Predictors of Red Cell Alloimmunization in Multitransfused Egyptian Patients With [beta]-Thalassemia.
There are 25 centers for thalassemia and sickle cell disease. These centers are affiliated with universities (primarily Cairo University and Ain Shams University, Egypt), with the Ministry of Health, and with the Egyptian Red Crescent.
Three thousand patients with thalassemia are treated in the hematology center at Cairo University Hospitals. Patients are transfused when their hemoglobin level drops below 8 g/dL (reference range, 14.0-17.5 g/dL).
Lifelong and frequent red blood cell transfusions remain the main treatment for severe cases. Development of red blood cell (RBC) alloantibodies and autoantibodies can complicate transfusion therapy. Some alloantibodies are hemolytic and may cause hemolytic transfusion reactions and limit the availability of compatible RBC units for transfusions. Others are clinically insignificant. (1) Development of RBC autoantibodies is rare but can result in clinical hemolysis and in cross-matching problems. (2) Approaches for prevention and treatment of alloimmunization are matters of debate and range from the provision of RBCs matched for all major antigens to blood matched only for RBC alloantibodies that have already developed. The cost-benefit ratio of these strategies is still being debated because many alloantibodies are clinically insignificant. (3) The aim of this work was to determine the prevalence of alloimmunization in multitransfused Egyptian patients with thalassemia. An additional goal was to the determine risk factors that might influence alloimmunization with the hope that this study could help minimize some of the transfusion-associated risks in those patients.
MATERIALS AND METHODS
Records of 272 patients (male, n = 134, 49.3%; females, n = 138, 50.7%) with [beta]-thalassemia who were receiving regular blood transfusions, matched for ABO-Rh(D) were analyzed. Data included age, sex, blood group, medical history, number of transfusions, and age at first transfusion. Blood was prestorage leukoreduced in 109 patients (40.1%).
The antibody identification was performed using DiaMed-ID microtyping system (DiaMed, Cressier sur Morat, Switzerland). The serum was mixed with saline-suspended RBCs with the addition of low ionic strength saline and incubated at 37 C degrees for 15 minutes. Three reagent cells were used for the antibody screening procedure, and an anti-immunoglobulin G reagent was used for the antiglobulin phase. If the results from the screening procedure were positive, an extended panel of 12 cells with the following antigens was used to identify the antibody: C, c, D, E, e, Lewis (Lea, Leb), Kell (K), Kidd (JKa, JKb), Duffy (Fya, Fyb), M, N, S, s, P1, and Lutheran (Lua, Lub). Autoantibodies were detected by a positive direct Coombs test.
Statistical analyses were performed using SPSS Version 11 (IBM, Armonk, New York). Analyses included descriptive statistics, frequency distribution, mean, and standard deviation calculations. Comparative studies were performed using a paired t test. Potential association between parameters was assessed by performing a correction-matrix study.
Analysis of the Patients
Of the 272 patients in the study, 170 (62.5%) were younger than 12 years, 47 patients (17.3%) were between 12 and 18 years, and 55 patients (20.2%) were older than 18 years. Table 1 provides a summary of the patients' characteristics.
The alloimmunization incidence was 22.8% with 123 alloantibodies detected in 62 patients. Twenty-nine of 62 alloimmunized patients (46.8%) were younger than 12 years. Alloantibodies in different age groups were tabulated (Table 2).
Thirty-one patients were 6 years old or younger, with alloantibodies detected in 8 of those patients (25.8%) (Table 3).
The most common alloantibody was Rh-related (46 of 123; 37.4%), comprising anti-E (18 of 123; 14.6%), anti-D (11 of 123; 8.9%], anti-C (11 of 123; 8.9%), and anti-c (6 of 123; 4.9%), followed by anti-Kell, (32 of 123; 26%), [right arrow] antiMNS (12 of 123; 9.8%), [right arrow] anti-Kidd (11 of 123; 8.9%), [right arrow] anti-Duffy (10 of 123; 8.1%), [right arrow] anti-Le (7 of 123; 5.7%), [right arrow] anti-Lu (3 of 123; 2.4%), and [right arrow] anti-P1 (2 of 123; 1.6%).
The frequency of Kell and Rh alloantibodies was equal in children younger than 12 years (16 of 48; 33.3% each), followed by anti-Kidd (4 of 48; 8.3%).
