Please cite this article in press as: El Kababi S, et al. Red blood cell alloimmunizations in beta-thalassemia patients in Casablanca/Morocco:
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Original article
Red blood cell alloimmunizations in beta-thalassemia patients in Casablanca/Morocco: Prevalence and risk factors
Les allo-immunisations anti-érythrocytaires chez les malades bêta-thalassémiques à Casablanca/Maroc : prévalence et facteurs de risques
S. El Kababi a , M. Benajiba b , B. El Khalfi a , J. Hachim c,d , A. Soukri a,∗
aLaboratoryofPhysiopathology,GeneticsMolecularandBiotechnology(PGMB),FacultyofSciencesAinChock,ResearchCenterHealthandBiotechnology, UniversityHassanIIofCasablanca,Km8RouteEljadida,BP5366,Mearif,20100Casablanca,Morocco
bNationalBloodTransfusionCenter,Immuno-hematology,Bab-El-Irfane,ruem’fadel-Cherka,10000Rabat,Morocco
cHematology-Oncologypediatricdepartment,Children’sHospitalAbderrahimHarouchi,IbnRochdUniversityHospitalCenter,Casablanca,Morocco
dFacultyofMedicineandPharmacyofCasablanca,Hassan2University,Tarikbnouziadstreet,20360Casablanca,Morocco
a r t i c l e i n f o
Articlehistory:
Availableonlinexxx
Keywords:
-thalassemia Autoantibodies Frequency
Regularbloodtransfusion Alloimmunization
a b s t r a c t
Redbloodcellalloimmunizationisoneofthemajorchallengestoregulartransfusionsin-thalassemic patients.InMorocco,rarestudieshavefocusedonthishemoglobinopathy.
Objective.– Weaimedtostudytheprevalence andriskfactorsofred cellalloimmunizationin- thalassemicpatients.
Patientsandmethods.–Retrospectivestudyduring9years (2009–2018)was conductedon 160- thalassemicpatientstransfusedregularlyinpediatricdepartmentofchildren’shospitalinCasablanca, Morocco.Themainclinical,demographicandtransfusionalcharacteristicsofalloimmunizedandnon- alloimmunizedpatients werecompared. Red blood cellsunits transfused wereleukodeplatedand phenotypicallymatchedforRH-KELLsystemsandforothersystemsafterimmunization.Screeningand antibodyidentificationwereperformedbygel-filtrationmethodonBIO-RADcasedsusing3and11red bloodcellspanels.Todetectautoantibodies,autocontrolanddirectantiglobulintestswerecarriedout usingpolyspecificcoombs(IgG/C3d)gelcards.
Results.–Theprevalenceofalloimmunizationswas8.75%duringthestudyperiod.Seventeenalloanti- bodiesidentifiedweredirectedmainlyagainstantigensofKELLandRHsystems:KEL1(35.29%),RH3 (23.52%),RH1(11.76%),Kpa(11.76%),RH2(5.88%).Redbloodcellsautoantibodieshadbeendetectedin 6of14(42.85%)ofalloimmunizedpatientsversus12of146(11.76%)ofnon-alloimmunizedpatients (P<0.01).Presenceofautoantibodies,transfusionalinterval<3weeksandgenderwereassociatedwith highrateofredcellsalloimmunization.
Conclusion.–Thisstudyprovesthedataofliterature.Thepresenceofredcellautoantibodiesappearstobe amajorriskfactorsforalloimmunizationinthalassemicchildren,andcouldadvocatespecifictransfusion guidelines.
©2019Soci ´et ´efranc¸aisedetransfusionsanguine(SFTS).PublishedbyElsevierMassonSAS.Allrights reserved.
Motsclés: Bêta-thalassémie Auto-anticorps Lafréquence
Transfusionsanguinerégulière Allo-immunisation
r é s u m é
L’allo-immunisationdesglobulesrougesest l’undesdéfismajeurspourles transfusionssanguines régulièreschezles patients-thalassémiques.AuMaroc, raressontles étudesfocaliséessur cette hémoglobinopathie.
∗ Correspondingauthor.
E-mailaddress:ab.soukri@gmail.com(A.Soukri).
https://doi.org/10.1016/j.tracli.2019.06.004
1246-7820/©2019Soci ´et ´efranc¸aisedetransfusionsanguine(SFTS).PublishedbyElsevierMassonSAS.Allrightsreserved.
Objectif.–Évaluerlaprévalenceetlesfacteursderisquesdesallo-immunisationsanti-érythrocytaires danslesbêta-thalassémies.
Patientsetméthodes.–Étuderétrospectivedurant9années(2009–2018)aétémenéechez160bêta- thalassémiquestransfusésrégulièrementauservicedepédiatriedel’hôpitald’enfantàCasablanca/Maroc.
