Free ASCP MLS Blood Banking Exam Mock Test – Part 60: Antibody Screening and Identification

• In gel technology (microcolumn agglutination):

  • Positive reaction: Red blood cells are agglutinated and remain at the top of the gel or microtube.

  • Strong (4+) reaction: Forms a solid layer of agglutinated cells at the top.

  • Negative reaction: Cells form a pellet at the bottom after centrifugation.

  • Weak/mixed reaction: Cells are dispersed in the gel column.

Other options:

• a) Pellet at the bottom → Negative reaction.

• b) Dispersed throughout the gel → Weak or mixed reaction.

• d) Suspended at mid-point → Typically a weaker positive (1+ to 2+).

• Antibodies that react only with enzyme-treated red cells are typically those whose target antigens are enhanced by enzyme treatment.

• Rh and Kidd system antibodies often show stronger reactivity or are only detectable with enzyme-treated cells because enzymes remove steric hindrance and expose more antigen sites.

• This is a useful clue in antibody identification.

Other options:

• b) Duffy – Duffy antigens are destroyed by enzymes, so anti-Fyᵃ/Fyᵇ would not react with enzyme-treated cells.

• c) MNS – M, N, S, s antigens are destroyed by enzymes.

• d) Lutheran – Lutheran antigens are generally not enhanced by enzymes.

Enzyme treatment of red cells (using papain, ficin, or bromelin) modifies surface glycoproteins, which can enhance or destroy antigenic sites depending on the blood group system.

Enhanced by enzymes:

• Rh system (e.g., anti-D, anti-E, anti-C)

• Kidd system (anti-Jkᵃ, anti-Jkᵇ)

• Lewis system (anti-Leᵃ, anti-Leᵇ)

• P system (anti-P1)

Destroyed or weakened by enzymes:

• Duffy system (anti-Fyᵃ, anti-Fyᵇ)

• MNS system (anti-M, anti-N, sometimes anti-S, anti-s)

Other options:

• (b) Duffy and MNS → These are destroyed, not enhanced.

• (c) Lutheran only → Enzyme effect is variable and not diagnostically useful.

• (d) ABO antibodies → Enzymes do not significantly affect ABO reactions.

• IgM antibodies are typically direct agglutinins that can cause visible clumping of red cells in saline at room temperature or below (immediate-spin phase).

• They do not require incubation at 37°C or antiglobulin reagent to be detected.

• Examples: anti-M, anti-Leᵃ, anti-P1, and some anti-A,B.

Other options:

• b) 37°C incubation – Enhances some IgM reactions but is more critical for IgG detection.

• c) Antiglobulin (AHG) phase – Mainly detects IgG antibodies.

• d) Enzyme phase – Can enhance some IgM reactions but is not the primary phase for IgM detection.

• The Indirect Antiglobulin Test (IAT) is designed to detect free (unbound) IgG antibodies present in the patient’s serum or plasma that can react with red cell antigens.

• In this test, screening red cells with known antigen profiles are incubated with the patient’s serum. If the serum contains antibodies, they will bind to the antigens on these cells.

• After washing away unbound antibodies, antihuman globulin (AHG) is added — if antibodies are present on the red cells, agglutination occurs, indicating a positive test.

Other options:

• (a) Detects complement on red cells → that’s the Direct Antiglobulin Test (DAT).

• (c) Hemolysis from cold agglutinins → detected by cold agglutinin testing, not IAT.

• (d) Platelet-bound antibodies → detected by platelet antibody assays, not IAT.

• The autocontrol tests the patient’s serum against patient’s own red cells.

• A positive autocontrol suggests:

  • Autoantibodies (in autoimmune hemolytic anemia)

  • Alloantibodies with recent transfusion (passively acquired or reacting with transfused cells)

  • Drug-induced antibodies

Other options:

• b) Blood group type → Done by forward/reverse grouping.

• c) Crossmatch compatibility → Done with donor cells, not autocontrol.

• d) Plasma protein concentration → Not related to autocontrol.

• Enzyme treatment (ficin, papain, bromelin) removes sialic acid residues and alters the red cell membrane, which:

  • Enhances reactions of certain antibodies (Rh, Kidd)

  • Destroys some antigens (M, N, Fyᵃ, Fyᵇ)

• This helps in antibody identification by changing reactivity patterns.

Other options:

• b) Destroy all red cell antigens → False; only certain antigens are destroyed.

• c) Eliminate IgG antibodies → False; enzymes act on red cells, not serum antibodies.

