Continuing Education Activity

The Duffy blood group system, regulated by the ACKR1 gene, includes the key antigens Fy^a and Fy^b, which are expressed on red blood cells as well as on the linings of blood vessels, lungs, kidney tubules, and Purkinje cells in the brain. These antigens function as chemokine receptors, which are vital in modulating inflammation and immune responses. Antibodies directed against Duffy antigens can cause hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. The Duffy-null phenotype, characterized by the absence of both Fy^a and Fy^b antigens, protects against Plasmodium vivax but has also been associated with increased susceptibility to certain infections, such as HIV. Understanding and recognizing a patient’s Duffy status is essential to ensure safe transfusion practices, assess prenatal risk, and guide personalized care.

This activity reviews the Duffy blood group system and its clinical implications in transfusion medicine, hematology, and infectious diseases. This activity also explores the genetic diversity and immunological function of Duffy antigens, including their roles in immune modulation and malaria resistance. In addition, this activity also helps healthcare professionals integrate laboratory findings into clinical practice to support safer, more informed decision-making and promote personalized, equitable healthcare delivery. This activity also emphasizes the importance of interprofessional collaboration among healthcare providers to develop skills in interpreting Duffy antigen testing, managing transfusion-related complications, and applying this knowledge to various clinical scenarios.

Objectives:

• Identify the key components of the Duffy blood group system, including the Fy^a and Fy^b antigens and their role in immune response.

• Implement appropriate transfusion protocols based on a patient’s Duffy antigen status to minimize the risk of hemolytic transfusion reactions.

• Select appropriate laboratory tests to evaluate Duffy antigen status and interpret results in clinical practice.

• Collaborate with the interprofessional healthcare team to ensure safe transfusion practices and accurate risk assessment in patients with different Duffy phenotypes.

Introduction

Blood group antigens are substances found on the surface of red blood cells (RBCs) that serve as markers, distinguishing different blood types. Common blood group antigens include the ABO system, the Rhesus (Rh) factor, Duffy, and Kell. The Duffy blood group system is determined by the ACKR1 gene (OMIM 613665), which is located on chromosome 1q23.2.[1] The 2 main codominant alleles, FY*A and FY*B, produce the Fy^a and Fy^b antigens, which are expressed on the surface of RBCs, vasculature endothelial cells, alveolar epithelial cells, collecting tubules in the kidneys, and the surface of Purkinje cells in the brain. Duffy antigens are absent on platelets, lymphocytes, monocytes, and granulocytes.[2][3] Single-nucleotide differences in the ACKR1 coding region result in a change in a single amino acid, which determines the presence of the Fy^a and Fy^b antigens. Fy^a contains a guanine at nucleotide 125 and a glycine at amino acid 42. Alternatively, Fy^b has an adenine at nucleotide 125 and an aspartate at amino acid 42.[4] 

Patients lacking Fy^a and Fy^b antigens are considered Duffy-null. These individuals are typically heterozygous for a genetic variation, rs2814778-C (c.-67T>C), in the ACKR1 promoter region, which results in the loss of Duffy antigens on the surface of RBCs without affecting their expression on other cells. In most Duffy-null individuals, a mutation in the FY*B allele, sometimes referred to as FY*B^ES or the erythrocyte silent allele, specifically the 67T>C mutation (or position 46, depending on the numbering system) near the gene's transcription initiation site, causes this phenotype. This mutation disrupts a binding site for the erythroid transcription factor GATA1, which is crucial for expressing the ACKR1 gene in erythroid cells. This mutation prevents the expression of Duffy glycoproteins on erythrocytes while allowing the expression of Fy^b on nonerythroid cells.[4] Rarely, the mutation may also involve the FY*A allele.

The Duffy antigens, also known as the atypical chemokine receptor 1 (ACKR1), are glycoproteins that function as chemokine receptors. They bind to chemokines released during inflammation and attract immune system cells to areas of damage. These chemokines include acute inflammation (C-X-R) and chronic inflammation (C-C) chemokines, interleukin (IL)-8, and regulated on activation, normal T expressed and secreted (RANTES). The ACKR1, previously the Duffy antigen receptors for cytokines (DARC) or CD234, is the primary attachment site for the malarial parasite Plasmodium vivax. Patients who are phenotypically Fy(a−b−) are resistant to infection.