Les principales caractéristiques cliniques, démographiques et transfusionnelles des patients allo- immunisésetnon-allo-immunisésontétécomparées.Lesconcentrésdeglobulesrougestransfusésont étédéleucocytésphénotypésdanslessystèmesRH-KELLetdanslesautressystèmesaprèsimmunisation.
Ledépistageetl’identificationdesalloanticorpsontétéréaliséspardestechniquesengel/filtrationsur cartesBIO-RADutilisantdespanelsà3età11hématies.Ladétectiondesautoanticorpsestréaliséeavec unautocontrôleetletestdirecteàl’antiglobulineenutilisantlecoombspolyspecifique(IgG/C3d)sur cartegel.
Résultats.–Laprévalencedesallo-immunisationsétaitde8,75%.Dix-septalloanticorpsidentifiésétaient dirigésprincipalementcontrelesantigènesdessystèmeKELLetRH:KEL1(35,29%),RH3(17,64%),RH1 (11,76%),Kpa(11,76%),RH2(5,88%).Lesautoanticorpsanti-érythrocytairesontétédétectéschez6parmi 14patientsallo-immunisés(42,85%)contre12parmi146(11,76%)patientsnonallo-immunisés(p<0,01).
Laprésencedesautoanticorps,l’intervalletransfusionnels<3semainesetlesexeétaientassociésautaux élevésdesallo-immunisations.
Conclusion.–Cetteétudeprouvelesdonnéesdelalittérature.Laprésencedesautoanticorpsérythrocy- tairessembleêtrelefacteurderisquemajeurd’allo-immunisationchezlesenfantsatteintsdethalassémie etpourraitjustifierdesrecommandationsspécifiquesenmatièredetransfusion.
©2019Soci ´et ´efranc¸aisedetransfusionsanguine(SFTS).Publi ´eparElsevierMassonSAS.Tousdroits r ´eserv ´es.
1. Introduction
Beta-thalassemia is an autosomal recessive hemoglobinopathy, resulting from insufficient production of ß-globin chains [1]. This disorder is characterized by variable severity anemia and various complications that occur in the first life cycle [2].
Currently, the transfusion therapy associated with chelators remains the conventional treatment of beta-thalassemia and has contributed to the reduction of mortality associated with this dis- ease. Despite that improved pre-storage screening methods have resulted in a dramatic decrease in the frequency of transfusion- transmitted infections [3], the development of anti-red blood cell (RBC) antibodies (both allo- and autoantibodies) remains a major problem causing delayed hemolysis, blood transfusion reactions, complicated RBC cross-matching, shortening of transfused cells in vivo survival and delays of provision of safe transfusions [4].
Knowing the factors influencing RBCs alloimmunization is essential in order to understand the challenges of preventing this com- plex phenomenon. The literature reports various frequencies of alloimmunization ranging from 4% to 45% in thalassaemia patients, depending on the homogeneity of the donor–recipient population, RBC phenotype matching policy and age at transfusion initiation.
In Morocco, it is estimated that 5% of population have the beta- thallassemia, which assumed to be a major public health problems [5]. While several measures that have been taken to improve the management of thalassemic patients in our country, there is a paucity data as regarding RBC immunizations in Moroccan tha- lassemic patients.
The primary aim of the current study was to determine and to compare the prevalence of alloimmunization of subjects in a hematology oncology pediatric center (HOPC) of children hospi- tal in Casablanca/Morocco during 9 years (from January 1st, 2009 to December 31st, 2018). We also assessed the potential clini- cal, transfusional and immunohematological risk factors associated with RBC alloimmunistaion.
2. Materialsandmethods
2.1. Patients
This study included all 160 patients with
-thalassemia receiv- ing regular transfusions at the Haematology Oncology Pediatric
Center (HOPC) of the Children’s hospital in Casablanca/Morocco, during 9 years (from January 2009 to December 2018). All these patients were from Casablanca and its sides. The consent was obtained from each subject or their legal guardians before enroll- ment in the study and from Casablanca’s Researches Ethics of the University Hospital Center Ibn Rochd. The clinical, demographic and transfusionnal data were collected from the files of the patients in HOPC and the immunohematological data from the registers at the Casablanca blood transfusion center (CBTC). All thalassemic patients benefit from an immunohematological follow-up in the context of the systematic surveillance of polytransfused subjects at (CBTC), including:
•
blood grouping ABO, Rhesus (1, 2, 3, 4, 5), and KEL1. Before the first transfusion, an extended erythrocyte phenotype (Fya, Fyb, Jka, Jkb, S and s) was made;
•
research for irregular antibody (RIA) before and after every trans- fusion (8 to 15 days) in order to reveal antibodies with a low titer;
•
direct antiglobulin testing (DAT) is not done systematically but is necessary for each positive RIA and in case of poor transfusion performance.
Decision to transfuse was taken when the hemoglobin (Hb) level was less than 7 g/dL after exclusion of anemia caused by sepsis or viral infections and when the Hb level was more than 7 g/dL with complications like poor growth, fractures, or facial changes.
