Free ASCP MLS Blood Banking Exam Mock Test – Part 58: The Rh Blood Group System

The D antigen of the Rh system is the most immunogenic blood group antigen after the ABO antigens.
It causes antibody formation in a high percentage of Rh-negative individuals exposed to Rh-positive red cells.
By comparison, K (Kell) is highly immunogenic but less so than D, while Fyᵃ and Jkᵇ are less immunogenic.

• RhIG (Rh immune globulin) is given to Rh-negative women who are not yet immunized to prevent anti-D formation.

• If a woman already has a high anti-D titer (e.g., 1:4096), she is already sensitized, so giving RhIG won’t help.

• Other scenarios:

  • First-trimester abortion → RhIG is indicated to prevent sensitization.

  • Husband is Rh-positive → RhIG may be needed if fetal status is unknown.

  • Positive DAT → indicates sensitization; RhIG not indicated in already sensitized women.

• Rh antibodies (IgG) typically cause extravascular hemolysis via the reticuloendothelial system, not intravascular complement-mediated lysis.

• They can cause:

  • Delayed hemolytic transfusion reactions

  • Hemolytic disease of the newborn (HDFN)

  • In vivo sensitization of red cells

• They are poor complement activators, unlike some IgM antibodies (e.g., anti-A, anti-B).

The Wiener theory proposes that a single gene complex produces a single “agglutinogen” with multiple specificities.

Its notation uses superscripts and the letter “R” (for Rh) to denote the gene complex, such as:

• R¹ for the complex producing Rh1 (D), Rh2 (C), and Rh4 (c) specificities.

• r for the complex producing Rh4 (c) and Rh5 (e) specificities.

• R² for the complex producing Rh1 (D), Rh3 (E), and Rh4 (c) specificities.

• Anti-f (also called anti-ce) reacts with red cells that have c and e on the same haplotype (cis position).

• The f antigen is present when the Rh haplotype is ce (little c and little e together on one chromosome).

• Genotype rr = dce/dce → both chromosomes are ce → f antigen strongly expressed.

• R₁R₁ (CDe/CDe) → no c, so no f.

• R₂R₂ (cDE/cDE) → no e, so no f.

• R₁R₂ (CDe/cDE) → c and e are on opposite chromosomes (trans) → f antigen negative.

The clinical designation “Rh-negative” refers specifically to the absence of the D antigen on the red blood cells.

A person can be Rh-negative (D-negative) but still have other Rh antigens like C, c, E, and e. The term does not mean the absence of all Rh antigens (which is the very rare Rh null phenotype).

Rh system antigens (D, C, c, E, e) are carried on non-glycosylated transmembrane proteins that pass through the red cell membrane multiple times. Unlike many other blood group antigens, they are proteins, not carbohydrates, and they lack significant sugar chains (glycans).

• Delayed hemolytic transfusion reactions (DHTRs) usually occur days to weeks after transfusion.

• They are caused by IgG antibodies, such as anti-Rh (e.g., anti-D, anti-E).

• IgG antibodies do not strongly fix complement, so red cells are mostly removed extravascularly by macrophages in the spleen and liver.

• Intravascular hemolysis (option a) is more typical of ABO incompatibility, where IgM and complement are involved.

Routine Rh typing often starts with an immediate-spin phase. If this is negative for the D antigen, it could mean the patient is truly D-negative or has a weak D phenotype (with a reduced number of D antigen sites).

To detect weak D, the test must proceed to the indirect antiglobulin test (IAT) phase. Anti-D is allowed to bind to the red cells, and then anti-human globulin (AHG) is added to cross-link the antibodies, causing agglutination of weak D-positive cells.

Let’s solve this step by step.

We are given the reactions of red blood cells with Rh antisera:

• Anti-D: 4+ → D is present

• Anti-C: 3+ → C is present

• Anti-E: 0 → E is absent

• Anti-c: 3+ → c is present

• Anti-e: 3+ → e is present

The positive reactions tell us which antigens are present on the RBCs.

• So the person has D, C, c, e antigens.

• E is absent.

Looking at possible genotypes:

• a) DCe/DcE → This would give D, C, E, c, e antigens → But E is absent, so wrong.

• b) DcE/dce → This would give D, c, E, e → Missing C, wrong.

• c) Dce/dce → This would give D, c, e only → Missing C, wrong.

