Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=yhem20 Hematology ISSN: (Print) 1607-8454 (Online) Journal homepage: https://www.tandfonline.com/loi/yhem20 RBC alloimmunization and double alloantibodies in thalassemic patients Azita Azarkeivan, Mohammad Hossein Ahmadi, Sima Zolfaghari, Mojgan Shaiegan, Shirin Ferdowsi, Negar Rezaei & Parvin Lotfi To cite this article: Azita Azarkeivan, Mohammad Hossein Ahmadi, Sima Zolfaghari, Mojgan Shaiegan, Shirin Ferdowsi, Negar Rezaei & Parvin Lotfi (2015) RBC alloimmunization and double alloantibodies in thalassemic patients, Hematology, 20:4, 223-227, DOI: 10.1179/1607845414Y.0000000189 To link to this article: https://doi.org/10.1179/1607845414Y.0000000189 Published online: 17 Aug 2014. Submit your article to this journal Article views: 785 View related articles View Crossmark data Citing articles: 1 View citing articles
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untitledFull Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=yhem20
Hematology
RBC alloimmunization and double alloantibodies in thalassemic patients
Azita Azarkeivan, Mohammad Hossein Ahmadi, Sima Zolfaghari, Mojgan Shaiegan, Shirin Ferdowsi, Negar Rezaei & Parvin Lotfi
To cite this article: Azita Azarkeivan, Mohammad Hossein Ahmadi, Sima Zolfaghari, Mojgan Shaiegan, Shirin Ferdowsi, Negar Rezaei & Parvin Lotfi (2015) RBC alloimmunization and double alloantibodies in thalassemic patients, Hematology, 20:4, 223-227, DOI: 10.1179/1607845414Y.0000000189
To link to this article: https://doi.org/10.1179/1607845414Y.0000000189
Published online: 17 Aug 2014.
Submit your article to this journal
Article views: 785
View related articles
View Crossmark data
1Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Thalassemia Clinic, Tehran, Iran, 2Hematology, Ghazvin Medical University, Ghazvin, Iran, 3Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran, 4Immunology Lab, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran, 5Hematology, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran, 6Serology Lab, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
Purpose: Alloimmunization is a common consequence of chronic blood transfusion. Double alloantibody production may complicate the condition of such patients especially for finding matched blood. In this study, we evaluated the frequency of alloantibodies in thalassemic patients with previous history of transfusion reactions. Samples and methods: This study was performed on 441 multiply transfused thalassemia patients Antibody screening test was carried out using three cell-panel by gel method. Positive patients were followed up for antibody identification using 11-cell panel. Direct combs’ test was performed to detect auto antibodies. Results: In a total of 441 cases (362 thalassemia major and 79 intermedia), 234 were males (53.1%) and 207 females (46.9%); mean age 22 years, range 3-61 years. Alloimmunization was detected in 50(11.3%) patients, including 37(74%) patients with one alloantibody, 8(16%) with two antibodies, 4(8%) patients with unknown antibodies and one patient (2%) with autoantibody. The most common alloantibodies were anti-Rh antibodies (-E/e/C/c/Cw) (26%), anti-K (28%), anti-D (16%), and anti-Colton (4%). Double antibodies were detected in eight out of 50 patients, including: Anti-D+anti-C (8%), anti-D+anti-E (2%), anti-Kell+anti-D (2%), and anti-Kell+KPa (2%). A significant association was observed between the transfusion reaction history and the alloantibody detection results (p< 0.05). Conclusion: Antibody production against RBC antigens makes hard condition in regular blood transfusion. Double antibodies production may more complicate this situation. Thus, it is advisable to phenotype patients and matches the red cells in multiply transfused thalassemia patients.
Keywords: Antibody screening, Thalassemia, Transfusion reaction, Alloantibody, Double antibody
Introduction Thalassemia is a congenital hemolytic anemia; with chronic blood transfusion.1 Sometimes transfusion is associated with significant clinical risks. Complication associated with blood transfusion therapy may be classified based on time of onset as acute and late transfusion reactions or based on etiol- ogy as immunological and non-immunological. Acute immunological reactions are associated with an immune response to antigens on blood cells or plasma proteins and include acute hemolytic transfu- sion reaction, febrile non-hemolytic transfusion reaction, allergic and anaphylactic, while non-
immunological reactions include transfusion- related sepsis, circulatory overload, and non-immune hemolytic.2–5 Although blood transfusions are lifesa- vers for thalassaemia patients, they may be associated with some complications especially erythrocyte alloimmunization. Some alloantibodies may cause hemolytic transfusion reactions and limits the possi- bility of safe transfusion.6 Results from a number of studies have confirmed various frequencies of alloanti- body and autoantibody formation in multi-transfused patients.7–9 Reported alloimmunization rates ranged from 4 to 50% in thalassaemia.7,8,10 Identification of antigens and transfusion of fully compatible blood may prevent alloimmunisation. The primary aim of this study was to determine the prevalence of alloim- munization in Iranian thalassemic patients. We assessed alloantibody and autoantibody rates and the
Correspondence to: Azarkeivan Azita, Pediatric Hematology Oncology, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran. Email: [email protected]
© W. S. Maney & Son Ltd 2015 DOI 10.1179/1607845414Y.0000000189 Hematology 2015 VOL. 20 NO. 4 223
effect of splenectomy status, transfusion initiation age, and transfusion reactions.
