Diagnosis: Hereditary Red Blood Cell Enzymopathy (Most Likely Pyruvate Kinase Deficiency)
This patient has a chronic hemolytic anemia with microcytosis, reticulocytosis, splenomegaly, and negative Coombs test—a constellation highly suggestive of a hereditary red blood cell enzymopathy, most likely pyruvate kinase (PK) deficiency, which requires specific enzyme assays and molecular testing for confirmation. 1
Clinical Reasoning
The key diagnostic features pointing away from acquired causes include:
- Negative direct Coombs test definitively excludes autoimmune hemolytic anemia (AIHA), which would be the primary acquired cause to consider in an adult with hemolysis 2, 3
- Microcytic anemia (MCV 70) is unusual for most hemolytic anemias but characteristic of certain enzymopathies, particularly PK deficiency 1
- Chronic presentation with splenomegaly suggests longstanding hemolysis rather than acute acquired process 4
- Post-cholecystectomy status indicates prior gallstone formation, a common complication of chronic hemolysis from congenital disorders 4
The elevated indirect bilirubin (17 mg/dL), reticulocytosis (6%), and anisocytes confirm active hemolysis 2, 5. Normal liver function tests exclude hepatocellular dysfunction as the cause of jaundice 6.
Essential Immediate Investigations
Hemolysis Confirmation Panel
- Complete hemolysis markers: LDH (elevated), haptoglobin (decreased), and peripheral blood smear review for specific morphology 1, 2
- Fractionated bilirubin to confirm predominantly unconjugated hyperbilirubinemia 6
- Bone marrow examination may show erythroid hyperplasia if diagnosis remains unclear 7
Red Blood Cell Enzyme Assays
- PK enzyme activity assay by spectrophotometry is the reference test for biochemical diagnosis, with activity typically <20% of normal in PK deficiency 1
- Critical timing: Sample must be collected >50 days after any transfusion to avoid donor RBC contamination (estimated 7-14% contamination at 50 days) 1
- Sample handling: Use EDTA anticoagulant, store at 4°C for maximum 14 days, and perform purification on α-cellulose/microcrystalline cellulose column 1
- Reticulocyte correction: Results must account for reticulocyte count by calculating ratio of PK activity to hexokinase activity, as reticulocytes have higher enzyme levels 1
- Glucose-6-phosphate dehydrogenase (G6PD) assay to exclude G6PD deficiency, another common enzymopathy 2, 4
Molecular Testing
- PKLR gene sequencing by Sanger method is highly recommended to confirm diagnosis when PK activity is decreased 1
- Next-generation sequencing (NGS) panels are reliable alternatives, particularly useful in recently transfused patients or when shipping times are prolonged 1
- Testing for intragenic PKLR deletions if standard sequencing shows only one or no mutations despite clinical suspicion 1
Exclusion of Other Hereditary Causes
- Hemoglobin electrophoresis at pH 7.1 to exclude hemoglobin M variants and other hemoglobinopathies 1, 4
- Osmotic fragility test or eosin-5-maleimide (EMA) binding test to exclude hereditary spherocytosis, though spherocytes should be evident on smear 2, 4
- Family history and consanguinity assessment (more common in cytochrome b5 reductase deficiency) 1
Differential Diagnoses to Exclude
Hemoglobin M Variants
- Can present with jaundice, hemolytic anemia, and cyanosis 1
- β-globin variants manifest after 6-9 months when β chains replace fetal γ chains 1
- Diagnosis by hemoglobin electrophoresis at pH 7.1 1
Unstable Hemoglobin Variants
- HbChile (β28 Leu→Met) presents with chronic methemoglobinemia and hemolytic anemia 1
- Can develop very high methemoglobin levels (>90%) after stressor events 1
Hereditary Spherocytosis
- Characterized by spherocytes on smear, family history, and negative Coombs test 2
- Would show increased osmotic fragility 4
Acquired Causes (Lower Probability Given Negative Coombs)
- Drug-induced hemolysis: Detailed medication and toxin exposure history essential 1, 2
- Infection-related hemolysis: Consider hepatitis A, which can cause acute hemolysis with indirect hyperbilirubinemia, reticulocytosis, and negative Coombs test 7
- Microangiopathic hemolytic anemia: Would show schistocytes on peripheral smear 2
Critical Pitfalls to Avoid
- Do not perform enzyme assays within 50 days of transfusion, as donor RBC contamination invalidates results 1
- Do not interpret low-normal PK activity as excluding disease without correcting for reticulocyte count, as young RBCs have higher enzyme activity 1
- Do not assume Gilbert syndrome with this degree of hyperbilirubinemia (17 mg/dL)—Gilbert syndrome never reaches such levels 6
- Do not overlook the microcytosis—this is atypical for most hemolytic anemias and should prompt consideration of enzymopathies 1
- Do not delay molecular testing if enzyme activity is decreased, as genotyping confirms diagnosis and enables genetic counseling 1
Management Plan
Immediate Supportive Care
- Folic acid supplementation (1-5 mg daily) for chronic hemolysis to prevent megaloblastic crisis 4
- Monitor for complications: Iron overload (ferritin, transferrin saturation), gallstones (already addressed with cholecystectomy), and leg ulcers 4
- Transfusion support only if symptomatic anemia or hemoglobin drops to critical levels 1, 4
Definitive Treatment Considerations
- Splenectomy may be curative or significantly reduce transfusion requirements in PK deficiency, though decision requires specialist evaluation 1, 4
- Avoidance of oxidative stressors if G6PD deficiency confirmed (certain drugs, fava beans, infections) 2, 4
- Emerging therapies: Gene therapy and PK activator treatments are in development for PK deficiency 1
Specialist Referral
- Immediate hematology consultation for enzyme assay interpretation, molecular testing coordination, and long-term management planning 1
- Genetic counseling once diagnosis confirmed, particularly important in severe cases for family planning 1