Red cell Alloimmunization and its Correlation with Clinical Spectrum & Transfusion Profile in Pediatric Thalassemia Major Patients: Experience from a Tertiary Care Pediatric Center in Northern India

Abstract

Background

Thalassemia is the most frequent congenital cause of anemia globally, categorized by anomalous hemoglobin production. Lifelong PRBC transfusion with iron chelation therapy is the only therapeutic option available for the majority. Long-term recurrent blood transfusion has its hindrances like RBC alloimmunization. As there is inadequate data on alloimmunization in the pediatric thalassemia major population, our study aims to find out its prevalence and correlation with the clinical spectrum & transfusion profile.

Methodology

Alloantibody screening was done using 3 cell screening panel that includes antigens D, C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, Lea, Leb, P1, M, N, S, s, Mia, Dia, and Xga. The presence of an unexpected antibody in the patient’s serum was considered a positive antibody screen for alloantibody. All samples were further examined to identify antibody specificity using 11 cell identification panels.

Results

6% (5/82) of the included patients demonstrated presence of one or more alloantibodies Alloimmunization was significantly greater in children having their first transfusion after one year of age [OR (95% CI) = 1.42(1.36-1.49), p = 0.02]; receiving > 12 transfusions per year [OR (95% CI) = 1.26(1.12–1.40), p = 0.03] and having > 150 ml/kg/year annual packed RBC consumption [OR (95%CI) = 1.13 (1.08–1.19), p = 0.05]. Total number of transfusions > 100 was also found to be positively associated with alloimmunization [OR (95% CI) = 1.22 (1.14–1.32), p = 0.04].

Conclusion

In the present study, alloimmunization was found to be 6% in multitransfused pediatric thalassemia patients. Our observation re-emphasizes the prerequisite for RBC antigen typing ahead of the first transfusion and early institution of transfusion therapy after diagnosis to decrease alloimmunization.

Introduction

Thalassemia is one of the most common inherited causes of anaemia globally, characterized by abnormal hemoglobin production, which necessitates frequent blood transfusions placing an immeasurable emotional, psychological, and economic burden on their families. India contributes the highest number of thalassaemic children with 10,000–15,000 thalassaemic babies being born annually [1]. In developing countries like India, lifelong PRBC (packed red blood cell) transfusion with iron chelation therapy is the only available therapeutic modality for majority, however, long-term repeated blood transfusion has its disadvantages like RBC alloimmunization, which is the process of development of alloantibody by the recipient’s immune system against a foreign (donor) RBC (red blood cell) antigen which is lacking in recipient’s RBC [2]. Preventing RBC alloimmunization in thalassemia children is a critical aspect of their care as it poses a significant challenge in the management of thalassemia, in the form of delay in the provision of compatible blood, reduced life span of transfused cells in recipient resulting in increased frequency of transfusion and thus leading to accelerated iron overloading of tissues. These alloantibodies also might lead to delayed hemolytic or serologic transfusion reactions [3]. Among the contributing factors for alloimmunization, the recipient’s immunity status, age at first transfusion, and total units of blood received per year are the major contributors [4]. Understanding the association between risk factors and preventive strategies is essential for improving the overall management and long-term outcomes of thalassemia patients facing this immunologic complication.

This involves the meticulous matching of blood donors and recipients by extended RBC phenotyping to minimize antigenic disparities and the use of leuko-reduced blood [5]. Worldwide literature study suggests prevalence of alloimmunization is approximately 5–11% in thalassemia patients [6]. However, there is limited data on alloimmunization in the pediatric population and its correlation with the clinical spectrum & transfusion profile especially in thalassemia patients of the pediatric age group from the Indian subcontinent. Although there are few studies from India depicting the alloimmunization rate in thalassemia patients, none of them was conducted in an exclusive pediatric population. Hence the present study was contemplated to find out the alloimmunization rate and its correlates in an exclusive pediatric cohort.

