Rh Blood Group System

Etiology and Epidemiology

The first report of the Rh blood group was in 1939 when Levine and Stetson described a pregnant woman that developed postpartum hemorrhage requiring transfusion from her husband. Even though she and her husband were ABO compatible, she developed pain and discolored urine after the transfusion. Agglutination occurred when her blood was remixed with her husband's blood. Levine and Stetson tested her blood with multiple ABO-matched donors and noted agglutination with 80% of the donor samples. The authors concluded that the patient had been isoimmunized with an unknown antigen from her fetus resulting in the incompatible blood transfusion.[3] 

Landsteiner and Wiener would describe the offending antigen as Rhesus (Rh) factor because of the agglutination in their experiments using rhesus monkey blood, assuming rhesus RBCs expressed the same antigens as humans. Their work would also describe the autosomal dominant inheritance pattern of the Rh factor.[4] Later, it was discovered that humans and rhesus monkeys do not share the same RBC antigens, though the name persists. The anti-Rh antibody produced from the Rh factor would subsequently be renamed anti-D.[5]

Pathophysiology

The Rh blood group system comprises 56 antigens; the five most clinically significant antigens are D, C, c, E, and e. The Rh blood group is encoded by two tightly linked loci on chromosome 1p34-36. The RHD gene encodes the RhD antigen, and the RHCE gene encodes RhCE antigens. SMP1, a sequence of unknown significance, separates RHD and RHCE.[6] RHD and RHCE encode eight haplotypes of the Rh antigens in various combinations. The Rh proteins are hydrophobic transmembrane proteins embedded in the RBC phospholipid bilayer with an extracellular expression of the antigens. Interestingly, the RhD and RhCE proteins are very similar, with the first 41 amino acids identical.[7] 

Rh proteins require the presence of Rh-associated glycoprotein (RhAG) for proper assembly in the RBC membrane.[8] Though RhAG and the Rh proteins are similar in structure, the gene locus for RhAG is located on chromosome 6p12-21. The combination of the Rh proteins and RhAG is termed the Rh family. Various other accessory glycoproteins comprise the Rh structure, including LW glycoprotein, integrin-associated protein, glycophorin B, and band 3 glycoprotein, encoded on chromosomes 19, 3, 4, and 17, respectively. The Rh proteins, RhAG, and accessory proteins are collectively termed the Rh complex.[1] The function of the Rh complex is unclear. Studies of different Rh phenotypes indicate that the Rh complex plays a role in the RBC membrane integrity and may be involved with ammonium transport across the RBC membrane.[6]

Various Rh complex phenotypes exist due to point and nonsense mutations, rearrangements, and nucleotide deletions. Of these phenotypes, a lack of D antigen (D-negative), weak D, partial D, RhCE variants, and Rh_null have clinical implications.

Lack of D Antigen

The lack of D antigen (D-negative) occurs for various reasons closely linked to ethnicity. Commonly identified in White populations, the deletion of RHD or mutations resulting in a premature stop codon produces the D-negative phenotype. In other people, the lack of D-antigen may be due to mutations preventing gene expression. For example, in African populations, the presence of a pseudogene leads to base pair duplication preventing gene expression.[6] Patients lacking D-antigen warrant special consideration to prevent alloimmunization; if exposed to D-antigen through transfusion or pregnancy, they may develop anti-D antibodies, as seen when a D-negative parturient has a D-positive fetus.[9]

Weak D

Approximately 1% of D-positive individuals type as weak D (historically known as Du), characterized by weak or absent RBC agglutination by anti-D antibodies during routine serologic testing. In weak D individuals, the D antigen usually requires enhancement with anti-human globulin (AHG) owing to a quantitative decrease in RhD protein. The weak D phenotype is a quantitative change in the D-antigen epitope caused by a defect in transcribing the RHD gene.[1] Various weak D genotypes exist; Types 1, 2, and 3 are the most common and produce sufficient D antigen epitopes to be managed as D-positive.[10] Female patients of childbearing age who are positive for a weak D phenotype should undergo additional genotyping to determine if immunoprophylaxis during pregnancy is necessary.[11]