Ten patients, accounting for 34.5% of all Rh-negative patients (n = 29), developed anti-D antibodies. Eighty percent (8 of 10 patients) of all anti-D antibodies developed in patients older than 18 years. One Rh-positive patient developed anti-D antibodies. Anti-D alloimmunization increased in O-negative patients (5 of 8; 62%), compared with B-negative (2 of 8; 25%; P = .31) and A-negative (3 of 11; 27%l P = .18), although the difference was not significant. Alloimmunization in the various blood groups was also tabulated (Table 4).
Fifty-three percent (33 of 62 patients) of alloimmunized patients had only one alloantibody (anti-Kell, 48.5% [16 of 33]; followed by anti-E, 24.2% [8 of 33]) (Table 5).
Fourteen of 62 patients (22.6%) had 2 antibodies, and more than 2 alloantibodies were detected in 24.2% (15 of 62) of the patients, with 53.3% (8 of 15) in patients older than 18 years.
Autoantibodies occurred in 1.5% (4 of 272) of all patients. Males had a higher antibody incidence (40 of 134; 29.9%), when compared with females (26 of 138; 18.8%; P = .047).
Alloimmunization incidence was not influenced by the age at which transfusion was started (P = .18), but it increased with the number of units transfused (P = .01).
Patients who had a splenectomy had a higher rate of alloimmunization (40 of 125; 32%), compared with those who did not have a splenectomy (24 of 147; 16.3%; P = .003).
Patients who received unfiltered blood had a higher alloimmunization rate than did those who always received leukoreduced blood (P < .001) (Table 6).
Thalassemia is reported to be common in Mediterranean, Turkey, Iran, India, equatorial or near-equatorial regions of the Arabian Peninsula, and Southeast Asia, especially Thailand, Cambodia, and southern China. (3)
Only a few studies have investigated the frequency and causes of alloimmunization in Egyptians. In the present work, we examined the prevalence and the common alloantibodies in multitransfused Egyptian patients with thalassemia. We also evaluated the risk factors that affected the alloimmunization incidence.
We demonstrated an alloimmunization incidence rate of 22.8%, with 123 alloantibodies detected in 62 patients. Egyptians have a large racial mix within the population. The high rate of alloimmunization demonstrated in our study is probably caused by the antigenic difference between donors' RBCs and the recipient's blood. Similar rates of alloimmunization were found among Asian population (22% and 20.8%). (1) The high incidence reported in the Singer et al (1) study was attributed to RBC antigenic difference of blood donors being white, and most of the recipients being Asians. Lower alloimmunization rates of 4.9% to 10% were reported in thalassemia patients in countries with more homogenous populations, in Greece, Italy, Iran, Pakistan, and India. (4-10)
The immunomodulatory role of white blood cells in transfused blood could have contributed to the high incidence of alloantibodies observed in our group of patients. Leukocytes in blood components may cause an enhanced B-cell function that may result in increased alloimmunization to red blood cell antigens. (11) Sixty percent of our patients were exposed to nonleukoreduced blood.
Some studies have demonstrated a significant effect of leukoreduction in multitransfused patients, showing lower alloantibody incidence rates, whereas others demonstrated no significance. (1,12)
Our patients who received unfiltered blood had a higher alloimmunization rate than did those who always received leukocyte-reduced blood. The standard of leukoreduction in Egypt is fewer than 5 X [10.sup.6]/[micro]L donor white blood cells.
An overall normal CD4:CD8 ratio was noted previously (13) in thalassemia patients transfused with poststorage leukoreduced blood. However, a marked, absolute lymphocytosis, mostly driven by an increase in B lymphocytes, predominantly in splenectomized patients, was reported with both nonleukoreduced and leukoreduced blood. (14) Lymphocytosis is associated with an increase in the serum immunoglobulins, immune complexes, and cells bearing surface immunoglobulins, which are the result of the immunomodulatory effect of blood transfusion, the recipient's immunologic condition, and splenectomy. (15) In our study, patients who had a splenectomy had a higher alloimmunization rate. Splenectomy may further enhance the immune response to the transfused foreign antigens that are not filtered efficiently. (1)
Many Egyptian patients with thalassemia require splenectomy. Optimizing the clinical and transfusion strategies, early in life, may help delay or even eliminate the need for splenectomy. Chou and colleagues (16) found a significantly increased risk of alloimmunization in young splenectomized patients, which merits consideration and deserves further studies.
In an Iranian study, (6) however, there was no significant difference in the frequency of splenectomy between alloimmunized and nonalloimmunized patients.
