Highlighting the hematology in immunohematology

Immunohematology is a division of hematology related to antigen-antibody reactions and accompanying changes in our blood. Moreover, the study of immunohematology amalgamates all five of the major disciplines in medical laboratory science: blood banking, chemistry, hematology, microbiology, and immunology.¹ This article briefly highlights the role of blood group antigens (BGAs) as structural red blood cell (RBC) membrane and transport proteins, their association with hemostatic abnormalities, and in the case of antigen suppression or absence (null phenotypes), their relationship to hematological diseases and/or RBC morphological abnormalities.

Internationally, we currently recognize 36 blood groups systems and 352 known antigen specificities.² Twenty-three blood groups systems are intrinsic red cell surface proteins.³ Some affect the morphological shape and integrity of the RBC membrane, including Diego (carried by Band 3), Rh-associated glycoprotein (RhAG), Rh, Gerbich (carried by glycophorins C and D), XK protein (carries Kx antigen), and Duffy.^3,4 The lipid bilayer and the membrane cytoskeleton are linked by the interaction of Band 3 and RhAG with ankyrin. Glycophorin, XK, Rh, and Duffy interact with protein 4.1R. Linkages by Band 3 and RhAG seem to be the chief component of membrane cohesion.

Band 3

Band 3, the red cell anion exchanger 1 (AE1), passes through the lipid bilayer 14 times.³ It comprises about one-fourth of all protein in the red cell membrane, links the lipid bilayer to the membrane skeleton, transports HCO3- ions out in exchange for Cl-, and promotes preservation of the membrane surface ratio.^5,6 Band 3 also supports carbohydrate based blood group systems such as ABH and Lewis. Mutations in the Band 3 gene (SLA4A1) result in red cell morphological abnormalities such as spherocytes (20 to 35 percent of hereditary spherocytosis cases)⁷, ovalocytes, or elliptocytes. Red cells become dehydrated and lose surface area-to-volume ratio which effects deformability. Consequently, extravascular hemolytic anemia may ensue as macrophages in the spleen prematurely remove red cells. The direct antiglobulin test (DAT) is positive in acquired immune-mediated spherocytosis, unlike a negative result seen in hereditary spherocytosis. SLA4A1 mutations have likewise been associated with abnormal secretion of acid by the kidney (renal tubule acidosis) such as that found in homozygous individuals with Southeast Asian Ovalocytosis (SEO).⁸ Interestingly, since Band 3 is a receptor for malaria, individuals with SEO are resistant to Plasmodium falciparum infection. Clinically, individuals affected by SEO generally experience negligible red cell loss in spite of reduced red cell elasticity.

RhAG

Rh-associated glycoprotein (RhAG) BGAs complex with Rh proteins (RhD, RhCE) in the red cell membrane ankyrin complex, and associate with glycophorin A and B (GPA, GPB), Landsteiner-Wiener (LW; ICAM-4) glycoprotein, and ultimately Band 3.⁷ This complex is responsible for linking to the red cell skeleton via ankyrin and protein 4.2, and supports the integrity of the red cell membrane. In addition, RhAG is involved in the transport of gases (possibly CO₂) and cations across the red cell membrane.

RhD and RhCE are multipass lipoproteins that transverse the RBC membrane 12 times and require the presence of RhAG for antigen expression. Reduced or absent expression of Rh BGAs (Rh_mod or Rh_null, respectively) is associated with a rare genetic disorder known as Rh deficiency syndrome. Clinically, Rh_null individuals present with a compensated hemolytic anemia, and examination of the peripheral blood smear reveals the characteristic presence of stomatocytes. Because of the association with RhAG, Rh_null cells lack all LW BGAs and have reduced glycophorin B (S and s antigens); yet, expression of the Diego system antigens are unaffected (Band 3). By the same token, mutations in RhAG may cause absence of the high prevalence antigens U (GPA) and Fy5 (Duffy).

Glycophorins

Glycophorins (sialoglycoproteins), which carry the MNS (glycophorin A/GPA and glycophorin B/GPB) and Gerbich (glycophorin C/GPC and glycophorin D/GPD) BGAs, are rich in sialic acid (N-acetylneuraminic acid) and account for much of the net negative surface charge responsible for the natural repulsion of RBCs. Approximately 75 percent of the RBC’s sialic acid is found in GPA (contains M, N and Ena antigens); and in fact, Band 3 and GPA are the most abundant glycoproteins on the red cell surface.⁵ Expression of Wr^b antigen from the Diego system requires a functional GPA; consequently, the rare Wr(a-b-) phenotype results when variations occur in both Band 3 and GPA.

