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Kheiri Ardahaei F, Yari F, Amo Hossein B. Compairing the Immune Response Induced in Peripheral Blood Mononuclear Cells by RhD+ Red Blood Cells and their Membrane Extract in Culture Medium. bloodj 2025; 22 (4) :297-304
URL: http://bloodjournal.ir/article-1-1604-en.html
Compairing the Immune Response Induced in Peripheral Blood Mononuclear Cells by RhD+ Red Blood Cells and their Membrane Extract in Culture Medium
Faeze Kheiri Ardahaei , Fatemeh Yari * , Behnaz Amo Hossein
Keywords: In vitro, Immunizaltion, Antibody Response, Red Blood Cells
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References:
  1. Avent ND, Reid ME. The Rh blood group system: a review. Blood 2000; 95(2): 375-87. [DOI:10.1182/blood.V95.2.375] [PMID]
  2. Huang CH, Liu PZ, Cheng JG. Molecular biology and genetics of the Rh blood group system. Semin Hematol 2000; 37(2): 150-65. [DOI:10.1016/S0037-1963(00)90040-4] [PMID]
  3. Flegel WA. The genetics of the Rhesus blood group system. Blood Transfus 2007; 5(2): 50-7.
  4. Wagner FF, Flegel WA. Review: the molecular basis of the Rh blood group phenotypes. Immunohematology 2004; 20(1): 23-36. [DOI:10.21307/immunohematology-2019-419] []
  5. Xie X, Fu Q, Bao Z, Zhang Y, Zhou D. Clinical value of different anti-D immunoglobulin strategies for preventing Rh hemolytic disease of the fetus and newborn: A network meta-analysis. PLoS One 2020; 15(3): e0230073. [DOI:10.1371/journal.pone.0230073] [PMID] []
  6. Nadarajan VS. Serological analysis of Rh antigens: how far can we go? Annals of Blood 2023; 8. [DOI:10.21037/aob-23-30]
  7. Kim HY, Stojadinovic A, Izadjoo MJ. Immunization, hybridoma generation, and selection for monoclonal antibody production. Methods Mol Biol 2014; 1131: 33-45. [DOI:10.1007/978-1-62703-992-5_3] [PMID]
  8. Leenaars M, Hendriksen CF. Critical steps in the production of polyclonal and monoclonal antibodies: evaluation and recommendations. ILAR J 2005; 46(3): 269-79. [DOI:10.1093/ilar.46.3.269] [PMID]
  9. Tomimatsu K, Shirahata S. Antigen-specific in vitro immunization: a source for human monoclonal antibodies. Methods Mol Biol 2014; 1060: 297-307. [DOI:10.1007/978-1-62703-586-6_15] [PMID]
  10. Kato M, Yan H, Tsuji NM, Chiba T, Hanyu Y. A method for inducing antigen-specific IgG production by in vitro immunization. J Immunol Methods 2012; 386(1-2): 60-9. [DOI:10.1016/j.jim.2012.08.019] [PMID]
  11. Wijkhuisen A, Savatier A, Cordeiro N, Léonetti M. Production of antigen-specific human IgGs by in vitro immunization. BMC Biotechnol 2016; 16: 22. [DOI:10.1186/s12896-016-0253-1] [PMID] []