• d) Stop complement fixation → False; complement can still be fixed.

• The reactions only at immediate spin/room temperature with no reactivity at 37°C or AHG indicate a cold-reactive IgM antibody.

• Anti-Leᵃ fits this pattern:

  • Naturally occurring IgM

  • Reacts best at lower temperatures

  • Often shows variable reactivity (only some cells positive) because Lewis antigen expression depends on secretor status and adsorption from plasma

• The other options are typically IgG and reactive at AHG phase:

  • a) Anti-Jkᵇ – IgG, AHG reactive

  • c) Anti-C – IgG, AHG reactive

  • d) Anti-Fyᵃ – IgG, AHG reactive

• When a patient has multiple antibodies, using enzyme-treated panel cells can help because:

  • Fyᵃ antigen is destroyed by enzymes, so anti-Fyᵃ will not react with enzyme-treated cells.

  • Rh antigens are enhanced by enzymes, so anti-D will still react (often more strongly).

• This differential reactivity can help separate and identify antibodies in a mixture.

Other options:

• b) Cold autoadsorption – Used for cold autoantibodies, not alloantibody mixtures.

• c) Albumin enhancement – An older method; less effective than enzymes or PEG for complex mixtures.

• d) Room temperature only – Would miss IgG antibodies and not help separate anti-D and anti-Fyᵃ.

• Antibody identification is confirmed when the patient’s serum reaction pattern matches the antigen profile of the panel cells:

  • All antigen-positive cells react

  • All antigen-negative cells do not react

• This pattern must be consistent across the panel, and the antibody should be ruled out against other possible specificities.

Other options:

• b) One random cell is positive → Insufficient; could be non-specific.

• c) Negative control fails → Indicates a problem with testing, not confirmation.

• d) All cells react at immediate-spin → Suggests a cold autoantibody or non-specific reactivity, not a specific antibody.

• Clinically significant antibodies are those capable of causing:

  • Hemolytic transfusion reactions

  • Hemolytic disease of the fetus and newborn (HDFN)

• These are typically IgG antibodies that react at 37°C and are detected in the antiglobulin (AHG) phase.

• Examples: Anti-D, anti-K, anti-Fyᵃ, anti-Jkᵃ.

Other options:

• b) IgM, reactive at 4°C only → Usually not clinically significant.

• c) Naturally occurring → Many naturally occurring antibodies (e.g., anti-Leᵃ, anti-P1) are not significant.

• d) Found only in cord blood → Incorrect; clinically significant antibodies are found in adult serum/plasma.

• The pre-warm technique is used to minimize interference from cold autoantibodies (e.g., anti-I) by:

  • Prewarming patient serum, reagent red cells, and saline to 37°C before combining them.

  • Maintaining 37°C throughout the test to prevent cold antibody binding.

• This allows detection of clinically significant IgG alloantibodies without cold autoantibody masking.

Other options:

• b) Longer incubation at 37°C – That’s standard for IgG detection, not specifically “pre-warm.”

• c) Heating serum to 56°C – Used to inactivate complement, not for cold antibody avoidance.

• d) Warming patient’s red cells to elute cold autoantibodies – Elution is a different process.

• In solid phase adherence assays (e.g., for antibody screening/identification):

  • Negative result: Indicator red cells do not adhere to the coated well surface and form a pellet at the bottom after centrifugation.

  • Positive result: Indicator red cells adhere and form a monolayer or diffuse pattern across the well.

Other options:

• b) Diffuse pattern throughout the well → Positive reaction.

• c) Red blood cell clumps symmetrically located → Positive reaction (agglutination).

• d) Red supernatant → Indicates hemolysis, not a typical result in solid phase.

• When an antibody to a high-incidence antigen is suspected (pan-reactive serum), the first confirmatory step is to test the serum against rare reagent red cells that are known to lack the antigen.

• If the serum does not react with these antigen-negative cells, it confirms the antibody specificity.

• Examples: k– cells for anti-k, Kp(b–) cells for anti-Kpᵇ, etc.

Other options:

• a) Test with patient’s own cells – Autocontrol may already be done; patient’s cells likely have the antigen if it’s high incidence.

• c) Perform an elution – Used if DAT is positive, not for serum antibody identification.

• d) Test family members’ cells – Not a first step; used for genetic studies if needed.

• Enzyme panels (e.g., ficin- or papain-treated red cells) are used in antibody identification to:

  • Confirm antibodies that are enhanced by enzymes (e.g., Rh, Kidd)

  • Rule out antibodies whose target antigens are destroyed by enzymes (e.g., Duffy, MNS)

• This helps narrow down possible antibody specificities.

