Indian Journal of Pathology and Microbiology

: 2013  |  Volume : 56  |  Issue : 4  |  Page : 378--383

Red cell alloimmunization and infectious marker status (human immunodeficiency virus, hepatitis B virus and hepatitis C virus) in multiply transfused thalassemia patients of North India

Raj Nath Makroo, Jatinder Singh Arora, Mohit Chowdhry, Aakanksha Bhatia, Uday Kumar Thakur, Antony Minimol 
 Department of Transfusion Medicine, Indraprastha Apollo Hospitals, National Thalassemia Welfare Society, New Delhi, India

Correspondence Address:
Raj Nath Makroo
Department of Transfusion Medicine, Gate No. 9, Indraprastha Apollo Hospitals, Sarita Vihar, Delhi - Mathura Road, New Delhi - 110 076


Background: Patients with thalassemia major are largely transfusion dependent and are thus exposed to a variety of risks such as transmission of infectious diseases, iron overload and alloimmunization. This study was performed to determine the prevalence of human immune deficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and red cell antibodies among multiple-transfused thalassemic patients in and around the national capital region. Materials and Methods: The Department of Transfusion Medicine, Indraprastha Apollo Hospitals, conducted this study in collaboration with the National Thalassemia Welfare Society over a period of 1 year starting February2011. Blood samples from the patients were tested for blood group, red cell alloantibody/ies, anti-HIV, anti-HCV and hepatitis B surface antigen (HBsAg) by ELISA and for the respective viral ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) by nucleic acid testing (NAT). Results: A total of 462 thalassemics which consists of 290 males and 172 females were tested. The overall alloimmunization rate was 4.1% and anti-Kell was the most common antibody identified. Thirteen cases (2.8%) were positive for HBsAg by ELISA, 107 (23.1%) were reactive for anti HCV and 11 (2.38%) for anti HIV antibodies. Further screening and discriminatory assays by NAT confirmed the presence of HBV DNA in 11 cases, HIV RNA in 7 cases and HCV RNA in 48 cases. Conclusion: In spite of advances in Immunohematology and infectious marker testing in recent years, the rates of alloimmunization and infectious marker positivity remains high among multiply transfused patients like thalassemics. Provision of safe and adequate blood supply to these patients is a key to improving their quality-of-life and longevity.

How to cite this article:
Makroo RN, Arora JS, Chowdhry M, Bhatia A, Thakur UK, Minimol A. Red cell alloimmunization and infectious marker status (human immunodeficiency virus, hepatitis B virus and hepatitis C virus) in multiply transfused thalassemia patients of North India.Indian J Pathol Microbiol 2013;56:378-383

How to cite this URL:
Makroo RN, Arora JS, Chowdhry M, Bhatia A, Thakur UK, Minimol A. Red cell alloimmunization and infectious marker status (human immunodeficiency virus, hepatitis B virus and hepatitis C virus) in multiply transfused thalassemia patients of North India. Indian J Pathol Microbiol [serial online] 2013 [cited 2021 Jun 15 ];56:378-383
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Patients with Thalassemia major are largely transfusion dependent. Repeated blood transfusion exposes these patients to a variety of risks such as transmission of infectious diseases, iron over load and alloimmunization. It is therefore important that before embarking on transfusion therapy for these patients, certain basic investigations be carried out. Besides ABO and RhD grouping, an extended red cell antigen typing, which includes at least Rh (CcEe) and Kell antigens and screening for any irregular red cell antibodies should ideally be performed in all cases. Wherever possible Rh (DCcEe) and Kell matched units should be provided for these patients. Besides antibody screening should preferably be repeated before each transfusion episode and if positive an attempt should be made to identify and characterize the antibodies. Thereafter, it is important to always transfuse blood units lacking the antigens against which alloantibodies have developed. [1]

It is the prime responsibility of a facility to offer its patients the safest possible blood for transfusion that is free from various transfusion transmissible infections (TTI's). Although measures such as adoption of strict donor selection criteria, encouragement and maintenance of voluntary non remunerative pool of blood donors and temporary or permanent deferral of those with high-risk behavior judged by the use of questionnaires are a routine practice globally, the final decision on whether or not to use a blood/blood product for transfusion relies on the results of infectious marker tests. The drug and cosmetics act that governs transfusion services in India mandates testing for several infectious agents such as human immune deficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) etc. Although tests for HBV were in place in the early eighties itself, testing for HIV has been enforced since 1989 and more recently since June 2001, testing for HCV has become mandatory. [2] Different centers use different screening modalities and kits that differ in their sensitivities and specificities. In spite of extensive screening protocols, none of the transfusion services across the globe can ensure 100% blood safety from any of these infectious diseases.