  12. Kheiri Ardahaei F, Yari F. Effective Factors in Creating Immunization Against a Desired Antigen in the Culture Medium. J Iran Blood Transfus 2024; 21(4): 330-41. [Article in Farsi] [DOI:10.61186/bloodj.21.4.333]
  13. Howe JG, Stack G. Relationship between immunogenicity and protein structure at amino acid substitution sites of blood group antigens. Blood 2025; 146(4): 504-17. [DOI:10.1182/blood.2024025071] [PMID]
  14. Hendrickson JE, Tormey CA, Shaz BH. Red blood cell alloimmunization mitigation strategies. Transfus Med Rev 2014; 28(3): 137-44. [DOI:10.1016/j.tmrv.2014.04.008] [PMID]
  15. de Almeida R, Nakamura CN, de Lima Fontes M, Deffune E, Felisbino SL, Kaneno R, et al. Enhanced immunization techniques to obtain highly specific monoclonal antibodies. MAbs 2018; 10(1): 46-54. [DOI:10.1080/19420862.2017.1331804] [PMID] []
  16. Kanof ME, Smith PD, Zola H. Isolation of whole mononuclear cells from peripheral blood and cord blood. Curr Protoc Immunol 2001; Chapter 7: 7.1.1-7.1.8.
  17. Dargahi T, Yari F, Rezaei N. The source of HLA molecules on platelets: Does platelets adsorb soluble HLA molecules from their environment? Iran J Ped Hematol Oncol 2019; 9(4): 236-43. [DOI:10.18502/ijpho.v9i4.1572]
  18. KÖHler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256(5517): 495-7. [DOI:10.1038/256495a0] [PMID] []
  19. Borrebaeck CA. Development of in vitro immunization in murine and human hybridoma technology. J Pharm Biomed Anal 1987; 5(8): 783-92. [DOI:10.1016/0731-7085(87)80096-1] [PMID]
  20. Steeghs L, Kuipers B, Hamstra HJ, Kersten G, van Alphen L, van der Ley P. Immunogenicity of outer membrane proteins in a lipopolysaccharide-deficient mutant of Neisseria meningitidis: influence of adjuvants on the immune response. Infect Immun 1999; 67(10): 4988-93. [DOI:10.1128/IAI.67.10.4988-4993.1999] [PMID] []
  21. Stack G, Tormey CA. Estimating the immunogenicity of blood group antigens: a modified calculation that corrects for transfusion xposures. Br J Haematol 2016; 175(1): 154-60. [DOI:10.1111/bjh.14175] [PMID]
  22. Zimmermann M, Rose N, Lindner JM, Kim H, Gonçalves AR, Callegari I, et al. Antigen Extraction and B Cell Activation Enable Identification of Rare Membrane Antigen Specific Human B Cells. Front Immunol 2019; 10: 829. [DOI:10.3389/fimmu.2019.00829] [PMID] []
  23. Hendrickson JE, Tormey CA. Understanding red blood cell alloimmunization triggers. Hematology Am Soc Hematol Educ Program 2016; 2016(1): 446-51. [DOI:10.1182/asheducation-2016.1.446] [PMID] []
  24. Burnet Institute. Methods of Delivery to Antigen-Presenting Cells:  Development of New and Improved Vaccines. Molecular Pharmaceutics 2007; 4(1): 1-3. [DOI:10.1021/mp060102h] [PMID]