Other options:

• b) To measure antibody titers → Done by serial dilution, not enzyme panels.

• c) To detect complement components → Detected by polyspecific AHG, not enzyme panels.

• d) To confirm weak D antigen → Done by indirect antiglobulin test with anti-D, not enzyme panels.

• A positive antibody screen means the patient’s serum contains unexpected antibodies (alloantibodies or autoantibodies) directed against red cell antigens other than the expected ABO antibodies.

• This requires further testing (antibody identification panel) to determine the antibody specificity before transfusion.

Other options:

• b) Complement in plasma → Not directly; complement may be detected if bound to red cells, but the screen is for antibodies.

• c) Abnormal clotting factors → Unrelated to antibody screening.

• d) Autoantibodies to white cells → Detected by different tests (e.g., HLA antibodies), not the red cell antibody screen.

• Mixed field reactions (a dual population of agglutinated and non-agglutinated red cells) often indicate:

  • Recent transfusion (donor cells and patient cells circulating together)

  • Bone marrow transplant (donor and recipient hematopoiesis)

  • Chimerism

• This occurs because the patient’s antibody may react only with transfused/donor cells, not their own.

Other options:

• b) Plasma contamination → Unlikely to cause mixed field.

• c) Instrument malfunction → Would affect all reactions uniformly.

• d) Sample hemolysis → Causes free hemoglobin, not mixed field agglutination.

• Antibody identification panels use Group O red cells from multiple donors, each with known antigen profiles for a wide range of blood group systems (Rh, Kell, Kidd, Duffy, MNS, etc.).

• Using Group O cells avoids interference from ABO antibodies.

• The known antigen patterns allow the lab to match the patient’s antibody reactivity against the antigen positives and negatives to identify the specific antibody.

Other options:

• b) Random donor blood samples → No; they are carefully selected and characterized.

• c) Group A red cells only → No; must be Group O to avoid ABO interference.

• d) Pooled platelet samples → Unrelated; platelet antibodies are tested separately.

• The patient’s phenotype is Jk(a–b–) → Kidd null.

• If their serum is nonreactive with phenotypically similar cells (also Jk(a–b–)), it suggests the antibody is anti-Jkᵃ or anti-Jkᵇ, because:

  • Jk(a–b–) cells lack Kidd antigens, so they won’t react with anti-Jkᵃ or anti-Jkᵇ.

  • If the antibody were against Rh, Kell, or Duffy, it would still react unless those cells were also antigen-negative for those systems.

• The Donath-Landsteiner test is specifically diagnostic for paroxysmal cold hemoglobinuria (PCH).

• It detects the biphasic hemolysin (IgG anti-P) that binds to red cells in the cold and fixes complement, then causes hemolysis upon warming.

Other options:

• a) Warm Autoimmune Hemolytic Anemia – Diagnosed by DAT showing IgG ± C3.

• b) Paroxysmal Nocturnal Hemoglobinuria – Diagnosed by flow cytometry for CD55/CD59.

• d) Cold Agglutinin Disease – Diagnosed by high-titer cold agglutinins and DAT positive for C3.

• Antibody screening cells are group O red cells from selected donors with known antigen profiles for major blood group systems (e.g., Rh, Kell, Kidd, Duffy, MNS, etc.).

• This allows the lab to determine antibody specificity when a patient’s serum reacts with the screening cells.

• Using cells with known antigens helps identify which antigen the patient’s antibody is targeting.

Other options:

• b) Randomly selected blood samples → False; they are carefully selected.

• c) Group O Rh-positive blood only → False; screening cells include both Rh-positive and Rh-negative to detect anti-D and other Rh antibodies.

• d) Autoantibody-positive samples → False; screening cells must not have autoantibodies.

• Zeta potential is the negative charge on the surface of red blood cells that causes them to repel each other, which can interfere with antibody binding.

• LISS works by reducing the ionic strength of the medium, thereby decreasing the zeta potential. This allows antibodies to approach the red cell surface more easily and enhances the rate and strength of agglutination.

Other reagents:

• Bovine albumin – Enhances agglutination by dehydrating the red cell surface to bring cells closer, but does not reduce zeta potential.

• Polyethylene glycol (PEG) – Concentrates antibodies in the solution by removing water (macromolecular enhancement), not by reducing zeta potential.

• Proteolytic enzymes – Modify red cell antigens to enhance or destroy antibody reactivity; they do not affect zeta potential.