Materials and methods This descriptive study was conducted in Adult Thalassemia Clinic related to the Iranian Blood Transfusion Organization and Qazvin thalassemia clinic. Written consent was provided for each patient. Clinical transfusion records of 441 thalassemic patients who fulfilled the criteria were analyzed for the presence of alloimmunization and autoimmuniza- tion. Age, history of splenectomy, and transfusion reactions were also recorded. The whole blood sample was taken in ethylene diamine tetra acetic acid (EDTA) tubes. Antibody screening test was carried out using three-cell-panel (ID-Dia cell 1, 2, 3; ID-Dia cell 1P, 2P, 3P) by gel method (Dia Med Company, Deputy of Rontgen Company, Iran) based on standard instructions; the result of agglutination and the pattern of reaction were recorded. Antibody identification test was performed for positive samples by gel method (Panel cell and Extra panel cell) accord- ing to the manufacturer instructions. The test was repeated in cases with double (mixed) alloantibodies. The results were analyzed using the χ2 test with SPSS 16.0. P values of less than 0.05 were considered statisti- cally significant.
Results This study was performed on 441 multiply transfused thalassemic patients (234 male (53.1%) and 207 female (46.9%); mean age 22 years, range 3–61 years). There were 362 (82.1%) patients with thalasse- mia major and 79 (17.9%) with thalassemia intermdia (According to International Thalassemia Federation, thalassemia major if the patient started transfusion protocol before 2 years old, and thalassemia interme- dia if transfusion started after 2 years old.) (Table 1). Two hundred and twenty-two (50.3%) of the thalas-
saemia major patients in this analysis had been sple- nectomized. The average age at which splenectomy occurred was 12.11± 7.6 years (Table 2). The mean age of starting transfusion in thalassemia
major and intermedia patients was 43.08± 73.51 months and the mean of blood receiving time was 3.5 years (Table 3). Three hundred and fifty-six
(80.7%) patients were receiving regular blood transfu- sion at an interval of 3–4 weeks, whereas 85 (19.3%) patients were under alternative treatments such as hydroxyurea capsules and blood transfusion only for severe anemia. Indeed for thalassemia major patients we had fixed periodic transfusion or regular transfu- sion, but in thalassemia intermedia patients we had transfusion just at time of severe anemia.
Alloimmunization was detected in 50 (11.3%) patients, including 37 patients with thalassemia major and 13 patients with thalassemia intermedia (Table 4). Mean age of patients with alloimmunization was 25.28± 7.77 years and 31 (62%) patients under- went splenectomy (18 patients with thalassemia major and 13 patients with thalassemia intermedia).
In the positive group, 37 (74%) patients had one alloantibody, 4 (8%) patients with unknown anti- bodies, 1 patient (2%) with autoantibody, and 8 (16%) patients showed double antibodies.
The most common alloantibodies were anti-K (in 28% cases), anti-rhesus (Rh) antibodies (D/E/e/C/ c/Cw) in 26%, anti-D in 16%, and anti-Colton in
Table 1 Frequency of patients according to sex and type of thalassemia
Type of thal
TotalMale Female
Major (%) 188 (42.6%) 174 (39.5%) 362 (82.1%) Intermedia (%) 46 (10.4%) 33 (7.5%) 79 (17.9%) Total (%) 234 (53.1%) 207 (46.9%) 441 (100.0%)
Table 2 Frequency of patients according to type of thalassemia and splenectomy
Type of thal
TotalYes No
Major (%) 165 (37.4%) 197 (44.7%) 362 (82.1%) Inter media (%) 57 (12.9%) 22 (5.0%) 79 (17.9%) Total (%) 222 (50.3%) 219 (49.7%) 441 (100.0%)
Table 4 Results of antibody screening by gel method according to type of thalassemia
Antibody screening test (gel method) Major Intermedia Total (%)
Negative (%) 325 (73.7) 66 (15) 391 (88.7) Positive (%) 37 (8.4) 13 (2.9) 50 (11.3) Total (%) 362 (82.1) 79 (17.9) 441 (100)
Table 3 The age and age of start transfusion and age of splenectomy of patients
Age (years)
Age of splenectomy
(years)
No Valid 441 441 222= Yes Missing 0 0 219=No
Mean 22.88 43.08 12.11 Median 22.00 12.00 10.00 Mode 24 12 7 Standard
deviation 9.308 73.512 7.620
Azarkeivan et al. RBC alloimmunization and double alloantibodies in thalassemic patients
Hematology 2015 VOL. 20 NO. 4224
4%. Double antibodies were found in eight of fifty patients, including Anti–D+Anti-C (8%), Anti-D+ Anti-E (2%), Anti-Kell+Anti-D (2%), and Anti- Kell+Anti-KPa (2%). The frequency of antibodies demonstrated in Table 5. Transfusion reaction frequency was 16.6% totally.
