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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 6  |  Issue : 2  |  Page : 211-217

Prevalence of unexpected red cell antibodies in healthy donor population in a tertiary care center in South Kerala


Department of Transfusion Medicine, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala, India

Date of Submission05-Jun-2021
Date of Decision09-Jul-2021
Date of Acceptance14-Sep-2021
Date of Web Publication30-Nov-2021

Correspondence Address:
Dr. Debasish Gupta
Department of Transfusion Medicine, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/gjtm.gjtm_60_21

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  Abstract 


Background and Objectives: Apart from naturally occurring red cell antibodies Anti-A and Anti-B in human plasma, there are two types of unexpected red cell antibodies: Alloantibodies and auto-antibodies. The presence of these antibodies, alone or in combination, makes difficulties with compatibility testing, thereby delaying in the issue of a compatible blood unit or may reduce posttransfusion red blood cells life span. Antibody screening is mandatory as laid down by the Drug and Cosmetic Act 1940 and Directorate General of Health Services guidelines. This goal of this paper was to study the prevalence of Unexpected Red Cell Antibodies in Healthy Donor Population in a Tertiary Care Centre in South Kerala. Methods: The sample size chosen for this study was 7000 randomly chosen healthy nonremunerated voluntary donors who attended in house and blood donation camps over a period between November 26, 2017, and February 15, 2019. Results: The prevalence of unexpected red cell antibodies was found to be 1 in 1000. Male blood donations were more than female and blood donors among 18–30 years of age were greater in number. There were no cases of direct antiglobulin test positivity encountered. Male: Female ratio is 2.5:1 and males had naturally occurring alloantibodies, whereas females had a previous history of pregnancy. Anti-M and Anti-Lewis antibodies were the most common alloantibodies followed by Anti-Rh (D and C) antibodies and Anti-IH in the current study. Benign cold agglutinins (BCA) were found predominately in the younger male population with a significant seasonal variation noted in the prevalence of these antibodies. Conclusion: All antibodies identified had the ability to cause immune hemolytic transfusion reactions in the recipients. BCA-positive cases were seen more during the winter season and rainy seasons when compared to hot climatic conditions.

Keywords: Allo-immunization, benign cold agglutinins, direct antiglobulin test, healthy donor population, unexpected red cell antibodies


How to cite this article:
Gayathri A M, Gupta D. Prevalence of unexpected red cell antibodies in healthy donor population in a tertiary care center in South Kerala. Glob J Transfus Med 2021;6:211-7

How to cite this URL:
Gayathri A M, Gupta D. Prevalence of unexpected red cell antibodies in healthy donor population in a tertiary care center in South Kerala. Glob J Transfus Med [serial online] 2021 [cited 2022 Aug 10];6:211-7. Available from: https://www.gjtmonline.com/text.asp?2021/6/2/211/331615




  Introduction Top


Naturally occurring Anti-A and Anti-B are the only red cell antibodies that are commonly found in human serum or plasma. All other antibodies are called “unexpected red cell antibodies.”[1] There are two types of unexpected red cell antibodies: Alloantibodies and auto-antibodies. Alloimmunization occurs because of red cells antigenic differences between donor and recipient in previous transfusions or between mother and fetus. Auto-antibodies are those produced against one's own antigens. The presence of these antibodies, alone or in combination, makes difficulties with compatibility testing, thereby delaying in the issue of a compatible blood unit or may reduce post transfusion red blood cells (RBC) life span.[2]

The compatibility test comprises ABO/Rh determination, antibody screen, and cross-match. Type and crossmatch technique/immediate spin crossmatch is routinely practised now which only detects ABO incompatibility between donor RBCs and recipient serum/plasma. Type and screen method is performed only when the recipient has unexpected alloantibodies to detect additional RBC incompatibility.[3] Studies conducted based on these unexpected antibodies have largely concentrated on multiply transfused patient populations or antenatal women. Alloimmunization in these groups has a reported incidence up to 60%, with an up to four-fold increased risk of multiple antibodies compared to the risk of single antibodies.[4] However, such studies related to the healthy donor population are not done extensively in India. The incidence of RBC alloimmunization depends on the demography and characteristics of the population being studied. The specificity, Ig class, thermal range, and concentration of the antibody can predict its clinical significance, as well as patient's individual immune response, is also significant factors. The balance between sensitivity and specificity can be influenced by the methods and technologies selected. It is not possible to detect all potentially clinically significant antibodies or to avoid detecting all clinically insignificant antibodies.[5]

When unexpected antibodies are present, as indicated by positive screening tests, they must be identified. At a minimum, this involves testing the patient's serum against a panel of fully phenotyped reagent red cell samples as well as the patient's own cells.[6] A recent study suggests that a positive direct antiglobulin test (DAT) result in a healthy blood donor may be a marker of risk of future development of malignancy.[6] All these points toward the need of type and Screening systems to be followed routinely in transfusion practices rather than Type and Matching systems. Antibody screening is mandatory as laid down by the Drug and Cosmetic Act 1940 and Directorate General of Health Services guidelines.[7]

Aims and objectives

Our Institute is supported by 100% voluntary blood donations. This study is aimed to know the prevalence of Unexpected Red Cell Antibodies in Healthy Donor Population in a Tertiary Care Center in South Kerala, India.


