In multiple myeloma, excess plasma proteins cause rouleaux formation, leading to false agglutination in reverse typing.
Performing a saline replacement (removing the patient’s serum and replacing it with saline) disperses the rouleaux, allowing true agglutination reactions to be accurately observed.

The other options are for different problems:

• a) Cold autoadsorption: Removes cold autoantibodies from the patient’s serum.

• c) Enzyme treatment: Enhances the reactivity of some antibodies (like in the panel) but doesn’t resolve rouleaux in reverse typing.

• d) DTT: Destroys IgM antibodies to identify IgG antibodies; doesn’t address protein-related stacking.

Let’s break down the genotype Sese Lele AO HH:

• HH → normal H antigen production.

• AO → A antigen will be produced in secretors.

• Sese → heterozygous for secretor gene → Se present → Secretor positive (H and A antigens in saliva).

• Lele → heterozygous for Lewis gene → Le present → can make Lea and Leb.

In a Secretor (Se+):

• H, A in saliva (due to Se).

• Lewis: since Se is present, most Le(a–b+) phenotype, so Leb in saliva (and trace Lea possible, but Leb dominates).

Thus saliva contains: Leb, A, H.

ABO antibodies (also called isohemagglutinins) are naturally occurring IgM antibodies that react best at room temperature.

• IgM is efficient at agglutination and complement activation, making it important in transfusion reactions.

• Some IgG antibodies may also be present—especially in group O individuals—but IgM predominates in the ABO system.

The lectin Dolichos biflorus specifically agglutinates A₁ cells but does not react with A₂ cells. Therefore, it is used to differentiate between A₁ and A₂ subgroups of blood group A.

• Ulex europaeus → reacts with H antigen, useful for identifying group O or Bombay phenotype.

• Arachis hypogaea (peanut lectin) → reacts with T antigen, not used in ABO typing.

• Vicia graminea → agglutinates A₁, A₂, and some A₃ cells, but is not specific for differentiating A₁ from A₂.

To be a secretor of ABH antigens in saliva, an individual must inherit:

1. At least one H (FUT1) gene to produce the H antigen on red blood cells and tissues.

2. At least one Se (FUT2) gene. The Se gene codes for a fucosyltransferase that adds fucose to a precursor substance in body fluids like saliva, creating soluble H antigen. This soluble H substance can then be converted to A or B antigens if the corresponding gene is present.

• H only: Allows for ABH on red cells but not in secretions (non-secretor).

• Se only: Is insufficient without the H gene to make the precursor structure.

• Le and Se: The Lewis (Le) gene is responsible for creating Lewis antigens in secretions, but secretor status is independently determined by the Se gene in conjunction with the H gene.

The H antigen is the precursor on red blood cells from which A and B antigens are formed.
All people (except those with the Bombay phenotype) have H antigen on their red cells.

Incorrect options:

• a) A antigen: Present only in group A and AB.

• b) B antigen: Present only in group B and AB.

• d) No antigen:  Incorrect — group O has H antigen.

The ABO blood group system is determined by the presence of specific sugars on red blood cells.

• Blood Group A has the N-acetylgalactosamine sugar attached to the core structure.

• Blood Group B has the galactose sugar.

• Fucose is the foundational sugar present in all ABO groups (O, A, and B).

• N-acetylglucosamine is a component of the core oligosaccharide chain but is not the immunodominant sugar for type A.

a) Rh HDFN is clinically more severe than ABO HDFN
✓ True — ABO HDN is usually mild; Rh HDN can cause severe anemia/hydrops.

b) The direct antiglobulin test is weaker in Rh HDFN than ABO HDFN
✗ False — DAT is typically stronger in Rh HDN; in ABO HDN it’s often weak/mixed-field.

c) Rh HDFN occurs in the first pregnancy
✗ False — Rh HDN usually requires sensitization from a previous pregnancy.

d) The mother’s antibody screen is positive in ABO HDFN
✗ False — In ABO HDN, the antibody screen is often negative because anti-A/B are naturally occurring and not IgG in all cases; only IgG causes HDN, but screen isn’t usually positive unless other antibodies are present.