The thalassemic patients were transfused according to institu- tional transfusion policy to keep target Hb level 9–11.5 g/dL. All patients received leukodeplated, RBC units that were phenotypi- cally matched for RH1 to RH5, and KEL1. After alloimmunization, patients were supposed to receive RBC units matched for the concerned antigens and for Fya, Fyb, Jka, Jkb, S and s, if
≥2 alloanti- bodies were identified. RBC must be compatibilized (compatibility test-direct matching) before leukodepletion.
3. Methods
3.1.1. Antibody detection and identification
The research for irregular agglutinins (RIA) is performed for any
demand for labile blood products (LBP). Before the first transfusion
Please cite this article in press as: El Kababi S, et al. Red blood cell alloimmunizations in beta-thalassemia patients in Casablanca/Morocco:
Fig.1. Frequenciesofalloimmunizedandnon-alloimmunizedpatientsbyrangeageduringstudyperiod.
RIA was performed in saline medium at 22
◦C (for irregular natural antibodies screening) and at 37
◦C. Red cell antibody screening and identification were carried out by indirect Coombs test – hemag- glutination in gel/filtration on BIO-RAD cards using commercial cell panels of known antigen constituent: 3 cell panels for screen- ing (Diacell, Bio-rad) and extended panel of 11 cells (ID-DiaPanel panel, Bio-RAD) for identification stage and completed when nec- essary with a 11-cell enzyme-treated panel (papain and/or trypsin).
Both tests have been performed by the same procedure. Fifty micro- liters of test cell reagent was pipette to the microtube. Then, 25
L the plasma sample was added to the microtube of the ID card. ID- Card has been incubated at 37
◦C for 15 minutes in ID-incubator.
The card was centrifuged for 10 minutes in the ID-Centrifuge. Anti- bodies screening results are interpreted as a positive or negative based on the presence or absence of hemagglutination. The plasma sample gave a positive result with “ID-Diacell”, it was tested with
“DI-DiaPanel”. Positive result indicates the presence of irregular antibodies directed to the antigens on the test cells. The specificity of antibodies was obtained when the reaction pattern of the sample against DiaPanel cells was interpreted according to the antibody identification tables that provided by the manufacturer. Internal quality controls for screening are provided by Diagast (sera CQI).
The search for autoantibodies was performed by direct Coombs test in gel card containing polyspecific (anti-IgG/C3d) and specifics antihuman globulin.
3.1.2. Extended phenotyping
The extended phenotype was carried out manually according to the manufacturer’s recommendations (gel/filtration or tubes). It was performed before the first transfusion and if the transfusion episode was more than 4 months for immunized patients.
Internal quality controls, were selected from the identification panel to validate each antigen.
3.1.3. Risk factors for alloimmunization
The following criteria were studied and compared between alloimmunized and non-alloimmunized patients: clinical and demographic records, allo- and autoantibodies, splenic sequestra- tion, age at first transfusion and interval transfusion.
3.1.4. Statistical analysis
Results were analyzed statistically with IBM SPSS statistics ver- sion 23 (property of SPSS 2010, Inc., IBM Company) software.
Patients with and without allo-alloantibodies were compared to find significant differences for relevant variables. T test was used for continuous variables and Chi
2test for nominal variables.
P-values < 0.05 were considered statistically significant.
4. Results
4.1. Patient characteristics
The 160 thalassemic patients enrolled in the HOPC between 2009 and 2018, were divided between 88 females (55%) and 72 males (45%). The sex ratio was 1.22 in favor of the women. The mean
±SD age for all patients was 10.22
±5.301 (range: 1–25) (Fig. 1). Age at the start of diagnosis was ranging from 3 months to 4 years.
The diagnosis of thalassemia was confirmed by standard hemoglobin electrophoresis.
Molecular biology techniques were not used. A total of 115 (71.87%) patients were diagnosed as thalassemic major (TM) and 45 patients (28.13%) were thalassemic intermediate (TI). Age at initiation transfusion ranged between 6 months and 4 years.
In accordance with current guidelines, RH1 to RH5 and KEL1 antigens are respected to as far as possible in all transfusion patients. Especially, in case of positive RIA or presence of clin- ical or biological sign of hemolysis (transfusional innefficiency, hemoglobinuria
...), the transfusion was insured by compatible expanded phenotype RBCs in following systems (Duffy, Kidd and MNS). RBC must be compatibilized (compatibility test-direct matching) before leukodepletion. RIA is prescribed for any patient likely to be transfused and for post-transfusion follow-up.
The cross-match was realized for all transfused units using indi-
rect antiglobuline test. Blood group O (50%) was the most common
in thalassemia patients, followed by both group A, B (20%) and AB
(10%). The Rhesus phenotypes DCcee, Dccee DCCee and DccEe were
most predominant, with the respective frequencies of 27.63, 19.73,
17.76 and 15.78% (Table 1).
