Laboratory investigations of haemolytic anaemia

Table of contents

Introduction

Hemolytic anemia occurs when red blood cells (RBCs) are destroyed (hemolysis) faster than the bone marrow can produce them. Normally, RBCs have a lifespan of about 120 days, but in hemolytic anemia, they are broken down prematurely. This leads to a shortage of red blood cells, impairs oxygen transport, and causes various symptoms.

Causes of Hemolytic Anemia

Intrinsic Causes (Inherited)
- Membrane Defects:
  - Hereditary Spherocytosis: Caused by a defect in membrane proteins (e.g., spectrin, ankyrin), leading to fragile, spherical RBCs prone to destruction.
  - Hereditary Elliptocytosis: Another inherited membrane disorder with elliptical RBCs that are more prone to hemolysis.
- Enzyme Deficiencies:
  - G6PD Deficiency: RBCs are susceptible to oxidative damage without G6PD enzyme activity, leading to episodic hemolysis when exposed to oxidative stress.
  - Pyruvate Kinase Deficiency: A glycolytic pathway enzyme deficiency causing reduced ATP production in RBCs, leading to hemolysis.
- Hemoglobinopathies:
  - Sickle Cell Anemia: Mutation in the beta-globin gene leads to sickle-shaped RBCs prone to hemolysis.
  - Thalassemia: Genetic defects in globin chain production cause imbalanced globin synthesis and hemolysis.
Extrinsic Causes (Acquired)
- Immune-Mediated Hemolysis:
  - Autoimmune Hemolytic Anemia (AIHA): The body’s immune system produces antibodies against RBCs. It can be warm AIHA (IgG antibodies) or cold agglutinin disease (IgM antibodies).
  - Alloimmune Hemolysis: Due to antibody formation against foreign RBC antigens from transfusions or fetal-maternal blood incompatibility (e.g., Rh incompatibility in hemolytic disease of the newborn).
- Non-Immune Causes:
  - Microangiopathic Hemolytic Anemia (MAHA): RBCs are mechanically damaged as they pass through small blood vessels. Seen in conditions like disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura (TTP), and hemolytic uremic syndrome (HUS).
  - Infections: Malaria, where the parasite infects and destroys RBCs, and Clostridium infections which produce toxins causing hemolysis.
  - Toxins and Chemicals: Snake venom, drugs (such as dapsone), and other chemicals can induce hemolysis.
  - Hypersplenism: An enlarged spleen can lead to increased RBC destruction, as seen in liver disease or other conditions affecting spleen function.

Symptoms

Symptoms of hemolytic anemia may vary depending on the severity and speed of hemolysis but can include:

Fatigue and weakness
Shortness of breath
Pale or jaundiced skin (due to elevated bilirubin from RBC breakdown)
Dark urine (from excreted hemoglobin)
Enlarged spleen (splenomegaly), as the spleen becomes overactive in removing damaged RBCs
Increased heart rate (to compensate for reduced oxygen-carrying capacity)

Classification of Hemolytic Anemia

Intrinsic Hemolytic Anemia (Hereditary):

These are usually inherited conditions that involve defects within the RBCs.

Membrane Defects:
- Hereditary Spherocytosis: Characterized by the presence of spherocytes—smaller, spherical RBCs that are fragile and prone to hemolysis due to defects in membrane proteins such as spectrin or ankyrin. The diagnosis can be confirmed by increased osmotic fragility testing and may be treated with splenectomy.
- Hereditary Elliptocytosis: Caused by defects in cytoskeletal proteins that lead to the production of elliptical-shaped RBCs. It is often less severe than spherocytosis.
Enzyme Deficiencies:
- Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency: An X-linked recessive disorder leading to impaired antioxidant protection in RBCs. Hemolysis occurs during oxidative stress (e.g., infections, certain medications, or fava beans). Diagnosis involves G6PD enzyme assay, often performed during a non-hemolytic phase to avoid false negatives.
- Pyruvate Kinase Deficiency: An autosomal recessive disorder that leads to decreased ATP production in RBCs, causing them to become rigid and hemolyze. Diagnosis is confirmed with enzyme assays.
Hemoglobinopathies:
- Sickle Cell Disease: A genetic disorder caused by a mutation in the beta-globin gene, producing abnormal hemoglobin (HbS). When deoxygenated, HbS polymerizes, causing RBCs to become sickle-shaped and leading to vaso-occlusive crises and hemolysis.
- Thalassemia: A group of inherited disorders characterized by reduced or absent synthesis of one or more globin chains, leading to ineffective erythropoiesis and hemolysis. Diagnosis is made via hemoglobin electrophoresis.