The A antigen is formed when the A transferase enzyme (encoded by the A allele of the ABO gene) adds N-acetylgalactosamine to the H antigen on red blood cells.

• B antigen is formed by B transferase, which adds galactose instead.

• Enzymes like glycosidases or glucosidases are not involved in this antigen synthesis.

Mixed-field agglutination (small agglutinates mixed with free cells) is characteristic of the A₃ subgroup.

• A₁ and A₂: show uniform agglutination.

• Aₓ: very weak or no agglutination with anti-A, but may adsorb and elute anti-A.

• A₃: mixed-field pattern with anti-A and anti-A,B.

In this case, the forward typing shows clear AB antigens (4+ with anti-A and anti-B), but the reverse typing shows no reaction with A₁ or B cells—meaning no ABO antibodies are detected.
This happens when the patient has low or absent immunoglobulins, such as in hypogammaglobulinemia, leading to a lack of naturally occurring anti-A or anti-B antibodies in the serum.

The other options are incorrect because:

• a) Acquired B: Would show a weak or mixed field reaction with anti-B, not a 4+ reaction. The reverse type would be normal (no anti-B).

• b) Cold autoantibody: Would typically cause panagglutination (false-positive reactions) in the forward type and unexpected reactions in the reverse type, not the clean, specific pattern seen here.

• d) A_x subgroup: Would show a weak reaction with anti-A in the forward type, not a 4+ reaction.

All three phenotypes — O_h (Bombay), group O, and Lu(a–b–) — arise due to homozygosity for recessive alleles of autosomal genes that prevent normal antigen expression.

Incorrect options:

• a) Identical sex-linked dominant genes:  Not sex-linked.

• b) Identical sex-linked recessive genes:  None are on sex chromosomes.

• c) Identical autosomal dominant genes: Require two recessive alleles, not one dominant.

In reverse ABO grouping, we test the patient’s serum with known reagent red cells (A₁ and B cells) to detect naturally occurring antibodies —

• Group A → has anti-B

• Group B → has anti-A

• Group O → has anti-A and anti-B

• Group AB → has no antibodies

The A₂ phenotype is a subgroup of A that differs from A₁ primarily in the quantity and structure of A antigens on the red blood cell (RBC) surface.

Incorrect options:

• b) Lack the H antigen: ❌
  A₂ cells actually have more H antigen, not less.

• c) Are strongly reactive with anti-A₁: ❌
  A₂ cells do not react with anti-A₁.

• d) Have more A antigen sites: ❌
  A₂ has fewer, not more, A antigen sites.

The H antigen is a carbohydrate chain found on red blood cells in people of all blood types. The ABO genes code for enzymes that modify this H antigen:

• The A gene adds an N-acetylgalactosamine sugar to the H antigen, creating the A antigen.

• The B gene adds a galactose sugar to the H antigen, creating the B antigen.

• The O gene is inactive and makes no modification, leaving the H antigen unchanged, which is then identified as the O blood type.

A₃ is one of the rare weak subgroups of A.
It shows very weak expression of the A antigen on red cells — less than A₂, but not completely absent.

Analysis of the other options:

• b) No reaction with anti-A or anti-B: This describes the Bombay (Oₕ) phenotype, not A₃.

• c) Strong reaction with both anti-A and anti-B: This describes the standard AB blood group phenotype.

• d) Agglutination with anti-H only: This describes the Group O phenotype, which has no A or B antigens and therefore has a high concentration of H antigen.

The Bombay phenotype occurs when an individual is homozygous for a defective FUT1 gene (the H gene).
This prevents the formation of the H antigen on red cells, which is the precursor to the A and B antigens.
Without H antigen, even if the A or B transferase genes are present, A and B antigens cannot be made. The individual will type as O in forward grouping but will have anti-H in addition to anti-A and anti-B.

