Hemolytic Anemias

Diagnosis

Indications for Testing

• Suspicion of hemolysis based on clinical and laboratory presentation
  • Increased reticulocytosis, lactate dehydrogenase, and bilirubin
  • Decreased haptoglobin
  • Anemia with deformed and fragmented erythrocytes on peripheral smear in some disorders

Laboratory Testing

• CBC with peripheral smear – initial screening
  • Platelet count
    • Decreased – consider disseminated intravascular coagulation (DIC); Hemolysis, Elevated Liver enzyme levels, Low Platelet count (HELLP) syndrome; thrombotic thrombocytopenia purpura (TTP); hemolytic uremic syndrome (HUS); atypical HUS (aHUS) 
      • Order D-dimer, ADAMTS13 activity
    • Normal – consider vasculitis, mechanical factors
  • 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 – 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, aHUS, mechanical cardiac valve, vasculitis, malignant hypertension
      • Order D-dimer testing
        • Increased D-dimer – DIC
        • Normal D-dimer and clinical presentation consistent with thrombotic microangiopathy (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 – aHUS
            • Positive Shiga toxin and history of diarrheal illness – classical HUS
    • Polychromasia
      • Without other reproducible morphologic abnormality
        • Consider one or more of the following tests: pyruvate kinase (PK), hexokinase (HK), glucose phosphate isomerase (GPI)
      • With or without platelet decrease
        • Consider paroxysmal nocturnal hemoglobinuria testing
    • Sickle cells – consider hemoglobin evaluation by high-performance liquid chromatography (HPLC)
      • Abnormal red cells (eg, sickle cells and target cells) – may indicate hemoglobinopathy
      • Do not repeat hemoglobin electrophoresis in patients who have a prior result and who do not require therapeutic intervention or monitoring of hemoglobin variant levels (Choosing Wisely: 20 Things Physicians and Patients Should Question, 2017; American Society for Clinical Pathology)
    • Stomatocytes – hereditary stomatocytosis likely
    • Basophilic stippling levels
      • Acquired – serum lead levels testing
      • 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 toxin or drug
    • Agglutination
      • Consider testing with direct Coombs (+) (anti-thyroglobulin)
        • Cold agglutinin disease (IgG-/C3d+)
        • Warm autoimmune hemolytic anemia (IgG+/C3d-)
    • Unusual red cell inclusions – if infection is suspected, consider evaluation for Babesia, malaria, Bartonella spp
• Reticulocyte count – usually elevated; does not give specific diagnosis
• Molecular testing – can be performed if confirmation is necessary or other testing does not confirm diagnosis
  • Genes involved in hereditary membrane disorders

    Genes Involved in Hereditary Membrane Disorders

    Condition	Genes	Inheritance
    Hereditary spherocytosis	ANK1, SLC4A1, SPTB, EPB42	AD/AR
    Hereditary elliptocytosis/pyropoikilocytosis	SPTA1, SPTB, EPB41	AD/AR
    Hereditary stomatocytosis	PEIZO1	AD
    AD, autosomal dominate; AR, autosomal recessive
  • Genes involved in RBC enzyme deficiency

    Genes Involved in Red Blood Cell (RBC) Enzyme Deficiency

    Condition	Genes	Inheritance
    Glucose 6 phosphate dehydrogenase deficiency	G6PD	XR
    Pyruvate kinase deficiency	PKLR	AR
    Glucose phosphate isomerase deficiency	GP1	AR
    Glutathione reductase deficiency	GSR	AR
    Phosphoglycerate kinase deficiency	PGK1	XL
    Triosephosphate isomerase deficiency	TPI1	AR
    Adenylate kinase 1	AK1	AR
    Pyrimidine 5’ nucleotidase	NT5C3	AR
    Hexokinase 1	HK1	AR
    AR, autosomal recessive; XL, x-linked; XR, x-linked recessive

Differential Diagnosis

See Classification in Background

Background

Epidemiology

• Prevalence – varies by etiology of hemolysis
  • Common – autoimmune hemolytic anemia, G6PD deficiency, 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

• Hereditary
  • Red cell enzyme defects
    • G6PD deficiency
    • PK deficiency
  • RBC membrane defects
    • HS
    • Stomatocytosis
    • Hereditary elliptocytosis
    • Hereditary pyropoikilocytosis
  • Hemoglobin synthesis abnormalities
    • Hemoglobinopathies
      • Sickle cell disease and other qualitative hemoglobin disorders
      • Alpha (α) thalassemia, beta (β) thalassemia
• Acquired
  • Immune
    • Infectious
      • Cytomegalovirus (CMV)
      • Epstein-Barr virus (EBV)
      • Hepatitis C virus (HCV)
      • HIV
      • Leptospira spp
      • Plasmodium spp
      • Mycoplasma pneumoniae
      • Parvovirus
      • Babesia microti
    • 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
      • TTP
      • HUS
      • aHUS
      • DIC
      • HELLP syndrome
  • Nonimmune
    • Environmental factors (physical disruption)
      • Mechanical valves
    • PNH

Clinical Presentation

• Clinical presentation of specific hemolytic disorders
  • Cell membrane disorders (mechanical weakness or fragility of erythrocyte membrane skeleton)

