The Role of Flow Cytometry in the Diagnosis of Paroxysmal Nocturnal Hemoglobinuria in the Clinical Laboratory

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Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell disorder closely related to aplastic anemia. Hemolytic anemia and life-threatening thromboses are common features in many patients. Rapid diagnosis is highly desirable and flow cytometry plays a key role in the laboratory investigation of PNH. By demonstrating absence of cell membrane glycosylphosphatidylinositol-anchored proteins from granulocytes or red cells, a definitive diagnosis of PNH can be established. This can have a considerable impact on patient management and outcome. As with all rare diseases, internal and external quality assurance is essential for good laboratory practice and to fulfill the requirements of national laboratory accreditation schemes.

Section snippets

Flow cytometry: general considerations

The availability of a diagnostic flow cytometry assay means that for patients who have suspected PNH, a definitive diagnosis can be established rapidly, resulting in improved patient management and prognosis. As PNH is a rare condition, it is essential to demonstrate deficiency of at least two GPI-linked antigens from red cells and granulocytes to establish a diagnosis confidently. For granulocytes, this is achieved best by multicolor cytometry, although more complex combinations allow

Diagnosis of paroxysmal nocturnal hemoglobinuria by flow cytometry: clinical value

Once a diagnosis of PNH is established on the basis of clinical findings and flow cytometry results, serial monitoring of the peripheral blood PNH clone can provide important information that may influence patient management [28]. Patients who have type III red cells (greater than 20%) almost always show signs of intravascular hemolysis, whereas patients who have granulocyte PNH clones (>50%) have a significant risk of thrombosis, and prophylactic anticoagulation is an important consideration

Internal and external quality assurance

As with any diagnostic flow cytometry assay, internal and external quality assurance forms an integral part of good clinical laboratory practice. Essential steps in establishing the validity of the assay should include extensive characterization of the antibody test combinations on normal peripheral blood samples and then validated on patients who have a confirmed diagnosis of PNH. As screening for PNH by flow cytometry is a clinical diagnostic test, but may be requested infrequently, it is

Summary

PNH is a rare disease in which detection of GPI-deficient blood cells (PNH clones) by flow cytometry is an essential component of the diagnostic process. Recently, the role of flow cytometry in this disorder has been extended to play a pivotal role in the clinical trial and assessment of treatment response of a novel monoclonal antibody therapy for PNH [36]. Eculizumab is a monoclonal antibody against the complement component C5 and has been used successfully to inhibit intravascular hemolysis

References (36)

  • C. Hall et al.

    Primary prophylaxis with warfarin prevents thrombosis in paroxysmal nocturnal hemoglobinuria

    Blood

    (2003)
  • Hill A, Platts PJ, Smith A, et al. The incidence and prevalence of paroxysmal nocturnal hemoglobinuria (PNH) and...
  • P. Hillmen et al.

    Natural history of paroxysmal nocturnal hemoglobinuria

    N Engl J Med

    (1995)
  • M. Bessler et al.

    Paroxysmal nocturnal hemoglobinuria (PNH) is caused by somatic mutations in the PIG-A gene

    EMBO-J

    (1994)
  • C. Hall et al.

    The glycosylphosphatidylinositol anchor and paroxysmal nocturnal haemoglobinuria/aplasia model

    Acta Haematol

    (2002)
  • S.J. Richards et al.

    The application of flow cytometry to the diagnosis of paroxysmal nocturnal hemoglobinuria

    Cytometry

    (2000)
  • C.E. van der Schoot et al.

    Deficiency of glycosyl-phosphatidylinositol-linked membrane glycoproteins of leukocytes in paroxysmal nocturnal hemoglobinuria, description of a new diagnostic cytofluorometric assay

    Blood

    (1990)
  • J.M. Navenot et al.

    Expression of glycosyl-phosphotidylinositol-linked glycoproteins in blood cells from paroxysmal nocturnal hemoglobinuria patients. A flow cytometry study using CD55, CD58 and CD59 monoclonal antibodies

    Leuk Lymphoma

    (1996)

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