In the ABO blood group system, transfusion compatibility depends on the antibodies present in the recipient’s plasma and the antigens on donor red cells.

Incorrect options:

• a) Only group O:  Too restrictive; AB can receive from A, B, AB, and O.

• c) Group A and B only:  AB can also receive from O.

• d) Group O and AB only:  Missing A and B donors; incomplete.

• At birth (a): Newborns have very low levels of ABO antibodies. The antibodies they have are primarily IgG antibodies that have been passively transferred from the mother across the placenta.

• Within the first few weeks of life (b): This is the correct answer. As infants are exposed to ABO-like antigens from food, bacteria, and other environmental sources, their immune systems begin to produce their own IgM anti-A and/or anti-B antibodies. These antibodies typically reach detectable levels by 3-6 months of age.

• During the fetal stage (c): The production of ABO antibodies does not occur in the fetus. The immune system is not sufficiently stimulated or developed to produce them at this stage.

• Only after transfusion (d): This is incorrect. While a transfusion can strongly boost antibody levels, the natural ABO antibodies appear without any exposure to foreign red blood cells.

A2 phenotype characteristics:

• Weaker expression of A antigen than A1 → cells react weakly with anti-A,B ✅

• Serum may contain anti-A1, but not always → so “always contains anti-A1” ❌

• Dolichos biflorus lectin reacts with A1 only, so A2 cells do not react ✅

• Most common A subgroup → A1 is most common (~80%), A2 ~20% ❌

Both a and c are correct statements, but the defining feature is the weak reaction with anti-A,B.

In the ABO blood group system, the A and B alleles are codominant, and the O allele is recessive.

• The A allele instructs the cell to produce A antigens.

• The B allele instructs the cell to produce B antigens.

• The O allele produces no antigen.

A person with the AO genotype has one A allele and one O allele.

• The A allele is expressed, leading to the production of A antigens on the red blood cells.

• The O allele is recessive and does not produce an antigen.

The “acquired B” phenomenon occurs when bacterial enzymes (e.g., from E. coli or Clostridium) in the patient’s system deacetylate the group A sugar (N-acetylgalactosamine).

• This removal of an acetyl group changes the A antigen’s structure so that it closely resembles the group B sugar (galactose).

• As a result, some anti-B reagents may cross-react with this altered A antigen, making the patient’s red cells appear to be AB.

Forward typing suggests AB (reacts with anti-A and anti-B), but reverse typing is inconsistent — there’s no reaction with B cells (should have anti-A if truly AB). The weak reaction with A1 cells may be due to a cold autoantibody.
The acquired B antigen occurs in some group A individuals (due to bacterial enzymes modifying the A antigen), causing false anti-B reaction in forward typing, while the person’s own anti-B remains absent in reverse typing.

The H antigen is the base structure (precursor) from which the A and B antigens are formed on red blood cells (RBCs).

Incorrect options:

• a) Group A₁:  Most H converted → least H left.

• b) Group A₂:  Intermediate H level.

• c) Group B:  Moderate H level.

• Forward typing suggests group AB (reacts with anti-A and anti-B).

• Reverse typing is abnormal: no reaction with A1 cells (should have anti-B if truly AB) and weak reaction with B cells.

• This pattern is characteristic of a weak A subgroup (e.g., A_x or A_el), where:

  • The A antigen is very weak (may react with some potent anti-A reagents).

  • The individual produces anti-A1 in their serum, which reacts with A1 cells but not with their own weak A RBCs.

  • The weak reaction with B cells may be due to the anti-A1 being weak or due to another factor.

Mixed-field agglutination (or “mixed-field reactivity”) is characterized by the presence of two distinct populations of red blood cells in a single test sample: one population that is agglutinated and one that is not. This is a critical observation in the blood bank.

