Asian Journal of Transfusion Science
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Year : 2016  |  Volume : 10  |  Issue : 2  |  Page : 161-163
Direct antiglobulin test positivity in multi-transfused thalassemics

1 Department of Transfusion Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Transfusion and Apheresis Services, Max Superspeciality Hospital, Shalimar Bagh, New Delhi, India

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Date of Web Publication8-Aug-2016


Introduction: Red cell allo- and auto-immunization is a well recognized problem in multi-transfused thalassemic patients. We conducted this study on 301 multi-transfused thalassemic patients under the Thalassemia Transfusion Programme of Advanced Pediatric Centre of PGIMER. Aims and Objectives: The study was designed to determine the frequency of alloimmunization and autoimmunization in multi-transfused thalassemic patients and to establish the specificity of alloantibody to red cell antigens, if alloimmunization is detected. Materials and Methods: The antibody screening was performed by the conventional tube technique using commercially available three cell screening panel (Diamed Switzerland) by saline, low ionic strength solution (LISS) and albumin indirect antiglobulin test (IAT). Samples with alloantibodies were then tested with red cell identification panel to determine the alloantibody specificity. Autoantibody screening was performed by direct antiglobulin test (DAT) during pre-transfusion testing. Results: Of the 301 patients, 52 (17.28%) were found to have antibodies (-allo and –autoantibodies). A total of 11 red cell alloantibodies were detected in 10 patients and the specificities were anti-Kell in 6(54.5%), anti-D in 2(18.2%), anti-c in 1(9.1%) and a combination of anti-E (9.1%) and anti-Jkb in 1 (9.1%) patients. DAT was positive in 48 (15.9%) patients. The frequency of autoantibody was significantly higher in alloimmunized group as compared to non-alloimmunized group (60% V/s 14.4%). Also, the pre-transfusion hemoglobin was significantly lower in the immunized group (8.5 gm/dl V/s 9.0 gm/dl; p=0.03) than the non-immunized group. Conclusion: Based on these observations, we suggest antigen typing of all thalassemia major patients for ABO, Rh and Kell antigens before initiating transfusion therapy. Also, screening for allo- and auto-antibodies at regular intervals should be done prior to each transfusion.

Keywords: Allommunization, antiglobulin test, multi-transfused thalassemics

How to cite this article:
Jain A, Agnihotri A, Marwaha N, Sharma RR. Direct antiglobulin test positivity in multi-transfused thalassemics. Asian J Transfus Sci 2016;10:161-3

How to cite this URL:
Jain A, Agnihotri A, Marwaha N, Sharma RR. Direct antiglobulin test positivity in multi-transfused thalassemics. Asian J Transfus Sci [serial online] 2016 [cited 2022 Aug 18];10:161-3. Available from:

   Introduction Top

Immune abnormalities have been reported in thalassemia major patients receiving regular allogenic blood transfusions. Thalassemic patients have a predisposition toward the formation of autoantibodies directed against various body tissues. The pathogenic mechanism behind these is not yet clear, still derangements in the immune system resulting from iron overload,[1],[2] splenectomy,[1] use of iron chelators,[3] repeated exposure to allogenic antigens in blood,[2] immunosuppressive viruses and liver damage following hepatitis [4] are considered to be important factors. Few case reports have described hyperhemolysis of red cells in the presence of positive direct anti-globulin test (DAT) resulting in markedly increased transfusion requirements above baseline.[5],[6] These erythrocytic antibodies implicated in red cell hemolysis are either IgG or IgM in nature, although their exact specificity is rarely defined. These autoantibodies are often formed in the presence of alloantibodies and require immunosuppressive therapy if the patient becomes symptomatic. The clinical implications of presence of autoantibodies in multitransfused thalassemics are not well understood, as only few studies have evaluated the association of thalassemia and red cell autoimmunization.[7],[8],[9]

   Materials and Methods Top

We conducted this study on 301 multi-transfused thalassemic patients under the Thalassemia Transfusion Program of our institute. For each patient, 3-5 ml of plain vial (clotted) and 2 ml of ethylenediaminetetraacetic acid (EDTA) anticoagulant sample was collected. The serum was separated and stored at −80°C in three aliquots, which were thawed at room temperature later and used for antibody screening. The EDTA sample was used for putting up the DAT. The antibody screening was performed by the conventional tube technique (CTT) using commercially available three cell screening panel (Biorad, Switzerland) by saline indirect antiglobulin test (IAT), low ionic strength solution (LISS) and albumin IAT.[10] Samples with alloantibodies were then tested with eleven red cell identification panel (Biorad, Switzerland) to determine the alloantibody specificity. Since the patients' sera were stored at −80° C and tested later after thawing, the autocontrol could not be put up at the time of antibody screening. Hence, the autoantibody detection was performed by DAT only during the pre-transfusion testing using the polyspecific anti-human globulin (AHG). Further, distinction of autoantibody positive cases was done using monospecific anti-IgG AHG and anti-C3d AHG by CTT. The statistical analysis was performed using Number Cruncher Statistical System (NCSS) software (Kaysville, Utah, USA).

