Transfusional Approach in Multi-Ethnic Sickle Cell Patients: Real-World Practice Data From a Multicenter Survey in Italy

Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder characterized by recurrent acute vaso-occlusive crises (VOCs and anemia). Gold standard treatments are hydroxycarbamide (HC) and/or different red blood cell (RBC) transfusion regimens to limit disease progression. Here, we report a retrospective study on 1,579 SCD patients (median age 23 years; 802 males/777 females), referring to 34 comprehensive Italian centers for hemoglobinopathies. Although we observed a similar proportion of Caucasian (47.9%) and African (48.7%) patients, Italian SCD patients clustered into two distinct overall groups: children of African descent and adults of Caucasian descent. We found a subset of SCD patients requiring more intensive therapy with a combination of HC plus chronic transfusion regimen, due to partial failure of HC treatment alone in preventing or reducing sickle cell-related acute manifestations. Notably, we observed a higher use of acute transfusion approaches for SCD patients of African descent when compared to Caucasian subjects. This might be related to (i) age of starting HC treatment; (ii) patients' low social status; (iii) patients' limited access to family practitioners; or (iv) discrimination. In our cohort, alloimmunization was documented in 135 patients (8.5%) and was more common in Caucasians (10.3%) than in Africans (6.6%). Alloimmunization was similar in male and female and more frequent in adults than in children. Our study reinforces the importance of donor-recipient exact matching for ABO, Rhesus, and Kell antigen systems for RBC compatibility as a winning strategy to avoid or limit alloimmunization events that negatively impact the clinical management of SCD-related severe complications. Clinical Trial Registration ClinicalTrials.gov, identifier: NCT03397017.

Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder characterized by recurrent acute vaso-occlusive crises (VOCs and anemia). Gold standard treatments are hydroxycarbamide (HC) and/or different red blood cell (RBC) transfusion regimens to limit disease progression. Here, we report a retrospective study on 1,579 SCD patients (median age 23 years; 802 males/777 females), referring to 34 comprehensive Italian centers for hemoglobinopathies. Although we observed a similar proportion of Caucasian (47.9%) and African (48.7%) patients, Italian SCD patients clustered into two distinct overall groups: children of African descent and adults of Caucasian descent. We found a subset of SCD patients requiring more intensive therapy with a combination of HC plus chronic transfusion regimen, due to partial failure of HC treatment alone in preventing or reducing sickle cell-related acute manifestations. Notably, we observed a higher use of acute transfusion approaches for SCD patients of African descent when compared to Caucasian subjects. This might be related to (i) age of starting HC treatment; (ii) patients' low social status; (iii) patients' limited access to family practitioners; or (iv) discrimination. In our cohort, alloimmunization was documented in 135 patients (8.5%) and was more common in Caucasians (10.3%) than in Africans (6.6%). Alloimmunization was similar in male and female and more frequent in adults than in children. Our study reinforces the importance of donor-recipient exact matching for ABO, Rhesus, and Kell antigen systems for RBC compatibility as a winning strategy to avoid or limit alloimmunization events that negatively impact the clinical management of SCD-related severe complications.

INTRODUCTION
Sickle cell disease (SCD) is one of the most common inherited red cell disorders worldwide, recognized by the World Health Organization (WHO) and the United Nations as a "global health problem" (1,2). About 300,000 infants are born each year with SCD; ∼90,000 in Nigeria, 44,000 in India, 40,000 in the Democratic Republic of Congo, 12,000 in the United States (US), 2,000 in Europe, and 300 in the United Kingdom (UK) (3). Although SCD is part of the rare disease group, more than 10,000 SCD patients live in the UK and more than 25,000 in France, making SCD much more common than cystic fibrosis or hemophilia A (4,5). The median life expectancy is now more than 60 years in Europe (6), but about 80% of patients with SCD in African regions die during childhood (7).
Treatment options for SCD are still limited, particularly in low-income settings. Hydroxycarbamide (HC) is the gold standard treatment for both children and adults with SCD (8). Hydroxycarbamide reduces painful vaso-occlusive events (VOCs) and acute chest syndrome as well as the need for red blood cell (RBC) transfusion. Until now, RBC transfusion has largely been used for clinical management of both acute and chronic sickle cell-related complications (9,10). Despite the known indications for RBC transfusion and HC therapy (9,(11)(12)(13)(14)(15)(16), treatment of patients with SCD in clinical practice is still not homogeneous.
Over the course of a lifetime, patients with SCD might be exposed to different RBC transfusion regimens, ranging from simple transfusion on-demand or manual/automatized RBC exchange to manage acute VOCs or to limit disease progression and organ damage (3,17). Alloimmunization to RBC blood group antigens carries an increased risk of hemolytic transfusion reactions (DHTR), potentially delaying the identification of compatible RBC units (18,19). In Europe, Italy presents a distinctive epidemiologic European niche for SCD, characterized by endemic SCD population mainly in Southern Italian regions and multi-ethnic SCD population localized in Central and Northern Italian regions (20)(21)(22)(23). This affects the distribution of SCD genotypes, mainly represented by Sβ 0 /Sβ + or SS genotype in patients of Caucasian descent, while SS or SC genotypes characterize patients of African descent. To better understand the scenario of transfusion regimens and alloimmunization of SCD patients in Italy, we carried out a national survey involving the National Comprehensive Reference Centers for SCD and the Italian Society of Thalassemia and Hemoglobinopathies (SITE), in collaboration with the Italian Society of Transfusion Medicine and Immunohematology (SIMTI) and the Italian Association of Hematology and Pediatric Oncology (AIEOP).