• The phase of reactivity helps characterize an antibody:

  • Immediate spin/room temperature → Typically IgM, cold-reactive

  • 37°C incubation → Can be IgG or IgM

  • Antiglobulin (AHG) phase → Typically IgG, clinically significant

• This information helps predict clinical significance and guide identification.

Other options:

• b) Its molecular weight – Not determined by serologic phase.

• c) Its age of development – Not directly indicated.

• d) Its enzyme sensitivity – Determined by comparing untreated vs. enzyme-treated cells, not phase alone.

• Anti-Jkᵃ (Kidd system) is typically:

  • IgG → reactive at AHG phase

  • Enhanced by enzyme treatment (ficin, papain)

• This pattern helps distinguish it from antibodies like anti-Fyᵃ, which is destroyed by enzymes.

Other options:

• a) Anti-M – Usually IgM, reactive at immediate spin/room temperature, not AHG phase.

• b) Anti-Leᵃ – Usually IgM, reactive at immediate spin; Lewis antigens are destroyed by enzymes.

• c) Anti-Fyᵃ – IgG, AHG reactive, but destroyed by enzyme treatment.

• Chloroquine diphosphate is used to dissociate IgG from red cells without damaging most blood group antigens.

• It is particularly useful for phenotyping red cells that are coated with IgG (positive DAT) in autoimmune hemolytic anemia or after transfusion.

• After chloroquine treatment, the cells can be tested with antisera to determine their phenotype.

Other options:

• a) Ficin – A proteolytic enzyme that destroys certain antigens (M, N, Fyᵃ, Fyᵇ).

• b) Dithiothreitol (DTT) – Destroys Kell system antigens and disrupts IgM.

• d) Polyethylene glycol (PEG) – An enhancement medium for antibody detection, not for removing IgG from cells.

The Kidd (Jk) system antibodies, particularly anti-Jkᵃ and anti-Jkᵇ, are among the most common causes of delayed hemolytic transfusion reactions. These antibodies are notorious for falling to undetectable levels in the plasma between exposures, making pre-transfusion screening difficult, but they rapidly reappear (anamnestic response) after a transfusion, causing delayed hemolysis.

Why the others are incorrect:

• (a) Lewis: Usually IgM, naturally occurring, and clinically insignificant.

• (c) P: Rarely causes transfusion reactions (mainly involved in paroxysmal cold hemoglobinuria).

• (d) I: Associated with cold agglutinins in Mycoplasma infections, not DHTRs.

A major crossmatch tests the recipient’s serum against donor red cells to detect any incompatibility.

• If it’s incompatible at the antiglobulin (AHG) phase, it indicates the presence of an IgG antibody in the recipient’s serum that reacts with an antigen on the donor’s RBCs — typically a clinically significant unexpected antibody.

Let’s review the options:

• a) ABO typing error → usually causes immediate spin (IS) incompatibility, not AHG phase.

• b)  Correct — unexpected IgG alloantibody reacting with donor antigen at AHG phase.

• c) DAT on recipient’s cells → affects autocontrol, not crossmatch with donor cells.

• d) Bacterial contamination → causes hemolysis or discoloration, not serologic AHG incompatibility.

• Dosage effect occurs when an antibody reacts more strongly with red cells that are homozygous for the corresponding antigen than with cells that are heterozygous.

• Common in systems like Rh, Duffy, Kidd, MNS.

• Example: Anti-Jkᵃ reacts more strongly with Jk(a+b–) cells than with Jk(a+b+) cells.

Other options:

• b) Prozone phenomenon – Weaker reactions at high antibody concentrations due to antibody excess.

• c) Zeta potential – Related to distance between red cells, not antigen strength.

• d) Complement fixation – Binding of complement, not related to antigen dose.

• Gel technology (microcolumn agglutination) uses a dextran gel matrix in microtubes to trap agglutinated red cells.

• The primary advantage is that it eliminates the saline washing step required in traditional tube testing for the antiglobulin phase.

• This reduces hands-on time, potential washing errors, and improves standardization.

Other options:

• a) Requires no centrifugation → False; centrifugation is required in gel testing.

• c) Uses less reagent → Not necessarily the primary advantage.

• d) Provides faster results → Can be faster due to no washing, but not always.

• If a patient’s serum reacts strongly with all panel cells, it suggests:

  • Autoantibody (reacting with a common antigen on all cells, including the patient’s own cells)

  • Antibody to a high-incidence antigen (present on nearly all red cells except very rare null phenotypes)

• Further testing (autocontrol, DAT, adsorption/elution) is needed to distinguish between these.