  Materials and Methods Top


Place and duration of study

Using a cross-sectional study design, randomly chosen 7000 voluntary donors coming for blood donation during a period from November 26, 2017, to February 15, 2019, were screened for the presence of unexpected red cell antibodies. Donor selection was done as per national guidelines.

Ethics

The project was cleared by the Institutional Ethics Committee of SCTIMST vide No. SCT/IEC/1075/AUGUST-2017, dated 17.10.2017

Sample type

Two ml ethylenediaminetetraacetic acid sample was collected for performing DAT, along with other routine samples.

Technology, techniques, and methods

All donors were screened for red cell antibodies by DAT and In DAT (IAT) primarily along with routine “O” papanisation during reverse grouping using conventional tube technique (CTT). For IAT, pooled “O” cells (from 3 phenotyped donors cells with R1R1, R2R2, and rr, respectively) were used and check cells were used for validation of both DAT and IAT results. All negative DAT and IAT readings were confirmed microscopically for suspicion of weak positivity. Auto-control using donor cells and serum is required for checking the validity of the test as well as confirmation of auto-agglutinins after incubation of the same at different temperatures (4°C, 22°C, and 37°C).

Antibody screening, identification, and titer

If IAT is positive, then antibody screening and identification are done using 3 cell panel (ReaCell panel, REAGENS Kit. Wysocki u. 1. Budapest, Hungary) and 11 cell panel (Panel A reagent red cells, Ortho Clinical Diagnostics, Inc. 1001 US Highway 202, Raritan, NJ 08869 USA and ID-DiaPanel-P, Biorad, Diamed GmbH, 1785 Crossier FR, Switzerland), respectively. The double dilution method with phosphate-buffered saline (PBS) gives IgM + IgG antibody titer. To distinguish IgG from IgM antibodies, DTT treatment is done by treating one part of serum with one part of DTT followed by 1 h incubation at 37°C and later performing double dilution method (with PBS) to estimate IgG titer alone. Secretor status was also performed in three donors with anti-IH and anti-Lewis antibodies. If DAT is positive then the possibility of alloimmunization is done after auto-adsorption. Donors with unexpected antibodies were called back for notification and another fresh sample were taken for retesting and given proper counseling as well as follow-up of the donor was also performed.

Benign cold agglutinins

The prevalence of benign cold agglutinins (BCA) among the blood donor population was estimated and the effect of seasonal change on the prevalence of BCA was also studied. The Chi-square test was used to find out the significance in the gender- and age-wise distribution of occurrence of BCA.

Categorization of participants

The categorization is done according to the gender and age group of the participants. The seasonal variation is also considered in the benign cold antibody session.

Statistical analysis

Percentage and mean values calculated in MS Excel and test of association performed using online software OpenEpi.


  Results Top


The sample size chosen for this study was 7000 healthy nonremunerated voluntary donors who were randomly chosen over a period between November 26, 2017, and February 15, 2019. Among the total subjects, 5046 (72.08%) were males and 1954 (27.2%) were females. The age-wise distribution based on gender is shown in [Graph 1]. The males are more among all age groups and the number of blood donors is more in the 18–30 years category and minimal donors below 50 years of age.



Among the 7000 samples analyzed only 7 cases were positive for clinically significant unexpected red cell antibodies. Hence, the prevalence of alloimmunization in the healthy voluntary donor population in the current study is 1/1000. There was no case of DAT positive in this study.

Out of the 7 positive cases of abnormal red cell antibodies, 2 cases were anti-M (28.57%), 2 cases were anti-Lewis (28.57%), 1 case of anti-D, 1 case of anti-C, 1 case of anti-IH. Anti-IH was determined by special technique as mentioned in [Table 1]. The testing of the prevalence with respect to age group or sex is not possible because of this very low prevalence value. The details of the 7 cases that showed unexpected reactions are given in [Table 2].
Table 1: Reactivity patterns of cold autoantibody anti-IH in a 19-year-old first-time blood donor having A negative blood group

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Table 2: Brief of significant red cell antibodies among blood donors during the study period

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Benign cold agglutinins

Cold autoantibodies are often detected in healthy individuals, with the majority being benign and IgM in nature. These antibodies react best at 4°C. The prevalence of BCA at 4°C for auto-control as well as with pooled O cells were 56.40% and 65.24% respectively and 31.16% of donors showed reaction in both auto-control and pooled O cells. The Chi-square test using R and C table in OpenEpi (online software) was used to find out the significance among the gender- [Graph 2] and age-wise distribution [Graph 3] of occurrence of BCA. It was found that there was a statistically significant difference observed in gender distribution (P < 0.001) and age-wise distribution (P < 0.001) stating that the male blood donors had more prevalence of BCA than female blood donor and also the age group of 18–30 years had more BCAs.