Materials and methods

This single-centric, prospective, cross-sectional study was conducted over 2 years from August 2020 to August 2022 at Dr. Baba Saheb Ambedkar Medical College and Hospital, New Delhi after obtaining Ethical committee approval. The primary objective was to study the prevalence of red cell alloimmunization in pediatric beta thalassemia major patients aged 0–18 years. Secondary objectives were to determine the pattern of positive alloantibodies and to study the correlation of clinical spectrum & transfusion profile of these children with alloimmunization. All Beta Thalassemia major patients of age < 18 years who were registered in the thalassemia daycare and receiving regular blood transfusions only from the blood bank of Baba Saheb Ambedkar Hospital during the two-year study period have been included. All the registered thalassemia patients were transfused leukodepleted PRBC (done as per standard transfusion policy and blood bank protocols). Thalassemic children with other hemoglobinopathies, autoimmune hemolytic, and immunodeficiency disorders have been excluded from this study. The work of Dhawan et al. observed that the prevalence of alloantibodies was 5.64% [7]. Considering this result as a reference, the least essential trial size with a 5% margin of error and a 5% level of significance, was calculated to be 82 patients.

Transfusion protocol: As per institutional protocol, thalassemia patients were transfused at 10–15 ml/kg at an interval of 2 to 4 weeks with a target to keep the hemoglobin between 9 and 11 gm/dl. All included patients had been receiving their blood transfusions as ABO and Rh D cross-matched leukodepleted blood from a single blood bank i.e. blood bank of Baba Saheb Ambedkar Hospital (the hospital where the study was conducted). Leukodepletion was carried out at pre storage stage as per our blood bank policy for all the patients. Patients developing alloimmunization were provided with corresponding antigen-negative cross-matched blood for further transfusions.

Alloantibody detection: 2 ml venous blood collected in an Ethylene Diamine tetra acetate tube was centrifuged for 10 min at 1500 rpm to obtain plasma for cross-matching and antibody screening. All included patients were routinely tested for ABO and RhD grouping using the ABO-Rh forward/Reverse grouping Gel cards. Samples not for immediate testing were stored at 2–6˚ C after separation for a maximum of 48 h. The Alloantibody screening was done using 3 cell screening panel (Diacell, Biorad) that includes antigens D, C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, Lea, Leb, P1, M, N, S, s, Mia, Dia, and Xga. After identifying the appropriate microtube of a Column agglutination test (CAT) card with the patient’s name/ hospital identification no, aluminum foil was removed from as many microtubes as needed. 50 µl each of the ready-to-use I-II-III screening cells was pipetted into the appropriate microtube labeled I, II & III. 25 µl of test plasma was added to all the microtubes. The microtubes were incubated for 15 min at 37˚C in the CAT incubator. The card in the CAT centrifuge was centrifuged for 10 min. Results were interpreted based on charts provided by the panel.

The presence of an unexpected antibody in the patient’s serum was considered a positive antibody screen for alloantibody. All alloantibody screen-positive samples were further examined to identify antibody specificity using 11 cell identification panels (Diapanel, Biorad) using the same methodology as above. This panel carried D, C, E, c, e, M, N, S, s, Fy^a, Fy^b,Jk^a, Jk^b,Kk; Lu^a,Lu^b,Le^a, Le^b, p1 antigens to identify clinically significant alloantibodies. Results were interpreted according to the reaction on the Antigram charts provided with the panel. The pattern of positive and negative reactions identifies the antigen against which the antibodies were formed. Clinico epidemiological data of the included patients were also collected during their day-care visits.

Data were analyzed using SPSS version 26 (SPSS Inc., Chicago, Illinois, USA). Descriptive statistics included the computation of percentages, means, and standard deviations [8]. Logistic regression model was used to identify the correlation of clinical spectrum and transfusion profile with alloimmunization and corresponding Odd’s ratio (OR) with a 95% confidence interval (CI) was calculated. A p-value ≤ 0.05 was considered significant.

