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Serological characteristics and immunohematological difficulties in autoimmune hemolytic anemia patients: A retrospective analytical study from South India

 Department of Transfusion Medicine, Jawaharlal Institute of Post Graduate Medical Education and Research, Puducherry, India

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Date of Submission04-Nov-2021
Date of Decision29-Dec-2021
Date of Acceptance11-Jan-2022
Date of Web Publication26-Sep-2022


INTRODUCTION: Autoimmune hemolytic anemia (AIHA) is a condition in which there is decreased survival of red blood cells (RBC) due to the destruction of RBC by autoantibodies. AIHA is classified into warm, cold, and mixed according to temperature sensitivity. The antibodies may be immunoglobulin G, immunoglobulin M, immunoglobulin A, or complement proteins, and hemolysis may be intravascular or extravascular. The present study was done to find out serological characteristics of AIHA patients in our population.
MATERIALS AND METHODS: During the study, a total of 112 patients' samples were analyzed. All immunohematology workup, including blood grouping, direct Coombs test (DCT), indirect Coombs test (ICT), monospecific DCT, and alloantibody identification, were done.
RESULTS: A maximum number of patients were in the age group of 16–45 years (69.6%). Primary AIHA (56.25%) is more common than secondary AIHA. Females (73.2%) were more affected than men, mostly due to the prevalence of autoimmune disease more in females. Warm AIHA (58%) was more common, followed by mixed (33%) and cold (9%). Grouping discrepancy was seen in 30 (26.8%) cases.
CONCLUSION: Warm AIHA is more common in our population followed by mixed and cold types. Blood group discrepancy seen in good proportion of AIHA patients, therefore meticulous immunohematological work up plays an essential role in these patients.

Keywords: Autoantibody, autoimmune hemolytic anemia, coombs test

How to cite this URL:
Sahoo D, Anuragaa S. Serological characteristics and immunohematological difficulties in autoimmune hemolytic anemia patients: A retrospective analytical study from South India. Asian J Transfus Sci [Epub ahead of print] [cited 2023 Mar 31]. Available from:

   Introduction Top

Autoimmune hemolytic anemia (AIHA) is the clinical condition in which antibodies of immunoglobulin G (IgG) and/or immunoglobulin M (IgM) bind to red cell surface antigens and initiate red cell destruction via the complement system and the reticuloendothelial system. The autoantibodies are directed against the red blood cells (RBC) membrane proteins and self-proteins.[1] The incidence of AIHA is 1:1000–100,000 in the general population in western countries with prevalence of 17:100,000.[2] The antibody may belong to any immunoglobulin class or complement protein. Destruction of RBC may be extravascular or intravascular depending upon the immunoglobulin class and type of AIHA. Mostly AIHA is primary that is idiopathic. Various other medical conditions such as systemic lupus erythematosus (SLE), leukemia, lymphoma, infections, and autoimmune disorders can lead to secondary AIHA. Sometimes AIHA can also be due to certain drugs taken by the patient. Therefore, it is very essential to know the detailed history of the patient to diagnose AIHA. They can be classified into warm, cold, and mixed according to the temperature sensitivity.[3] Some of the patients with AIHA may have difficulty in blood grouping. Blood grouping discrepancies are encountered during immunohematology workup. These patients may also develop alloantibody due to repeated transfusion due to anemia.[4] A detailed and meticulous workup is needed to diagnose immunoglobulin class, type of AIHA for further patient management. Various difficulties are encountered during the pretransfusion testing, and there is increased turnaround time. The presence of alloantibody makes the immunohematology work up even more complex, and it is essential to give the patient an antigen-negative unit. The aim of the present study is to assess serological characteristics and immunohematological difficulties faced in AIHA patients.

   Materials and Methods Top

This is a retrospective study done in the department of Transfusion Medicine in a tertiary care hospital of South India. All patient details were obtained from hospital records. All patient samples were sent under the provisional diagnosis of anemia, suspecting primary or secondary AIHA. Patients from January 2019 to April 2021 were included in this study with clinical and laboratory features of suggesting AIHA. A total of 112 patients were included in this study.

Immunohematology work up

All the immunohematology workup, including blood grouping, Direct Coombs test (DCT) and Indirect Coombs test (ICT), detection and identification of auto and alloantibody, solving blood grouping discrepancy were done in our Transfusion Medicine department.