Although Fy^a and Fy^b are the main determinants of the Duffy system, a total of 5 antigens are present in the Duffy blood group system: Fy^a, Fy^b, Fy3, Fy5, and Fy6.[5][6][7] Anti-Fy^a and anti-Fy^b are the most clinically significant antibodies. These antibodies are the leading causes of immediate or delayed hemolytic transfusion reactions (HTRs). Anti-Fy3 is a less common cause of delayed HTRs. Anti-Fy^a is the most common cause of hemolytic disease of the fetus and newborn (HDFN), whereas anti-Fy3 and anti-Fy^b are uncommon causes of HDFN.[8]

Antibodies against Duffy blood group antigens are primarily immunoglobulin G (IgG), and IgM type is uncommon. These antibodies typically develop following exposure to Duffy antigens during blood transfusions, pregnancy, or organ transplantation. Anti-Fy^a antibodies are the most common among Duffy antibodies.[9][10]

Most Duffy antibodies react at body temperature, which can cause hemolysis and make them clinically significant. These antibodies can lead to both acute and delayed types of HTRs and HDFN.[11][12] The Duffy blood group system demonstrates a dosage phenomenon, where individuals with a homozygous phenotype have more antigens per RBC than those with a heterozygous phenotype. As a result, Duffy antibodies react more strongly in patients with homozygous phenotypes, Fy(a−b+) or Fy(a+b−), than in those with the heterozygous phenotype Fy(a+b+).[13]

The predominant Duffy phenotypes are Fy(a+b−), Fy(a+b+), Fy(a−b+), and Fy(a−b−). The Fy^a and Fy^b antigens are most commonly found in White patients and patients of Asian descent, and least commonly in Black patients. Additionally, 67% of Black individuals have the Duffy-null phenotype, which is rare in White patients (see the Table below).[14][15][16]

An additional minor Duffy phenotype, the Fy^x or [Fy(b+x)], is a variant caused by a missense mutation in ACKR1. The FY*X allele encodes the Fy^b antigen and weakly expresses Fy^b, which may not be detected by anti-Fy^b.

Table. Worldwide Duffy Blood Group Antigenic Frequencies 

Function

ACKR1 is a non-signaling chemokine receptor found on RBCs, capillaries, and postcapillary venule endothelial cells. Structurally related to the G-protein–coupled receptor (GPCR) family, it primarily functions as a chemokine scavenger during inflammatory processes. ACKR1 helps regulate inflammation by binding, storing, and releasing chemokines, which influence both the intensity of the inflammatory response and the recruitment of neutrophils.[17]

ACKR1 interacts with chemokines from the C-C and C-X-C families, such as IL-8 (CXCL8), melanoma growth stimulatory activity-α (CXCL1), monocyte chemotactic protein-1 (CCL2), and RANTES (CCL5). ACKR1 also facilitates neutrophil migration across the endothelium during inflammation.[18] Despite its ability to modulate local inflammation, studies in animals and observations in individuals with the Duffy-null phenotype (who typically have a baseline lower absolute neutrophil count [ANC]) suggest that ACKR1 is not essential for normal immune function. 

Issues of Concern

Nearly 25% of patients who are Duffy-null have an ANC below the established lower limit of normal values. However, Duffy-null status is clinically insignificant with regard to immune function, and patients with a decreased neutrophil count due to Duffy-null status exhibit normal neutrophil function and response to infection.[19][20][21] Despite this, established laboratory reference ranges do not account for Duffy-null individuals. This lack of appropriate reference ranges and the failure to recognize Duffy-null as a normal variant can lead to unnecessary tests, such as bone marrow biopsies, discontinuation of medications, or exclusion from clinical trials, where reference ranges for ANC are often based on Duffy-positive patients.[16]

Clinical Significance

Duffy Antigens, Hemolytic Transfusion Reactions, and Hemolytic Disease of the Fetus and Newborn

Duffy antibodies, particularly anti-Fy^a and anti-Fy^b, can lead to HTRs and HDFN. As discussed earlier, surface antigens on RBCs determine a patient's blood type. Patients lacking certain antigens may be exposed through allogeneic blood transfusion, pregnancy, or organ transplantation, leading to the development of antibodies against Fy^a or Fy^b, which increases their risk of hemolysis. Fetal antigens inherited from the father can be recognized as foreign by the mother's immune system, resulting in alloimmunization, primarily anti-Fy^a.[9][22] These alloantibodies can cross the placenta, potentially causing HDFN.