The purpose of this study was to determine the prevalence of HIV, HBV, HCV and red cell antibodies among multiple-transfused thalassemic patients in and around the national capital, thereby contributing important and powerful data for epidemiological reasons.

 Materials and Methods

The Department of Transfusion Medicine, Indraprastha Apollo Hospitals, conducted this study in collaboration with the National Thalassemia Welfare Society (NTWS) over a period of 1 year starting February 2011. Thalassemics registered with the NTWS receive transfusions at various centers, both government run and private, in and around Delhi, that differ in their infrastructure, leukoreduction facilities, immunohaematology services and infectious marker screening methodologies.

Pre test counseling, consent and sample collection

The study protocol was approved by our Institutional Review Board and the Ethics Committee at the Indraprastha Apollo Hospitals. Parents of all thalassemic children (or the patient himself / herself in case over 18 years of age) were explained in details the nature and the significance of this testing as well as the medical and social implications of the results thereof. Those willing to participate in the study were required to sign the consent form. Medical and transfusion history were taken from the patient/patient's guardians. Appropriate samples were collected for testing.

Sample testing

All the samples were tested in the Department of Transfusion Medicine at the Indraprastha Apollo Hospitals. Blood grouping and antibody screening was done on the fully automated immunohematology analyzer (Galileo: Immucor Inc., Norcross GA). Blood grouping is based on hemagglutination technique. Any discrepancy or NTD (no type determined) results of blood grouping were resolved manually using the tube techniques. Antibody screening was performed using the capture R ready screen cells (4-cell panel) with solid phase red cell adherence (Capture) technology. The screening cell panels covered the various clinically significant antibodies with homozygous expression of the most important ones. In case of a positive antibody screen, further testing was performed to precisely characterize the irregular antibody/ies and to determine their specificities in case of alloantibodies.

Screening for infectious markers (HIV, HBV and HCV) was performed using the ELISA technique (Evolis Walk away system; BioRad) as well as by nucleic acid testing (NAT) for HIV-1, HCV and HBV viral ribo nucleic acid (RNA) or deoxy ribo nucleic acid (DNA) using the Procleix Ultrio NAT (Chiron). For ELISA testing, fourth generation kits were used for anti-HIV (Genedia HIVAg-Ab), third generation kits for anti-HCV (SP Nanbase) and hepatitis B surface antigen (HBsAg) (Monolisa HBsAg Ultra). All the samples reactive / positive in ELISA were retested in duplicate by ELISA and only those samples found reactive / positive on repeat testing were considered as ELISA positives.

All NAT positive samples were further tested with discriminatory NAT assays separate for HIV, HBV and HCV. Infectious marker positivity was taken as a positive result in either ELISA or NAT or both.

Statistical analysis

The data was tabulated in a Microsoft Excel sheet (Microsoft Corporation Ltd., Redmond, USA). Statistical analysis was done using Chi-square test and p values were calculated. p < 0.05 was considered to be significant.


A total of 462 transfusion dependent thalassemia patients were studied in this series. Of these, 290 were males and 172 females. The age of the patients at the time of sample collection ranged from 7 months to 38 years. Most of the patients (nearly 70%) were diagnosed with thalassemia and started transfusion therapy within the 1 st year of life itself, although the age of the patients, at the time of diagnosis, ranged from 45 days to 7 years. The interval between successive transfusions varied from 10 days to 2 months.

The most common ABO group was B (43.7%) followed by O (29.6%) and A (17.3%). Nearly 95% of the patients were RhD positive.

Antibody screening was positive in 20 cases (4.3%), which were further worked-up to identify the exact specificity of the antibody. The results of the same are provided in [Table 1]. Warm reacting autoantibodies were identified in one patient while all the other 19 had alloantibodies, with or without autoantibodies in their plasma. With an overall alloimmunization rate of 4.1%, alloantibodies against the Rh and Kell antigens were the most frequent. Anti-Kell was the most common antibody identified (nine cases).{Table 1}

Any statistically significant correlation could not be established between the patient's age, gender, frequency of transfusion or age of onset of transfusion and the prevalence of alloantibodies.

Out of the 462 patients, 13 cases (2.8%) were positive for HBsAg by ELISA, 107 (23.1%) were reactive for anti-HCV and 11 (2.38%) for anti-HIV antibodies. Of these, 4 patients demonstrated dual reactivity for anti-HCV and anti-HIV antibodies while 2 cases showed anti-HCV along with HBsAg.

