Rh Blood Group System PDF

Rh is the most important blood group system after ABO in transfusion medicine. One of the most complex of all RBC blood group systems with more than 50 different Rh antigens. Antigens of Rh system ○ Terms “D positive” and “D negative” refer only to presence or absence of the Rh...

Rh is the most important blood group system after ABO in transfusion medicine. One of the most complex of all RBC blood group systems with more than 50 different Rh antigens. Antigens of Rh system ○ Terms “D positive” and “D negative” refer only to presence or absence of the Rh antigen D on the red blood cell. Terms “Rh pos” and “Rh neg” are old terms, although blood products still labeled as such. Early name “Rho” less frequently used. ○ D Ag: only expressed on RBCs Not platelets Not WBCs Not tissue cells ○ Residual RBCs may be present in products (like platelets) which is why Rh type is recorded on unit ○ Four additional antigens: C, c, E, e. Named by Fisher for next letters of alphabet according to precedent set by naming A and B blood groups. Major alleles are C/c and E/e. ○ MANY variations and combinations of the 5 principle genes and their products, antigens, have been recognized. ○ ​The Rh antigens and corresponding antibodies account for majority of unexpected antibodies encountered. ○ Rh antibodies stimulated as a result of transfusion or pregnancy, they are immune (not naturally occurring). Rh system ○ Mid 1940’s: 4 more antigens described ○ C, c, E, e ○ Now ~50 Rh antigens ○ No such thing as ‘d’ antigen (amorph) ○ C 70% population ○ c 80% population ○ E 30% population ○ e 98% population History ○ Key observation by Levine and Stetson in 1939 that delivery of stillborn fetus and adverse reaction in mom to blood transfusion from father were related. Syndrome in fetus is now referred to as hemolytic disease of the fetus and newborn (HDFN). Syndrome had complicated pregnancies for decades causing severe jaundice and fetal death, “erythroblastosis fetalis”. Erythroblastosis fetalis (HDFN) linked with Anti-Rh by Levine in 1941. ○ Rh system IDENTIFIED in 1940. Immunized animals to Rhesus monkey RBCs.  Antibody agglutinated 100% of Rhesus and 85% of human RBCs. Reactivity paralleled reactivity of sera in women who delivered infant suffering from hemolytic disease. Later antigen detected by rhesus antibody and human antibody established to be dissimilar but system already named. ○ Clinical significance D antigen, after A and B, is the most important RBC antigen in transfusion practice. Individuals who have D antigen DO NOT have anti-D. Antibody produced through exposure to D antigen through transfusion or pregnancy. Immunogenicity of D greater than that of all other RBC antigens studied. Has been reported that >80% of D neg individuals who receive single unit of D pos blood can be expected to develop immune anti-D (as little as 0.1 mL of blood) Testing for D is routinely performed so D neg will be transfused with D neg. Inheritance and nomenclature ○ Two systems of nomenclature developed prior to advances in molecular genetics. ○ Reflect serologic observations and inheritance theories based on family studies. ○ Because these are used interchangeably it is necessary to understand the theories well enough to translate from one to the other. ○ Two additional systems developed so universal language available for use with computers. Fisher-Race CDE terminology ○ Fisher Race Suggested that antigens are determined by 3 pairs of genes which occupy closely linked loci (proven incorrect) Actually, current theory is that there are two closely linked genes: RhD and RhCE Fisher Race (theory) Each gene complex carries D or its absence (d), C or c, E or e. Each gene (except d, which is an amorph) causes production of an antigen. The order of loci on the gene appears to be “DCE” but many authors prefer to use “CDE” to follow alphabet. Inherited from parents in linked fashion as “haplotypes” The gene d is assumed to be “present” when D is absent. Three loci carry the Rh genes are so closely linked that they never separate but are passed from generation to generation as a unit or gene complex.  