Exercise 4 Rh Phenotyping

Textbook: Blaney, Chapter 5

Skills: 20 Points

Objectives:

1. List 4 reasons for determining an individuals Rh phenotype.

2. State the antigens of the Rh blood group system.
3. Describe the allele “d”.
4. Define the following terms: “amorph, dominant, codominant, heterozygous, and homozygous as they relate
to inheritance of the Rh antigens.
5. Define the terms “phenotype” and “genotype” as they relate to blood group antigens.
6. Briefly state the procedure whereby an individual’s Rh phenotype is determined.
7. Recall each of the 8 Rh genes in both Fisher-Race and Wiener notation.
8. Given an Rh phenotype list the potential genotypes in both Wiener and Fisher-Race notations.
10. Determine the Rh phenotype and probable genotype of two (2) patient samples.

Discussion

In this procedure, commonly tested antigens of the Rh system will be studied. Normally the only Rh antigen
identified in routine pretransfusion testing is the D antigen. Four additional Rh antigens which may be tested
for are: C, E, –c, and ç. Phenotyping for these antigens may be useful in: family studies, resolution of antibody
problems, population studies, cases of disputed parentage, and to predict whether the sexual partner of a woman
with Rh antibodies is likely to transmit genes that will result in offspring negative for the particular antigen.

The Rh system initially appears simple and straightforward since it involves only five (5) antigens (C, –c, E, ç,
and D), but it is in fact one of the more complicated human blood group systems known. The gene complex
is inherited and individuals may be analyzed in terms of separate antigens: D or its absence (d), C or –c, and E
or ç. Gene D is dominant to its allele d, because gene d is an amorph which makes no detectable antigenic
product. The alleles C and –c, and E and ç are codominant and if both alleles are present, both will be
expressed.

Wiener theorized that the Rh antigens displayed by an individual are the result of only a pair of genes, one
inherited from each parent. If these are identical, the individual is homozygous for that gene and all the
products of the gene will be expressed in a double dose on the person's red cell. When the genes inherited from
each parent are not identical, the person is heterozygous for two different genes and, since the Rh genes are
codominant, the products of both will be expressed on the person's red cells. Antigens that are products of both
genes will be present in double dose, while those produced by only one of the pair of genes will be present in
single dose.

EXAMPLE:
DCe/DCE (slash separates the gene) transmitted from each parent
antigen D = homozygous, present on both sides
antigen C = homozygous, present on both sides
antigens ç and E = heterozygous, present in onseide only

Rh Phenotyping MLAB 2431• 1

A phenotype is the assortment of antigens actually detectable on an individual's red cells using selected
antiserums. In many blood group systems, the phenotype is an exact expression of the genotype.
Unfortunately, this is not the case in the Rh system. Since any one antigen may derive from any of several
genes, identifying antigens does not always allow the genotype to be deduced with certainty. Presumptions
regarding the most probable genotype rest on knowledge of the frequency with which particular antigenic
combinations derive from a single gene complex. To determine the genotype with more certainty, it is
necessary to do family studies which are not always possible. The following is an example of a phenotype and
the possible genotypes which could be present.

EXAMPLE:

Phenotype (antigens detected on the red cells) is: D, C, c, e

Possible Genotypes Possible Genotypes
(Fisher-Race) (Wiener)
DCç/d–cç R1/r
DCç/D–cç R1/Ro
D–ce/dCç Ro/r'

In the general population, the most common D positive genotype is R1/r. The most common D negative
genotype is rr.

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Principle

The Rh phenotype is determined by testing the patient red blood cells with the five standard antiserums: anti-D,
anti-C, anti-–c, anti-E, and anti-ç. If the antigen to which the antiserum is directed is present, agglutination of
the red blood cells will occur. No agglutination of the red blood cells indicates the absence of the antigen.
Based on the reactions with these serums, certain statistical assumptions are made, not always correctly. For
instance, if a cell reacts with anti-C but not with anti-–c, the antigen C is assumed to be present in a double dose
(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 a
specific blood group antigen. Although human source anti-serums are still available, the move is to provide
these reagents from clones.

Agglutination of an individual's red cells by a specific antiserum indicates the presence of the corresponding
antigen. No agglutination indicates its absence.