Partial D

The partial D phenotype is a qualitative change to the D antigen epitope caused by RHD gene conversions, point mutations, or expression of a low-incidence antigen.[6] The majority of patients with partial D phenotype will type D-positive. Notably, these patients may form anti-D antibodies if exposed to D-positive RBCs and require special consideration for transfusion and during pregnancy.[10]

RhCE Variants  

RhCE polymorphisms occur due to single or multiple nucleotide substitutions in RHCE. The E and e alleles differ only by a proline to alanine substitution. However, the C and c polymorphic alleles have four different amino acid substitutions.[12] The expression of these different alleles significantly impacts patients receiving chronic transfusions as they may develop alloantibodies more frequently. Notably, patients with sickle cell disease are at particular risk for alloimmunization.[13]

Rh_null  

Rh_null phenotype contains two classifications, amorph and regulator. Amorph Rh_null occurs from a mutation of RHCE, leading to nonfunctional proteins on a D-negative background. Regulator Rh-null is the most common phenotype due to an RHAG mutation that produces dysfunctional RhAG.[6] Clinically, Rh_null patients have shortened RBC life spans, characteristic RBC morphology on a peripheral blood smear and compensated hemolytic anemias. Transfusion support can be challenging because Rh_null individuals can become sensitized to multiple Rh antigens, including high-frequency antigens. Some alloimmunized Rh_null patients can develop anti-RH29 antibodies; however, this antibody does not react with Rh_null RBCs.[14]

Specimen Requirements and Procedure

The blood sample collected in EDTA is called whole blood and is used for ABO and Rh typing. EDTA is the preferred anticoagulant for hematological testing because it allows the best preservation of cellular components and morphology of blood cells.[15] The minimum volume of blood required for Rh typing ranges from 0.5 to 4 mL of whole blood, depending on the laboratory and the age of the patient.

Diagnostic Tests

The direct antiglobulin test (DAT) demonstrates the in vivo coating of RBC surfaces with immunoglobulin antibodies or complement protein C3. The DAT is most commonly utilized for the serologic investigation of potential antibody-mediated hemolysis.[16] Clinical situations warranting a DAT include acute or delayed hemolytic transfusion reactions due to antibody incompatibility, hemolytic disease of the fetus and newborn, and antibody-mediated drug-induced hemolysis. While a DAT is not routinely performed for otherwise uncomplicated pretransfusion testing, it may be informative in cases where the auto-control is reactive to confirm the presence of self-reactive antibodies.[17] 

The DAT is performed by directly adding antihuman globulin (AHG) reagent to a sample of the patient’s RBCs and observing for agglutination with a positive reaction occurring if the RBCs are already coated such that AHG can bind. All positive DAT samples should be tested with an inert control such as saline or 6% albumin before concluding a DAT test is positive.[16] Various preparations of AHG sera are available for blood bank testing; the choice of AHG preparation is dictated by the application, direct or indirect, and whether testing is being performed to detect RBC sensitization by IgG, complement, or both.[18]

The indirect antiglobulin test (IAT) is performed to detect in vitro antibody binding to RBCs, regardless of the antibody’s ability to fix complement. Laboratory indications include antibody detection as in crossmatch and antibody screening, antibody identification, antibody titration, and RBC phenotyping. Antiglobulin testing may be performed using test tubes, capillary tubes, microtiter plates, or gel microtube techniques.[19] To standardize antiglobulin sera and confirm true-negative antiglobulin reactions, two types of quality control RBCs are customarily used, those coated with IgG and those coated with C3b, C3d, or both. Rh antibodies are usually used to sensitize RBCs with IgG.[17]

Quality control cells for the antiglobulin test are called check cells or Coombs control cells. In a true-negative test, free active antiglobulin reagent should remain. Control cells, sensitized with IgG or C3, are added to all negative tests and centrifuged. Hemagglutination of check cells confirms the presence and reactivity of the AHG reagent, thus validating a negative test result. If the control cells fail to agglutinate in any tube, the tests must be repeated because they are invalid and may have yielded false-negative results.[20]

Although the antiglobulin test is extremely sensitive, a negative test does not exclude the possible presence of antibodies on RBCs. A negative reaction can occur with small quantities of bound IgG and C3.[16] In addition, AHG sera may possess greater activity against some subclasses of IgG than others. Consequently, certain AHG sera may produce negative results with RBCs coated by a particular IgG subclass.[21]