We also found that the incidence of alloimmunization was not influenced by the age at which transfusions were started. Similar results were noted in an Iranian study. (6) Singer and coworkers, (1) however, found that transfusion at ages younger than 1 to 3 years may have offered some immune tolerance and protection against alloimmunization in thalassemia patients. Female gender has been known as a risk factor for alloimmunization. (17) However, our results did not demonstrate higher immunization rates in females; in fact, males had a higher alloimmunization rate in our study. Similar data have been observed previously. (18-20) In our study, alloimmunization increased with the number of units transfused. The number of units transfused is an important predictor of alloimmunization in patients who receive longterm transfusion support, including patients with sickle cell disease. (20-22)
Our data demonstrated that the most frequent alloantibodies were anti-Rh (37.4%; 46 of 123), and anti Kell (26%; 32 of 123). Similarly, Ameen and colleagues (18) showed rates of red blood cell alloimmunization of 30% among Arabs, and most antibodies were against antigens in the Kell and Rh systems. In most Western countries, the most common alloantibodies in thalassemia patients are directed against C, E, and Kell antigens. (20) Anti-D alloimmunization was also frequently reported, even though most patients receive units cross-matched for ABO and Rh(D) blood groups world wide. (16)
It is not mandatory in Egypt to test apparently Rh(D) negative donors for weak D.
The automated gel method for blood group serology was introduced to the Cairo University Blood Bank 2 years ago. Since then, Rh(D) typing has been performed with the gel technique in parallel with the immediate spin tube test.
The high incidence of anti-D antibodies in Rh-negative patients observed in our study is likely related to transfusions of units with weak D antigens. Therefore, all Rh-negative units of blood should be retested during antihuman globulin phase to rule out that weaker reaction. According to the AABB standards, weak-D testing is mandatory in blood donors but is not required when testing a patient or a pregnant woman. (23)
A high incidence of anti-D antibodies has been reported in individuals of mixed ethnicity because of the common occurrence of D variants. Hussein and Teruya (24) demonstrated an anti-D antibody incidence of 63.5% in Egyptian Rh-negative children with thalassemia who received units for which weak D was not tested.
In our study, one Rh-positive patient developed anti-D antibodies. Genotyping of blood groups has simplified the identification of different D variants. Anti-D alloimmunization has been documented for patients with partial D and weak D types 4.0, 4.2 (DAR), 11, and 15. (25,26) Patients with weak D phenotype should be typed as D-negative, although that is a matter of debate. The most frequent weak D types 1, 2, and 3 can safely receive D-positive blood without complications. (25)
There are few reports on the effect of phenotypematching strategy on the incidence rates of alloimmunization in thalassemia. Currently some centers routinely provide extended antigen-matched units. (20) Matching RBCs was shown to be effective in some studies. (1,27) However, in a multicenter study, institutions that performed extended matching routinely did not have significant differences in alloimmunization rates. (20)
The costs and feasibility of providing antigen-matched blood may limit the implementation of that approach in our center. Providing blood matched only for Rh and Kell antigens can reduce alloimmunization by 63.5% and achieve economic benefits. Transfusion support using a limited number of blood donors for a designated patient has been proposed previously as an effective approach in patients with thalassemia. (19)
Transfusing matched blood only for RBC alloantibodies that have already been developed would reduce costs and increase the feasibility of providing matched blood.
In summary, the rate of alloimmunization in our group of multitransfused patients with thalassemia was quite high. Our data suggest that the number of transfusions, RBC antigenic differences between the donor and recipient, recipient splenectomy, and component leukoreduction influence RBC alloimmunization.
Transfusion of leukoreduced, phenotypically matched cells for selective antigens may help reduce expenses and risks of alloimmunization in those patients.
(1.) Singer ST, Wu V, Mignacca R, Kuypers FA, Morel P, Vichinsky EP. Alloimmunization and erythrocyte autoimmunization in transfusion dependent thalassemia patients of predominantly Asian descent. Blood. 2000; 96(10):3369-3373.
(2.) Cianciulli P, Sorrentino F, Morino L, et al. Radiotherapy combined with erythropoietin for the treatment of extramedullary hematopoiesis in an alloimmunized patient with thalassemia intermedia. Ann Hematol. 1996; 72(6): 379-381.
(3.) Barrai L, Rosito A, Cappellozza G, et al. [beta]-Thalassemia in the Po Delta: selection, geography, and population structure. Am J Hum Genet. 1984; 36(5): 1121-1134.
(4.) Blumberg N, Ross K, Avila E, Peck K. Should chronic transfusions be matched for antigens other than ABO and Rh (D). Vox Sang. 1984; 47(3):205-208.
