8.3: Immune-Mediated Hemolytic Anemias

Hemolytic anemias can be caused by antibodies that can be directed against self (auto-antibodies) or foreign (allo-antibodies) antigens. Antibodies implicated vary in immunoglobulin class and optimal temperature of reactivity.

Table 1. Comparison of Warm and Cold Reacting Antibodies.¹

	Warm	Cold
Immunoglobulin Class	IgG	IgM (Exception: autoanti- P is IgG)
Optimal Temperature	37℃	<30℃ (often at 4℃) Pathological cold agglutinins react closer to body temperature
Mechanism of Hemolysis	Extravascular IgG or C3b attachment to macrophages	Extravascular or Intravascular Complement-mediated
Specificity	anti-Rh (Broad specificity)	Autoanti-I Autoanti-i Autoanti-P

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Warm Auto-Immune Hemolytic Anemia (WAIHA)

Associated Conditions:^2,3

Lymphoproliferative diseases (e.g. Chronic lymphocytic leukemia)

Other autoimmune diseases: systemic lupus erythematosus, rheumatoid arthritis

Ingestion of certain drugs

Some non lymphoid neoplasms

Some inflammatory diseases

Affected age: Usually old age (>40 years old).¹

Antibody Specificity: Panreactive, polyclonal anti-Rh (IgG).³

Pathophysiology:

Often due to a Pan-reacting antibody against the Rh blood group system causing extravascular hemolysis. The antibodies bind to the red blood cells, resulting in their removal by macrophages in the spleen. Incomplete phagocytosis results in the removal of only some of the red blood cell membrane allowing the rest to reform. This reformation changes the red blood cell shape, and it becomes as spherocyte. Red blood cells can also be coated with complement along with IgG antibodies as another mechanism of opsonization and removal from circulation.³

Laboratory Findings for WAIHA:^1-3

CBC: WBC: Normal to increased PLT: Normal to increased Hb: Decreased RETIC: Increased	PBS: Normochromic Normocytic Spherocytes Polychromasia +/- nRBCs	BM: Erythroid hyperplasia Erythrophagocytosis by macrophages
Other Tests: DAT: Positive (IgG & C3b) Osmotic Fragility: Increased Antibody Screen: Positive with all cells Autocontrol: Positive Bilirubin: Increased Haptoglobin: Decreased LD: Increased Hemoglobinuria: Positive Hemosiderinuria: Positive

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Cold Agglutinin Disease (CAD)

Associated Conditions:²

Primarily idiopathic

B-cell lymphoproliferative neoplasms

Mycoplasma pneumoniae (anti-I)

Infectious mononucleosis (anti-i)

Affected age: > 50 years old.²

Antibody Specificity: Autoanti-I and autoanti-i.²

Autoantibody is an IgM antibody that reacts optimally below body temperature, usually around 4℃². Pathological cold agglutinins will react closer to body temperature (around 30℃).

Can be polyclonal (i.e. infections) or monoclonal (Monoclonal is more pathogenic).¹

Pathophysiology:

Under cold temperatures (circulation in the extremities), the autoantibodies bind to the red blood cells causing them to agglutinate. As the autoantibodies are strong complement activators, complement (C3b) also binds the red blood cells.^1,2,4

When the cells return to body temperature (central circulation), the autoantibody unbinds allowing cells to separate and leaves C3b behind remaining on the red blood cell. This leads to complement-mediated hemolysis by macrophages in the liver (extravascular hemolysis).^1,2,4

Can cause acrocyanosis and hemolysis is self-limiting.⁵

Laboratory Findings for CAD:^1,2,4

CBC: RBC: Decreased WBC: Normal PLT: Normal RETIC: Increased MCV, MCH, MCHC: Falsely increased (due to cold-agglutination)	PBS: Agglutination at room temperature (Not present if sample is heated to 37℃) Spherocytes Normohromic Normocytic +/- nRBCs	BM: Erythroid hyperplasia
Other Tests: DAT: Positive for C3b, but negative for IgG or IgM IAT: Reactive at < 25℃ Screen Cells: Positive Autocontrol: Positive Cord cells: Negative (If autoanti-I, otherwise positive if autoanti-i) Bilirubin: Increased Haptoglobin: Decreased Hemoglobinemia Hemoglobinuria (Acute) Hemosiderinuria (Chronic)

