Hemolytic Anemias

Diagnosis

Indications for Testing

  • Anemia with 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
    • Cells noted may help with diagnosis
      • Spherocytes – hereditary spherocytosis or elliptocytosis, immune-mediated hemolytic anemias
        • Examination of peripheral smear
          • Fast, easy screen
        • If considered hereditary – red blood cell (RBC) surface protein band 3 testing
          • Highly sensitive and specific for disease
        • If considered acquired – direct Coombs testing
          • IgG+ – autoimmune hemolytic anemia
          • C3+ – cold agglutinins disease, paroxysmal cold  hemoglobinuria (PCH)
            • Confirm PCH with Donath Landsteiner testing
      • Schistocytes/fragmented cells – suggests microangiopathic RBC destruction
        • Consider DIC, TTP, HELLP, HUS, mechanical cardiac valve, vasculitis, malignant hypertension
        • Order D-dimer testing
          • Increased D-dimer – DIC
          • Normal D-dimer and clinical presentation consistent with TMA
            • Pregnant – consider HELLP
            • Not pregnant – order ADAMTS13 activity or E. coli Shiga-like Toxin by EIA (dependent on presentation)
              • ADAMTS13 activity <10% – TTP
              • Normal test results – atypical HUS
              • Positive Shiga toxin – HUS
      • Polychromasia
        • Without other reproducible morphologic abnormality
          • Consider one or more of the following tests: pyruvate kinase, hexokinase, glucose phosphate isomerase
        • With or without platelet decrease
      • Sickle cells – consider hemoglobin evaluation by high-performance liquid chromatography (HPLC)
        • Abnormal red cells (eg, sickle cells and 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 hemoglobinopathies, glucose-6 phosphate dehydrogenase (G6PD) deficiency, chemical and toxin exposure
        • Consider G6PD testing or isopropanol heat stability of the signals
        • If G6PD deficiency has been ruled out – Heinz bodies may implicate presence of a toxin or drug
      • Agglutination
        • Consider testing for direct Coombs (+)
      • Unusual red cell inclusions
  • 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

Epidemiology

  • 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 individuals of African, Kurdish or Sephardic Jewish, Arab, Mediterranean, Southeast Asian, and Asian-Indo/Pakistani descent
    • Sickle cell disease most often found in Africans

Classification

  • Infectious
  • Autoimmune (Bass, 2014)
    • Warm antibody disease
      • Primary
      • Secondary associated with
        • Lymphoproliferative disorders (eg, non-Hodgkin lymphoma)
        • Nonlymphoid malignancies (eg, ovarian cancer)
        • Chronic inflammatory disorders (eg, ulcerative colitis)
        • Drugs
    • Cold agglutinin syndrome
      • Primary
      • Secondary associated with
        • Lymphoproliferative disorders
        • Infections (eg, mycoplasma)
        • PNH
    • Mixed antibody disease
      • Primary
      • Secondary often associated with rheumatic disease
    • Drug-induced (>150 drugs)
      • Drug-dependent
      • Drug-independent
  • Microangiopathic red cell destruction
  • 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)
    • Mechanical valves
  • 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
        • Mutations may occur in ANK1, EPB42, SLC4A1, SPTA1, SPTB genes
      • Clinical presentation
        • Range of severity from asymptomatic to severe disease
        • Anemia, splenomegaly, jaundice, gallstones (particularly in pediatric cases)
        • Reticulocytosis (after prior anemia)
    • Hereditary elliptocytosis or hereditary pyropoikilocytosis
      • Incidence – 1-2/10,000 in Caucasians
      • Inheritance – autosomal dominant
        • Involves gene mutations associated with defects in spectrin and some bands
      • Clinical presentation
        • Usually no significant hemolysis – may be asymptomatic
    • Dehydrated hereditary stomatocytosis  (xerocytosis)
      • Incidence – 1/50,000 in Caucasians
      • Inheritance – autosomal dominant
      • Clinical presentation – asymptomatic to moderately severe disease
    RBC enzyme defects
    • Overview
      • >20 disorders recognized
      • X-linked forms – G6PD deficiency, phosphoglycerate kinase 1 deficiency
      • Deficiencies of enzymes involved with
        • Glycolysis
        • Hexose monophosphate shunt
        • Glutathione metabolism
        • Nucleotide metabolism
      • Usually associated with normocytic normochromic hemolytic anemia with no specific abnormalities of RBC morphology – occasional bite cells can be seen
      • Severity of hemolysis is variable – may be a result of an external stressor (eg, infection, administration of drugs, or ingestion of some foods)
      • Non-hematological manifestations possible – myopathy, neurological dysfunction, intellectual disability
      • Diagnosis is based on detection of reduced specific enzyme activity and/or molecular genetic testing to identify causative mutation(s)
    • G6PD deficiency
      • Incidence
        • 400 million worldwide – most common enzyme deficiency worldwide
      • Ethnicity
        • African
        • Kurdish Jewish
        • Sephardic Jewish
        • Arab
        • Mediterranean
        • Southeast Asian
        • Asian-Indo/Pakistani
        • Northern Europeans (0.1%)
      • 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 in individuals of African descent is caused by the G6PDA allele
        • Female mutation carriers may have hemolytic episodes even if G6PD enzyme studies are normal, because deficient as well as non-deficient red blood 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 (PK) deficiency
      • Incidence
        • 1/20,000 in Caucasians
        • Most common cause of congenital non-spherocytic hemolytic anemia
      • Ethnicity – more common in Mediterranean populations
      • Inheritance – autosomal recessive
        • Carriers are recognized by erythrocytes – PK activity ~50% of normal
        • >180 different mutations
      • Clinical presentation
    Acquired hemolytic anemias – immune-mediated
    • Incidence – 1-3/100,000 annually
    • Age – 30s-40s
    • Sex – M<F
    • Common types
      • Warm and cold antibody disease
        • IgG – warm antibody disease
        • IgM – most common for cold antibody disease
    • 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
        • >150 drugs have been implicated –  cephalosporins, NSAIDs are most common
    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
Test Name and Number Recommended Use Limitations Follow Up
Osmotic Fragility, Erythrocyte 2002257
Method: Spectrophotometry