• d) DCe/dce → This would give D, C, c, e → Matches perfectly.

• Rh-negative is required to avoid further hemolysis from maternal anti-D.

• Group O is universally compatible with the mother and fetus regardless of ABO type.

• Irradiated to prevent transfusion-associated graft-versus-host disease.

• CMV safe (CMV-negative or leukoreduced) to protect the immunocompromised fetus.

The weak D test is an indirect antiglobulin test (IAT). After incubating the patient’s red cells with anti-D reagent, anti-D IgG antibodies bind to the weak D antigens. These antibodies are not visible as agglutination. Anti-human globulin (AHG) is then added. AHG cross-links the anti-D IgG molecules on adjacent red cells, causing visible agglutination, which confirms a weak D positive result.

• Rh antibodies (like anti-D, anti-C) are not naturally occurring; they form only after exposure to foreign Rh antigens through transfusion or pregnancy.

• They are typically IgG, which can cross the placenta and cause hemolytic disease of the fetus and newborn (HDFN).

• They are warm-reactive and do not usually fix complement, unlike some IgM antibodies.

Let’s analyze step by step.

• Mother: Rh-negative → genotype dd (homozygous for absence of D).

• Father: Rh-positive → could be DD (homozygous) or Dd (heterozygous).

• Baby: Rh-negative → genotype dd.

For the baby to be dd, it must inherit one d from each parent.

• Mother provides d → always.

• Father must provide d → so he cannot be DD, because DD has no d to pass on.

Let’s solve this step by step.

We are given the donor’s Rh typing results:

• anti-D: +

• anti-C: +

• anti-E: 0

• anti-c: +

• anti-e: +

Step 1: Determine which antigens are present

• D⁺ → has D

• C⁺ → has C

• E⁰ → does not have E

• c⁺ → has c

• e⁺ → has e

So phenotype: D⁺ C⁺ E⁰ c⁺ e⁺

Step 2: Match phenotype with Rh genotypes

Common Rh haplotypes:

We need D⁺ C⁺ E⁰ c⁺ e⁺ →

• Has D → haplotype is R₁, R₂, or R₀

• Has C → could be R₁ or R₂

• Does not have E → not R₂

• Has c and e → must pair with r (r = dce)

✅ So genotype = R₁r

• Fisher-Race system expresses the Rh genotype using the antigens themselves: D, C, E, c, e.

• For example:

  • R₁ = D C e

  • R₂ = D c E

• The Weiner system, in contrast, uses numbers (1, 2, 3, 4, 5) to represent the same antigens.

Let’s analyze:

• Warm autoantibodies react at 37°C and are usually IgG.

• The most common specificity of warm autoantibodies is against Rh antigens (D, C, E, c, e).

• Other antigens:

  • I → usually cold antibody (reacts <30°C)

  • P → usually cold antibody

  • Leᵃ → usually not clinically significant

The term “Dᵘ” is an older, historical designation that is now obsolete. It was used to describe samples that were D-negative in the immediate-spin phase of typing but became D-positive after an indirect antiglobulin test (IAT).

This phenomenon is what we now classify under the broader category of Weak D. Modern practice uses “Weak D” to describe this quantitative reduction in D antigen sites.

• R₁r (D C e / d c e) is the most common Rh phenotype among Caucasians.

• It results in red cells that are D positive, C positive, c positive, E negative, e positive.

• Other phenotypes (R₀r, R₂r, rr) are less frequent in this population

• In donors (not patients), if initial testing with anti-D is negative, the blood bank must perform weak D (Du) testing to ensure the red cells are truly D-negative.

• This is because a donor with weak D expression could stimulate anti-D in an Rh-negative recipient if transfused as Rh-negative.

• For recipients in transfusion, weak D testing is not required (they can be treated as Rh-negative if initial anti-D is negative to avoid giving Rh-positive blood).

• But for donor labeling, weak D testing is standard to prevent mislabeling a weakly D-positive unit as Rh-negative.

The rosette test is a screening method used on a D-negative maternal blood sample after delivery.
It detects the presence of fetal D-positive red cells by causing them to form visible “rosettes” around reagent D-positive indicator cells.
A positive rosette test indicates a suspected large fetomaternal hemorrhage, which would require further quantification (e.g., with a Kleihauer-Betke test) to determine the correct dose of Rh immune globulin needed.