Study Design
This is a retrospective multicenter national study (ClinicalTrials.gov identifier NCT03397017). Data from the clinical records of eligible patients were collected from 2015 to 2018 through a standard web-based application (www.SITE-italia.org) encrypted by the Central Server. The recording of personal, therapy, and complications data of patients with sickle cell anemia included in the National Transfusion Treatment Survey was undertaken by the responsible investigator or sub-investigators selected by each center, following registration on the site. The operator can subsequently access the patient's clinical data and perform updates to follow clinical evolution over time. The study did not involve any additional tests compared to routine patient management.
The study aimed to identify the lifelong therapeutic approaches, focusing on transfusion regimens, in clinical management of a large multi-ethnic Italian cohort of patients with SCD. The study was coordinated by SITE, in collaboration with SIMTI and AIEOP. A total of 34 Italian reference centers from 15 Italian regions were involved. Data were collected through the National Comprehensive Reference Centers for SCD. The study was approved by the Ethics Committee of Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico of Milan, Italy, the coordinating center of the study.
The inclusion and exclusion criteria were, respectively, the presence or absence of a biochemical and/or genetic diagnosis of SCD and the capability or not to give informed consent. Written informed consent was obtained from all included patients after the study aims and protocol had been explained. When necessary, a Cultural Linguistic Mediator could be engaged to assist in the consent process. Besides demographic data, every center collected data concerning the type and sequence of therapy, acute transfusion therapy (ATR), chronic transfusion therapy (CTR, i.e., regular transfusion regimen for more than 1 year), or HC, the main indications for ACR and CTR, and details on alloimmunization (number and systems of antibodies) over the previous 4 years of treatment.

Statistical Analysis
Descriptive statistics of continuous variables were expressed using medians and interquartile range (IQR: 25th-75th percentile); dichotomous variables were summarized as counts and percentages. The Shapiro-Wilk test was used to test the normality of distribution. As no normal distribution was found, the differences between groups were analyzed with the Wilcoxon signed-rank test. Two-or multiple-proportion comparisons between variables were performed with the Z-test. All tests were two-sided, and p-values < 0.05 were considered as statistically significant. Data management and graphs were created with Excel (Microsoft, Seattle, WA, USA), and statistical analysis was performed using R version 3.5.1 for Windows (R Core Team, Vienna, Austria).

SCD Italian Patients Cluster Into Two Overall Distinct Groups: Children of African Descent and Adults of Caucasian Descent
Our study shows that Italian SCD patients overall cluster into two distinct groups: children of African and adults of Caucasian descent ( Figure 1A). We collected data from 1,579 patients, of whom 802 were male (51%) and 777 were female (49%) with a median age of 23 (IQR 10-41) years (Supplementary Figure S1). No significant difference in age was found between males and females (p = 0.18); the median age was 21.5 (IQR 10-40) years in males and 25.3 (IQR 10-42) years in females. Ethnicity was equally distributed between Caucasians (47.9%) and Africans (48.7%), with a minority of African American (2.8%) and Asian (0.4%) SCD patients. Ethnicity was not defined for 0.2% of patients (Table 1). In African patients, the most common genotype was homozygosity for HbS (SS; 71.3%). Compound heterozygosity with β-thalassemia mutations was 2.3% for S/β0thalassemia, 3.9% for S/β+-thalassemia, and 20.8% for SC disease. Otherwise, the most prevalent genotype in Caucasians was S/β-thalassemia (74.5%; in particular, S/β 0 -thalassemia 34.8% and S/β + -thalassemia 39.7%). SS genotype was present in 21.4% and SC in 0.5% of SCD patients of Caucasian descent. The median age of African SCD patients was 12 (IQR 7-22) years compared with 38 (IQR 25-49) years in Caucasian patients (p < 0.001).