    • Hereditary spherocytosis
      • Prevalence – 1/1,000-3,000 in Caucasians
      • Inheritance – variable
        • Autosomal dominant variants in ankyrin and α-spectrin – 75% of cases
        • Autosomal recessive or de novo variants – 25% of cases
        • Variants may occur in ANK1, EPB42, SLC4A1, 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
      • Incidence – 1/2,000-4,000 in Caucasians
      • Inheritance – autosomal dominant
        • Involves gene variants associated with defects in spectrin – SPTA1, SPTB, and EPB41
      • Clinical presentation
        • Usually no significant hemolysis – may be asymptomatic
    • Hereditary pyropoikilocytosis
      • Incidence – uncommon
      • Inheritance – autosomal recessive (felt to be a subset of hereditary elliptocytosis)
        • Same gene variants as hereditary elliptocytosis, homozygous/compound heterozygotes
      • Clinical presentation – usually presents in infancy or early childhood
    • Dehydrated hereditary stomatocytosis (xerocytosis)
      • Incidence – 1/50,000 in Caucasians
      • Inheritance – autosomal dominant
        • Gene variants in PIEZO1
      • Clinical presentation – asymptomatic to moderately severe disease
  • RBC enzyme defects

    • Overview
      • >20 disorders recognized
      • ​Deficiencies of enzymes include (Gallagher, 2015)
        • G6PD
        • Phosphofructokinase M (PFK)
        • HK
        • GPI
        • PK
        • Triosephospate isomerase (TPI)
        • Phosphoglycerate kinase (PGK)
        • Pyrimidine 5’-nucleotidase (P5N)
      • 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)
      • Nonhematological 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 variant(s)
    • G6PD deficiency
      • Prevalence
        • 400-600 million worldwide
        • Most common enzyme deficiency worldwide
      • Ethnicity
        • African
        • Kurdish Jewish
        • Sephardic Jewish
        • Arab
        • Mediterranean
        • Southeast Asian and Asian-Indo/Pakistani
        • Northern Europeans (0.1%)
      • Inheritance
        • X-linked recessive
        • >400 G6PD gene variants have been described
          • Some are benign
          • Others result in intermittent or chronic hemolytic anemia
          • >99% of G6PD deficiency in individuals of African descent are caused by the G6PDA allele
        • Most affected individuals have moderate G6PD deficiency (~10% normal activity), resulting in acute hemolytic anemia in response to oxidative stress
        • Rare variants cause severe G6PD deficiency (<10% normal activity), resulting in chronic nonspherocytic hemolytic anemia in absence of oxidative stressors
        • Female variant carriers may have hemolytic episodes even if G6PD enzyme studies are normal, because deficient as well as nondeficient RBCs coexist in various proportions
          • Female presentation also dependent on presence or absence of imbalanced x-activation
      • 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
  • Enzymatic hemolytic anemias

    Enzymatic Hemolytic Anemias

    Deficiency	Incidence/Ethnicity	Inheritance	Gene	Clinical Presentation
    Pyruvate kinase	1/20,000 in Caucasians Mediterranean populations	AR Carriers recognized by erythrocytes – activity ~50% of normal	PKLR >180 different variants	Neonatal anemia Mild to severe hemolysis
    Glucose phosphate isomerase	Rare	AR	GPI	Infancy/early childhood Moderate to severe hemolytic anemia Rare – neurologic disease (eg, developmental delay)
    Phosphofructokinase (glycogen storage disease VII, Tauri disease)	Rare Ashkenazi Jewish	AR	PFKM	Adolescence/adulthood Exertional myopathy Mild, well-compensated hemolytic anemia
    Triosephospate isomerase	Rare 240 cases	AR	TPI1	Infancy Cardiomyopathy Progressive neurologic impairment
    Phosphoglycerate kinase	Rare	X-linked	PGK1	CNS disorders Developmental delay Myopathy Some patients lack myopathy or CNS disorder
    Hexokinase	Rare	AR	HK1	Neonatal/adulthood Neonatal severe anemia
    Pyrimidine 5’-nucleotidase	Rare	AR	NT5C3A	Mild to moderate hemolytic anemia with basophilic stippling Splenomegaly
    AR, autosomal recessive; CNS; central nervous system
  • Acquired hemolytic anemias – immune mediated

    • Incidence – 1-3/100,000
    • 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 autoimmune hemolytic anemia
      • Antibody generally directed broadly against Rh antigens
      • Symptoms usually a result of anemia
        • Slow, insidious onset
        • May have jaundice
      • Cold agglutinin hemolysis
        • Episodic acute hemolysis with hemoglobinuria
        • Acrocyanosis in response to cold and vasoocclusive phenomena of fingers, toes, ears, and nose present in some patients
        • 90% of patients have monoclonal B-cell lymphoproliferative bone marrow disorders
      • Drugs – immune mediated
        • Severity depends on the drug and varies from mild to severe hemolysis
        • >150 drugs have been implicated – cephalosporins, nonsteroidal anti-inflammatory drugs (NSAIDs) are most common
  • Paroxysmal cold hemoglobinuria

    • Incidence – rare
    • Age – 5 years (median)
    • Caused by a biphasic antibody (Donath-Landsteiner antibody) and inherited CD59 deficiency
    • Clinical presentation
      • Pallor, hemoglobinuria, and mild jaundice
      • Patients usually have a history of recent viral infection