   Results Top

In our study, the frequency of alloimmunization was 3.3% (10 out of 301 patients). A total of 11 red cell alloantibodies were detected in 10 patients and the specificities were: Anti-K in 6 (54.5%), anti-D in 2 (18.2%), anti-C in 1 (9.1%), and a combination of anti-E and anti-Jk b in 1 (9.1%) patient. These alloantibodies were detected by all the three techniques used (saline, LISS, and albumin IAT). DAT was found to be positive in 48 out of 301 patients (15.9%). These were IgG in nature in 27 (56.3%) cases, C3d in 16 (33.3%), and blended (both IgG and C3d) in 5 (10.4%) cases [Table 1]. DAT positivity was associated with alloimmunization in six patients (12.5%). The higher DAT positivity (60%; 6 out of 10) in the alloimmunized group was found to be statistically significant (P = 0.001) than the non-alloimmunized group (14.4%; 42 out of 291). We also found that of the 48 patients with positive DAT, alloantibody formation was present in 6 (12.5%) cases, whereas in 253 patients in whom DAT was negative, alloimmunization was present in 4 (1.6%) cases. This rate of alloimmunization in patients with positive DAT was significantly higher when it was compared to the alloimmunization rate in the DAT negative patients (P = 0.0001).
Table 1: Details of DAT positive cases (n = 48) in alloimmunized and non-alloimmunized group

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Direct antiglobulin test with monospecific AHG revealed that the greater portion of the alloimmunized patients (3 out of 10) were having complement coating on their red cells as compared to that in the non-alloimmunized group (13 out of 291), that is, 30% versus 4.5%, respectively (P = 0.0004).

   Discussion Top

A positive DAT in our study indicated either the presence of autoantibodies or the presence of residual donor cells coated with antibodies. However, using the three IAT based techniques (saline, LISS, and albumin) and the standard commercial three cell panel alloantibodies were detected only in 10 patients. Thus, DAT positivity may be more suggestive of the presence of weak or low titer autoantibodies, though follow-up studies will be required for the definitive diagnosis. We detected a positive DAT in 15.9% of our study population and none of them had any features of hyperhemolysis necessitating therapeutic intervention. Similar findings were reported by Amen et al.,[9] who found the frequency of autoimmunization to be 11%, with 52% of the autoantibodies detected being IgG in nature and none of them caused significant hemolysis. Singer et al.[7] found antibodies in 25% of thalassemics, having IgG specificity in 68.8% and C3d specificity in 32.2% of patients, respectively. The presence of antibodies in these studies was detected by performing DAT. We also found that alloimmunization in patients with a positive DAT (6/48: 12.5%) was significantly higher (P = 0.0001) compared to alloimmunization rate in DAT negative patients (4/253: 1.6%). A similar observation was made by Amen et al.[9] where they found that alloantibodies were present in 95% of the patients with autoantibodies (20 out of 21 DAT positive cases). A study on multitransfused patients found that patients with thalassemia major showed a significant lymphocytosis, with mainly B-cell changes consistent with ongoing B-cell stimulation associated with chronic exposure to red cell antigens.[11] This B-cell stimulation is accompanied by an increase in serum immunoglobulin, immune complexes, and cells expressing surface immunoglobulin. These immune system alterations in thalassemia patients might explain the increased chances of autoantibodies and alloantibodies being present together.

