EXERCISE №11 – IMMUNOGENETICS OF THE BLOOD GROUPS. ALLOANTIGENS ON HUMAN ERYTHROCYTES


1. Human blood group systems

Human blood group antigens are alloantigens – they trigger an immune response in other individuals of the same species due to genetic variations. Examples: ABO(H), Rhesus (Rh), Lewis, MN, etc.


2. ABO(H) system


2.1. History

- Karl Landsteiner discovered antigens A and B in 1901 (groups A, B, O).

- Decastello and Sterley added group AB in 1902.


2.2. Antigens and antibodies**

- Antigens (agglutinogens) on erythrocyte membrane.

- Antibodies (agglutinins) in plasma, mainly IgM, appear in first months after birth.

- Most accepted hypothesis for natural antibodies: cross-reactivity with intestinal bacteria that have structures similar to A, B, H antigens.


| Blood type | Genotype | Agglutinogen (on RBC) | Agglutinin (in plasma) |

|------------|----------|----------------------|------------------------|

| A | I^A I^A or I^A I^O | A | Anti-B (β) |

| B | I^B I^B or I^B I^O | B | Anti-A (α) |

| AB | I^A I^B | A and B | None |

| O | I^O I^O | H | Anti-A (α) + Anti-B (β) |

| O_h (Bombay) | hh (plus any I) | None | Anti-A, Anti-B, Anti-H |


2.3. Genetics of ABO(H) system

- Multiple allelism – I gene on chromosome 9 has 3 alleles: I^A, I^B, I^O.

- I^A and I^B are codominant; both dominate over I^O.

- H gene on chromosome 19 (independent but functionally related).


| Allele | Product | Function |

|--------|---------|----------|

| H | Fucosyltransferase 1 (FUT1) | Adds fucose → forms H antigen |

| h | Non-functional | No H antigen formed |

| I^A | Glycosyltransferase A | Adds N-acetylgalactosamine to H → A antigen |

| I^B | Glycosyltransferase B | Adds galactose to H → B antigen |

| I^O | Non-functional | H antigen remains unmodified (blood type O) |


2.4. Biosynthesis of A, B, H antigens

- These are oligosaccharides (sugar chains) on glycolipids/glycoproteins.

- All share a core oligosaccharide. The last sugar added determines the antigen.


2.4.1. H antigen synthesis

- Individuals with HH or Hh make FUT1 → adds L-fucose to core → H antigen.


2.4.2. A and B antigen synthesis

- I^A allele → Transferase A → adds N-acetylgalactosamine to H → A antigen

- I^B allele → Transferase B → adds galactose to H → B antigen

- I^O I^O → no functional enzyme → H antigen remains unchanged (blood type O)


2.4.3. Bombay phenotype (O_h)

- Genotype hh → no H antigen → cannot make A or B even with I^A or I^B.

- These people have anti-A, anti-B, AND anti-H antibodies → cannot receive any normal blood (only other Bombay donors).


#### 3. Secretor status

- Ability to secrete ABO antigens into body fluids (saliva, tears, sweat, urine, semen, breast milk).

- Determined by Se gene on chromosome 19.

- Se (dominant) → encodes fucosyltransferase 2 (FUT2) → works in epithelial cells → secretor.

- se (recessive) → non-functional → non-secretor.

- ~80% of people are secretors.


#### 4. Rhesus (Rh) system

- Most complex blood group system. Most important antigen = D antigen.

- Rh+ = has D antigen. Rh– = no D antigen.

- D antigen is 40× more immunogenic than other Rh antigens.


4.1. History

- Landsteiner & Wiener (1940): immunized rabbits with Macacus rhesus monkey RBCs → produced antibodies that agglutinated some human RBCs.


4.2. Immunogenetics

- Two closely linked genes on chromosome 1: RhD (encodes D antigen) and RhCE (encodes C and E antigens).

- No natural antibodies against Rh factor – anti-D are always immune (IgG) , arise only after incompatible transfusion or pregnancy.


4.3. Immune conflict: mother-fetus (Hemolytic disease of the newborn / Erythroblastosis fetalis)

- Model: Rh– mother, Rh+ father, Rh+ fetus.