An Rh-positive person making anti-D is rare but possible in cases of partial D (formerly called D mosaic).

• In partial D, the individual has an altered RHD gene that produces a D antigen missing some epitopes of the complete D antigen.

• If exposed to normal D-positive RBCs (via transfusion or pregnancy), they can make anti-D targeting the missing epitopes.

• This is not due to gene deletion (which would cause no D antigen), trans effect, or simple gene inhibition, but rather a variant D antigen that lacks parts of the complete D protein.

• Rh-negative individuals lack the D antigen.

• If transfused with Rh-positive (D+) blood, they are very likely to produce anti-D.

• Anti-D is immunogenic and commonly forms after exposure to D-positive RBCs.

• Other antibodies (anti-E, anti-C, anti-e) are less likely unless the donor blood had other mismatched Rh antigens, but anti-D is the primary expected antibody.

The G antigen is a part of the Rh system that is present when a red blood cell has either the C antigen (encoded by the RHCE gene) or the D antigen (encoded by the RHD gene).
This is because the amino acid sequence that codes for the G antigen is present on both the C and D proteins.

Let’s carefully work this out.

Step 1: Analyze the parents’ Rh phenotypes

Mother: D+C+E–c–e+

• Antigens present: D, C, e

• Antigens absent: E, c

• Serum contains anti-c → confirms she lacks c

• Most probable genotype: R₁r (DCe/dce)

Father: D+C+E–c+e+

• Antigens present: D, C, c, e

• Antigens absent: E

• Most probable genotype: R₁r’ (DCe/Dce)

Step 2: Baby is Rh-negative

• Rh-negative = dd → lacks D antigen entirely.

• Baby’s genotype cannot contain D, so it must inherit d from both parents.

Step 3: Check which alleles parents can pass

• Mother (R₁r = DCe/dce) → can pass R₁ (DCe) or r (dce)

• Father (R₁r’ = DCe/Dce) → can pass R₁ (DCe) or r’ (Dce)

• To get dd, the baby must receive r alleles from both parents.

• Only the mother’s r (dce) provides d

• Father must pass a d allele → only r’ (Dce)? Wait, r’ has D → cannot give d

• Father cannot give a d allele? That seems tricky.

• Homozygous for D means the individual has two D-positive alleles.

• Using Fisher-Race/Weiner notation:

  • R₂ = D c E

  • R₂R₂ → both alleles have D → homozygous D

• R₁r → one D-positive (R₁) and one D-negative (r) → heterozygous

• rr → no D antigen → homozygous D-negative

• R₀r → one D-positive (R₀) and one D-negative (r) → heterozygous

• Among Rh antigens, D is the most immunogenic, meaning it is most likely to provoke an antibody response in an Rh-negative individual.

• Other antigens like C, c, E, and e are less immunogenic.

• This is why anti-D is the main cause of hemolytic disease of the fetus and newborn (HDFN) and why Rh-negative individuals are given RhIG prophylaxis.

The Wiener notation R₂ corresponds directly to the Fisher-Race haplotype DcE.

This haplotype produces the following antigens:

• D from the RHD gene

• c and E from the RHCE gene

The other common correspondences are:

• R₁ = DCe

• R₀ = Dce

• r = ce

The Kleihauer-Betke (KB) calculation estimates fetomaternal hemorrhage (FMH) volume using this formula:

$$\text{FMH (mL)}=\%\text{fetal cells}\times \text{maternal blood volume (mL)}/100$$

• Maternal blood volume is roughly 5000 mL.

• Fetal cells = 0.3% → 0.003 × 5000 mL = 15 mL

So the estimated FMH = 15 mL of whole blood.

The Rh null phenotype is a rare condition where red blood cells lack all Rh antigens (D, C, c, E, e). This can occur due to a “regulator” type mutation (most common) that silences the entire Rh complex or an “amorph” type that affects the RhCE and RhD genes directly. This condition is associated with a chronic hemolytic anemia called Rh null disease.

The most common mechanism for weak D expression is an inherited quantitative variant, where the individual has a genetically altered RHD gene that produces a complete D protein, but in a reduced number on the red cell surface. This low density of D antigens is insufficient to cause agglutination in the immediate-spin phase of typing but can be detected with the more sensitive indirect antiglobulin test (IAT).