Testing Procedures

Rh typing detects the presence or absence of the D antigen on the surface of RBCs and is performed analogously to ABO forward typing. During Rh typing, the RBCs of the patient are combined with reagent anti-D antibodies, and the resulting presence or absence of agglutination confers the Rh “positive” or “negative” status, respectively.[22] While not routinely required for pretransfusion testing, serologic testing to detect weak D or partial D phenotypes should occur in cases of ambiguous Rh typing or discrepancies with a patient’s historical typing result.[23] In cases of an apparently negative Rh type, the serologic assessment for weak or partial D requires adding AHG reagent to enhance otherwise undetectable agglutination. Agglutination in this context suggests the presence of a weak or partial D phenotype; definitive distinction requires subsequent molecular genotyping to predict the true D antigen phenotype.[24]

Various methods for Rh blood group testing may be employed. Serological testing is most commonly based on hemagglutination reactions with RBC antigens against specific antibodies.[25] The hemagglutination process occurs in 2 stages. The first stage, often called RBC sensitization, combines paratope and epitope in a reversible reaction that follows the law of mass action and has an associated equilibrium constant. Noncovalent attractions hold together the antigen and antibody. During the second stage, multiple RBCs with bound antibodies form a stable latticework through antigen-antibody bridges formed between adjacent cells. This latticework is the basis of all visible agglutination reactions.[26]

Reagents that detect the D antigen in the slide, tube, microplate, automated, and gel tests often have different formulations and performance characteristics. Various anti-D reagents may contain different antibody clones, potentiators, additives, or diluents, and reagents that contain the same antibody clone may vary in antibody dilution or preservative. Hence, instructions for testing may differ and must be consulted and followed carefully for accurate testing.[27]

Slide Testing

A glass slide containing a drop of 40% to 50% serum or plasma suspension of RBCs and a drop of anti-D is mixed and placed on a heated Rh viewing box tilted continuously for 2 minutes to observe for agglutination. To rapidly warm the materials on the slide at 37°C, the Rh viewing box is kept at a temperature between 40°C and 50°C. The result is positive when the patient sample shows agglutination and the control shows suspension.[28] The sensitivity of this method is low and can be easily affected by multiple factors, thus making standardization difficult.[29]

Tube Testing

The test tube method can be utilized for emergency and first-time blood group typing. The test tube method is quicker, more sensitive, and uses fewer reagants than the slide method.[28] In the test tube method, a patient’s antibody-containing plasma and reagent RBCs known to express specific antigens are combined in a test tube. Alternatively, a patient’s RBCs may be combined with reagent antibodies of known specificity. The mixture undergoes a series of centrifugation and incubation steps with stepwise quantitation of agglutination. The strongest agglutination, a 4+ reaction, results in an effectively nondissociable single clump of RBCs when the tube is gently agitated, whereas the weakest agglutination, a negative reaction, results in complete dissociation into individual RBCs by the same assessment. The intermediate strength reactions fall in a spectrum in between these extremes.[30]

Gel Testing

The gel testing method is a widely available alternative serologic testing method that utilizes dextran acrylamide gel-containing microtube columns built into small plastic cards.[31] Antibodies containing plasma and reagent RBCs are combined in a chamber above the columns, and the card is subsequently centrifuged to force the RBCs down through the permeable gel, which acts like a size-selective sieve. The gel is typically impregnated with AHG to facilitate agglutination. Depending on the degree of agglutination, the RBCs have varying mobility as they travel down the column, and their visible stopping point quantifies the reaction. Gel method testing can be automated and has the advantage of a more objective scale for quantifying results than tube testing.[25]

Microplate Agglutination Testing

The microplate technology uses automated platforms to detect serum antibodies and RBC surface antigens.[26] The reactants are centrifuged and incubated in microplates, and the ABO/RH(D) blood type is read through an automated system. The antiserum must indicate that it is formulated for automation or microplate Rh testing and may require the blood to be collected in a specific anticoagulant.[25] 

Interfering Factors

False positive or negative results can be caused by improper technique, contaminated materials, omission of reagents or antisera, delays in reading tests, inadequate incubation time and temperature, inappropriate centrifugation, inappropriate or prolonged storage of red cells, and autoantibodies.[32]

