Global Journal of Transfusion Medicine

: 2019  |  Volume : 4  |  Issue : 2  |  Page : 208--213

Investigation of HLA-DRB1*15:03 and HLA-DRB1*09:01 alleles to determine predisposition to development of ABO antibodies (Anti-A and Anti-B) amongst blood donors of type “O” in Southern Iran

Bentolhoda Mozafari1, Ali Akbar Pourfathollah2, Leila Kohan1,  
1 Department of Biology, Islamic Azad University, Arsanjan Branch, Arsanjan, Iran
2 Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

Correspondence Address:
Dr. Ali Akbar Pourfathollah
Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran


Introduction: Agglutination in red blood cells can be associated with a number of complications. These complications are related to the amount of antibody titer produced. Some genetic factors, such as HLA-DRB1*15:03 and HLA-DRB1*09:01 alleles, are involved in the production of antibody titers. Aims and Objectives: The aim of this study was to investigate the causative allele of the high and dangerous titer antibodies (anti-A and anti-B) among blood donors in Bandar Abbas. Methods: Antibody A and B levels were measured by titration on 121 serum samples of blood type O donors in 2 incubations at room temperature and anti-human globulin tube method. Titers higher than 512 and the same number of participants with a titer of <64 were selected and whether or not genotypes 901 and 1503 were found. After titration, sample DNAs were extracted, and then, amplification-refractory mutation system–polymerase chain reaction was used to determine allelic polymorphism. Results: The antibody titer was found to be elevated in this study, but there was no significant relationship between the presence or absence of genotype 901 (HLA-DRB1*9:01) and genotype 1503 (HLA-DRB1*15:03) and the high titer of anti-A and anti-B antibodies. Discussion and Conclusion: Finally, studies have shown that a critical titer should be determined for antibodies to the ABO blood system in order to provide blood transfusion safety. However, the higher the titers of anti-A and anti-B, the safety of transfusion medicine is also provided.

How to cite this article:
Mozafari B, Pourfathollah AA, Kohan L. Investigation of HLA-DRB1*15:03 and HLA-DRB1*09:01 alleles to determine predisposition to development of ABO antibodies (Anti-A and Anti-B) amongst blood donors of type “O” in Southern Iran.Glob J Transfus Med 2019;4:208-213

How to cite this URL:
Mozafari B, Pourfathollah AA, Kohan L. Investigation of HLA-DRB1*15:03 and HLA-DRB1*09:01 alleles to determine predisposition to development of ABO antibodies (Anti-A and Anti-B) amongst blood donors of type “O” in Southern Iran. Glob J Transfus Med [serial online] 2019 [cited 2019 Dec 7 ];4:208-213
Available from:

Full Text


Blood is a physiological fluid that plays an important role in the human body. The most important role considered is to provide a substrate for the transport of oxygen and nutrients to the cells of the body, as well as the transfer of waste material, which can also have important immunological functions.[1] One of the side effects of blood transfusions is the occurrence of immunological reactions in which about 8% of red blood cell (RBC) donors may produce new antibodies after a period of transmission.[2] Overall, >108 million units of blood are donated each years worlldwide which is used for replacing lost blood or for other clinical conditions. There is no reaction between donor and recipient in majority of cases. Hemolytic reactions occur with shock, renal failure, and ultimately death.[3],[4],[5] The difference between RBCs and erythrocytes is due to their surface antigens, on which safe blood transfusion depends. However, Rh is also important for blood transfusion.[6] ABO blood group classification systems include A, B, AB, and O, which are represented by Rh+ or Rh−.[7] Each of these blood groups also produces antibodies that are of the immunoglobulin M (IgM), IgG, and IgA class. In general, the predominant antibody is in the serum of individuals with IgM Class A and B and the predominant antibody in IgG Class O.[8] In the ABO blood group system, some factors, including platelets, express the ABO blood group antigens to a minor extent, which can be both intrinsic to the platelet structure and an acquired structure derived from plasma. Platelet components are usually present in the blood plasma of the donor population.[9],[10] Due to the low plasma volume of platelet packages and the low ABO antigen content in platelets, unlike ABO RBCs, ABO compatibility is not observed in platelet infusion and platelet injection is performed without ABO compatibility.[11],[12] Some patients with blood group A or B who received platelets from group O have developed RBC lysis following platelet administration.[13]

The main histocompatibility complex contains the most information for the proper delivery of antigens. In humans, this complex is known as human leukocyte antigens (HLAs), whose Class II function is to bind to alien antigenic fragments and supply them to TCD4+ cells. Class II consists of at least 9 functional genes, some of which include DRB1/3/4/5, DQB1, and DPA1.[14],[15]