Combined Transfusion Approaches With Hydroxycarbamide Were Used to Limit HC-Resistant Sickle Cell-Related Clinical Manifestations
Full data on therapeutic management of SCD patients were collected for 1,364 out of the 1,579 patients (Table 2; Figure 1B; Supplementary Figure S1). The following therapeutic regimens were identified: (i) HC: chronic hydroxycarbamide therapy for more than 1 year; (ii) CTR: chronic transfusion regimen; (iii) ATR: acute transfusion approach; and (iv) no treatment (Figure 2A). When we considered therapeutic strategies in function of age, we found that blood transfusion approaches were used throughout patient's journey ( Figure 2B). A total of 292 patients (21.4%) were on CTR, 196 (14.4%) on ATR, and 497 on HC (36.4%), of whom 100 needed transfusions for acute VOCs. Three hundred and seventy-nine (27.8%) patients never received long-term therapy. In both ATR and CTR clinical setting, donor-recipient exact matching for serological ABO, Rhesus and Kell antigen for RBC compatibility was carried out according to international and Italian guidelines, released in 2014 (9,13,(24)(25)(26)(27).
For 250 out of 1,364 patients (18.3%), it was possible to follow the temporal sequence of change in therapy (   4.6 years (IQR 2.2-11.1 years). This was due to the severity of sickle cell-related clinical presentation, which was only partially controlled by HC therapy. The main indications for CTR in these 60 patients were, respectively, VOCs (number of events: 33), anemia (n = 25), ACS (n = 13). HC-resistant pain (n = 6), splenic sequestration (n = 4), primary (n = 2) or secondary (n = 3) stroke prevention. Combined therapy (CTR+HC) was started at a median age of 20.  (Figure 2A). Two out of 1,364 patients with SCD displayed hyperhemolysis reaction after RBC transfusion (1 African SS child: 5 years of age; 1 African SS adult: 23 years of age). Collectively, our data indicate that acute and chronic transfusion approaches were chosen to limit disease progression in patients of both Caucasian and African descent. RBCs from Caucasian donors were used. The donor-recipient exact matching for ABO, Rhesus and Kell antigen for RBC compatibility strategy limits alloimmunization, which might further complicate clinical management of patients with SCD (9,18,19,28).

Low Immunization Events Characterize the Italian Multi-Ethnic Cohort of SCD Patients
In our study, alloimmunization was documented in 135 (8.5%) out of 1,579 patients with SCD. Sixty-one of these 135 patients (45.2%) were already alloimmunized at their arrival at the comprehensive SCD center. Four of them (6.6%) developed alloimmunization against new RBC antigen (Supplementary Figure S3A) 36 of these 135 patients (27%) developed alloantibodies after they arrived at the Italian referral center (Supplementary Figure S3A). Data on alloantibody status in relation to the timing of arrival at the SCD center were not available for the other 38 alloimmunized SCD patients.
Fifty-one alloimmunized patients were African [median age 20 years (IQR 11-28 years)], corresponding to 6.6% of the African group; 78 patients were Caucasian [median age 41 years (IQR 31-51 years)], representing 10.3% of the Caucasian group; five patients were African American [median age 35 years (IQR 30-37 years)] and for one patient (age 29 years) ethnicity was not reported. Thus, alloimmunization was more frequent in adults than in children with SCD (p < 0.001). The frequency of alloimmunization was similar in female and male patients with SCD.
RBC-specific antibodies were detected in 124 patients, of whom 70 (41 males, 29 females) had a single antibody and 43 (16 males and 27 females) had multiple antibodies (59.5%, 28.6, 9.5, and 2.4%, respectively, with two, three, four, and six antibodies). Specific antibodies were not identified in 11 patients (3 males and 8 females) (Supplementary Figure S3C). A higher proportion of Caucasian patients had multiple antibodies compared with patients of other ethnicities. In the Caucasian group, a single antibody was prevalently found in males (24/40, 60%), while there were no significant differences between genders in the African group. When genotypes were considered, we observed alloimmunization to be more frequently associated with SS genotype in both African and African American patients, whereas SS and Sβ 0/+ genotypes were equally associated with alloimmunization in SCD patients of Caucasian descent (Supplementary Figure S3B).
Eleven patients (8.1%) with alloimmunization developed a delayed hemolytic transfusion reaction (DHTR) (8 females and 3 males) with a median age of 39 years (IQR: 18.5-49). Six of these patients had SS genotype (4 Africans, 2 Caucasians), and five were Caucasian with Sβ genotype. In 4 out of the 11 patients, a single antibody (D, C, Cw, Jsb) was detected, and in another 4 of the 11 (all females, 3 Caucasians, and 1 African) multiple antibodies (2-4 different specificities) were detected. In 2 patients, antibodies were not clearly identified. Taken together, these data indicate that low alloimmunization events recorded in both African and Caucasian patients might most likely be related to the donorrecipient exact matching for ABO, Rhesus and Kell antigen for RBC compatibility.