Hemolytic complications, allergic symptoms, and febrile reactions during transfusion occurred in 7 (1.6%), 25 (5.7%), and 41 (9.3%) patients, respectively. A considerable association observed between transfu- sion reactions occurrence and alloantibody presence (P= 0.0001). There was no significant association between alloantibody formation and gender, type of thalassemia, regular blood transfusion, and splenect- omy (P> 0.05), (Table 6).
Discussion Alloimmunization, a reaction of the immune system to foreign antigens, is one of the major complications of transfusions, particularly in patients who are
chronically transfused.11–13 In our patients, the pre- transfusion donor vs. recipient matching was targeted for ABO and Rh ‘D’ antigens. All the patients were being transfused a non-leukodepleted blood. In Iran, selection of donors is based on medical and demo- graphic factors and screening tests for blood transfu- sion transmitted infections perform for all blood units.14 It has been recognized that if blood were matched only for ABO and Rh ‘D’ groups, a high rate of alloimmunization would be expected.7 In our study, the frequency of alloimmunization was 11.3% with a significant association between history of trans- fusion reactions and alloantibody development. This study has demonstrated that the most frequent anti- bodies (single or double antibodies) are against Kell and subgroups of Rh, which is similar to other studies.15–17
There are also a few similar studies in Iran. In one study that was performed on 711 thalassemia patients in Shiraz (in southern Iran), 38 (5%) patients had red cell antibodies.18 In a study by Shamsian et al.19 in Tehran (Mofid Children’s Hospital), red cell alloim- munization was found in 7.4% of patients (9 of 121). The blood alloantibodies were anti-k and anti-D. In southern Iran, alloantibodies were observed only among 5.3% of patients.18 In another study, Sadeghian et al.20 indicated that the frequency of alloimmunization in thalassemia patients in northeast Iran is 2.87%. They identified 12 alloantibodies in nine patients that all were against Rh blood group antigens (D, C and E). The most common alloantibodies were Anti-D (88.88%) and followed by Anti-C and Anti-E. Higher frequency of alloimmunization was observed in female, Rh negative and splenectomized patients. Cheng et al.15 reported that 23% (88 of 382) of Chinese thalassemia major patients have RBC antibodies. Anti-E (42, 39.3%), anti-Mia/Mur (33, 30.85%), anti-c (14, 13.1%), and anti-Jka (seven,
Table 5 Frequency of detected alloantibodies and calculation of percent among total and positive patients
Antibody screening test results Frequency Actual percent
Positive (N= 50) Anti Kell 14 28.0 Anti D 8 16.0 Anti E 4 8.0 Anti c 4 8.0 Anti cw 3 6.0 Anti e 1 2.0 Anti D and C 4 8.0 Anti D and E 1 2.0 Anti colb 2 4.0 Anti C 1 2.0 Anti Kell and E 1 2.0 Anti Kell and KPa 1 2.0 Anti Kell and D 1 2.0 Unknown 4 8.0 Auto antibody 1 2.0
Table 6 Statistical analysis and Relation between different variables and their correlation to Antibody screening test results (NS=Not significant)
Antibody screening test result Relating factors Negative results N= 391 (%) Positive results N= 50 (%) P value (correlation)
Sex Male 210 (89.7) 24 (10.3) NS Female 181 (87.4) 26 (12.6) 0.446
Type of thalassemia Major 325 (89.8) 37 (10.2) NS Intermedia 66 (83.5) 13 (16.5) 0.113
Regular transfusion Yes 320 (89.5) 36 (10.1) NS No 71 (83.5) 14 (16.5) 0.097
History of reaction Hemolytic 0 (0) 7 (100) 0.000* Allergic 25 (100) 0 (0) Febrile 34 (82.9) 7 (17.1) No reaction 332 (90.2) 36 (9.8)
Splenectomy Yes 191 (86) 31 (14) NS No 200 (91.3) 19 (8.7) 0.080
*In our analytical method, we analyzed the relation between the transfusion reaction (yes or no) and positive alloantibodies, and was positive correlation. NS, not significant.
