Principles of the Rh System Principles of the Rh System Complex system composed of 50 different antigens 5 Major antigens: D, C, ¢ E,e 2" most important blood group after ABO. Rh pos or Rh neg refers to presence ot absence of D antigen With Rh antigens, and any other blood group other than ABO, people who lack the antigens, do not regularly have the corresponding antibody —have to be exposed. D antigen has greater immunogenicity than all other RBC antigens. 80% of all D negative persons who receive a D positive transfusion will develop anti-D 1) History Contribution of Levine and Stetson — in 1930's ABO matching done but transfusion still resulted in morbidity and mortality. Hemolytic transfusion reaction reported in OB patient who delivered a stillborn infant and was transfused with father’s blood to which she had a reaction. Levine and Stetson isolated an antibody ftom her ‘serum that reacted with his blood. They postulated that father and baby carried a common factor that she was exposed to during pregaaney and made an antibody. Contribution of Landsteiner and Wiener - Reported on an antibody produced by rabbits & guinea pigs who were transfused with Rhesus monkey blood that agglutinated 85% of human RBCs and the OB patient who had a reaction. They named it Rh, Later it was found that the 2 antibodies were different but the system had already been named. Their antibody was renamed LW 2) Terminologies 4 systems-convey serologic & genetic information Fisher-Race Wiener Rosenfield ISBT ae se A. Fisher-Race 1.) Genetic Theory- 3 closely linked loci which control sets of alleles Definition ‘Codominant- each inherited gene expresses its corresponding antigen on the rbeFisher-Race Genette theory cont Genotype- determined by combo of matemal and paternal haplotypes Phenotype- antigens expressed on the RIC that can be detected serologically Nomenclature- meant to convey gene and gene product (antigens) Shorthand System B. Wiener Genetic theory- Rh genes code for a specific “agglutinogen” that gives rise to 3 serological factors (antigens) which can be demonstrated on the RBC, or (in other words) a single, complex loci with multiple alleles, Agglutinogen considered the phenotypic expression of the haplotype. Nomenclature -Symbols for “serological factors”Conversion of Weiner to Fisher-Raee~ Table 7-3 Gene Agglutinogen Blood Factors | Shorthand Fisher-Race antigens Rh” Rho Rho,hr* hr” Ro Dee Rh Rh, Rho,th* hr" Ri DCe Rh Rho Rho, hr’ rh” Ra DeE Re th a oo oh . Rosenfield Genetic theory- none, Numeric system meant to convey serological observations, . ISBT- Intemational Society of Blood Transfusion 6 digit computer number for each antigen -Rh system assigned to #4 Predicting Probable Genotypes Phenotype- the expression of observed reactions when red cells are tested with antisera Genotype- derived based on frequency of combinations Uses of Rh phenotypes and genotypes a. Probability studies for parentage and population studies b. Predicting HDN in offspring of an Rh Negative women with an Rh antibody3. Molecular Genetics RH genes - only 2 closely linked genes control the expression of Rh ‘* RHD codes for the presence or absence of D and RHCE for Ce, cE, ce, CE. Each gene has 10 exons Rh locus located on chromosome 1 RHAG gene - another gene important to Rh antigen expression, . It is a coexpressor and must be present for successful expression of the Rh antigen. Product is similar to Rh proteins but is glycosylated Product called Rh-associated glycoprotein Within RBC membrane, it forms complexes with Rh antigens Mutations result in missing or altered RhD and RhCE proteins. RHAG locus located on chromosome 6 Rh-Positive Phenotypes ‘Inherits one or two RHD genes * Two RHCE genes are inherited from each parent Rh-Negative Phenotypes ‘Most common causes atises from 3 mutations most often found in 3 different ethnicities. «European Ethnicity ~ common possess no RHD gene but have two RHCE genes * African Ethnicity - RHD pseudogene, sequence identical except for mutations in exon 5 and 6 * Asian Ethnicity — Da (discussed further in weak D)Se 4. Biochemistry Biochemical structure of Rh antigens- gene product is a nonglycosylated protein. Rh antigens are transmembrane polypeptides so they are an integral part of the RBC membrane, ‘Number of Rh antigen sites varies with genotypes a, comparison to ABO system Acells—1x 10° D cells 4500-33,000 (homozygous) K cells ~ 6000 (homozygous) b. comparison to Various Phenotypes — 5. Variations of the Rho (D) antigen Weak D-weak reactivity with D antisera Significance of weak D~ AABB standards require Donor specimens to be tested for weak D and labeled as D Positive if it is. Recipients are not requited 10 be tested . Causes of weak D — multiple causes @ Cin Trans to RHD — position effect/gene interaction effect b. Weak D-- gene codes for weakened expression of D antigen.. Molecularly, mutations in