10.3 Antiglobulin Testing, Elution, Adsorption, and Special Antibody Procedures

Antiglobulin Testing, Elution, Adsorption, and Special Antibody Procedures

The Antiglobulin Test, originally conceptualized by Carlo Moreschi in 1908 and reintroduced to modern medicine in 1945 by Robin Coombs, Arthur Mourant, and Robert Race, remains the cornerstone of clinical immunohematology. The technique enables the laboratory detection of non-agglutinating, monomeric IgG antibodies and complement components bound to human red blood cells. By bridging the biophysical and electrical forces that naturally keep circulating erythrocytes separated, antiglobulin testing allows medical technologists to detect both in vivo erythrocyte sensitization (the Direct Antiglobulin Test, DAT) and in vitro antigen-antibody interactions (the Indirect Antiglobulin Test, IAT).

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1. Biophysical Principles of Antiglobulin Testing (The Coombs Technique)

Agglutination of erythrocytes occurs in two distinct, sequential stages:

1. First Stage (Sensitization): Physical attachment of antibody molecules (or complement cleavage products) to specific antigenic epitopes on the red blood cell membrane. Sensitization occurs without visible clumping and is governed by non-covalent forces (electrostatic interactions, hydrogen bonding, hydrophobic effects, and van der Waals forces).
2. Second Stage (Lattice Formation): Cross-linking of sensitized red blood cells into a stable, three-dimensional macroscopic lattice structure visible as agglutination.

                                  ┌──────────────────────────────────────────────┐
                                  │      The Biophysical Agglutination Gap       │
                                  └──────────────────────┬───────────────────────┘
                                                         │
         ┌───────────────────────────────────────────────┴───────────────────────────────────────────────┐
         ▼                                                                                               ▼
  ┌──────────────────────────────┐                                                ┌──────────────────────────────┐
  │  Pentameric IgM Agglutination │                                                │   Monomeric IgG Sensitization│
  └──────────────┬───────────────┘                                                └──────────────┬───────────────┘
                 │                                                                               │
  • Reach: 30 to 35 nm                                                            • Reach: Only 14 nm
  • Can directly bridge the 25-nm                                                 • CANNOT bridge the 25-nm gap!
    intercellular gap                                                             • Coats RBCs (Sensitization) without
  • Direct visible macroscopic                                                      lattice formation
    agglutination in saline                                                       • Requires AHG to bridge the gap!

Zeta Potential and Intercellular Distance

• Electrical Double Layer: Erythrocyte membranes carry a strong negative net surface charge due to carboxyl groups of sialic acid (N-acetylneuraminic acid, NANA) on transmembrane sialoglycoproteins (predominantly Glycophorin A). When suspended in an electrolytic solution such as $0.9\%$ physiological saline, cations ($Na^+$) form an immovable Stern layer around the cell, followed by a diffuse outer ionic cloud.
• Zeta Potential ($\zeta$): The electrical potential difference measured between the shearing plane of the red cell and the neutral bulk electrolytic solution is termed the zeta potential, typically measuring approximately $-15\text{ mV}$ in standard saline. This negative charge exerts mutual electrostatic repulsion, maintaining a minimum physical separation distance of approximately $25\text{ nm}$ ($250\text{ \AA}$) between adjacent red blood cells.
• The Molecular Reach of Immunoglobulins:
  • IgM Pentamers: Possess a wide molecular span of $30\text{ to }35\text{ nm}$ ($300\text{--}350\text{ \AA}$). Because this reach exceeds the $25\text{-nm}$ intercellular gap, IgM antibodies easily span adjacent red cells, causing direct visible agglutination in saline at room temperature.
  • IgG Monomers: Possess a molecular span of only $14\text{ nm}$ ($140\text{ \AA}$). Although IgG Fab arms bind specific membrane antigens with high affinity, IgG cannot bridge the $25\text{-nm}$ physical gap between adjacent cells. Consequently, sensitized IgG-coated red blood cells remain suspended and unagglutinated.
• The AHG Bridging Principle: Antihuman Globulin (AHG / Coombs reagent) consists of bivalent antibodies produced in animals (or hybridoma cell cultures) directed against human globulins (anti-IgG and/or anti-complement). The bivalent Fab arms of AHG bind to the exposed $Fc$ domains of IgG molecules (or $C3d$ fragments) coating adjacent red cells, bridging the $25\text{-nm}$ gap and producing visible macroscopic lattice agglutination.