Azarkeivan et al. RBC alloimmunization and double alloantibodies in thalassemic patients
Hematology 2015 VOL. 20 NO. 4 225
6.55%) were the commonest antibodies reported. Bhatti et al.21 showed that 4.97% of 161 patients with thalassemia major were alloimmunized. RBC alloantibodies belonged mainly to the Rh system although other antibodies such as anti-K, anti-Jsb, and anti-Jka were detected. There was no definite relation between alloantibodies formation and the age of start blood transfusion, number of blood trans- fusions, and patient’s race. In another study, Ameen et al.,9 found a high frequency of red cell alloimmuni- zation in 57 (30%) of 190 transfusion-dependent Arab thalassemia patients. The most common clinically sig- nificant alloantibodies were against antigens in the Kell and Rh systems. Anti-K in 41 (72%) patients and anti-E in 26 (45.6%) were reported. Autoantibody was detected in 21 (11%) patients. They found the majority of alloimmunized patients formed first alloantibodies between age 2 and 10 years (58%). These researchers believe that performing an accurate compatibility assessment for donor and recipient blood units and using post storage leukode- pleted blood are important factors to reduce alloim- munization in high prevalence alloantibodies among thalassemia patients. In this study, anti-D antibody was detected in 12
patients. One of the reasons for this alloimmunization is transfusion of some red blood cells with rhesus D incompatible with thalassemia patients due to false- negative results in weak D typing of blood donors. Anti-D antibody was also reported in other studies. Sadeghian et al.20 reported high prevalence of Anti- D (88.88%) but in Karimi et al.’s18 study the frequency of Anti-Rh (47.7%) was very common. In this study, double antibodies were detected in 8 of
the 50 patients (16%), including Anti-D+Anti- C(8%), anti-D+Anti-E (2%), anti-Kell+Anti-D (2%), and anti-Kell+KPa (2%). Wang et al.10 ana- lyzed 30 individuals with thalassemia who had regular transfusions for periods ranging from 0·5 to 20 years. Of the 30 patients, 11 (37%) were found to carry alloantibodies. All alloantibodies were clinically significant specificities, including four cases of anti-E, two of anti-E+ c, two of anti-‘Mia’, one of the anti- ‘Mia’+ E, one of anti-D, and one of anti-S. In Bilwani et al.’s22 study, irregular red cell alloantibodies were found in 9.2% (9 of 44). Three (33.3%) patients developed anti-K while two (22.2%) had non-specific antibody. One patient each developed anti-D (11.1%) and anti-E (11.1%). Two had anti-D (11.1%) and anti-C, while the other one (11.1%) developed anti-E and anti-K.
Conclusion Alloimmunization is a common consequence in patients that need persistant blood transfusion. Antibodies against Rh subgroups and Kell groups
are more frequent antibodies in thalassemia patients receiving a chronic blood transfusion. Decreasing the rate of alloantibody production may be possible by the mean of identifying RBC phenotype specially Rh/Kell/Kidd, and Duffy subgroups for each patient before start chronic transfusion, and doing a careful cross match with evaluating the blood group of a donated unit (especially for Rh subgroups and Kell antigens).
Acknowledgements The authors wish to thank Mr Mohammad Hossein Lotfi and Dr Ayoub (from Diamed Company) and Mrs Mehmanchy (Roantgen Company, Iran) for their technical help.
Disclaimer statements Contributors All authors know the roles and agreed with their position on this manuscript.
Funding None.
Ethics approval The Iranian Blood Transfusion Organization Research Committee approved the study.
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2 Prati D. Benefits and complications of regular blood transfusion in patients with beta-thalassaemia major. Vox Sang. 2000;79(3): 129–37.
3 Pahuja S, Pujani M, Gupta SK, Chandra J, Jain M. Alloimmunization and red cell autoimmunization in multitrans- fused thalassemics of Indian origin. Hematology 2010;15(3): 174–7.
4 Goodnough LT. Risks of blood transfusion. Anesthesiol Clin N Am. 2005;23(2):241–52.
5 Rabani A, et al. Clinical evaluation of 413 Thalassemic patients. TUMJ. 2000;58(3):35–41.
6 Pineda A, et al. Trends in the incidence of delayed hemolytic and delayed serologic transfusion reactions. Transfusion 1999;39(10): 1097–103.
7 Spanos T, et al. Red cell alloantibodies in patients with thalasse- mia. Vox Sang. 1990;58(1):50–5.
8 Singer ST, et al.Alloimmunization and erythrocyte autoimmuni- zation in transfusion-dependent thalassemia patients of predomi- nantly Asian descent. Blood 2000;96(10):3369–73.
9 Ameen R, et al. RBC alloimmunization and autoimmunization among transfusion-dependent Arab thalassemia patients. Transfusion 2003;43(11):1604–10.