Other options:

• b) Antibody to a low-frequency antigen → Would cause reactions with only a few cells, not all.

• c) Typing error → Unlikely to cause uniform strong reactions.

• d) Clerical mistake → Possible but not the most likely serologic interpretation.

• Reacts with all panel cells → suggests an antibody against an antigen present on virtually all reagent cells.

• Negative autocontrol → rules out an autoantibody (which would react with the patient’s own cells).

• This pattern is classic for an alloantibody to a high-prevalence antigen (e.g., anti-k, anti-Kpᵇ, anti-Ytᵃ, etc.).

Other options:

• a) Warm autoantibody → Would typically have a positive autocontrol.

• b) Multiple alloantibodies → Possible, but less likely to react with every single panel cell unless they cover all antigens.

• d) Rouleaux → Causes aggregation at immediate spin, not AHG phase, and usually disperses in saline.

• Anti-Leᵃ is typically IgM and can fix complement effectively.

• When fresh serum (with active complement) is used in testing, anti-Leᵃ can cause in-vitro hemolysis as complement proceeds to the membrane attack complex.

• This is commonly seen in antibody screening or crossmatch if serum is not inactivated.

Other options:

• a) Anti-K – Usually IgG, may bind complement but rarely causes visible in-vitro hemolysis.

• c) Anti-E – IgG, complement binding possible but in-vitro hemolysis uncommon.

• d) Anti-Fyᵃ – IgG, may bind complement but in-vitro hemolysis rare.

• Neutralization is a specific test where soluble Lewis substance (from human saliva or commercial preparation) is added to the patient’s serum.

• If the antibody is anti-Leᵇ, it will be neutralized and will no longer react with Le(b+) red cells.

• This confirms specificity without relying on complex adsorption/elution.

Other options:

• b) Treatment of panel cells with ficin – Lewis antigens are destroyed by enzymes, but this doesn’t confirm specificity as clearly as neutralization.

• c) Testing with cord blood cells – Cord cells lack Lewis antigens, so they would be negative, but this doesn’t confirm anti-Leᵇ specifically.

• d) Adsorption and elution studies – More complex; neutralization is simpler and definitive for Lewis antibodies.

• Rouleaux is the stacking of red cells (like coins) due to high serum proteins (e.g., in multiple myeloma, inflammation), which can be mistaken for agglutination.

• In the saline replacement technique:

  1. Observe for agglutination.

  2. Replace serum with saline.

  3. Resuspend and re-examine.

• Rouleaux will disperse in saline, while true agglutination will remain.

Other options:

• a) Enhance IgG antibodies – No; enhancement uses LISS, PEG, or enzymes.

• c) Identify cold agglutinins – Not the primary use; cold agglutinins are identified by temperature reactivity.

• d) Remove fibrin strands – Fibrin can cause false positives, but saline replacement is mainly for rouleaux.

• In the indirect antiglobulin test (IAT), under-centrifugation may fail to bring together sensitized red cells, resulting in a false-negative because agglutination is not visible.

• Proper centrifugation is needed to promote contact between red cells coated with antibody and the antihuman globulin reagent.

Other options:

• a) Over-centrifugation – Can cause false-positives by packing cells too tightly.

• c) Dirty glassware – More likely to cause false-positives due to contaminants.

• d) Use of EDTA plasma – EDTA chelates calcium and prevents complement activation, but IgG antibodies can still be detected; not a common cause of false-negative IAT.

• Enhancement media like LISS (Low Ionic Strength Solution) and PEG (Polyethylene Glycol) are used to reduce the incubation time and increase the sensitivity of antibody detection by promoting antigen-antibody binding.

• LISS – Reduces ionic strength, increasing the rate of antibody uptake.

• PEG – Concentrates antibodies by excluding water, enhancing reactivity.

Other options:

• b) Destroy antibodies → False; they enhance detection.

• c) Reduce serum viscosity → PEG actually increases viscosity.

• d) Inhibit complement activity → Not their primary purpose.

• In antibody identification, the “rule-out” method involves:

  • Comparing the patient’s reaction pattern with the antigen profiles of panel cells.

  • Excluding any antigen as a target if the patient’s serum reacts negatively with a cell that is positive for that antigen.

  • The antibody must be directed against an antigen that is positive in all reacting cells and negative in all non-reacting cells.

Other options:

• b) Using only positive reactions – That’s part of it, but ruling out requires negative reactions too.