In our study, 14 out of 48 (29%) DAT positive patients had undergone splenectomy. Similarly, Singer et al.,[7] and Amen et al.,{19} observed that 56% and 28.6% of the patients with autoantibodies, respectively, had undergone splenectomy. The immune dysfunction which occurs after repeated blood transfusion and altered deformability profile of erythrocytes in splenectomized patients, may account for this observation.[7] It was a striking finding that the two patients with anti-D alloimmunization, always received Rh (D) negative packed red blood cell (PRBC) transfusion and they were Rh (D) negative. One plausible reason for the development of anti-D alloantibody in these patients is the possible transfusion of PRBCs which were actually “weak D,” but were not detected serologically. Although, all the Rh (D) negative donor unit samples were subjected to “weak D” testing by CTT using a monoclonal blend (IgG + IgM) of anti-D antisera by an IAT. If the result is negative then only the PRBC units are labeled as Rh (D) negative, but if the result of “weak D” testing is positive then the PRBC units are labeled as Rh (D) positive. However, in spite of “weak D” testing some weaker variants of D antigen may not be detectable and thus could result in anti-D production when transfused to D negative individuals. Earlier studies have demonstrated that red blood cells from subjects with the weak D type 1 phenotype and weak D type 2 phenotype may cause primary alloimmunization of anti-D.[12],[13]

Based on the observations in our study we suggest that the thalassemic patients should also be typed for Rh (C, c, E, e) and Kell antigens before the initiation of transfusion. They should be screened for allo- and auto-antibodies at regular intervals, preferably prior to each transfusion. Once an alloantibody is detected, the antigen negative blood should be transfused, even if the titer of the antibody subsequently declines.

However, even to optimize the transfusion policies, further prospective studies are required to know the clinical significance of red cell autoantibodies, especially in relation to serial estimation of pre-transfusion hemoglobin, transfusion interval and hemolytic transfusion reactions. It will also help to determine the role of co-existing auto- and allo-antibodies in determining transfusion requirements and the effect of giving antigen matched blood in the presence of autoantibodies to prevent alloimmunization.

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Conflicts of interest

There are no conflicts of interest.

   References Top

Kapadia A, de Sousa M, Markenson AL, Miller DR, Good RA, Gupta S. Lymphoid cell sets and serum immunoglobulins in patients with thalassaemia intermedia: Relationship to serum iron and splenectomy. Br J Haematol 1980;45:405-16.  Back to cited text no. 1
Grady RW, Akbar AN, Giardina PJ, Hilgartner MW, de Sousa M. Disproportionate lymphoid cell subsets in thalassaemia major: The relative contributions of transfusion and splenectomy. Br J Haematol 1985;59:713-24.  Back to cited text no. 2
Lederman HM, Cohen A, Lee JW, Freedman MH, Gelfand EW. Deferoxamine: A reversible S-phase inhibitor of human lymphocyte proliferation. Blood 1984;64:748-53.  Back to cited text no. 3
Dwyer J, Wood C, McNamara J, Williams A, Andiman W, Rink L, et al. Abnormalities in the immune system of children with beta-thalassaemia major. Clin Exp Immunol 1987;68:621-9.  Back to cited text no. 4
Mizon P, Cossement C, Mannessier L, Caueir MT, Rose C, Goudemand J. Severe hemoloysis related to an association of erythrocyte allo-and autoantibodies in athalassemia patient. Nouv Rev Fr Hematol 1994;36:363-6.  Back to cited text no. 5
Saran RK, editor. Transfusion Medicine, Technical Manual. 2nd ed. New Delhi, India: Directorate General of Health Services, Govt. of India; 2003. p. 101-8.  Back to cited text no. 6
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. 7
Spanos T, Karageorga M, Ladis V, Peristeri J, Hatziliami A, Kattamis C. Red cell alloantibodies in patients with thalassemia. Vox Sang 1990;58:50-5.  Back to cited text no. 8
Amen R, Shemmari SA, Humood SA, Chawdhury RI, Eyaadi OA, Bashir AA. RBC alloimmunization and autoimmunization among transfusion-dependent thalassemia patients. Blood 2001;97:3999-4000.  Back to cited text no. 9
Brecher ME, editor. Technical Manual. 14th ed. Bethesda, Maryland (USA): American Association of Blood Banks, AABB Puublications; 2003. p. 687-97.  Back to cited text no. 10
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;105:817-25.  Back to cited text no. 11
Mota M, Fonseca NL, Rodrigues A, Kutner JM, Castilho L. Anti-D alloimmunization by weak D type 1 red blood cells with a very low antigen density. Vox Sang 2005;88:130-5.  Back to cited text no. 12
Flegel WA, Khull SR, Wagner FF. Primary anti-D immunization by weak D type 2 RBCs. Transfusion 2000;40:428-34.  Back to cited text no. 13

Correspondence Address:
Neelam Marwaha
Department of Transfusion Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh - 160 012
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-6247.164268

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