DISCUSSION
Our study highlights the complexity of the clinical management of SCD manifestations throughout the patients' journey. The peculiar multi-ethnic profile of the Italian SCD population, with a roughly equal proportion of Caucasian and African patients, is an added value to the analysis of real-life data. In our series, Caucasian SCD patients were generally older than African patients. This is most likely related to the efficacy of the prenatal program for hemoglobinopathies that started in Italy in the 1980s (29,30). In contrast, African SCD patients were mainly clustered in pediatric and young adult populations, reflecting the voluntary migration fluxes of the last two decades (22,23,31). Indeed, the presence of elderly Caucasian SCD patients represents a unique SCD cohort, providing important information on the combinatory effects of aging and SCD on disease natural history (32,33).
In our study, the transfusion approach was similar in SS and S/β patients in terms of ATR and CTR regimens, and the percentage of patients with S/β+-and S/β • who were treated with ATR and CTR was similar. Both CTR and ATR were used more frequently in SS patients compared with either S/β+ or S/β •patients (34). The main reasons for ATR were similar to those described by Rees et al. (35).
Notably, we found that SS African patients were treated with ATR more than Caucasian patients with S/β genotypes. This might be related to (i) patients' low social status; (ii) patients' limited access to family practitioners, generally related to troubles in speaking Italian. All these factors might contribute to the higher access to an emergency department for African SCD patients during acute events when compared to Caucasian SCD subjects (10).
Simple transfusions were the main transfusion strategy used during CTR, and single top-up transfusions were the main one in the acute setting. Manual and automatic exchange transfusions were used more in CTR than in ATR. This might be related to: (i) difficulties in vein access during VOCs; (ii) absence of expertise; (iii) overnight acute events (36,37).
Of note, we found a subset of patients still symptomatic for acute SCD-related clinical manifestations under treatment with HC, highlighting the biocomplexity of SCD that lies beneath red cell sickling and dehydration (38)(39)(40)(41)(42)(43)(44). Indeed, HC partially acts on sickle cell-related inflammatory vasculopathy and chronic inflammation (4,(45)(46)(47). Thus, SCD patients considered partial responders to HC require a more intensive treatment, which has until now been represented by the combination of HC and chronic transfusion programs. In our cohort, combination therapy of CTR and HC was administered in a similar percentage of African and Caucasian SS patients. The percentage of SS patients who required long-term combination treatment was higher than the proportion with S/β • -S/β+-genotypes. Considering chronic therapies (CTR and HC), we confirmed that SC patients needed less intensive treatment than other SCD genotypes (14). Thus, our data highlight the presence of a subset of SCD patients with severe clinical manifestations resistant to HC treatment. These might now be eligible for new US Food and Drug Administration and European Medicines Agency therapeutic options such as crizanlizumab, a humanized antibody against P-selectin, or voxelotor, a novel oral anti-sickling agent, alone or in combination with HC (41,42,(48)(49)(50)(51)(52).
In our cohort, alloimmunization was identified in SCD patients aged between 18 and 40 years; those aged <18 years had a FIGURE 3 | Number of children/adults in SCD patients by antibody system. The pie charts report the overall distribution of the specific antibodies for children and adults. Data are shown as column chart that reports, for each system and differentiating for children/adults, the number of African, Caucasian and African-American patients with the specific antibody.
lower alloimmunization events than those aged ≥18 years, most likely because they had a shorter transfusion history. A higher proportion of Caucasian patients (10.3%) than African patients (6.6%) had alloimmunization, and among these, Caucasian patients again were older than African patients (median 41 vs. 20 years).
