Immune Hemolytic Anemias (IHAs)
– Premature destruction of RBCs by the immune process mediated by antibody and/or complement.
– Presence and severity of anemia depend on:
▪ The severity of hemolysis.
▪ The ability of BM to compensate for RBC losses.
– Initial confirmation of immune mechanism
▪ Demonstration of attachment of Ab or complement to the patient's RBC.
– Diagnosis of anemia:
▪ Reduced Hb and Hct, increased reticulocytes and/or unbound bilirubin
▪ Reduced haptoglobin
Classification of IHAs
• Based on stimulus for antibody production
– Autoimmune hemolytic anemia.
– Drug-induced hemolytic anemia.
– Alloimmune hemolytic anemia.
• The determination process is important because each type requires specific treatment.
Autoimmune hemolytic anemia (AIHA)
– Shortened RBC survival
– Due to the production of autoantibodies against RBC antigens
– Ab-induced reactions include
▪ Sensitization, agglutination, hemolysis.
– Further classified
▪ Warm-antibody autoimmune HA (WAIHA)
▪ Cold-antibody AIHA (cold agglutinin disease/CAD)
▪ Mixed-type AIHA (both warm-reacting and cold-reacting autoantibodies).
Drug-induced hemolytic anemia
– Drugs bind to the RBC membrane or change it.
– Classification based on the patient's red blood cell and drug response in the vitro test system
▪ Drug-dependent
▪ Drug-independent.
Alloimmune hemolytic anemia
– Antibody (Ab) produced to RBC antigen (Ag) that individual lacks
▪ Do not react with individual's own RBCs
▪ HDFN-mother creates Abs against Ags on fetal RBCs
▪ Transfusion reactions when the recipient makes Abs to Ags on transfused (donor cells).
Characteristics of Agglutinin in Hemolytic Anemia
|
|
Warm-Reacting
Antibodies |
Cold-Reacting
Antibodies |
|
Immunoglobulin
class |
IgG IgA (Usually
with IgG) |
IgM IgG (PCH only) |
|
Optimal
reactivity |
37oC |
<30oC,
usually <10oC |
|
Mechanism of
Hemolysis |
-Extravascular |
-Intravascular,
complement-mediated lysis. -Extravascular,
Attachment of membrane-bound IgG or C3b to macrophages receptors |
|
Specificity |
Usually broad
Specificity anti-Rh |
-Usually
autoanti-I -Occasionally
autoanti-i -PCH: autoanti-P |
Sites and Factors that Affect Hemolysis
Intravascular or extravascular hemolysis
– Depends on:
▪ Class of Ab
▪ Ability to fully activate the complement cascade.
– Extravascular hemolysis
▪ Most common
▪ RBC sensitized with Ab or complement
▪ Sensitized cells phagocytized by macrophages in the spleen or liver.
– Intravascular hemolysis
▪ Complement cascade activated C9 (MAC) leads to RBC lysis.
Immune-mediated extravascular hemolysis. Erythrocytes sensitized with antibody or complement (C3b) attach to macrophages via specific cell receptors for these immune proteins.
Factors affecting the rate of hemolysis in IHAs
|
Factor |
Effect |
|
Class of Immunoglobulin coating the erythrocytes |
For IgG subclasses, the affinity of macrophage receptor and rate of
hemolysis is greatest for IgG3 and IgG1 |
|
Quantity of Ig molecules per erythrocyte |
Immunoglobulins coat a cell with a high density (larger number)of
corresponding antigens more heavily than a cell with low density (fewer)
antigens |
|
The ability of the Ig to activate complement |
IgM and IgG can activate complement; for IgG, the ability to activate
the complement is IgG3>IgG1>IgG2 |
|
The thermal amplitude of the antibody |
Warm-reacting (37oC)antibodies can cause hemolysis but
cold-reacting (0-4 oC)antibodies usually do not. |
|
The activity level of macrophages |
Suppression of Fc receptor decreases the rate of hemolysis |
|
Complement components on the membrane |
Macrophages have receptors for C3b but no effective receptor for C3d |
Mechanisms of Hemolysis
• Based on the presence of IgM, IgG, and/or complement in RBC
• Three types:
– IgG-mediated
– Complement-mediated
– IgM-mediated
IgG-Mediated Hemolysis
• IgG is attached to the RBC membrane Ags through the Fab region.