10 Wang LY, et al. Alloimmunization among patients with transfu- sion-dependent thalassemia in Taiwan. Transfus Med. 2006; 16(3):200–3.
11 Arinsburg S, et al. The significance of a positive DAT in thalas- semia patients. Immunohematology 2010;26(3):87–91.
12 Azarkeivan A, et al. Evaluation of transfusion reactions in tha- lassemic patients referred to the Theran adult thalassemia clinic. J Zanjan Univ of Medival Sci Health Serv. 2008.
13 Walker R, Lin D, Hartrick MB. Alloimmunization following blood transfusion. Arch Pathol Lab Med. 1989; 113(3):254–61.
14 Gharehbaghian A, Abolghasemi H, Namini MT. Status of blood transfusion services in Iran. Asian J Transfus Sci. 2008; 2(1):13.
15 Cheng C, Lee C, Lin C. Clinically significant red blood cell anti- bodies in chronically transfused patients: a survey of Chinese
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thalassemia major patients and literature review. Transfusion 2012;52(10):2220–4.
16 Guirat-Dhouib N, et al. High frequency of autoimmunization among transfusion-dependent Tunisian thalassaemia patients. Transfus Apher Sci. 2011;45(2):199–202.
17 Azarkeivan A, et al. Blood transfusion and alloimmunization in patients with thalassemia: multicenter study. Pediatr Hematol Oncol. 2011;28(6):479–85.
18 Karimi M, et al. RBC alloimmunization in blood transfusion- dependent beta-thalassemia patients in southern Iran. Int J Lab Hematol. 2007;29(5):321–6.
19 Shamsian BS, et al. Frequency of red cell alloimmunization in patients with?-major thalassemia in an Iranian Referral Hospital. Iranian J Pediatr. 2008;18(2):149–53.
20 Sadeghian MH, et al. Alloimmunization among transfusion- dependent thalassemia patients. Asian J Transfus Sci. 2009; 3(2):95.
21 Bhatti FA, et al. Red cell immunization in beta thalassaemia major. JCPSP. 2004;14(11):657–60.
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Azarkeivan et al. RBC alloimmunization and double alloantibodies in thalassemic patients
Hematology 2015 VOL. 20 NO. 4 227
Hematology
RBC alloimmunization and double alloantibodies in thalassemic patients
Azita Azarkeivan, Mohammad Hossein Ahmadi, Sima Zolfaghari, Mojgan Shaiegan, Shirin Ferdowsi, Negar Rezaei & Parvin Lotfi
To cite this article: Azita Azarkeivan, Mohammad Hossein Ahmadi, Sima Zolfaghari, Mojgan Shaiegan, Shirin Ferdowsi, Negar Rezaei & Parvin Lotfi (2015) RBC alloimmunization and double alloantibodies in thalassemic patients, Hematology, 20:4, 223-227, DOI: 10.1179/1607845414Y.0000000189
To link to this article: https://doi.org/10.1179/1607845414Y.0000000189
Published online: 17 Aug 2014.
Submit your article to this journal
Article views: 785
View related articles
View Crossmark data
1Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Thalassemia Clinic, Tehran, Iran, 2Hematology, Ghazvin Medical University, Ghazvin, Iran, 3Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran, 4Immunology Lab, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran, 5Hematology, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran, 6Serology Lab, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
Purpose: Alloimmunization is a common consequence of chronic blood transfusion. Double alloantibody production may complicate the condition of such patients especially for finding matched blood. In this study, we evaluated the frequency of alloantibodies in thalassemic patients with previous history of transfusion reactions. Samples and methods: This study was performed on 441 multiply transfused thalassemia patients Antibody screening test was carried out using three cell-panel by gel method. Positive patients were followed up for antibody identification using 11-cell panel. Direct combs’ test was performed to detect auto antibodies. Results: In a total of 441 cases (362 thalassemia major and 79 intermedia), 234 were males (53.1%) and 207 females (46.9%); mean age 22 years, range 3-61 years. Alloimmunization was detected in 50(11.3%) patients, including 37(74%) patients with one alloantibody, 8(16%) with two antibodies, 4(8%) patients with unknown antibodies and one patient (2%) with autoantibody. The most common alloantibodies were anti-Rh antibodies (-E/e/C/c/Cw) (26%), anti-K (28%), anti-D (16%), and anti-Colton (4%). Double antibodies were detected in eight out of 50 patients, including: Anti-D+anti-C (8%), anti-D+anti-E (2%), anti-Kell+anti-D (2%), and anti-Kell+KPa (2%). A significant association was observed between the transfusion reaction history and the alloantibody detection results (p< 0.05). Conclusion: Antibody production against RBC antigens makes hard condition in regular blood transfusion. Double antibodies production may more complicate this situation. Thus, it is advisable to phenotype patients and matches the red cells in multiply transfused thalassemia patients.