The net negative charge of glycophorins also plays a significant role in protecting RBCs from mechanical damage, caused by adhesion to nearby red cells or the endothelium, and microbial attack.⁷ Glycophorins serve as receptors for viruses such as influenzas A and B, and Sendai virus, as well as for Plasmodium falciparum and an exotoxin produced by Escherichia coli. In effect, sialic acid is critical to the mechanical invasion of merozoites into RBCs; consequently, individuals whose red cells lack GPA and GPB (MkMk genotype) are resistant to invasion by Plasmodium.

GPC and GPD (Gerbich BGAs) help maintain RBC membrane integrity via protein 4.1 and contribute to the RBC’s total net negative charge. The Gerbich system has three high frequency BGAs, Ge2, Ge3, and Ge4. Protein 4.1 deficient RBCs have reduced levels of GPC and GPD and result in the null phenotype (Ge:-2,-3,-4 or Leach phenotype). This gene mutation is associated with hereditary elliptocytosis, and may produce a mild compensated anemia because of decreased in vivo red cell survival.

XK protein

Chromosome X carries the loci for the XK protein (bears the Kx antigen) and for the rare X-linked disorder that leads to McLeod syndrome found only in males. Covalently linked by a disulfide bond, the Kell and XK glycoproteins may conceivably function as a complex. The biological function of the Kell glycoprotein is unknown; however, it belongs to the family of zinc endopeptidases that cleaves big endothelin-3, a vasoconstrictor. Although the biological mechanism of McLeod syndrome is not clearly elucidated, studies have demonstrated that normal function and structure of muscle depends on normal presence of XK protein. Clinical features of McLeod syndrome include myopathy, CNS manifestations, elevated plasma creatinine kinase (CK) levels, as well as the presence of acanthocytes on peripheral blood smear. Absence of XK protein, along with severe reduction in the single-pass Kell transmembrane glycoprotein, is credited with consequent compensated hemolytic anemia. In contrast, Kell null red cells (Ko) have increased expression of Kx antigen and normally shaped red cells. Finally, McLeod syndrome has been associated with chronic granulomatous disease (CGD), retinitis pigmentosa, and Duchenne muscular dystrophy whose genes are also found on the X chromosome. Avoiding transfusion in patients with McLeod syndrome and CGD is imperative as alloantibodies in these individuals may be stimulated to produce (anti-Kx+Km), which sets the stage for nearly impossible feat of finding compatible red cell donors.

DARC

The glycoprotein carrying the Duffy BGAs, Duffy antigen receptor for chemokines (DARC), is a multipass transmembrane glycoprotein. Two alleles found in people of European and Asian ancestry, Fy^a and Fy^b, produce the antigens Fy^a and Fy^b, respectively. A more common third allele, Fy is found in people of African origin. When inherited in the homozygous state (erythroid-specific GATA-1 binding site mutation), Fy fails to code for Duffy glycoprotein and red cells phenotype as null Fy(a-b-). The Fy coding region is like the Fy^b gene, other than it prevents normal expression of the gene in RBCs. Although Africans lack the Duffy glycoprotein on their RBCs, DARC is expressed on cells from other tissues; therefore, do not produce antibody to Fy^b.

DARC glycoprotein is a RBC receptor for chemokines, which are chemical messengers that attract leukocytes to inflammation sites. Absence of DARC is related to a condition known as benign ethnic neutropenia, associated with low neutrophil counts in as many as 98 percent of Africans.9 Linked to changes in neutrophil margination patterns, the low neutrophil count appears to cause no significant clinical concerns. DARC is also a receptor for the invasion of RBCs with Plasmodium vivax and Plasmodium knowlesi. Africans with the Duffy null phenotype are consequently resistant to malarial infection.

GPI-anchored proteins

Some BGAs insert into the RBC membrane via a glycosylphosphatidylinositol (GPI) anchor (GPI-linked). These include antigens from the Cartwright, Dombrock, Cromer, JMH, and CD59 blood group systems. Two of these systems are related with complement regulation. Cromer is located on decay accelerating factor (DAF, CD55) which inhibits assemblage of C3 and C5 converting enzymes of the classical and alternative pathways. CD59 inhibits complement by binding to the membrane attack complex and preventing C9 polymerization. Conditions associated with GPI anchor defects, most notably paroxysmal nocturnal hemoglobinuria (PNH), have reduced expression of all GPI-linked antigens, although physiological changes are mainly associated with the absence or marked deficiency of CD55 and CD59. Dysregulation of complement results in chronic intravascular hemolysis, bone marrow failure, hemoglobinuria, and high risk of thrombosis. Flow cytometry employing antibodies to GPI-anchored proteins has replaced previously utilized diagnostic tests for PNH such as the sucrose hemolysis test.

