Asian Journal of Transfusion Science
Home About Journal Editorial Board Search Current Issue Ahead of print Back Issues Instructions Subscribe Login  Users: 841 Print this page  Email this page Small font sizeDefault font sizeIncrease font size 

ORIGINAL ARTICLE Table of Contents   
Year : 2017  |  Volume : 11  |  Issue : 2  |  Page : 115-120
Red cell alloimmunization in repeatedly transfused patients

Department of Immunohaematology and Blood Transfusion, M. P. Shah Government Medical College, Jamnagar, Gujarat, India

Click here for correspondence address and email

Date of Submission09-Jan-2016
Date of Acceptance19-Jul-2016
Date of Web Publication11-Sep-2017


Introduction: Repeated blood transfusions can result in the production of alloantibodies against one or more red cell antigens, which complicates subsequent transfusions. Aims: The study was done to find incidence of various red cell alloantibodies; to determine the type of alloantibody; to identify the factors such as frequency of transfusion, splenectomy status, donor ethnicity and gender and their association with the development of antibody in repeatedly transfused patients. Materials and Methods: This study was carried out in Dept. of IHBT, Shree M. P. Shah Medical College, Jamnagar, Gujarat. Blood was taken from the patients of thalassemia major, sickle cell disease, chronic renal failure, post partum haemorrhage, aplastic anemia, Myelodysplastic syndrome with more than 10 red cell transfusions. The plasma/serum was used for antibody screening and antibody identification test. Three cell antibody screening was performed using antihuman globulin gel cards (ID-Card LISS/Coombs) and three cell panel (ID-DiaCell I,II,III-Asia). Those with positive antibody screening were analyzed further for antibody identification test using eleven cell panel (Set ID-Dia Panel). Results: Antibody screening and identification was done in 2 consecutive set of samples (n = 300) which showed, nine (9) patients (3%) were alloimmunized. All repeatedly transfused patients had developed alloantibody before the starting of study period, no patient developed new alloantibody during study period. Conclusions: Alloantibodies should be identified in repeatedly transfused patients and should be given corresponding antigen negative blood unit which will minimize the antibody mediated destruction of transfused red cells.

Keywords: Alloantibodies, antibody identification, antibody screening

How to cite this article:
Bhuva DK, Vachhani JH. Red cell alloimmunization in repeatedly transfused patients. Asian J Transfus Sci 2017;11:115-20

How to cite this URL:
Bhuva DK, Vachhani JH. Red cell alloimmunization in repeatedly transfused patients. Asian J Transfus Sci [serial online] 2017 [cited 2022 May 25];11:115-20. Available from:

   Introduction Top

Alloimmunization to red blood cell (RBC) antigens resulting from the genetic disparities between donor and recipient is one of the risks of blood transfusion. Repeated blood transfusions can result in the production of alloantibodies against one or more red cell (RBC) antigens, which complicate subsequent transfusions. Alloantibodies can interfere in the cross-match testing and therefore can cause delay in obtaining compatible blood and also sometimes associated with delayed type of hemolytic transfusion reaction.

The probability of alloimmunization depends on the number and frequency of transfusions, antigen immunogenicity, and recipient's immune response. The influence of ethnic and antigenic pattern differences between donors and recipients has also been reported.[1]

Various diseases that require repeated red cell transfusions are thalassemia major (THAL), sickle cell diseases (SCDs), aplastic anemia (AA), myelodysplastic syndrome (MDS), chronic myeloproliferative disease and other malignancy, chronic renal failure (CRF).

Antigen matched transfusion would effectively prevent alloimmunization. To do so, the patient's ABO, Rhesus, Kell, Kidd, and Duffy systems should be typed at diagnosis or before the institution of transfusion therapy. Blood to be transfused should always be matched at least with ABO, Rhesus, and Kell system.[2] Further, using of leukocyte filter during transfusion may prevent alloimmunization due to white blood cells.

Management part of red cell alloimmunization cases include detection, identification of antibodies, and finally providing antigen negative blood for further transfusions. This study was designed to determine the prevalence of red cell alloimmunization in repeatedly transfused patients and will contribute to a better comprehension of the problem and will help to find the ideal approach for the future policy in Jamnagar population and our institute.