The following outlines potential Duffy blood group phenotypes and their associated likelihood of antibody production:

• Patients with the Fy(a+b–) phenotype can produce anti-Fy^b antibodies.
• Patients with the Fy(a–b+) phenotype may develop anti-Fy^a antibodies.
• Patients with the Fy(a+b+) phenotype do not produce anti-Fy^a or anti-Fy^b antibodies.
• Patients with the Duffy-null phenotype may produce anti-Fy^a antibodies, but they usually do not form anti-Fy^b, as Fy^b can be expressed in other tissues.

In individuals with the Fy(a–b–) phenotype, additional antibodies such as anti-Fy3, anti-Fy4, anti-Fy5, and anti-Fy6 may develop. These patients should also receive blood that is Duffy antigen-negative. While IgG antibodies, particularly IgG1, are responsible for most Duffy-related HTRs, IgM antibodies have been rarely reported.

To ensure safe and effective transfusion outcomes, patients with antibodies against the Duffy blood group system and fetuses affected by HDFN must receive RBCs that are both Duffy antigen-negative and crossmatch-compatible.

Duffy Antigens and Malaria

Duffy status is critical in determining survival from Plasmodium infections.[23] The 2 most common global causes of malaria infections include P vivax and P falciparum. Malaria begins when an infected Anopheles mosquito bites a human, injecting sporozoites into the skin. These sporozoites then enter the bloodstream and eventually travel to the liver, where they replicate and mature into merozoites. The merozoites are released into the circulation, where they infect red blood cells (RBCs), causing their lysis and continuing the cycle of infection.

ACKR1 serves as the binding site on RBCs for P vivax and P knowlesi. The ligand-receptor interaction between the merozoites and host erythrocytes mediates the invasion of RBCs. The P vivax Duffy-binding protein (PvDBP) on the surface of the merozoite interacts with ACKR1 on the reticulocyte surface, forming a junction that facilitates parasitic invasion.[24] Studies implicate that the intraerythrocytic killing of malarial parasites involves platelet factor-4 in erythrocytes expressing ACKR1. This process leads to platelet factor-4 accumulation via endocytosis, resulting in the lysis of the parasitic digestive vacuoles.[25]

Between 80% and 100% of the population in sub-Saharan Africa is Duffy-null.[26][27][28][29] Individuals who are Duffy-null are resistant to infection by P vivax and P knowlesi. However, infection can still occur, suggesting the involvement of Duffy-independent pathways, such as transferrin receptor 1 (TfR1 or CD71) and CD98, an amino acid transporter, which may facilitate infection.[30] The protective effect of the Duffy-null phenotype does not include P falciparum, which can infect individuals with all phenotypic variants of the Duffy blood group system.[25][31][32] 

Duffy Antigens and HIV

Patients who are Duffy-null or ACKR1-negative have an increased susceptibility to HIV infection but experience slower disease progression once infected.[33][34] This paradox may be explained by differences in how ACKR1 influences inflammation and viral interactions throughout the stages of infection.

Studies reveal a 3-fold increase in both the risk and rate of HIV acquisition, particularly among individuals of African descent with the Duffy-null genotype who have the lowest neutrophil levels. This suggests that the high prevalence of the Duffy-null trait may contribute to the disproportionate impact of HIV in African populations.

In individuals who are ACKR1-positive, the Duffy antigen binds a wide range of proinflammatory chemokines, including CCL2, CCL5, and CXCL1. People of African ancestry with low neutrophil counts often have elevated levels of inflammatory markers such as IL-8 and granulocyte colony-stimulating factor, which are associated with increased inflammation.[35] Notably, in laboratory studies, IL-8 has been shown to enhance HIV-1 transmission. In contrast, higher neutrophil counts are associated with resistance to HIV acquisition. Although CCL5 can suppress HIV, the proinflammatory environment created by the binding of other chemokines to ACKR1 outweighs its effects, potentially exacerbating disease progression.