Compairing the Immune Response Induced in Peripheral
Blood Mononuclear Cells by RhD+ Red Blood Cells and their
Membrane Extract in Culture Medium

Faezeh Kheiri Ardahaei1, Fatemeh Yari1         , Behnaz Amo Hossein



1Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran

Received: 2025/11/13
Accepted: 2026/01/07
 





     http://dx.doi.org/10.61186/bloodj.21.4.333

    



Citation:
Kheiri Ardahaei F, Yari F, Amo Hossein B. Compairing the Immune Response Induced in Peripheral Blood Mononuclear Cells by RhD+ Red Blood Cells and their Membrane Extract in Culture Medium.  J Iran Blood Transfus. 2025: 22 (4): 297-304
    



Correspondence: Yari F., Professor of Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine.
P.O.Box: 14665-1157, Tehran, Iran.
Tel: (+9821) 82052237
E-mail: f.yari@
1- Acridine Orange
1- Biological safety cabinet
1- Platelet Concentrate
2- Food and Drug Administration
3- Normal Skin Flora
4- Platelet Rich Plasma-Platelet Concentrate
5- Eosin-Methylene blue
6- Thioglycolate
1- Acridine Orange
1- Biological safety cabinet
1- Platelet Concentrate
2- Food and Drug Administration
3- Normal Skin Flora
4- Platelet Rich Plasma-Platelet Concentrate
5- Eosin-Methylene blue
6- Thioglycolate
tmi.ac.ir  		 A B S T R A C T
Background and Objectives
The Rh blood group system is one of the most significant and complex antigenic systems of blood group. The production of specific antibodies against RhD is of great importance for diagnostic and therapeutic applications. This study aims to evaluate the immune response generated in peipheral blood mononuclear cells (PBMCs) by red blood cells containing the RhD antigen and their membrane extracts in a cell culture medium.
Materials and Methods
In this experimental study, PBMCs were isolated from two whole blood units with O- blood type using a density gradient method and Ficoll reagent. After a 40-minute exposure to L-leucine methyl ester (2.5 mM) and subsequent washing, the cells were exposed to red blood cells containing the RhD antigen or their membrane extract in a culture medium. Their ability to induce the production of specific antibodies was assessed using the ELISA method.
Results
Following immunization of PBMCs with O+ red blood cells or their membrane extract, antibody production was quantitatively assessed using ELISA method. The mean concentration of total antibodies was 159.3±51.19 ng/mL in response to membrane extract (n=4) and 136.5±38.9 ng/mL following stimulation with O+ RBC (n=2). In addition, the concentration of RhD-specific antibodies was measured as 38.17±10.58 ng/mL in response to membrane extract and 18.75±2.95 ng/mL in response to O+ RBC cells. The data indicate that both antigenic forms are capable of inducing an immune response.  Wilcoxon method showed that the differences between two groups of antigens in both evaluated total antibodies and RhD-specific antibodies were not significant.
Conclusions 
Based on the findings of this study, it seems that the membrane extract of RBC plays a more effective role in the immunogenicity against the RhD antigen in compare to O+ RBC cells but the difference were not significant. Based on the data, the use of both membrane extracts and the whole red blood cells could be helpful in the context of immunization strategies in culture conditions.
Key words: In vitro, Immunizaltion, Antibody Response, Red Blood Cells
 
Copyright © 2025 Journal of Iranian Blood Transfusion, Published by Blood Transfusion Research Center.
This work is licensed under a Creative Common Attribution-Non Commercial 4.0 International license.







 