Keywords: Antibody screening, Thalassemia, Transfusion reaction, Alloantibody, Double antibody
Introduction Thalassemia is a congenital hemolytic anemia; with chronic blood transfusion.1 Sometimes transfusion is associated with significant clinical risks. Complication associated with blood transfusion therapy may be classified based on time of onset as acute and late transfusion reactions or based on etiol- ogy as immunological and non-immunological. Acute immunological reactions are associated with an immune response to antigens on blood cells or plasma proteins and include acute hemolytic transfu- sion reaction, febrile non-hemolytic transfusion reaction, allergic and anaphylactic, while non-
immunological reactions include transfusion- related sepsis, circulatory overload, and non-immune hemolytic.2–5 Although blood transfusions are lifesa- vers for thalassaemia patients, they may be associated with some complications especially erythrocyte alloimmunization. Some alloantibodies may cause hemolytic transfusion reactions and limits the possi- bility of safe transfusion.6 Results from a number of studies have confirmed various frequencies of alloanti- body and autoantibody formation in multi-transfused patients.7–9 Reported alloimmunization rates ranged from 4 to 50% in thalassaemia.7,8,10 Identification of antigens and transfusion of fully compatible blood may prevent alloimmunisation. The primary aim of this study was to determine the prevalence of alloim- munization in Iranian thalassemic patients. We assessed alloantibody and autoantibody rates and the
Correspondence to: Azarkeivan Azita, Pediatric Hematology Oncology, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran. Email: [email protected]
© W. S. Maney & Son Ltd 2015 DOI 10.1179/1607845414Y.0000000189 Hematology 2015 VOL. 20 NO. 4 223
effect of splenectomy status, transfusion initiation age, and transfusion reactions.
Materials and methods This descriptive study was conducted in Adult Thalassemia Clinic related to the Iranian Blood Transfusion Organization and Qazvin thalassemia clinic. Written consent was provided for each patient. Clinical transfusion records of 441 thalassemic patients who fulfilled the criteria were analyzed for the presence of alloimmunization and autoimmuniza- tion. Age, history of splenectomy, and transfusion reactions were also recorded. The whole blood sample was taken in ethylene diamine tetra acetic acid (EDTA) tubes. Antibody screening test was carried out using three-cell-panel (ID-Dia cell 1, 2, 3; ID-Dia cell 1P, 2P, 3P) by gel method (Dia Med Company, Deputy of Rontgen Company, Iran) based on standard instructions; the result of agglutination and the pattern of reaction were recorded. Antibody identification test was performed for positive samples by gel method (Panel cell and Extra panel cell) accord- ing to the manufacturer instructions. The test was repeated in cases with double (mixed) alloantibodies. The results were analyzed using the χ2 test with SPSS 16.0. P values of less than 0.05 were considered statisti- cally significant.
Results This study was performed on 441 multiply transfused thalassemic patients (234 male (53.1%) and 207 female (46.9%); mean age 22 years, range 3–61 years). There were 362 (82.1%) patients with thalasse- mia major and 79 (17.9%) with thalassemia intermdia (According to International Thalassemia Federation, thalassemia major if the patient started transfusion protocol before 2 years old, and thalassemia interme- dia if transfusion started after 2 years old.) (Table 1). Two hundred and twenty-two (50.3%) of the thalas-
saemia major patients in this analysis had been sple- nectomized. The average age at which splenectomy occurred was 12.11± 7.6 years (Table 2). The mean age of starting transfusion in thalassemia
major and intermedia patients was 43.08± 73.51 months and the mean of blood receiving time was 3.5 years (Table 3). Three hundred and fifty-six
(80.7%) patients were receiving regular blood transfu- sion at an interval of 3–4 weeks, whereas 85 (19.3%) patients were under alternative treatments such as hydroxyurea capsules and blood transfusion only for severe anemia. Indeed for thalassemia major patients we had fixed periodic transfusion or regular transfu- sion, but in thalassemia intermedia patients we had transfusion just at time of severe anemia.
Alloimmunization was detected in 50 (11.3%) patients, including 37 patients with thalassemia major and 13 patients with thalassemia intermedia (Table 4). Mean age of patients with alloimmunization was 25.28± 7.77 years and 31 (62%) patients under- went splenectomy (18 patients with thalassemia major and 13 patients with thalassemia intermedia).
In the positive group, 37 (74%) patients had one alloantibody, 4 (8%) patients with unknown anti- bodies, 1 patient (2%) with autoantibody, and 8 (16%) patients showed double antibodies.