- Primary sensitization – fetal Rh+ RBCs enter mother’s blood (usually at birth, miscarriage, invasive procedures, trauma). Mother produces IgM (does not cross placenta) and B memory cells.

- Next pregnancy with Rh+ fetus – even small fetal RBCs activate memory B cells → rapid production of IgG anti-D (crosses placenta) → destroys fetal Rh+ RBCs → hemolytic disease.

- Prevention: Anti-D immunoglobulin (RhoGAM) given to mother within 48–72 hours after birth/dangerous event. It destroys fetal RBCs in mother’s blood before her immune system reacts → no memory formed.


#### 5. Lewis system

- Gene on chromosome 19, alleles Le and le. Antigens: Le^a and Le^b.


#### 6. Other blood group systems

MNSs, P, Duffy, etc.


#### 7. Method for determining blood groups (agglutination test)


7.1. Materials

- Test sera: anti-A, anti-B, anti-A,B, anti-D

- Well plate, pipettes, stirring rods


7.2. Procedure

- One drop of each serum in separate wells.

- Add one drop of test blood to each.

- Mix, observe for agglutination (clumping) = red dots/particles against clear background.


Interpretation examples:

- Agglutination with anti-A and anti-A,B but not anti-B → blood type A

- Also agglutination with anti-D → Rh+


#### 8. Blood transfusion rules (Ottenberg's rule)

- Donor's RBCs must not be agglutinated by recipient's plasma.


| Blood type | Can donate to | Can receive from |

|------------|---------------|------------------|

| O (universal donor) | A, B, AB, O | O only |

| A | A, AB | A, O |

| B | B, AB | B, O |

| AB (universal recipient) | AB only | A, B, AB, O |

| Bombay (O_h) | Only Bombay | Only Bombay |


- Rh+ can receive from Rh+ or Rh–.

- Rh– can receive only from Rh– (transfusion of Rh+ will cause anti-D formation → dangerous next time).

- Note: Up to ~300 mL, donor's antibodies are diluted enough to usually not cause reaction.


#### 9. Applications of blood group typing

Blood transfusions, transplants, paternity testing.


#### 10. Biological functions of blood group antigens

- Cell recognition & intercellular communication

- Receptors for viruses, bacteria, toxins

- Embryonic development & tissue differentiation

- Ontogenetic markers

- Strong immunogens


Example: Duffy-negative individuals are resistant to Plasmodium vivax (malaria tertiana) because the parasite needs the Duffy antigen to enter RBCs.


### Figure 1 – Biosynthesis of H, A, B antigens

This shows the sugar chain assembly line:

1. Precursor substance (core oligosaccharide with terminal galactose)

2. H gene (FUT1) adds fucose to galactose → H antigen

3. I^A transferase adds N-acetylgalactosamine to H → A antigen

4. I^B transferase adds galactose to H → B antigen

5. If I^O → no addition → remains H antigen (blood type O)


Why this matters: The Bombay phenotype (hh) breaks the chain at step 2 – no H, so no A or B possible, even with working transferases.


### Figure 2 – Immune conflict "mother-fetus" and prevention

This diagram (described in text) shows:

- Left side (sensitization) : Rh– mother carries Rh+ fetus. At birth, fetal RBCs enter mother → mother makes anti-D (IgM, no placental crossing) + memory B cells.

- Right side (next pregnancy) : Rapid IgG anti-D production → crosses placenta → attacks fetal RBCs.

- Prevention box: Anti-D immunoglobulin injected into mother within 72 hours – binds to fetal RBCs in mother's blood, marking them for destruction before mother's own immune system sees them.


Key insight: First Rh+ baby is usually fine. The second Rh+ baby is at risk. This is why Rh– mothers get RhoGAM after every pregnancy/miscarriage/abortion.


### Figure 3 & 4 – Agglutination test

- No agglutination = blood lacks that antigen (e.g., no clumping with anti-B means no B antigen).

- Agglutination = antigen present.

- Interpretation example from text: Agglutination with anti-A, anti-A,B, and anti-D but not anti-B = A Rh+.


