4. Rh Phenotyping - Austin Community College

1 MLAB 2431 1Rh PhenotypingExercise 4Rh PhenotypingTextbook: Blaney, Chapter 5 Skills: 20 4 reasons for determining an individuals Rh the antigens of the Rh blood group the allele d . the following terms: amorph, dominant, codominant, heterozygous, and homozygous as they relateto inheritance of the Rh the terms phenotype and genotype as they relate to blood group state the procedure whereby an individual s Rh phenotype is each of the 8 Rh genes in both Fisher-Race and Wiener an Rh phenotype list the potential genotypes in both Wiener and Fisher-Race Determine the Rh phenotype and probable genotype of two (2) patient samples. DiscussionIn this procedure, commonly tested antigens of the Rh system will be studied. Normally the only Rh antigenidentified in routine pretransfusion testing is the D antigen. Four additional Rh antigens which may be testedfor are: C, E, , and.

2 Phenotyping for these antigens may be useful in: family studies, resolution of antibody cproblems, population studies, cases of disputed parentage, and to predict whether the sexual partner of a womanwith Rh antibodies is likely to transmit genes that will result in offspring negative for the particular Rh system initially appears simple and straightforward since it involves only five (5) antigens (C, , E, , cand D), but it is in fact one of the more complicated human blood group systems known. The gene complexis inherited and individuals may be analyzed in terms of separate antigens: D or its absence (d), C or , and E cor . Gene D is dominant to its allele d, because gene d is an amorph which makes no detectable antigenicproduct. The alleles C and , and E and are codominant and if both alleles are present, both will be theorized that the Rh antigens displayed by an individual are the result of only a pair of genes, oneinherited from each parent.

3 If these are identical, the individual is homozygous for that gene and all theproducts of the gene will be expressed in a double dose on the person's red cell. When the genes inherited fromeach parent are not identical, the person is heterozygous for two different genes and, since the Rh genes arecodominant, the products of both will be expressed on the person's red cells. Antigens that are products of bothgenes will be present in double dose, while those produced by only one of the pair of genes will be present insingle dose. EXAMPLE:DCe/DCE (slash separates the gene) transmitted from each parentantigen D = homozygous, present on both sidesantigen C = homozygous, present on both sidesantigens and E = heterozygous, present in on sei de onlyMLAB 2431 2Rh PhenotypingA phenotype is the assortment of antigens actually detectable on an individual's red cells using selectedantiserums.

4 In many blood group systems, the phenotype is an exact expression of the , this is not the case in the Rh system. Since any one antigen may derive from any of severalgenes, identifying antigens does not always allow the genotype to be deduced with certainty. Presumptionsregarding the most probable genotype rest on knowledge of the frequency with which particular antigeniccombinations derive from a single gene complex. To determine the genotype with more certainty, it isnecessary to do family studies which are not always possible. The following is an example of a phenotype andthe possible genotypes which could be present. EXAMPLE:Phenotype (antigens detected on the red cells) is: D, C, c, ePossible GenotypesPossible Genotypes(Fisher-Race)(Wiener)DC /d R/r c1oDC /D R/R c1oDe/dC R/r' cIn the general population, the most common D positive genotype is R/r.

5 The most common D negative1genotype is 2431 3Rh PhenotypingPrincipleThe Rh phenotype is determined by testing the patient red blood cells with the five standard antiserums: anti-D,anti-C, anti-, anti-E, and anti- . If the antigen to which the antiserum is directed is present, agglutination of cthe red blood cells will occur. No agglutination of the red blood cells indicates the absence of the on the reactions with these serums, certain statistical assumptions are made, not always correctly. Forinstance, if a cell reacts with anti-C but not with anti-, the antigen C is assumed to be present in a double dose c(homozygous for C). The exceptions to this are rare, but may be significant in paternity testing. In the past the reagent antiserums were made from individuals who have developed an antibody against aspecific blood group antigen. Although human source anti-serums are still available, the move is to providethese reagents from clones.

6 Agglutination of an individual's red cells by a specific antiserum indicates the presence of the correspondingantigen. No agglutination indicates its 2431 4Rh Bank Reagent Rack (See Procedure 3) , Anti-E, Anti-e, Anti-, Rh control c Bank plastic squares (biowipes) test bottle of saline10. to the ABO/D typing laboratory to set up tubes for the ABO/D a tube for the each Rh typing sera with the patient's first and last initial and the Rh anti-sera to be tested.(anti-E, anti-e, anti-C and anti-c ). place one drop of the correct typing sera in the properly labeled tube for that one (1) drop of the 4-6% patient cell suspension to each forward type tube and to each of the Rh typingtubes (anti-e, anti-E, anti-c and anti-C ). for 15-20 seconds. resuspend cell button and examine for macroscopic agglutination. the agglutination reaction and immediately record the results while observing the tube reaction in your YOUR REACTIONS WITH YOUR INSTRUCTOR by respinning the tubes and having instructorverify the ResultsRecord the reactions of the individual's cells with each appropriate antiserum.