Results

A group of 82 [Male: 55 (67.1%)] pediatric beta thalassemia major patients of age 11.2 ± 3.6 years were involved in this work. Table 1 summarizes the demographic and transfusion features of the study population. Approximately three-fourths of them received their first transfusion before one year of age. The cohort received 15.5 ± 2.1 blood transfusions per year at an interval of 21.1 ± 9.5 days. 80% of the study participants have undergone > 100 total blood transfusions with an annual packed red cell consumption rate of 158.6 ± 22.2 ml/kg/year. Splenectomy was done in two (2.44%) children. The spreading of ABO blood types was A [17(20.7%)], B [32(39%)], O [24(29.2%)], and AB [9(10.9%)] among the study subjects. Three children (3.7%) had a Rh-negative phenotype.

Table 1.

Demographic and transfusion characteristics of the study cohort

Variable	Mean ± SD	Study cohort (n = 82)
		Number	Percentage
Age (in years)	11.2 ± 3.6
< 10		58	70.7
> 10		24	29.3
Gender	--
Male		55	67.1
Female		27	32.9
Age at first transfusion (months)	11.6 ± 9.7
< 12		60	73.2
> 12		22	26.8
No of blood transfusions/year	15.5 ± 2.1
> 12		63	76.8
< 12		19	23.1
Interval Between two consecutive blood transfusions (in days)	21.1 ± 9.5
< 21		71	86.5
> 21		11	13.4
Annual consumption of packed red cells (ml/kg/year)	158.6 ± 22.2
> 150		60	73.1
< 150		22	26.8
Total Transfusion duration (in months)	118 ± 4
> 100		66	80.4
< 100		16	19.5
Total number of transfusions	114 ± 6
> 100		61	74.3
< 100		21	25.6
Splenectomy status	--
Yes		2	2.4
No		80	97.6
Pretransfusion Haemoglobin (g/dl)	7.4 ± 0.5	--	--
Pretransfusion Total Bilirubin (mg/dl)	1.2 ± 0.2	--	--
Pretransfusion Unconjugated Bilirubin (mg/dl)	0.8 ± 0.06	--	--
Serum Ferritin	2459 ± 104	--	--

Prevalence and specification of Alloantibody: Out of 82 patients, 5 (6%) were found to be positive for a total of 7 RBC alloantibodies, and details of them are illustrated in Table 2. Two out of these five patients (40%) had dual alloantibodies. Among 7 alloantibodies, the majority [n = 5 (71.4%)] were of the Rh blood group system (Anti-E = 14.2%, Anti-D = 14.2%, Anti-C = 28.5%, Anti-Cw = 14.2%). Among the rest two alloantibodies, [n = 1(14.2) %] confined to the Kell and Kidd blood group system each.

Table 2.

Clinical and transfusion-related details of the patients who developed alloantibodies

Sl no	Age (years)	Sex	Blood Group ABO/Rh	Age at first transfusion (in months)	Number of blood transfusions/ year	Interval Between two consecutive blood transfusions (in days)	Annual consumption of packed red cells (ml/kg/year)	Total Transfusion duration (in months)	Total number of transfusions	Splenectomy status	Specificity Alloantibody
1	13.2	F	O + ve	17	22	15	232	265	247	No	Anti Cw
2	11.4	M	B -ve	11	21	15	246	116	188	No	Anti D
3	6.2	M	AB + ve	18	11	30	114	58	56	No	Anti C and K
4	10.5	F	A + ve	16	25	14	264	121	244	No	Anti E
5	12.8	M	O + ve	18	24	14	272	144	276	No	Anti C, Anti Jkb

Four out of these five alloimmunized children had their first transfusion after one year of age, received > 12 blood transfusions/year, and had > 150 ml/kg/year packed RBC consumption rate. Furthermore, these four patients had > 100 number of total transfusions. However, none of the five subjects had undergone splenectomy.

