Antibody screening and identification are fundamental procedures in immunohematology used to detect unexpected red cell antibodies in patient serum or plasma. These tests are essential for ensuring blood transfusion safety, investigating transfusion reactions, and preventing hemolytic disease of the newborn.
This chapter focuses on the principles, techniques, and interpretation of antibody detection and identification using various testing methodologies.
Topics Covered in This Chapter
- Principles of antibody detection and clinical importance
- Screening cell panels and reagent preparation
- Phases of testing: immediate spin, 37°C incubation, and antiglobulin phase
- Enhancement media: LISS, PEG, albumin, enzymes
- Autocontrol and antibody titration
- Differentiating autoantibodies and alloantibodies
- Interpretation of antibody panels and rule-out strategies
- Complex antibody identification cases and troubleshooting
Why It Matters
Accurate antibody detection prevents incompatible transfusions and ensures the selection of antigen-negative blood for transfusion.
Understanding antibody patterns allows laboratory professionals to solve serologic puzzles, especially when dealing with multiple antibodies or warm/cold autoantibodies.
Learning Outcomes
After studying this section, learners should be able to:
- Resolve common serologic discrepancies.
- Describe the principle and purpose of antibody screening and identification.
- Perform and interpret results from antibody panels.
- Differentiate between autoantibodies and alloantibodies.
- Apply enhancement techniques to improve antibody detection.
60 MCQs (4541 – 4600):
- The main purpose of an antibody screen is to:
a) Detect unexpected antibodies in a patient’s serum or plasma
b) Confirm ABO group accuracy
c) Identify Rh phenotype
d) Test crossmatch compatibility only - Antibody screening cells are prepared from donors with:
a) Known antigen profiles
b) Randomly selected blood samples
c) Group O Rh-positive blood only
d) Autoantibody-positive samples - The indirect antiglobulin test (IAT) is used in antibody screening to detect:
a) Complement on red cells
b) Unbound antibodies in patient serum
c) Hemolysis caused by cold agglutinins
d) Platelet-bound antibodies - A positive antibody screen indicates the presence of:
a) Unexpected red cell antibodies
b) Complement in plasma
c) Abnormal clotting factors
d) Autoantibodies to white cells - What type of cells are typically used in antibody identification panels?
a) Group O reagent red cells with known antigen profiles
b) Random donor blood samples
c) Group A red cells only
d) Pooled platelet samples - Which phase of testing best detects IgM antibodies?
a) Immediate-spin phase
b) 37°C incubation
c) Antiglobulin (AHG) phase
d) Enzyme phase - Which phase of testing best detects IgG antibodies?
a) Room temperature
b) 37°C and AHG phases
c) Immediate-spin
d) Saline phase - An autocontrol is used to determine:
a) Presence of autoantibodies coating the patient’s own red cells
b) Blood group type
c) Crossmatch compatibility
d) Plasma protein concentration - If the autocontrol is positive, the cause may be:
a) Alloantibody
b) Autoantibody or medication-induced reaction
c) Contaminated reagents
d) Weak D antigen - Enhancement media such as LISS and PEG are used to:
a) Speed up antigen–antibody binding
b) Destroy antibodies
c) Reduce serum viscosity
d) Inhibit complement activity - Enzyme-treated red cells are useful because they:
a) Enhance reactions of some antibodies (e.g., Rh, Kidd)
b) Destroy all red cell antigens
c) Eliminate IgG antibodies
d) Stop complement fixation - Which antibodies are typically enhanced by enzyme treatment?
a) Rh, Kidd, Lewis, and P
b) Duffy and MNS
c) Lutheran only
d) ABO antibodies - Which antibodies are destroyed by enzyme treatment?
a) Duffy, M, and N
b) Kidd and Kell
c) Lewis and P
d) Rh and Lutheran - A single strong reaction with all panel cells suggests:
a) Autoantibody or antibody to high-incidence antigen
b) Antibody to a low-frequency antigen
c) Typing error
d) Clerical mistake - Mixed field reactions during antibody testing can indicate:
a) Recent transfusion or bone marrow transplant
b) Plasma contamination
c) Instrument malfunction
d) Sample hemolysis - An antibody showing stronger reaction with homozygous cells than heterozygous cells exhibits:
a) Dosage effect
b) Prozone phenomenon
c) Zeta potential
d) Complement fixation - The “rule-out” method in antibody identification means:
a) Excluding antigens that show negative reactions
b) Using only positive reactions to identify antibodies
c) Ignoring weak reactions
d) Eliminating all antibodies detected - The antibody identification panel is usually composed of:
a) 10–11 group O cells with known antigen profiles
b) 5 randomly selected group A cells
c) Pooled reagent red cells
d) Autologous cells only - The phase at which an antibody reacts provides information about:
a) Its immunoglobulin class and temperature range
b) Its molecular weight
c) Its age of development
d) Its enzyme sensitivity - A positive antibody screen but negative autocontrol indicates:
a) Alloantibody
b) Autoantibody
c) Cold autoantibody
d) Antibody to complement only - A positive antibody screen and positive autocontrol suggest:
a) Alloantibody
b) Autoantibody or drug-induced antibody
c) ABO discrepancy
d) Sample contamination - What is the purpose of using enzyme panels in antibody identification?