Table1
FrequencyofRhesusphenotypesinthalassemiapatients.
Phénotype Effective Frequency(%)
DCcee 52 27.63
Dccee 30 19.73
DCCee 27 17.76
DccEe 20 15.78
ddccee 13 9.86
DCcEe 07 5.96
ddCcee 05 3.28
4.2. RBCalloantibodies
Data regarding alloantibody persistence and specificity were collected between the BTC of Casablanca registry and the HOP Cen- ter at Casablanca Children’s Hospital. During 9 years from 2009 to 2018, the red cell alloantibodies were reported in 14 of 160 (8.75%) patients, among them 35.71% (n = 5) were males and 64.29% (n = 09) were females. The mean
±SD age of patients who developed red cell alloantibody was 12
±4.11. A total of 16 alloantibodies were reported in 14 patients. The most frequently identified alloanti- bodies were directed against following antigens: KEL 1 (in 6 cases) (35.29%) RH 3 (in 4 cases) (23.52%), RH1 (in 2 cases) (11.76%), Kpa (in 2 cases) (11,76%), RH2 (in 1 cases) (5.88%), following by MNS1 (in one cases) (5.88%) and Jka (in one cases) (5.88%) antigens (Table 2).
Among the 14 alloimmunized patients, 12 (85.71%) had a single RBC alloantibody, 1 (7.14%) had 2 alloantibodies and 1 (7.14%) had 3 alloantibodies. Anti-KEL1 antibody was identified single in 5 sub- jects and associated in 1 subjects, anti-RH1 antibody was identified single in children who have 11years old and associated in girl who have 10 years old.
Among 14 patients who reported having alloantibodies, 7 (50%) were already alloimmunized (single antibody) before at the time of beginning this study (Table 3).
Two patients of them formed additional antibodies. Ten alloan- tibodies appeared during the study period: 3 anti-KEL 1, 3 anti-RH3, 1 anti-RH1, 1 anti-RH2, 1 anti-jka and 1 anti-Kpa.
The comparison of prevalence of alloimmunizations between 2009 and 2018 is shown in Fig. 2 and Table 3. From January 1st, 2009 to December 31st, 2012, a total of 80 thalassemia patients were reported and diagnosed, among them 7 patients were already alloimmunized against a single RBC antigen, (prevalence of alloim- munization was 10.4%). Two new cases of alloimmunization were detected during 2011–2012 (11.25%). The incidence of alloimmu- nization was 2.5. From January 1st 2013 to December 31st 2018, the number of patients increased to 160, among them 14 cases of alloimmunizations were recorded in total. The prevalence and
incidence of alloimmunization decreased to P = 8.75% and IC = 0.6 at the end of the year 2018.
4.2.1. RBCs Autoantibodies
Six patients (42.85%) among 14 alloimmunized patients had autoantibodies. Patients who were RBC alloimmunized were more likely to had a RBC autoantibody, alloimmunized 6/14 (42.85%) vs.
non-alloimmunized 12/146 (8.21%), P < 0.001.
4.2.2. Risk factors
The clinical and immuno-haematological features for the 14 patients with proven RBC alloimmunization were compared to those for the 146 non-alloimmunized patients including gen- der and age of patients, age at transfusion initiation, transfusion interval < 3 weeks and > 3 weeks, splenectomy sequestration and presence of autoantibodies (Table 4).
On univariate analysis, statistically significant risk factors for alloimmunization were the presence of red cell autoantibodies (42.85% vs. 8.21%; P < 0.001), transfusion interval < 3 weeks (71.43%
vs. 35.61%; P = 0.01) and gender (64.29% of females vs. 35.71% of males in non-alloimmunized patients).
5. Discussion
In our study, which is the first study on red cell alloimmu- nization in patients with thalassemia in Casablanca/Morocco, the overall prevalence of RBC alloimmunization during 9 years was 8.75%. Worldwide, several studies have reported the variable fre- quencies of alloimmunization but in general, the specificities of the antibodies detected were comparable in the different studies.
The main risk factors for alloimmunization in our cohort were the presence of red cell autoantibodies.
The alloimmunization was associated with gender and interval between transfusion. The rate was higher in the group receiving transfusion at intervals < 3 weeks than patients transfused at inter- val > 3 weeks. Age at data analysis, and age at first transfusion were not significantly different for alloimmunized compared with non- alloimmunized patients.
5.1. Prevalence of alloimmunization
The literature reports various frequencies of alloimmunization ranging from 4% to 45% in thalassaemia patients, depending on the homogeneity of the donor–recipient population, age at trans- fusion initiation, RBC phenotype matching policy, the resources available in a particular country for antibody detection and vary- ing blood collection. Studies originating in Europe have reported
Table2
RBCalloantibodyspecificityinalloimmunizedthalassemicpatients.