### Figure 5 – Duffy antigen and malaria

- Duffy antigen is a receptor on RBCs.

- Plasmodium vivax (malaria tertiana) uses it to enter the cell.

- People with Duffy-negative phenotype (common in West Africa) are resistant to this form of malaria – a classic example of natural selection maintaining a "deficiency" because it confers survival advantage.


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## Part 3: Simple Analogy – The Package Delivery System


Imagine your red blood cells are mailboxes in a neighborhood.


### The ABO system = mailbox labels


- H antigen = a basic blank label on every mailbox (in most people).

- Transferase A = a worker with a red sticker. If he's present (I^A allele), he puts a red sticker (antigen A) on the label.

- Transferase B = a worker with a green sticker. If he's present (I^B allele), he puts a green sticker (antigen B).

- Blood type A = red sticker only.

- Blood type B = green sticker only.

- Blood type AB = both red and green stickers.

- Blood type O = no worker to add stickers → just the blank H label.


Antibodies = neighborhood watch. If you have red sticker (type A), you have "anti-green" watchmen. If they see a green sticker (type B blood), they attack.


Bombay phenotype (hh) = your house has no blank labels at all. You can't put red or green stickers even if you have the workers. Your watchmen attack any sticker (red, green, OR blank H) → you can only get mail from other houses with no labels (other Bombay donors).


### The Rhesus system = a separate "priority mail" flag


- Rh+ = you have a little red flag on your mailbox (D antigen).

- Rh– = no flag.

- No natural antibodies = no one is born hating flags. Only if an Rh– person receives flag-positive blood (or has an Rh+ baby) do they develop anti-flag antibodies (IgG).


### Mother-fetus conflict = the accidental key incident


- First pregnancy (Rh– mom, Rh+ baby) : During birth, some baby blood (with flags) gets into mom's bloodstream. Mom's immune system sees the flags for the first time and makes memory cells (but only IgM – can't cross placenta, so baby is safe). This is like a security camera recording the shape of a new key.

- Second pregnancy with another Rh+ baby : Even a tiny bit of baby blood triggers the memory cells → rapid production of IgG anti-flag (small enough to cross the placenta). These attack the baby's flag-positive RBCs → hemolytic disease.

- Prevention (RhoGAM) : Within 72 hours after first birth, we inject mom with ready-made anti-flag antibodies. These grab onto the baby's flag-positive cells in mom's blood and remove them before mom's own immune system ever sees them → no memory cells formed → next baby is safe. Like sending in a cleanup crew to erase all evidence of the key before the security system learns it.


### Blood transfusion rules (Ottenberg) = mailing compatibility


- Donor's mailboxes (RBCs) must not be attacked by recipient's watchmen (antibodies) .

- Type O = blank label (H only). No red or green stickers. Can give to anyone because no one attacks blank labels. But can only receive blank labels back.

- Type AB = both stickers. Has no watchmen (no antibodies). Can receive from anyone. But can only give to AB because others would attack the mismatched sticker.

- Rh– can only receive Rh– blood. If they receive Rh+ once, they'll make anti-flag antibodies, and the second Rh+ transfusion will be dangerous.


### Duffy & malaria = the locked door

- Malaria parasite (*Plasmodium vivax*) needs the Duffy antigen (like a specific doorknob) to enter RBCs.

- People without Duffy antigen = no doorknob = parasite cannot get in = resistant to malaria tertiana.

- This is evolution in action – in malaria-prone regions, being "Duffy-negative" is an advantage, so that gene spread.