Extrinsic Hemolytic Anemia (Acquired):

These causes are external to the RBCs and can be classified as immune-mediated or non-immune-mediated.

Immune-Mediated Hemolysis:
- Autoimmune Hemolytic Anemia (AIHA): The immune system mistakenly produces antibodies against its RBCs. AIHA can be classified into:
  - Warm AIHA: Usually IgG-mediated and occurs at body temperature, often associated with autoimmune disorders (e.g., lupus).
  - Cold Agglutinin Disease: Usually IgM-mediated and occurs at lower temperatures, causing agglutination and hemolysis in the peripheral circulation.
- Alloimmune Hemolysis: Results from immune responses to foreign RBC antigens, often seen in:
  - Transfusion Reactions: When incompatible blood is transfused.
  - Hemolytic Disease of the Newborn (HDN): Due to maternal antibodies targeting fetal RBCs.
Non-Immune Causes:
- Microangiopathic Hemolytic Anemia (MAHA) is characterized by the destruction of RBCs in small blood vessels, leading to fragmented RBCs (schistocytes). Causes include:
  - Thrombotic Thrombocytopenic Purpura (TTP): A condition with a deficiency in ADAMTS13, leading to excessive platelet aggregation.
  - Hemolytic Uremic Syndrome (HUS): Often associated with infection (e.g., E. coli) leading to acute renal failure and hemolysis.
- Infections: Certain infections can lead to hemolysis, such as:
  - Malaria: The parasite infects and destroys RBCs.
  - Clostridium: Can produce toxins that cause hemolysis.
- Toxins: Various toxins and chemicals can cause hemolysis:
  - Lead Poisoning: Lead interferes with heme synthesis.
  - Drugs: Certain medications (e.g., penicillin, dapsone) can induce hemolysis.
- Hypersplenism: An enlarged spleen sequesters and destroys RBCs excessively, which can occur in liver diseases or hematologic disorders.

Laboratory Investigations for Hemolytic Anemia

A comprehensive laboratory workup is crucial for diagnosing hemolytic anemia, identifying its cause, and guiding treatment. Here’s a detailed exploration of each laboratory investigation:

Complete Blood Count (CBC)

The CBC provides an initial assessment of anemia.

Hemoglobin (Hb): Typically low in hemolytic anemia, indicating decreased RBC mass. Values can vary based on the severity of hemolysis.
Hematocrit (Hct): Also low, reflecting decreased blood volume occupied by RBCs.
Mean Corpuscular Volume (MCV): This may be normal, low (microcytic), or high (macrocytic), depending on the underlying cause and the body’s compensatory mechanisms.
Mean Corpuscular Hemoglobin (MCH) and Mean Corpuscular Hemoglobin Concentration (MCHC): Low in iron deficiency or hemoglobinopathies cases. Normal or elevated MCHC may be seen in hereditary spherocytosis.

Reticulocyte Count

Elevated Reticulocyte Count: Indicates a compensatory increase in RBC production due to hemolysis. A significantly elevated reticulocyte count (greater than 2% or RPI > 2) suggests active hemolysis. In contrast, a low reticulocyte count despite anemia indicates inadequate bone marrow response, potentially pointing to aplastic anemia or ineffective erythropoiesis.