Furthermore, in individuals with ACKR1 expression, RBCs act as carriers for HIV, thereby facilitating viral transfer to CD4+ target cells, particularly during established infection when viral loads are high.[36] Experts believe that HIV bound to ACKR1 is more stable and infectious than free virus, enhancing infection efficiency and potentially contributing to faster disease progression.

In contrast, Duffy-null individuals who lack ACKR1 on RBCs may have a lower inflammatory response and fewer RBC-bound HIV reservoirs. This could contribute to a slower progression of HIV once infection occurs, helping to explain why individuals with the Duffy-null phenotype may be more susceptible to acquiring HIV but tend to experience a slower disease course over time.

Duffy Antigens and SARS-CoV-2

The Duffy blood group system may influence the presence and severity of SARS-CoV-2 infections.[37] ACKR1 regulates the activity of proinflammatory chemokines on endothelial cells in postcapillary venules, which have a key role in directing neutrophils to sites of inflammation. Additionally, ACKR1 facilitates the transport of chemokines across the endothelium, promoting neutrophil migration into the lungs. These mechanisms enhance the inflammatory response by supporting leukocyte extravasation, which may contribute to lung inflammation and increased disease severity in SARS-CoV-2 infections.[38]

At baseline, the Duffy-null genotype is associated with benign neutropenia in individuals of African, Middle Eastern, and West Indian descent. This variation is considered a normal genetic trait and does not cause clinically significant disorders. However, the proinflammatory effects of the Duffy-null phenotype can enhance leukocyte migration into the lungs. In the absence of the Duffy system to modulate cytokine activity, the effects of cytokines are more pronounced than in a Duffy-positive state. Compared to other patients, those with the erythroid Duffy-null genotype have a 17% higher risk of mortality, as well as fewer ventilator-free and organ-failure-free days.[37] 

Role of the Duffy Antigen Receptor for Chemokines in Organ Transplantation

A mouse model study demonstrates that ACKR1-negative mice exhibit enhanced chemokine production and an increased inflammatory response, leading to both immunological and non-immunological injuries due to excessive leukocyte recruitment.[39][40] This finding is supported by data from the United Network for Organ Sharing (UNOS) database, which reveals higher rates of delayed graft function and allograft failure in Duffy-null patients.[41] The proposed mechanism suggests that ACKR1 acts as a "chemokine sink," reducing inflammation associated with delayed graft function by limiting the impact of excess chemokines.

Recent data indicate greater antibody-mediated renal allograft rejection and reduced graft survival in patients who are ACKR1-positive compared to those who are ACKR1-negative.[42] 

ACKR1 and Atherosclerosis

ACKR1 has a crucial role in the inflammatory process and the development of atherosclerosis. On RBCs, ACKR1 regulates inflammation, leukocyte migration, and endothelial function. CXCL2 presented by ACKR1 facilitates unidirectional neutrophil diapedesis.[43][44]

Recent studies highlight the role of a high-fat diet in inducing endothelial dysfunction due to increased CCL2 binding to RBCs expressing ACKR1. This interaction promotes endothelial cell and leukocyte communication, potentially facilitating the development of atherosclerotic plaque. In an experimental animal model, Wan et al demonstrated that mice lacking ACKR1 due to gene knockout had smaller atherosclerotic plaques.[45][46] The role of ACKR1 in regulating chemokine levels and leukocyte trafficking suggests its potential as a therapeutic target for diseases associated with inflammation and immune dysregulation, such as cancer, infectious diseases, and other inflammatory conditions.

Enhancing Healthcare Team Outcomes

The Duffy blood group system, encoded by the ACKR1 gene, includes antigens expressed on RBCs and other tissues, such as endothelial cells. The primary antigens, Fy^a and Fy^b, are determined by a single-nucleotide variation and are clinically significant in transfusion medicine, as antibodies against them can lead to HTRs and HDFN.

