Hemolytic Anemias

Indications for Testing

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

Laboratory Testing

CBC with peripheral smear – initial screening
- Platelet count
  - Decreased – consider DIC, HELLP, TTP, HUS
    - Order D-dimer, ADAMTS activity
  - Normal – consider vasculitis, mechanical factors
- Cells noted may help diagnosis
  - Spherocytes – hereditary spherocytosis, elliptocytosis, immune-mediated hemolytic anemias
    - Consider testing for osmotic fragility
      - (+) – membrane defect
      - (+) acquired – direct Coombs
  - Schistocytes/fragmented cells – microangiopathic
  - Polychromasia without morphologic abnormality
    - Consider testing for one or more of the following: pyruvate kinase, hexokinase, glucose phosphate isomerase
  - Sickle cells – consider testing for HPLC
    - Abnormal red cells such as sickle cells, target cells may indicate hemoglobinopathy
  - Stomatocytes – hereditary stomatocytosis likely
  - Basophilic stippling levels
    - Acquired – lead levels
    - Non-acquired – 5’ nucleotidase test
  - Heinz body stain positive – suggests hereditary hemoglobinopathies, G6PD deficiency
    - Consider G6PD testing or isopropanol heat stability of the signals
  - Agglutination
    - Consider testing for direct Coombs (+) – cold agglutinin disease (C3d+ only); warm autoimmune hemolytic anemia (IgG+/C3d-)
  - Polychromasia only with possible lowered pH
    - Consider PNH testing
  - Unusual red cell inducers
    - If infection suspected – consider evaluation for Babesia, malaria
Reticulocyte count – usually elevated; does not give specific diagnosis

Differential Diagnosis

See classification in 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

Epidemiology

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

Classification

Molecular defect hemoglobinopathies
- Sickle cell anemia and other qualitative hemoglobin disorders
- α-thalassemia, β-thalassemia
Abnormality in RBC membrane structure or RBC enzymatic defects
- G6PD
- PK deficiency
- HS, stomatocytosis
- Hereditary 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

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 – autosomal dominant
    - May involve 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 – frequent in persons of African descent
      - 50% in Kurdish Jewish males
      - 0.1% in northern Europeans
  - Inheritance
    - X-linked recessive
    - ~350 different mutations
    - Mutation protects against malaria
  - Clinical presentation
    - Most patients are asymptomatic
    - 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
  - Inheritance
    - Autosomal recessive
    - More common in the Mediterranean population
    - 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 and jaundice, necessitating exchange transfusion
Acquired hemolytic anemias – immune-mediated
- Incidence – 1-3/100,000 annually
- Age – more common in women during 4th and 5th decades
- 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
- Median age – 5 years
- Caused by a biphasic antibody (Donath-Landsteiner antibody)
- Clinical presentation
  - Pallor, hemoglobinuria and mild jaundice
  - Patients usually have a history of recent viral infection

Tests

Tests generally appear in the order most useful for common clinical situations

Test name: CBC with Platelet Count & Automated Differential

ARUP #: 0040003

Methodology: Automated Cell Count with Flow Cell Differential

Use:

Identify presence of hemolysis and anemia

Cells noted on peripheral smear may help diagnosis

Test name: Reticulocytes, Percent & Number

ARUP #: 0040022

Methodology: Flow Cytometry

Use:

Identify increased RBC production (suggestive of increased RBC loss)

Test name: Osmotic Fragility, Erythrocyte

ARUP #: 2002257

Methodology: Spectrophotometry

Use:

Assess presence or absence of spherocytes (which have very little redundant membrane) and roughly gauge quantity in RBC population

Recommend testing during a state of prolonged homeostasis with stable hematocrit

Use in conjunction with Wright stain

Diagnose hereditary spherocytosis

Limitations:

For patients with acute hemolysis, a normal test result cannot exclude an abnormality since osmotically labile cells may be hemolyzed and not present

Does not distinguish between spherocytes in hereditary spherocytosis and acquired autoimmune hemolytic anemia

Test name: Heinz Body Stain

ARUP #: 0049090

Methodology: Supravital Stain

Use:

Use as a nonspecific screen for inherited disorders in conjunction with clinical information

Detect unstable hemoglobins or inherited defects in erythrocyte oxidative pathways (eg, G6PD deficiency)

If inherited disorder (eg, G6PD deficiency) has been ruled out, Heinz bodies may implicate a toxin or drug

