What lab tests are used to diagnose hemolytic anemias and hereditary spherocytosis?

Laboratory Testing for Hemolytic Anemias and Hereditary Spherocytosis

Initial Hemolysis Confirmation

Begin by confirming hemolysis with the following laboratory markers: elevated reticulocyte count, elevated lactate dehydrogenase (LDH), decreased or absent haptoglobin, elevated indirect (unconjugated) bilirubin, and peripheral blood smear examination for red blood cell morphology abnormalities 1.

• Complete blood count (CBC) with differential and reticulocyte count are essential first-line tests 2, 3, 4.
• Peripheral blood smear review identifies specific morphologic abnormalities such as spherocytes, schistocytes, or bite cells 1, 4.
• Elevated LDH and decreased haptoglobin indicate intravascular hemolysis 1, 4.
• Elevated indirect bilirubin reflects increased red cell breakdown 1, 4.

Critical Branching Point: Direct Antiglobulin Test (DAT)

Perform the direct antiglobulin test (DAT, also called direct Coombs test) before initiating any treatment to differentiate immune from non-immune hemolysis 1.

• A positive DAT indicates immune-mediated hemolysis and requires evaluation for autoimmune hemolytic anemia, lymphoproliferative disorders, autoimmune diseases, infections, and drug-induced causes 1.
• The indirect antiglobulin test detects free autoantibodies in serum 1.
• A negative DAT directs evaluation toward hereditary causes including membranopathies (hereditary spherocytosis, hereditary elliptocytosis) and enzymopathies 1.

Specific Testing for Hereditary Spherocytosis

When hereditary spherocytosis is suspected based on negative DAT, spherocytes on smear, family history, and chronic hemolysis:

Screening Tests

• Mean corpuscular volume (MCV) and mean sphered cell volume (MSCV) difference (ΔMCV-MSCV) >10 fL has 82.75% sensitivity and 95.9% specificity for hereditary spherocytosis diagnosis 5.
• Mean corpuscular hemoglobin concentration (MCHC) >35 g/dL supports spherocytosis but has lower sensitivity (44.82%) 5.
• Mean reticulocyte volume (MRV) combined with MSCV helps differentiate hereditary spherocytosis from immune hemolytic anemia: ΔMCV-MSCV >10 fL and ΔMRV-MSCV <25 has 68.9% sensitivity and 98.8% specificity 5.

Confirmatory Tests

• Eosin-5'-maleimide (EMA) binding test by flow cytometry is highly sensitive and specific for hereditary spherocytosis 6, 7.
• Osmotic fragility test (OFT), particularly the incubated version, demonstrates increased red cell fragility in spherocytosis 6, 5, 7.
• Flow cytometric osmotic fragility test provides quantitative assessment 5.
• Acid glycerol lysis time test (AGLT) and cryohemolysis test are alternative screening methods 7.

No single screening test detects all hereditary spherocytosis cases; some laboratories use combinations such as AGLT plus EMA binding 7.

Advanced Diagnostic Testing

• SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins identifies specific protein deficiencies in atypical cases 7.
• Next-generation sequencing (NGS) gene panels analyzing 68+ hemolytic anemia-related genes serve as the gold standard for hereditary hemolytic anemias when initial testing is equivocal 1.

Enzymopathy Evaluation (When Spherocytosis is Excluded)

If membranopathy testing is negative but hemolysis persists:

• Pyruvate kinase (PK) activity by spectrophotometry is the reference method for PK deficiency, the most common glycolytic enzyme defect 2, 1.
• Critical interpretation caveat: falsely normal PK levels occur with markedly elevated reticulocyte count, recent blood transfusions, incomplete platelet/leukocyte removal, or kinetically abnormal mutant enzymes 2, 1.
• PKLR gene sequencing (Sanger or NGS) confirms PK deficiency when enzyme assays are equivocal 2, 1.
• Glucose-6-phosphate dehydrogenase (G6PD) enzyme activity testing identifies G6PD deficiency, particularly in patients with oxidative stress-triggered hemolysis 1, 4.

Essential Ancillary Laboratory Studies

• Serum ferritin, transferrin saturation, and iron studies exclude iron deficiency as a confounding factor 2.
• Vitamin B12 and folate levels rule out megaloblastic causes of macrocytic anemia 2.
• Hemoglobin electrophoresis excludes hemoglobinopathies such as sickle cell disease and thalassemia 3, 4.
• Paroxysmal nocturnal hemoglobinuria (PNH) flow cytometry screening should be considered in cases with clinical suspicion 2, 1.

Clinical Context Requirements

• Obtain clinical history including neonatal jaundice, transfusion history, drug exposures, and family history of anemia or splenectomy 1, 6.
• Document timing of last blood transfusion, as this affects enzyme assay interpretation 1.
• Family investigation strengthens hereditary spherocytosis diagnosis, as most cases follow autosomal dominant inheritance 6.