Introduction

Pyruvate kinase deficiency (PKD) is an inherited blood disorder that is uncommon and leads to a form of chronic anaemia caused by the premature breakdown of red blood cells. Although the disorder affects approximately 1 in 100,000 to 1 in 300,000 individuals worldwide, its true incidence may be less than this since diagnosis is challenging.¹ PKD is characterised by a deficiency of the pyruvate kinase enzyme, a vital component in energy production in red blood cells. The resulting energy shortage makes the cells susceptible and causes them to be destroyed prematurely.²

This article provides an overview of the diagnosis of PKD, the most significant laboratory tests, the challenges involved, and the implications for the creation of future treatment.

What is pyruvate kinase deficiency?

Pyruvate kinase deficiency is an inherited disease in which a lack or dysfunction of the enzyme pyruvate kinase disrupts the energy-generating process in red blood cells. In a normal process, the enzyme finishes the final step in the breakdown of glucose (glycolysis) to form adenosine triphosphate (ATP)—the energy currency of the cell. In PKD, the deficient production of ATP results in premature red blood cell destruction and subsequently chronic hemolytic anaemia. The clinical course is extremely variable, and a few patients experience mild symptoms, whereas others develop severe anaemia that requires repeated medical interventions.³

Common clinical features

The clinical features of PKD are subtle yet significant. These include:

• Chronic fatigue and weakness: Chronic fatigue is a universal symptom of the reduced oxygen-carrying capacity of the blood
• Jaundice: Yellowing of the skin and eyes, caused by an accumulation of bilirubin from the haemolysis of the red blood cells
• Splenomegaly: Splenic enlargement, which results from its increased activity in filtering out the destroyed red blood cells
• Gallstones: The development of gallstones is a frequent complication associated with high levels of bilirubin
• Iron overload: Excess iron may be stored even in those patients who do not require regular transfusions, as the body absorbs additional iron to replace the chronic anaemia
• Bone health issues: The disease may result in decreased bone density over the years, resulting in osteopenia or osteoporosis⁴

As these symptoms are also associated with other forms of haemolytic anaemia, the diagnosis process has to be meticulous and systematic to properly diagnose PKD.

The diagnosis process: Step by step

The diagnosis of PKD typically comes after a combination of clinical evaluation and a set of laboratory tests. The aim is to confirm the presence of haemolytic anaemia, rule out more common disorders, and then use special tests to diagnose PKD.

Clinical evaluation

Diagnosis begins with a thorough clinical evaluation by a healthcare provider. This evaluation includes:

Medical history

A detailed history of chronic anaemia, bouts of neonatal jaundice, and any background history of unexplained pallor or fatigue. Family history may not always be informative even though PKD is inherited due to its autosomal recessive inheritance.

Physical examination

An examination for physical signs such as pallor, jaundice of the eyes or skin, and splenomegaly. Gallstones may also be detected on examination.⁴

This initial step is to raise a clinical suspicion of PKD, especially if the symptoms are chronic.

Basic laboratory tests^3,5

After clinical evaluation, basic blood tests are requested to assess for signs of haemolytic anaemia. These are:

Complete blood count (CBC)

CBC is used to measure the haemoglobin level, which is typically low in PKD patients. It also provides information regarding the red blood cell count and reticulocyte count (an indicator of how actively the bone marrow is producing new cells).

Peripheral blood smear

This test involves examining a tiny drop of blood under a microscope to assess the shape and appearance of the red blood cells. Although PKD abnormalities may be subtle, certain abnormal cell shapes and a moderate quantity of "burr cells" (echinocytes) may be observed.

Markers of haemolysis

Biochemical tests such as bilirubin, lactate dehydrogenase (LDH), and haptoglobin levels are utilised. Elevated indirect bilirubin and LDH levels and low haptoglobin levels establish that haemolysis has occurred.

Iron studies

These examine the body's iron status by measuring serum ferritin and transferrin saturation. Ferritin increase may indicate iron overload, a common complication of chronic haemolysis.

While these tests validate that haemolysis is occurring, they are not sufficient to diagnose PKD by themselves.

Specialised diagnostic testing^1,3

Once the preliminary tests indicate haemolysis, additional, more specific tests must be conducted to diagnose PKD.

Pyruvate kinase enzyme activity test

The pyruvate kinase enzyme activity test is the standard for diagnosing PKD. Essentially, this test identifies how effectively the enzyme functions in red blood cells. Normal enzyme activity ranges from 8 to 18 IU/g haemoglobin. The activity levels in individuals with PKD are typically below 30% of normal. Keep in mind that some factors, such as recent blood transfusions or a high number of young red blood cells (reticulocytes), can sometimes lead to a normal test result even if the enzyme is deficient.