An offspring of a DCe/dce individual will inherit EITHER DCe or dce from the parent, never dCe as this would indicate crossing over which does not occur in Rh system in human. With the exception of “d” each allelic gene controls presence of respective antigen on RBC. The gene complex DCe would cause production of the D, C and e antigens on the red cells (codominant). If the same gene complex were on both paired chromosomes (DCe/DCe) then only D, C and e would be present on the cells (D+C+c-E-e+). If one chromosome carried DCe and the other was DcE this would cause D, C, c, E and e antigens to be present on red blood cells (D+C+c+E+e+). Each antigen except d is recognizable by testing red cells with specific antiserum. ○ Weiner ○ Postulated that one gene, on each chromosome pair, controls the entire express of Rh system (proven incorrect). ○ One gene produces a structure on the red cell called an agglutinogen (antigen). ○ Eight (8) major alleles (agglutinogens): R0, R1, R2, Rz, r, r’, r” and ry. ○ Each agglutinogen has 3 factors (antigens or epitopes) The three factors are the antigens expressed on the cell. For example the agglutinogen R0= Rh0 (D), hr’ (c), hr” (e) ○ Each agglutinogen can be identified by its parts or factors that react with specific antibodies (antiserums). ○ Superscripts (Rh1) refer to genes ○ Subscripts (Rh1) refer to the agglutinogen (antigens) ○ For example, the Rh1 gene codes for the Rh1 agglutinogen made of D, C, e Usually, this can be written in shorthand, leaving out the “h” DCe is written as R1 Weiner and Fisher Race ○ The two theories are the basis for the two notations currently used for the Rh system.  ○ Immunohematologists use combinations of both systems when recording most probable genotypes. ○ You MUST be able to convert a Fisher-Race notation into Wiener shorthand, i.e., Dce (Fisher-Race) is written R0. ○ Given an individual’s phenotype you MUST determine all probable genotypes and write them in both Fisher-Race and Wiener notations. ○ R1r is the most common D positive genotype (31% Caucasian). ○ rr is the most common D negative genotype (15% Caucasian). Rosenfield ○ In 1962 proposed a nomenclature based ONLY on serologic (agglutination) reactions. ○ Antigens are numbered in the order of their discovery and recognition as belonging to the Rh system. ○ No genetic assumptions made ○ The phenotype of a given cell is expressed by the base symbol of “Rh” followed by a colon and a list of the numbers of the specific antisera used. ○ If listed alone, the Antigen is present (Rh:1 = D Ag) ○ If listed with a “-”, antigen is not present (Rh:1, -2, 3, 4, -5 = DcE) ○ If not listed, the antigen status was not determined ○ Adapts well to computer entry ○ R1r 33% in white ○ rr 15% in white ○ R0R0 0.1% in white International society of blood transfusion ○ Abbreviated ISBT--created to standardize blood group system nomenclature. ○ Assigned 6 digit number for each antigen. First 3 numbers indicate the blood group system, eg., 004 = Rh Last 3 numbers indicates the specific antigen, eg., 004001 = D antigen. ○ For recording of phenotypes, the system adopts the Rosenfield approach Phenotype versus Genotype ○ The phenotype is the result of the reaction between the red cells and antisera ○ The genotype is the genetic makeup and can be predicted using the phenotype and by considering the race of an individual ○ Only family studies can determine the true genotype ○ Five reagent antisera available.  Only anti-D required for routine testing. Other typing sera used for typing RBCs to resolve antibody problems or conduct family studies. ○ Agglutination reactions (positive and negative) will represent the phenotype. ○ No anti-d since d is an amorph. ○ Use statistical probability to determine most probable genotype (based on frequencies) ○ Molecular testing becoming more popular: Cannot use anti-sera on recently transfused individuals, molecular testing can differentiate. Anti-sera not available for some antigens, molecular testing being developed for all blood group genes. D zygosity can be determined. Fetal genotyping for D can be done on fetal DNA present in maternal plasma. Monoclonal reagents from different manufacturers react differently with variant D