Rh Blood Group System PDF

Chapter 7 **The Rh Blood Group System** **OBJECTIVES:** Explain the derivation of the term Rh. 1. Differentiate Rh from LW blood group systems. 2. Compare and contrast the Fisher-Race and Wiener theories of Rh inheritance. 3. Translate the five major Rh antigens, haplotypes, and predict...

Chapter 7 **The Rh Blood Group System** **OBJECTIVES:** Explain the derivation of the term Rh. 1. Differentiate Rh from LW blood group systems. 2. Compare and contrast the Fisher-Race and Wiener theories of Rh inheritance. 3. Translate the five major Rh antigens, haplotypes, and predicted haplotypes, from one nomenclature to another, including Fisher-Race, Wiener, Rosenfield, and ISBT. 4. Define the basic biochemical structure of Rh. 5. Compare and contrast the genetic pathways for the regulator type of Rhnull and the amorphic Rhnull. 6. Describe and differentiate five mechanisms that result in weakened expression of D on red blood cells. 7. List one instance in which the weak-D status of an individual must be determined. 8. List and differentiate four types of Rh typing reagents, and provide two advantages of each type. 9. Define three characteristics of Rh antibodies. 10. Describe three symptoms associated with an Rh hemolytic transfusion reaction. 11. Compare and contrast Rhnull and Rhmod and describe the role of RhAG in Rh antigen expression. 12. List four Rh antigens (excluding DCcEe), and give two classic characteristics of each antigen. 13. Determine the most probable genotype of an individual when given the individual's red blood cell typing results, haplotype frequencies, and ethnicity. The Anti -- rhesus were renamed Anti -- LW. The LW antigen is expressed more strongly on D+ than D- red cells. Fisher and Race postulated three closely linked loci producing D/d, C/c and E/e Anti -- d has never been found and does not exist. Rh genes are codominant. Rh~nall~ phenotype written as \_\_\_ /\_\_\_. Wiener theory the Rh-Hr terminology , there different genetically theory involving only one gene locus.(table 7-1 ) table7-4.jpg Tippet theory two loci, one producing D antigen and other gene producing C/c and E/e (this theory was validated by molecular genetic studies. **Haplotypes, genotypes and phenotypes** table 7-2. There are only two Rh gene loci RHD and RHCE. The three pairs of allele can comprise eight possible haplotypes. There are eight haplotypes can be paired to form 36 different genotypes. Only 18 different phenotypes can be recognized by serological test with Anti -- D Anti -- C, Anti -- c, Anti -- E and Anti -- e. The most common Rh phenotype is D+, C+, c+, E- and e+. The alternative numerical terminology of (ISBT) International Society of Blood Transfusion:- D is RH1; C is RH2; E is RH3; c is RH4 and e is RH5, example : RH:1,2,-3,4,5 = D+,C+,E-,c+,e+. **Biochemistry and Molecular Genetics** The Rh phenotypes are controlled by two genes RHD which encoded the D antigen and RHCE which encoded the C/c and E/e antigens on chromosome ***one***. The RHD and RHCE encode proteins of 417 amino acids. The RhD and RhCcEe proteins differ by between 31 and 35 amino acids. The Rh protein cross the membrane of RBC 12 times providing six extracellular loops N -- and C -- termini are inside the cytosol. The Rh proteins are not glycosylated. The Rh proteins are associated with a glycoprotein called the Rh -- associated glycoprotein (RhAG). The RhAG is glycosylated with a single N -- linked sugar on the first extracellular loop. The RhAG producing from gene RHAG on chromosome 6 and cold coexpressor. The RhAG does not carry any Rh antigen, but RhAG is essential for expression of Rh antigen and if RhAG is absence lead to absent of Rh antigen expression. **The D antigen (RH1)** The D antigen is the most important Rh antigen after A and B in the field of transfusion medicine. The 30% of the D -- recipient of D+ makes the Anti -- D. Molecular Basis of The D Polymorphism. The D negative phenotype results from homozygosity for deletion of RHD. Some of D negative have RHD gene but not active because mutation in exon 6, this inactive RHD called RHD Ψ; not produce D protein. The D antigen has a 30 epitopes. **Weak D :variations of the Rh~0~ (D) antigen expression** D Variant of D exist caused by mutation with in RHD gene and it ranked into three main classes:- 1. **Genetic Weak D (formerly D^u^)** D antigen presents all epitopes but weakly expression. The D^u^ individual cannot make Anti -- D when immunized by normal complete antigen. Weak D is associated with amino acid substitution in the membrane -- spanning or cytosolic domains of the Rh D protein, which are not exposed to the outside of the membrane. 