Polymorphisms are widely found in the antigen-binding domain of these molecules, but in HLA-DR, they are restricted to the DRβ chain (DRB1, DRB3, DRB4, and DRB5 genes) and DRα. Rheumatoid arthritis is one of the best-known examples for understanding the genetic association between HLA-II alleles and autoimmune disorders.[16],[17] One of these alleles is HLA-DRB1*09:01, which is more common in people with RBC alloantibody who produce anti-E.[18] In addition, the HLA-DRB1*15:03, HLA-DRB1*11, and HLA-DRB1*09:01 alleles are associated with alveolarization in patients with thalassemia. As a result, the frequency of the HLA-DRB1*15:03 allele is significantly different among thalassemic patients.[19] Given the importance of anti-blood group antibody titers in the present study, the HLA-DRB1*15:03 and HLA-DRB1*09:01 alleles were used to determine the alleles predisposing to high titration and dangerous antigen–antibody production. Blood group samples were collected from blood donors with O blood group in Bandar Abbas, Southern Iran.



In this cross-sectional, case–control study, between 2018 and 2017, 121 blood donors were enrolled in the Blood Transfusion Center in Bandar Abbas city with O blood type. Blood samples were collected from healthy individuals with ethical considerations and their complete consent. Samples were randomly selected.

Determination of antibody level by titration

Titration was done using two fold serial dilution method in test tubes from 1 to 1024 and continued further if needed. Pooled A1 cells and pooled B cells negative by Direct antiglobulin test (DAT) were used for titration of test serum. The cells were negative for Rh (D) while testing for allo Anti-D. The DAT test was performed on them and then washed to make the supernatant completely transparent. The 2%–5% RBC suspension was prepared by Alsever's solution. After serial dilution, the following procedures were used for each sample according to AABB guidelines:

Room temperature (RT) incubation methodTube anti-human globulin or indirect antiglobulin test (IAT) method.

The phenomenon of prozone was important in our study and could have caused errors in our experiment, as reactions at higher serum concentrations appeared to be weaker than higher dilutions which was taken care of by doing further dilutions as needed.

Hemagglutination tests used RT and IAT techniques to measure IgM and IgG, respectively, but IgM and IgG antibodies can, however, cause RBC agglutination at RT and can also activate the complement system at 37°C.

DNA analysis

We used the salting-out method to extract DNA from the collected samples. We investigated the quantity and quality of extracted DNAs using 0.8% agarose gel electrophoresis and NanoDrop device (Thermo Scientific; NanoDrop 2000). Amplification-refractory mutation system–polymerase chain reaction (ARMS-PCR) method was used to evaluate HLA-DRB1*09:01, HLA-DRB1*15:03, and β-actin target genes.

In the present study, DNA was extracted from the collected blood samples using the salting-out method and then extracted. First, it collects blood from the patients in the tubes and lysates the RBCs using a cell lysis solution (sucrose, MgCl2, Tris-HCl, Triton ×100) as well as a centrifuge, and white blood cells were separated from them.

To the resulting precipitate 1000 μL of lysate solution (sodium dodecyl sulfate [SDS], ethylenediaminetetraacetic acid, Tris) was added. The proteins were then digested using proteinase K and precipitated by adding 10% SDS solution and sodium chloride, which were reduced by ethanol 70% aqueous polarity and DNA appeared. Finally, we collected the resulting DNA for subsequent steps. The extracted DNA was examined at a wavelength of 260–280 nm, and the purity of the extracted DNA was between 1.6 and 2, which was desirable for extraction.

Primer design and analysis of amplification-refractory mutation system–polymerase chain reaction for DRB1 15:03 and DRB1 09:01 alleles

In this study, three primer pairs were designed using AlleleID software (Premier Biosoft, California, USA) for the genes of DRB1 09:01, DRB1 15:03, and β-Actin as internal control genes and then for specificity analysis at NCBI Blast Site. The specification of the primers is given in [Table 1].{Table 1}

ARMS-PCR reaction mixture was prepared in a final volume of 20 μL for both alleles. To amplify the HLA-DRB1*09:01 allele PCR program to perform the ARMS-PCR procedure included 5 min of initial denaturation at 95°C, 30 cycles of replication (denaturation of 60 s and 95°C, binding of 90 s and 55°C, and replication at 120 s and 72°C) and the final replication was 7 min and 72°C. Concentrations used for the ARMS-PCR reaction included: each PCR microtube contained 1 μl of each primer (10 pmol/l), 0.5 μl of master mix, and 1 μl of DNA (50 ng), which reached a final volume of 20 μl. PCR program conditions for ARMS-PCR HLA-DRB1*15:03 allele also included 5-min initial denaturation at 95°C, 30 cycles of replication step (denaturation of 60 s and 95°C, binding of 90 s and 55°C, and replication at 2 min and 72°C) and the final replication was 7 min and temperature 72°C. The concentrations used to perform the ARMS-PCR reaction in this allele were similar to those of HLA-DRB1*09:01.