Here’s why the other options are less accurate:

• a) Polyagglutination: While some forms of polyagglutination (like T-activation) can cause mixed-field agglutination, it is not the most common cause. Polyagglutination is a specific phenomenon where red cells are agglutinated by most normal adult sera. “Recent transfusion” is a far more frequent and classic cause of mixed-field agglutination in routine testing.

• c) Weak subgroup of B: Weak subgroups (e.g., A_x, A_m) typically cause weak or delayed agglutination overall, not a clear mixed-field pattern with two distinct cell populations.

• d) Clerical error only: This is incorrect. While a clerical error (e.g., mislabeling) could theoretically lead to a mixed-field result, it is not the intrinsic cause.

The patient is a subgroup of A (e.g., A₂ or weaker) and has anti-A₁ in the serum.
To find a compatible unit, you must locate A₂ (or weaker A) blood, since A₁ red cells would react with the patient’s anti-A₁.

• About 80% of group A individuals are A₁.

• About 20% are A₂ (or weaker A subgroups).

So, the probability of finding an A₂ unit = 1 in 5.

The H gene encodes an enzyme called α-1,2-fucosyltransferase, which adds L-fucose to the terminal galactose of the precursor substance on red blood cells. This addition creates the H antigen, which serves as the foundation for the A and B antigens in the ABO blood group system.

Anti-A,B is a reagent antibody that reacts with both A and B antigens. Its primary purpose is to help detect weak subgroups of A (or B).

• In forward typing, some subgroups of A (like A_x) may react very weakly or not at all with anti-A but will show a clear reaction with the more potent anti-A,B.

• This stronger reaction with anti-A,B helps identify the presence of a weak A antigen that might otherwise be missed, confirming the patient belongs to a subgroup of A rather than being group O.

In the ABO blood group system, the antigens present on red blood cells (RBCs) determine which antibodies are found in the serum.
Antibodies are always against the antigens that the person does not have.

Incorrect options:

• a) Group A:  Has anti-B only.

• b) Group B: Has anti-A only.

• c) Group AB: Has no antibodies (both A and B antigens are self).

The A₁ antigen is a subtype of the A antigen found on red blood cells of most individuals with blood group A or AB.
It reacts with both anti-A (which detects all A antigens) and anti-A₁ (which specifically detects the A₁ subtype).

Incorrect options:

• b) Anti-B and anti-A: 
  A₁ cells do not react with anti-B (they have no B antigen).

• c) Anti-H only: 
  A₁ cells have less H antigen, not “only” H antigen.

• d) Anti-A₂ and anti-B: 
  There is no reagent called anti-A₂, and A₁ cells lack B antigen.

Let’s break this down:

• Red cells:

  • 4+ with anti-A → A antigen present.

  • 0 with anti-B → B antigen absent.

  • 4+ with anti-A,B (confirms strong A antigen).

• Serum (reverse grouping):

  • 0 with A₁ cells → no anti-A₁ (expected since patient is group A).

  • 4+ with B cells → strong anti-B present.

This is a classic group A pattern:

• A antigen on red cells.

• Anti-B in serum.

The core structure (the H antigen) is the same. The difference lies in the final sugar added by the specific enzyme (glycosyltransferase) a person inherits:

• The A gene adds the sugar N-acetylgalactosamine.

• The B gene adds the sugar galactose.

This single sugar difference is what distinguishes A antigens from B antigens.

The O blood group is the most common ABO phenotype globally. Individuals with O type have no A or B antigens on their red blood cells, only the H antigen, and they naturally produce both anti-A and anti-B antibodies in plasma.

Lectins are plant extracts that act like antibodies by agglutinating specific red cell antigens.
In ABO grouping, certain lectins are used to differentiate subgroups based on antigen specificity.

Here is the role of the other lectins listed:

• b) Ulex europaeus: This lectin is specific for the H antigen. It is used to identify Group O cells, which have the highest concentration of H substance.