Correlation of alloimmunization with Clinical spectrum and transfusion profile:

Table 3 depicts the various transfusion characteristics and clinical parameters of patients with and without alloimmunization. On multivariate logistic regression analysis, five parameters namely: age at first transfusion, number of transfusions/years, annual rate of packed red cell consumption, total number of transfusions till date, and serum ferritin were found to have a statistically significant association with RBC alloimmunisation. In our study, out of 5 patients who developed alloimmunization, 4 patients had their first transfusion after 1 year of age i.e. significantly higher rate [18% (4/22)] of alloimmunization compared to those with their first transfusion before one year of age [1.6% (1/60) [OR (95% CI) = 1.42 (1.36–1.49), p = 0.02].

Table 3.

Univariate and multivariate logistic regression analysis for clinical and transfusion factors associated with alloimmunization in pediatric Thalassemia major patients

Variables	Alloantibody		Univariate analysis		Multivariate analysis
	Present (N = 5)	Absent (N = 77)	Odd’s ratio (OR) (95% CI)	p value	Odd’s ratio (OR) (95% CI)	p value
Gender			0.72 (0.68–0.77)	0.10
Male	3 (60%)	52 (67.5%)
Female	2 (40%)	25 (32.4%)
Age at first transfusion (months)			13.11  (12.42-13.88)	0.04	1.42 (1.36–1.49)	0.02
>12	4 (80%)	18 (23.3%)
<12	1 (20%)	59 (76.6%)
No of blood transfusions/year			1.2 (1.02–1.41)	0.04	1.26 (1.12–1.40)	0.03
> 12	4 (80%)	59 (76.2%)
< 12	1 (20%)	18 (23.3%)
Interval Between two consecutive blood transfusions (in days)			0.59 (0.42–0.64)	0.06
< 21	4 (80%)	67 (87.1%)
> 21	1 (20%)	10 (12.9%)
Annual consumption of packed red cells (ml/kg/year)			1.54 (1.35–1.72)	0.05	1.13 (1.08–1.19)	0.05
> 150	4 (80%)	56 (72.7%)
< 150	1 (20%)	21 (27.2%)
Total Transfusion duration (in months)			0.96 (0.88–1.04)	0.06
> 100	4 (80%)	62 (80.5%)
< 100	1 (80%)	15 (19.5%)
Total number of transfusions			1.40 (1.34–1.46)	0.03	1.22 (1.14–1.32)	0.04
> 100	4 (80%)	57 (74%)
< 100	1 (20%)	20 (26%)
Splenectomy status			7.50 (6.21–8.42)	0.16
Yes	0 (0%)	2 (2.5%)
No	5 (100%)	75 (97.5%)
Pretransfusion Hb (g/dl)	7.6 ± 0.4	7.8 ± 0.3	0.91 (0.78–1.04)	0.10
Pretransfusion Total Bilirubin (mg/dl)	1.4 ± 0.02	1.2 ± 0.01	1.15 (1.01–1.2)	0.12
Pretransfusion Unconjugated Bilirubin (mg/dl)	0.84 ± 0.04	0.82 ± 0.03	1.12 (1.04–1.19)	0.18
Serum Ferritin	2984 ± 104	2262 ± 106	1.30 (1.05–1.37)	0.03	1.14 (1.02–1.25)	0.05

Alloimmunization was significantly greater in children receiving > 12 transfusions per year [OR (95% CI) = 1.26(1.12–1.40), p = 0.03] and having > 150 ml/kg/year annual packed RBC consumption [OR (95%CI) = 1.13 (1.08–1.19), p = 0.05]. Likewise, the whole number of blood transfusions > 100 was found to be positively associated with alloimmunization [OR (95% CI) = 1.22 (1.14–1.32), p = 0.04]. Serum ferritin was significantly higher in alloimmunized patients (p = 0.05). No significant association was noticed between other parameters such as gender, the interval between consecutive transfusions, total duration of transfusions to date, splenectomy status, pre-transfusion hemoglobin, and pre-transfusion bilirubin with alloimmunization.

