Hemolytic Anemias

  • Diagnosis
  • Algorithms
  • Background
  • Lab Tests
  • References
  • Related Content

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

Hemolytic Anemias Testing Algorithm

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

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

Osmotic Fragility, Erythrocyte 2002257
Method: Spectrophotometry

Limitations

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

Limitations

False positives – congenital dyserythropoietic anemia, Southeast Asian ovalocytosis

Heinz Body Stain 0049090
Method: Supravital Stain

Limitations

Test results are unreliable in infants <6 months

Glucose-6-Phosphate Dehydrogenase 0080135
Method: Quantitative Enzymatic

Limitations

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

Limitations

Only the G6PD A-allele (A376G and G202A mutations together on the same chromosome) and theG6PD 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

Limitations

Sequencing may detect variants of unknown clinical significance

Not detected by sequencing deep intronic mutations, regulatory region mutations, and large deletions/duplications

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

Limitations

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 alpha-globin gene cluster may appear to have a normal number of alpha-globin gene copies

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

Rare syndromic or acquired forms of alpha-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

Limitations

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

Limitations

Diagnostic errors can occur due to rare sequence variations

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

Other beta- and alpha-globin variants are not identified

Pyruvate Kinase 0080290
Method: Quantitative Enzymatic

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

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

Cold Agglutinins 0050175
Method: Semi-Quantitative Hemagglutination

Antibody Detection, RBC 0010004
Method: Hemagglutination

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

Limitations

Does not detect mutations in genes not tested and large exonic deletions/duplications

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

CBC with Platelet Count and Automated Differential 0040003
Method: Automated Cell Count/Differential

Comments

Initial test for evaluation of hemoglobinopathy

Reticulocytes, Percent & Number 0040022
Method: Flow Cytometry

Comments

Identify increased RBC production

Lactate Dehydrogenase, Serum or Plasma 0020006
Method: Quantitative Enzymatic

Comments

Identify the presence of hemolysis

Bilirubin, Total, Serum or Plasma 0020032
Method: Spectrophotometry

Comments

Identify the presence of hemolysis

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

Comments

Determine presence of sickle cell disease

Haptoglobin 0050280
Method: Quantitative Immunoturbidimetry

Comments

Use to assess presence of intravascular hemolysis

Hemoglobin, Plasma 0020058
Method: Quantitative Spectrophotometry

Comments

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

Comments

Assess presence of anemia

Donath Landsteiner 0013039
Method: Hemolysis

Comments

Determine if Donath-Landsteiner antibodies are present 

Diagnose paroxysmal cold hemoglobinuria

ADAMTS13 Activity 0030056
Method: Chromogenic Assay

Paroxysmal Nocturnal Hemoglobinuria (PNH), High Sensitivity, RBC and WBC 2005006
Method: Quantitative Flow Cytometry

Comments

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

 Analytical sensitivity – limit of detection is 0.1% for WBCs and 0.005% for RBCs

Guidelines

Bolton-Maggs P, Stevens R, Dodd N, Lamont G, Tittensor P, King M, General Haematology Task Force of the British Committee for Standards in Haematology. Guidelines for the diagnosis and management of hereditary spherocytosis. Br J Haematol. 2004; 126(4): 455-74. PubMed

General References

An X, Mohandas N. Disorders of red cell membrane. Br J Haematol. 2008; 141(3): 367-75. PubMed

Bass G, Tuscano E, Tuscano J. Diagnosis and classification of autoimmune hemolytic anemia. Autoimmun Rev. 2014; 13(4-5): 560-4. PubMed

Cappellini M, Fiorelli G. Glucose-6-phosphate dehydrogenase deficiency. Lancet. 2008; 371(9606): 64-74. PubMed

Crowther M, Chan Y, Garbett I, Lim W, Vickers M, Crowther M. Evidence-based focused review of the treatment of idiopathic warm immune hemolytic anemia in adults. Blood. 2011; 118(15): 4036-40. PubMed

Delaunay J. The molecular basis of hereditary red cell membrane disorders. Blood Rev. 2007; 21(1): 1-20. PubMed

