Abstract
INTRODUCTION:
Alloimmunization, a dreadful complication of blood transfusion, varies between populations and geographical regions. The antibody prevalence of the South Indian population was studied as the data were scarce. Rh–Kell matching is traditionally followed, but other alloantibodies may have higher prevalence because of geographical variation.
MATERIALS AND METHODS:
It is a retrospective study on alloantibody prevalence in a tertiary care hospital in southern India from April 2021 to March 2023. The frequency, prevalence, and proportion of them are studied.
RESULTS:
Out of 6640 patients, 1128 (16.98%) were Rh D negative. One hundred and thirty-three patients have 155 alloantibodies, among which anti-D is the most common (1.02%), followed by anti-C (0.21%) and S (0.19%), with none having anti-Kell alloantibodies. Here, 1.58% of alloantibodies are formed against Rh and Kell blood group system, whereas 1.94% are against Rh MNS and system.
CONCLUSION:
Anti-M and anti-S are common alloantibodies prevalent in the southern India population besides Rh. The focus should be given to Rh–MNS-matched blood in our population.
Keywords: Alloantibody, antibody identification, antibody screening, anti-M, anti-S, Rh–Kell
Introduction
Blood transfusion is a lifesaving management, but it has complications and adverse reactions. One of the significant issues is alloimmunization. Alloimmunization occurs when the donor and recipient red blood cells’ (RBCs) antigenic profile differs posttransfusion. Transplantation and pregnancy can also lead to alloimmunization by the same phenomenon. Even if all alloantibodies are focused against the lacking antigen in the patient/recipient, most are not harmful. It can be classified as immunoglobulin (Ig) M type and IgG type. Among these, IgG-type alloantibodies are usually clinically significant. They can cause hemolytic transfusion reactions (acute or delayed) and hemolytic disease of the fetus and newborn (HDFN). It is often a dreadful situation when we cannot rule out the presence of alloantibodies or find a compatible unit for the patient in emergencies.
Alloantibody prevalence varies depending on the population, demographic characteristics, and the features of the geographical area under study. It can be mitigated by indirect antiglobulin crossmatching, using phenotype-matched units by either partial or extended matching.[1] In India, Rh and Kell matching was used as a traditional protective measure to reduce alloimmunization following Western literature. Many studies suggest that other alloantibodies are more prevalent than anti-Kell in unmatched transfusions.[2,3,4,5] The present study aims to determine the prevalence and proportion of alloantibodies in high-risk patients.
Materials and Methods
Study design
This study was a single-center retrospective observational study conducted in the department of transfusion medicine at a tertiary care hospital-based blood bank in southern India. It is a 2500-bedded hospital with all super- and subspecialties. The study period was from April 2021 to March 2023.
Sampling
The data were collected retrospectively. In total, 6640 blood samples were received for indirect antiglobulin tests from various specialties, which include high-risk patients such as Rh-negative pregnancy, autoimmune disorders, and patients requiring multiple or chronic transfusion who are more prone to form alloantibodies. Among these, 1435 were from the obstetrics department covering pregnant females, and 5205 were from nonpregnant patients. The data of these samples were collected from the immunohematology registers in a decoded manner and documented in the online data sheet after masking the personal identity. It was then separated for pregnant and nonpregnant individuals and tabulated. The results were calculated using the high-risk population as a denominator in and around Pondicherry territory.
Principle of tests
The samples submitted for high-risk patients’ direct and indirect antiglobulin tests were worked up and entered in the immunohematology register. The workup includes ABO blood grouping, Rh typing by forward and reverse method, and thermal amplitude test by conventional tube technique. Direct and indirect antiglobulin tests, antibody screening, identification, and antiglobulin crossmatch were made by column agglutination technique in Ortho Clinical Diagnostics (New Jersey, USA). The procedures were done as per standard operating protocol. Our center follows type and screen protocol for elective surgery and stable nonbleeding patients requiring transfusion within 48 h. Emergencies are dealt with antihuman globulin crossmatch unless life-threatening or dead. Emergencies are issued with immediate spin or saline crossmatch compatible blood products. The samples with positive antibody screen or incompatible on crossmatching were also worked up, entered into the same register, and made an online copy of it. In the case of multiple alloantibodies, adsorption and elution studies with select cells (D + C−, D − C+, c + E−, etc.) and autoantibody with alloantibody are solved by either auto- or alloadsorption. Since our donor units are not phenotyped, if any alloantibody is detected, crossmatch compatible units will be phenotyped and, if found to be negative, will be issued for transfusion.
Calculation of proportion and prevalence
The results were shown by the frequency of alloantibodies denoted in numbers, followed by prevalence and proportion of specific alloantibodies in percentage. The prevalence of antibodies was calculated by dividing the number of antibodies by the total population denoted in percentage whereas proportion by the number of each antibody by the total number of antibodies in percentage.