Discussion

Regular transfusion therapy is the mainstay in the management of thalassemia major patients with RBC alloimmunization being one of the main limitations of multiple blood transfusions [9]. The major reason for alloimmunisation is postulated to be antigenic dissimilarities between donor and recipient RBC. This could be well explained by the presence of 45 blood group systems and over 362 diverse blood group antigens (International society of Blood Transfusion, 2023) which result in an assortment of varied RBC antigens in recipients, leading to the formation of alloantibodies after multiple transfusions [10]. Our study intended to realize the rate of occurrence of alloimmunization and its correlation with transfusion profile in pediatric thalassemia major patients.

In our study, 6% (5/82) of the included patients demonstrated the presence of one or more alloantibodies. Previous Indian studies from various parts of the country have reported the prevalence of alloimmunization to be less than 10%, ranging from 5.6 to 8.6% [Table 4]. Studies conducted in several other nations have, however, revealed a notable variance in the degree of alloimmunization among patients with diverse ethnic backgrounds, ranging from 3.1 to 37% [7]. Heterogeneity of population increases the occurrence of alloimmunization due to RBC antigenic discrepancy between the recipient and the blood donor as documented in the study by Singer et al. from the USA where they found 20.8% of alloimmunization in the recipients of Asian ethnicity owing to their variance in the racial background from the white donors [11]. Even very high alloimmunization rates of 30% and 42.5% in transfusion-dependent thalassemia patients have also been reported in studies from Kuwait and Israel respectively [12].

Table 4.

RBC alloimmunization rates and its clinical correlation in Thalassemia patients in previous Indian studies

Study	Year	Demographic Region	Prevalence	Major Clinical & Transfusion Correlates for Alloimmunization
Dhawan et al. [7]	2014	Punjab	5.64%	Age at first transfusion
Elhence P el al. [13]	2014	North India	8.6%	Patient age
Datta et al. [14]	2015	Eastern india	5.6%	Age at first transfusion
Handa et al. [15]	2020	India	7%	No associations
Sahu et al. [16]	2020	Odisha	7.5%	➣ Age at first transfusion ➣ No. Of units transfused/ year
Yadav et al. [17]	2023	Uttar pradesh	6.6%	➣ Age at first transfusion ➣ Transfusion interval time ➣ No. of units transfused/ year
Present Study	2022	Delhi	6%	➣ Age at first transfusion ➣ Number of transfusions/year ➣ Annual rate of packed red cell consumption ➣ Total number of transfusions to date ➣ Serum ferritin

Two studies from northern India by Yadav et al. [17] (From Uttar Pradesh) and Dhawan et al. [7] (from Punjab) have reported 6.6% and 5.6% alloimmunization in 255 and 319 thalassemia patients respectively, consisting of both pediatric as well as adult patients in the study population. A slightly lower prevalence of alloimmunization in our study might be because of the inclusion of only pediatric patients. There is always a possibility of these alloantibody-negative children becoming positive in the future as alloimmunization is positively correlated with the number of transfusions, as shown in our study as well as several other studies [18].

In our study, the majority (71.4%) of the alloantibodies are directed against the Rh blood group system, which agrees with various other studies. We also investigated the correlation of alloimmunization with the transfusion profile of the thalassemia patients. In the current study, patients who started receiving transfusions early in life i.e. during infancy had a significantly lower rate of alloimmunization (1.6%) compared to those who had their first transfusion after one year of age (18%). In 1986, a study by Floss and coworkers demonstrated that infants do not produce alloantibodies even on exposure to many red and white cell antigens [19]. Hence, A lower risk of alloimmunization in patients starting transfusion at an early age might be because the immature immune system of the infant is incapable of mounting a strong immune response to foreign RBC antigens or acquired immune tolerance to red cell alloantigen [20]. However, Pahuja et al. reported a contradictory result in their study with an alloimmunization rate of 30% even in patients who started transfusion before 1 year [21].