Results, Reporting, and Critical Findings

A patient or donor RBC sample previously found to be positive but now determined to be negative, or vice versa, should always be investigated to rule out identification, clerical, or recording errors.[32] A new sample should be obtained and tested. If the discrepancy is between current and historical test results, the difference may be due to the testing method employed, phase of testing (DAT or IAT), type of reagent (polyclonal vs. monoclonal), or manufacturer.[33] Different reagents often contain different antibody clones that may demonstrate varying reactions with RBCs with weak or partial Rh antigens. Knowledge of the ethnicity of the donor or patient can be helpful when investigating a typing discrepancy because some partial and weak phenotypes are more common in a specific ethnic group. Typing with several reagents from different manufacturers may be helpful.[34]

Clinical Significance

Hemolytic Disease of the Newborn

Hemolytic disease of the newborn (HDN), or erythroblastosis fetalis, is a clinically important and potentially life-threatening condition where antibodies from an Rh-negative gravida attack an Rh-positive fetus.[35] Despite being described by a French midwife in 1609, HDN was not understood until the 1950s.[36] Before the onset of immunoprophylactic therapies, 1% of all pregnancies resulted in fetal death from HDN. HDN is currently estimated to affect 3 to 8 of every 100,000 pregnancies. 

HDN results from maternal antibodies attacking fetal red blood cells. Typically, it is the second Rh-positive fetus that is affected. The first fetus inherits the paternal D antigen following an autosomal dominant pattern, and maternal and fetal blood mixing occurs during the pregnancy. This mixing most commonly happens during labor and delivery but can theoretically occur at any time during the pregnancy. Once mixing occurs, the gravida begins producing anti-D antibodies. This constitutes alloimmunization, as the gravida is D-negative.

The initial antibodies produced are IgM, which cannot cross the placental barrier. However, when isotype switching occurs, IgG antibodies are produced. The general absence of maternal and fetal blood mixing during the first pregnancy and the delay of IgG antibody production make it unlikely that the first D-negative pregnancy is affected. However, in the subsequent D-negative pregnancy, IgG antibodies cross the placenta and attack the D antigens on fetal RBCs. This leads to hemolysis that may result in jaundice, anemia, kernicterus, and hydrops fetalis. Intrauterine death may occur without intrauterine blood transfusion, and any surviving fetus may have developmental delays, hearing loss, and hypotonia.[35]

Hemolytic Transfusion Reactions

One of the more serious complications of autologous blood transfusion is a hemolytic transfusion reaction (HTR). HTR is rare, with an incidence of 1:70,000 per unit transfused.[37][38]

HTRs are caused when there is an immunological mismatch between the RBCs from the blood donor and the transfusion recipient, resulting in extravascular or intravascular immune-mediated hemolysis.[37] An intravascular HTR is typically more clinically severe than extravascular hemolysis.[39]

HTRs are classified by their severity and onset time, early or late. HTRs greatly vary in severity from mild, clinically insignificant hemolysis presenting weeks after the transfusion to very sudden and life-threatening intravascular hemolysis leading to shock, renal failure, disseminated intravascular coagulation, and death. The main determining factor of the reaction's onset and severity is the antibody's class, such as IgG, IgM, IgG subclass, or complement binding, the antigens targeted, and their titer concentration.[40]

The most severe type of HTR occurs during an ABO mismatch, typically due to clerical error. Patients who create AB antigens do so because of molecular mimicry to antigen presentation of gastrointestinal bacteria. These antibodies are typically IgM but may also be IgG. Severe reactions occur when IgM binds to ABO-typed antigens on RBCs and subsequently fixes complement, creating a sudden, massive intravascular rupture of RBCs. Hemolysis releases large amounts of hemoglobin, resulting in shock, renal failure, and DIC leading to hemorrhage and hypercoagulability with thrombosis.