The most common alloantibodies were anti-K (in 28% cases), anti-rhesus (Rh) antibodies (D/E/e/C/ c/Cw) in 26%, anti-D in 16%, and anti-Colton in
Table 1 Frequency of patients according to sex and type of thalassemia
Type of thal
TotalMale Female
Major (%) 188 (42.6%) 174 (39.5%) 362 (82.1%) Intermedia (%) 46 (10.4%) 33 (7.5%) 79 (17.9%) Total (%) 234 (53.1%) 207 (46.9%) 441 (100.0%)
Table 2 Frequency of patients according to type of thalassemia and splenectomy
Type of thal
TotalYes No
Major (%) 165 (37.4%) 197 (44.7%) 362 (82.1%) Inter media (%) 57 (12.9%) 22 (5.0%) 79 (17.9%) Total (%) 222 (50.3%) 219 (49.7%) 441 (100.0%)
Table 4 Results of antibody screening by gel method according to type of thalassemia
Antibody screening test (gel method) Major Intermedia Total (%)
Negative (%) 325 (73.7) 66 (15) 391 (88.7) Positive (%) 37 (8.4) 13 (2.9) 50 (11.3) Total (%) 362 (82.1) 79 (17.9) 441 (100)
Table 3 The age and age of start transfusion and age of splenectomy of patients
Age (years)
Age of splenectomy
(years)
No Valid 441 441 222= Yes Missing 0 0 219=No
Mean 22.88 43.08 12.11 Median 22.00 12.00 10.00 Mode 24 12 7 Standard
deviation 9.308 73.512 7.620
Azarkeivan et al. RBC alloimmunization and double alloantibodies in thalassemic patients
Hematology 2015 VOL. 20 NO. 4224
4%. Double antibodies were found in eight of fifty patients, including Anti–D+Anti-C (8%), Anti-D+ Anti-E (2%), Anti-Kell+Anti-D (2%), and Anti- Kell+Anti-KPa (2%). The frequency of antibodies demonstrated in Table 5. Transfusion reaction frequency was 16.6% totally.
Hemolytic complications, allergic symptoms, and febrile reactions during transfusion occurred in 7 (1.6%), 25 (5.7%), and 41 (9.3%) patients, respectively. A considerable association observed between transfu- sion reactions occurrence and alloantibody presence (P= 0.0001). There was no significant association between alloantibody formation and gender, type of thalassemia, regular blood transfusion, and splenect- omy (P> 0.05), (Table 6).
Discussion Alloimmunization, a reaction of the immune system to foreign antigens, is one of the major complications of transfusions, particularly in patients who are
chronically transfused.11–13 In our patients, the pre- transfusion donor vs. recipient matching was targeted for ABO and Rh ‘D’ antigens. All the patients were being transfused a non-leukodepleted blood. In Iran, selection of donors is based on medical and demo- graphic factors and screening tests for blood transfu- sion transmitted infections perform for all blood units.14 It has been recognized that if blood were matched only for ABO and Rh ‘D’ groups, a high rate of alloimmunization would be expected.7 In our study, the frequency of alloimmunization was 11.3% with a significant association between history of trans- fusion reactions and alloantibody development. This study has demonstrated that the most frequent anti- bodies (single or double antibodies) are against Kell and subgroups of Rh, which is similar to other studies.15–17
There are also a few similar studies in Iran. In one study that was performed on 711 thalassemia patients in Shiraz (in southern Iran), 38 (5%) patients had red cell antibodies.18 In a study by Shamsian et al.19 in Tehran (Mofid Children’s Hospital), red cell alloim- munization was found in 7.4% of patients (9 of 121). The blood alloantibodies were anti-k and anti-D. In southern Iran, alloantibodies were observed only among 5.3% of patients.18 In another study, Sadeghian et al.20 indicated that the frequency of alloimmunization in thalassemia patients in northeast Iran is 2.87%. They identified 12 alloantibodies in nine patients that all were against Rh blood group antigens (D, C and E). The most common alloantibodies were Anti-D (88.88%) and followed by Anti-C and Anti-E. Higher frequency of alloimmunization was observed in female, Rh negative and splenectomized patients. Cheng et al.15 reported that 23% (88 of 382) of Chinese thalassemia major patients have RBC antibodies. Anti-E (42, 39.3%), anti-Mia/Mur (33, 30.85%), anti-c (14, 13.1%), and anti-Jka (seven,
Table 5 Frequency of detected alloantibodies and calculation of percent among total and positive patients
Antibody screening test results Frequency Actual percent
Positive (N= 50) Anti Kell 14 28.0 Anti D 8 16.0 Anti E 4 8.0 Anti c 4 8.0 Anti cw 3 6.0 Anti e 1 2.0 Anti D and C 4 8.0 Anti D and E 1 2.0 Anti colb 2 4.0 Anti C 1 2.0 Anti Kell and E 1 2.0 Anti Kell and KPa 1 2.0 Anti Kell and D 1 2.0 Unknown 4 8.0 Auto antibody 1 2.0
Table 6 Statistical analysis and Relation between different variables and their correlation to Antibody screening test results (NS=Not significant)
Antibody screening test result Relating factors Negative results N= 391 (%) Positive results N= 50 (%) P value (correlation)
Sex Male 210 (89.7) 24 (10.3) NS Female 181 (87.4) 26 (12.6) 0.446
Type of thalassemia Major 325 (89.8) 37 (10.2) NS Intermedia 66 (83.5) 13 (16.5) 0.113
Regular transfusion Yes 320 (89.5) 36 (10.1) NS No 71 (83.5) 14 (16.5) 0.097
History of reaction Hemolytic 0 (0) 7 (100) 0.000* Allergic 25 (100) 0 (0) Febrile 34 (82.9) 7 (17.1) No reaction 332 (90.2) 36 (9.8)
Splenectomy Yes 191 (86) 31 (14) NS No 200 (91.3) 19 (8.7) 0.080
*In our analytical method, we analyzed the relation between the transfusion reaction (yes or no) and positive alloantibodies, and was positive correlation. NS, not significant.
