23: Immune Hemolytic Anemia: IgM & IgG

• Immune hemolytic anemia is when the body's immune system attacks and destroys its own red blood cells.

Types of Antibodies Involved

• Warm antibodies are active at body temperature (37°C) and are usually IgG.
• Cold antibodies are most active at lower temperatures and are usually IgM.

IgM-Mediated Hemolysis

• IgM antibodies efficiently activate the classical complement pathway, leading to intravascular hemolysis.
• After IgM binds to red blood cells, C1q to C3b binds to IgM, resulting in the formation of the membrane attack complex (MAC).
• The MAC creates pores in the red blood cell membrane, causing cell lysis.

IgG-Mediated Hemolysis

• IgG antibodies are less efficient at activating the complement pathway.
• IgG-mediated hemolysis primarily occurs extravascularly, mainly in the spleen.
• Macrophages in the spleen recognize red blood cells coated with IgG via Fc receptors, leading to phagocytosis and destruction of the red blood cells.

Typical Laboratory Findings in Immune Hemolytic Anemia

• Increased reticulocyte count occurs as the bone marrow tries to compensate for red blood cell destruction.
• Elevated indirect bilirubin is a breakdown product of heme.
• Increased lactate dehydrogenase (LDH) is released from damaged red blood cells.
• Decreased haptoglobin occurs because haptoglobin binds free hemoglobin, so levels decrease when red blood cells are destroyed.
• Anemia results in reduced red blood cell count and hemoglobin levels.

Direct Antiglobulin Test (DAT)

• The DAT is crucial for diagnosing immune hemolytic anemia.
• It detects antibodies or complement proteins attached to the surface of red blood cells.
• A positive DAT indicates that the red blood cells are coated with antibodies (IgG) or complement components (C3d).
• The DAT helps confirm that the hemolysis is immune-mediated.

Warm Autoimmune Hemolytic Anemia (WAIHA)

• Immunoglobulin Class: IgG (usually)
• Optimal Temperature: 37°C
• DAT Proteins Detected: IgG and/or C3d
• Complement Activation: Yes (sometimes)
• Type and Site of Hemolysis: Extravascular (spleen)
• Autoantibody Specificity: Rh antigens (usually)

Cold Agglutinin Disease (CAD)

• Immunoglobulin Class: IgM
• Optimal Temperature: 4°C
• DAT Proteins Detected: C3d
• Complement Activation: Yes
• Type and Site of Hemolysis: Intravascular & Extravascular
• Autoantibody Specificity: I/i antigens

Paroxysmal Cold Hemoglobinuria (PCH)

• Immunoglobulin Class: IgG (Donath-Landsteiner)
• Optimal Temperature: 4°C
• DAT Proteins Detected: C3d
• Complement Activation: Yes
• Type and Site of Hemolysis: Intravascular
• Autoantibody Specificity: P antigen
• Biphasic; binds P Ag on RBCs at cold temps with partial complement activation, and hemolysis occurs when temp returns to 37°C

Mixed-Type Autoimmune Hemolytic Anemia

• Immunoglobulin Class: IgG, IgM
• Optimal Temperature: 4° to 37° C
• DAT Proteins Detected: IgG and/or C3d
• Complement Activation: Yes (variable)
• Type and Site of Hemolysis: Variable
• Autoantibody Specificity: Drug-related or red cell antigens

Pathophysiology of Autoimmune Hemolytic Anemia

• AIHA occurs when autoantibodies (often IgG or IgM) or complement proteins target red blood cells (RBCs), leading to their destruction.
• The DAT detects these antibodies or complement bound to the RBCs, confirming the immune-mediated hemolysis.

Laboratory Findings in Autoimmune Hemolytic Anemia

• Hemoglobin is decreased due to increased RBC destruction.
• Reticulocyte Count is increased because the bone marrow compensates by producing more immature RBCs.
• MCV, MCHC are Within Normal Limits: RBC size and hemoglobin concentration remain normal.
• RDW is increased due to greater variability in RBC size due to new and destroyed cells.
• Serum Bilirubin is increased because unconjugated bilirubin rises from hemoglobin breakdown.
• LDH: increased because it is released from lysed RBCs during hemolysis.
• Haptoglobin is decreased as it binds free hemoglobin from lysed RBCs.
• Blood Film shows Spherocytes are predominant due to antibody-mediated membrane loss, with few schistocytes, which are more indicative of mechanical hemolysis.

DAT and Immune Cause Identification

• A positive DAT confirms the presence of antibodies or complement on RBCs, directly linking the findings to immune mechanisms, distinguishing AIHA from non-immune causes of hemolysis.
• The Indirect Antiglobulin Test (IAT) may also assist in identifying circulating autoantibodies.

Acute Hemolytic Transfusion Reactions

• Occur minutes to hours after transfusion
• Most commonly due to transfusion with ABO-incompatible donor cells

Delayed Hemolytic Transfusion Reactions

• Occur days to weeks after transfusion
• Due to second exposure to antigen and a subsequent increase in Ab titer
• Happens after a second transfusion or second pregnancy
• Most often Duffy and Kidd antigens are involved

Cause of Hemolytic Disease of the Fetus and Newborn (HDFN)

• Maternal IgG alloantibodies cross the placenta and destroy fetal RBCs.
  • Rh: Rh-negative mother with anti-D antibodies attacks D-positive fetal RBCs.
  • ABO: Type O mother’s anti-A/B antibodies affect Type A/B fetus.

Pathophysiology of Hemolytic Disease of the Fetus and Newborn (HDFN)

• Extravascular hemolysis occurs as antibody-coated RBCs are destroyed in the spleen/liver.