Bloodgroupsystem Antibodiesspecificities Numberofalloantibodiesspecificities
Total Frequencyofeachspecificities% Singlealloantibodies Associatedalloantibodies RH
Anti-E 3 17.64 1 2
Anti-C 1 5.88 1 –
Anti-D 2 11.76 1 1
KELL
Anti-K 6 35.29 5 1
Anti-Kpa 2 11.76 1 1
Kidd
Anti-jka 1 5.88 1 –
MNSs
Anti-M 1 5.88 1 –
17 11 5
RBC:redbloodcell.
Please cite this article in press as: El Kababi S, et al. Red blood cell alloimmunizations in beta-thalassemia patients in Casablanca/Morocco:
Table3
RBCalloimmunizationnotificationsinoncologyhematologypediatricdepartmentfrom2009to2018.
2009–2010 2011–2012 2013–2014 2015–2016 2017–2018
RBCAbs T.Pts RBCAbs T.Pts RBCAbs T.Pts RBCAbs T.Pts RBCAbs T.Pts
Numberofreports 7 67 9 80 11 10 13 142 14 160
Prevalence 10.4 11.25 11 9.1 8.75
Incidence 2.9 2.5 1.8 1.4 0.6
RBC:redbloodcell;Abs:antibodies;T.Pts:totalpatients.
Fig.2.Variationofnumberofbeta-thalassemicpatientsandalloantibodyprevalencefrom2009to2018.
Table4
Comparisonofalloimmunizedandnon-alloimmunizedthalassemiapatients.
Alloimmunizedpatients(n=14) Non-alloimmunizedpatients(n=146) P-value
Sex Males5 (35.71%) Males67 (45.89%) P=0.01
Females9 (64.29%) Females79 (54.11%)
Age(years)mean±sd 12.27±2.57 10.31±4.38 NS
Minimum 7 1
Maximum 19 25
Ageatinitiationoftransfusion(months)Mean±sd 35.84±29.74 25.76±28.46 NS
Transfusioninterval
<3weeks 10(71.28%) 52(35.61%) P=0.01
>3weeks 4(28.57%) 94(64.39%)
Splenectomy 2/14(14.28%) 3/146(2.05%) NS
Presenceofautoantibodies 6/14(42.85%) 12/146(8.22%) P<0.01
NS:nonsignificant.
alloimmunization rates of about 5% to 25%, [6] whereas in the Mid- dle East and India rates of 4% to 30% have been observed [7,8].
In the present study, we reported a decreased rate and inci- dence (IC) of RBC alloimmunisations in thalassemia patients during 9 years from 2009 to 2018.
The prevalence and the incidence at the beginning of the study from 2009 until 2018, were respectively P = 10.4%, IC = 2.9 and P = 8.75% and IC = 0.6. The relatively decrease the frequency of alloimmunization is probably due to the respect processing, and matching techniques/protocols (transfusion by leukodepleted blood matched for RRH1 to RH5 and Kel1antigens. A study
conducted by Spanos et al. [6] showed a 3.7% rate of RBC alloimmu- nization recipients of RBCs matched at ABO, RH, and KEL, compared with a 22.6% rate in recipients receiving only ABO-matched and RH1-matched RBCs.
Unlike patients with thalassemia, patients with sickle cell dis-
ease (SCD) often not begin transfusion therapy early in life and
having rates of alloimmunisation ranging from 2.6% to 65% [4]. As
thalassemia patients, RBC alloimmunisation rate in SCD patients is
influenced by homogenity and the extent of RBC antigen match-
ing between recipients and donors. In the United Kingdom (UK),
United States (US) and Kuwait, rates reported for alloimmunization
in patients with SCD are 18–65.45% with ABO and D matching alone [8–10], 5–11% with additional limited phenotype matching for C, E, and K antigens [9]. However, in Jamaica and Uganda, alloimmu- nization rates in patients with SCD are lower, at 2.6–6.1% with ABO and D matching alone. T he lower incidence of RBC sensitization in these reports is probably influenced by homogeneity between recipients and donors of African origin, compared to the UK and US where donors of African descent represent a minority [11].
In the case of other pathologies, various studies have shown that the risk of anti-erythrocyte alloimmunization is higher in patients with autoimmune diseases. In patients with autoimmune haemolytic anemia who had been transfused, the frequency of RBCs alloantibodies was ranged from 32 to 75% in many studies [9].
Finally, data on an increased risk of anti-erythrocyte alloimmu- nization in certain diseases, such as cirrhosis, acute myeloblastic leukemia [12], are contradictory. Studies have shown that dia- betes, neoplasia, is a risk factor for alloimmunization, while lymphoproliferative diseases and atherosclerosis “protect” from alloimmunization [13].