Functional testing of red blood cell sensitivity to osmotic stress

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

Testing should be performed during a state of prolonged homeostasis with stable hematocrit

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

 
RBC Band 3 Protein Reduction in Hereditary Spherocytosis 2008460
Method: Qualitative Flow Cytometry

Highly sensitive and specific for the diagnosis of  hereditary spherocytosis

False positives – congenital dyserythropoietic anemia, Southeast Asian ovalocytosis

Should be used as a screening test

 
Heinz Body Stain 0049090
Method: Supravital Stain

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

Positive in unstable hemoglobins or inherited defects in erythrocyte oxidative pathways (eg, G6PD deficiency) as well as with many chemical and toxin exposure

Test results are unreliable in infants <6 months

 
Glucose-6-Phosphate Dehydrogenase 0080135
Method: Quantitative Enzymatic

Preferred initial test for screening for G6PD deficiency

Reduced sensitivity for detection of G6PD deficiency in presence of hemolytic crises; neonates; presence of high reticulocyte count; after blood transfusion; heterozygous females

Rare diagnostic errors may occur due to primer-site mutations

 
Glucose-6-Phosphate Dehydrogenase (G6PD) 2 Mutations 0051684
Method: Polymerase Chain Reaction/TaqMAN

Preferred genetic test for individuals of African descent

Detects the single most common pathogenic G6PD mutation (the A- allele) in individuals of African descent

99% clinical sensitivity in individuals of African descent

Only the G6PD A- allele (A376G and G202A mutations together on the same chromosome) and the G6PD A+ allele (A376G variant in isolation) are detected

Analytical sensitivity may be affected by rare primer or probe site mutations

 
Glucose-6-Phosphate Dehydrogenase Deficiency (G6PD) Sequencing 2007163
Method: Polymerase Chain Reaction/Sequencing

Preferred genetic test for individuals of high-risk ethnic backgrounds other than those of African descent

Appropriate  test for symptomatic individuals of African descent who do not carry the A- allele

Detects most G6PD deficiency-causing mutations

>98% clinical sensitivity

Deep intronic mutations, regulatory region mutations, and large deletions/duplications are not detected

Rare diagnostic errors can occur due to primer site mutations

 
Hemoglobin Evaluation Reflexive Cascade 2005792
Method: High Performance Liquid Chromatography/Electrophoresis/RBC Solubility/Polymerase Chain Reaction/Fluorescence Resonance Energy Transfer/Sequencing

Optimal test for the initial and confirmatory diagnosis of any suspected hemoglobinopathy or thalassemia