The prevalence of IgM antibodies reacting at 22°C were 0.46% for auto-control and 0.69% for pooled O cells, respectively [Graph 4]. Here also, the prevalence was more with males compared to females.



Another interesting finding observed in the prevalence of BCA was the seasonal variations observed in the frequency of cases. Kerala is located close to the equator and hence experiences a tropical climate. Trivandrum, the capital city of Kerala, is a coastal city with temperatures stay fairly consistent throughout the year. The overall average daily temperature does not drop <27°C or rise >29°C in the year. In the present study, the BCA cases were more during monsoon seasons and the winter season [Graph 5]. The cases during the summer season were relatively lower when compared to the cold weather. During August to September 2018, Kerala was hit by heavy rainfall and floods and may be due to this, the cases of BCA were more during this time interval which was otherwise the spring season in Kerala with a warm weather.



As per the Institute policy, the plasma and platelet components from the donors who had unexpected red cell antibodies were discarded and the red cell concentrates were washed and issued for transfusion.


  Discussion Top


Various worldwide literature calls out the importance of donor unexpected antibody screening and also a topic of debate whether to make this mandatory for the recipient safety. In our study, since all antibodies detected had potential to cause HTR, unexpected red cell antibody screening should be an imperative element in assuring transfusion safety.

Red cell all-immunization rates

The incidence of RBC alloimmunization depends largely upon the demography of the population studied.[8] Global literature state that the reported incidence of erythrocyte allo-antibodies in the donor population varies from 0.32% to 2.4%.[2],[3],[4],[5],[9],[10],[11],[12],[13],[14],[15],[16] REDS III study (Recipient Epidemiology and Donor Evaluation Study) gives inference of a positive antibody screen noted overall in 0.5% of donations and 0.77% of blood donors at the four centers in the US included in the study over the 4.5-year study period, utilizing methodologies ranging from solid phase to gel card to tube testing.[13]

In the present study, we followed the conventional tube technique (CTT) which is less sensitive than column agglutination test (CAT)/solid phase hematological procedures. CTT has been the mainstay for antibody detection in pretransfusion testing for many years and believed to be the gold standard, it has got its own limitations.[17] The endpoints of the reaction are unstable; reading and grading require a high level of expertise leading to inter-observer variation. CAT has an objective reading phase; its results are standardized and reproducible. The lack of washing phase in CAT decreases the potential for false weak or negative reactions and makes it ideal for automation. However, the incidence of false positives is more with CAT when compared to conventional tube methods.[18],[19],[20]

From the current study on healthy voluntary donors, the prevalence of alloantibodies is 0.1%. Our institute follows a stringent selection of blood donors attending our camp and in-house blood donations.

Nature of alloantibodies

A study on the Shaoguan area describes that the frequency of unexpected antibodies in females was higher than that in male and Rh blood group antibodies (47.6%) such as Anti-D, Anti-E, and Anti-c were common.[21] Another US study on male military veterans, the prevalence of red cell antibodies was in the increasing order of K, E, D, Lea, Fya, c, C, P1, Jka, and Leb.[20] Another study from Minnesota, the most frequent alloantibodies were E, Lea, K, D, Leb, M, P1, Fya, C, and c in the order.[9] A study on the frequency of allo-immunization in the Kuwaiti population describes that five most frequently identified alloantibodies were Anti-D, Anti-E, Anti-K, Anti-Lea and Anti-Leb in the descending order.[15] Malaysian study on prevalence and specificities of red cell alloantibodies among blood recipients shows that Anti-E antibody comprised the most common alloantibody followed by the Anti-Lea antibodies and the Anti-M antibody.[10] A study done in the Southern Thai population showed the following antibodies frequency wise as most common – Anti-Mia, Anti-E, Anti-Lea, Anti-c and Anti-Leb.[11]

Several Indian studies mostly in the Northern population are available in the literature. 0.09% prevalence of unexpected red cell antibodies was noted among healthy donors in the Delhi population.[16] The most common alloantibodies were of the MNS system (39.13%) followed by Anti-D (13%) and Anti-Lewis (10.8%). Another similar study involving blood donors in northern India showed the prevalence of 0.05% alloantibodies was observed.[21] A tertiary care center in northern India reported positive screening cells and pooled O cells in 0.27% of donors, 66.08% of these donors had autoantibodies, 1 had autoantibodies with underlying alloantibody Anti-Jka (0.001%), and 0.09% had alloantibodies alone in their plasma. Anti-M was the most common antibody (18.94%) identified, followed by Anti-D (9.25%).[22] In Indian literature on red cell alloantibodies, males have a higher incidence when compared to females (mostly with a history of pregnancy). In the Indian scenario, female blood donations are fewer when compared to male blood donations and hence this may be a reason that the female population with positive red cell alloimmunization maybe the tip of the iceberg phenomenon.