The RHCE gene on chromosome 1 encodes the RhCE protein, which carries the C/c and E/e epitopes.
The four common antigen combinations (ce, cE, Ce, CE) result from polymorphisms in the RHCE gene.
RHD encodes the D antigen only.

Let’s work this out carefully.

We are told the patient’s red cells react positively with all Rh antisera: anti-C, anti-D, anti-E, anti-c, and anti-e.

This means the red cells express all five common Rh antigens: D, C, E, c, e.

Now let’s recall the common Rh haplotypes:

To express all five antigens, the genotype must be a combination that covers D, C, E, c, e.

• R₁R₁ (DCe/DCe): D, C, e only → missing c and E →

• R₁R₂ (DCe/DcE): D, C, c, E, e → covers all five antigens

• R₀R₀ (Dce/Dce): D, c, e → missing C and E →

• R₂R₂ (DcE/DcE): D, c, E → missing C and e →

• Anti-D antibodies in an Rh-negative mother can cross the placenta and bind to Rh-positive fetal red cells, causing their destruction.

• This leads to hemolytic disease of the fetus and newborn (HDFN/HDN).

• It is not related to ABO incompatibility, iron deficiency, or polycythemia.

• Rh antibodies are typically IgG and warm-reactive, reacting optimally at body temperature (37°C).

• Detection usually occurs during the indirect antiglobulin test (IAT) after 37°C incubation, which allows antibodies to bind red cells, followed by AHG addition.

• They do not react well at immediate-spin, room temperature, or saline-only phases.

A person with a partial D phenotype lacks some parts of the normal D antigen.
If they are exposed to normal D-positive blood through transfusion or pregnancy, their immune system may see the “missing part” as foreign and can make anti-D.
Routine typing may still show them as D-positive or weakly positive, but their immune response is like that of a D-negative person.

Let’s reason carefully:

Given:

• Mother: A, D-negative, has anti-D

• Newborn: O, D-negative, DAT 4+ (strongly positive)

Step 1: Interpret the findings

• DAT (Direct Antiglobulin Test) 4+ indicates coating of the baby’s RBCs with antibody, usually maternal IgG.

• Maternal antibody is anti-D, which reacts only with D-positive RBCs.

• But the baby is reported D-negative, so anti-D should not react.

Step 2: Identify the questionable test

• Maternal ABO type → plausible (A)

• Newborn DAT → plausible (4+)

• Paternal D type → not given; could be D-positive or D-negative

• Newborn D type → reported as D-negative, but strong anti-D (from mother) has coated RBCs → this is inconsistent

Step 1 – Understand the scenario

• Recipient: CDe/CDe (common Rh genotype, also written as DCe/DCe).
  This means the recipient’s Rh antigens are:

  • D: positive

  • C: positive

  • E: negative (since CDe has C and e, not E)

  • c: negative (since CDe has C, not c)

  • e: positive (since CDe has e)

• Donor: Rh-negative red cells → no D antigen.
  Most likely Rh-negative genotype in donors: dce/dce (also called rr).
  This donor has:

  • D: negative

  • C: negative

  • E: negative

  • c: positive

  • e: positive

Step 2 – Compare donor vs recipient antigens
Recipient (CDe/CDe): D+ C+ E− c− e+
Donor (dce/dce): D− C− E− c+ e+

So the donor has c antigen, which the recipient lacks.
Recipient lacks E as well, but donor also lacks E, so no E exposure.

Step 3 – Which antibody is most likely?
After transfusion, recipient is exposed to donor’s c antigen → may make anti-c.
Anti-d is not possible because “d” is not a real antigen — it’s the absence of D.
Anti-e? Recipient is e+, so won’t make anti-e.
Anti-E? Donor is E−, so no E exposure.

Thus, anti-c is the most likely.

One standard vial of RhIG (300 µg) covers a fetomaternal hemorrhage (FMH) of 15 mL of whole blood or 30 mL of red blood cells.

• Step 1: Determine the FMH volume in mL of whole blood: 48 mL (given).

• Step 2: Divide the total FMH volume by the volume one vial covers: 48 mL / 15 mL per vial = 3.2 vials.

• Step 3: Always round up to the next whole vial to ensure adequate protection.

• e is the most common Rh antigen in all populations, including Caucasians.