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Paroxysmal Cold Hemoglobinuria (PCH)

Associated Conditions:^2,3

Can develop following viral infections or upper respiratory infections

Affected age: Primarily in children. ²

Antibody Specificity: autoanti-P (IgG, polyclonal, binds optimally at 4-20℃, reactive at 37℃).^1,4,5

Pathophysiology:^1,4,5

Attachment of autoanti-P to cells do not cause the cells to agglutinate but does result in an intravascular, complement-mediated hemolysis.

Autoanti-P is a biphasic antibody meaning that it activates only partial complement at cold temperatures (<37℃) and full complement at warmer temperatures (37℃) leading to hemolysis.

Laboratory Findings for PCH:^2-4

CBC: WBC: Normal Hb: Decreased RETIC: Increased

PBS: Spherocytes Polychromasia +/- nRBCs Some Schistocytes

Other Tests: DAT: Positive for C3d only LD: Increased Haptoglobin: Decreased Hemoglobinemia Hemoglobinuria Donath-Landsteiner Test is Positive: Control incubated at 37℃: Hemolysis absent Patient sample incubated at 37℃ only: Hemolysis absent Patient sample incubated at 4℃ and 37℃: Hemolysis present

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Table 2. Comparative Table of Warm and Cold Immune-Related Hemolytic Anemias

	WAIHA	CAD	PCH
Age (years old)	>40	>50	Children (After viral infection)
Antibody Class	IgG	IgM	IgG
Antibody Specificity	Anti-Rh (most-often)	Anti-I, Anti-i	Anti-P
Optimal Binding Temperature	37℃	4℃	<20℃
DAT Reactivity	IgG	IgG, C3d	C3d
Donath-Landsteiner Test	N/A	Negative	Positive
Type of Hemolysis	Extravascular	Extravascular	Intravascular

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Drug-Induced Immune Hemolytic Anemia

Immune hemolytic anemias can also be induced when certain drugs are administered into the body. There are four mechanisms in which they are able to do this:

1. Autoantibody Induction^1,4,6

Most commonly caused by methyldopa.

Mechanism mimics that found in warm autoimmune hemolytic anemia. The drug induces the production of warm-reactive antibodies against the red blood cell membrane (self-antigens). Antibodies bind at 37℃ and affected red cells are removed by the spleen through extravascular hemolysis.

2. Drug Adsorption (Hapten)^1,4,6

Most commonly caused by penicillins.

The drug is non-specifically adsorbed onto the red blood cells and antibodies are produced against the drug itself. As red blood cell pass through the spleen, they are removed by macrophages.

3. Immune Complex Formation (Innocent Bystander)^1,4,6

Most commonly caused by quinidine.

An IgG or IgM antibody is produced against the drug when it loosely binds to the red blood cells (antibody-drug immune complex). The immune complex induces the activation of complement, leading to the formation of membrane attack complexes and intravascular hemolysis.

4. Membrane Modification^1,6

Most commonly caused by cephalosporins.

Drug modifies the red blood cell membrane causing it to become “sticky”. This results in red blood cells becoming coated with many plasma proteins. No hemolysis occurs, but DAT testing will be positive.

Table 3. Comparison of Mechanisms Leading to Drug-Related Immune Hemolytic Anemia

	Autoantibody Induction	Drug Adsorption	Immune Complex	Membrane Modification
Drug Example	Methyldopa	Penicillins	Quinidine	Cephalosporins
Antibody Class	IgG	IgG	IgG or IgM	N/A, due to plasma proteins
DAT	IgG Positive C3 Negative	IgG Positive C3 Negative	C3 Positive IgG Variable	Positive, due to plasma proteins
Eluate	Positive	Usually Negative	Usually Negative	Usually Negative
Type of Hemolysis	Extravascular	Extravascular	Intravascular	No hemlysis

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Alloimmune Hemolytic Anemias

Hemolytic anemias can also occur with there is sensitization of red blood cells due previous exposure to another individual’s red blood cells.