Statistical analysis
The results were calculated using Microsoft Office Excel version 2013.
Results
Out of 6640 samples from high-risk patients suspecting alloantibodies, 1128 (16.98%) were from Rh D-negative individuals. One hundred and thirty-three patients (2%) were found to have 155 alloantibodies, of which 69 are Rh D negative. Out of these, anti-D is the most prevalent alloantibody (1.02%), C being the second-most common (0.21%), followed by S (0.19%), M (0.16%), and E (0.15%), as mentioned in Table 1. Among 1128 Rh D-negative patients, anti-D is the most common alloantibody (5.85%), followed by C (0.88%), G (0.44%), S (0.08%), and Lea (0.17%). Twenty-one individuals have multiple alloantibodies with D with C (0.16%) followed by D with G (0.04%) and C with E (0.03%). Anti-D with S, E with Jka, Leb with E, M with S, D with C, and G (0.01% each) were also seen [Table 2]. Here, 1.58% of alloantibodies are formed against RH and Kell, whereas 1.94% are formed against Rh and MNS blood group system.
Table 1.
Alloantibody prevalence in high-risk patients
| Antibody | n | Percentage | Prevalence |
|---|---|---|---|
| D | 68 | 43.87 | 1.02 |
| C | 14 | 9.03 | 0.21 |
| S | 13 | 8.39 | 0.19 |
| M | 11 | 7.09 | 0.16 |
| E | 10 | 6.45 | 0.15 |
| c | 8 | 5.16 | 0.12 |
| Lea | 8 | 5.16 | 0.12 |
| Leb | 7 | 4.51 | 0.10 |
| G | 5 | 3.22 | 0.07 |
| Fyb | 3 | 1.93 | 0.04 |
| Fya | 2 | 1.29 | 0.03 |
| Jka | 2 | 1.29 | 0.03 |
| Jkb | 2 | 1.29 | 0.03 |
| N | 1 | 0.64 | 0.01 |
| e | 1 | 0.64 | 0.01 |
| Total | 155 | 100 | 2.33 |
Table 2.
Frequency of multiple alloantibodies in high-risk patients
| Alloantibody (multiple) | Frequency |
|---|---|
| D, C | 11 |
| D, G | 3 |
| c, E | 2 |
| D, S | 1 |
| E, Jka | 1 |
| Leb, E | 1 |
| M, S | 1 |
| D, C, G | 1 |
| Total | 21 |
Of 1435 samples from pregnant females, 1033 (71.98%) were from the Rh D-negative population. Eighty-three (5.78%) patients had alloantibodies, whereas 66 were Rh D negative. Anti-D was the most common alloantibody (4.45%), present in all 60 Rh D-negative patients either alone or in combination with anti-D except two cases with only clinically significant Lea. Anti-C is the second-most common (0.69%), followed by M, S, G, E (0.27%), and Lea (0.20%). Among 1033 Rh D-negative patients, anti-D is the most common alloantibody (6.2%), followed by C (0.87%), G (0.38%), S (0.09%), and Lea (0.19%). Fourteen individuals had multiple alloantibodies. D with C (0.62%) was the most common followed by D with G (0.13%), D with S, Leb with E, D with C and G the third (0.06% each) [Table 3] which were confirmed by adsorption and elution studies protocol of it was given in Figure 1.
Table 3.
Alloantibody prevalence in pregnant patients
| Frequency | Proportion | Prevalence | |
|---|---|---|---|
| D | 64 | 65.30 | 4.45 |
| C | 10 | 10.20 | 0.69 |
| M | 4 | 4.08 | 0.27 |
| S | 4 | 4.08 | 0.27 |
| G | 4 | 4.08 | 0.27 |
| E | 4 | 4.08 | 0.27 |
| LE A | 3 | 3.06 | 0.20 |
| JK A | 1 | 1.02 | 0.06 |
| JK B | 1 | 1.02 | 0.06 |
| Small E | 1 | 1.02 | 0.06 |
| LE B | 1 | 1.02 | 0.06 |
| FY B | 1 | 1.02 | 0.06 |
| Total | 98 | 100 | 6.82 |
Figure 1.
Flowchart to solve patients with anti-D, C specificity to detect anti-G
In the 5205 non-pregnant individuals, 50 (0.96%) patients have been found to have alloantibodies. Out of these anti S being the most common, M & c shares the second common, followed by Leb & E, Lea, C and D [Table 4]. Seven individuals had multiple alloantibodies with small C with E, D with C, D with G, M with S, E with Jka.
Table 4.