antigens, molecular test specific. ○ Typing sera continue to be the “gold standard” but this will change in the future Rh phenotyping ○ Uses Parentage testing Predicting hemolytic disease of the fetus and newborn (HDFN) Confirmation of Rh antibody specificity Locating compatible blood for recipients with Rh antibodies. ○ Protocol Mix unknown RBCs with Rh antisera Agglutination indicates presence of antigen on cell and determines phenotype. Use published frequencies and subject information to predict genotype. Genotype frequencies ○ Refer to textbook. ○ Genotypes are listed as “presumptive” or “most probable”. ○ Genotypes will vary in frequency in different racial groups. ○ Dce R0 46% blacks ○ DCe R1 common in white Weak expression of D ○ Not all D positive cells react equally well with anti-D. ○ RBCs not immediately agglutinated by anti-D must be tested for weak D ( AHG). Incubate cells with anti-D at 37°C, coating of D antigens will occur if present. Wash X4 add AHG ○ RBCs not immediately agglutinated by anti-D must be tested for weak D. AHG will bind to anti-D coating cells if present. If negative, individual is D negative  If positive, individual is D positive w ○ Three mechanisms 1. Genetic 2. Position effect 3. Mosaic Results in differences from normal D expression Quantitative (inherited weak D or position effects) Qualitative (mosaic D; could produce Anti-D) ○ Weak D genetic Inheritance of D genes which result in lowered densities of D Antigens on RBC membranes, gene codes for less D. Quantitative decrease in D antigen ○ Position effect C trans - position effect; The D gene is in trans to the C gene, eg., C and D are on OPPOSITE sides: Dce/dCe C and D antigen arrangement causes steric hindrance which results in weakening or suppression of D expression (functionally normal). ○ Partial D Absence of a portion or portions of the total material that comprises the D antigen. Known as “partial D” (old term “D mosaic”). If the patient is transfused with D positive red cells, they may develop an anti-D alloantibody* to the part of the antigen (epitope) that is missing *alloantibody- antibody produced with specificity other than self ○ Significance Donors Labeled as D positive Weak D substantially less immunogenic than normal D Weak D has caused severe HTR in patient with anti-D Patients If weak D due to partial D can make antibody to portion they lack. If weak D due to suppression or genetic expression theoretically could give D positive Standard practice to transfuse with D negative Weak D testing on donors by transfusion service not required (done at donor center).  Weak D testing on patients not required except in certain situations (Neg newborn; fertility). Compound antigens ○ Compound antigens are epitopes which occur due to presence of two Rh genes on the same chromosome, cis position. ○ Gene products include not only products of single gene but also a combined gene that is also antigenic. (f, rh1, etc.) ○ f antigens occur when c and e are found in cis (Example: dce/dce) r(cde) gene makes c and e but also makes f (ce). ONLY OCCURS when c and e are in the CIS position. f antigen will NOT be present in trans position. ○ rh1 or Ce antigens occur when C and e are in cis (example: dCe/dce) ○ Antibodies rarely encountered but if individual had anti-f would only react with f positive cells, not cells positive for c or e in trans only. ○ f cells clearly marked on antigram of screen and panel cells. G antigen ○ Genes that code for C or D also code for G ○ G almost invariably present on RBCs possessing C or D ○ Anti-G mimics anti-C and anti-D. ○ Anti-G activity cannot be separated into anti-C and anti-D. D deletion ○ Very rare ○ Individuals inherit only part or all of the Rh genes (or are unable to express) ○ May be at Ee or Cc (or both) C or E deletion really ○ Must be homozygous for rare deletion to be detected. ○ No reaction when RBCs are tested with anti-E, anti-e, anti-C or anti-c ○ Requires transfusion of other D-deletion red cells, because these individuals may produce antibodies with single or separate specificities. ○ Written as D- - or -D- ○ (partial deletion) Rh null ○ Red cells have no Rh antigen sites ○ Genotype written ---/--- ○ The lack of antigens causes the red cell membrane to appear abnormal leading to: Stomatocytosis Hemolytic anemia ○ 2 Rh null phenotypes: Regulator type – gene inherited, but not expressed Amorph type – RHD gene is absent, no expression of RHCE gene ○ Complex antibodies may be produced requiring use of rare, autologous or compatible blood from siblings. LW  ○ Discovered at same time as Rh antigen. ○ LW detected on cells of Rhesus monkeys and human RBCs in same proportion as D antigen. Thought was the same antigen but discovered differences. Named LW in honor of Landsteiner and Wiener. ○ Rare individuals lack LW yet have normal Rh antigens. ○ Can form allo anti-LW. Reacts more strongly with D pos than D neg cells; therefore, can look like an anti-D Keep in mind when D pos individual appears to have anti-D Cw ○ Variant Rh antigen ○ Low frequency antigen found in only 1-2% of Whites and very rare in Blacks ○ Most individuals who are C+ are Cw+ ○ Antibodies to these antigens can be naturally occurring and may play a role in HFDN and HTR Rh antibodies ○ Except for rare examples of anti-E and anti-Cw which may be naturally occurring, most occur from immunization due to transfusion or pregnancy. ○ Clinically significant ○ Associated with HTR and HDFN. ○ Characteristics IgG but may have MINOR IgM component so will usually NOT react in saline suspended cells (IS). ○ Characteristics May be detected at 37°C but most frequently detected by IAT/AHG. Enhanced by testing with enzyme treated cells. ○ Order of immunogenicity: D > c > E > C > e ○ Do not bind complement, extravascular destruction. ○ Anti-E most frequently encountered antibody followed by anti-c. anti-E - 70% population ee → produces anti-E Small amount of population can produce anti-c but it’s very immunogenic Only 20% of people can make it but if they are exposed they are very likely to produce antibody ○ Anti-C rare as single antibody. ○ Anti-e rarely encountered as only 2% of the population is antigen negative (allo-Ab) ○ Detectable antibody persists for many years and sometimes for life. ○ Anti-D may react more strongly with R2R2 cells than R1R1 due to higher density of D antigen on cells. R1 DCe/DCe R2 DcE/DcE - stronger D expression because no big C there to weaken the D antigen Concomitant Rh antibodies  ○ Antibodies which often occur TOGETHER. Sera containing anti-D may contain anti-G (anti-C +/-D) Anti-C rarely occurs only, most often with anti-D. Anti-ce (-f) often seen in combination with anti-c. ○ MOST IMPORTANT is R1R1 who make anti-E frequently make anti-c. Patients with anti-E should be phenotyped for c antigen. If patient appears to be R1R1 should be transfused with R1R1 blood. Anti-c frequently falls below detectable levels. Detection of D antigens ○ Four types of anti-D reagents High Protein - Faster, increased frequency of false positives; requires use of Rh control tube, converts to weak D testing IgM (Low protein/Saline reacting) - Low protein (fewer false positives); long incubation times; cannot convert to weak D testing High protein anti-D ○ IgG anti-D potentiated with high protein and other macromolecules to ensure agglutination at IS. ○ May cause false positives with RBCs coated with antibody. ○ Diluent control REQUIRED. ○ False positives due to autoagglutinins, abnormal serum proteins, antibodies to additives and using unwashed RBCs. ○ Can be used for weak D test. IgM anti-D (low protein/saline) ○ Prepared from predominantly IgM antibodies, scarce due to difficulty obtaining raw material. ○ Reserved for individuals giving false positive with high protein anti-seras. ○ Newer saline anti-sera require incubation at 37. ○ No negative control required unless AB positive. ○ CANNOT be used by slide test OR weak D test Chemically modfiied ○ IgG converted to saline agglutinin by weakening disulfide bonds at hinge region, greater flexibility, increases span distance. ○ Stronger reactivity than IgM antibodies. ○ Can be used for slide, tube and weak D test. ○ Negative control unnecessary unless AB positive. Monoclonal anti-D ○ Prepared from blend of monoclonal IgM and polyclonal IgG. IgM reacts at IS IgG reacts at AHG (weak D test) ○ Most frequently utilized reagent. ○ Used for tube, slide and weak D test. ○ Negative control unnecessary unless AB positive. Control for low protein reagents ○ Diluent used has protein concentration equaling human serum.  ○ False positives due to immunoglobulin coating of test RBCs occurs no more frequently than with other saline reactive anti-sera. ○ False positives do occur, patient will appear to be AB positive on forward type. ○ Must run saline or manufacturer’s control to verify. Precautions for Rh typing ○ MUST follow manufacturer’s instructions as testing protocols vary. ○ Cannot use IAT unless explicitly instructed by manufacturer. ○ Positive and negative controls must be tested in parallel with test RBCs. QC performed daily for anti-D QC for other anti-seras performed in parallel with test since these are usually not tested each day, only when necessary. Sources of error: false positive ○ Spontaneous agglutination ○ Contaminated reagents ○ Use of wrong typing sera ○ Autoagglutinins or abnormal serum proteins coating RBCs. ○ Using anti-sera in a test method other than that required by the manufacturer Sources of error: false negatives ○ Use of wrong anti-serum ○ Failure to add anti-serum to test ○ Incorrect cell suspension ○ Incorrect anti-serum to cell ratio ○ Shaking tube too hard ○ Reagent deterioration ○ Failure of anti-serum to react with variant antigen ○ Anti-serum in which the antibody is directed against compound antigen, often problem with anti-C. Summary ○ Rh system second to ABO in transfusion medicine. ○ Correct interpretation of D is essential to prevent immunization of D negative which may result in HDFN. ○ Most polymorphic of all blood group systems. ○ Of the five antigens only D testing is required. Continues to grow ○ Last decade has led to abundance of information detailing genetic diversity of the Rh locus. ○ Has exceeded all estimates predicted by serology. ○ Well over 100 RhD and more than 50 different RhCE have been documented. ○ New alleles are still being discovered. ISBT ○ International Society of Blood Transfusions ○ Assigned numbers to blood groups ○ Defined blood group system vs. blood group collection  ○ ISBT recognizes 36 blood group systems with nearly 350 antigens. Some VERY rare only found in certain ethnic groups. Some associated with diseases or resistance to infection; certain functions Most important are ABO and D High incidence”, “public” or “high frequency” antigens are those present on almost every person’s red blood cells (Present in >99% of population) “Low incidence”, “private” or “low frequency” antigens are present on very, very few individuals red blood cells (Present in 10,000 May cause hemolytic anemia when present in high titers ○ I antibodies Can be detected in serum of most normal adults if serum is tested at 4°C Associated with atypical pneumonia caused by M. pneumoniae , cold agglutinin titers are used to monitor the disease. ○ Disease associations Anti-i Infectious mononucleosis (EBV) Cytomegalovirus Alcoholic cirrhosis;  Reticulosis (increase in RES cells) Myeloid leukemia This antibody is rarely encountered ○ Clinical signficance Usually benign Clinically significant examples seen in Cold Agglutinin Syndrome (CAS) ○ Antibodies are of high titer (1000>) ○ High thermal amplitude ○ Cause hemolytic anemia ○ Transfuse blood through blood warmer Cannot cause HDFN – TWO REASONS Antibody class is IgM Antigen not well developed on fetal RBCs; 2 years to develop Does not cause HTR ○ Serological confirmation of anti-I Test serum against 3 adult group O RBCs and 3 group O cord RBCs and an auto-control Adult cells and auto-control = positive Cord RBCs = negative/very weak positive Reactivity enhanced using enzyme treated cells; just like what? ○ Pre-warmed technique Pre-warmed technique will eliminate reactivity of most examples of anti-I Original reactions at AHG Pre-warmed 1 – no other alloantibody present. Pre-warmed 2 – alloantibody present which must be identified. ○ Cold autoabsorption of anti-I Very strong examples of anti-I may react at AHG and require cold autoabsorption or rabbit erythrocyte stroma test (REST) to rule out presence of other antibodies. Collect EDTA blood samples, place