Extravascular HTRs occur when the antibody targeting the RBCs results in opsonization. Opsonized cells are sequestered by the macrophages of the reticuloendothelial system, primarily in the liver and spleen. Extravascular hemolysis is a comparatively slower and more progressive process, as each RBC may make several passes through the reticuloendothelial system before being phagocytosed. Unlike intravascular hemolysis, the hemoglobin is released inside the sequestering macrophage, where it is processed normally. The clinical manifestations of extravascular hemolysis are typically milder, resulting in hyperbilirubinemia and fever. However, more severe complications, such as kidney failure, may also occur.[39]

Immune-mediated Hemolytic Anemia

Immune-mediated hemolytic anemia occurs when autoantibodies bind to RBCs, resulting in hemolysis and possible anemia. The autoantibodies are generally IgG, although IgM and IgA have also been reported.[41] Particular subclasses of antibodies are more destructive than others; IgG1 and IgG3 can fix complement at a high rate and are very destructive. The RBCs are mainly cleared through the reticuloendothelial system, but intravascular hemolysis may occur in severe cases.

Like many autoimmune disorders, most immune-mediated hemolytic anemias are associated with an underlying or triggering condition. Common triggering conditions are viral infections, autoimmune disorders, immunodeficiency, or pregnancy. Rarer instances of medication interactions, spider bites, sickle cell disease, babesiosis, and organ transplants have been documented.[42]

Quality Control and Lab Safety

All blood centers, hospital blood banks, and collection centers must establish and implement a quality management system guided by requirements of established standards, guidelines, and principles. It is the responsibility of the management to ensure that each personnel has a clear job description which includes lines of authority and responsibility.[43] Each personnel must be adequately trained and assessed to be competent in the specified task before being allowed to carry out the task independently. Records of training and assessment of competency are established and systematically maintained.[44]

All critical equipment that impacts the quality of tests or blood components prepared must be operated within their defined specifications. Materials used in laboratory tests and processing must be appropriately validated. All reagents shall be used and controlled according to the manufacturer's instructions. All anti-sera must be visually inspected for contamination, such as discoloration, cloudiness, turbidity, or particulate matter. The visual inspection results, reagent lot number, expiry date, inspection date, and the individual performing the inspection must be documented. The expiry date should be checked on each reagent used.[45] Do not use reagents beyond the expiry date. The reactivity of blood grouping reagents shall be confirmed daily by control tests with known antigen-positive and negative red cells.[46]

Under CLIA regulations, all laboratories that perform non-waived testing must enroll in and perform proficiency testing (PT) using one of the CMS-approved PT providers. The PT programs are required to provide a minimum of 5 samples per testing event, with 3 testing events per year. The minimum acceptable score for satisfactory participation in immunohematology PT is 100% for each analyte and overall testing event score for the ABO group and Rh(D) typing.[47]

The blood centers, hospital blood banks, and collection centers must establish policies and procedures on safety and security. Relevant committees should maintain and continually improve safety and security in the organization.[48] Access to the laboratory area must be restricted to authorized personnel only. Staff must wear white coats on entering the laboratory and remove them before leaving the laboratory. Keep the laboratory clean and tidy and retain only necessary items. Do not store food or personal items in the laboratory, especially in the main area of the laboratory. Wash hands with soap and water before leaving the laboratory. Eating, drinking, smoking, and applying cosmetics are strictly prohibited in the laboratory. Prevent the formation of aerosols or splashing of materials. Consider all specimens as potentially infectious. Any spillage, waste, or reusable materials must be appropriately decontaminated by bleach solution before disposal. Dispose of all needles and lancets into secured, puncture-proof containers located as close as possible to where they are to be used, and then handle them as infected materials.[45]

Enhancing Healthcare Team Outcomes

Patients with known Rh antibodies require additional testing to receive transfusions safely. This includes knowledge of transfusion reactions, compatibility testing, and, in some cases, genotyping. Healthcare practitioners should be attuned to the possibility of patients developing new and rare antibodies, especially those undergoing chronic transfusion. There should be established protocols and guidelines to ensure safe transfusion practices for patients with different Rh blood group phenotypes. This may involve additional consultation with a hematologist. Clinicians must ensure that transfusions are necessary as each transfusion may lead to the alloimmunization of Rh antigens.

Communication is essential for ensuring safe transfusion practices. Healthcare team members must confirm that the correct blood product is administered to the correct patient at the correct time. Likewise, all healthcare team members should know the patient's transfusion history, allergies, and other relevant medical information.