For diagnosing clinicians

The need for early diagnosis

Early diagnosis is essential for improved patient management and prognosis.1

  • Thrombosis and renal failure are leading causes of death.2,3
Survival of patients with PNH compared to control
Survival of patients with PNH compared to control2
Study description: Researchers followed 80 consecutive patients with PNH referred to Hammersmith Hospital in London, United Kingdom.2 The patients were treated with supportive measures such as oral anticoagulant therapy after established thromboses and transfusions.
PNH, paroxysmal nocturnal haemoglobinuria
  • Diagnosis is typically delayed from 1 to more than 10 years.4

Identify patients with PNH early within high-risk groups2-23

aAnaemia, neutropenia, thrombocytopenia or pancytopenia; bUnusual sites include hepatic veins and arteries (Budd-Chiari syndrome), other intra-abdominal veins and arteries (portal, splenic, splanchnic), cerebral sinuses and dermal veins; cDetects PNH cells down to a 0.01% clone size
IDA, iron deficiency anaemia; LDH, lactate dehydrogenase; MDS, myelodysplastic syndrome
The information on this page is intended as an educational resource for healthcare professionals. It does not replace a healthcare provider’s professional judgement or clinical diagnosis.
Nearly 1 in 5 patients (19%) with haemolytic anaemia have a PNH clone23 Almost half of patients (45%) with aplastic anaemia have a PNH clone23 In PNH, 40–67% of deaths are due to venous or arterial thrombosis20

High-sensitivity flow cytometry is the gold standard for PNH testing: rapid, accurate and highly reliable1,11,24,25

1. Prepare your sample
Specimen source – Peripheral blood (not bone marrow)

Sample volume – For RBCs, whole anti-coagulated peripheral blood should be diluted 1:100 with PBS and a minimum of 100 µl of the diluted sample should be used. For WBCs, a minimum of 1 mL; 3mL is adequate for most testing, though more may be needed if WBC count is very low

Maximum sample age – <48 h for RBCs and WBCs

Sample storage – 4°C after 24 h

Anticoagulant – EDTA is the preferred/most tested agent, but heparin or ACD are also acceptable

Cell lineages – RBCs in all cases, or at least in those samples positive for a PNH clone following WBC analysis. RBCs, neutrophils and monocytes should always be evaluated. No analysis of lymphocytes1,25

2. Request a clear report
  • It is essential for appropriate clinical decisions in combination with other clinical findings25
  • When ordering a PNH flow cytometry, request:25,26
    • Results for both white blood cell populations
    • PNH clone size (ie proportion [%] of GPI-deficient cells) in each lineage tested
      • For RBCs, total clone size as well as the proportions of Type II and Type III cells should be reported
    • Clear terminology: ‘PNH clone’, ‘minor PNH clone’ or ‘rare cells with PNH phenotype/GPI-deficiency’
    • Sensitivity level used for each lineage tested
    • Previous flow results to monitor change of clone size over time
    • Indications for further testing
3. Monitor your PNH patients
  • Clone size increased in 40% (10/25) of patients with PNH clone size between 0.11% and 10%27
  • International PNH Interest Group (IPIG) recommends routine monitoring every 6 to 12 months of patients who have an identified clone28
  • International Clinical Cytometry Society guidelines, consensus guidelines and the IPIG recommend continued monitoring of patients at higher risk for PNH5,25,28
ACD, acid citrate dextrose; EDTA, ethylenediaminetetraacetic acid; PBS, phosphate buffered saline, RBCs, red blood cells

  • References
    1. Sutherland DR et al. Cytometry B Clin Cytom 2018; 94: 23-48.
    2. Hillmen P et al. N Engl J Med 1995; 333: 1253-1258.
    3. Socié G et al; for the French Society of Haematology. Lancet 1996; 348: 573-577.
    4. Dacie JV, Lewis SM. Ser Haematol 1972; 5: 3-23.
    5. Dezern AE, Borowitz MJ. Cytometry B Clin Cytom 2018; 94: 16-22.
    6. Brodsky RA. Blood Rev 2008; 22: 65-74.
    7. Brodsky R. In: Hoffman R et al, eds. Hematology: Basic Principles and Practices. 4th ed. Philadelphia, PA: Elsevier Churchill Livingstone; 2005: 419-427.
    8. Rother RP et al. JAMA 2005; 293: 1653-1662.
    9. Hillmen P et al. Am J Hematol 2010; 85: 553-559.
    10. Rachidi S et al. Eur J Intern Med 2010; 21: 260-267.
    11. Kelly R et al. Ther Clin Risk Manag 2009; 5: 911-921.
    12. Hill A et al. Br J Haematol 2007; 137: 181-192.
    13. Adams T et al. Dig Dis Sci 2002; 47: 58-64.
    14. Rosse WF. Paroxysmal nocturnal hemoglobinuria. In: Hoffman Ret al, eds. Hematology: Basic Principles and Practice. 3rd ed. New York, NY: Churchill Livingstone; 2000: 331-342.
    15. Rother RP et al. Nat Biotechnol 2007; 25: 1256-1264. [Published correction appears in Nat Biotechnol 2007; 25: 1488].
    16. Nishimura J-I et al. Medicine 2004; 83193-207.
    17. Lee JW et al. Int J Hematol 2013; 97: 749-757.
    18. Brodsky RA. Blood 2014; 124: 2804-2811.
    19. Hill A et al. Blood 2013; 121: 4985-4996.
    20. Hillmen P et al. Blood 2007; 110: 4123-4128.
    21. Thomason et al. Am J Clin Pathol 2004; 122: 128-134.
    22. Killick SB et al. Br J Haematol 2016; 172: 187-207.
    23. Morado M et al. Cytom B Clin Cytom 2017; 92: 361-370.
    24. Sharma VR. Clin Adv Hematol Oncol 2013; 11: 1-11.
    25. Illingworth A et al. Cytometry B Clin Cytom 2018; 94: 49-66.
    26. Davis BH et al. CLSI H52-A2 Red Blood Cell Diagnostic Testing Using Flow Cytometry; Approved Guideline, 2nd ed. Wayne, PA: Clinical and Laboratory Standards Institute. 2014.
    27. Movalia M et al. Poster 1033 presented at ASH, San Diego, CA; 10-13 December, 2011.
    28. Parker C et al; for International PNH Interest Group. Blood 2005; 106: 3699-3709.