5.2. Antigen specificity
Regarding the alloantibody specificity, anti-KEL1 (35.29% of the alloantibodies) was the first most common antibody, following by anti-RH3 (17.64%), anti-RH1 (11.76%) and anti-Kpa (11.76). The least prevalent alloantibodies were anti-RH2, anti-Jka and anti-M, each being detected in just one case (5.88%). Likewise in European, Middle Eastern, and Indian studies, the most common antibodies detected were directed against KELL and RH antigens [14,15]. The same result was reported by viguensky [16], also Hoeltge et al. [17]
who counted 23.0% of anti-KEL1, 17.6% of anti-RH3 and 12.4% of anti-RH1 among 6996 antibodies in 4700 patients. Whilst Chinese patients with thalassemia are also likely to have antibodies directed against antigens in the Miltenberger family [18].
The anti-D alloantibodies was detected in two RH3-negative children. The reason for this antibodies development might be the fact that these patients were transfused by partial or weak D blood bags. This situation reflects the potential interest of performing red cell genotyping for blood donors [19]. This suggestion was con- firmed by study conducted in Morocco by El Housse et al. [20] who assured the alloimmunisation risk in Moroccan recipients of blood phenotyped in routinely as RhD - C/E+, and found Rh D variants as (Weak D type Del in 6.35%, partial D in 3.1%, and 3.1% (2/63) of gene variants whose type is not yet determined. The frequency of vari- ant RhD in individuals of African descent appears to be higher than in European populations, but is not well documented. RHD vari- ants may cause reduced expression of D antigen, termed weak D, altered D epitopes, termed partial D, or weak partial D expression [21]. Anti-Kpa (anti-KEL3) was present in 1 of 160 patients (0.62%), but this alloantibody was not considered dangerous and did not justify specific transfusion guidelines.
We found two alloantibody combinations (14.28% of the alloan- tibodies) both included the anti-RH3: a combination of two alloantibodies 6.25% anti- (E + D), and a combination of three anti- bodies 6.25% (E + K + kpa). The same result was reported by Achargui et al. [22] in Blood Transfusional Regional Center (BTRC) in Rabat including 425 various patients and pregnant women. They revealed that (15%) of immunized subjects developed a combination of alloantibodies. Whilst 84.4% have developed 2 alloantibodies, 14%
3 alloantibodies and 1.6% developed 4 alloantibodies.
The most immunogenic systems involved in alloimmunization was the RH and kell system in Our study and were in accordance with most reported studies, [18–21,23]. These findings are not unique to patients with thalassemia, similar results were reported in patients with SCD. Vichinsky et al. [9] reported that antibod- ies most prevalent in patients with SCD are those against antigens
of Kell and RH blood group. They found 68 RBC alloantibodies in 107 SCD patients whose anti-KEL 1 was found in 18 cases (26%), anti-RH3 in 16 cases (24%), anti-RH2in 11 cases (11%). In another Brazilian study in 108 sickle cell patients, Dias Zanette et al. [10]
found 56 patients with antibodies (51%). Anti-RH3 specificity was detected in 39.3% of patients, anti-RH2 specificity in 16.1% and anti- KEL1 specificity in 21.4% [24].
As regards alloantibody associations, Rosse et al. [10] showed in one study that 45% of immunized patients had developed a single RBCs antibody, while 17% of immunized patients had four or more anti-erythrocyte antibodies.
In this 9-year retrospective study, 7 new patients have developed single alloantibodies and 2 cases among the already immunized patients before the beginning of the study, have devel- oped additional alloantibodies. This shows that regular exposure to transfusion may increase the risk to develop additional antibodies.
The study of alloantibodies, produced by the patients of this cohort, showed that 14 negative antigenic patients produced the corresponding antibody. Therefore, some of them have escaped at one time or another for reasons of shortage or urgency, to the basic transfusion instruction (RH/KEL phenocompatibility) or these anti- bodies could have appeared after transfusion of an RBC unit from a donor mistyped as antigen (corresponding) – negative, as may occur for some very weak antigen variants. This situation reflects the potential interest of performing red cell genotyping for blood donors. Therefore, in Morocco a laboratory of molecular genetics is in the process of being established in both BTRC in Casablanca and BTNC in Rabat. Also, promoting the strategy of targeted blood dona- tion to apprehend needs in compatible units. An Egyptian study reported a lower RBC alloimmunization rates (8.3%) in patients with thalassemia receiving blood from a limited number of donors, compared to those who are not in a limited donor program (21.6%) [25].
5.3. Auto-immunisation
Our findings showed higher levels of autoantibody production among alloimmunized patients (46.2% vs. 8.21%; P < 0.001). This result agrees with other literature data and suggests the presence of red cell autoantibodies as a major risk factor for alloimmunization in both thalassemia and SCD patients [12,26–28]. The pathogenesis of autoimmunization following transfusions is not well understood.
Some authors suggested that the binding of alloantibodies to the transfused erythrocytes could lead to conformational changes in antigenic epitopes, which would stimulate production of autoan- tibodies and specially in splenectomized patients [29]. Another possible explanation would be that Genetic determination may be involved in autoantibody formation, as stated by Ofosu et al. in their study of the major histocompatibility complex in SCD patients [30].