Alloantibody prevalence among nonpregnant patients
| Antibody | Frequency | Percentage | Prevalence (total among 5205 cases) |
|---|---|---|---|
| S | 9 | 16.36 | 0.17 |
| M | 7 | 12.72 | 0.13 |
| c | 7 | 12.72 | 0.13 |
| E | 6 | 10.90 | 0.11 |
| Leb | 6 | 10.90 | 0.11 |
| Lea | 5 | 9.09 | 0.09 |
| C | 4 | 7.27 | 0.07 |
| D | 4 | 7.27 | 0.07 |
| Fya | 2 | 3.63 | 0.03 |
| Jkb | 1 | 1.81 | 0.01 |
| Jka | 1 | 1.81 | 0.01 |
| Fyb | 1 | 1.81 | 0.01 |
| G | 1 | 1.81 | 0.01 |
| N | 1 | 1.81 | 0.01 |
| Total | 55 | 100 | 1.05 |
Discussion
Alloimmunization is the term used to describe the antibody formation to nonself-RBC antigens, usually IgG type. Alloantibodies are caused by transfusion, transplantation, and pregnancy. Alloimmunization depends on the characteristics of RBCs and transfusion recipients. Among these biochemical and conformational properties of RBC include RBC antigen characteristics, number of transfusions, pregnancies, mode of exposure, and number of antigens per red cell. Patient factors such as age, gender, diagnosis, disease severity, comorbidities, inflammation, immune status and human leukocyte antigens also plays important role in alloimmunisation.[6] The rate of alloimmunization is 0.11% to 0.58% in the hospital-based population.[7] Risk becomes higher if conditions coexist in an individual. The rate of alloimmunization is higher in chronically transfused patients such as sickle cell anemia and thalassemia between 4.8% and 11.4%.[7] Alloantibodies’ formation is higher in previously alloimmunized individuals.[2] The alloimmunization can delay getting compatible blood since antigen-negative antiglobulin crossmatch compatible unit is to be transfused lifelong even if the future antibody screen shows negative antibody screening. In this study, our alloimmunization rate was higher than previous studies since our study population includes samples from high-risk groups. Since most of the samples were from the obstetrics department, our rate of alloimmunization was similar to the study done by Sidhu et al., where the prevalence of red cell antibodies in the pregnant population is 2.67% out of that 80% of their study participants are Rh negative.[8] This coincides with our study, where we had 71.98% Rh-negative pregnant females among pregnant females who developed anti-D with or without anti-C and anti-G. Our analysis is also comparable with the study done by Agrawal et al., where the alloimmunization rate is 2.71% in multi-transfused individuals,[9] similar to our alloimmunization rate (2%). Among the alloantibodies, most of the antibodies were formed against the Rh blood group system followed by MNS, which was similar to a study done by Thakral et al.,[2] Philip et al.,[3] Kaur et al.,[4] and Tiwari et al.[5] in various regions of our country.
In the pregnant females, 65.3% of our study population had anti-D, whereas Gottvall and Filbey found that allo-anti-D is present in 60% of their pregnant study population.[10] The higher rate may be because most of the samples submitted are from the obstetrics department as a routine antenatal screening for D-negative mothers. There was also a significant increase in the degree of alloimmunization with an increase in the gravida status.[11] HDFN caused by anti-K is generally severe since K-antigens are present in early RBC progenitors, hemolyzed by anti-K, causing anemia and reticulocytopenia by suppressing erythropoiesis.[12,13] Since Kell is immunogenic and there is a higher prevalence of anti-Kell antibodies in the Western population, it has been followed traditionally to match Rh and Kell antigens, so the alloantibody formation could be minimized. No anti-Kell alloantibodies were found in our study. In our population, Kell antigen’s prevalence is only 0.15%, whereas almost 100% carry k-antigen.[14] This possibly explains the lower prevalence of anti-Kell, supported by other studies from various parts of India. Next to the Rh blood group system, most of the alloantibodies are formed against the MNS system.[15] M, N, S, and s antigens have a prevalence of 84.57%, 58.51%, 49.47%, and 93.09% of antigens in southern India, respectively.[14] Hence, matching Rh and MNS blood group antigens as a prophylactic partial matching in unimmunized multi-transfused patients will be better than traditional Rh and Kell matching. Rh and MNS matching has been shown to reduce the alloimmunization induced by transfusion by up to 75% in a study done by Gogri et al. in the Western Indian population at the National Institute of Immunohematology.[16] In the present study, 81.3% of alloantibodies are against Rh and MNS blood group system, which includes alloimmunization not only by transfusion but also by other factors such as pregnancy and transplantation in our study. The rate of alloimmunization is higher in the Rh blood group system, followed by the MNS system. As the anti-Kell proportion is very low, it is time to screen and match M, S, and Rh during partial matching of blood group antigens for chronically transfused individuals and individuals with a higher rate of alloimmunization rather than traditional Rh and Kell matching, which has been followed till date.
Conclusion
The present study has shown a higher prevalence of anti-S and anti-M in our population next to Rh antibodies. Anti-Kell antibodies were the least. However, the result of our study can be validated with a larger sample size from different parts of South India.
Conflicts of interest
There are no conflicts of interest.
Funding Statement
Nil.
References
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