Rh Phenotyping MLAB 2431• 3

Reagents

1. Blood Bank Reagent Rack (See Procedure 3)

– Anti-–c, Rh control
2. Anti-C, Anti-E, Anti-e,
3. Blood Bank plastic pipets
4. Blotting squares (biowipes)
5. 12x75 test tubes
6. Serofuge
7. Heat Block
8. Agglutination viewer
9. Squirt bottle of 0.85% saline
10. Sharpie

Procedure
1. Refer to the ABO/D typing laboratory to set up tubes for the ABO/D type.
2. Label 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 ).
3. CAREFULLY place one drop of the correct typing sera in the properly labeled tube for that anti-sera..
4. Place one (1) drop of the 4-6% patient cell suspension to each forward type tube and to each of the Rh typing
tubes (anti-e, anti-E, anti-c and anti-C ).
5. Centrifuge for 15-20 seconds.
6. Gently resuspend cell button and examine for macroscopic agglutination.
7. Grade the agglutination reaction and immediately record the results while observing the tube reaction in your hand.
8. CONFIRM YOUR REACTIONS WITH YOUR INSTRUCTOR by respinning the tubes and having instructor
verify the results.

Recording Results

Record the reactions of the individual's cells with each appropriate antiserum. RECORD THE REACTIONS BY
HOLDING 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 provided.
Use the attached chart for assistance.

Rh Phenotyping MLAB 2431• 4

Interpretation of Results

Once the results of antigen typings have been obtained, the most probable genotype must be determined. At
first this will be confusing and difficult, but interpretation will get easier with practice. The following example
may help.

Results obtained:
anti-D anti-C anti-c anti-E anti-e
4+ 4+ 4+ 0 4+

Positive reactions indicate the presence of the antigen, negative reactions indicate the absence of the antigen.
The phenotype of this patient (antigens detectable on the rbc) is: D, C, c, e.

An Rh gene complex consists of a D or d, C or c, E or e antigens is inherited from each parent. When a patient
is D positive the phenotype of the person is either D/d (heterozygous for D) or D/D ( homozygous for D). Make
a separate column for each possible genotype, the other Rh antigens will be added to this “base” type.

Heterozygous D Homozygous D
D /d D /D

The results from the Rh phenotype indicates that the patient is C+ and c+, the individual is heterozygous for
the C antigen, so place a C or c on either side of the Dd as follows. It is critical to remember to list ALL
possible combinations.
DC /dc DC /Dc (only combination possible)
Dc /dC

The next antigen to “plug in” is E. The patient is E=e+ indicating that this individual is homozygous for e, so
e will be present on both sides.

DCe /dce DCe /Dce

Dce /dCe

You must now convert the Wiener notations to Fisher-Race using the chart supplied, also indicate the
frequencies in which these genotypes occur:

DCe /dce R1r 33%

Dce /dCe R0r’ <0.1%
DCe /Dce R1R0 2%

One thing to remember is that frequencies given are for the white population. In the black population the R0
genotype is very common. It is critical to know the race of the individual when determining most probable
genotypes.

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Interpreting Results

Memorize this chart!

Antigens Fisher-Race Wiener

D,c,e D–cç Ro
D,C,e DCç R1
D–cE D–cE R2
D,C,E DCE Rz
–c,ç d–cç r
C,ç dCç r'
–c,E d–cE r"
C,E dCE ry

In interpreting reactions for genotypes, always remember that if the D antigen is present it does not mean that
it is homozygous. Figure the genotype with D on one side and d on the other (heterozygous for D). Use the
chart on the following page and select the genotype with the highest frequency. For example, if the D, C, –c and
ç antigens are present, there are three (3) possible genotypes:

Fisher-Race Wiener % frequency from

following table
1. DCç/d–cç R1/r 33%
2. DCç/D–cç R1/Ro 2%
3. D–ce/dCç Ro/r' 0.01-0.1%

The most probable genotype is R1r since this has the highest statistical probability.