Aims and objectives

  • To initiate pretransfusion antibody screening on patient's sample before cross-match to initiate safe transfusion practice
  • To find out incidence of various RBC alloantibodies as well as autoantibodies in repeatedly transfused patients
  • To determine the type of antibody present in multiply transfused patients
  • To identify the factors such as frequency of transfusion, splenectomy status, donor ethnicity, and gender and their association with the development of antibody in repeatedly transfused patients
  • To identify common RBC alloantibodies.

   Subjects and Methods Top

This prospective study was carried out for duration of 2 years, i.e., August 2011–July 2013 in the Department of Immunohaematology and Blood Transfusion, Shree M. P. Shah Government Medical College and G. G. Government Hospital, Jamnagar, Gujarat. Written consent was taken for each patient. This study was approved by the Ethical Committee of the Shree M. P. Shah Medical College.

Patient selection

  • Patients of THAL major, SCD, CRF, postpartum hemorrhage, AA, MDS with >10 transfusions
  • Those who require blood transfusion at 2–4 weeks interval
  • Repeatedly transfused patients with difficulty in cross-matching to find out matched blood
  • Those patients having positive direct Coombs test (DCT) and suspected to have alloantibodies, having increased requirement of blood units.

Patients having regular transfusion at our hospital were included in this study. These data were collected from patient's clinical record files.

Patient exclusion criteria

  • Patients with known connective tissue disorders were excluded
  • Patients whose clinical history and regular follow-up were not possible.

Clinical transfusion records of the patients who fulfilled the inclusion and exclusion criteria were given a questionnaire and were reviewed for the demographic data, i.e., age, sex, ethnicity, blood group, age at the start of first transfusion, number of packed cell units received, frequency of transfusion, splenectomy status, history of transfusion reactions, if any, use leukocyte filter.

Sample collection

A volume of 3–4 ml of blood was collected in plain and ethylenediaminetetraacetic acid tubes from multiply transfused patients after they met the inclusion and exclusion criteria.

The plasma/serum was used for antibody screening and antibody identification test.

The red cells were used for ABO grouping, Rh grouping, antigen phenotyping, and DCT.

Laboratory methods

Using column agglutination technology, plasma/serum was analyzed for detection of red cell alloantibodies, and again, after 6 months of interval, blood sample was collected for detection of antibody to RBC antigen.

Three-cell antibody screening was performed using antihuman globulin gel cards (ID-Card LISS/Coombs) and three-cell panel (ID-DiaCell I, II, III-Asia). Those with positive antibody screening were analyzed further for antibody identification test using eleven-cell panel (Set ID-Dia Panel).

The red cells were used for ABO grouping, Rh grouping, and DCT during antibody screening.

The entire test was done using Bio-Rad ID microtyping system.

Criteria for completion of test/further testing

Antibody identification is considered complete if:

  • The panel results are consistent with one or more clear-cut antibody specificities satisfying the conditions for identification
  • The patient types negative for corresponding antigen
  • The direct antiglobulin test (DAT) is negative.

   Results Top

Out of total 300 patients, there were 178 males and 122 females who received regular blood transfusions. The age range of the patients was from 1 year to 80 years. There were 223 Hindu, 52 Muslim, 25 Sindhi patients.

Distribution of patients according to diagnosis is demonstrated in [Table 1].
Table 1: Distribution of patients by diagnosis

Click here to view

The packed cell units transfused were between 10 and 549 units (mean – 101.98 units). Only 4 (1.33%) patients were using leukocyte filter during blood transfusion. The low rate of using leukocyte filter during blood transfusion can be due to lack of awareness and cost-effectiveness. Sixteen (5.33%) patients had transfusion reactions in the form of mild itching, fall in blood pressure, fever, chills, rigors, and breathlessness. 10–550 transfusion episodes were tracked to capture transfusion reaction. In the present study, 12 (4%) out of total 300 patients had splenectomy, but no one was alloimmunized.

