Hemolytic Anemias

  • Diagnosis
  • Algorithms
  • Background
  • Lab Tests
  • References
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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 – atypical HUS
              • 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
      • 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 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 Hemoglobinopathies, Unstable Hemoglobinopathies, Thalassemias. For information about hemolytic anemias associated with mechanical RBC destruction and thrombocytopenia refer to Thrombotic Microangiopathies.

Epidemiology

  • 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

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
      • 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
    • Nonimmune
      • Environmental factors (physical disruption)
        • Mechanical valves
      • PNH

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

Recommended Use 

Confirms diagnosis of hereditary spherocytosis when hemolytic anemia and spherocytes are present

Clinical sensitivity – 93%

Analytical sensitivity/specificity – unknown

Limitations 

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

Osmotic Fragility, Erythrocyte 2002257
Method: Spectrophotometry

Recommended Use 

Functional testing of red blood cell sensitivity to osmotic stress; do not use to distinguish between spherocytes in hereditary spherocytosis and acquired autoimmune hemolytic anemia

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

Heinz Body Stain 0049090
Method: Supravital Stain

Recommended Use 

Use as a nonspecific screen for hemolysis due to drugs/toxins, enzyme deficiencies, thalassemias, and unstable hemoglobins

Limitations 

Test results are unreliable in infants <6 months

Glucose-6-Phosphate Dehydrogenase 0080135
Method: Quantitative Enzymatic

Recommended Use 

Preferred initial test for screening for G6PD deficiency

Clinical sensitivity – >99% 

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 variants

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

Recommended Use 

Preferred genetic test for individuals of African descent

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

Clinical sensitivity – 99% in individuals of African descent

Analytical sensitivity/specificity – 99% 

Limitations 

Only the G6PD A- allele (A376G and G202A variants 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 variants

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

Recommended Use 

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 variants

Clinical sensitivity – >98%

Analytical sensitivity/specificity – 99%

Limitations 

Does not detect deep intronic variants, regulatory region variants, and large deletions/duplications

Sequencing may detect variants of unknown clinical significance

Rare diagnostic errors can occur due to primer site variants

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

Recommended Use 

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

Genes include – HBB (β-globin), HBA1, and HBA2 (α-globins)

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

Refer to Additional Technical Information document for further content

Sensitivity/specificity – varies, depending on test components

Limitations 

Cascade may not detect all Hb variants

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

The phase of identified variants may not be determined

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

Recommended Use 

Effective test for screening and follow up of individuals with hemoglobinopathies

 Can be ordered individually or may be performed as part of Hb cascade testing

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

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

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

Recommended Use 

Confirm suspected HbS, HbC, or HbE variants

Clinical sensitivity – sickle cell disease >70%; other hemoglobinopathies vary by ethnicity

Analytical sensitivity – 99%

Limitations 

Detects only the 3 most common missense variants in the 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

Recommended Use 

Preferred initial test for screening for pyruvate kinase deficiency

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

Recommended Use 

Identify antibodies as cause of hemolysis

Cold Agglutinins 0050175
Method: Semi-Quantitative Hemagglutination

Recommended Use 

Identify antibodies as cause of hemolysis

Antibody Detection, RBC 0010004
Method: Hemagglutination

Recommended Use 

Identify antibodies as cause of hemolysis

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

Recommended Use 

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

Genes include – ADA, AK1, ALDOA, ANK1, CYB5R3, EPB41, EPB42, G6PD, GCLC, GPI, GSR, GSS, HK1, NT5C3A, PFKL, PFKM, PGK1, PIEZO1, PKLR, SLC4A1, SLC1B1, SLCO1B3, SPTA1, SPTB, TPI1, UGT1A1, UGT1A6, UGT1A7

Refer to Additional Technical Information document for further content

Clinical sensitivity – unknown

Analytical sensitivity/specificity – 99%

Limitations 

Does not detect variants in genes not tested, large exonic deletions/duplications, deep intronic or regulatory region variants

Alpha-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 variant detection in PGK1

Guidelines

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

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, 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

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 Rapid Testing 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: September 2016

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