(5.) Walker RH, Lin DT, Hartrick MB. Alloimmunization following blood transfusion. Arch Pathol Lab Med. 1989; 113(3):254-261.
(6.) Karimi M, Nikrooz P, Kashef S, Jamalian N, Davatolhagh Z. RBC alloimmunization in blood transfusion dependent B thalassemia patients in southern Iran. Int J Lab Hematol. 2007; 29(5):321-626.
(7.) Bhatti FA, Salamat N, Madeem A, Shabbir N. Red cell immunization in [beta]-thalassemia major. J Coll Physicians Surg Pak. 2004; 14(11):657-560.
(8.) Bilwani F, Kakepoto GN, Adil SN, Usman M, Hassan F, Khurshid M. Frequency of irregular red cell alloantibodies in patients with thalassemia major: a bicenter study. J Pak Med Assoc. 2005; 55(12):563-565.
(9.) Azarkeivan A, Ansari S, Ahmadi MH, et al. Blood transfusion and alloimmunization in patients with thalassemia: multicenter study. Pediatr Hematol Oncol. 201128(6):479-485.
(10.) Gupta R, Singh DK, Singh B, Rusia U. Alloimmunization to red cells in thalassemics: emerging problem and future strategies. Transfus Apher Sci. 2011; 45(2):167-170.
(11.) Lannan KL, Sahler J, Spinelli SL, Phipps RP, Blumberg N. Transfusion immunomodulation--the case for leukoreduced and (perhaps) washed transfusions. Blood Cells Mol Dis. 2013; 50(1):61-68.
(12.) Wang LY, Liang DC, Liu HC, et al. Alloimmunization among patients with transfusion- dependent thalassemia in Taiwan. Transfus Med. 2006; 16(3):200-203.
(13.) Grady RW, Akbar AN, Giardina PJ, Hilgartner MW, de Sousa M. Disproportionate lymphoid cell subsets in thalassemia major: the relative contributions of transfusion and splenectomy. Br J Haematol. 1985; 59(4):713-724.
(14.) Hodge G, Lloyd JV, Hodge S, Story C, Han P. Functional lymphocyte immunophenotypes observed in thalassaemia and haemophilia patients receiving current blood product preparations. Br J Haematol. 1999; 10(3):817-825.
(15.) Smit Sibinga CT. Immune effects of blood transfusion. Curr Opin Hematol. 1999; 6(6):442-445.
(16.) Chou ST, Liem RI, Thompson AA. Challenges of alloimmunization in patients with haemoglobinopathies. Br J Haematol. 2012; 159(4):394-404.
(17.) Bauer MP, Wiersum-Osselton J, Schipperus M, Vandenbroucke JP, Briet, E. Clinical predictors of alloimmunization after red blood cell transfusion. Transfusion. 2007; 47(11):2066-2071.
(18.) Ameen R, Al-Shemmari S, Al-Humood S, Chowdhury RI, Al-Eyaadi O, Al-Bashir A. RBC alloimmunization and autoimmunization among transfusion dependent Arab thalassemia patients. Transfusion. 2003; 43(11):1604-1610.
(19.) el-Danasoury AS, Eissa DG, Abdo RM, Elalfy MS. Red blood cell alloimmunization in transfusion-dependent Egyptian patients with thalassemia in a limited donor exposure program. Transfusion. 2012; 52(1):43-47.
(20.) Thompson AA, Cunningham MJ, Singer ST, et al; Thalassemia Clinical Research Network Investigators. Red cell alloimmunization in a diverse population of transfused patients with thalassaemia. Br J Haematol. 2011; 153(1):121-128.
(21.) Fluit CR, Kunst VA, Drenthe-Schonk AM. Incidence of red cell antibodies after multiple blood transfusion. Transfusion. 1990; 30(6):532-535.
(22.) Rosse WF, Gallagher D, Kinney TR, et al. Transfusion and alloimmunization in sickle cell disease: the cooperative study of sickle cell disease. Blood. 1990; 76(7):1431-143 7.
(23.) Carson TH, ed. Standards for Blood Banks and Transfusion Services. 28th ed. Bethesda, MD: AABB; 2012.
(24.) Hussein E, Teruya J. Weak D types in the Egyptian population. Am J Clin Pathol. 2013; 139(6):806-811.
(25.) Cruz BR, Chiba AK, Moritz E, Bordin JO. RHD alleles in Brazilian blood donors with weak D or D-negative phenotypes. Transfus Med. 2012; 22(2):84-89.
