Diagnosis and monitoring

In order to diagnose and monitor patients with PNH, two types of assessment are needed1:

• 1 blood tests designed to diagnose and measure intravascular haemolysis;
• 2 flow cytometry to definitively confirm the diagnosis of PNH.

Under what clinical conditions can PNH be suspected? 27

PNH is a disease with various clinical forms and non-specific symptoms. It is therefore tough to diagnose, explaining why it is not diagnosed on average for 1 to 10 years7. The following clinical presentations can identify patients at risk for whom a diagnosis of PNH would be relevant.

• Intravascular haemolysis
- indicated by haemoglobinuria or high levels of plasma haemoglobin.

• Unexplained haemolysis with one of the following factors:
- iron deficiency;
- abdominal pain or dysphagia;
- thrombosis;
- neutropenia and/or thrombocytopenia.

• Another non-infectious, Coombs-negative acquired haemolytic anaemia without schistocytes.

• Atypical thrombosis:
- unusual sites:

hepatic veins (Budd-Chiari syndrome),
other intraabdominal veins (portal, splenic, splanchnic),
cerebral venous sinuses,
dermal veins;

- with signs of haemolytic anaemia;
- with unexplained cytopenia.

• Myelosuppression:

- suspected or confirmed bone marrow failure or hypoplasia;
- refractory cytopenia with dysplasia of a single line;
- other cytopenia of unknown origin despite adequate investigation.

Clinical picture suggestive of PNH  

Blood tests to perform 1

It is important to monitor these parameters, which are markers of intravascular haemolysis:

• anaemia and increased reticulocytes in the blood indicate peripheral destruction of RBCs;

• LDH is an excellent marker for haemolysis. In case of RBC lysis, these enzymes are released and their blood concentration increases. Monitoring them is therefore important for effective monitoring of PNH;

• the amount of free bilirubin, as a degradant of haemoglobin, increases in the event of haemolysis;

• conversely, free plasma haptoglobin decreases with haemolysis, since this protein combines with free haemoglobin to be eliminated by the reticuloendothelial system10.

Confirming diagnosis: Flow cytometry 27

Diagnosis is based on the detection of a deficiency in GPI-anchored proteins at the surface of at least two lines of blood cells. The results are expressed as a percentage of abnormal cells. The latter establishes the size of the PNH cell clone.

Flow cytometry is the reference diagnostic tool, evidencing and quantifying the total deficiency (type III cells) or partial deficiency (type II cells) in GPI- anchored protein cells.

The clinician can choose between two types of analysis:

• routine analysis is used to diagnose haemolytic PNH. It can detect a PNH clone with a minimum size of 1%;

• high sensitivity analysis is recommended to detect a PNH clone in patients with myelosuppression. It can detect a clone with a minimum size of 0.01%.

It is advisable to perform the test on neutrophils and on at least one other cell line. A monocyte analysis confirms the results. Detecting the clone in two lines increases diagnostic confidence.

However, limiting analyses to RBCs is not advisable. Haemolysis and blood transfusions can lead to a significantly underestimated PNH clone size or the diagnosis being missed altogether. This analysis is nevertheless useful as it is the best option to identify type II cells.

To identify PNH leukocytes, it is advised (routine analysis), if not essential (high sensitivity analysis) to use two markers characterising GPI anchors. One of the most relevant markers is FLAER (Fluorescein-labelled proaerolysin), an inactivated bacterial protein that specifically binds to the leukocyte GPI anchor. One marker is sufficient to detect the PNH clone on RBCs, preferably an anti-CD59 antibody.

Flow cytometry must be carried out within 24 to 48 hours of taking the blood to avoid impairing the antigen expression on the surface of leukocytes.

The flow cytometry report must contain:

• • the clone size of each cell line tested;
• • breakdown of RBCs into types I, II and III;
• • the sensitivity level used;
• • the results of previous tests to monitor changes in clone size.

Type III and type II PNH clone on RBCs


Type III (blue) and type II (green) PNH clone on granulocytes
 

Monitoring of PNH patients 1,27

Patients diagnosed with PNH must be regularly monitored through lab work, clinical tests and flow cytometry.

Regular lab work will be useful to assess the extent of intravascular haemolysis. LDH is a relevant marker for this.

A suitable radiological assessment is necessary where underlying thrombosis is suspected.

Monitoring serum creatinine helps monitor renal function.

If the disease is stable, an annual diagnosis using flow cytometry is sufficient. However, any changes in clinical and biological parameters should lead to closer monitoring. Indeed, an increase in PNH clone size in a patient with myelosuppression may indicate progression to haemolytic PNH.

Summary

> Flow cytometry must be performed in all patients with:

• • Coombs-negative haemolytic anaemia;
• • haemoglobinuria;
• • unexplained thrombosis;
• • bone marrow failure;
• • myelodysplastic syndrome with refractory anaemia;
• • unexplained cytopenia.

> Flow cytometry must be performed on at least two cell lines, including neutrophils, with at least two different markers.

> PNH patients require regular clinical and biological monitoring. LDH monitoring is essential as a relevant marker of haemolysis.