Chapter 9 Detection and Identification of Antibodies

• Antibodies can be regular ("expected," like ABO system antibodies) or irregular ("unexpected", due to RBC antigen exposure from transfusion, transplant, or pregnancy)
• AABB standards mandate antibody screens for pre-transfusion testing, prenatal risk assessment for HDFN, evaluating mothers for RhIG prophylaxis, and donor testing

Antibody Screen Techniques:

• Tube Technique:
  • Uses serum or plasma with homozygous RBC reagents for Rh, Kidd, Duffy, MNSs, and Lutheran antigens
  • Flexible and inexpensive with common lab equipment
  • Has unstable reactions and subjective grading
• Gel Technique:
  • Employs dextran acrylamide gel and screening cells in 0.8% LISS
  • Eliminates the washing step with stable and standardized reactions for up to 24 hours
  • Facilitates mixed field reaction detection and automatic interpretation
  • Requires incubators and centrifuges
• Solid Phase Technique:
  • Uses indicator cells instead of AHG with automation
  • Requires smaller sample sizes and LISS that changes color with serum or plasma addition
  • Requires positive and negative controls for each batch

Reagents to Enhance Reactions:

• 22% albumin reduces zeta potential, increasing RBC agglutination chances
• Low Ionic Strength Solution (LISS) contains glycine in albumin solution and increases antibody uptake by RBCs during sensitization
• Polyethylene Glycol (PeG) in LISS removes water, increasing antibody concentration and RBC sensitization, but is unsuitable for patients with high plasma protein levels (e.g., multiple myeloma)

Anti Human Globulin (AHG):

• AABB standards mandate that reagent contains anti-IgG for pre-transfusion compatibility testing
• Polyspecific (polyvalent or broad spectrum) Coomb's serum is used
• AHG should be verified using Coomb's control cells

Antibody Screen Interpretation:

• Agglutination or hemolysis at any testing stage indicates a positive result

Factors for investigators to consider:

• Reaction Phase:

  • IgM reacts best at low temperatures
  • IgG reacts optimally at 37°C

• Autologous Control:

  • A positive control indicates autoantibody or medication-related antibody presence: recent transfusions may cause false positives from alloantibodies on transfused cells
  • A negative autologous control alongside positive antibodies indicates alloantibodies

• Multiple Screening Cell Reactions:

  • Similar phase and strength reactions suggest single antibody specificity, differing reactions suggest multiple antibodies

• Hemolysis or Mixed-Field Agglutination:

  • Anti-Leª, Anti-Leb, Anti-PP¹Pk, and Anti-Vel can cause in vitro hemolysis
  • Mixed-field reactions are linked to Anti-Sdª and Lutheran antibodies

• True Agglutination vs. Rouleaux:

  • Rouleaux, which appear as "stacked coins" under microscopy, can result from multiple myeloma or high-molecular-weight plasma expanders like dextran
  • It does not interfere with the AHG phase
  • It disperses unlike agglutination, with saline addition

• Screening reagents and methods target clinically significant antibodies

• A negative result provides 95% confidence that no clinically significant antibodies are present

Sensitivity Factors of Antibody Screen:

• Cell-to-Serum Ratio:
  • Excess antibody causes prozone, excess antigen causes postzone
  • Both give false-negative results
• Temperature:
  • IgG reacts at 37°C
  • IgM reacts at 21 to 25°C
• Incubation Length:
  • Saline requires 45 mins to 1 hour
  • Potentiators need 10 minutes
• pH:
  • Most antibodies react between 6.8 and 7.2

Antibody Identification Process:

• Identify and determine an antibody's clinical significance after detection
• Requires patient history: age, sex, race, diagnosis, transfusion and pregnancy history, and medications
• Identification uses 11 to 20 "O" cells with varied antigen expressions, including homozygous Rh, Duffy, Kidd, and MNSs
• These cells are positive for low-frequency and negative for high-frequency antigens
• Exclude negative results with homozygous positive antigens and logically evaluate panel results

Evaluation of Panel Results:

• Reaction Phase and Strength: Homozygous cells react more strongly than heterozygous cells and varying strengths indicate multiple antibodies

Antigens with Variable Expression Includes:

• I, P1, Leª, Vel, Ch/Rg and Sda
• Phase Consistency: Reactions at different phases may indicate multiple antibodies
• Homozygous cells react faster than heterozygous cells
• IgM antibodies usually react at the immediate spin phase and are less significant
• IgG Abs are clinically significant and easily detected during AHG phase

Serum Reactivity Factors:

• Serum reactivity should match known specificities
• If a single alloantibody is present, the reactivity pattern is precise
• Rule out common RBC antibodies, testing selected cells for commonly encountered antibodies
• Autologous Controls: A negative one indicates alloantibodies and a positive one indicates autoantibodies or allo/autoantibody mixtures
• Confirm suspected antibody through at least three positive and three negative antigen reactions; a p-value of ≤0.05 validates results
• Check for missing antigen corresponding to the antibody to confirm results
• Options for DAT-positive, non-phenotypable patients include gentle heat elution, chloroquine diphosphate, and glycine/EDTA reagent to remove antibody coating
• Reticulocyte typing can be done for recently transfused individuals
• Flow cytometry to detect trace antigens and PCR for fetal antigen typing, requiring positive controls with heterozygous antigen expression

Techniques for Antibody Identification:

• Use extra cells with minimal overlapping antigens and extended cell panels to identify multiple antibodies
• Enzymes (ficin, papain, trypsin, bromelin) can destroy antigens or improve expression
• Neutralization uses soluble substances to block antibodies that inhibit agglutination

Adsorption Techniques:

• Antibodies are removed from serum via target antigen
• Commercial reagents: human platelet concentrate (for Bg-like antibodies) and rabbit erythrocyte stroma (REST) (for I, H, IH antigens)
• Autoadsorption uses patient's RBCs to remove autoantibodies (patients should not have been transfused in the past 3 months)
• Homologous is used when insufficient RBCs are available or the patient has been recently transfused, match cells lacking the antigens
• Differential adsorption is used when the patient's phenotyping is complicated by a positive DAT, recent transfusion, or multiple antibodies (use R1R1, R2R2, and rr cells, one negative for Jkª, K, Jkb)

DAT (Direct Antiglobulin Test)

• Detects antibodies on RBCs in hemolytic transfusion reactions (HTRs), HDFN, autoimmune and drug-induced hemolytic anemia
• Detects in vivo RBC sensitization
• Elution releases and concentrates antibodies by modifying forces between antigen and antibody and changing the RBC surface
• Total elution releases all bound antibodies and destroys RBC antigens
• Partial elution removes antibody, preserving RBC antigens for phenotyping or preparing cells for autoadsorption
• Elution methods depend on temperature, pH, and solvents
• Techniques:
  • Gentle heat (45°C for partial, 56°C for total)
  • Lui freeze (-18°C)
  • Acid elution (glycine at pH 3.0) for non-ABO abs and organic solvents (dichloromethane, xylene, ether) to reduce surface tension
• Last wash should match the eluate and test negative; method best for non-ABO abs but is time-consuming

Antibody Titration:

• Evaluated by flow cytometry, radioimmunoassay, or enzyme-linked immunoassay to determine the antibody level
• Titer is the highest dilution with observed agglutination
• Assign scores based on reaction strength and calculate the total score
• A change of two tubes or score change of 10+ is significant
• Use cells with homozygous or heterozygous target antigens; lack other antigens to avoid issues
• Titer studies track IgG antibodies in pregnant patients with potential HDFN
• Elevated titers may indicate the need for intrauterine transfusion
• RhIg titers are rarely above 4

HTLA (High Titer, Low Avidity):