Result of antibody screening and identification

A total of nine RBC alloantibodies were detected in 9 out of total 300 patients, and none developed red cell alloantibody during the study period.

The incidence of red cell alloantibodies in two consecutive sets of samples of repeatedly transfused patients was 3% and 3% respectively. Interval between two tests was 6 months, and the patients were transfused between two intervals.

A total of nine samples showed to have alloantibody, 5 (55.55%) belonged to Kell blood group and 4 (44.44%) belonged to Rhesus system. Out of five Kell alloantibodies, four were anti-K and one was anti-Kp a. Out of four Rhesus alloantibodies, three were anti-D and one was anti-E [Table 2].
Table 2: Data of patients with alloantibodies (n=9)

Click here to view

Out of 178 males, 5 (2.81%) developed alloantibodies, and out of 122 females, 4 (3.28%) developed alloantibodies [Graph 1].

Out of 52 Muslims, 5 (9.61%); of 25 Sindhis, 2 (8%); of 223 Hindus, 2 (0.9%) developed alloantibodies [Graph 2].

Rate of alloimmunization in THAL major patients was 3.2% and in CRF was 5%. Other patients did not produce alloantibody.

Number of packed cells transfused in alloimmunized patients was between 18 and 152 units (mean – 86 units).

Out of nine alloimmunized patients, six patients (66.67%) were DCT positive for IgG only while three were negative both for IgG and C3d [Table 2].

   Discussion Top

The red cell alloantibodies develop in a variable number of multiply transfused patients. In such circumstances, transfusion therapy may become significantly complicated. Effects of alloimmunization may include difficulty in finding compatible RBC units because of the presence of clinically significant RBC antibodies, transfusion reactions, or platelet refractoriness. The present study is an effort to characterize blood group alloantibody formation in the patient population.

Rate of alloimmunization

The present study showed low rate of alloimmunization 3% which can be explained by homogeneity between the donor and the recipient population.[3]

In the present study, rate of alloimmunization was consistent with other studies done internationally [4],[5],[6] and various studies from India [Table 3].[3],[7],[9],[11]
Table 3: Rate of alloimmunization in various studies

Click here to view

Antibody specificity (type of antibody)

In the present study, a total of nine samples showed presence of alloantibodies, four anti-K, three anti-D, one anti-Kp a, and one anti-E. Kell blood group consisted of majority of alloantibodies with 55.55% followed by Rhesus blood group with 44.44%.

The present study showed alloantibodies against Kell blood group are most common, i.e., 55.55%, which showed similar results, i.e. Khalid et al.[12] (47.06%), Bilwani et al.[13] (38.85%), and Roopam et al.[7] (80%).

Rate of red cell autoantibodies

Red cell autoantibodies are antibodies that are directed against patient's own RBC antigen. Patients may show persistent positive DCT for both IgG and C3d and can result in significant hemolysis and increased requirement for transfusions.

Out of total 300 patients, 10 patients (3.33%) were DCT positive for IgG but not for C3d, and out of nine alloimmunized patients, six were positive for DCT for IgG only. There was no evidence of autoimmune hemolytic anemia in these patients. Positive DAT did not interfere in finding compatible blood. Positive DAT may indicate alloantibodies in a recipient's circulation, reacting with antigens on recently transfused donor red cells.

The present study showed 3.33% rate of autoantibodies which shows similar to other studies reported.[11]


In the present study, out of 300 patients of repeatedly transfused, there were 178 males and 122 females. It was observed that there was increased incidence of THAL.

In the present study, the rate of alloimmunization was higher in females (3.28%) comparable with males (2.81%) which is similar to other studies.[3],[7],[11]

There was no alloimmunization in multiparous women.

The association between sex and alloimmunization was statistically not significant (P = 0.9122).


In the present study, the age range of the patients was from 1 year to 80 years with mean age of 12.76 years. Majority of the patients in this study were more than 8 years – 55.7%.

In the present study showed that out of 300 patients of repeatedly transfused, nine showed presence of alloantibody. The age of alloimmunized individuals ranged between 6 and 45 years (mean – 14.33 years) showed results similar to other studies.[7],[13]

Religion and caste

In the present study, out of 300 cases, 223 were Hindus, 52 were Muslims, and 25 were Sindhis.