Peripheral Blood Smear

Microscopic Examination: This is essential for identifying characteristic RBC abnormalities.
- Spherocytes: Indicative of hereditary spherocytosis and immune-mediated hemolysis.
- Schistocytes: Fragmented RBCs found in MAHA.
- Target Cells: Often seen in liver disease and hemoglobinopathies.
- Bite Cells: Associated with G6PD deficiency and seen in oxidative stress situations.
- Heinz Bodies: Aggregates of denatured hemoglobin seen in G6PD deficiency and other oxidative hemolytic anemias.

Lactate Dehydrogenase (LDH)

Elevated LDH: LDH is an enzyme released during cell breakdown. High levels indicate hemolysis and can help assess the severity of hemolytic anemia. In cases of significant hemolysis, LDH can be markedly elevated.

Indirect (Unconjugated) Bilirubin

Elevated Indirect Bilirubin: As RBCs are destroyed, hemoglobin is released and metabolized to bilirubin. Elevated levels of indirect bilirubin indicate increased hemolysis and may lead to jaundice if significantly elevated.

Haptoglobin Levels

Decreased Haptoglobin: Haptoglobin binds free hemoglobin in the circulation. In hemolytic anemia, the increased destruction of RBCs leads to a decrease in haptoglobin levels as it becomes depleted. Low haptoglobin is a key indicator of intravascular hemolysis.

Direct Antiglobulin Test (DAT or Coombs Test)

Positive DAT: A positive test indicates that antibodies or complement proteins are bound to RBCs, supporting a diagnosis of immune-mediated hemolysis. This test helps differentiate between immune-mediated (e.g., AIHA) and non-immune causes of hemolysis.
Negative DAT: Suggests that hemolysis is more likely due to intrinsic or non-immune extrinsic factors.

Hemoglobin Electrophoresis

Identification of Hemoglobin Variants: This test separates different types of hemoglobin and is crucial for diagnosing hemoglobinopathies such as sickle cell disease and thalassemia. Abnormal migration patterns on electrophoresis can indicate specific disorders.

Enzyme Assays

G6PD Enzyme Assay: Confirms the diagnosis of G6PD deficiency. It is essential to perform this test during a non-hemolytic period to ensure accurate results.
Pyruvate Kinase Activity: Assay can diagnose pyruvate kinase deficiency, revealing reduced enzyme activity.

Osmotic Fragility Test

Increased Osmotic Fragility: This test assesses the susceptibility of RBCs to hemolysis in hypotonic solutions. Increased fragility indicates hereditary spherocytosis, confirming the diagnosis when clinical and laboratory findings suggest this condition.

Flow Cytometry for Paroxysmal Nocturnal Hemoglobinuria (PNH)

Detection of GPI-Anchor Deficiency: Flow cytometry detects the absence of glycosylphosphatidylinositol (GPI) anchors on blood cells, a hallmark of PNH. This condition leads to complement-mediated hemolysis and can be confirmed by analyzing the surface markers on RBCs.

Bone Marrow Examination

Bone Marrow Aspirate and Biopsy: In certain cases, a bone marrow examination may be performed to evaluate the erythropoietic activity and check for infiltration by malignant cells or to assess for aplastic anemia. Hypercellularity with increased erythroid precursors indicates a compensatory response to hemolysis.

Management and Treatment Considerations

Management of hemolytic anemia depends on the underlying cause:

Autoimmune Hemolytic Anemia: May require corticosteroids, immunosuppressive therapy, or splenectomy.
G6PD Deficiency: Focus on avoiding oxidative stressors.
Sickle Cell Disease: Pain management, hydroxyurea, and blood transfusions during crises.
Thalassemia: Regular blood transfusions and iron chelation therapy.
Microangiopathic Hemolytic Anemia: Treat underlying conditions (e.g., TTP or HUS) with specific therapies like plasma exchange.