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## Final Summary Table – Blood Group Systems


| System | Key Antigens | Key Antibodies | Genetics | Clinical Importance |

|--------|--------------|----------------|----------|----------------------|

| ABO(H) | A, B, H | Anti-A, Anti-B (IgM, natural) | I gene (chr 9): I^A, I^B, I^O; H gene (chr 19) | Transfusion mismatch → acute hemolysis |

| Rhesus | D (main) | Anti-D (IgG, immune only) | RhD, RhCE on chr 1 | Hemolytic disease of newborn |

| Lewis | Le^a, Le^b | Usually none | Le gene (chr 19) | Can change during pregnancy |

| Duffy | Fy^a, Fy^b | Immune antibodies | Duffy gene | Malaria resistance |

| MNSs | M, N, S, s | Can form immune antibodies | Multiple genes | Transfusion reactions |



To easily remember the different blood groups and their corresponding agglutinins (antibodies), use the following mnemonic:

  • A: Anti-B (β)

  • B: Anti-A (α)

  • AB: None (no agglutinins)

  • O: Anti-A + Anti-B (both α and β)

  • O_h (Bombay phenotype): Anti-A, Anti-B, Anti-H (all three)

Mnemonic:

  • Think of it as ‘A-B-C, O-AB’ where:   - A has B antibodies,   - B has A antibodies,   - AB has None (like the letter O)   - O has antibodies to both A and B.   - Bombay is the rare exception with all. This visualization helps to remember that A and B blood types have antibodies against the other, AB has neither, and O can recognize both.

To differentiate the Bombay blood group (O_h) from blood group O, a special test called the H antigen test should be conducted. This test specifically checks for the presence or absence of the H antigen on the red blood cells.

Why this test is necessary:

  • H antigen Presence: Individuals with blood group O have a functional H antigen, which serves as the foundation for A and B antigens on red blood cells. Thus, type O blood has the H antigen, but lacks A and B antigens.

  • Bombay Phenotype Absence: Individuals with the Bombay phenotype (hh) lack the functional H antigen altogether. Therefore, they cannot form A or B antigens, even if they carry the alleles for them (I^A or I^B).

Test Procedure:

  • The blood sample is tested for the presence of the H antigen. If the H antigen is detected, the blood group is determined to be O. If it is absent, the blood group is identified as Bombay (O_h).

In the context of blood types, the antibodies present in plasma belong to specific immunoglobulin (Ig) classes, primarily IgM and IgG. Here are the details:

  1. Blood Groups and Corresponding Antibodies
       - Type A
         - Antigen: A
         - Antibody: Anti-B (β) - IgM
       - Type B
         - Antigen: B
         - Antibody: Anti-A (α) - IgM
       - Type AB
         - Antigen: A and B
         - Antibody: None
       - Type O
         - Antigen: H
         - Antibodies: Anti-A (α) + Anti-B (β) - IgM
       - Type O_h (Bombay phenotype)
         - Antigen: None
         - Antibodies: Anti-A, Anti-B, Anti-H - IgM

  2. General Characteristics of Antibodies
       - IgM: Typically, the natural antibodies against blood group antigens (Anti-A and Anti-B) are primarily of the IgM class. They are large pentameric structures that are effective in agglutination.
       - IgG: While not typically involved in the ABO system for blood group typing, IgG antibodies can form in response to sensitization, such as in blood transfusions or during pregnancy.

In cases of Rh incompatibility combined with ABO(H) compatibility, the conflict may be stronger due to the presence of striking differences in blood types between the mother and fetus, especially when the mother is Rh-negative and the fetus is Rh-positive. In this situation, the maternal immune system reacts against the Rh-positive fetal blood cells by producing anti-D antibodies. This immunological response can occur during the first pregnancy or subsequent pregnancies if the mother has been sensitized.

The ABO(H) compatibility or incompatibility plays a role as follows:

  • If the mother has type O blood (which has anti-A and anti-B antibodies) and the fetus is type A or B (which possess A or B antigens), there might be a lesser immune response to the fetal RBCs than in Rh incompatibility alone. However, should the fetus be Rh+ (regardless of ABO compatibility), the potential for sensitization remains significant, leading to hemolytic disease of the newborn (HDN).

  • Conversely, if the mother is blood type A or B and the fetus is type O, the ABO incompatibility may not elevate the risk of severe HDN compared to Rh incompatibility, but the presence of anti-D antibodies can still pose a critical risk if the fetus is Rh+. Therefore, Rh incompatibility tends to exacerbate the conflict because IgG antibodies (anti-D) can cross the placenta and initiate hemolysis in the fetal circulation, especially in subsequent pregnancies.