Rh Phenotyping MLAB 2431• 6

 Rh Genotypes and Their Serological Reactions
Genotypes Reactions with anti- Caucasian
Fisher-Race Wiener D C E c e Frequency (%)
1
DCe/dce R /r + + 0 + + 33
DCe/Dce R1 /R0 + + 0 + + 2
Dce/dCe R0 /r' + + 0 + + †
DCe/DCe R1/R1 + + 0 0 + 18
DCe/dCe R1 /r' + + 0 0 + *
DcE/dce R2/r + 0 + + + 11
Dce/dcE R0 /r" + 0 + + + †
DcE/Dce R2 /R0 + 0 + + + *
DcE/DcE R2 /R2 + 0 + + 0 2
DcE/dcE R2 /r" + 0 + + 0 *
DCe/DcE R1/R2 + + + + + 12
DCe/dCE R1 /ry + + + 0 + ‡
DCe/dcE R1 /r" + + + + + 1
DcE/dCE R2 /ry + + + + 0 ‡
DcE/dCe R2 /r' + + + + + *
DCE/DCE Rz/Rz + + + 0 0 ‡
DCE/DCe Rz/R1 + + + 0 + *
DCE/DcE Rz/R2 + + + + 0 †
DCE/dCe Rz/r' + + + 0 + ‡
DCE/Dce Rz/R0 + + + + + †
DCE/dcE Rz/r" + + + + 0 ‡
DCE/dce Rz/r + + + + + *
DCE/dCE Rz/ry + + + 0 0 ‡
Dce/dCE R0 /ry + + + + + ‡
Dce/dce R0 /r + 0 0 + + 2
Dce/Dce R0 /R0 + 0 0 + + †
dCe/dce r'/r 0 + 0 + + *
dCe/dCe r'/r' 0 + 0 0 + ‡
dcE/dce r"/r 0 0 + + + *
dcE/dcE r"/r" 0 0 + + 0 †
dCe/dcE r'/r" 0 + + + + †
dCE/dce ry/r 0 + + + + ‡
dCE/dcE ry/r" 0 + + + 0 ‡
dCE/dCe ry/r' 0 + + 0 + ‡
dCE/dCE ry/ry 0 + + 0 0 ‡
dce/dce r/r 0 0 0 + + 15

* = less than 1 percent but greater than 0.1 percent

† = less than 0.1 percent but greater than 0.01 percent
‡ = less than 0.01 percent

Rh Phenotyping MLAB 2431• 7

 Comparison of Wiener and Fisher-Race Concepts of
the Rh Blood Group System
Approximate
Wiener Concept Fisher-Race Concept Frequency in
Caucasoids of
Gene New York
Gene Agglutinogen Blood Factors Blood Factors
Complex City (%)
R0 Rho Rh0, hr', hr" Dce D, c, e 2.5
R1 Rh1 Rh0, rh', hr" DCe D, C, e 51.2
R2 Rh2 Rh0, rh", hr' DcE D, c, E 16.5
Rz Rhz Rh0, rh', rh" DCE D, C, E 14.9
r rh hr', hr" dce c, e 13.4
r' rh' rh', hr" dCe C, e 1.1
r" rh" rh", hr' dcE c, E 0.4
ry rhy hr', hr" dCE C, E 0.02

Rh Phenotyping MLAB 2431• 8

 Name_________________________________ Date_________________________________

Exercise 4: Rh Phenotyping
Recording Results
A. ABO/D Typing
Forward Type D Type Weak D Test Reverse Type

Patient Name ID Number Anti-A Anti-B Anti-D *D Ctrl Wk D T cells Wk D ctrl T cells A Cells B Cells Interpretation
1.

2.

B. Rh Phenotype - circle the most probable genotype. COPY D TYPING from chart above DO NOT repeat. Instructor verified _______

Reactions with anti- Most probable genotype

Name D C E –c –e Rh ctrl Phenotype Fisher-Race Wiener
Example 3+ 3+ O 3+ 3+ O D,C,–c–e DC–e/d–ce R1r

1.
#

2.
#

Rh Phenotyping MLAB 2431• 9

Name_________________________________

Rh Phenotyping
Study Questions

1. List the antigens of the Rh system. (2.5 points)

2. State three situations in which testing for the Rh antigens (phenotyping) provides useful information. (1.5
points).
a.

b.

c.

3. Is the allele to D actually detected? Explain. (1 point)

4. What does it mean to say that the Rh antigens C and c or E and e are “codominant”. (1 point)

5. What is meant by the terms homozygous and heterozygous? (2 pts)

6. Define phenotype. (1 point)

Rh Phenotyping MLAB 2431• 10

7. Define genotype. (1 point)

8. State the most common D positive genotype in both Fisher-Race and Wiener notations. Provide the same
for the most common D negative genotype. (2 points)

Fisher-Race Wiener
D positive
D negative

9. How are presumptions regarding the most probable genotype determined? (1 point)

10. Fill in the following chart and memorize!

Fisher Race Wiener

a. R1
DcE b.
c. Rz
dce d.
e. R0
dcE f.
g. r’
dce h.

Rh Phenotyping MLAB 2431• 11

11. For the following phenotypes, list all possible genotypes. State each ones' statistical probability as given for
Caucasians. (Use Fisher-Race and Wiener.) Circle the most probable genotype. Point values vary.

a. D, C, E, ç (3 points)

b. d, –c, ç (1 point)

c. D, E, –c, ç (3 points)

12. State 2 situations in which a D control must be run AND THE REASON FOR RUNNING IT.(2 points)

Rh Phenotyping MLAB 2431• 12