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2. Antiglobulin Reagents: Polyspecific vs. Monospecific

                               ┌─────────────────────────────────────────────────────────┐
                               │             Antihuman Globulin Reagents                 │
                               └────────────────────────────┬────────────────────────────┘
                                                            │
                            ┌───────────────────────────────┴───────────────────────────────┐
                            ▼                                                               ▼
                 Polyspecific AHG Reagent                                        Monospecific AHG Reagents
  • Blend of anti-human IgG AND anti-C3d/C3b                    ┌───────────────────────────┴───────────────────────────┐
  • Manufactured from rabbit polyclonal or                      ▼                                                       ▼
    murine monoclonal hybridomas                         Anti-IgG (Heavy Chain)                    Anti-C3b / Anti-C3d
  • Routine screening for DAT and pre-transfusion        • Contains no light-chain activity        • Contains no antibody
    compatibility testing                                  (avoids non-specific binding)             activity against IgG
                                                         • Detects IgG warm auto/allo-             • Critical for Cold Agglutinin
                                                           antibodies and HDFN                       Disease, PCH, & drug complexes

Polyspecific Antihuman Globulin

• Composition: Polyspecific AHG contains antibodies against both human IgG (heavy chain-specific) and human complement components (specifically $C3d$ and $C3b$). Reagents may be prepared as a blend of rabbit polyclonal antisera or murine monoclonal hybridoma antibodies (e.g., clone 17-2 anti-IgG blended with clone BRIC-8 anti-C3d).
• Primary Application: Polyspecific AHG is employed for the initial screening phase of the Direct Antiglobulin Test (DAT). A positive result indicates that either IgG, complement, or both are bound to the circulating red cells in vivo.

Monospecific Antihuman Globulin

When a polyspecific DAT is positive, the medical technologist must immediately repeat the test using monospecific reagents to determine the precise immunologic mechanism:

1. Monospecific Anti-IgG: Contains antibodies reactive specifically with the gamma ($\gamma$) heavy chain of human IgG molecules. It contains no light-chain reactivity, ensuring it will not non-specifically cross-react with circulating IgM or IgA.
2. Monospecific Anti-C3b/-C3d: Contains antibodies directed against the $C3d$ or $C3b$ cleavage fragments of the third component of human complement. It contains no reactivity against human immunoglobulins. Anti-C3d is especially vital because when complement is activated in vivo, circulating regulatory proteins (Factor I and Factor H) rapidly cleave cell-bound $C3b$ into inactive $C3bi$ and finally into the stable, non-cleavable membrane-bound $C3d$ fragment, which persists on the red cell surface throughout its lifespan.

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3. The Direct Antiglobulin Test (DAT)

The Direct Antiglobulin Test (DAT) detects in vivo sensitization—immunoglobulins or complement components that attached to the patient's red blood cells while circulating inside the vascular system.

                                  ┌──────────────────────────────────────────────┐
                                  │          DAT Diagnostic Algorithm            │
                                  └──────────────────────┬───────────────────────┘
                                                         │
                                                         ▼
                                            Perform Polyspecific DAT
                                                         │
                                ┌────────────────────────┴────────────────────────┐
                                ▼                                                 ▼
                          Negative Result                                   Positive Result
                   (Add Check Cells; must be 2+)                                  │
                                                                                  ▼
                                                                Perform Monospecific DAT Panel
                                                                (Anti-IgG and Anti-C3d testing)
                                                                                  │
         ┌────────────────────────────────────────────────────────────────────────┼────────────────────────────────────────────────────────┐
         ▼                                                                        ▼                                                        ▼
  Anti-IgG Positive / Anti-C3d Negative                    Anti-IgG Negative / Anti-C3d Positive                    Anti-IgG Positive / Anti-C3d Positive
  • Warm AIHA (20% to 40%)                                 • Cold Agglutinin Disease (CAD)                          • Warm AIHA (50% to 60%)
  • HDFN (Rh, Kell, ABO)                                   • Paroxysmal Cold Hemoglobinuria (PCH)                   • Severe active HTR
  • Hemolytic Transfusion Reaction (HTR)                   • Drug-induced immune complex mechanism                  • Drug-induced autoantibody
  • Drug-adsorption (Penicillin mechanism)                 • Delayed complement-mediated HTR                          mechanism (Methyldopa)