• High-frequency antibodies with weak but persistent reactions at high dilutions (2048+)
• Includes Anti-Ch, Anti-Rg, Anti-Csª, Anti-Ykª, Anti-Kna, Anti-McCa, and Anti-JMH
• Usually clinically insignificant but may mask significant antibodies
• Compatible blood: Matching blood is determined by antigen frequency in donor population and antibody's clinical impact
• Use random, crossmatch-compatible blood for antibodies not reducing antigen-positive RBC survival (e.g., Anti-M, -N, -P1, Leª, Leb)
• Transfuse antigen-negative units if there is a clinically significant antibody or a history of one
• Use crossmatch at AHG phase to prove compatibility , antigen type if little sample is available
• Formulas
  • Units to be typed = (Units requested)/(Frequency of antigen-negative individuals)
    • Units to be typed = (Units requested)/(Multiplied frequencies of negative antigens)
• Match donor ethnicity to fit antigen frequencies in recipient's race

Resolving Difficulties in Cases With Multiple Antibodies:

• Multiple Abs suspected when several screening/panel RBCs react with varying strengths, but auto control is negative
• Perform selective cell panels, enzyme techniques for antibody separation, and neutralization/absorption
• Suspect when most screening and panel cells are positive while auto control is negative
• Use cells lacking the antibodies to resolve, confirm with reference lab, phenotype, and perform absorption studies
• Choose autologous or family donations for transfusion

Resolving Difficulties in Cases With Low-Frequency Antibodies:

• This type of antibody is suspected when only one cell becomes positive: crossmatch incompatible or DAT-positive in newborns with compatible maternal blood
• Use extended panels to detect rare antibodies

Resolving Difficulties in Cases With Cold Reacting Autoantibodies:

• Most adult samples contain low cold autoantibodies (Anti-I, -H, -IH)
• Antibodies interfere with antibody screen, panel is positive at immediate spin, weaker at 37°C/AHG phase
• Resolved with monospecific Anti-IgG Coomb's, pre-warming, and autoadsorption at 4°C
• REST absorption could be used if the patient has a strong reactant or if the patient was recently transfused
  • REST adsorbed serum is not suitable for crossmatch as anti-B may be removed
• Use DTT, 2-Mercaptoethanol to break disulfide bonds and denature IgM

Resolving Difficulties in Cases With Warm Reacting Autoantibodies:

• Associated with chronic lymphocytic leukemia, lymphoma, systemic lupus erythematosus and other autoimmune diseases
• Use partial elution, ZZAP, "autologous adsorption," and phenotyping for target antigen if the patient has not been recently transfused
• Select least incompatible blood for crossmatching

For allogeneic vs homologous/differential situations:

• If the patient has been recently transfused, choose blood lacking alloantibody antigen for transfusion

Important Points:

• Antibody screens detect unexpected antibodies (0.2-2% of the population)
• Antibodies are classified as immune (RBC stimulation), passive (blood products/derivatives), or naturally occurring (environmental)
• Also classified as alloantibodies (directed to one's own antigens)
• Significant antibody: Shortens survival of RBCs with the antigen, IgG reacts best at 37°C/AHG, and causes HTRs and HDN
• Screening cells: Group O cell suspensions that are commercially prepared and phenotyped for common and clinically important RBC antigens
• Homozygous: Double antigen dose (two genes for same antigen)
• Heterozygous: Single dose of two different antigens (each gene codes for different antigens)
• Dosage exhibited in systems such as Kidd, Duffy, Lutheran, Rh and MNSs: homozygous expression has stronger reactions Enhancement Reagents:
• LISS and PEG solutions are added to promote binding
• Coombs' control cells: RBCs coated with human IgG and confirms proper AHG reagent and washing
• Gel/Solid: Gel and solid methods are alternatives to tube and can be automated Antibody Exclusion:
• Rules out possible antibodies, based on cell reactions Conclusive Identification:
• Antibody identified when serum reacts positively with 3+ antigen-positive cells
• Also reacts negatively with 3+ antigen-negative cells, and the patient's RBCs phenotype negative
• DAT: Detects cells with Abs in vivo, elution releases Abs from cells for ident.
• Determine the number of random donor units screened for patients possessing an antibody
  • Divide the count of desired antigen-negative units for transfusion by the antigen-negative individuals' proportion
• Antibody Titre: test serial two-fold dilutions of serum against antigen-positive RBCs: the reciprocal of the highest serum dilution showing agglutination