We further observed that increased transfusion (both the number of transfusions/year and total number of transfusions received to date) is significantly associated with a higher rate of alloimmunization. In fact, four out of our five alloantibody-positive children had been receiving > 12 transfusions/year and had already received > 100 total transfusions at the time of screening for alloimmunization. Yadav et al. had also reported similar findings in their study. However, several other studies did not find any significant correlation between the number of transfusions with alloimmunization [17]. Theoretically, an increase in the number of transfusions exposes the patient to a vast array of RBC antigens, some of which might trigger alloantibody formation which might be the plausible explanation for the positive correlation between the number of transfusions with alloimmunization [18]. Likewise, in concordance with the study by Yadav et al., in our study also the patients receiving > 150 ml/kg/year packed RBC had significantly higher alloimmunization rates [17]. No other study till date investigated the relationship between annual red cell consumption with alloimmunization in pediatric patients. The degree of transfusions, the complete number of antigens for every RBC, and the immune response provoked by the antigen were found to be the main contributing factors for alloimmunization, as the blood group antigen dosage for each RBC may fluctuate from a mere few to millions of antigens [12]. In this study, there was no substantial variance in the alloimmunization rate that was found among males and females. Numerous studies stated a higher post-transfusion alloimmunization rate in female thalassaemics, an outcome that could not be established in a meta-analysis by Verduin et al. [22]

Yadav et al. [17] and Elhence et al. [13] showed a higher alloimmunization rate in splenectomized than non-splenectomized patients. This alteration is projected to be due to conformational variations in the RBC membrane which boost immunomodulation ending up in a higher risk of RBC alloimmunisation. However, in this study, no noteworthy correlation was detected between splenectomy and the production of alloantibodies, which is in line with the previous study by Handa et al [23].

Yadav et al. reported greater alloimmunization risk as the duration between consecutive transfusions decreases to less than 30 days [17]. However, in our study, we could not notice any statistically significant correlation. This might be because the mean cut-off interval between transfusions is 21 days in our study. The results might have been significant if it was kept a bit longer e.g. 30 days as done by Yadav et al. [17]. Few other studies have reported an increase in the risk of alloimmunization as the duration of transfusion increases and the interval between transfusions decreases [14, 15]. As ours was an exclusive pediatric cohort and hence the total duration for which patients received transfusion was lower which might have led to the discrepancy.

Gender did not have any significant association with alloimmunization in our study which is similar to the findings reported by various other studies. Likewise, splenectomy status did not have any correlation with alloimmunization in the current study. In support of the study by Yadav et al., alloantibody-positive children had significantly higher serum ferritin in contrast to alloantibody-negative patients in our study [17]. This study further corroborates the positive correlation observed between the increased number of transfusions/increased annual RBC consumption with alloimmunization, as all factors eventually lead to iron overload. The strength of our study is it comprises a highly heterogeneous population including patients from various regions of the country who migrated to Delhi, in contrast to most studies in which the patients typically belong to a particular state or region. Moreover, all the studies conducted in India have included both pediatric as well as adult thalassemia patients, whereas our study has been conducted exclusively in pediatric age group patients. Furthermore, we have attempted to correlate multiple transfusion parameters to know the risk factors for alloimmunisation. Unlike previous studies which had examined two or three factors related to alloimmunization, we analyzed a total of eight clinical & transfusion characteristics and four biochemical parameters concerning alloimmunization. However, smaller sample size is a limitation of this study. A larger sample size would have allowed better correlations and strengthened the findings.

Conclusion

Red cell alloimmunization is an important complication in β-thalassemia major patients. In the present study, alloimmunization was found to be 6% in multitransfused pediatric thalassemia patients. Our observation re-emphasizes the prerequisite for RBC antigen typing ahead of the first transfusion and early institution of transfusion therapy after diagnosis to decrease alloimmunization.

Authors’ Contribution

SP conceptualized the study, designed the study, and collected all data. KP performed statistical analysis, derivation of results, and interpretation of data. BKB and JBK contributed to the critical revision of the final manuscript for important intellectual content. SS performed the final editing of the manuscript. KP approved the final version and agreed to be accountable for all aspects of the work. KP is the corresponding author of this manuscript and is responsible for coordinating the work from its inception to publication and can be approached for access to raw data.

Funding

None.

Declarations

Conflict of interest

There is no financial or non-financial conflict of interest between the authors.