• c) Phaseolus vulgaris: This is the lectin from the kidney bean. It is anti-B but is not commonly used in routine blood grouping to differentiate subgroups of B.

• d) Vicia graminea: This lectin is specific for the N antigen of the MNS blood group system, not the ABO system.

ABO antibodies are not naturally present at birth. They begin to develop after exposure to environmental antigens (like bacteria and food) and are typically detectable around 3-6 months of age. Their levels rise until adult levels are reached around 5-10 years of age.

• A 2-year-old child is still within the age range where ABO antibodies may be present but are often low or undetectable. It is not unexpected to find weak or absent ABO antibodies in a patient this young.

• All the other options (30-year-old, 16-year-old, 45-year-old) are well beyond the age when ABO antibodies are consistently present.

The “acquired B” phenomenon is a rare, transient condition in which group A₁ red cells appear to react weakly with anti-B serum, even though the person genetically lacks the B antigen.

Incorrect options:

• b) Iron deficiency anemia: Affects RBC morphology, not ABO antigens.

• c) Vitamin B₁₂ deficiency: Causes megaloblastic changes, no antigen alteration.

• d) Plasma protein disorders:  Affect plasma viscosity, not RBC antigen structure.

• Group O mothers naturally have IgG anti-A and anti-B antibodies that can cross the placenta.

• Mothers who are group A or B typically have IgM antibodies that do not cross the placenta.

• Therefore, ABO HDN almost exclusively occurs when a group O mother has a baby with a different blood group (A or B).

The other options are incorrect:

• a) It is usually mild and rarely requires exchange transfusion.

• b) It can occur in a first-born child, unlike Rh disease.

• c) It is rarely severe enough to cause stillbirth.

• The H antigen (coded by the FUT1 gene) is the precursor to A and B antigens.

• A person with hh genotype (Bombay phenotype) cannot produce the H antigen, so even if they have the A and/or B genes, they cannot make A or B antigens.

• Here, genotype is hh, AB → they have A and B transferase genes but no H substrate to convert into A or B antigens.

Individuals with the Bombay (Oₕ) phenotype lack the H antigen, which is the precursor for the A and B antigens.

• Their serum contains potent anti-H, in addition to anti-A and anti-B.

• Since all other blood groups (O, A, B, AB) express the H antigen, transfusing any of them would cause a severe hemolytic transfusion reaction.

• The only compatible red blood cells are from another Bombay phenotype donor, as these cells also lack the H antigen and will not be attacked by the patient’s anti-H.

The ABO blood group system is determined by specific antigens (A and B) present on the surface of red blood cells (RBCs). The corresponding antibodies are present in the plasma, but the classification itself is based on the antigens on RBCs, not antibodies or enzymes.

The H antigen is synthesized by the enzyme fucosyltransferase, which is encoded by the FUT1 gene. This gene is located on chromosome 19. Mutations in FUT1 can lead to the Bombay (hh) phenotype, where the H antigen is absent, preventing expression of A or B antigens.

• Reverse grouping is the specific method used to detect and identify ABO antibodies present in a person’s serum or plasma. It works by mixing the patient’s serum with known reagent red blood cells (Type A and Type B cells) and observing for agglutination.

Here’s why the other options are incorrect:

• a) Forward grouping: This is the opposite test. It detects ABO antigens on a person’s own red blood cells.

• c) Crossmatching: This is a pre-transfusion test designed to check for compatibility between a specific donor’s red cells and a specific recipient’s serum. While it detects antibodies, its purpose is broader than just identifying ABO type.

• d) DAT (Direct Antiglobulin Test): This test detects antibodies or complement proteins that are already bound to a person’s red blood cells inside the body.

In reverse ABO grouping, the patient’s serum (plasma) is tested against known reagent red cells (A₁ and B cells) to detect naturally occurring antibodies (anti-A or anti-B).
A discrepancy occurs when the serum reactions don’t match the expected pattern based on the cell (forward) grouping.