• c) Ignoring weak reactions – Weak reactions should not be ignored; they may be significant.

• d) Eliminating all antibodies detected – Incorrect; the goal is to identify, not eliminate.

Check cells are used as a quality control to verify that the anti-human globulin (AHG) reagent was present and active. If the AHG test was negative and the check cells also fail to agglutinate, it means the AHG reagent was not present to bridge the antibodies on the check cells.

The most common reasons for this are:

• AHG was not added.

• The test cells were over-washed, removing all the AHG reagent before the check cells were added.

• Anti-Fyᵃ is well known for showing a dosage effect — it reacts more strongly with Fy(a+b–) cells (homozygous) than with Fy(a+b+) cells (heterozygous).

• This can cause variable reactivity in antibody identification panels depending on the zygosity of the Fyᵃ antigen on panel cells.

Other options:

• a) Anti-K – Does not show dosage; K antigen expression is similar in heterozygotes and homozygotes.

• c) Anti-A – No dosage effect; ABO antibodies are not dosage-dependent.

• d) Anti-Leᵃ – Lewis antigens are adsorbed, not intrinsic, so no dosage effect.

• Cold autoantibodies (e.g., anti-I) react best at temperatures below 37°C and can cause unwanted agglutination during the room temperature phase of testing.

• This interference can:

  • Mask the presence of clinically significant alloantibodies

  • Cause panreactivity in antibody screening/identification panels

  • Lead to difficulties in crossmatching

• Resolved by using prewarmed techniques or adsorption.

Other options:

• b) Weakening antibody binding → No, they cause strong agglutination.

• c) Destroying red cell antigens → No, enzymes or chemicals destroy antigens, not cold autoantibodies.

• d) Masking ABO reactions → Unlikely; ABO reactions are strong and usually unaffected.

• Once an antibody is identified, the next step is to select donor units that are negative for the corresponding antigen and then perform a crossmatch to confirm compatibility.

• This ensures the transfused red cells will not be destroyed by the patient’s antibody.

Other options:

• a) Perform a DAT on donor units – Not standard; DAT is not used for donor screening.

• c) Type the patient’s cells for the corresponding antigen – This should have been done during antibody identification to confirm the patient lacks the antigen.

• d) Perform an antibody titer – Used in HDFN monitoring, not routine pre-transfusion testing.

• The serum reacts with all panel cells but not the autocontrol, indicating an alloantibody to a high-incidence antigen (not an autoantibody).

• The next step is to perform an antibody identification panel to determine the specificity of the antibody.

• The panel will help identify which high-incidence antigen is the target (e.g., k, Kpᵇ, Jsᵇ, Ytᵃ, etc.).

Other options:

• a) Report warm autoantibody → Incorrect; autocontrol is negative.

• b) Perform cold autoadsorption → Used for cold autoantibodies, not relevant here.

• d) Treat serum with DTT → Used to differentiate IgM/IgG or destroy Kell antigens; not the next step here.

• Anti-Jkᵃ (Kidd system antibody) is notorious for causing delayed hemolytic transfusion reactions (DHTR).

• Kidd antibodies often drop to undetectable levels between exposures, leading to negative pre-transfusion screens.

• Upon re-exposure to the antigen, a rapid anamnestic response causes hemolysis days after transfusion.

Other options:

• b) Anti-Leᵃ – Usually not clinically significant; rarely causes DHTR.

• c) Anti-M – Typically cold-reactive and not significant.

• d) Anti-N – Rarely clinically significant.

• Positive antibody screen + Positive autocontrol suggests the antibody is reacting with both donor/panel cells and the patient’s own cells.

• Common causes:

  • Autoantibody (as in autoimmune hemolytic anemia)

  • Drug-induced antibody

  • Passively acquired antibody from recent transfusion or IVIG

  • Alloantibody with recent transfusion (reacting with transfused cells still in circulation)

Other options:

• a) Alloantibody → Usually negative autocontrol.

• c) ABO discrepancy → Unrelated to antibody screen/autocontrol.

• d) Sample contamination → Unlikely to cause this specific pattern.

• Positive antibody screen + Negative autocontrol typically indicates an alloantibody (an antibody directed against foreign red cell antigens, not self).

• The antibody is reacting with donor/panel cells but not with the patient’s own cells.

• This is the most common scenario in pre-transfusion testing.

Other options:

• b) Autoantibody → Usually causes a positive autocontrol.

• c) Cold autoantibody → Can cause positive screen and often positive autocontrol if strong.