• About 98% of people express the e antigen.

• D is also high (~85% in Caucasians), but e is higher.

• C (~68%), E (~29%) are less frequent.

The phenotype D+C+E-c+e+ means the patient has the D, C, and e antigens, but lacks E.

Let’s break down the common genotypes:

• R₁ (DCe) has D, C, and e.

• r (ce) has c and e.

• R₀ (Dce) has D, c, and e.

• R₂ (DcE) has D, c, and E.

The patient is E-, which rules out any genotype containing E, such as R₂ (DcE).
The patient is C+, which rules out R₀r (Dce/ce), as this would not produce the C antigen.

The genotype R₁r (DCe/ce) perfectly matches the phenotype: it produces D, C, and e, and does not produce E (or big C).

Rh antibodies, particularly anti-D, are a classic cause of delayed hemolytic transfusion reactions (DHTR).
A patient with a previous exposure (e.g., pregnancy or transfusion) may have a low, undetectable level of antibody. Upon re-exposure to the antigen during a transfusion, a secondary immune response occurs, leading to the production of IgG antibodies that cause extravascular hemolysis of the transfused red cells days to weeks later.

Let’s solve this step by step.

Step 1: Analyze antigen reactions

• Anti-D (+) → D present

• Anti-C (+) → C present

• Anti-E (+) → E present

• Anti-c (+) → c present

• Anti-e (+) → e present

• Anti-f (0) → f absent → meaning c and e are not together on the same haplotype

Recall: f antigen = c + e on the same chromosome

So this person has all five common Rh antigens individually, but no f, meaning c and e are on different haplotypes.

Step 2: Check the haplotype combinations

1. R₁R₂ (DCe/DcE)

   • Antigens: D, C, c, E, e

   • c and e are on different haplotypes →  f negative

   • Matches perfectly

2. R₂r (DcE/dce)

   • Antigens: D, c, E, e → C absent →

3. R₀r (Dce/dce)

   • Antigens: D, c, e → C absent →

4. R₂R₂ (DcE/DcE)

   • Antigens: D, c, E → e absent →

• The Rh system was discovered in 1940 by Landsteiner and Wiener.

• They immunized rabbits and guinea pigs with red cells from Rhesus monkeys and found antibodies that reacted with human red cells.

• This led to the identification of the Rh (Rhesus) antigen in humans.

Rh antibodies are clinically significant IgG antibodies that react optimally at body temperature (37°C). This is why crossmatching and antibody detection tests include a 37°C incubation phase. Their reactivity at 37°C is a key reason they can cause Hemolytic Transfusion Reactions and Hemolytic Disease of the Fetus and Newborn.

The most common cause of the Rh-negative phenotype (D-negative) in humans is the complete deletion of the RHD gene.
The RHCE gene remains intact and determines C/c and E/e antigens.
A deletion of both RHD and RHCE is extremely rare and not the usual cause of the standard Rh-negative phenotype.

Let’s carefully analyze this scenario.

Step 1: Mother’s information

• Rh-positive → D antigen present

• Red cell phenotype not fully given, but she has anti-c → she lacks c antigen → c-negative

• Anti-c titer: 32 at AHG → sensitized, but baby not affected → no hemolysis

Mother’s most probable genotype: R₁R₁ (DCe/DCe) or R₁R₀ (DCe/Dce) if she is C-positive, c-negative. But key point: she is c-negative, so she can produce anti-c.

Step 2: Baby is unaffected and DAT-negative

• If the father carried c, baby could be c-positive, and mother’s anti-c could cause HDN.

• Since baby is unaffected → baby is c-negative.

Step 3: Determine father’s genotype

• For baby to be c-negative, father must lack c antigen on both haplotypes → father’s genotype likely R₁R₁ (DCe/DCe) or similar → must be c-negative.

Looking at options:

a) rr (dce/dce) → c-positive → 
b) r’r (Dce/dce) → c-positive → 
c) R₁r (DCe/dce) → c-positive (because of r = dce) → 
d) R₂r (DcE/dce) → c-positive →

Hmm, none of the given options are fully c-negative. Let’s check each carefully:

• R₁ = DCe → C+, e+

• R₂ = DcE → c+, E+

• r = dce → c+, e+

• r’ = Dce → c-, e+

If the father is r’r (Dce/dce):

• r’ = c-negative

• r = c-positive

• Child could inherit r’ (c-) → baby could be c-negative → possible

• RhIG contains passive anti-D antibodies.

