Hemolytic Anemias

  • Diagnosis
  • Algorithms
  • Background
  • Lab Tests
  • References
  • Related Topics
  • Videos

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
  • Molecular testing
    • Molecular testing can be performed if confirmation necessary or other testing does not confirm diagnosis

Differential Diagnosis

  • See classification in Clinical Background section

Hemolytic anemias result from premature destruction of red blood cells (RBCs). For information on hemolytic anemias as a result of hemoglobin synthesis abnormalities, refer to the following topics HemoglobinopathiesUnstable HemoglobinopathiesThalassemias. For information about hemolytic anemias associated with acquired immune defects refer to Thrombotic Microangiopathies.


  • Prevalence
    • Varies by 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


  • Hereditary
    • RBC membrane defects
      • G6PD deficiency
      • PK deficiency
    • Red cell enzyme 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

Clinical Presentation of Specific Hemolytic Disorders

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.

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


False positives – congenital dyserythropoietic anemia, Southeast Asian ovalocytosis

Osmotic Fragility, Erythrocyte 2002257
Method: Spectrophotometry


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

Heinz Body Stain 0049090
Method: Supravital Stain


Test results are unreliable in infants <6 months

Glucose-6-Phosphate Dehydrogenase 0080135
Method: Quantitative Enzymatic


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


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


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

Sequencing may detect variants of unknown clinical significance

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


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


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


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


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


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

α-globin and β-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

Related Tests


Bolton-Maggs PH B, Stevens RF, Dodd NJ, 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 GF, Tuscano ET, Tuscano JM. Diagnosis and classification of autoimmune hemolytic anemia. Autoimmun Rev. 2014; 13(4-5): 560-4. PubMed

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

Crowther M, Chan YL Tracey, Garbett IK, Lim W, Vickers MA, Crowther MA. 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 ND, Koepsell SA, Tharp DR, Cohn CS. 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 RD, Yaish HM, Nussenzveig RH, Reading S, Agarwal AM, Eggert LD, Prchal JT. Acute kernicterus in a neonate with O/B blood group incompatibility and a mutation in SLC4A1. Pediatrics. 2013; 132(2): e531-4. PubMed

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

Sirdah M, Reading S, Perkins SL, Shubair M, Aboud L, Prchal JT. 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 SL, Shubair ME, Aboud L, Roper D, Prchal JT. 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 AM, Naidoo K, Lorenzo FR, Whisenant J, Nussenzveig RH, Agarwal N, Coetzer TL, Prchal JT. 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

Best, Hunter, PhD, Medical Director, Molecular Genetics; Director, High Complexity Platforms-NGS 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

Mao, Rong, MD, Medical Director, Molecular Genetics and Genomics at ARUP Laboratories; Associate Professor of Clinical Pathology and Co-director, Clinical Medical Genetics Fellowship Program, 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

Smock, Kristi J., MD, Medical Director, Hemostasis/Thrombosis at ARUP Laboratories; Associate 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: April 2016