2. **Partial D** Many D variant arise from missense mutation in RHD. The partial D antigen is missing some D epitopes are expressed normally or weakly expression. The partial D individual can make antibody to those epitopes they lacks, when exposed to complete D antigen. The partial D is usually associated with amino acid change in the exposed extracellular loops of the RhD protein. Partial D individual can produce Anti -- D. There are seven partial D antigen designated by roman numbers I up to VII and classified by using monoclonal anti-D (MAb-D). 3. **C Trans** Haplotype of C allele can be interfering with expression of D antigen when the individual inherited the allele carrying D gene is trans to the allele carrying C gene such as Dce/dCe which lead to weak expression of D antigen. **Clinical significant of Anti -- D** Anti -- D is IgG and can cause severe HTRs and severe HDFN. The D positive blood must never be given to patient with Anti -- D. Maternal Anti -- D are fetal death at about 17th (after 4 months) week of pregnancy and lead to hydrops and jaundice which lead to kernicterus which leads to permanent cerebral damage or death. Mother can immunize by contamination of fetal D+ cells via trans placental hemorrhage during delivery. Anti -- D immunization can be prevented by infuse dose of Anti -- D immunoglobulin to the D negative mother immediately during 73h from delivery of D+ baby or administrated to D negative pregnant Women antenatal. Prophylactics Anti -- D administrate for D antigen negative and weak expression of D antigen for abstract patients. Pro -- D neutralization D positive fetal cells from the maternal circulation. The Anti -- D in women with D variant antigen can cause severe HDFN in fetus with complete D antigen. Rh antibodies **don't** bind complement but can cause extravascular hemolysis. **Detection of Rh antibodies and antigen** 1. Anti-D can be detected by test of serum against D positive cells ( antibody screen test ). 2. The anti-D reagent suspended in **high-protein-based** or **low-protein-based**, **saline based** , **chemically modified** , **monoclonal** or **blends of monoclonal**. A. **Saline based anti-D** contain IgM which can used for coated RBCs by IgG antibody , but cannot used for weak D typing , however this reagent is limited availability and cost of production. B. **High-protein-based** is derived human plasma which contain high-protein molecular and used to optimize reaction in the stander slide and rapid tube test and can be detect weak D antigen , but can give false positive reaction. C. **The chemical reagents** used for slide and tube testing and do not recurred separate, may can give false positive reaction. D. **Monoclonal antibody reagent** is derived from hybridomas cell, is composed of blend anti-IgM and anti-IgG used for immediate and indirect antiglobulin testing. Table 7-3  3. Patients samples are tested in duplicate by direct agglutination with IgM monoclonal Anti -- D to give a negative result called category DVI cells, antiglobulin test for detecting weak D variant should not be carried out, DVI will be found to be D negative and will receive D negative blood. 4. Donors sample should be detecting weak form of D antigen by using the antiglobulin reagent. 5. Prediction of Rh D phenotype from fetal DNA - Obtain sample by amniocentesis. - Obtain sample by chorionic villus sampling. Both methods are highly invasive and carry a risk of miscarriage and transplacental hemorrhage, which lead to boost any maternal antibody that lead to risk of HDFN. A much better source is the tiny quantity of cell -- free fetal DNA present in maternal plasma and derived from the placenta. The test detects of RHD and several specific regions of RHD are present by (PCR) Polymerase Chain Reaction. 6. A newborn of weak D antigen with maternal anti-D may cause block antigen D and give negative result of D antigen typing which called blocking phenomena, DAT in this case positive and in this cases elution should be administrate to retyping D antigen. **C, c, E, e Antigen (RH2, RH4, RH3, RH5)** C, c, E and e are products of alleles of RHCE. The e and c antigen are high frequency in population. The C and E are present by different percentage in population. **Clinical Significance of CcEe.** All Rh antibodies should be considered to have the potential to cause HTRs and HDFN. Anti -- c is clinically the most harm after Anti -- D often cause severe HDFN. Anti -- C, Anti -- E and Anti -- e rarely cause HDFN when they do the disease is mild but not always. **Other Rh Antigen** The 49 Rh antigen recognized by the ISBT. The 20 antigens have a frequency between 1% to 99% and the 21 are rare antigens and 8 antigens have very high frequency. **Compound Antigens : ce, Ce, CE, cE (RH6, RH7, RH22, RH27) and G (RH12)** The F antigen is expressed one RBCs when c and e are found in same haplotype, and when the individual absente of both antigens in same haplotype can produce anti-ce( anti-F ) which only react with d**ce** or D**ce** complex with c and e in cis, anti-F can cause HDN and HTR. Anti -- Ce react with DCe/DcE. Anti -- ce is common component of Anti -- c and Anti -- e sera. Most Anti -- C and Anti -- C +D sera contain some Anti -- Ce. Anti -- CE and Anti -- cE are rare antibodies. Anti -- G react with red cells have D or C antigens. Anti -- G is often present in sera containing Anti -- D plus Anti -- C and can confuse serological investigations of HDFN. **C^w^, C^x^ and MAR (RH8, RH9,RH51)** C^w^ is a relatively low frequency antigen in all population, can be expressed with C and c. C^x^ is a rare antigen 0.1% to 0.3%. C^w^ and C^x^ are usually produced by DCe complexes produce a weakened form of C. C^w^ is associated with Glu41Arg substitution. C^x^ is associated with Ala36Thr substitution. These substitutions responsible for weakness of C. MAR is high frequency antigen is abolished by either the C^w^ or C^x^. **VS, V (RH20,RH10)** VS has frequency of about 30% - 40%. VS is represented by a (Leu245Val) substitution in CcEe protein and is associated with weak e. V is represented by a (Leu245Val) and also has (Gly336Cys) substitution. VS + V -- phenotype also usually D -- and abnormal C. **Rh -- deficient phenotypes Rh~null~ and Rh~mod~** Rh~null~ no Rh antigens express and produce Anti -- Rh29. Rh~null~ has two type of inheritance:- 1. Homozygosity for inactive Rh haplotypes lake of RHD and homozygous for RHCE containing inactivating mutation. So that neither Rh protein can be produced. 2. Normal active RHD and RHCE genes but homozygosity for inactivating mutation in RHAG ( the amorphic type ), The absence of RhAG leads to Rh antigens disappeared. Rh~mod~ phenotype some mutation in RHAG which lead to low -- level expression of all antigens. The Rh~null~ has a mild hemolytic anemia and for transfusion should be select only Rh~null~ blood. The Rh~mod~ has less severity clinical symptoms. The Rh~null~ and Rh~mod~ exhibit other blood group antigen like S,s and U antigen. The Rh~null~ RBCs are negative for FY5. **Putative Function of the Rh Proteins and RhAG.** Rh proteins and RhAG termini in the cytosol are characteristic of membrane transports like ammonium transport. Rh proteins and band 3 functions as an oxygen / carbon dioxide gas exchange channel. **Landsteiner-Wiener (LW)** LW^a^ and LW^b^ (Gln70Arg). The LW glycoprotein is intercellular adhesion molecule-4 (ICAM-4). LW and CD47 could function to maintain red cells in close contact with the endothelia of the capillary surface. Anti -- LW^ab^ produced from LW -- null phenotype and Rh~null~ which also LW(a-b-). LW antibodies are not considered clinically significant. - The Rh antibody was so named on the basis of antibody production by guinea pigs and rabbits when transfused with rhesus monkey RBCs. - Historically, Rh was a primary cause of HDN, erythroblastosis fetalis, and a significant cause of haemolytic transfusion reactions. - Fisher-Race DCE terminology is based on the theory that antigens of the system are produced by three closely linked sets of alleles and that each gene is responsible for producing a product (or antigen) on the RBC surface. - A person who expresses no Rh antigens on the RBC is said to be Rh~null~, and the phenotype may be written as \_\_\_\_/\_\_\_\_\_. - In the Wiener Rh-Hr nomenclature, it is postulated that the gene responsible for defining Rh actually produces an agglutinogen that contains a series of blood factors, in which each factor is an antigen recognized by an antibody. - It is currently accepted that two closely linked genes control the expression of Rh; one gene (RHD) codes for the presence or absence of D, and a second gene (RHCE) codes for the expression of CeEe antigens. - In the Rosenfield alpha/numeric terminology, a number is assigned to each antigen of the Rh system in order of its discovery (Rh1 = D, Rh2 = C, Rh3 = E, Rh4 = c, Rh5 = e). - Rh antigens are characterized as nonglycosylated proteins in the RBC membrane. - The most common genotype in whites is R^1^r (31%); the most common genotype in blacks is R^0^r (23%), followed by R^0^R^0^ at 19%. - The Rh antigens are inherited as codominant alleles. - A partial-D individual is characterized as lacking one or more pieces or epitopes of the total D antigen and may produce alloantibody to the missing fraction if exposed to RBCs with the complete D antigen. - Blood donor units for transfusion are considered Rh-positive if either the D or weak-D test is positive; if both the D and weak-D tests are negative, blood for transfusion is considered Rh-negative. - Most Rh antibodies are IgG immunoglobulins and react optimally at 37^0^C or following antiglobulin testing; exposure to less than 0.1mL of Rh-positive RBCs can stimulate antibody production in an Rh-negative person. - Rh-mediated haemolytic transfusion reactions usually result in extravascular haemolysis. - Rh antibodies are IgG and can cross the placenta to coat fetal (Rh-positive) RBCs.