Laboratory Findings in Hemolytic Disease of the Fetus and Newborn (HDFN)

• Positive DAT confirms antibody-coated RBCs.
• Decreased Hemoglobin, increased reticulocytes and bilirubin, and spherocytes in the blood film.
• Treatment for severe cases require intrauterine transfusion.
• Prevention: RhoGAM prevents maternal anti-D antibody formation.

Drug Adsorption (Hapten Mechanism)

• The drug binds strongly to the red blood cell (RBC) membrane.
• Antibodies (usually IgG) bind to the drug on the RBC surface.
• This leads to RBC destruction, primarily extravascular hemolysis.

Immune Complex Formation

• The drug binds to an antibody in the plasma, forming an immune complex.
• These complexes deposit on the RBC membrane, activating complement.
• Complement activation leads to intravascular hemolysis.

Membrane Modification

• The drug modifies the RBC membrane, causing plasma proteins (including antibodies) to bind.
• This can lead to a positive DAT and RBC destruction.
• The mechanism isn't fully understood, but it can result in both intravascular and extravascular hemolysis.

Drug-Dependent Antibodies Pathophysiology

• Antibodies react with the drug only when it is bound to the red blood cell membrane or when the drug forms a complex with the antibody in the plasma.
• Mechanisms include drug adsorption (hapten), immune complex formation, and membrane modification.
• Hemolysis ceases when the drug is discontinued.

Drug-Dependent Antibodies Lab Findings

• Positive DAT, but only in the presence of the drug.
• Antibody testing is positive only when the drug is present in the testing system.

Drug-Independent Antibodies Pathophysiology

• The drug induces the production of an autoantibody that reacts with red blood cell antigens even after the drug is discontinued.
• The mechanism is similar to warm autoimmune hemolytic anemia (WAIHA).
• Hemolysis can persist for a period even after drug cessation.

Drug-Independent Antibodies Lab Findings

• Positive DAT, even after the drug is discontinued.
• Antibody testing reveals an autoantibody that reacts with red blood cells in the absence of the drug.
• The autoantibody often has specificity against Rh antigens.

Complete Blood Count (CBC) Use

• Hemoglobin/Hematocrit: Decreased in hemolytic anemia to assess the severity of the anemia.
• Reticulocyte Count: Elevated, indicating the bone marrow's response to the anemia.
• MCV (Mean Corpuscular Volume): Can be increased due to reticulocytosis (reticulocytes are larger).
• MCHC (Mean Corpuscular Hemoglobin Concentration): Can be increased in some cases, particularly with spherocytes.

Peripheral Blood Film Examination Clues

• Spherocytes: Small, round RBCs without central pallor; characteristic of antibody-mediated hemolysis (especially warm AIHA).
• Schistocytes: RBC fragments; suggest microangiopathic hemolytic anemia (MAHA), but can be seen in some cases of severe hemolysis.
• Agglutination: Clumping of RBCs; strongly suggests cold agglutinin disease.
• Polychromasia: Bluish-tinged RBCs, indicating reticulocytes.

Biochemical Tests Findings

• Bilirubin (Indirect/Unconjugated): Elevated due to heme breakdown.
• LDH (Lactate Dehydrogenase): Elevated due to RBC lysis.
• Haptoglobin: Decreased or absent, as it binds free hemoglobin.
• Urine Hemoglobin: Present in intravascular hemolysis.
• Serum Free Hemoglobin: Elevated in intravascular hemolysis.

Direct Antiglobulin Test (DAT) Results

• Positive DAT: Confirms immune-mediated hemolysis.
  • IgG: Suggests warm AIHA or drug-induced hemolysis.
  • C3d: Suggests cold agglutinin disease, PCH, or sometimes warm AIHA or drug-induced hemolysis.
  • IgG + C3d: Can be seen in warm AIHA or drug-induced hemolysis.
• Negative DAT: Hemolysis is likely not immune-mediated (consider other causes like mechanical hemolysis, enzyme deficiencies, etc.).
  • A weakly positive or negative DAT can occur in some cases of immune hemolysis.

Indirect Antiglobulin Test (IAT) / Antibody Screen Use

• Identifies antibodies in the patient's serum.
• Helps determine antibody specificity.
• Important for identifying alloantibodies in the case of hemolytic transfusion reactions.
• In cold agglutinin disease, the IAT will detect cold agglutinins.

Warm Autoimmune Hemolytic Anemia (WAIHA) Indicators

• History: Underlying autoimmune disorder possible.
• Blood Film: Spherocytes.
• DAT: Positive for IgG and/or C3d.
• IAT: May detect warm-reacting autoantibodies.

Cold Agglutinin Disease (CAD) Indicators

• History: Cold exposure, recent Mycoplasma pneumoniae infection.
• Blood Film: RBC agglutination.
• DAT: Positive for C3d only.
• IAT: Detects cold agglutinins (usually IgM).

Paroxysmal Cold Hemoglobinuria (PCH) Indicators

• History: Recent viral infection.
• DAT: Positive for C3d only.
• Donath-Landsteiner antibody- P test is positive.

Drug-Induced Immune Hemolytic Anemia Indicators

• History: Medication history is key!
• DAT: Positive (IgG, C3d, or both).
• Antibody testing: May be positive only in the presence of the drug (drug-dependent) or may reveal a drug-independent autoantibody.

Hemolytic Transfusion Reaction Indicators

• History: Recent transfusion.
• DAT: Positive (IgG and/or C3d).
• IAT: Will identify the alloantibody causing the reaction.