Blood grouping

As a general routine first blood grouping was done irrespective of previously known groups. All grouping in our laboratory were done by conventional tube technique using commercially available antisera anti-A, anti-B, anti-D, and anti-H (Tulip diagnostic Pvt. Ltd Goa, India) and reverse grouping using in-house pooled A, B, and O cell. All grouping discrepancy in suspected AIHA patients were resolved before proceeding to the next diagnostic test by warm saline wash and warming the sample at 37°C.

Direct Coombs test

After identifying the patient blood group, DCT was performed using a coombs card containing polyspecific anti-human globulin (AHG) (IgG + C3d) according to the manufacturer's instruction (Biorad diagnostics Switzerland). The column agglutination technique was used to do DCT, and results were interpreted accordingly. In all DCT positive cases, further monospecific distinction (IgG and C3d) were done. Furthermore, monospecific coombs test for IgG, IgM, IgA, C3d, and C3c was done.

Thermal amplitude testing

This test is done to diagnose the type of AIHA (cold, warm, or mixed). Diagnosis is made based upon the reactivity of the antibody at 4° C, 22°C, and 37°C. Three test tubes are taken with patient's serum and 5% in-house O pooled cells in the ratio of 2:1 of serum and cells. They were incubated at three different temperatures mentioned. Results were interpreted according to the reactivity of the sample at the different temperature. Reactivity only at 37°C was considered as warm type, reactivity only at 4°C cold type. And reactivity at all temperature was mixed type AIHA.

Antibody screening and identification

Commercially available 3 and 11 cell panel reagents (Biorad Diagnostics Switzerland) were used for antibody screening and identification. Positive results were decoded using antigram, and the alloantibody was identified.

Statistical analysis

All categorical variables were expressed as number and percentage. All continuous variables were expressed as mean with standard deviation or median with interquartile range according to normality. The association among categorical variables was tested using Chi-square test. All statistical test was carried out in SPSS version 19. The P < 0.05 was considered statistically significant.

   Results Top

A total of 112 patients were included in our study, from a minimum age of 3 years to a maximum age of 75 years. Age distribution, gender distribution, and type of AIHA (primary, secondary) have been shown in [Table 1]. Secondary AIHA was due to SLE, lymphoma, infection, sepsis. A total of 82 (73.2%) females and 30 males (26.8%) were diagnosed with AIHA. Forty-nine females (59.8%) and 14 (46.7%) males were diagnosed with primary AIHA [Figure 1]. Thirty-three (40.2%) females and 16 (53.3%) males were secondary AIHA due to other causes.
Figure 1: Gender distribution in primary and secondary autoimmune hemolytic anemia

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In 112 patients, 30 (26.8%) patients had blood grouping discrepancies [Table 2]. All grouping discrepancy was sorted with warm saline wash and 37°C incubation. For one patient, the discrepancy was sorted with DTT treatment. Grouping discrepancy was maximum in patients having mixed AIHA. Sixteen cases of mixed AIHA were having grouping discrepancy followed by warm 13 cases and cold one case. Based on the serological and thermal amplitude testing (temperature sensitivity) AIHA was classified into cold, mixed, and warm type. Among primary AIHA patients four cases were cold, 33 cases were warm while 26 were mixed type. In secondary AIHA patients six were cold, 32 cases were warm while 11 were mixed type. Number of female patients with warm AIHA was 47, cold 6 and mixed AIHA 29. Males with warm AIHA were 18, cold and mixed AIHA were 4 and 8 [Figure 1] and [Figure 2].
Table 1: Patient characteristics (n=112)

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Table 2: Serological characterization (n=112)

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Figure 2: Gender distribution in warm cold and mixed autoimmune hemolytic anemia