1. Hemolytic Transfusion Reactions⁷

Hemolytic transfusion reactions occur when there is an incompatibility between the patient’s blood (contain alloantibodies) and the transfused cells. Alloantibodies present in the patient’s blood binds the antigens on the transfused cells and this results in hemolysis. Transfusion reactions are classified as being acute or delayed.

Table 4. Comparison of Acute and Delayed Transfusion Reactions.

	Acute	Delayed
Time	Immediate; minutes to hours	Days to weeks
Related Blood Groups	ABO	Other non-ABO blood groups
Symptoms	Fever, chills, back pain, pain at infusion site, difficulty breathing, hypotension, urticaria, tachycardia	Usually show no clinical symptoms but may develop a fever
Type of Hemolysis	Intravascular	Extravascular
DAT	Negative (if all transfused cells have all been hemolyzed)	Positive for IgG and/or C3d (Can be negative depending on time of sample collection)
Other Laboratory Findings	Hemoglobinemia Hb: Decreased Bilirubin: Increased after a few days Haptoglobin: Decreased	Hemoglobinuria Hb: Variable Bilirubin: Increased Eluate is positive for offending antibody.

2. Hemolytic Disease of the Fetus and Newborn (HDFN)

Hemolysis that occurs in the fetus or newborn due to incompatibility between the mother’s alloantibodies and the fetus’s/newborn’s blood groups.

Mother’s immune system can become sensitized and produce alloantibodies against the blood group antigens that she lacks during a previous pregnancy or transfusion. If the fetus/newborn contains the blood group antigens that the mother has alloantibodies against, HDFN can develop. During pregnancy, alloantibodies are able to pass through the placenta and bind to the red blood cells in the fetus/newborn resulting in hemolysis of the fetal red blood cells.^4,6

Newborns appear jaundiced and have high levels of bilirubin at birth.^4,7 The peripheral blood smear will show increased spherocytes, polychromasia, and increased nucleated red blood cells (normoblastemia).

Alloantibodies can be produced against Rh, ABO, and other blood groups.⁷

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References:

1. Smith LA. Hemolytic anemia: immune anemias. In: Clinical laboratory hematology. 3rd ed. New Jersey: Pearson; 2015. p. 348-71.

2. Barcellini W, Fattizzo B. Clinical Applications of Hemolytic Markers in the Differential Diagnosis and Management of Hemolytic Anemia. Dis Markers [Internet]. 2015 Dec 27 [cited 2018 Jun 26];2015:635670. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4706896/

3. Berentsen S. Role of complement in autoimmune hemolytic anemia. Transfus Med Hemotherapy [Internet]. 2015 Sep 7 [cited 2018 Jun 27];42(5):303–10. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4678321/

4. Przekop KA. Extrinsic defects leading to increased erythrocyte destruction – immune causes. In: Rodak’s hematology clinical applications and principles. 5th ed. St. Louis, Missouri: Saunders; 2015. p. 411-25.

5. Packman CH. The clinical pictures of autoimmune hemolytic anemia. Transfus Med Hemotherapy [Internet]. 2015 Sep 11 [cited 2018 Jun 26];42(5):317–24. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4678314/

6. Harmening DM, Yang D, Zeringer H. Hemolytic anemias: extracorpuscular defects. 5th ed. Philadelphia: F.A. Davis Company; 2009. p. 250-79).

7. Landis-Piwowar K, Landis J, Keila P. The complete blood count and peripheral blood smear evaluation. In: Clinical laboratory hematology. 3rd ed. New Jersey: Pearson; 2015. p. 154-77.