Among the 13 cases positive for HBsAg by ELISA, 11 were also positive by NAT. Further discriminatory assays confirmed the presence of HBV DNA in all the 11 cases.

Out of the 11 cases reactive for anti-HIV antibodies on ELISA, 9 were positive by NAT. Further discriminatory assays revealed HIV RNA in 7 out of these 9 cases. Of the remaining two NAT positive cases, one showed the presence of HCV RNA (ELISA showed dual reactivity for HIV and HCV antibodies) and the other showed the presence of HBVDNA.

Among the 107 cases reactive for anti-HCV antibodies on ELISA, 50 were also positive by NAT. Of these 48 cases showed HCV RNA on discriminatory assay and one case each of HIV RNA and HBV DNA was identified, both of which were dual infections of HCV with HIV and HBsAg respectively on ELISA.

An overall NAT positivity of 14.9% was observed, with a total yield of 6 cases, 3 cases each of HBV DNA and HCV RNA. Among these 6 yield cases, 3 were completely negative by ELISA while the other three yielded an additional infection, besides the one detected by ELISA.

It was further observed that 10 of the 11 HIV reactive cases (90.9%), began transfusions after the testing was made mandatory in 1989. As for HCV 26 (24.3%) started transfusions after June 2001. All the 13 HBsAg positive cases began their transfusions after the commencement of screening for HBsAg.

The age of the patient and the frequency of transfusion showed a significant correlation with infectious marker positivity (P < 0.001) [Figure 1]. However, the marker positivity rates among male patients did not differ significantly from those among females (P = 0.29).{Figure 1}


The therapeutic options available for management of thalassemia major are largely based on lifelong transfusion dependence and iron chelation. [3],[4] Bone marrow transplants are now being promoted in advanced centers as a treatment modality, but the success rates in India are currently unknown.

Among the various inherent risks associated with repeated transfusions lie the risks of contracting TTI's such as HIV, HBV and HCV, among others, and that of formation of alloantibodies against red blood cell antigens by the recipients. [1]

A safe blood supply does not only imply thorough testing for infectious markers, but also protection from alloimmunization. An ideal approach would be to antigen type each and every thalassemic child prior to commencement of transfusion therapy, at least for the Rh and Kell antigens against which the alloantibodies are formed most commonly. Antigen typing at this time is by far the most reliable and once transfusion therapy has started, variable inconsistencies in the phenotypes may be observed due to admixture with the donor's red cells recently received by the patient. As also demonstrated by Singer et al., [1] wherever possible atleast Rh and Kell matched units should be provided. Unfortunately, most transfusion services in India have as yet not been able to provide Rh and kell matched units successfully to their multi-transfused patients. The major reason for this is the lack of molecular blood grouping facilities to accurately determine the Rh phenotypes in these patients. Besides, antibody screening should be carried out at regular intervals to detect any irregular antibodies that the patient may have developed. However, lack of trained manpower and facilities in a resource-limited country like ours make such special provisions for these children difficult.

We have observed in our study an overall alloimmunization rate of 4.1%, which is fairly similar to that observed by Pahuja et al.(3.7%) [5] but lower than that reported in other Indian literature [Table 2]. [4],[6] {Table 2}

The rate of alloimmunization in patients of thalassemia major in different parts of the world ranges from 2.8% to 37% [Table 3]. [1],[7],[8],[9],[10],[11],[12],[13]{Table 3}

Most parents /guardians of the patients did not recollect the exact number of transfusions received by the patient. However, neither the age of the patient at the time of study, age at the onset of transfusion therapy nor the frequency of transfusion, all of which provide a rough estimate of the number of transfusion episodes that the patient must have undergone, showed a statistical correlation with the alloimmunization rates. This is contrary to the finding of Michail-Merianou et al., [14] who found a statistically significant difference between the frequency of alloimmunization in patients starting transfusion therapy early (<12 months) and those starting transfusions at an older age (>12 months). Most literature reports antibodies against the Rh and Kell blood group antigens as the most frequent. [1],[4],[5],[8],[11] In our study, we identified only antibodies against the Rh and Kell antigens, predominantly anti K and anti E. In the Indian population, nearly 97% are Kell negative [15],[16] and the most common Rh phenotype is R1R1 (DCCee), [16] both of which are relatively higher than for the Caucasians. The fact that Kell and "E" are absent in a large proportion of the Indian population and both antigens are highly immunogenic, could account for the corresponding antibodies being relatively more common.