Clinical Indications for the DAT

1. Autoimmune Hemolytic Anemia (AIHA):
   • Warm AIHA (WAIHA): Accounts for 70% to 80% of all autoimmune hemolytic cases. Mediated by warm-reactive IgG autoantibodies (optimally reactive at $37^\circ\text{C}$). The monospecific DAT pattern reveals IgG alone in 20% to 40% of patients, and both IgG and C3d in 50% to 60% of patients (C3d alone is seen in ~10%). Sensitized cells undergo extravascular phagocytosis by splenic reticuloendothelial macrophages via $Fc\gamma$ receptors, generating microspherocytes.
   • Cold Agglutinin Disease (CAD): Accounts for 15% to 20% of AIHA cases. Mediated by cold-reactive IgM autoantibodies (predominantly auto-anti-I) that bind red cells in cool peripheral extremities ($<30^\circ\text{C}$) and fix complement through $C3b$. When erythrocytes return to the central core circulation ($37^\circ\text{C}$), the IgM antibody dissociates from the cell, but the covalently bound complement remains behind. Endogenous plasma Factor I and Factor H cleave $C3b$ down to $C3d$. Consequently, the monospecific DAT in CAD is positive for C3d ONLY and negative for IgG.
   • Paroxysmal Cold Hemoglobinuria (PCH): Caused by the biphasic IgG Donath-Landsteiner auto-anti-P. The antibody binds and fixes complement at $4^\circ\text{C}$, but elutes at $37^\circ\text{C}$. The DAT is characteristically positive for C3d ONLY.
2. Hemolytic Disease of the Fetus and Newborn (HDFN):
   • Performed on neonatal cord blood or infant peripheral blood to detect maternal IgG alloantibodies (anti-D, anti-K, anti-c, or immune anti-A,B) coating fetal erythrocytes in utero.
   • Monospecific DAT is strongly positive for IgG.
3. Hemolytic Transfusion Reactions (HTR):
   • Occurs when recipient alloantibodies coat transfused donor red blood cells (or transfused donor antibodies coat recipient red cells).
   • Demonstrates a pathognomonic mixed-field agglutination pattern under the microscope (small agglutinated clumps of sensitized donor cells interspersed among a vast background of unagglutinated recipient cells).
4. Drug-Induced Immune Hemolytic Anemia (DIIHA):
   • Drug Adsorption / Hapten Mechanism (e.g., high-dose IV penicillin, ampicillin, cefazolin): The drug binds firmly to membrane proteins. The patient produces high-titer anti-drug IgG antibodies that bind specifically to the membrane-bound drug. DAT is positive for IgG only; eluates are non-reactive with normal panel cells unless pre-incubated with the drug.
   • Immune Complex / "Bystander" Mechanism (e.g., quinidine, ceftriaxone, stibophen): Drug and anti-drug antibody form circulating immune complexes that loosely adsorb to the red cell membrane, activate the classical complement cascade, and dissociate, leaving complement fixed to the membrane. DAT is positive for C3d only.
   • Autoantibody Induction Mechanism (e.g., $\alpha$-methyldopa [Aldomet], L-dopa, fludarabine): The drug alters suppressor T-cell regulation, inducing the synthesis of true erythrocyte autoantibodies identical to Warm AIHA (often with broad Rh-like specificity). DAT is strongly positive for IgG; eluate and serum react against all normal panel red cells even in the complete absence of the offending drug.
   • Non-Immunologic Protein Adsorption (NIPA) / Membrane Modification (e.g., cephalosporins such as cephalothin [Keflin], tazobactam): The drug modifies the erythrocyte membrane, causing non-specific adherence of all circulating plasma proteins (albumin, IgG, IgM, IgA, complement, fibrinogen). DAT is positive for IgG and C3d; eluate is completely non-reactive; true in vivo hemolysis is typically absent.

