Hemolytic Anemias

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 mechanical RBC destruction and thrombocytopenia, refer to Thrombotic Microangiopathies.

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

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
    • 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, 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, 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
        • 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
        • If considered acquired – serum 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 toxin or drug
      • Agglutination
        • Consider testing with direct Coombs (+) (anti-thyroglobulin)
      • Unusual red cell inclusions
  • Reticulocyte count – usually elevated; does not give specific diagnosis
  • Molecular testing
    • Molecular testing can be performed if confirmation is necessary or other testing does not confirm diagnosis

Differential Diagnosis

See classification in Clinical Background section


  • 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
    • 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

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


Not typically used as a screening test, but has been proposed as a screening test by many authorities

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

Heinz Body Stain 0049090
Method: Supravital Stain


Results are unreliable in infants <6 months

Glucose-6-Phosphate Dehydrogenase 0080135
Method: Quantitative Enzymatic


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


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

Analytical sensitivity may be affected by rare primer- or probe-site variants

Diagnostic errors can occur due to rare sequence variations

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


Sequencing may detect variants of unknown clinical significance

Diagnostic errors can occur due to rare sequence variations

Regulatory region mutations, deep intronic mutations, and large deletions/duplications will not be detected

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

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

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


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

Other β- and α-globin variants are not identified

Diagnostic errors can occur due to rare sequence variations

Pyruvate Kinase 0080290
Method: Quantitative Enzymatic


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


Bolton-Maggs PH, 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

Choosing Wisely. An initiative of the ABIM Foundation. [Accessed: Nov 2017]

General References

Barcellini W. Immune Hemolysis: Diagnosis and Treatment Recommendations. Semin Hematol. 2015; 52(4): 304-12. 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

Gallagher PG. Diagnosis and management of rare congenital nonimmune hemolytic disease. Hematology Am Soc Hematol Educ Program. 2015; 2015: 392-9. PubMed

Grace RF, Zanella A, Neufeld EJ, Morton H, Eber S, Yaish H, Glader B. Erythrocyte pyruvate kinase deficiency: 2015 status report. Am J Hematol. 2015; 90(9): 825-30. PubMed

Höchsmann B, Schrezenmeier H. Congenital CD59 Deficiency. Hematol Oncol Clin North Am. 2015; 29(3): 495-507. PubMed

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

Naik R. Warm autoimmune hemolytic anemia. Hematol Oncol Clin North Am. 2015; 29(3): 445-53. PubMed

Shanbhag S, Spivak J. Paroxysmal cold hemoglobinuria. Hematol Oncol Clin North Am. 2015; 29(3): 473-8. 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

References from the ARUP Institute for Clinical and Experimental Pathology®

Agarwal AM, Nussenzveig RH, Reading NS, Patel JL, Sangle N, Salama ME, Prchal JT, Perkins SL, Yaish HM, Christensen RD. Clinical utility of next-generation sequencing in the diagnosis of hereditary haemolytic anaemias. Br J Haematol. 2016; PubMed

Christensen RD, Agarwal AM, Nussenzveig RH, Heikal N, Liew MA, Yaish HM. Evaluating eosin-5-maleimide binding as a diagnostic test for hereditary spherocytosis in newborn infants. J Perinatol. 2015; 35(5): 357-61. PubMed

Christensen RD, Yaish HM, Nussenzveig RH, Agarwal AM. Siblings with severe pyruvate kinase deficiency and a complex genotype. Am J Med Genet A. 2016; 170(9): 2449-52. PubMed

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

Reading S, Sirdah MM, Shubair ME, Nelson BE, Al-Kahlout MS, Al-Tayeb JM, Aboud LN, Shaban MA, Luzzatto L, Prchal JT. Favism, the commonest form of severe hemolytic anemia in Palestinian children, varies in severity with three different variants of G6PD deficiency within the same community. Blood Cells Mol Dis. 2016; 60: 58-64. 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 and Genomics; Co-Scientific Director, NGS and Biocomputing; Director, High Complexity Platforms-NGS, at ARUP Laboratories; Assistant Professor of Clinical Pathology, University of Utah

Blaylock, Robert C., MD, Medical Director, Blood Services, and Phlebotomy and Support Services, Immunohematology Reference, Clinical Laboratory and Transfusion Services at the University of Utah Hospitals and Clinics at ARUP Laboratories ; Associate Professor of Clinical Pathology, University of Utah

Couturier, Marc Roger, PhD, D(ABMM), Medical Director, Microbial Immunology, Parasitology and Fecal Testing, and Infectious Disease AntigenTesting at ARUP Laboratories; Associate 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, FACMG, Medical Director, Molecular Genetics and Genomics, Medical Director, Pharmacogenomics at ARUP Laboratories; Professor of Clinical Pathology, University of Utah

Mao, Rong, MD, FACMG, Laboratory Section Chief, Molecular Genetics and Genomics, at ARUP Laboratories; Professor of Clinical Pathology, Adjunct Associate Professor, Pediatrics, and Co-Director, Clinical Molecular 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: January 2018