• d) Antibody to complement only → Rare; would require specific complement component testing.

• In warm autoimmune hemolytic anemia (WAIHA), the most common DAT finding is IgG alone or IgG with complement (C3d).

• About 20–30% of cases show IgG + C3, and many others show IgG alone.

• The autoantibody is usually IgG (often IgG1 or IgG3) which can activate complement.

Other options:

• a) IgG only – Common, but IgG + C3 together is also very common.

• b) C3 only – More typical of cold agglutinin disease.

• c) IgM only – Rare in WAIHA; IgM is associated with cold antibodies.

The antibody screening test (also called antibody detection test) is designed to detect the presence of unexpected antibodies in a patient’s serum or plasma that may react with donor red cells.

• a) Identifying the specific antibody → done by the antibody identification panel, not the screen.

• b) ✅ Correct — this is the primary purpose of the antibody screen.

• c) ABO type → determined by forward and reverse grouping, not the screen.

• d) Antibody titer → used for monitoring known antibodies (e.g., anti-D in pregnancy).

• Enzyme treatment (ficin, papain, bromelin) destroys certain antigens:

  • Duffy (Fyᵃ and Fyᵇ)

  • MNS system antigens (M, N, S, s)

• This is useful for antibody identification — loss of reactivity after enzyme treatment suggests anti-Fyᵃ, anti-Fyᵇ, anti-M, anti-N, etc.

Other options:

• b) Kidd and Kell → Kidd is enhanced, Kell is unaffected by enzymes (but destroyed by DTT).

• c) Lewis and P → Lewis is destroyed, but P (globoside) is not destroyed by enzymes.

• d) Rh and Lutheran → Rh is enhanced, Lutheran is variable but generally not destroyed.

• Dithiothreitol (DTT) is a reducing agent that breaks disulfide bonds, which:

  • Destroys Kell system antigens (K, k, Kpᵃ, Kpᵇ, Jsᵃ, Jsᵇ, etc.)

  • Used to confirm anti-Kell specificity by showing loss of reactivity after DTT treatment

  • Also used in phenotyping when cells are coated with IgG (e.g., in autoimmune hemolytic anemia)

Other options:

• a) Destroy IgG antibodies → False; DTT destroys IgM by breaking pentamer structure, not IgG.

• b) Enhance reactivity of weak antibodies → Not its primary use; enzymes or PEG are used for enhancement.

• d) Neutralize complement → Not its function; EDTA is used to chelate calcium and prevent complement activation.

• In a delayed hemolytic transfusion reaction (DHTR), the patient’s previously formed antibody is anamnestic—it rises after exposure to transfused red cells.

• These antibodies are typically IgG, which coat the donor RBCs and lead to extravascular hemolysis.

• Therefore, the post-transfusion DAT is usually positive for IgG only.

Why not the other options:

• a) Negative – Rare during the active hemolysis phase.

• b) Mixed-field positive – Seen when both donor and patient cells are present, not typical of DHTR.

• d) Positive with complement only – Complement-mediated hemolysis is uncommon in DHTR; IgG-mediated extravascular hemolysis predominates.

• A positive autocontrol means the patient’s serum reacts with their own red cells.

• Common causes:

  • Autoantibodies (as in warm autoimmune hemolytic anemia)

  • Medication-induced antibodies (e.g., drug-dependent antibodies)

  • Recent transfusion (alloantibodies reacting with transfused cells still in circulation)

Other options:

• a) Alloantibody – Can cause positive autocontrol only if transfused cells are still present.

• c) Contaminated reagents – Unlikely to systematically cause positive autocontrol.

• d) Weak D antigen – Does not cause positive autocontrol.

• Reacts with 16/16 panel cells: This means the antibody is directed against a high-frequency antigen (present on almost all red cells).

• Negative autocontrol: Rules out an autoantibody.

• No reactivity with ficin-treated cells: Ficin destroys certain high-frequency antigens in the Rh system, like e. The loss of reactivity after ficin treatment is a classic characteristic of anti-e.

• Check cells (also known as Coombs control cells) are IgG-sensitized red cells added to negative antiglobulin tests.

• They should agglutinate if:

  • The AHG reagent was added

  • The AHG reagent is active

  • Washing was adequate (no residual unbound protein neutralized the AHG)

• If check cells fail to agglutinate, the test is invalid.

Other options:

• a) Ensure patient’s serum was added → Not the purpose; that’s checked during initial steps.

• b) Confirm washing was adequate → Part of it, but the main purpose is broader: to verify AHG reactivity.