Genetic testing

DNA testing is used to diagnose PKD by identifying mutations in the PKLR gene that holds the instructions for making the pyruvate kinase enzyme. To confirm that the low enzyme activity is genetic in nature. The test is not influenced by factors that may affect enzyme activity, such as recent transfusions. A blood sample is drawn to examine the DNA for alterations in the PKLR gene. A positive test verifies that the enzyme deficiency is indeed due to an inherited defect. Genetic testing is conclusive and is required for interpretation of the condition, guiding treatment, and providing genetic counselling to family members.

Additional tests

In certain instances, additional testing is performed to rule out other causes of haemolytic anaemia. These tests are:

• Osmotic fragility test: To exclude other red blood cell membrane diseases
• G6PD activity test: To ensure that another enzyme deficiency (G6PD deficiency) is not the cause of the haemolysis
• Haemoglobin electrophoresis: To detect abnormal variants of haemoglobin that can present similarly to PKD

All these are part of a further workup to ensure that the diagnosis of PKD is accurate.

Future treatment: Looking to the future⁵

In addition to refining diagnostic methods, research is on the march to treatments that go to the root of PKD's cause. Two avenues hold promise:

PK activators

PK activators are a class of drugs that are used to activate the remaining activity of the pyruvate kinase enzyme in red blood cells. One of the drugs in this class that has shown promise in clinical trials is a drug known as mitapivat. Mitapivat works by stimulating the generation of energy in red blood cells, a move that can boost haemoglobin levels and reduce the severity of anemia. The therapy is an upgrade in that it treats the underlying cause of the enzyme deficiency and not the symptoms.

Gene therapy

Gene therapy is an emerging approach that addresses the genetic mutation responsible for PKD. By replacing or fixing the defective PKLR gene, gene therapy could restore normal enzyme function, offering a long-term solution or cure. Though in the research stage, early research in animal models has been encouraging, and clinical trials are underway.

Personalised medicine

Technological advances in genetic diagnosis and next-generation sequencing are facilitating access to personalised medicine for PKD. This approach enables personalising treatment based on the individual patient's unique genetic characteristics, theoretically able to determine which patients are likely to benefit most from treatments like PK activators.

The importance of a correct diagnosis

A correct diagnosis of PKD is significant for several reasons:

• Early intervention: Early detection of PKD makes it possible to manage it in good time, thereby preventing iron overload—a condition in which excessive iron accumulation in organs like the liver and heart
• Guided treatment: As soon as the diagnosis is established, healthcare providers can choose the best treatment for the patient, such as ongoing monitoring, blood transfusions, or splenectomy (splenectomy) when and if the case necessitates it
• Family counseling: Since PKD is hereditary, a definite diagnosis is essential for genetic counselling. This notifies families about future children's risk and other carriers or affected relatives in the family
• Access to new therapies: Accurate diagnosis opens doors to new therapies, such as PK activator clinical trials and gene therapy, for improved future outcomes

A simplified diagnostic pathway

In short, the diagnosis of pyruvate kinase deficiency typically takes this route:

• Clinical evaluation: Physicians evaluate symptoms like persistent fatigue, jaundice, and splenomegaly, as well as a comprehensive patient history
• Basic blood tests: Procedures such as the complete blood count (CBC), reticulocyte count, and bilirubin, LDH, and haptoglobin measurements serve to ensure that haemolytic anaemia exists
• Specialised testing: A specific blood test is used to quantify the activity of the pyruvate kinase enzyme. Reduced enzyme activity, if confirmed by genetic testing of the PKLR gene, confirms the diagnosis of PKD
• Exclusion of other conditions: Additional tests rule out other causes of hemolytic anemia, and the diagnosis is thus definite⁶

Summary

Pyruvate kinase deficiency (PKD) is a rare genetic blood disorder that results in chronic anaemia as a result of the premature breakdown of red blood cells. While its symptoms are subtle and rather diverse, modern testing—combining routine blood tests, an enzyme activity test, and genetic testing—is now able to make an accurate diagnosis. It is essential to diagnose it early because this allows for appropriate management to prevent complications such as iron overload and gallstones, and permits new treatments such as PK activators and gene therapy. Advances in technology for diagnostics and targeted therapies promise improved quality of life and cures. Clinicians are encouraged to include PKD in the evaluation of unexplained chronic anaemia to offer timely and effective therapy.