DAT Procedural Steps

• Specimen Collection: Blood collected in EDTA (lavender top) is preferred over clotted red-top tubes. EDTA chelates ionized calcium ($Ca^{2+}$) and magnesium ($Mg^{2+}$), which are mandatory cofactors for the activation of the $C1$ and $C3$ convertase complexes, thereby preventing in vitro complement uptake during refrigerated storage that could cause false-positive complement DAT results.
• Procedure: A 2% to 5% red cell suspension is washed 3 to 4 times with large volumes of fresh $0.9\%$ saline. AHG reagent is added, the tube is centrifuged at $1,000\times g$ for 15 to 20 seconds, and read immediately for IgG. For complement evaluation, tubes showing no immediate agglutination are incubated at room temperature for 5 to 10 minutes, re-centrifuged, and read.

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4. The Indirect Antiglobulin Test (IAT) and Enhancement Media

The Indirect Antiglobulin Test (IAT) detects in vitro sensitization—antibodies present in patient serum or plasma that bind to reagent red blood cells during controlled laboratory incubation at $37^\circ\text{C}$.

                                  ┌──────────────────────────────────────────────┐
                                  │          IAT Procedural Continuum            │
                                  └──────────────────────┬───────────────────────┘
                                                         │
   1. Incubation Phase (37°C)        2. Washing Phase (Saline)         3. AHG Addition & Read        4. Validation Phase
  ┌───────────────────────────┐     ┌───────────────────────────┐     ┌───────────────────────┐     ┌───────────────────┐
  │ Patient Serum + Reagent   │ ──► │ Wash 3 to 4 times with    │ ──► │ Add AHG reagent;      │ ──► │ Add Check Cells   │
  │ RBCs + Potentiator        │     │ fresh 0.9% saline to      │     │ Centrifuge & grade    │     │ to all NEGATIVE   │
  │ (LISS: 10-15 min;         │     │ remove all trace unbound  │     │ agglutination         │     │ tubes; must yield │
  │  PEG / Albumin: 30 min)   │     │ free serum globulins      │     │ macroscopically       │     │ >= 2+ clumping!   │
  └───────────────────────────┘     └───────────────────────────┘     └───────────────────────┘     └───────────────────┘

Clinical Indications for the IAT

1. Unexpected Antibody Screening: Detection of clinically significant red cell alloantibodies in pre-transfusion patients and prenatal women.
2. Antibody Identification Panels: Delineation of specific antibody specificities using 8 to 20 typed panel red cells.
3. Major Pre-Transfusion Crossmatch: Testing recipient serum against prospective donor red blood cells.
4. Red Blood Cell Antigen Phenotyping: Typing patient or donor red cells for specific blood group antigens (e.g., $Fy^a, Jk^a, S$) using commercial antisera that require an antiglobulin phase.
5. Weak D Testing: Confirmation of weak D expression in blood donors and neonates.

Enhancement Media (Potentiators)

To accelerate antibody-antigen association and shorten required incubation times, various chemical potentiators are employed during the $37^\circ\text{C}$ incubation phase:

• Low Ionic Strength Saline (LISS):
  • Lowers the ionic strength of the incubation medium from $0.17\text{ M}$ ($0.9\%\text{ NaCl}$) to $0.03\text{ M}$.
  • Mechanistically reduces the shielding effect of the $Na^+$ and $Cl^-$ ionic cloud surrounding erythrocytes, diminishing the zeta potential and accelerating the initial association rate ($K_a$) of IgG antibodies.
  • Shortens mandatory incubation time from 30–60 minutes down to 10 to 15 minutes at $37^\circ\text{C}$.
• Polyethylene Glycol (PEG):
  • A neutral, water-soluble polymer that sterically excludes free water molecules from the incubation mixture, concentrating antibody molecules around the red cell surface.
  • Demonstrates exceptional sensitivity for detecting low-titer, clinically significant warm IgG antibodies while suppressing cold-reactive IgM autoantibodies.
  • Technical Requirement: PEG promotes non-specific cellular aggregation at room temperature. Therefore, tubes incubated with PEG must NEVER be centrifuged or read after the $37^\circ\text{C}$ incubation; they must proceed directly to 3 to 4 warm saline washes before AHG addition.
• Proteolytic Enzymes (Ficin and Papain):
  • Cleave terminal sialic acid residues from Glycophorin A and B, stripping negative surface charges and eliminating steric hindrance.
  • Enhance reactivity of Rh, Kidd, Lewis, and P system antibodies.
  • Destroy antigenic epitopes of the Duffy ($Fy^a, Fy^b$) and MNS ($M, N, S$) blood group systems.