After the PCR reaction, the PCR products were electrophoresed for rs11053646 and rs1050283 polymorphisms for 25 min at 93 V. As can be seen, all bands have good resolution and no extra band or smear [Figure 1].{Figure 1}

Statistical analysis

HLA-DRB1*15:03 and HLA-DRB1*09:01 alleles are identified among donor populations as predisposing alleles to the production of high and dangerous titers of the main blood group antigens (anti-A and anti-B). The blood of Bandar Abbas with O blood type was analyzed using Chi-square statistical analysis (χ2) and logistic regression with a 95% confidence interval (SPSS version 16, IBM, USA).


General characteristics of the population under study

In this study, the age of the participants ranged from 18 to 60 years. Their mean age was 35.04 ± 8.02 years. Furthermore, out of 121 participants, 90 (74.4%) were male and 31 (25.6%) were female.

Anti-A titer and genotype HLA-DRB1*09:01

The findings showed that out of 121 individuals, 84 had HLA-DRB1*09:01 genotype and 37 did not. The Chi-square test results also showed that there was a significant relationship between the presence or absence of HLA-DRB1*09:01 gene and the high or low anti-A titers (P = 0.032). In other words, 20.2% of those with genotype HLA-DRB1*09:01 and 10.8% of those without genotype HLA-DRB1*09:01 had high A titers [Table 2]. Therefore, there is a significant relationship between those with genotype HLA-DRB1*09:01 and those without those with high A titers.{Table 2}

Anti-A titer and genotype HLA-DRB1*15:03

The results showed that out of 121 individuals, 58 had HLA-DRB1*15:03 genotype and 63 did not. The results showed that there was no significant relationship between the presence or absence of HLA-DRB1*15:03 gene and the high or low level of anti-A (P = 0.353). In other words, 20.7% of those with genotype HLA-DRB1*15:03 and 14.3% of those without genotype HLA-DRB1*15:03 had high A titers [Table 2].

Anti-B titer and genotype HLA-DRB1*09:01

Of the 99 individuals with anti-B, 12 had genotype 901 and 87 did not. The results of the Chi-square test (χ2) showed that there was no significant relationship between the presence or absence of HLA-DRB1*09:01 gene and the high or low antibody B titers [Table 2] (P = 0.250). Among those with genotype HLA-DRB1*09:01 and those without genotype HLA-DRB1*09:01, 25% and 12.6%, respectively, had high B titers. Therefore, those with genotype HLA-DRB1*09:01 and those without those with high titers of anti-B are no different.

Anti-B titer and genotype HLA-DRB1*15:03

The results of this titer showed that out of 99 individuals with anti-B, 37 had genotype HLA-DRB1*15:03 and 62 did not. Furthermore, there was no significant relationship between the presence or absence of HLA-DRB1*15:03 gene and the high or low antibody titer (P = 0.292). Among those with genotype HLA-DRB1*15:03 and those without genotype HLA-DRB1*15:03, 18.9% and 11.3%, respectively, had high titers of anti-B [Table 2].

The logistic regression results also show that the HLA-DRB1*09:01 and HLA-DRB1*15:03 genes are influenced by high and low titers because their significance is <0.05. In the corresponding interpretation, for each unit increase in the HLA-DRB1*09:01 gene polymorphism, the odds of high titer over low titer in anti-A despite the HLA-DRB1*15:03 gene being constant is 0.104. However, in the HLA-DRB1*15:03 gene, the chances of high titers are about three times higher.

It was also observed from the results of anti-B that by increasing each unit of the HLA-DRB1*09:01 gene, the odds of high titers were four times higher than those of low titers, which is also true for HLA-DRB1*15:03 gene.


Antibodies are one of the immune system factors that provide good conditions for survival and life. Antibodies to the bloodstream may alter some of the immunological conditions that may be incompatible with certain pregnancies or blood transfusions, such as agglutination or hemolytic conditions. In addition to alloimmunization previous contact is the source of antibody production. In many cases, there is a natural (native) antibody that is involved in antibody production. Antibodies also have physiological and homeostasis roles. The abnormal antibody titers in some immunological reactions can be indicated by some genetic factors in the blood group system, including the HLA-DRB1*15:03 and HLA-DRB1*09:01 alleles: Further, alleles can be associated with the production of blood antigens, such as HLA-DRB1*15:03, which is a major risk factor for alloimmunization in patients with multiple blood transfusions such as sickle cell anemia. On the other hand, HLA-DRB1*09:01 is protective against alloantibody formation.