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All 112 patients were tested for IgG, IgM, IgA, complement C3d, and C3b. Single autoantibody were present in 39 patients. Thirty (26.8%) patients were positive for IgG, and 9 (8%) for C3d. The remaining 73 patients nine (8.1%) patients were positive for all IgG, IgM, IgA, C3d, and C3c. Sixteen (14.3%) patients were positive for IgG, IgM, C3d. Forty-five (40.2%) patients were positive for IgG and C3d, two patients (1.8%) had Ig G, C3d, C3c, one patient had only C3d, C3c. Out of 112 cases, 69 patients had positive ICT. All ICT positive cases were worked up to rule out the presence of alloantibodies. Most of the cases were due to autoantibody. In 3 and 11 cells, there was pan reactivity. Six patients had alloantibody with autoantibody. Auto adsorption was done initially in these cases. The remaining serum was tested with 3 cell and 11 cell panels. Alloantibody was identified using 3, 11 cell panels and sorting out the pattern with antigram. Three patients had anti-C, 1 patient anti-D, 1 patient anti-M, and 1 patient anti-Jkb. Most of the patients in our study group were having blood group O+. Blood group distribution in our study was 44 cases (39.3%) O RhD positive, 31 (27.7%) B RhD positive, 22 (19.6%) A RhD positive, 9 (8%) AB RhD positive, 2 (1.8%) O RhD Negative, 3 (2.7%) B RhD negative and 1 (0.9%) A RhD negative.

   Discussion Top

The mean age in our study was 31 years. In the pediatric setting, a total of 16 cases (14.3%) were belonging to pediatric age group which is similar to the study by Naithani et al.[5] Most of our patients (69.6%) were in the 16–45 year age group. This was similar to the study conducted by Das et al. in which 66% was <40 years.[6] This study revealed that primary AIHA is more than secondary AIHA. In our study total, number of patients with primary AIHA was 62, which was more than the secondary AIHA. In 50 patients with secondary AIHA, most of the cases were due to SLE. AIHA is one of the hematological abnormalities in SLE.[7] A study conducted in north India showed secondary AIHA more common than primary.[6] The present study showed female preponderance in AIHA. Our study population included 82 females and 30 males. This is because autoimmune disorders like SLE are more common in females. A study from the Swedish national register showed that autoimmune disorder is 60% more common in women.[8] This may be attributed reason why the incidence of AIHA is more in females.

Blood grouping discrepancy is a common and significant problem in AIHA. It is very essential and mandatory to perform both forward and reverse grouping in AIHA. If grouping discrepancy persists, various techniques are performed to solve the discrepancy. Blood group identification is the first and foremost step in AIHA workup. In this study, 26.8% of our patients had grouping discrepancies similar to Zhu et al. In a study done by Zhu and his colleagues in ABO, grouping discrepancy in AIHA showed that 31.6% of AIHA patients were having difficulty in typing their blood group.[9] Blood group distribution in AIHA patients was similar to its prevalence in our population. There was no significance of blood group distribution in AIHA patients.[10] AIHA can occur in persons with any blood group. But blood group antigens play a role in AIHA. In warm autoantibodies are directed against Rh antigens and in cold against I/i antigens.

In our study, AIHA classification was done depending on thermal amplitude testing, DCT subtype, and clinical correlation. Maximum cases (58%) were due to warm AIHA. Auto control in patients with warm AIHA is reactive at 37°C and AHG phase. In cold AIHA patients these antibodies reactive at 4°C–6°C, while mixed cases show both the features of both warm and cold. The majority of cases in both primary and secondary AIHA were due to warm AIHA. A study was done by Chen et al. on 450 hospitalized patients also showed that 97.3% was due to warm AIHA.[11] Followed by warm AIHA, most of the cases were due to mixed AIHA 33% and cold AIHA 9%. Antibody to what Ig type is also essential in AIHA. Thirty-nine (34.8%) of our patients had only a single antibody, either IgG or C3d. A study performed by Issitt et al. show similar results, 43.7% of their AIHA patients had a single autoantibody.[12] Remaining 73 patients in our study showed multiple autoantibodies. In multiple antibodies majority of our patients were positive for IgG and C3d (40.2%). Only 9 patients (8%) were positive for Ig A along with other antibodies IgM, IgG, and C3d. A study done by Sokol et al. showed only 5 cases solely positive for Ig A in 13 years of follow-up.[13]

Alloantibody may also be present in combination with autoantibody. Certain patients are transfused often due to severe hemolysis, default in treatment, loss of follow-up, or acute exacerbations due to stress or infection. Because of repeated transfusion, chances of alloimmunization are more in AIHA. In our study, 6 (5.3%) patients were having alloantibody. In our study, alloimmunization rate was less. Some studies show alloimmunization to be from 12% to 40%.[14] In a study by Issitt et al. some autoantibodies also mimicked like alloantibodies.[12] If a patient has both allo and autoantibody, the immunohematology workup becomes tedious to identify the alloantibody involved. It is very essential and compulsory to give that antigen-negative unit to the patient; otherwise the patient will develop a hemolytic transfusion reaction. The drawback in our study was DCT negative AIHA was not included in our study. Subclass identification of IgG into IgG1 or IgG3 was not done. The strength of the study is that immunohematological work up was completely done in all these patients. We could have sub grouped all DCT positive cases to IgG, IgM, IgA, C3d, and C3c. Also grouping discrepancies could be resolved in all case.