Limitations:

Test results are unreliable in infants <6 months

Test name: Glucose-6-Phosphate Dehydrogenase

ARUP #: 0080135

Methodology: Quantitative Enzymatic

Use:

Confirm etiology of congenital non-spherocytic hemolytic anemia

Determine safety of anti-malarial drugs prior to use

Limitations:

Patients who have recently received transfusions have normal donor cells that may mask G6PD-deficient erythrocytes

Test name: Glucose-6-Phosphate Dehydrogenase (G6PD) 2 Mutations

ARUP #: 0051684

Methodology: Polymerase Chain Reaction/TaqMAN®

Use:

Use to determine if deficiency due to mutation

99% clinical sensitivity in individuals of African descent

Limitations:

Only the 2 G6PD mutations targeted (A376G and G202A) will be detected; analytical sensitivity may be affected by rare primer or probe site mutations

Test name: Hemoglobin Evaluation with Reflex to Electrophoresis and/or RBC Solubility

ARUP #: 0050610

Methodology: High Performance Liquid Chromatography/Electrophoresis/RBC Solubility

Use:

Detect common thalassemic hemoglobinopathies (eg, HbCS)

Limitations:

Some mutations are electrophoretically silent; false positives may occur for RBC solubility in hemoglobin S

Test name: Hemoglobin S, Evaluation with Reflex to RBC Solubility

ARUP #: 0050520

Methodology: High Performance Liquid Chromatography

Use:

Determines presence of hemoglobin S

Test name: Pyruvate Kinase

ARUP #: 0080290

Methodology: Quantitative Enzymatic

Use:

Determine levels of PK in suspected anemia

Screen for carriers of hemolytic anemia

Limitations:

Elevated serum PK levels may be seen in disorders of shortened erythrocyte survival

Patients who have recently received transfusions have normal donor cells that may mask PK-deficient erythrocytes

Test name: Direct Coombs (Anti-Human Globulin)

ARUP #: 0013008

Methodology: Hemagglutination

Use:

Identify antibodies as cause of hemolysis

Test name: Cold Agglutinins

ARUP #: 0050175

Methodology: Semi-Quantitative Hemagglutination

Use:

Identify antibodies as cause of hemolysis

Test name: Antibody Detection, RBC

ARUP #: 0010004

Methodology: Hemagglutination

Use:

Identify antibodies as cause of hemolysis

Additional Tests Available

Test name: Haptoglobin

ARUP #: 0050280

Methodology: Quantitative Immunoturbidimetric

Comments:

Assess presence of intravascular hemolysis

Test name: Hemoglobin, Plasma

ARUP #: 0020058

Methodology: Quantitative Spectrophotometry

Comments:

Increase concentration indicative of acute intravascular destruction of erythrocytes

Not of clinical value in the diagnosis of chronic hemolytic disorders

Test name: Hematocrit

ARUP #: 0040080

Methodology: Automated Cell Counter

Comments:

Assess presence of anemia

Test name: Hemosiderin, Urine

ARUP #: 0020222

Methodology: Semi-quantitative Microscopic Stain

Comments:

Order for suspected intravascular hemolysis within the past week

Test name: Donath Landsteiner

ARUP #: 0013039

Methodology: Hemolysis

Comments:

Determine if Donath-Landsteiner antibodies are present

Diagnose paroxysmal cold hemoglobinuria

Test name: Blood Group (ABO & 8 Minor Antigens) Genotyping by Microarray

ARUP #: 2004739

Methodology: Polymerase Chain Reaction/ASH Microarray

Comments:

Patients with multiple transfusions with or without subsequent hemolytic reaction

Test name: Blood Group (9 Minor Antigens) Genotyping by Bead Array

ARUP #: 2004743

Methodology: Polymerase Chain Reaction/Bead Array

Comments:

Patients with multiple transfusions with or without subsequent hemolytic reaction

Test name: Blood Group (5 Minor Antigens) Genotyping by Bead Array

ARUP #: 2004741

Methodology: Polymerase Chain Reaction/Bead Array

Comments:

Patients with multiple transfusions with or without subsequent hemolytic reaction

References

General

ARUP Institute for Clinical and Experimental Pathology®

Prchal JT, Gregg XT. Red cell enzymes. Hematology Am Soc Hematol Educ Program. 2005; :19-23.PubMed

Medical Reviewers

Comprehensive Review: July 2011
Last Update: July 2011