Patients with autoantibodies may have a higher transfusion rate and often require immunosuppressive drugs. In addition to that, these autoantibodies can lead to reduction of the life span of trans- fused RBCs and, therefore, lead to accelerated hemolysis. Presence of autoantibodies in alloimmunized patients interfere in the pre- transfusion testing and have a hidden effect on alloantibodies and engender a panagglutination problems, leading to non-detection of alloantibodies.
5.4. Risk factors for alloimmunization
In order to develop strategies to mitigate RBC alloimmuniza-
tion, factors influencing the development of RBC alloantibodies
must be considered. According to Olivier Garraud [31], these factors
are linked with donor characteristics (antigens vary in immuno-
genicity), BC characteristics (pre-storage, leukoreduction, age of
blood, transfusion Immuno Modulation (TRIM)) and recipients
Please cite this article in press as: El Kababi S, et al. Red blood cell alloimmunizations in beta-thalassemia patients in Casablanca/Morocco:
characteristics (genetic susceptibility, causal disease and associ- ated treatment).
5.4.1. Donor factors
Differences in alloimmunization are often attributed to the eth- nic or racial disparity of the donor recipient population. The relative homogeneity of donor and recipient populations in India [32], Iran [33] and Pakistan [34] contribute to lower alloimmunization rates, which were respectively (8.6%), (5.3%) and (6.48%) for transfused thalassemic patients compared to more heterogeneous popula- tions, such as Arab (30%) [8], USA and UK (20.8%) [32,35], and USA Asians (20.8%) [12]. In a French study conducted by Noisette et al. [36], they have shown that, in sickle cell patients living in metropolitan France, the risk of immunization has increased because of the ethnic polymorphism of the blood group anti- gens, between donors (95% are Caucasian origin) and recipients (African-Caribbean origin). This hypothesis seems to be valid in our homogenous population and may be the reason of low (moderate) rate of alloimmunization in our study.
5.4.2. RBC phenotype matching policy
RBC alloimmunization rate is influenced by the extent of RBC antigen matching between recipients and donors. In the United States, to partly account for ethnic differences between donors and recipients, the use of RHCE and Kell negative RBC units has led to a reduction in the number of alloantibodies formed and to lower alloimmunization rates in SCD patients [37]. However, even with this matching alloimmunization rates are still higher than those seen in Uganda and Jamaica (6.10 and 2.60%, respec- tively) where donors and recipients are ethnically similar and in the case of the former even after limiting RBC matching to just ABO and RH1 [11]. Furthermore, a recent study about 182 SCD patients transfused from American African donors, have shown that despite phenotype matching for these antigens, RBC alloim- munization of SCD patients was reduced but not prevented [38]
and RH genotyping revealed variant alleles in 87% of individuals. In thalassemia, there are limited data on the impact of RBC phenotype matching policy on alloimmunization rates [12]. However, in a mul- ticentre study, sites that routinely performed extended matching did not have significantly different alloimmunization rates com- pared to those institutions that practice standard matching for ABO and RH1 antigens [35]. Some Authors argue that these results were due to the extreme diversity of Rh genes, and that molecu- lar genotyping might be necessary for better RBC matching [21,38].
Our data (decreased rate of alloimmunization during study period) support the contention that transfusion of RH (RH1 to RH5) and Kell-matched red cells, has significantly reduced the immunization rate in patients with multiply transfused thalassemia.
5.4.3. Host factors
5.4.3.1. Age at initiation transfusion. Younger age at the time of ini- tial RBC exposure correlates in some studies with a lower likelihood of alloimmunization [16,39]. In our cohort, statistically, age at first transfusion did not appear to be a significant risk factor for alloim- munization (mean age at start transfusion was 35.84
±29.74 vs.
25.76
±28.46 in non-alloimmunized patients).
This non-significance may be due to the small number of alloimmunized group. The immunologic mechanisms behind these observations are not fully understood, although tolerance to RBC antigens has been reported to occur in a murine model of RBC alloimmunization [40]. Several hypotheses have been evoked to explain these findings [26,29–33,41]:
•
a lower ability to produce antibodies in young children due to immunological immaturity;
•
induction of tolerance to erythrocyte antigens by repeated early transfusions.
These findings are not unique to patients with thalassemia, with similar trends reported in patients with SCD. However, a study con- ducted by Benamor et al. [26] about thalassemic and SCD patients, support these findings. They reported a lower rate of alloimmuniza- tion in thalassemic patients whose mean age is lower than that of SCD patients despite a higher consumption of red blood cell con- centrates. In another study in the United States, 29% of transfused SCD children developed alloantibodies versus 47% of SCD adults [42]. However, some studies have not found a significant differ- ence between age at transfusion initiation and alloimmunization [28].