a) To confirm or eliminate enzyme-sensitive or enzyme-enhanced antibodies
b) To measure antibody titers
c) To detect complement components
d) To confirm weak D antigen - Antibodies that are clinically significant are usually:
a) IgG, reactive at 37°C and AHG phase
b) IgM, reactive at 4°C only
c) Naturally occurring
d) Found only in cord blood - Which antibody may show variable reactions due to dosage?
a) Anti-K
b) Anti-Fyᵃ
c) Anti-A
d) Anti-Leᵃ - A cold autoantibody can interfere with testing by:
a) Causing false agglutination at room temperature
b) Weakening antibody binding
c) Destroying red cell antigens
d) Masking ABO reactions - To resolve a cold autoantibody interference, the serum can be:
a) Warmed or prewarmed testing methods used
b) Cooled to enhance binding
c) Diluted with saline
d) Frozen before use - Which antibody commonly causes delayed hemolytic transfusion reactions?
a) Anti-Jkᵃ
b) Anti-Leᵃ
c) Anti-M
d) Anti-N - An antibody that reacts only with enzyme-treated red cells may belong to which system?
a) Rh or Kidd
b) Duffy
c) MNS
d) Lutheran - Antibody identification is confirmed when:
a) All positive reactions match known antigen patterns
b) One random cell is positive
c) Negative control fails
d) All cells react at immediate-spin - In antibody screening, a negative result means:
a) No unexpected antibodies detected under test conditions
b) The patient has no antibodies at all
c) The red cells lack antigens
d) The test was invalid - 1. During an antibody identification panel, a technologist observes 2+ reactions with 3 of 10 cells at immediate spin (IS) and room temperature (RT), with no reactions at 37°C or with antihuman globulin (AHG). What is the most likely antibody?
a) Anti-Jkᵇ
b) Anti-Leᵃ
c) Anti-C
d) Anti-Fyᵃ - A patient’s serum is reactive at the antiglobulin phase with all panel cells, including the autocontrol. The patient was transfused 6 months ago. What is the optimal adsorption method to remove the autoantibody and check for underlying alloantibodies?
a) Allogeneic adsorption using R₁R₁ and R₂R₂ cells
b) Autoadsorption using the patient’s ZZAP-treated red cells
c) Cold autoadsorption using the patient’s untreated red cells
d) Adsorption using enzyme-treated random donor cells - In the process of identifying an antibody, the use of dithiothreitol (DTT) is helpful to:
a) Destroy IgG antibodies to confirm IgM specificity
b) Enhance the reactivity of weak antibodies
c) Destroy Kell system antigens for phenotyping
d) Neutralize complement in the serum - A patient’s serum reacts weakly positive (1+) with 16 of 16 group O panel cells at the AHG phase. The autocontrol is negative. Tests with ficin-treated panel cells show no reactivity. Which antibody is most likely responsible?
a) Anti-Ch
b) Anti-k
c) Anti-e
d) Anti-Jsᵇ - The primary purpose of the antibody detection (screening) test is to:
a) Identify the specific antibody present in the serum
b) Detect the presence of unexpected, clinically significant antibodies
c) Confirm the ABO type of the patient
d) Determine the titer of a known antibody - A major crossmatch that is incompatible at the antiglobulin phase is most likely due to:
a) An ABO typing error on the donor unit
b) An unexpected antibody in the recipient’s serum reacting with donor antigens
c) A positive direct antiglobulin test (DAT) on the recipient’s cells
d) Bacterial contamination of the donor unit - When check cells (IgG-sensitized red cells) are added to a negative antiglobulin test and no agglutination occurs, what is the most likely explanation?
