Hemolytic Anemias

Diagnosis

Indications for Testing

• Deformed and fragmented erythrocytes on peripheral smear; suspicion of hemolysis based on clinical presentation
  • Increased reticulocytosis, lactate dehydrogenase, and bilirubin
  • Decreased haptoglobin

Laboratory Testing

• CBC with peripheral smear – initial screening
  • Platelet count
    • Decreased – consider disseminated intravascular coagulation, Hemolysis, Elevated Liver enzyme levels, Low Platelet count syndrome (HELLP), Thrombotic thrombocytopenia purpura, hemolytic uremic syndrome
      • Order D-dimer, ADAMTS activity
    • Normal – consider vasculitis, mechanical factors
  • Cells noted may help diagnosis
    • Spherocytes – hereditary spherocytosis or elliptocytosis, immune-mediated hemolytic anemias
      • Consider testing for osmotic fragility (usually positive)
        • If considered membrane defect – molecular analysis
        • If considered acquired – direct Coombs
    • Schistocytes/fragmented cells – suggests microangiopathic RBC destruction
    • Polychromasia without morphologic abnormality
      • Consider one or more of the following tests: pyruvate kinase, hexokinase, glucose phosphate isomerase
    • Sickle cells – consider hemoglobin evaluation by high-performance liquid chromatography (HPLC)
      • Abnormal red cells such as sickle cells, target cells may indicate hemoglobinopathy
    • Stomatocytes – hereditary stomatocytosis likely
    • Basophilic stippling levels
      • If considered acquired – lead levels testing
      • If considered nonacquired – 5’ nucleotidase testing
    • Heinz body stain positive – suggests hereditary hemoglobinopathies, glucose-6 phosphate dehydrogenase (G6PD) deficiency
      • Consider G6PD testing or isopropanol heat stability of the signals
      • If G6PD deficiency has been ruled out, Heinz bodies may implicate a toxin or drug
    • Agglutination
      • Consider testing for direct Coombs (+) – cold agglutinin disease (C3d+ only); warm autoimmune hemolytic anemia (IgG+/C3d-)
    • Polychromasia only with possible lowered pH
      • Consider paroxysmal nocturnal hemoglobinuria testing
    • Unusual red cell inclusions
      • If infection suspected – consider evaluation for Babesia, malaria, Bartonella spp
• Reticulocyte count – usually elevated; does not give specific diagnosis

Differential Diagnosis

• See classification in Clinical Background

Clinical Background

Hemolytic anemias result from premature destruction of red blood cells (RBCs). For information on types of hemolytic anemias, refer to the following topics

• Hemoglobinopathies
• Unstable hemoglobinopathies
• Thalassemias

Epidemiology

• Prevalence
  • Prevalence depends on etiology of hemolysis
    • Common – autoimmune hemolytic anemia, glucose-6 phosphate dehydrogenase (G6PD) deficiency, pyruvate kinase (PK) deficiency, hereditary spherocytosis (HS)
    • Rare – paroxysmal nocturnal hemoglobinuria (PNH)
• Sex – M:F, equal
• Ethnicity – higher prevalence of G6PD deficiency in persons of African, Kurdish or Sephardic Jewish, Arab, Mediterranean, Southeast Asian, and Asian-Indo/Pakistani descent; sickle cell disease most often found in Africans

Classification

• Molecular defect hemoglobinopathies
  • Sickle cell disease and other qualitative hemoglobin disorders
  • α-thalassemia, β-thalassemia
• Abnormality in RBC membrane structure or RBC enzymatic defects  
  • G6PD deficiency
  • PK deficiency
  • HS, stomatocytosis  
  • PNH
• Environmental factors (physical disruption) or antibodies
  • Autoimmune (acquired) cold agglutinin
  • Drug-induced
  • Warm antibody-induced
  • Lymphoproliferative disorder or disseminated malignancy
    • Chronic lymphocytic leukemia
  • Mechanical valves
  • Microangiopathic red cell destruction
    • Thrombotic thrombocytopenic purpura (TTP) and hemolytic uremia syndrome (thrombotic microangiopathies)
    • Disseminated intravascular coagulation (DIC)
    • HELLP syndrome (Hemolysis, Elevated Liver enzyme levels, Low Platelet count)
• Infectious  
  • Cytomegalovirus
  • Epstein-Barr virus
  • Hepatitis C virus
  • HIV
  • Leptospira spp
  • Plasmodium spp
  • Mycoplasma pneumoniae
  • Toxins

Pathophysiology

• Processes cause breaking up (hemolysis) of RBCs into various sizes and shapes
• Manifested by elevated reticulocyte counts, low or absent haptoglobin levels, and variable presence of hemosiderin in urine
• Peripheral blood smear demonstrates evidence of RBC fragmentation, including spherocytes, target cells, and Howell-Jolly bodies

Clinical Presentation of Specific Hemolytic Disorders

Cell Membrane disorders (mechanical weakness or fragility of erythrocyte membrane skeleton)