 
Type of Study: Research | Subject: Imunology
References
1. Avent ND, Reid ME. The Rh blood group system: a review. Blood 2000; 95(2): 375-87. [DOI:10.1182/blood.V95.2.375] [PMID]
2. Huang CH, Liu PZ, Cheng JG. Molecular biology and genetics of the Rh blood group system. Semin Hematol 2000; 37(2): 150-65. [DOI:10.1016/S0037-1963(00)90040-4] [PMID]
3. Flegel WA. The genetics of the Rhesus blood group system. Blood Transfus 2007; 5(2): 50-7.
4. Wagner FF, Flegel WA. Review: the molecular basis of the Rh blood group phenotypes. Immunohematology 2004; 20(1): 23-36. [DOI:10.21307/immunohematology-2019-419] []
5. Xie X, Fu Q, Bao Z, Zhang Y, Zhou D. Clinical value of different anti-D immunoglobulin strategies for preventing Rh hemolytic disease of the fetus and newborn: A network meta-analysis. PLoS One 2020; 15(3): e0230073. [DOI:10.1371/journal.pone.0230073] [PMID] []
6. Nadarajan VS. Serological analysis of Rh antigens: how far can we go? Annals of Blood 2023; 8. [DOI:10.21037/aob-23-30]
7. Kim HY, Stojadinovic A, Izadjoo MJ. Immunization, hybridoma generation, and selection for monoclonal antibody production. Methods Mol Biol 2014; 1131: 33-45. [DOI:10.1007/978-1-62703-992-5_3] [PMID]
8. Leenaars M, Hendriksen CF. Critical steps in the production of polyclonal and monoclonal antibodies: evaluation and recommendations. ILAR J 2005; 46(3): 269-79. [DOI:10.1093/ilar.46.3.269] [PMID]
9. Tomimatsu K, Shirahata S. Antigen-specific in vitro immunization: a source for human monoclonal antibodies. Methods Mol Biol 2014; 1060: 297-307. [DOI:10.1007/978-1-62703-586-6_15] [PMID]
10. Kato M, Yan H, Tsuji NM, Chiba T, Hanyu Y. A method for inducing antigen-specific IgG production by in vitro immunization. J Immunol Methods 2012; 386(1-2): 60-9. [DOI:10.1016/j.jim.2012.08.019] [PMID]
11. Wijkhuisen A, Savatier A, Cordeiro N, Léonetti M. Production of antigen-specific human IgGs by in vitro immunization. BMC Biotechnol 2016; 16: 22. [DOI:10.1186/s12896-016-0253-1] [PMID] []
12. Kheiri Ardahaei F, Yari F. Effective Factors in Creating Immunization Against a Desired Antigen in the Culture Medium. J Iran Blood Transfus 2024; 21(4): 330-41. [Article in Farsi] [DOI:10.61186/bloodj.21.4.333]
13. Howe JG, Stack G. Relationship between immunogenicity and protein structure at amino acid substitution sites of blood group antigens. Blood 2025; 146(4): 504-17. [DOI:10.1182/blood.2024025071] [PMID]
14. Hendrickson JE, Tormey CA, Shaz BH. Red blood cell alloimmunization mitigation strategies. Transfus Med Rev 2014; 28(3): 137-44. [DOI:10.1016/j.tmrv.2014.04.008] [PMID]
15. de Almeida R, Nakamura CN, de Lima Fontes M, Deffune E, Felisbino SL, Kaneno R, et al. Enhanced immunization techniques to obtain highly specific monoclonal antibodies. MAbs 2018; 10(1): 46-54. [DOI:10.1080/19420862.2017.1331804] [PMID] []
16. Kanof ME, Smith PD, Zola H. Isolation of whole mononuclear cells from peripheral blood and cord blood. Curr Protoc Immunol 2001; Chapter 7: 7.1.1-7.1.8.
17. Dargahi T, Yari F, Rezaei N. The source of HLA molecules on platelets: Does platelets adsorb soluble HLA molecules from their environment? Iran J Ped Hematol Oncol 2019; 9(4): 236-43. [DOI:10.18502/ijpho.v9i4.1572]
18. KÖHler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256(5517): 495-7. [DOI:10.1038/256495a0] [PMID] []
19. Borrebaeck CA. Development of in vitro immunization in murine and human hybridoma technology. J Pharm Biomed Anal 1987; 5(8): 783-92. [DOI:10.1016/0731-7085(87)80096-1] [PMID]
20. Steeghs L, Kuipers B, Hamstra HJ, Kersten G, van Alphen L, van der Ley P. Immunogenicity of outer membrane proteins in a lipopolysaccharide-deficient mutant of Neisseria meningitidis: influence of adjuvants on the immune response. Infect Immun 1999; 67(10): 4988-93. [DOI:10.1128/IAI.67.10.4988-4993.1999] [PMID] []
21. Stack G, Tormey CA. Estimating the immunogenicity of blood group antigens: a modified calculation that corrects for transfusion xposures. Br J Haematol 2016; 175(1): 154-60. [DOI:10.1111/bjh.14175] [PMID]
22. Zimmermann M, Rose N, Lindner JM, Kim H, Gonçalves AR, Callegari I, et al. Antigen Extraction and B Cell Activation Enable Identification of Rare Membrane Antigen Specific Human B Cells. Front Immunol 2019; 10: 829. [DOI:10.3389/fimmu.2019.00829] [PMID] []
23. Hendrickson JE, Tormey CA. Understanding red blood cell alloimmunization triggers. Hematology Am Soc Hematol Educ Program 2016; 2016(1): 446-51. [DOI:10.1182/asheducation-2016.1.446] [PMID] []
24. Burnet Institute. Methods of Delivery to Antigen-Presenting Cells:  Development of New and Improved Vaccines. Molecular Pharmaceutics 2007; 4(1): 1-3. [DOI:10.1021/mp060102h] [PMID]
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