In the present study, nine patients were alloimmunized; two Hindus (one Rajput, one Koli), two Sindhis, and five Muslims. Hence, alloimmunization rate was more in Muslims and Sindhis as compared to Hindu patients.

Till now, to the best of our knowledge, there has not been any study in alloimmunization regarding religion and caste in India.

Age at the first transfusion

In case of THAL major, early detection in a patient may help in treatment and preventing subsequent allo and autoantibody formation in repeatedly transfused patients.

Immune response may be affected by patient's age at the start of transfusion. Transfusion at early age (<3 years) may offer some immune tolerance and protection against alloimmunization in THAL patients.[5]

In the present study, alloimmunized patients received the first transfusion ranged from 4 months to 44 years (mean – 7.85 years), and results were similar with other studies.[15],[16]

Number of packed cells transfused

In the present study, number of packed cell units transfused ranged from 18 to 152 units (mean – 78.89 units). All alloimmunized patients received more than ten transfusions correlating with other studies.[7],[12],[13],[14],[16]

Bhatti et al.[17] and Chaudhary et al.[3] showed no correlation between the number of transfusions and the alloimmunization rate. In the present study, statistical association between number of packed cell units transfused and alloimmunization was not significant (P = 0.7894). The result of the present study for association of number of packed cell units transfused as a risk factor for alloimmunization was not established.


The absence of spleen may enhance the immune response to the infused foreign antigens, which are not effectively filtered. Splenectomy may enhance or promote immune reactions as there is absence of an efficient filtering system for removal for damaged RBC.

Singer et al.[18] found from their study that patients who had a splenectomy had a higher alloimmunization rate.

In the present study, nine patients were alloimmunized, but none had splenectomy. Out of total 300 patients, 12 had splenectomy. No association between alloimmunization and splenectomy (P = 0.8089) was found out in the present study similar to other studies.[9],[16]

Leukocyte filter

Another important aspect that has emerged is the role of contaminating leukocytes of the allogeneic blood transfusion in causing immunomodulatory effects in the recipient. Contaminating leukocytes downregulate T-helper cell Type 1 immune responses and drive the recipient toward T-helper cell Type 2 responses. Such skewing toward Type 2 immunity may enhance alloantibody formation.[19]

In the present study, only 4 (4–1.33%) patients were using leukocyte filter, none of them were alloimmunized, the difference was not statistically significant (P = 0.2622).

In the present study, however, it was not established that leukocyte filter would prevent alloimmunization probably due to a small number of patients, in whom leukocyte filters were used during transfusions.

   Conclusion Top

It is concluded here that red cell alloimmunization should not be overlooked in repeatedly transfused patients. It should always be considered if the patient repeatedly suffers from hemolytic transfusion reactions, difficulty in finding compatible blood during cross–match, or patients not able to maintain hemoglobin at desired level in spite of regular transfusions.

It is also concluded here that regular screening for development of alloantibodies in repeatedly transfused patients would add toward better management of these patients. With the screening and identification technique, the alloantibodies should be identified and patients should be given corresponding antigen negative blood unit which will minimize the antibody-mediated destruction of transfused red cells.

Several factors might contribute to red cell alloimmunization such as heterogeneity of population, difference in age at first transfusion, antigenic difference between the donor and the recipients, recipient's immune status, immunomodulatory effects of allogenic blood transfusion on recipient's immune status, and splenectomy.

Obtaining RBC antigenic phenotype on all repeatedly transfused patients, providing leukodepleted blood-matched for antigens of ABO, Rhesus, and Kell systems in patients who have a lifelong transfusion dependency could be effective against RBC alloimmunization.