Not recommended for routine carrier screening in healthy adults for  purposes of reproductive decision making

Cascade reflex testing may include electrophoresis, solubility testing, and/or molecular analyses of the globin genes

A faculty hematopathologist personally directs and interprets each stage of testing to completion

A comprehensive report is provided

Do not use for the follow-up of an individual with a known diagnosis

Sensitivity/specificity – varies, depending on test components

Cascade may not detect all Hb variants

Regulatory region mutations and sequence variants in genes other than HBB, HBA1, and HBA2 will not be detected

The phase of identified mutations may not be determined

Specific breakpoints of large deletions/duplications will not be determined, and it may not be possible to distinguish mutations of similar size

Individuals carrying both a deletion and duplication within the α-globin gene cluster may appear to have a normal number of α-globin gene copies

Sequencing of both HBA1 and HBA2 genes may not be possible in individuals harboring large α-globin deletions on both alleles

Rare syndromic or acquired forms of α thalassemia associated with ATRX gene mutations will not be detected

Diagnostic errors can occur due to rare sequence variations

 
Hemoglobin Evaluation with Reflex to Electrophoresis and/or RBC Solubility 0050610
Method: High Performance Liquid Chromatography/Electrophoresis/RBC Solubility

Effective test for screening and follow up of individuals with hemoglobinopathies

Reflex pattern – if HPLC detects any abnormal peaks suggestive of a hemoglobin variant, then RBC Solubility and/or Capillary Electrophoresis will be added to aid in identification and confirmation of the variant

Sensitivity/specificity – varies, depending on test components

Quantitation of hemoglobin is recommended for a definitive diagnosis in infants 1 year and older

May not detect all hemoglobin variants

Diagnostic errors can occur due to rare sequence variations

 
Beta Globin (HBB) HbS, HbC, and HbE Mutations 0051421
Method: Polymerase Chain Reaction/Fluorescence Resonance Energy Transfer

Confirm suspected HbS, HbC, or HbE mutations

Clinical sensitivity

  • Sickle cell disease – >70%
  • Other hemoglobinopathies –vary by ethnicity

Analytical sensitivity – 99%

Diagnostic errors can occur due to rare sequence variations

Detects only the 3 most common missense variants in the β-globin gene

Other β- and α-globin variants are not identified

 
Pyruvate Kinase 0080290
Method: Quantitative Enzymatic

Preferred initial test for screening for pyruvate kinase deficiency

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

 
Direct Coombs (Anti-Human Globulin) 0013008
Method: Hemagglutination

Identify antibodies as cause of hemolysis

   
Cold Agglutinins 0050175
Method: Semi-Quantitative Hemagglutination

Identify antibodies as cause of hemolysis

   
Antibody Detection, RBC 0010004
Method: Hemagglutination

Identify antibodies as cause of hemolysis

   
Hereditary Hemolytic Anemia Sequencing, 28 Genes 2012052
Method: Massively Parallel Sequencing

Confirm etiology of hemolytic anemia in individuals with hemolysis or a family history of hemolytic anemia

Mutations in genes not tested and large exonic deletions/duplications are not detected

Alpha-globin and beta-globin genes are not analyzed due to high level of gene homology and frequency of large deletions

Small deletions or insertions may not be detected

Diagnostic errors can occur due to rare sequence variation

The presence of a highly homologous pseudogene may interfere with mutation detection in PGK1

 
Additional Tests Available
 
Click the plus sign to expand the table of additional tests.
Test Name and NumberComments
CBC with Platelet Count and Automated Differential 0040003
Method: Automated Cell Count/Differential

Initial test for evaluation of hemoglobinopathy

Reticulocytes, Percent & Number 0040022
Method: Flow Cytometry

Identify increased RBC production

Lactate Dehydrogenase, Serum or Plasma 0020006
Method: Quantitative Enzymatic

Identify the presence of hemolysis

Bilirubin, Total, Serum or Plasma 0020032
Method: Spectrophotometry

Identify the presence of hemolysis

Hemoglobin S, Evaluation with Reflex to RBC Solubility 0050520
Method: High Performance Liquid Chromatography

Determine presence of sickle cell disease

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

ADAMTS13 Activity 0030056
Method: Chromogenic Assay
Paroxysmal Nocturnal Hemoglobinuria (PNH), RBC and WBC 2005006
Method: Quantitative Flow Cytometry

Preferred test for initial diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) and quantification of PNH clones