a) The test is truly negative for antibody
b) The patient’s serum has neutralized the AHG reagent
c) The AHG reagent was not added, or the cells were over-washed
d) The centrifuge speed was set too low - A patient’s serum sample is reactive with all cells except the autocontrol when tested by polyethylene glycol-antihuman globulin (PEG-AHG). The next step should be to:
a) Report the presence of a warm autoantibody
b) Perform a cold autoadsorption
c) Perform an antibody identification panel
d) Treat the serum with DTT - Which of the following techniques is most useful for resolving the specificity of an antibody that reacts with all panel cells, including the autocontrol, when the patient has not been recently transfused?
a) Saline replacement technique
b) Cold allogeneic adsorption
c) Warm autoadsorption
d) Titration studies - A patient with a positive direct antiglobulin test (DAT) needs a phenotype. Which reagent can be used to remove IgG from the red cell surface without destroying antigens?
a) Ficin
b) Dithiothreitol (DTT)
c) Chloroquine diphosphate
d) Polyethylene glycol (PEG) - In a suspected case of a delayed hemolytic transfusion reaction, the post-transfusion DAT is often:
a) Negative
b) Mixed-field positive
c) Positive with IgG only
d) Positive with complement only - A patient’s serum contains a mixture of antibodies, including anti-D and anti-Fyᵃ. What technique may be helpful to identify other possible antibodies?
a) Testing against a selected cell panel treated with proteolytic enzymes
b) Performing a cold autoadsorption
c) Using albumin as an enhancement medium
d) Repeating the panel at room temperature only - The saline replacement technique is primarily used to:
a) Enhance the reactivity of IgG antibodies
b) Distinguish true agglutination from rouleaux
c) Identify the presence of cold agglutinins
d) Remove fibrin strands that may mimic agglutination - A 4+ positive reaction using gel technology will appear as red blood cells:
a) In a pellet at the bottom of the microtubule
b) Dispersed throughout the gel media
c) In a layer at the top of the gel media
d) Suspended at the mid-point of the gel media - A negative result using solid phase adherence assays will demonstrate indicator red cells as:
a) A red blood cell pellet in the bottom of the well
b) A diffuse pattern of red blood cells throughout the well
c) Red blood cell clumps symmetrically located throughout the well
d) A red supernatant, indicating lysis - Which of the following antibodies is most likely to show in-vitro hemolysis during testing with fresh serum?
a) Anti-K
b) Anti-Leᵃ
c) Anti-E
d) Anti-Fyᵃ - The “pre-warm” technique involves:
a) Warming all reagents and red cells to 37°C before mixing to avoid cold antibody interference
b) Incubating tests at 37°C for a longer period to enhance antibody uptake
c) Heating the patient’s serum to 56°C to inactivate complement
d) Warming the patient’s red cells to elute cold autoantibodies - To confirm the specificity of an antibody identified as anti-Leᵇ, what test should be performed?
a) Neutralization with Lewis substance
b) Treatment of panel cells with ficin
c) Testing the serum with cord blood cells
d) Adsorption and elution studies - A patient has an antibody that reacts at the AHG phase and is enhanced by enzyme treatment. This describes which of the following antibodies?
a) Anti-M
b) Anti-Leᵃ
c) Anti-Fyᵃ
d) Anti-Jkᵃ - A false-negative indirect antiglobulin test (IAT) can be caused by:
a) Over-centrifugation
b) Under-centrifugation
c) Dirty glassware
d) Use of EDTA plasma - When an antibody to a high-incidence antigen is suspected, the first step in confirmation should be to:
a) Test the patient’s serum with their own cells
b) Test the patient’s serum with rare reagent red cells known to lack the antigen
c) Perform an elution on the patient’s cells
d) Test the patient’s family members’ red cells - The primary advantage of using gel technology for antibody screening is that it:
a) Requires no centrifugation
b) Eliminates the need for saline washing
c) Uses less reagent than tube testing
d) Provides faster results than solid phase - A patient with warm autoimmune hemolytic anemia (WAIHA) has a positive DAT. The most common immunoglobulin and/or complement found on their red cells is:
a) IgG only
b) C3 only
c) IgM only
d) IgG and C3 - In antibody identification, “ruling out” an antigen means:
a) The antigen is present on the patient’s red cells
b) The patient’s serum did not react with a cell that is positive for that antigen
c) The patient’s serum reacted strongly with a cell that is negative for that antigen
d) The antigen is absent from the patient’s red cells - A patient’s serum reacts with all panel cells at the AHG phase. The autocontrol is negative. This pattern is most consistent with:
a) A warm autoantibody
b) Multiple alloantibodies
c) An antibody to a high-prevalence antigen
d) Rouleaux formation - The principle of the “check cell” system in the antiglobulin test is to:
a) Ensure the patient’s serum was added to the test
b) Confirm that washing was adequate to remove unbound protein
c) Verify that the AHG reagent was added and is reactive
d) Detect the presence of complement-sensitized red cells - A patient’s phenotype is determined to be C–E+c+e+; K–k+; Fy(a–b+); Jk(a–b–). When their serum is tested against phenotypically similar cells and found to be nonreactive, this suggests the antibody is likely directed against which system?