The present study also recommended extended phenotype-matched blood transfusion for antigen against Kell and Rhesus blood group system. This will minimize the antibody-mediated destruction of transfused red cells that result in reduction of transfusion needs for the patients. Less number of transfusions reduces the psychological and financial burden on the family and will increase the patient compliance.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

Vichinsky EP, Earles A, Johnson RA, Hoag MS, Williams A, Lubin B. Alloimmunization in sickle cell anemia and transfusion of racially unmatched blood. N Engl J Med 1990;322:1617-21.  Back to cited text no. 1
Hoffbrand AV. Genetic disorders of haemoglobin. In: Postgraduate Hematology. New York: Oxford University Press Inc.; 2001. p. 91-119.  Back to cited text no. 2
Shukla JS, Chaudhary RK. Red cell alloimmunization in multi-transfused chronic renal failure patients undergoing hemodialysis. Indian J Pathol Microbiol 1999;42:299-302.  Back to cited text no. 3
[PUBMED]  [Full text]  
Sirchia G, Zanella A, Parravicini A, Morelati F, Rebulla P, Masera G. Red cell alloantibodies in thalassemia major. Results of an Italian cooperative study. Transfusion 1985;25:110-2.  Back to cited text no. 4
Chow MP, Hu HY, Lyou JY, Lin JS, Yung CH, Lee A. Red cells, HLA and platelet antibody formation in patients with multiple transfusions. Acta Haematol 1994;92:57-60.  Back to cited text no. 5
Ansari S, Voosogh P, Mosthaghian S. Assessment of frequency of allo immunization and erythrocyte alloimmunisation in transfusion dependent thalassemia patients. Acta Med Iran 2008;46:137-40.  Back to cited text no. 6
Roopam J, Perkins J, Susan JT, Choudhury NA. A prospective study for detection and identification of red cell allo-antibodies in multiply transfused thalassemia major patients: 34th National Congress of Indian Society of Blood Transfusion and Immunohaematology; 2009. p. 20-2.  Back to cited text no. 7
Pahuja S, Pujani M, Gupta SK, Chandra J, Jain M. Alloimmunization and red cell autoimmunization in multitransfused thalassemics of Indian origin. Hematology 2010;15:174-7.  Back to cited text no. 8
Nikam SA, Dama SB, Saraf SA, Jawale CJ, Kirdak RV, Chondekar RP. Prevalence of red cell allo-immunization in repeatedly transfused patients with B-thalassemia in Solapur district, Maharashtra state, India. UGC-Sponsored National Level Workshop cum Seminar on “Bio-Resources for Bio-Industries and Economic Zoology” Organized by Department of Zoology, D.B.F. Dayanand College of Arts and Science, Solapur (M.S.); 2011. p. 24-5.  Back to cited text no. 9
Usman M, Moin S, Moinuddin M, Saeed A, Perveen R, Azmi MA. Frequency of red cell alloimmunization among patients with transfusion dependent beta thalassemia in Pakistan. Int J Hematol Oncol 2011;21:166-9.  Back to cited text no. 10
Sood R, Makroo RN, Riana V, Rosamma NL. Detection of alloimmunization to ensure safer transfusion practice. Asian J Transfus Sci 2013;7:135-9.  Back to cited text no. 11
[PUBMED]  [Full text]  
Khalid H, Younus M, Ikran N, Naseem L, Zaheer HA. Red cell alloimmunization in repeatedly transfused thalassemia major patients. Int J Pathol 2004;2:16-9.  Back to cited text no. 12
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;55:563-5.  Back to cited text no. 13
Gupta R, Singh B, Rusia U, Goyal S. Alloimmunization to red cells in thalassemics: Emerging problem and future strategies. Transfus Apheresis Sci 2011;45:167-70.  Back to cited text no. 14
Bashawri LA, Ahmed MS, AL-Qatary AA, Ahmed MA. Red cell alloimmunization in thalassemia patients. Bahrain Med Bull 2005;27:1-5.  Back to cited text no. 15
Chaudhari CN. Red cell alloantibodies in multiple transfused thalassaemia patients. Med J Armed Forces India 2011;67:34-7.  Back to cited text no. 16
Bhatti FA, Salamat N, Nadeem A, Shabbir N. Red cell immunization in beta thalassaemia major. J Coll Physicians Surg Pak 2004;14:657-60.  Back to cited text no. 17
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;96:3369-73.  Back to cited text no. 18
Clark JA, Tanley PC, Wallace CH. Evaluation of patients with positive direct antiglobulin tests and nonreactive eluates discovered during pretransfusion testing. Immunohematology 1992;8:9-12.  Back to cited text no. 19