• When given to an Rh-negative, unsensitized mother, it binds any fetal D-positive red cells in her circulation, preventing her immune system from recognizing them and forming active anti-D antibodies.

• It does not stimulate D antigen, treat the newborn, or increase antibody titers.

Partial D (also known as D mosaic) is a qualitative variant. The individual’s RHD gene codes for a D protein that is missing one or more parts (epitopes) of the complete D antigen.

If they are transfused with red cells carrying a normal, complete D antigen, their immune system may recognize the “missing” epitopes as foreign and produce anti-D against those specific parts.

In Wiener notation:

• R₁ represents a gene complex that produces antigens D, C, and e.

• r represents a gene complex that produces antigens c and e.

Therefore, an individual with the genotype R₁r has the following antigens on their red cells:

• From R₁: D, C, e

• From r: c, e

• The RHD and RHCE genes, which encode the Rh antigens (D, C, c, E, e), are located on chromosome 1.

• These genes are tightly linked and determine the expression of the Rh blood group system.

• Other chromosomes listed (4, 6, 19) are not involved in Rh antigen production.

Anti-D is the most common and most severe cause of HDFN.
This is because the D antigen is highly immunogenic, and D-negative individuals exposed to D-positive red cells (most commonly during pregnancy with a D-positive baby) are very likely to form the antibody.
While other Rh antibodies (like anti-c and anti-E) can cause HDFN, anti-D is involved in the vast majority of significant cases.

In Wiener notation:

• R₀ represents the haplotype that produces antigens D, c, and e.

• r represents the haplotype that produces antigens c and e.

Therefore, the genotype R₀r (Dce/ce) produces the following antigens:

• From R₀: D, c, e

• From r: c, e

The D antigen is inherited in an autosomal dominant pattern.

• If an individual inherits one RHD gene (from one parent), they will express the D antigen and be Rh-positive.

• An individual is Rh-negative only if they inherit two non-functional or deleted RHD genes (homozygous recessive for the absence of D).

Rhₙᵤₗₗ red cells lack all Rh antigens (D, C, c, E, e) due to the absence of the entire Rh antigen complex in the membrane.

The LW antigen (Landsteiner-Wiener) is closely associated with the Rh complex. When the Rh protein complex is missing (as in the Rhₙᵤₗₗ phenotype), the expression of LW antigens is also profoundly weakened or absent. Therefore, Rhₙᵤₗₗ cells typically type as LW(a-b-).

The other options (Duffy, Kell, Kidd null) are independent blood group systems not directly affected by the absence of the Rh complex.

• Rh antibodies (IgG) are warm-reactive and do not usually agglutinate red cells directly at room temperature or in saline.

• The indirect antiglobulin test (IAT) / AHG phase is required to detect IgG bound to red cells.

• Immediate-spin, saline, or enzyme-only phases are insufficient for detecting most Rh antibodies.

Let’s analyze:

• Weak D testing is done on donor RBCs that react weakly or not at all with anti-D during routine typing.

• The goal is to accurately classify D-positive units so that recipients do not form anti-D antibodies.

Key points:

• Donor weak D → considered D-positive for transfusion purposes to prevent alloimmunization in D-negative recipients.

• This prevents the recipient from making anti-D.

Proteolytic enzymes like papain (and ficin) are known to enhance the reactivity of Rh antibodies (e.g., anti-D, anti-c, anti-E) with their corresponding antigens.

Enzymes work by removing sialic acid residues and cleaving certain glycoproteins from the red cell surface, which reduces the zeta potential and allows IgG molecules to get closer together, facilitating agglutination.

• D antigen is the key determinant of Rh status:

  • Presence of D → Rh-positive

  • Absence of D → Rh-negative

• Other Rh antigens (C, c, E, e) do not determine overall Rh status, though they can be clinically significant.

• Rh null phenotype lacks all Rh antigens on red cells.

• Absence of Rh proteins affects the red cell membrane’s structural integrity, leading to:

  • Spherocytosis (spherical, fragile red cells)

  • Mild to moderate chronic hemolytic anemia

• It is not associated with increased antibody production, leukopenia, or plasma protein defects.