• d) Detect complement-sensitized red cells → Check cells are usually IgG-coated, not complement-coated.

• IgG antibodies typically react best after 37°C incubation to allow binding to red cells, followed by the antiglobulin (AHG) phase where anti-IgG causes agglutination.

• Most clinically significant alloantibodies (anti-D, anti-K, anti-Fyᵃ, anti-Jkᵃ) are IgG and require AHG testing for detection.

Other options:

• a) Room temperature – Mainly detects IgM antibodies.

• c) Immediate-spin – Detects IgM agglutinins.

• d) Saline phase – Also detects IgM, not IgG.

• In antibody identification, “ruling out” means that an antigen can be excluded as the target of the antibody if:

  • The patient’s serum does not react with a panel cell that has that antigen.

  • This indicates the antibody is not directed against that antigen.

Other options:

• a) The antigen is present on the patient’s red cells – That’s phenotyping, not ruling out in antibody ID.

• c) The patient’s serum reacted strongly with a cell that is negative for that antigen – This would contradict the rule-out.

• d) The antigen is absent from the patient’s red cells – That’s the likely reason the patient made the antibody, but it’s not the definition of “ruling out” in panel interpretation.

• An antibody identification panel typically consists of 10–11 group O reagent red cells from different donors, each with documented antigen profiles for the major blood group systems (Rh, Kell, Kidd, Duffy, MNS, Lewis, P, etc.).

• Using group O cells avoids interference from ABO antibodies.

• The known antigen patterns allow the technologist to match reactivity to specific antibodies by comparing positive and negative reactions.

Other options:

• b) 5 randomly selected group A cells → Too few cells and wrong blood group; would cause ABO interference.

• c) Pooled reagent red cells → Pooling loses the ability to identify specific antibodies based on reaction patterns.

• d) Autologous cells only → Used for autocontrol, not for antibody identification.

• A negative antibody screen means that no unexpected antibodies were detected under the specific test conditions used (e.g., immediate spin, 37°C, AHG phase).

• It does not guarantee the complete absence of all antibodies, as some may be:

  • Present in low titer

  • Directed against low-frequency antigens not present on screening cells

  • Only detectable with additional methods (e.g., enzymes, PEG)

Other options:

• b) The patient has no antibodies at all → False; ABO antibodies are expected and not detected in the screen.

• c) The red cells lack antigens → False; screening cells are selected to have common antigens.

• d) The test was invalid → False; a negative result is valid if controls are correct.

• The scenario describes a warm autoantibody (positive reactions with all panel cells and positive autocontrol).

• To detect underlying alloantibodies, the autoantibody must be removed by adsorption.

• Autoadsorption with ZZAP-treated patient cells is effective because:

  • ZZAP (a combination of dithiothreitol and cysteine-activated papain) removes IgG from red cells and enhances antigen–antibody reactions.

  • Using the patient’s own cells ensures no alloantigens are present to adsorb potential alloantibodies.

  • This method is preferred when the patient has not been recently transfused (transfusion >3 months ago ensures no donor red cells are present).

Other options:

• a) Allogeneic adsorption – Used if the patient has been recently transfused (to avoid adsorbing alloantibodies onto donor cells).

• c) Cold autoadsorption – Used for cold autoantibodies, not warm.

• d) Adsorption using enzyme-treated random donor cells – Could remove alloantibodies, not appropriate here.

• The antibody screen (indirect antiglobulin test) is used to detect unexpected antibodies (non-ABO antibodies) in a patient’s serum.

• It is a critical pre-transfusion test to identify clinically significant antibodies that could cause hemolytic transfusion reactions or HDFN.

Other options:

• b) Confirm ABO group accuracy → This is done by forward/reverse grouping.

• c) Identify Rh phenotype → This requires specific antigen typing with antisera.

• d) Test crossmatch compatibility only → The crossmatch is a separate test; antibody screening is done prior to crossmatch.

• Cold autoantibodies (e.g., anti-I) can cause interference by reacting at room temperature or below.

• To minimize this interference:

  • Use prewarmed techniques (warming serum and cells to 37°C before mixing)

  • Perform testing strictly at 37°C

  • Use adsorption with patient’s own cells to remove cold autoantibodies

• This allows detection of underlying clinically significant alloantibodies.

Other options:

• b) Cooled to enhance binding → Would worsen cold autoantibody interference.

• c) Diluted with saline → Not a standard method for resolving cold autoantibodies.

• d) Frozen before use → Freezing does not eliminate cold autoantibody activity.