It is noteworthy that recently there have been reports of death of a recipient of blood following ABO incompatible plasma, in that recipients have died during incompatible transfusions. In fact, donor plasma has a hemolytic effect on the recipient's red cells in a blood transfusion containing a high titer of the recipient's anti-RBC antibody.[20] In addition to the importance of high antibody titers in the production of platelet products, it is important that the plasma components of donors with O blood group have antibodies that may damage the RBCs and connective tissues of the recipients. For this reason, donors with blood type O are randomly titrated by age and sex to determine the best source for apheresis products and blood transfusions associated with anemia. There is evidence that HLA typing is associated with an increased response to RBC antigens.[21],[22],[23]

Finally, it is reported that the HLA-DRB1-15:03 allele is associated with an increased risk of global RBC allo-immunization. However, some individuals do not develop a vesicular allele despite repeated injections that may express specific genotypes that resist immunization against RBC antigens,[24] for example, individuals with the HLA-DRB1*09:01 genotype. They are resistant to immunization.[19] The present study also found that individuals with genotypes 901 and 1503 were highly correlated with anti-A and anti-B titers, as well as the high chance of a low titer in Anti A in the 1503 gene, which was 0.104. That is, in the 901 gene, the high titer is lower than the low anti-A titer. However, in the high 1503 titers, the gene is about three times lower in the low titers. A study by Darvishi et al. showed that there was no association between HLA-DRB1*09:01 and allo-immunization. In addition, the heterozygous combination of HLA DRB1*09:01 with DRB1*15:03 may cause some autoimmune diseases such as rheumatoid arthritis.[26] Other studies have shown that HLA-DRB1*09:01 is found to be negative in most alloantibody patients[19] and the development of multiple RBC antibodies in association with the HDR*15:03 allele which plays an important role in RBC alloantigen responses.[27] Studies have also shown that HLA-DRB1*15:03 is a major risk factor for allo-immunization in patients with a previous history of blood transfusion and sickle cell anemia.[28]

HLA genes are the most polymorphic genes in the genome of all species. The presence of certain specific HLA loci makes a person more resistant or susceptible to certain diseases than others. There are various opinions on the relationship between HLA and disease. The association between HLA alleles and susceptibility or resistance to some infectious diseases such as tuberculosis, malaria, and parasitic diseases such as cutaneous leishmaniasis and schistosomiasis has so far been identified.


In the present study, we investigated the alleles of HLA-DRB1*15:03 and HLA-DRB1*9:01 to determine the predisposing allele of high-titer production and dangerous antibodies to the main blood group antigens (anti-A and anti-B). The results showed that there was a significant relationship between the HLA-DRB1*15:03 and HLA-DRB1*9:01 alleles and the anti-A and anti-B antibodies. It is noteworthy that no similar study has been performed in Iran so far, and this is the first study to investigate the aforementioned alleles in order to determine the predisposing allele to produce high titers of antibodies to blood antigens.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


