cause of immunological disturbances in thalassaemia. The mechanisms suggested include toxic effects of high iron levels on lymphocyte function [26–28] and redistribution of B lymphocytes from the spleen and lymph nodes to the circulating pool [29].
Prevention of such serious events is possible by extended matching and typing of donor’s blood against the patient’s for all the possible antigens, but this process is cumbersome and costly. Therefore, every patient should be screened for unexpected/alloantibody to prevent any untoward effect of transfusion. Our β-TM patients can receive the most phenotypically matched RBC, so complications such as severe hemochromatosis and psychosocial problems may decrease and its cost-effective benefits will appear.
Conclusion
Alloimmunization is the major risk for all chronically transfused patients. We found a 17.5 % rate of alloantibody-formation in β-TM patients. The risk of alloimmunization is higher in patients with increased age and strong association with splenectomy.
Acknowledgments
We sincerely thank Department of Clinical Pathology, Nan Hospital for financial support. We also sincerely thank all co-workers in the Blood Bank Unit of Nan Hospital, for blood collection and their technical help. We especially thank those who participated and donated blood samples for this study. Finally we sincerely thank Asst. Prof. Dr. Ronald A. Markwardt, Faculty of Public Health, Burapha University, for his critical reading and correcting of the manuscript.
Conflict of interest
None.
Abbreviation
- β-TM patients
β-Thalassemia major and β-thalassemias/HbE patients
- DHTR
Delayed haemolytic transfusion reaction
- BMI
Body mass index
- WBC
White blood cell
- RBC
Red blood cell
- NRBC
Nucleated red blood cells
- Hb
Hemoglobin
- Hct
Hematocrit
- CGA
Column gel agglutination
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