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5. Coombs Control Cells (Check Cells): Mandatory Quality Assurance

                     ┌─────────────────────────────────────────────────────────────────┐
                     │          The Golden Rule of Antiglobulin Quality Control        │
                     └────────────────────────────────┬────────────────────────────────┘
                                                      │
                            ┌─────────────────────────┴─────────────────────────┐
                            ▼                                                   ▼
                 Check Cells AGGLUTINATE (>= 2+)                     Check Cells FAIL TO AGGLUTINATE (0)
  • Test is VALIDATED and ACCURATE.                                  • TEST IS COMPLETELY INVALID!
  • Proves:                                                          • Immediate Actions Required:
    1. Active AHG was added to the tube.                               1. DO NOT REPORT THE RESULT.
    2. AHG was not neutralized by residual serum.                      2. Discard the invalid test.
    3. Washing technique was adequate and complete.                    3. COMPLETELY REPEAT the procedure
                                                                          from the initial wash step.

Composition and The Golden Rule

Coombs Control Cells (commonly termed Check Cells) are commercial Group O Rh-positive erythrocytes that have been lightly sensitized in vitro with human IgG (anti-D).

• The Mandatory Rule: Check cells MUST BE ADDED TO EVERY NEGATIVE TEST TUBE in both Direct and Indirect Antiglobulin testing.
• Three Critical Operational Parameters Validated by Check Cells:
  1. Reagent Addition: Validates that AHG reagent was actually dispensed into the tube (detects technologist omission).
  2. Reagent Potency: Validates that the AHG reagent retains biological potency and was not degraded by heat, contamination, or expiration.
  3. Adequate Cell Washing: Validates that the red blood cells were adequately washed and that the AHG was NOT neutralized by trace residual unbound human globulins.

The Extreme Sensitivity of AHG Neutralization

Antihuman Globulin possesses an extraordinarily high binding affinity for free, uncomplexed human globulins. A minute trace of residual patient serum—as little as $1\text{ }\mu\text{L}$ of serum diluted $1:1000$ in saline—is capable of entirely binding and neutralizing an entire drop of AHG reagent! Once neutralized in solution, the AHG cannot bind cell-bound IgG, causing a false-negative result. Adding check cells unmasks this error: if free globulins neutralized the AHG, no uncomplexed anti-IgG remains to bind the check cells, and they fail to agglutinate.

Protocol for Non-Reactive Check Cells

If check cells fail to agglutinate ($<= 1+$ or $0$ reaction):

1. The test result is COMPLETELY INVALID and cannot be interpreted or released.
2. The technologist MUST REPEAT THE ENTIRE TEST PROCEDURE FROM THE BEGINNING, ensuring: (a) fresh, unadulterated $0.9\%$ saline is used, (b) tubes are decanted completely to a dry button between each of the 3 to 4 washes, (c) AHG is added immediately following the final wash, and (d) centrifugation timing and speed are calibrated.

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6. Technical Troubleshooting: False-Positive and False-Negative Antiglobulin Reactions

                                  ┌──────────────────────────────────────────────┐
                                  │      Antiglobulin Testing Error Sources      │
                                  └──────────────────────┬───────────────────────┘
                                                         │
         ┌───────────────────────────────────────────────┴───────────────────────────────────────────────┐
         ▼                                                                                               ▼
  ┌──────────────────────────────┐                                                ┌──────────────────────────────┐
  │   False-Positive Reactions   │                                                │   False-Negative Reactions   │
  └──────────────┬───────────────┘                                                └──────────────┬───────────────┘
                 │                                                                               │
  • Over-centrifugation                                                           • Inadequate washing (AHG neutralized!)
  • Bacterial contamination of saline/reagents                                    • Failure to add AHG reagent
  • Dirty glassware / colloidal silica                                            • Delayed addition of AHG (elution of IgG)
  • Polyagglutination (T-activation)                                              • Under-centrifugation
  • Reading complement after prolonged incubation                                 • Inactive / degraded / expired AHG
  • Refrigerated clotted red-top sample (in vitro C')                             • Prozone or postzone cell suspensions