1Chang YJ, Ho CY, Zhou XM, Yen HR. Determination of degree of RBC agglutination for blood typing using a small quantity of blood sample in a microfluidic system. Biosens Bioelectron 2018;102:234-41.
2Sawierucha J, Posset M, Hähnel V, Johnson CL, Hutchinson JA, Ahrens N. Comparison of two column agglutination tests for red blood cell antibody testing. PLoS One 2018;13:e0210099.
3Nilghaz A, Ballerini DR, Guan L, Li L, Shen W. Red blood cell transport mechanisms in polyester thread-based blood typing devices. Anal Bioanal Chem 2016;408:1365-71.
4Shrivastava SR, Shrivastava PS, Ramasamy J. Thank you for saving my life: Blood donation matters. J Res Med Sci 2016;21:23.
5Daniels G, Bromilow I. Essential Guide to Blood Groups. First edn. IBM, USA: Blackwell Pub.; 2007.
6Mujahid A, Dickert FL. Blood group typing: From classical strategies to the application of synthetic antibodies generated by molecular imprinting. Sensors (Basel) 2015;16. pii: E51.
7Huet M, Cubizolles M, Buhot A. Red blood cell agglutination for blood typing within passive microfluidic biochips. High Throughput 2018;7. pii: E10.
8van Furth R, Schuit HR, Hijmans W. The immunological development of the human fetus. J Exp Med 1965;122:1173-88.
9Consensus conference. Platelet transfusion therapy. JAMA 1987;257:1777-80.
10Quillen K, Sheldon SL, Daniel-Johnson JA, Lee-Stroka AH, Flegel WA. A practical strategy to reduce the risk of passive hemolysis by screening plateletpheresis donors for high-titer ABO antibodies. Transfusion 2011;51:92-6.
11de França ND, Poli MC, Ramos PG, Borsoi CS, Colella R. Titers of ABO antibodies in group O blood donors. Rev Bras Hematol Hemoter 2011;33:259-62.
12Ogasawara K, Ueki J, Takenaka M, Furihata K. Study on the expression of ABH antigens on platelets. Blood 1993;82:993-9.
13Romphruk AV, Cheunta S, Pakoate L, Kumpeera P, Sripara P, Paupairoj C, et al. Preparation of single donor platelet with low antibody titers for all patients. Transfus Apher Sci 2012;46:125-8.
14Moutsianas L, Jostins L, Beecham AH, Dilthey AT, Xifara DK, Ban M, et al. Class II HLA interactions modulate genetic risk for multiple sclerosis. Nat Genet 2015;47:1107-13.
15Balassa K, Andrikovics H, Remenyi P, Batai A, Bors A, Kiss KP, et al. The potential role of HLA-DRB1*11 in the development and outcome of haematopoietic stem cell transplantation-associated thrombotic microangiopathy. Bone Marrow Transplant 2015;50:1321-5.
16Raychaudhuri S, Sandor C, Stahl EA, Freudenberg J, Lee HS, Jia X, et al. Five amino acids in three HLA proteins explain most of the association between MHC and seropositive rheumatoid arthritis. Nat Genet 2012;44:291-6.
17Viatte S, Plant D, Raychaudhuri S. Genetics and epigenetics of rheumatoid arthritis. Nat Rev Rheumatol 2013;9:141-53.
18Tian L, Hou L, Wang L, Xu H, Xiao J, Ying B. HLA-DRB1*09:01 allele is associated with anti-E immunization in a Chinese population. Transfusion 2018;58:1536-9.
19Hoppe C, Klitz W, Vichinsky E, Styles L. HLA type and risk of alloimmunization in sickle cell disease. Am J Hematol 2009;84:462-4.
20Services USDoH. Fatalities Reported to FDA Following Blood Collection and Transfusion: Annual Summary for Fiscal Year; 2012.
21Hendrickson JE, Desmarets M, Deshpande SS, Chadwick TE, Hillyer CD, Roback JD, et al. Recipient inflammation affects the frequency and magnitude of immunization to transfused red blood cells. Transfusion 2006;46:1526-36.
22Reviron D, Dettori I, Ferrera V, Legrand D, Touinssi M, Mercier P, et al. HLA-DRB1 alleles and jk(a) immunization. Transfusion 2005;45:956-9.
23Chiaroni J, Dettori I, Ferrera V, Legrand D, Touinssi M, Mercier P, et al. HLA-DRB1 polymorphism is associated with kell immunisation. Br J Haematol 2006;132:374-8.
24Zalpuri S, Zwaginga JJ, van der Bom JG. Risk factors for alloimmunisation after red blood cell transfusions (R-FACT): A case cohort study. BMJ Open 2012;2. pii: e001150.
25Darvishi P, Sharifi Z, Azarkeivan A, Akbari A, Pourfathollah AA. HLA-DRB1*15:03 and HLA-DRB1*11: Useful predictive alleles for alloantibody production in thalassemia patients. Transfus Med 2019;29:179-84.
26Shimane K, Kochi Y, Suzuki A, Okada Y, Ishii T, Horita T, et al. An association analysis of HLA-DRB1 with systemic lupus erythematosus and rheumatoid arthritis in a Japanese population: Effects of *09:01 allele on disease phenotypes. Rheumatology (Oxford) 2013;52:1172-82.
27Maluskova A, Mrazek F, Pauliskova M, Kovarova P, Koristka M, Jindra P, et al. Association of HLA-DRB1 and HLA-DQB1 with red-blood-cell alloimmunization in the Czech population. Vox Sang 2017;112:156-62.
28Yari F, Sobhani M, Sabaghi F, Zaman-Vaziri M, Bagheri N, Talebian A. Frequencies of HLA-DRB1 in Iranian normal population and in patients with acute lymphoblastic leukemia. Arch Med Res 2008;39:205-8.