(26.) Felgel WA. http://www.ncbi.nlm.nih.gov/pubmed/17053462 How I manage donors and patients with a weak D phenotype. Curr Opin Hematol. 2006; 13(6):476-483.
(27.) Hmida S, Mojaat N, Maamar M, Bejaoui M, Mediouni M, Boukef K. Red cell alloantibodies in patients with haemoglobinopathies. Nouv Rev Fr Hematol. 1994; 36(5):363-366.
Eiman Hussein, MD; Nermeen Desooky, MD; Abeer Rihan, MD; Abeer Kamal, MD
Accepted for publication June 13, 2013.
From the Cairo University Blood Bank, Clinical Pathology Department, Cairo University, Cairo, Egypt.
The authors have no relevant financial interest in the products or companies described in this article.
Reprints: Eiman Hussein, MD, 6 Hussein Elezaby St, Higazy St Cairo Alexandria Desert Road, Cairo, Egypt (e-mail: eimanhussein@ ymail.com).
Table 1. Patient Characteristics Age, No. (%) Sex, No. (%) < 12 y 12-18 y > 18 y M F Patients, 170 47 55 134 138 n = 272 (62.5) (17.3) (20.2) (49.3) (50.7) Splenectomy, Thalassemia, No. (%) No. (%) Yes No Major Intermedia Patients, 125 147 207 65 n = 272 (46.0) (54.0) (76.1) (23.9) Transfusion Frequency, No. (%) Monthly 2 wk Weekly Patients, 182 60 30 n = 272 (66.9) (22.1) (11.0) Table 2. Alloimmunization by Age Group Age y Alloantibodies, Alloimmunized Patients, n = 123, No. (%) n = 62, No. (%) < 12 48 (39.0) 29 (46.8) 12-18 32 (26.0) 11 (17.7) > 18 43 (35.0) 22 (35.5) Age y Alloimmunized Patients Type of by Age Group, Alloantibody, No. (%) No. (%) n = 170 n = 48 < 12 29 (17.1) Rh, 16 (33.3) Kell, 16 (33.3) Kidd, 4 (8.3) Le, 4 (8.3) MNS, 4 (8.3) Fy, 2 (4.2) Lu, 1 (2.1) P1, 1 (2.1) n = 47 n = 32 12-18 11 (23.4) Rh, 12 (37.5) Fy, 7 (21.9) Kell, 4 (12.5) MNS, 4 (12.5) Kidd, 3 (9.4) Le, 1 (3.1) Lu, 1 (3.1) n = 55 n = 43 > 18 22 (40.0) Rh, 18 (41.9) Kell, 12 (27.9) Kidd, 4 (9.3) MNS, 4 (9.3) Le, 2 (4.7) Fy, 1 (2.3) Lu, 1 (2.3) P1, 1 (2.3) Table 3. Alloimmunization in Children Younger Than 6 years Alloantibodies, Patients, Type of No. No. (%) Alloantibodies, No. (%) 1 5 (62) Kell, 4 (80) Le, 1 (20) 2 2 (25) S and M, 1 (50) C and Le, 1 (50) 4 1 (12) P1, Le, Kell, and Kidd, 1 (100) Table 4. Alloimmunization Incidence by Blood Group Blood Patients With Alloimmunized Group Thalassemia, Patients, No. (%) n = 272, No. (%) A+ 82 (30.1) 17 (20.7) O+ 79 (29.0) 21 (26.6) B+ 66 (24.3) 10 (15.2) AB+ 16 (5.9) 4 (25.0) A- 11 (4.0) 3 (27.3) O- 8 (2.9) 5 (62.5) B- 8 (2.9) 2 (25.0) AB- 2 (0.7) 00 (0.0) Table 5. Single-Alloantibody Incidence Rate by Age Group Age y Single- < 12 12-18 > 18 Type of Alloantibody Alloantibody, Alloimmunized No. (%) Cases, No. (%) 33 (53) 19 (58) 10 (30) 4 (12) Anti-Kell, 16 (48.5) Anti-E, 8 (24) Anti-D, 3 (9) Anti-Fya, 2 (6) Anti-Le, 2 (6) Anti-c, 1 (3) Anti-Kidd, 1 (3) Table 6. Alloimmunization Incidence in Patients Receiving Leukoreduced and Nonleukoreduced Blood Type of Blood Patients, Alloimmunized Received n = 272, Patients, n = 62, No. (%) No. (%) Always leukoreduced 109 (40.1) 12 (19.4) Not always leukoreduced 163 (59.9) 50 (80.6)