In our study, the prevalence of unexpected antibodies (0.1%) was much lower than in other national and international studies. Moreover, the most common alloimmunization identified out of total 7 cases were Anti-M and Anti-Lewis antibodies (2 out of 7 each), followed by Rh antibodies (Anti-C and Anti-D, (1 out of 7 each) and Anti-IH (only one case) which is comparable with other Indian studies. Here, the males had higher incidence of red cell alloimmunization and all of them were devoid of a prior sensitization via blood transfusion indicating that all 5 cases were naturally occurring alloantibodies. Whereas females with unexpected antibodies belong to Rh antibodies (Anti-D and Anti-C) and result from a previous history of pregnancy.

First time donor versus Repeat donors

Myhre et al. reported that an average of 0.14% of the total units of blood yearly contain unexpected red cell antibodies and this group of donors includes mainly first-time donors (84%) but contains, in addition, a few repeat donors who had not given blood since their screening program began or who had been recently sensitized.[12] Since 25% of the total donor population in their study composed of first-time donors, the percentage of antibodies found in first-time donors is 0.56%. In our study, 57.14% of cases and 42.86% cases of unexpected red cell antibodies were seen in the first-time donors and repeat donors, respectively. Out of three repeat donors who came positive, two had naturally occurring alloantibodies and one was sensitized due to a history of pregnancy in the past.

Gender distribution of alloantibodies

In our study, we found that alloimmunization was common among males than females, but males had naturally occurring antibodies and females had alloantibodies as a result of past pregnancy. Male-to-female ratio of alloantibodies was found to be 8.25:1 in Garg et al. study[16] and 4.84:1 in Makroo et al. study.[22] These studies also pointed out the female allo-immunization resulted out from past pregnancy, whereas males had mostly naturally occurring alloantibodies followed by history of blood transfusion.

Direct antiglobulin test positivity among study population

In the present study, there was no case of auto-immunization or DAT positivity in a total of 7000 healthy blood donors studied. Issitt and Anstee[23] reported that of blood donors with a positive DAT and IgG coating the RBCs, 5%–10% will develop AIHA, 20%–25% will become DAT negative over time, and 60%–70% will remain DAT positive, but hematologically normal. The incidence of strongly positive DATs in healthy individuals was 1:3300 in a Canadian study.[24] Another French study in 1980, the incidence was found to be 1: 13,000.[25] The incidence of a positive DAT among the healthy blood donors was 0.04% in an Indian study done in the northern Indian population by Tiwari et al.[26] Makroo et al.[22] state that in their study, the prevalence of DAT positivity among healthy donors was 0.18% and in Kaur et al. study, it was 0.05%.[8]

Benign cold agglutinin distribution

Cold autoantibodies are often detected in healthy individuals, with the majority being benign which react more strongly at 0°C–4°C than at warmer temperatures. Pathological cold autoantibodies are characterized by wide thermal amplitude and/or high antibody titers.[27] Joshi et al. state that cold autoantibodies observed in their study were found among blood donors with no apparent health problems and may be due to exposure to infectious agents as antibody stimulants in past was open as the population in the city of Surat was exposed to vector-borne infections such as malaria, gastroenteritis, and infective hepatitis during the contemporary period of this study.[28] In the current study, we found the prevalence of BCAs at 4°C for auto-control as well as with pooled O cells were 56.40% and 65.24%, respectively, and 31.16% of donors showed reaction in both auto-control and O cells. Since Kerala is a tropical country with near perennial rainfall and vector-borne diseases like dengue, etc., as well as frequent viral upper respiratory infections, the occurrence of cold agglutinins may be associated with the above. Cold agglutinins active at 22°C are of very low prevalence (<1%) from the current study.


  Conclusion Top


Out of 7000 healthy voluntary blood donor population, the prevalence of unexpected red cell antibodies was found to be 1 in 1000. Male blood donations were more when compared to female and blood donors among 18–30 years of age were maximum in number in the study population. There were no cases of DAT positivity encountered in this study. Male: Female ratio is 2.5:1 and males had naturally occurring alloantibodies, whereas females had a previous history of pregnancy. Anti-M and anti-Lewis antibodies were most common alloantibodies followed by Anti-Rh (D and C) antibodies and Anti-IH in the current study. BCAs were found predominately in the younger male population with a significant seasonal variation noted in the prevalence of these antibodies.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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