Correspondence Address:
Dimel K Bhuva
Maliba Raktadan Kendra, Vilasini K. Desai Arogya Sankul, Near Vanchetna, Kakrapar Bypass, Tadkuva, Vyara, Tapi - 394 650, Gujarat
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-6247.214347

Rights and Permissions


  [Table 1], [Table 2], [Table 3]

This article has been cited by
1 Erythroid Differentiation of Induced Pluripotent Stem Cells Co-cultured with OP9 Cells for Diagnostic Purposes
Juhye Roh, Sinyoung Kim, June-Won Cheong, Su-Hee Jeon, Hyun-Kyung Kim, Moon Jung Kim, Hyun Ok Kim
Annals of Laboratory Medicine. 2022; 42(4): 457
[Pubmed] | [DOI]
2 Erythrocyte Antibody Due to Alloimmunization in Repeated Transfusion: A Meta-Analysis
Zelly Dia Rofinda, Eryati Darwin, Ellyza Nasrul, Irza Wahid
Open Access Macedonian Journal of Medical Sciences. 2022; 10(F): 257
[Pubmed] | [DOI]
3 Snapshot on physicians’ view on safe blood transfusion in multiply transfused patients in Nigeria
ChilotaC Efobi, AngelaO Ugwu, EstherI Obi, EdmundN Ossai, Sunday Ocheni
International Journal of Medicine and Health Development. 2021; 26(1): 50
[Pubmed] | [DOI]
4 Red-cell alloimmunization profile in multi transfused patients: Findings and insights of a blood transfusion service
M.L. Pereira Bueno, M.B. Mitestainer, J.A.R. Da Silva, B.D. Benites, F.M. Roversi
Transfusion Clinique et Biologique. 2021; 28(3): 258
[Pubmed] | [DOI]
5 The potential role of antibodies against minor blood group antigens in renal transplantation
Stephen G. Holt, Prasanti Kotagiri, Chris Hogan, Peter Hughes, Rosemary Masterson
Transplant International. 2020; 33(8): 841
[Pubmed] | [DOI]
6 Pathophysiology of Alloimmunization
Rubiraida Molina-Aguilar, Soledad Gómez-Ruiz, Jorge Vela-Ojeda, Laura Arcelia Montiel-Cervantes, Elba Reyes-Maldonado
Transfusion Medicine and Hemotherapy. 2020; 47(2): 152
[Pubmed] | [DOI]
7 Red cell alloimmunisation in patients receiving veno-venous extracorporeal membrane oxygenation (VV-ECMO)
Deepa R. J. Arachchillage, Steve Owen, Maria Anievas, Mihaela Gaspar, Mike Laffan
Intensive Care Medicine. 2020; 46(10): 1932
[Pubmed] | [DOI]
8 Red cell antigen phenotypes in blood donors & thalassaemia patients for creation of red cell antigen-matched inventory
Swati Kulkarni, Bhavika Choudhary, Harita Gogri, Jayashree Sharma, Manisha Madkaikar
Indian Journal of Medical Research. 2020; 152(3): 273
[Pubmed] | [DOI]
9 Analysis of red cell alloimmunization in multi transfused patients at a Tertiary care teaching hospital
Anjali Handa, Neetu Kukar, RamN Maharishi, Neha Syal, Harkiran Arora
Journal of Family Medicine and Primary Care. 2020; 9(6): 2907
[Pubmed] | [DOI]



    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  

   Subjects and Methods
    Article Tables

 Article Access Statistics
    PDF Downloaded83    
    Comments [Add]    
    Cited by others 9    

Recommend this journal

Association Contact us | Sitemap | Advertise | What's New | Copyright and Disclaimer | Privacy Notice

© 2006 - Asian Journal of Transfusion Science | Published by Wolters Kluwer - Medknow
Online since 10th November, 2006