Hereditary spherocytosis Prevalence – 1/2,000 in Caucasians Inheritance – variable May involve autosomal dominant mutations in ankyrin and spectrum – 75% of cases Autosomal recessive or de novo mutations – 25% of cases Clinical presentation Range of severity from asymptomatic to severe disease Anemia, splenomegaly, jaundice, gall stones (particularly in pediatric cases) Reticulocytosis (after prior anemia) Hereditary elliptocytosis Incidence – 1-2/10,000 in Caucasians Inheritance – autosomal dominant Involves spectrin and some band mutations Clinical presentation Usually no significant hemolysis; may be asymptomatic Hereditary stomatocytosis Incidence – 1/50,000 in Caucasians Inheritance – autosomal dominant Clinical presentation – asymptomatic to moderately severe disease

RBC enzyme defects

G6PD deficiency Incidence 400 million worldwide (most common enzyme deficiency worldwide) Ethnicity African Kurdish Jewish Sephardic Jewish Arab Mediterranean Southeast Asian Asian-Indo/Pakistani 0.1% in northern Europeans Inheritance – X-linked recessive Rare mutations cause severe G6PD deficiency (<10% normal activity), resulting in chronic nonspherocytic hemolytic anemia in the absence of oxidative stressors Most affected individuals have moderate G6PD deficiency (~10% normal activity) – associated with acute hemolytic anemia in response to oxidative stress >99% percent of G6PD deficiency among individuals of African descent is caused by the G6PD A- allele Female mutation carriers may have hemolytic episodes even if G6PD enzyme studies are normal because deficient as well as non-deficient red cells coexist in various proportions Several hundred G6PD gene variants have been described Some are benign; others result in intermittent or chronic hemolytic anemia Clinical presentation Most patients are asymptomatic Usually only symptomatic if residual enzyme activity is ≤10% Patients may present with fatigue, back pain, anemia, and jaundice as an indicator of hemolysis Hemolysis occurs when patient is exposed to environmental stressors Viral and bacterial infections (most common) Many oxidative stressors secondary to drug exposure, most commonly sulfa, nitrofurantoin, antimalarials, toluidine blue, methylene blue Pyruvate kinase deficiency Incidence 1/20,000 in Caucasians Most common cause of congenital non-spherocytic hemolytic anemia Ethnicity – more common in the Mediterranean population Inheritance – autosomal recessive Carriers are recognized by erythrocytes – PK activity ~50% of normal >180 different mutations Clinical presentation Degree of hemolysis varies from mild to life-threatening neonatal anemia

Acquired hemolytic anemias – immune-mediated

Incidence – 1-3/100,000 annually Age – 30s-40s Sex – M<F Common types Warm antibody disease and cold reactive antibody disease Usually caused by IgG or IgM antibodies reacting to RBCs Clinical presentation Warm antibody – generally directed broadly against Rh antigens Symptoms usually a result of anemia Slow, insidious onset May have jaundice Cold agglutinin hemolysis 90% of patients have monoclonal B-cell lymphoproliferative bone marrow disorders  Episodic acute hemolysis with hemoglobinuria Acrocyanosis in response to cold and vasoocclusive phenomena of the fingers, toes, ears, and nose present in some patients Drugs – immune-mediated Severity depends on the drug Varies from mild to severe hemolysis Most common drugs implicated – cephalosporins, NSAIDs

Paroxysmal cold hemoglobinuria

Incidence – rare Age – 5 years (median) Caused by a biphasic antibody (Donath-Landsteiner antibody) Clinical presentation Pallor, hemoglobinuria, and mild jaundice Patients usually have a history of recent viral infection

Indications for Laboratory Testing

• Tests generally appear in the order most useful for common clinical situations
• Click on number for test-specific information in the ARUP Laboratory Test Directory

Click the plus sign to expand the table of additional tests.

Test Name and Number	Comments
Haptoglobin 0050280 Method: Quantitative Immunoturbidimetry	Use to assess presence of intravascular hemolysis
Hemoglobin, Plasma 0020058 Method: Quantitative Spectrophotometry	Identify increased concentration, which is indicative of acute intravascular destruction of erythrocytes Not of clinical value in the diagnosis of chronic hemolytic disorders
Hematocrit 0040080 Method: Automated Cell Count	Assess presence of anemia
Hemosiderin, Urine 0020222 Method: Semi-Quantitative Microscopy	Order for suspected intravascular hemolysis within the past week
Donath Landsteiner 0013039 Method: Hemolysis	Determine if Donath-Landsteiner antibodies are present  Diagnose paroxysmal cold hemoglobinuria
Blood Group (ABO & 8 Minor Antigens) Genotyping by Microarray 2004739 Method: Polymerase Chain Reaction/ASH Microarray
Blood Group (9 Minor Antigens) Genotyping by Bead Array 2004743 Method: Polymerase Chain Reaction/Bead Array
Blood Group (5 Minor Antigens) Genotyping by Bead Array 2004741 Method: Polymerase Chain Reaction/Bead Array

Diagnostic Algorithm

  Hemolytic Anemias Testing Algorithm