7 RECORD THE REACTIONS BYHOLDING THE TUBE IN FRONT OF THE CORRECT COLUMN, visually inspect and write your interpretation . Determine the individual's phenotype and most probable genotype in the appropriate spaces. See example the attached chart for assistance. MLAB 2431 5Rh PhenotypingInterpretation of ResultsOnce the results of antigen typings have been obtained, the most probable genotype must be determined. Atfirst this will be confusing and difficult, but interpretation will get easier with practice. The following examplemay obtained:anti-Danti-Canti-canti-Eanti-e4 +4+4+04+Positive reactions indicate the presence of the antigen, negative reactions indicate the absence of the phenotype of this patient (antigens detectable on the rbc) is: D, C, c, Rh gene complex consists of a D or d, C or c, E or e antigens is inherited from each parent. When a patientis D positive the phenotype of the person is either D/d (heterozygous for D) or D/D ( homozygous for D).

8 Makea separate column for each possible genotype, the other Rh antigens will be added to this base DHomozygous DD /dD /DThe results from the Rh phenotype indicates that the patient is C+ and c+, the individual is heterozygous forthe C antigen, so place a C or c on either side of the Dd as follows. It is critical to remember to list ALLpossible combinations. DC /dcDC /Dc (only combination possible) Dc /dCThe next antigen to plug in is E. The patient is E=e+ indicating that this individual is homozygous for e, soe will be present on both sides. DCe /dceDCe /Dce Dce /dCeYou must now convert the Wiener notations to Fisher-Race using the chart supplied, also indicate thefrequencies in which these genotypes occur:DCe /dceRr33%1 Dce /dCeRr < /DceRR 2%10 One thing to remember is that frequencies given are for the white population.

9 In the black population the R0genotype is very common. It is critical to know the race of the individual when determining most 2431 6Rh PhenotypingInterpreting ResultsMemorize this chart! Antigens Fisher-RaceWienerD,c,eD R coD,C,eDC R1 DEDER c c2D,C,EDCERz, d r c cC, dC r',EdEr" c cC,EdCEryIn interpreting reactions for genotypes, always remember that if the D antigen is present it does not mean thatit is homozygous. Figure the genotype with D on one side and d on the other (heterozygous for D). Use thechart on the following page and select the genotype with the highest frequency. For example, if the D, C, and c antigens are present, there are three (3) possible genotypes:Fisher-RaceWiener% frequency fromfollowing /d R/r33% /D R/R 2% R/r' cThe most probable genotype is Rr since this has the highest statistical probability. 1 MLAB 2431 7Rh PhenotypingRh Genotypes and Their Serological ReactionsGenotypesReactions with anti-CaucasianFrequency (%)

10 Fisher-RaceWienerDCEceDCe/dceR/r++0++331 DCe/DceR/R++0++210 Dce/dCeR/r'++0++ 0 DCe/DCeR/R++00+1811 DCe/dCeR/r'++00+*1 DcE/dceR/r+0+++112 Dce/dcER/r"+0+++ 0 DcE/DceR/R+0+++*20 DcE/DcER/R+0++0222 DcE/dcER/r"+0++0*2 DCe/DcER/R+++++1212 DCe/dCER/r+++0+ 1yDCe/dcER/r"+++++11 DcE/dCER/r++++0 2yDcE/dCeR/r'+++++*2 DCE/DCER/R+++00 zzDCE/DCeR/R+++0+*z1 DCE/DcER/R++++0 z2 DCE/dCeR/r'+++0+ zDCE/DceR/R+++++ z0 DCE/dcER/r"++++0 zDCE/dceR/r+++++*zDCE/dCER/r+++00 zyDce/dCER/r+++++ 0yDce/dceR/r+00++20 Dce/DceR/R+00++ 00dCe/dcer'/r0+0++*dCe/dCer'/r'0+00+ dcE/dcer"/r00+++*dcE/dcEr"/r"00++0 dCe/dcEr'/r"0++++ dCE/dcer/r0++++ ydCE/dcEr/r"0+++0 ydCE/dCer/r'0++0+ ydCE/dCEr/r0++00 yydce/dcer/r000++15* = less than 1 percent but greater than percent = less than percent but greater than percent = less than percentMLAB 2431 8Rh PhenotypingComparison of Wiener and Fisher-Race Concepts ofthe Rh blood group SystemWiener ConceptFisher-Race ConceptApproximateFrequency inCaucasoids ofNew YorkCity (%)GeneAgglutinogenBlood FactorsGeneComplexBlood Factorso0 RRhRh, hr', hr"DceD, c, , rh', hr"DCeD, C, , rh", hr'DcED, c, , rh', rh"DCED, C, ', hr"dcec, 'rh'rh', hr"dCeC, "rh"rh", hr'dcEc, ', hr"dCEC, 2431 9Rh PhenotypingName_____ Date_____Exercise 4: Rh PhenotypingRecording ResultsA.