Beyond transfusion compatibility, Duffy antigens, also called ACKR1, are crucial in modulating inflammatory responses. ACKR1 is a non-signaling chemokine receptor that binds many proinflammatory chemokines, including CCL2, CCL5, and IL-8. By functioning as a "chemokine sink," ACKR1 sequesters excess chemokines, thereby regulating their availability and controlling the extent of leukocyte recruitment to sites of inflammation. ACKR1 also facilitates the transcytosis of chemokines across the endothelium, aiding neutrophil migration into tissues, such as the lungs, during respiratory inflammation. This regulatory role makes ACKR1 crucial in balancing an effective immune response and excessive inflammation. For example, altered Duffy antigen expression is associated with varying responses in conditions such as malaria, HIV infection, and possibly SARS-CoV-2, where inflammation is central to disease severity. Understanding the function of Duffy antigens in immune regulation and transfusion medicine is essential for optimizing patient care, especially in individuals with the Duffy-null phenotype.

Effective healthcare regarding Duffy blood group antigens requires clinical expertise, strategic planning, interprofessional communication, and coordinated care among healthcare professionals. Physicians and advanced practitioners must recognize the clinical significance of Duffy antigens, particularly in the context of transfusion safety, susceptibility to infectious diseases, and the risk of HDFN. Nurses play a crucial role in verifying the compatibility of the patient's identity and blood type with the donor blood. They also monitor for transfusion reactions and educate patients about potential adverse effects, as well as the need for Duffy antigen-negative blood in future transfusions for those with the Duffy-null phenotype. Blood bank personnel are essential to patient safety by performing blood typing, antibody identification, and crossmatching to ensure compatibility between donor and recipient blood. Pharmacists offer guidance on safe medication use and assist in supporting transfusion protocols.

Effective communication among healthcare team members, including laboratory staff, clinicians, and pharmacists, is crucial for accurately interpreting blood typing and antibody screens while maintaining consistent documentation. Coordinated care is vital in situations such as prenatal care, chronic transfusion management, or surgical preparation, where knowledge of a patient's Duffy status can significantly impact outcomes. In such cases, collaboration with specialists in hematology, infectious disease, obstetrics, pediatrics, neonatology, and blood banking ensures proper planning and intervention. By collaborating and leveraging shared expertise, healthcare teams can enhance patient safety, reduce the risk of adverse events such as HTRs, and deliver more patient-centered care.

Nursing, Allied Health, and Interprofessional Team Interventions

The Duffy blood group system requires coordinated care from nursing, allied health, and interprofessional healthcare teams. Nurses ensure safe transfusions by administering Duffy antigen-negative blood to patients with anti-Fy antibodies, preventing hemolytic reactions, and educating patients on post-transfusion care. Allied health professionals conduct phenotyping and antibody screening to identify compatible blood units and assess malaria susceptibility. The interprofessional team collaborates on targeted treatments, enhancing transfusion safety and improving patient outcomes through timely, evidence-based care.

Nursing, Allied Health, and Interprofessional Team Monitoring

Effective management of the Duffy blood group system in clinical settings depends on the collaborative efforts of nursing, allied healthcare professionals, and the interprofessional healthcare team to ensure patient safety and optimize outcomes. Nurses are crucial in monitoring patients with Duffy antibodies, ensuring that Duffy antigen-negative, crossmatch-compatible blood units are used during transfusions to prevent HTRs or HDFN. They also educate patients on the importance of adhering to transfusion protocols and monitor for delayed reactions, which can occur weeks after transfusion due to the body's unique immunogenicity.

Allied healthcare professionals, such as laboratory technologists, play a key role in accurately identifying Duffy phenotypes and antibodies through serological and molecular testing, providing essential data for informed treatment decisions. The interprofessional healthcare team, including physicians, advanced practice providers, pharmacists, and transfusion specialists, uses this information to tailor interventions, such as intrauterine transfusions for HDFN or selecting Duffy-negative blood for patients with the Duffy-null phenotype. This coordinated approach supports accurate diagnostics, safe transfusion practices, and improved patient outcomes across a range of clinical scenarios involving the Duffy blood group system.