Incorrect options:

• b) Lack of H antigen:  Associated with Bombay phenotype, affects forward grouping, not reverse.

• c) Weak D antigen:  Involves Rh typing, not ABO discrepancies.

• d) Bacterial contamination: Can cause hemolysis or unexpected reactions, but not a classic cause of reverse grouping discrepancy.

When a group O, Rh-positive mother gives birth to a group A, Rh-positive infant, the mother’s IgG anti-A (and possibly anti-B) antibodies can cross the placenta and attach to the baby’s A red blood cells.

This causes a positive direct antiglobulin test (DAT) and mild to moderate hemolysis, resulting in elevated bilirubin levels soon after birth.

Key points:

• ABO hemolytic disease often occurs in first pregnancies, unlike Rh incompatibility, which typically affects subsequent pregnancies.

• The hemolysis is usually mild, rarely requiring exchange transfusion.

• Rh incompatibility is ruled out because both mother and infant are Rh-positive

ABO blood grouping is based on:

• Forward typing: Testing the patient’s red blood cells with known anti-A and anti-B reagents to detect A and B antigens.

• Reverse typing: Testing the patient’s serum with known A₁ and B red cells to detect the expected anti-A and/or anti-B antibodies.

This dual testing confirms the ABO group by identifying both the antigens on RBCs and the naturally occurring antibodies in the plasma.

Individuals with the Bombay phenotype (Oh) lack the H antigen because they have hh genotype (no functional H gene). Since H antigen is the base structure required to form A and B antigens, these individuals:

• Do not express A, B, or H antigens on their red cells.

• Their serum contains anti-A, anti-B, and anti-H antibodies.

The Bombay phenotype (Oh) is a rare blood group that lacks the H antigen, which is the precursor for A and B antigens.

Analysis of the other options:

• b) AB group with weak reactivity: Incorrect. They have no A or B antigens, so they cannot be AB.

• c) B group with anti-A: Incorrect. They have no B antigen and their serum contains anti-B in addition to anti-A and anti-H.

• d) A₂ group with no anti-H: Incorrect. They are not group A, and crucially, they have a very strong anti-H in their serum.

• Forward grouping is the method used to detect ABO antigens on an individual’s own red blood cells. It involves testing the person’s red cells with known commercial antisera (anti-A, anti-B) to see which ones cause agglutination.

Here is a breakdown of the other terms:

• a) Reverse grouping: This is the method used to detect ABO antibodies in an individual’s serum. It involves testing the person’s serum with known commercial red cells (A1 cells, B cells) to confirm the presence of the expected antibodies.

• c) Coagulation test: This is unrelated to blood grouping. It is a test used to measure the blood’s ability to clot (e.g., PT, PTT).

• d) Antiglobulin test: This is a different test, also known as the Coombs test. It is used to detect antibodies that are coating red blood cells but do not directly cause agglutination.

• Infant: Group A

• Mother: Group O → risk of ABO HDFN (maternal anti-A can hemolyze infant’s A cells)

• Exchange transfusion: goal is to replace infant’s RBCs safely.

Key principles for selecting units in ABO HDFN:

1. RBCs should lack the antigen that maternal antibodies target → maternal anti-A is in plasma, so ideally RBCs should not express A antigen.

2. Plasma should not contain antibodies that can attack infant RBCs → AB plasma is “universal” (no anti-A or anti-B).

3. Rh compatibility: use Rh-negative if infant is not typed yet or at risk.

Options:

• a) Group A, Rh-positive RBCs → wrong, would be attacked by maternal anti-A.

• b) Group O, Rh-positive RBCs → safer, RBCs lack A/B antigens, but plasma may contain anti-A (risk).

• c) Group O, Rh-negative RBCs suspended in group AB plasma → ✅ correct: RBCs lack A/B (safe from maternal anti-A), AB plasma has no anti-A/B (safe for infant).