Michel M. Classification and therapeutic approaches in autoimmune hemolytic anemia: an update. Expert Rev Hematol. 2011; 4(6): 607-18. PubMed

Zanella A, Fermo E, Bianchi P, Valentini G. Red cell pyruvate kinase deficiency: molecular and clinical aspects. Br J Haematol. 2005; 130(1): 11-25. PubMed

Zantek N, Koepsell S, Tharp D, Cohn C. The direct antiglobulin test: a critical step in the evaluation of hemolysis. Am J Hematol. 2012; 87(7): 707-9. PubMed

Zeerleder S. Autoimmune haemolytic anaemia - a practical guide to cope with a diagnostic and therapeutic challenge. Neth J Med. 2011; 69(4): 177-84. PubMed

References from the ARUP Institute for Clinical and Experimental Pathology®

Christensen R, Yaish H, Nussenzveig R, Reading S, Agarwal A, Eggert L, Prchal J. Acute kernicterus in a neonate with O/B blood group incompatibility and a mutation in SLC4A1. Pediatrics. 2013; 132(2): e531-4. PubMed

Prchal J, Gregg X. Red cell enzymes. Hematology Am Soc Hematol Educ Program. 2005; 19-23. PubMed

Sirdah M, Reading S, Perkins S, Shubair M, Aboud L, Prchal J. Hemolysis and Mediterranean G6PD mutation (c.563 C>T) and c.1311 C>T polymorphism among Palestinians at Gaza Strip. Blood Cells Mol Dis. 2012; 48(4): 203-8. PubMed

Sirdah M, Reading S, Vankayalapati H, Perkins S, Shubair M, Aboud L, Roper D, Prchal J. Molecular heterogeneity of glucose-6-phosphate dehydrogenase deficiency in Gaza Strip Palestinians. Blood Cells Mol Dis. 2012; 49(3-4): 152-8. PubMed

Swierczek S, Agarwal A, Naidoo K, Lorenzo F, Whisenant J, Nussenzveig R, Agarwal N, Coetzer T, Prchal J. Novel exon 2 α spectrin mutation and intragenic crossover: three morphological phenotypes associated with four distinct α spectrin defects. Haematologica. 2013; 98(12): 1972-9. PubMed

Medical Reviewers

Agarwal, Archana Mishra, MD, Medical Director, Hemoglobin Laboratory in the Special Genetics Division; Associate Medical Director, Molecular Oncology at ARUP Laboratories ; Assistant Professor of Clinical Pathology, University of Utah

Grenache, David G., PhD, Medical Director, Special Chemistry; Co-Director, Electrophoresis and Manual Endocrinology; Chief Medical Director, Clinical Chemistry at ARUP Laboratories; Associate Professor of Clinical Pathology, University of Utah

Heikal, Nahla, MD, MS, Assistant Medical Director, Immunology and Hemostasis/Thrombosis at ARUP Laboratories; Assistant Professor of Clinical Pathology, University of Utah

Krautscheid, Patti, MS, LCGC, Genetic Counselor, Molecular Genetics and Special Genetics Laboratories at ARUP Laboratories

Lyon, Elaine, PhD, Medical Director, Genetics, and Co-Medical Director, Pharmacogenomics at ARUP Laboratories; Associate Professor of Clinical Pathology, University of Utah

Prchal, Josef T., MD, Medical Director, Special Genetics at ARUP Laboratories; Professor, Division of Hematology and Hematologic Malignancies, Dept. of Internal Medicine; Adjunct Professor in Human Genetics and Pathology; Huntsman Cancer Institute Investigator, University of Utah

Rodgers III, George M., MD, PhD, Medical Director, Hemostasis and Thrombosis at ARUP Laboratories; Professor of Internal Medicine and Adjunct Professor of Clinical Pathology, University of Utah

Wittwer, Carl T., MD, PhD, Medical Director and Technical Vice President, General Flow Cytometry at ARUP Laboratories; Professor of Clinical Pathology, University of Utah

Last Update: December 2015