at 37°C Harvest EDTA cells, wash with 37°C saline Place clotted blood sample at 4°C, separate serum Add 1 mL serum to 1mL RBCs, incubate at 4°C for 1 hour Harvest serum and test against screen cells, if negative, continue screen, if positive repeat absorption with new aliquot of RBCs. Lewis system (ISBT 007) ○ Identified in 1946 and named after antibody maker, Mrs. Lewis. ○ Major antigens Lea and Leb , other antigens include Lec, Led and Lex ○ The Lewis (Le, FUT3) gene is located on chromosome 19 at position 19p13.3 as is the secretor (Se, FUT2) gene ○ Antigens ARE NOT intrinsic to RBCs but are secreted into the plasma and absorbed from the plasma and inserted into RBC membrane. Only seen in plasma  Can get absorbed onto RBC membrane ○ Genetic control reside in single gene “Le” Amorph le, if homozygous will not have Lewis antigens Lea formed first, then modified to form Leb which is adsorbed onto RBC membrane preferentially over Lea ○ Both Lea and Leb soluble antigen in your plasma (Leb >>> Lea) ○ Lewis phenotype of RBC can be changed by incubating with plasma containing Lea or Leb glycolipid. ○ ○ Your true Lewis phenotype is detectable after 5 to 6 years of age ○ Antigens not on fetal cells – can antibodies cause HDFN? Ags absent or extremely weak at birth Expression of Leb gradual Birth Le(a-b-) 2 months: Le(a+b-); FUT3 increased activity 12-18 months: Le(a+b+); FUT2 increased 2-3 years Le(a-b+) ○ Lewis Ag cannot be used for paternity testing on infants ○ Antigen strength may decline dramatically during pregnancy. ○ Transiently Le (a-b-) may produce Lewis antibodies during pregnancy. ○ Antigens return after delivery and antibodies disappear. ○ Interaction of Le, Se, and H genes Le gene must be present for a precursor substance (type 1) to be converted to Lea, (FUT3) Se gene must be present for conversion to Leb, (FUT2) In general (for adults): Le (a+b-) RBCs are from ABH non-secretors (sese) Le (a-b+) RBCs are from ABH secretors (at least one Se) The le, h and se genes are amorphs and produce no detectable products. lele will not have Lewis antigens, but if Se present will have A, B and H in secretions Genotype se/se and have one Lewis gene (Le) will have Lea in their secretions but no A, B or H. (Leb?)  ○ Lewis antibodies naturally occurring, NOT clinically significant Almost always IgM React most often at RT Agglutination relatively fragile, easily dispersed May cause ABO discrepancy if reverse cells (A1 or B) have Lewis antigen. Occur almost exclusively in Le (a-b-) and production of anti-Lea AND –Leb not unusual Anti-Lea frequently encountered, anti-Leb rarely encountered. Although most react at RT reactivity may be seen at 37°C, but is weaker and may be weakly reactive at AHG Can bind complement and cause IN-VITRO hemolysis, most often with enzyme treated cells Antibodies NOT implicated in HDFN – TWO REASONS Antibodies are IgM and Antigens are poorly developed at birth; (5-6 years) Can be neutralized in-vitro by addition of Lewis Substance Le antigens are present in secretions Add to serum with Lewis antibodies and the antibodies will be bound to the soluble Lewis antigens Useful when multiple antibodies are present and one is a Lewis, eliminates the reactivity of the antibody Transfusion practice Transfused RBCs will acquire the Lewis phenotype of the recipient within a few days Lewis antibodies in patient will be neutralized by Lewis substance in donor plasma Lewis antibodies rarely cause in-vivo hemolysis It is not necessary to phenotype donors for Lewis antigens prior to transfusion, give crossmatch compatible P blood group (ISBT 003) ○ Discovered 1927 when Landsteiner immunized rabbits with human RBCs  Initially named “P” but as complexity of P blood group was discovered renamed “P1” RBCs lacking P1 are P2 Other P phenotypes exist but are rare(Lua>E>P1>c>M>Leb>C>Lea>Fya>S ○ ○ Kell antibodies Produced as a result of immune stimulation. IgG (react well at AHG) Clinically significant Anti-K is most common because the K antigen is extremely immunogenic Cause numerous cases of HTRs both immediate and delayed. Cause of severe HDFN (no anti-K Rx) Anti-k occurs much less frequently due to high frequency of antigen,