• d) Group A, Rh-positive RBCs suspended in AB plasma → RBCs still have A antigen → risk.

Ulex europaeus lectin, extracted from gorse seeds, is the specific reagent used to identify H antigen expression on red blood cells. It binds to the terminal sugar (L-fucose) that defines the H antigen.

Analysis of the other options:

• a) Dolichos biflorus lectin: This is used to identify the A₁ antigen, not the H antigen.

• c) A₁ cells: These are used in reverse grouping to detect the presence of anti-A₁ antibody in a patient’s serum. They are not a reagent for detecting antigens on cells.

• d) Anti-D reagent: This is used to determine the Rh(D) blood type, which is completely separate from the ABO and H systems.

The Bombay phenotype results from inheriting two defective (h) alleles of the FUT1 gene on chromosome 19. This prevents the production of the H antigen precursor. Since the A and B antigens cannot be formed without the H antigen, the individual tests as type O, but also lacks the H antigen itself, which is the defining characteristic of the Bombay (Oh) phenotype.

In the ABO blood group system, the most important and common subgroup is the division of blood group A into A₁ and A₂.

• A₁ (a): This is the most common and strongly expressed A antigen. Approximately 80% of group A or AB individuals are A₁.

• A₂ (b): This is the most common subgroup of A, characterized by weaker antigen expression compared to A₁. About 20% of group A or AB individuals are A₂. The number of A antigen sites on A₂ red cells is significantly lower than on A₁ cells.

• B₃ (c): This is not a standard or recognized subgroup in the ABO system.

• AB (d): This is a separate phenotype, not a subgroup of A.

• Group O means they have H antigen (but not A or B).

• Se gene (FUT2) allows H antigen to be secreted into body fluids.

• Le gene (FUT3) converts secreted H antigen into Leb antigen.

• A small amount of unconverted H antigen remains, and a trace of Lea may be present, but the dominant antigens in secretions are Leb and H.

• In a group O, Le, Se individual, Leb is the major Lewis antigen in secretions, and H substance is also present.

In ABO blood typing, the presence of agglutination or hemolysis after adding an antisera (like Anti-A or Anti-B) indicates a positive antigen–antibody reaction.

Analysis of the other options:

• a) Strong antibody presence: While hemolysis requires a strong reaction, this option is less precise. The key takeaway is that it signifies a positive reaction, not just the presence of a strong antibody.

• c) Test invalidity: This is incorrect. Hemolysis is a valid, expected, and interpretable result in tube-based ABO typing. It does not invalidate the test; it provides a positive result.

• d) Protein degradation: This is unrelated. Degraded reagents would more likely lead to weak or false-negative reactions, not hemolysis.

In ABO blood grouping, two types of tests are performed:

• Forward grouping (cell grouping): Patient’s RBCs are tested with known antisera (Anti-A, Anti-B).

• Reverse grouping (serum grouping): Patient’s serum is tested with known reagent RBCs (A cells, B cells).

Other options (incorrect):

• a) Incorrect centrifugation speed:
  May affect strength of agglutination but usually does not cause forward/reverse discrepancy specifically.

• c) Excess plasma volume:
  May cause rouleaux formation or non-specific reactions, but not a typical cause of ABO discrepancy.

• d) Use of outdated reagents:
  Can cause weak or false reactions, but this affects both forward and reverse reactions uniformly — not necessarily a discrepancy between them.

The A_x subgroup is characterized by very weak expression of the A antigen on red blood cells.

• Forward Typing (Cells):

  • Weak with anti-A: The A antigen is present but in very low numbers, leading to weak or negative agglutination with routine anti-A reagent.

  • 0 with anti-B: The B antigen is absent.

• Reverse Typing (Serum):

  • 0 with A₁ cells: The patient’s serum contains anti-A₁ antibody, which agglutinates common A₁ cells. This is the key serological clue.

  • 4+ with B cells: The serum contains the expected anti-B antibody.