Azarkeivan et al. RBC alloimmunization and double alloantibodies in thalassemic patients
Hematology 2015 VOL. 20 NO. 4 225
6.55%) were the commonest antibodies reported. Bhatti et al.21 showed that 4.97% of 161 patients with thalassemia major were alloimmunized. RBC alloantibodies belonged mainly to the Rh system although other antibodies such as anti-K, anti-Jsb, and anti-Jka were detected. There was no definite relation between alloantibodies formation and the age of start blood transfusion, number of blood trans- fusions, and patient’s race. In another study, Ameen et al.,9 found a high frequency of red cell alloimmuni- zation in 57 (30%) of 190 transfusion-dependent Arab thalassemia patients. The most common clinically sig- nificant alloantibodies were against antigens in the Kell and Rh systems. Anti-K in 41 (72%) patients and anti-E in 26 (45.6%) were reported. Autoantibody was detected in 21 (11%) patients. They found the majority of alloimmunized patients formed first alloantibodies between age 2 and 10 years (58%). These researchers believe that performing an accurate compatibility assessment for donor and recipient blood units and using post storage leukode- pleted blood are important factors to reduce alloim- munization in high prevalence alloantibodies among thalassemia patients. In this study, anti-D antibody was detected in 12
patients. One of the reasons for this alloimmunization is transfusion of some red blood cells with rhesus D incompatible with thalassemia patients due to false- negative results in weak D typing of blood donors. Anti-D antibody was also reported in other studies. Sadeghian et al.20 reported high prevalence of Anti- D (88.88%) but in Karimi et al.’s18 study the frequency of Anti-Rh (47.7%) was very common. In this study, double antibodies were detected in 8 of
the 50 patients (16%), including Anti-D+Anti- C(8%), anti-D+Anti-E (2%), anti-Kell+Anti-D (2%), and anti-Kell+KPa (2%). Wang et al.10 ana- lyzed 30 individuals with thalassemia who had regular transfusions for periods ranging from 0·5 to 20 years. Of the 30 patients, 11 (37%) were found to carry alloantibodies. All alloantibodies were clinically significant specificities, including four cases of anti-E, two of anti-E+ c, two of anti-‘Mia’, one of the anti- ‘Mia’+ E, one of anti-D, and one of anti-S. In Bilwani et al.’s22 study, irregular red cell alloantibodies were found in 9.2% (9 of 44). Three (33.3%) patients developed anti-K while two (22.2%) had non-specific antibody. One patient each developed anti-D (11.1%) and anti-E (11.1%). Two had anti-D (11.1%) and anti-C, while the other one (11.1%) developed anti-E and anti-K.
Conclusion Alloimmunization is a common consequence in patients that need persistant blood transfusion. Antibodies against Rh subgroups and Kell groups
are more frequent antibodies in thalassemia patients receiving a chronic blood transfusion. Decreasing the rate of alloantibody production may be possible by the mean of identifying RBC phenotype specially Rh/Kell/Kidd, and Duffy subgroups for each patient before start chronic transfusion, and doing a careful cross match with evaluating the blood group of a donated unit (especially for Rh subgroups and Kell antigens).
Acknowledgements The authors wish to thank Mr Mohammad Hossein Lotfi and Dr Ayoub (from Diamed Company) and Mrs Mehmanchy (Roantgen Company, Iran) for their technical help.
Disclaimer statements Contributors All authors know the roles and agreed with their position on this manuscript.
Funding None.
Ethics approval The Iranian Blood Transfusion Organization Research Committee approved the study.
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