5.4.3.2. Gender of recipients. Although female gender has been identified as a risk factor for alloimmunization in the general pop- ulation [43,44], studies of thalassaemia conducted by Thompson [35] and Dainasoury [25] do not reported a higher rates in women.
The causes are not fully understood. In our study, alloimmuniza- tion frequency was higher in females (sex ratio was 1.22 in favor to women). The authors assumed that the predominance of female subjects in alloimmunized individuals can be explained by the antigenic stimulation due to fetal red cells during pregnancy, but perhaps also to other unknown factors until today. In our children cohort, this result may be due to the already high number of females compared to males.
5.4.3.3. Transfusion interval. We reported significant correlation between interval of transfusion and alloantibody formation. The same result was found in other study who stated that alloim- munization rate was found to be less in the group receiving transfusion at intervals > to 3 weeks than those received transfusion at interval < to 3 weeks [14]. Therefore a greater exposure to RBC units may result in higher RBC alloimmunisation [45]. Schonewille et al. reported in study, which has been conducted on 653 non- chronically transfused patients, that 33.4% of patients produced additional red cell antibodies after repeat transfusion events [46].
5.4.3.4. Transfusion immunomodulatory.
5.4.3.4.1. Splenectomy. Splenectomy, which is typically per- formed in thalassaemia patients to alleviate hypersplenism or stabilize transfusion requirements [47], is a significant risk factor for alloimmunization according to Singer et al. [12], Thompson et al.
[35], El Danassoury et al. [25]. The mechanism by which removal of the spleen increases alloantibody formation is not clear. Shih Ching et al. [48] showed when the spleen is absent or removed, immune responses are altered. It has been postulated that post- splenectomy conformational changes in RBC membranes enhance immunomodulation that result in allosensitization [12]. Another study showed that although splenectomy is performed less fre- quently in thalassaemia [35], the significantly increased risk of alloimmunization in splenectomized younger patients is concern- ing and deserves further study. A Contradictory study performed by Koc¸ yi˘git et al. suggesting that splenectomy did not affect the incidence of alloimmunization [14]. In our study, the number of splenectomized patients was not significant. Only five patients in total were splenectomized and only 2 of the 14 alloimmunized patients underwent splenectomy. It could be assumed that non- splenectomy of our patients has contributed to the non-increased of alloimmunization in thalassemic children.
5.4.3.4.2. Leucocyte reduction. The immunomodulatory effect
of transfusion is more apparent with the use of non-leukoreduced
blood components. Several authors, advocate using pre-storage
leukoreduced blood which, apart from their beneficial effect on
the reduction of alloimmunization, reduce the transfusion reac- tions, transfusion-refractory states of platelets and transmission of infectious agents [12,43,49]. However, It is estimated that even in settings where blood components are pre-storage leukoreduced to less than 106 residual cells, nearly 10% of sickle cell disease patients become immunized to RBC antigens other than RH1 and nearly 30–50% when BCs are not leukoreduced [3,49].
The transfusion of leukodepleted blood in all our patients could have also contributed to the decrease rate of alloimmunization.
However, the precise mechanism and the host factors that mediate sensitization are not clear. CD4+ regulatory T cells (Tregs), which are prime regulators of immune responses, had reduced activity in alloimmunized thalassaemia patients compared to other chron- ically transfused patients who remain non-alloimmunized [50].
This suggests that the identification of molecular markers of hosts that predict a propensity to form alloantibodies could be useful in designing strategies to mitigate this risk [4].
Recently a new paradigm in alloimmunization has been rec- ognized. Immunized patients were no longer ascribed a status of
“good responders” but rather as “good presenters”, as their HLA receptors preferentially present certain blood group antigen epi- topes [31].
Previous studies have showed that the HLA II genotype of the patient is a key predictor of a person’s response to RBC antigens and is likely to influence predisposition to the RBC antibody responder status [51]. A study conducted by Noizat-Pirenne et al. [52] showed that RBC alloantibodies against Fya antigen was strongly associated with the DRB1 04 and DRB1 15 alleles. Compared to Fya, the ery- throcyte KEL antigen is highly immunogenic, probably because the potential KEL antigen–derived peptides can bind to multiple HLA molecules, as indicated by the wide variety of HLA II phenotypes found in individuals producing anti-KEL.
6. Conclusion
The prevalence of RBC alloimmunization was relatively low among the thalassemia patients followed in pediatric center of children’s hospital in Casablanca. However, more efforts need to be made to control autoimmunization and alloimmunization in thalassemia patients. Further improvements will require the imple- mentation of:
•
targeted blood donation strategy to overcome the deficiency of compatible units;
•
systematic red cell genotyping in thalassemia patients and donors;
•
a careful transfusion strategy for those with red cell autoanti- bodie.
Disclosureofinterest
The authors declare that they have no competing interest.
Acknowledgment
The authors would like to thank Dr Kamal Bouisk for his inter- vention to providing us the statistics and immunohematological registers at the regional blood transfusion Center in Casablanca.
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