Although the alloimmunization rate was not available due to the study's retrospective design, the low alloimmunization events in both African and Caucasian patients, might reflect the satisfactory level of donor-recipient exact matching for ABO Rhesus and Kell antigen for RBC compatibility, consistent with the Italian guidelines for children and adults released on 2014 (11,15). Antigen mismatch between donor and recipient is the basis for antibody formation, as the recipient recognizes those antigens as non-self and, thereby, an immune response might be elicited (53,54). Transfused SCD patients have the highest rates of RBC alloimmunization over their lifetimes (up to 40-50%), compared to fewer than 5% of other transfused individuals with either thalassemia major or myelodysplasia (55,56). Detection of alloantibodies may make locating compatible RBC units for future transfusion difficult, delayed, and sometimes impossible (54). Potential reasons for alloimmunization include (i) number of donor exposures; (ii) antigenic mismatches between donor RBCs and recipient RBCs related to ethnicity (mainly between Caucasian donors and African descent recipients); (iii) the inflammatory state of recipients (57). SCD is characterized by a chronic inflammatory state, worsening during acute VOCs (57,58). Thus, SCD patients transfused in their baseline state of health are thought to be less likely to become alloimmunized than patients transfused in a state of inflammation, such as during acute VOCs (58). Studies in SCD patients have shown that having an acute chest syndrome at the time of transfusion is a significant risk factor for becoming alloimmunized (53,58). In our cohort, the antibodies identified were mainly of RhCDE and Kell systems, as reported in studies of transfused SCD patients from the United States and Brazil (9). Different antibodies systems (i.e., MNS and Kidd) and different frequencies of alloimmunization have been recently described in SCD patients in French Guiana when compared to our cohort or those from the United States or Brazil (18). These might reflect the heterogeneity of donor and recipient populations in different geographical settings, which increases the complexity of data analysis and comparison.
Our study presents some limitations: (i) the retrospective design of the study, which did not allow us to evaluate the alloimmunization rate; (ii) lacking data on patients' transfusion history before their arrival to the comprehensive centers for hemoglobinopathies; (iii) no record on patients' mortality.
In conclusion, our study highlights that transfusion regimens are still crucial as intensive treatments for both acute and chronic SCD-related complications. In our cohort, HC was offered to symptomatic SCD children in agreement with national recommendations (10,15). This clearly favors a more extensive use of transfusion approaches in the pediatric setting in our cohort in case of acute complications. Since alloimmunizations remains a barrier for safe and effective transfusion of patients with SCD, a careful evaluation of RBC transfusion guidelines transfusion becomes a key factor, along with the implementation of transfusion protocols (such as phenotypically-matched RBCs: RhCDE and Kell), genotyping of patients and donors to identify RBC antigenic variants and maintenance of records on patient transfusion history (59,60). In addition, dissemination of the knowledge on management of DHTR in patients with SCD, might positively impact patients' outcome (61). These measures are expected to reduce the occurrence of alloimmunization as well as the incidence of delayed hemolytic transfusion reactions, optimizing the management of SCD subjects of different descent throughout the patients' journey.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics Committee of Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico of Milan, Italy. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

AUTHOR CONTRIBUTIONS
GG, LDF, and GLF contributed to the conceptualization and design of the study, acquisition and curation of the data, contributed to data analysis and interpretation, writing, critical appraisal and comments, and reviewing and editing. LS, DV, NM, PB, AV, MC, GL, and VV contributed to the conceptualization and design of the study, acquisition and curation of the data, and critical appraisal and comments.

FUNDING
Financial support for Medical Editorial Assistance was provided by Novartis Farma SpA. Novartis Farma SpA was not involved in the study design, collection, analysis, interpretation of data, and the writing of this article or the decision to submit it for publication.

ACKNOWLEDGMENTS
The authors thank Ray Hill, an Independent Medical Writer who provided medical writing assistance with this manuscript on behalf of Springer Healthcare, which was funded by Novartis Farma SpA, Italia in accordance with Good Publication Practice (GPP3) guidelines (http://www.ismpp.org/gpp3). Authors had full control of the content and made the final decision for all aspects of this article.