a) Rh
b) Kell
c) Duffy
d) Kidd - Which of the following reagents acts as an enhancement medium by reducing the zeta potential around red cells?
a) Bovine albumin
b) Low Ionic Strength Saline (LISS)
c) Polyethylene Glycol (PEG)
d) Proteolytic enzymes - The Donath-Landsteiner test is diagnostic for which condition?
a) Warm Autoimmune Hemolytic Anemia
b) Paroxysmal Nocturnal Hemoglobinuria
c) Paroxysmal Cold Hemoglobinuria
d) Cold Agglutinin Disease - After identifying an antibody, the next step before releasing blood for transfusion is to:
a) Perform a direct antiglobulin test on the donor units
b) Crossmatch units that are antigen-negative for the identified antibody
c) Type the patient’s cells for the corresponding antigen
d) Perform an antibody titer
📌 How to Use This Practice Set
- Answer each question before checking the key.
- Focus on why the correct answer is right and the others are wrong.
- Use this set as timed practice to simulate the real exam environment.
Answer Key
Answer Key:
- a) Detect unexpected antibodies in a patient’s serum or plasma
- a) Known antigen profiles
- b) Unbound antibodies in patient serum
- a) Unexpected red cell antibodies
- a) Group O reagent red cells with known antigen profiles
- a) Immediate-spin phase
- b) 37°C and AHG phases
- a) Presence of autoantibodies coating the patient’s own red cells
- b) Autoantibody or medication-induced reaction
- a) Speed up antigen–antibody binding
- a) Enhance reactions of some antibodies (e.g., Rh, Kidd)
- a) Rh, Kidd, Lewis, and P
- a) Duffy, M, and N
- a) Autoantibody or antibody to high-incidence antigen
- a) Recent transfusion or bone marrow transplant
- a) Dosage effect
- a) Excluding antigens that show negative reactions
- a) 10–11 group O cells with known antigen profiles
- d) Its enzyme sensitivity
- a) Alloantibody
- b) Autoantibody or drug-induced antibody
- a) To confirm or eliminate enzyme-sensitive or enzyme-enhanced antibodies
- a) IgG, reactive at 37°C and AHG phase
- b) Anti-Fyᵃ
- a) Causing false agglutination at room temperature
- a) Warmed or prewarmed testing methods used
- a) Anti-Jkᵃ
- a) Rh or Kidd
- a) All positive reactions match known antigen patterns
- a) No unexpected antibodies detected under test conditions
- b) Anti-Leᵃ
- b) Autoadsorption using the patient’s ZZAP-treated red cells
- c) Destroy Kell system antigens for phenotyping
- c) Anti-e
- b) Detect the presence of unexpected, clinically significant antibodies
- b) An unexpected antibody in the recipient’s serum reacting with donor antigens
- c) The AHG reagent was not added, or the cells were over-washed
- c) Perform an antibody identification panel
- c) Warm autoadsorption
- c) Chloroquine diphosphate
- c) Positive with IgG only
- a) Testing against a selected cell panel treated with proteolytic enzymes
- b) Distinguish true agglutination from rouleaux
- c) In a layer at the top of the gel media
- a) A red blood cell pellet in the bottom of the well
- b) Anti-Leᵃ
- a) Warming all reagents and red cells to 37°C before mixing to avoid cold antibody interference
- a) Neutralization with Lewis substance
- d) Anti-Jkᵃ
- b) Under-centrifugation
- b) Test the patient’s serum with rare reagent red cells known to lack the antigen
- b) Eliminates the need for saline washing
- d) IgG and C3
- b) The patient’s serum did not react with a cell that is positive for that antigen
- c) An antibody to a high-prevalence antigen
- c) Verify that the AHG reagent was added and is reactive
- d) Kidd
- b) Low Ionic Strength Saline (LISS)
- c) Paroxysmal Cold Hemoglobinuria
- b) Crossmatch units that are antigen-negative for the identified antibody
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Top 8 Medical Laboratory Scientist (MLS) Exams that are recognized globally and can help professionals validate their credentials and enhance their career opportunities:
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