   Conclusion Top

Warm AIHA is more common in our population followed by mixed and cold type. Blood group discrepancy seen in good proportion of AIHA patients, therefore meticulous immunohematological work up plays an essential role in these patients. The present study adds knowledge for a better understanding of the serological characteristics of AIHA patients.

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Conflicts of interest

There are no conflicts of interest.

   References Top

Packman CH. The clinical pictures of autoimmune hemolytic anemia. Transfus Med Hemother 2015;42:317-24.  Back to cited text no. 1
Barcellini W, Fattizzo B, Zaninoni A, Radice T, Nichele I, Di Bona E, et al. Clinical heterogeneity and predictors of outcome in primary autoimmune hemolytic anemia: A GIMEMA study of 308 patients. Blood 2014;124:2930-6.  Back to cited text no. 2
Das SS, Chakrabarty R, Zaman RU. Immunohematological and clinical characterizations of mixed autoimmune hemolytic anemia. Asian J Transfus Sci 2018;12:99-104.  Back to cited text no. 3
[PUBMED]  [Full text]  
Dara RC, Tiwari AK, Arora D, Mitra S, Acharya DP, Aggarwal G, et al. Alloimmunization in autoimmune hemolytic anemia patient: The differential adsorption approach. Asian J Transfus Sci 2017;11:53-7.  Back to cited text no. 4
[PUBMED]  [Full text]  
Naithani R, Agrawal N, Mahapatra M, Kumar R, Pati HP, Choudhry VP. Autoimmune hemolytic anemia in children. Pediatr Hematol Oncol 2007;24:309-15.  Back to cited text no. 5
Das SS, Nityanand S, Chaudhary R. Clinical and serological characterization of autoimmune hemolytic anemia in a tertiary care hospital in North India. Ann Hematol 2009;88:727-32.  Back to cited text no. 6
Fayyaz A, Igoe A, Kurien BT, Danda D, James JA, Stafford HA, et al. Haematological manifestations of lupus. Lupus Sci Med 2015;2:e000078.  Back to cited text no. 7
Ji J, Sundquist J, Sundquist K. Gender-specific incidence of autoimmune diseases from national registers. J Autoimmun 2016;69:102-6.  Back to cited text no. 8
Zhu JY, Lan JC, Hu LY, Meng QB, Luo HQ. Study on blood ABO typing in patients with autoimmune hemolytic anemia. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2004;12:525-7.  Back to cited text no. 9
Lambert JF, Nydegger UE. Geoepidemiology of autoimmune hemolytic anemia. Autoimmun Rev 2010;9:A350-4.  Back to cited text no. 10
Chen C, Wang L, Han B, Qin L, Ying B. Autoimmune hemolytic anemia in hospitalized patients: 450 patients and their red blood cell transfusions. Medicine (Baltimore) 2020;99:e18739.  Back to cited text no. 11
Issitt PD, Pavone BG, Goldfinger D, Zwikder H, Issitt CH, Tessel JA, et al. Anti-Wrb, and other autoantibodies responsible for positive direct antiglobulin tests in 150 individuals. Br J Haematol 1976;34:5-18.  Back to cited text no. 12
Sokol RJ, Booker DJ, Stamps R, Booth JR. Autoimmune hemolytic anemia due to IgA class autoantibodies. Immunohematology 1996;12:14-9.  Back to cited text no. 13
Branch DR, Petz LD. Detecting alloantibodies in patients with autoantibodies. Transfusion 1999;39:6-10.  Back to cited text no. 14

Correspondence Address:
Dibyajyoti Sahoo,
Department of Transfusion Medicine, Jawaharlal Institute of Post Graduate Medical Education and Research, Puducherry
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ajts.ajts_163_21


  [Figure 1], [Figure 2]

  [Table 1], [Table 2]


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