the RHD gene causes changes in the intracellular or transmembrane region of the RHD protein¢. Partial D~ Concept arose because some people who were D Positive formed anti-D unreactive to their own cells, * Original theory ~ missing epitopes of the D antigen ‘* Current theory — hybrid genes resulting from portions of the RHD gene being replaced by portions of the RHCE gene resulting in parts of the antigen not being expressed. d. Da-extremely low levels that cannot be detected by routine methods but antigens can adsorb and elute, ¢. D Epitopes on RhCE protein 6. Rh antibodies a. Characteristics - predominantly IGG b. Reactivity ¢. Formation of antibodies 4. Immunogenicity - D>e>E>C>e . Rhantibodies that commonly occur togetherRh antigen typing reagents a. Saline Reactive —first reagent available Igm Low protein based © High cost & limited availability # Cant be used for weak D testing b. High protein based- human plasma with high titer anti-D and other additives © Higher protein optimized reactions but caused false positives © Must run control © Short incubations © Can be used for weak D c. Low protein based/chemically modified - Anti-D in reagent modified by breaking disulfide bonds allowing molecule to relax © Allows a lower protein base so it has fewer false positives © Replaced need for Saline and High protein reagents 4. Monoclonals/Blends-derived from single clones of antibody producing cells * Uses a blend of monoclonals to ensure broad spectrum reactivity * Not human derived so lack all potential for transmitting disease 7. Clinical considerations Rh antibodies cause: a. HTR- causing extravascular destruction of immunoglobulin coated RBC’s b, HDN- often severe because Rh antigens are well developed at birth and antibodies ate primarily IgG which readily cross the placenta. ‘Rhogam - purified anti-D prevents sensitization. Rh Deficiency Syndromes Rh Null - RBCs have no Rh antigens, RBCs lacking Rh antigens have membrane abnormalities and causes a mild hemolytic anemia2 genetic mechanisms: © Regulator — mutation in RHAG gene ‘* Mutation in each of the RHCE Rh Mod ~ pattial suppression of RH gene expression caused by mutations in the RHAG gene. D deletion genes (-D-) — no Ce and or Ble reactivity but do posess D & G reactivity 8. Unusual phenotypes and rare alleles a, C* ~ Antigen found in 2% of whites and is very rare in blacks. Usually found on C+ RBCs. Can be formed without exposure. b. f{ce) - compound antigen. Expressed on rbe’s when both ¢ & ¢ are present on the same haplotypes or are in cis position, however fis a single entity. fis expressed by RHce protein. Ex. Dee/DCE DcE/DCe. Anti f only reacts with the first one. ¢. thy(Ce) — another compound antigen present when C & ¢ are in cis configuration 4. G— antigen present on most D positive and all C positive rbes. Anti G will zeact as if it were a combination of anti-C & anti-D. Important for obstetric patients. For transfusion give anti C and anti D negative units Hr — (RH17) antigen present on all rbes with the “common” Rh phenotypes. (RIRI, R2R2). Antibody reacts to entire protein resulting from the RHCE gene. £ Rh23,Rh30 and Rh40 — low incidence partial D variations. g. Rh:33 — low incidence antigen associated with a rare variant of the R° gene called Ro'*" which codes for normal amounts of ¢, reduced amounts of e and D bh. Rh43 (Crawford) —low-prevalence antigen on a variant Rhee protein found in African descent.Little e variants — partial litle e (similar to partial D) found in Some African or mixed ethnic individuals. Same phenomenon of being little e positive but making an anti-e. Y, Vs — found in about 30% of African-Americans LW -— original antigen discovered by Landsteiner and Weiner that they calied Rhco ) ) ‘gamma* Yologicals, inc. Como pepe with ssl gna aprox nena tS popiton "Tet rent wih eS "Most commen Ri] Comes Ri phsvoiypes etek aes iat erasers alloutiboty produced by | — that may be expected 2c ae fete ee =se| (CDE) ‘con | Wer pregnancy or transfusion tobe compatible : ‘CDefede ‘Rr t]ole}el + ‘CeDe Ri CDeleDe RR ane Tar, Rarer ‘GieteDe |r ofof+[+]o Ce Ri CDeACDe | RIRT ‘anti-¢ Ri CDelCis Rie anti-c+E Cede | RIE o eDECde Re anti-ce (4) cee | Ri | CDede | Rr Ra IR CD Eade | — Rr +[s]o[+ Cheede | RR one epucae | Fry DE vede Rr ° t]+]ofe sDEe Ror DBDs Re ‘anti-C or anti-Ce Rat, re |_| eDehedE Ber TTP poTe po] ae) | edeeDe |e aii or sn-Ce Cs epeiat | Rit antec DIOTET SPOT] ae |e ebeede | Rr acre far eDelede | _ me CeCDE | RIRF ° 0] come | mm | coece | ay ake % CDECds Re CDEDE | RIR? ofofo] coe | rm | cozeds | Rr ante & eps | Rie ER coECoE| eR mile & copce |_ Rw ane % 7s 7] edatede [or Fan of ane OWD TT Gin preganey) Grif na antec present) | | Sie | “rr ce 2 Gable | ro tami tees [re Cedeede |r Gaprogancy Ee Pr cdBede_ re fier e P| edBeas | 7 tas [9 7 *Dpegaia donne oad iL ewe | or cy sled te ce ty Caerede [Pr Sar _Dvnegative recipients, ear ame