• Fc receptors
– FcγR-I, -II, -III on splenic macrophages
– Bind to the Fc part of Ab connected to RBC
– Macrophage pits the Ag/Ab complex
•Fc receptors
– Damages RBC membrane
– RBC membrane reseals itself
– The spleen repeatedly passes —continues to lose membrane, forms spherocyte
▪ Phagocytized by splenic macrophages.
• Ab-sensitized RBCs can be entirely engulfed by:
– Macrophages
– PMN (FcγR-I, -III)
• NK cells (FcγR-III)— leads to ADCC (Ab dependent cell-mediated cytotoxicity)
• Spleen
– Lightly opsonized cells more efficiently removed
• Liver
– Removes heavily sensitized cells
• Splenomegaly is common.
Complement-Mediated Hemolysis
Role of complement
– Sensitization
▪ The only part of the complement cascade is activated and deposited on the RBC membrane
– Lysis of RBCs
▪ The entire complement system is activated and deposited on the RBC membrane.
• Initiation of complement activation
– Classic, alternate, lectin mechanisms
• Classic pathway
– Initiated by Ag/Ab reaction
▪ IgM
–Activates complement more efficiently
–Only requires one IgM molecule
▪ IgG (IgG1 and IgG3; occasional IgG2)
–Activation requires two IgG molecules
• Cascade initiated
–C1 sticks to the Fc region of IgG or IgM.
– Activates C4, C2, C3
– Activates the terminal components C5 to C9
▪ Membrane attack complex (MAC)
–Lytic attack to RBC membrane
–Intravascular hemolysis when complement activation C1 to C9 is complete.
• Cascade initiated
– Activations through C3
▪ RBC sensitized with C3b
– Macrophages completely or partially engulf the C3b receptor
▪ C3b on RBC divided by plasma C3b inactivator
▪ C3c dissociates from the membrane.
▪ C3d no receptors on macrophages
–Normal RBC survival
IgM-Mediated Hemolysis
• Macrophages lack receptors for the Fc portion of IgM.
– IgM is an efficient activator of complement
▪ Intravascular
–Complement activation through C9 and RBC hemolyzes.
• Macrophages lack receptors for the Fc portion of IgM.
– IgM is an efficient activator of complement.
▪ Extravascular
–Activation incomplete
–C3b coats RBCs and sensitized cells destroyed extravascularly through CR1 and CR3 receptors on macrophages.
– IgM is able to agglutinate cells in addition to activating complement.
Laboratory ID of Sensitized RBCs
• Two agglutination techniques
– Saline—detects IgM antibodies
– AHG test—detects IgG and/or complement
▪ Direct AHG (DAT)
–Detects RBCs coated in vivo
– Needed to differentiate AIHA from other types of HA
• Two agglutination techniques
– AHG test—detects IgG and/or complement
▪ Indirect AHG (IAT)
–Detects antigens in plasma or serum (in vitro)
– Specify alloimmunization or free autoAbs in the patient's serum.
• Negative DAT in AIHA
– Can result from
▪ Insufficient number of IgG molecules on RBCs
▪ Autoantibodies of IgA or IgM class
▪ Autoantibodies with low affinity for RBCs
• Negative DAT in AIHA
– Newer techniques more sensitive
▪ Enzyme-linked DAT
▪ Gel tests
▪ Flow cytometry
▪ Polybrene tests
• Positive DAT in a normal individual
– Healthy blood donors and hospitalized patients
▪ Positive DAT—No shortened RBC survival.
• Positive DAT in a normal individual
– Possible causes:
▪ Inefficient macrophage removal of sensitized RBCs
▪ Insufficient amount of Ab on the cell surface
▪ Subclass of Ab not recognized by macrophage
• Positive DAT in a normal individual
– Possible causes:
▪ The thermal amplitude of antibody (< 37°C)
▪ + DAT due to the presence of complement on RBCs
▪ Patients with hypergammaglobulinemia or receiving high-dose IV gamma globulin exhibit nonspecific binding.
 – Premature destruction of RBCs by the immune process mediated by antibody and/or complement. – Presen…){kind=link}
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