Systematic Error Analysis

1. False-Positive Reactions:

   • Inadequate Washing Technique in Clotted Samples: Using refrigerated clotted blood allows in vitro complement uptake, yielding a false-positive polyspecific or anti-C3d DAT. Always collect in EDTA.
   • Over-Centrifugation: Excessive speed or duration packs the red cell button so firmly that gentle shaking fails to disperse unagglutinated cells, mimicking true agglutination.
   • Bacterial Contamination: Contamination of saline reservoirs, tubes, or commercial reagents by bacteria can cause spontaneous red cell clump formation.
   • Colloidal Silica or Chemical Contaminants: Using dirty glassware, detergent-contaminated tubes, or collecting tubes with silicone gel separators can non-specifically aggregate red blood cells.
   • Extreme Cold Agglutinins: If cold-reactive autoantibodies are present and testing is performed below $37^\circ\text{C}$, cold autoagglutination can be misinterpreted as an antiglobulin reaction.

2. False-Negative Reactions:

   • Inadequate Washing: The single most common cause of false-negative AHG testing. Residual plasma globulins neutralize the AHG.
   • Omission of AHG Reagent: Technologist fails to add AHG; unmasked when check cells fail to agglutinate.
   • Delayed Addition of AHG: Allowing washed red cells to sit in saline for extended periods prior to adding AHG promotes spontaneous dissociation and elution of low-affinity bound IgG off the cell membrane into the supernatant, neutralizing the subsequently added AHG.
   • Under-Centrifugation: Inadequate centrifugal force fails to bring sensitized cells into physical contact to form a macroscopic lattice.
   • Improper Cell Suspension Concentration: Preparing a cell suspension that is too dense (>5%) creates a postzone effect (antigen excess), resulting in insufficient AHG molecules to cross-link cells; preparing a suspension that is too light (<2%) hinders macroscopic visualization.

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7. Antiglobulin Testing Procedural and Troubleshooting Reference Table

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8. Elution, Adsorption, Neutralization, Titration, and Enzyme or DTT Treatment

These special procedures (5% of the blood banking blueprint) are used when the routine DAT, IAT, and antibody panel cannot explain a result.

Elution: Removing Antibody From Red Cells

An eluate frees antibody bound to DAT-positive red cells so it can be identified with a panel. The cells are washed first, and the last wash is tested in parallel; it must be nonreactive to prove the eluate reactivity came from cell-bound antibody rather than unbound plasma antibody.

Adsorption: Removing Antibody From Plasma

• Warm autoadsorption: The patient's own red cells, often pretreated with ZZAP (dithiothreitol plus papain) or enzymes to remove bound autoantibody, adsorb the warm autoantibody from the plasma so underlying alloantibodies can be identified. This cannot be used if the patient was transfused within the last 3 months, because donor cells in the circulation could adsorb alloantibodies.
• Allogeneic (differential) adsorption: Phenotyped reagent cells of chosen types (for example R1R1, R2R2, and rr cells, one lacking Kidd antigens) remove the autoantibody while leaving alloantibodies to antigens those cells lack.
• Cold autoadsorption at 4 °C removes cold autoantibodies such as anti-I.

Neutralization: Inhibiting Antibody With Soluble Antigen

A neutralization test needs a dilution control (plasma plus saline), so loss of reactivity from dilution alone is not mistaken for neutralization.

Titration

• Serial twofold dilutions of plasma are tested against selected red cells. The titer is the reciprocal of the highest dilution giving a 1+ reaction. A score sums the graded reactions across all tubes.
• Comparing titers is meaningful only when samples are tested side by side. A difference of at least two dilutions (fourfold) is significant.
• Uses: Monitoring alloimmunized pregnancies (see the HDFN section), separating cold agglutinins of clinical significance, and recognizing high-titer, low-avidity (HTLA) antibodies (for example, anti-Chido and anti-Rodgers), which react weakly at very high dilutions.

Enzyme and Dithiothreitol (DTT) Treatment of Red Cells

• Proteolytic enzymes (ficin, papain) destroy M, N, Fya, Fyb, and (variably) S antigens, and they enhance Rh, Kidd, Lewis, I, and P1 antigens. Comparing untreated and enzyme-treated panels helps separate mixtures of antibodies.
• DTT destroys Kell system antigens and CD38. Treating reagent cells with DTT removes interference from the anti-CD38 drug daratumumab (used for multiple myeloma), which otherwise makes every panel cell react; K-negative units are then supplied.