What is the recommended test to diagnose pyruvate kinase deficiency?

How to Test for Pyruvate Kinase Deficiency

The diagnostic approach to pyruvate kinase (PK) deficiency requires a two-step process: start with RBC PK enzyme activity assay by spectrophotometry as the reference biochemical test, followed by confirmatory molecular testing of the PKLR gene via Sanger sequencing or NGS panels. 1

Initial Clinical Suspicion

Suspect PK deficiency in patients presenting with:

• Chronic anemia with splenomegaly and/or jaundice with normal or near-normal red cell morphology (this distinguishes it from membranopathies)
• Unexplained transfusion-dependent anemia
• Severe neonatal indirect hyperbilirubinemia
• High reticulocyte counts in splenectomized patients without diagnosis 1

Step 1: Essential Laboratory Workup

Before ordering specific PK testing, obtain:

Mandatory baseline tests:

• Complete blood count with reticulocyte count
• RBC morphology
• Markers of hemolysis: LDH, unconjugated bilirubin, haptoglobin (note: haptoglobin only useful after 6 months of age), reticulocyte count 1

Differential diagnosis exclusion:

• Rule out acquired hemolytic anemia (AIHA, PNH)
• Exclude membranopathies, congenital dyserythropoietic anemias (CDAs), unstable hemoglobins, and other RBC enzymopathies 1

Step 2: RBC PK Enzyme Activity Assay

This is the reference biochemical test with 98.7% consensus agreement among experts 1.

Critical Technical Requirements:

Sample specifications:

• Anticoagulant: EDTA is standard; ACD, CPD, or heparin acceptable 1
• Storage: Stable at 4°C for up to 21 days (maximum 14 days if calculating PK:HK ratio due to hexokinase instability) 1
• Cannot be performed on whole blood - requires purification on α-cellulose/microcrystalline cellulose column (buffy coat removal is alternative) 1

Critical Interpretation Caveats:

Transfusion interference:

• Wait minimum 50 days after last RBC transfusion before testing (reduces donor RBC contamination to 7-14%) 1
• If transfusion history unknown, interpret results with extreme caution 1

Reticulocyte correction:

• Reticulocyte count must be considered when interpreting results, especially with low-normal PK activity
• Compare with control sample having same reticulocytosis OR calculate PK:hexokinase ratio 1
• The PK:HK ratio has superior diagnostic sensitivity (98%) compared to PK enzyme activity alone (90%) 2

Common Pitfall:

The enzyme assay can yield false negatives in recently transfused patients or when reticulocyte correction is not performed. The PK:HK ratio addresses the reticulocyte issue more reliably than PK activity alone 2.

Step 3: Confirmatory Molecular Testing

When PK enzyme activity is decreased, PKLR gene sequencing is highly recommended (88.3% consensus) to confirm diagnosis 1.

Testing Strategy:

First-line molecular approach:

• Sanger sequencing of PKLR gene for patients with decreased PK activity 1

NGS panels are particularly indicated for:

• Neonates (if family study unavailable)
• Transfusion-dependent or recently transfused patients (where enzyme assay unreliable)
• Samples with prolonged shipping times 1

When Genotype-Phenotype Discrepancies Occur:

If suspected PK deficiency with only one or no mutations detected:

• Test for large intragenic PKLR deletions
• Consider KLF1 gene mutations (can cause PK deficiency phenotype)
• Re-evaluate other causes of hemolysis with specific tests or broader NGS platform 1

Important note: A significant minority of patients have occult mutations in non-coding regions of PKLR missed on standard genetic tests 3. Genetic testing may be required even when enzyme assay appears normal if clinical suspicion remains high 4.

Algorithmic Approach Summary:

1. Clinical suspicion → Obtain hemolysis markers + CBC + exclude other causes
2. If hemolysis confirmed → Order RBC PK enzyme activity (ideally PK:HK ratio)
   • Ensure ≥50 days since transfusion
   • Document reticulocyte count
3. If PK activity decreased → Confirm with PKLR gene sequencing
4. If genotype incomplete → Test for large deletions, KLF1 mutations, or use NGS
5. If recently transfused or neonate → Consider proceeding directly to NGS panel

The complementary use of enzyme activity and molecular testing maximizes diagnostic accuracy, with molecular testing becoming increasingly important given the availability of disease-modifying therapies like mitapivat that require confirmed diagnosis 1, 5, 6.