In spite of thorough blood testing absolute safety from TTI's cannot be promised anywhere in the world and a small but definite risk remains. With administrative lapses and economical crisis affecting several smaller transfusion centers, especially in suburban and rural India, quality assurance programs suffer major setbacks, further increasing the risk of contracting the disease during transfusion.

HCV is a leading cause of transfusion related hepatitis, the transmission primarily being parenteral. [17] In our study, we identified an alarming 107 cases (23.1%) seroreactive for anti-HCV antibodies on ELISA. Only 48 cases of these were confirmed as bearing HCV RNA after NAT Discriminatory assays. The remaining 59 cases could either represent a false positive ELISA result or spontaneous clearance of the viral RNA from the patient's serum. Another possible explanation for this non-conformity could be clearing of HCV RNA following treatment in the already diagnosed HCV cases. On questioning the patient's guardians / parents, we could elicit a known history of HCV infection in nearly 30, although the treatment history in many cases was not clear. Besides, there were several parents / guardians, who were unable to provide any specific history of any infectious marker testing or treatment whatsoever. A clear distinction between these three categories therefore could not be made in the current study. Besides there are similar reports in literature stating high number of ELISA reactive cases where viremia has not been detected. [18],[19]

It was also observed that though majority (75.7%) began transfusions before the testing for HCV antibodies was started in June 2001, 24.3% became HCV reactive despite blood being screened for HCV.

Similarly, HBsAg ELISA was positive in 13 cases (2.8%), 11 of which were confirmed by NAT. Five parents gave a positive history for HBV. All our HBsAg reactive thalassemics became positive despite receiving blood screened for HBsAg. Anti HIV antibodies were detected in 11 cases (2.38%), 7 out of which were confirmed by NAT and 6 had a positive history as well.

Transfusion Transmitted HIV is fortunately not a major cause of concern, owing to advanced testing techniques such as 4 th generation ELISA and NAT, better donor interview and screening methodology and increasing awareness about this infection in the general population. Despite this we observed that of the 11 HIV reactive cases in our study, 10 (90.90%) began transfusions after the testing was made mandatory in 1989 and only 1 person began transfusions before 1989.

In case of HBV, since an effective vaccine is available, vaccination prior to commencement of transfusion therapy and timely boosters, can effectively protect against transfusion transmitted HBV. [17] However, no such vaccine is yet available for HCV. As shown in [Table 4], the prevalence of HCV seropositivity in multiply transfused β-thalassemia patients has been observed to vary greatly from 11.1% to 63.8%. [20],[21],[22],[23],[24],[25],[26],[27],[28],[29] Our results are well within this range.{Table 4}

The prevalence of HIV, HBV and HCV in India is high and there is always a possibility of missing early window period and occult infections in blood donors. [30] Our study caters to thalassemics registered for transfusion at various centers in and around Delhi. Different centers use different modalities and kits that differ in their sensitivities and specificities for screening donated blood units for infectious markers. Further, NAT is not as yet a mandatory screening test in India and hence may or may not be available at different centers. This might contribute to the high infectious marker positivity observed in our study population.

In our study, we identified a strong positive correlation between the patient's age and infectious marker positivity. As is expected more the age of the patient, more is likelihood of being multiply transfused, thereby increasing donor exposure and chances of contracting TTI's. Although thalassemia management has drastically improved in recent years, unfortunately the services are not uniform in all the centers. It must also be noted that infections like HIV, HBV and HCV, although most commonly result from parenteral routes, iatrogenic spread through infected needles, IV sets and other equipments is also an important cause especially in these patients who undergo repeated hospitalization and investigations and thus, accounting for the increasing trend of TTI with age. A small dip seen towards the end of the graph [Figure 1] after the age of 25 years is probably due to the fact that there is a drastic fall in the sample size beyond 25 years of age in our study population.

Even though, several measures have been taken by the government and various NGO's in improving thalassemia care, complications associated with transfusion therapy such as immunization, TTI's and iron overload, limited access to safe, adequate and timely blood supply, limited pool of regular, repeat voluntary blood donors that constitute a safe blood supply, lack of proper clinical management, financial constraints, lack of awareness and social stigmas associated with the disease are still the common problems faced by our thalassemic children.

The very fact that a good percentage of infections has occurred in patients starting transfusions after the incorporation of mandatory screening tests indicates that there are definite pitfalls in the screening methodology and a vast scope for improvement in this area.


The authors would like to thank Immucor Inc., (Norcross, GA) for providing reagents for immunohaematological testing, Chiron Corporation (Hemogenomics) for providing kits and technical assistance for nucleic acid testing and BioRad for providing kits for ELISA testing.


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