Other BGA suppression/expression

Conditions associated with suppression (sometimes total) or modification of BGA expression include leukemia, Hodgkin’s lymphoma, PNH, autoimmune hemolytic anemia, thalassemia, and hematopoietic stress. Serological effects seen in immunohematology as a result include ABO typing discrepancies, conflicts in antigen typing results compared with historical, and the potential for an alloimmunizing event.

Concerning blood clotting protein levels, studies have shown individuals with non-group O blood types (A, B, AB) have higher concentrations of Factor VIII (FVIII) and von Willebrand factor (vWF). As a result, these individuals may be at increased risk of venous thromboembolic events (VTEs), myocardial infarction (MI), cerebrovascular ischemic events, and peripheral vascular disease (PVD).^10,11 In a study of 1.5 million healthy blood donors, greater than 30 percent of venous thromboembolic events were attributed to blood types other than group O.¹¹ Similarly, genetic variants have been identified in the ABO locus that are associated with venous thromboembolic events (VTEs).¹² Recently, patients whose blood group is other than O were found to be at a considerably higher risk for post-thrombotic syndrome (PTS) with deep vein thrombosis (DVT) recurrence.¹³

As can be demonstrated by this brief review, red cell blood group antigens have many functional roles, and with more research, these roles are likely to expand. As research elucidates function, opportunities will be revealed for therapeutic and perhaps pharmacologic intervention into the pathologies that affect the red cell membrane composition and cytoskeleton. The “hematology” of the human red blood cell may be likened to a molecular Rubik’s cube with an intricate array of antigens that literally affect the quality of life and even survival itself. Immunology provides us with the ultimate tool with which to probe this spectacular antigenic array and to learn how it predestines our resistance and susceptibility to infectious diseases, strokes, and an impressive constellation of other cardiovascular events.

REFERENCES

1. Immunohematology. (n.d.) Farlex Partner Medical Dictionary. (2012). Accessed February 18, 2018.
2. Fung, M; Eder, A; Spitalnik, S; and Westhoff, C. Technical Manual, 19th edition. AABB Press (2017).
3. Klein, Harvey G.; Anstee, David J. Mollison’s Blood Transfusion in Clinical Medicine, 12th edition. Wiley Blackwell (2014).
4. Mohandas N, Gallagher PG. Red cell membrane: past, present, and future. Blood. 2008;112(10):3939-3948.
5. Aoki, Takahiko. A comprehensive review of our current understanding of red blood cells (RBC) glycoproteins. Membranes. 2017; 7, 56.
6. Red blood cell membrane dynamics and organization. Up to Date (2011). Accessed February 18, 2018.
7. Daniels, G. Human Blood Groups, 3rd edition. Wiley-Blackwell (Apr 2013).
8. Keohane, E; Smith, L; and Walenga, J. Rodak’s Hematology Clinical Principles and Applications, 5th edition. Elsevier (2016).
9. SLC4A1 solute carrier family 4 member 1. Gene ID: 6521, updated on 4-Feb-2018. Accessed February 18, 2018.
10. Reid, M; Lomas-Francis, C; and Olsson, M. The Blood Group Antigen Facts Book, 3rd edition. Academic Press (2012).
11. Hoffbrand, A.V., Moss, P.A.H. Essential Hematology, 6th edition. Wiley-Blackwell (2011).
12. Vasan, S., Rostgaard, K., Majeed, A., et. al. ABO Blood Group and Risk of Thromboembolic and Arterial Disease. Circulation (February 2016).
13. Spiezia, L., Campello, E., Valle, F.D. et al. ABO blood group and the risk of post-thrombotic syndrome. Ann Hematol (2018).
14. Heit JA1, Armasu SM, Asmann YW, et.al. A genome-wide association study of venous thromboembolism identifies risk variants in chromosomes 1q24.2 and 9q. J Thromb Haemost (August 2012).
15. Spiezia, L., Campello, E., Dalla Valle, F., et.al. ABO blood group and the risk of post-thrombotic syndrome. Ann Hematol (February 2018).