Test for PNH (Flow Cytometry)

High-sensitivity flow cytometry* (HSFC) is the gold standard diagnostic test for PNH1,3,8

  • The ICCS recommends multiparameter HSFC to diagnose PNH1
  • HSFC is performed on peripheral blood—not bone marrow1,3
  • Labs should perform PNH HSFC on white blood cells (WBCs) (using >1 reagent) and on red blood cells (RBCs)1
    • Granulocytes using FLAER/CD24 combinations give most accurate estimate of PNH clone size1,3,7,9
    • Evaluation of RBCs alone may not be accurate due to hemolysis and the dilution effect of transfusions1,3,10,11

Clear reporting is essential for appropriate clinical decisions and should include1,9:

  • Clone size for each cell lineage (ie, granulocytes, monocytes, and RBCs)
  • Proportion of Type II and III (percentage of GPI-deficient cells) as well as total RBCs
  • Sensitivity level used (0.01% high-sensitivity analysis is ideal)
  • All previous flow results in order to monitor clonal expansion

Sample of lab reports

Sample of lab reports

Sample of lab reports

  • The International PNH Interest Group (IPIG) recommends routine monitoring every 6 to 12 months of patients who have an identified clone3

* Detects PNH cells down to a 0.01% clone size.

Labs should perform PNH HSFC on RBCs, and on WBCs using >1 reagent1,2

  • Evaluation of RBCs alone may underreport clone size due to hemolysis and the dilution effect of transfusions1,3-5
  • “Routine” CD55- and/or CD59-based approaches are neither accurate nor sensitive below the 1%-2% clone size1
  • Granulocytes give most accurate estimate of PNH clone size1,3,6
    • FLAER/CD24 combinations recommended to detect PNH granulocytes2
  • Low RBC clone size compared to WBC clone size indicative of intravascular hemolysis1

PNH lab sample

  • The International PNH Interest Group (IPIG) recommends that high-sensitivity flow cytometry for PNH be performed3:
    • After establishment of a PNH diagnosis:
      • Every 6-12 months
    • In patients with AA or RA-MDS:
      • At diagnosis and yearly, regardless of clone identification
Doctor Patton video

Listen to Jeff Patton, MD, discuss the importance of a full clinical assessment in diagnosing PNH.

Clinical Diagnostic Cheat Sheet

For more information about performing a full clinical assessment to diagnose PNH, download the Clinical Diagnostic Cheat Sheet.

References: 1. Borowitz MJ, Craig FE, DiGiuseppe JA, et al; for Clinical Cytometry Society. Cytometry Part B. 2010;78B:211-230. 2. Sutherland DR, Keeney M, Illingworth A. Cytometry Part B. 2012;82B:195-208. 3. Parker C, Omine M, Richards S, et al; for International PNH Interest Group. Blood. 2005;106:3699-3709. 4. Hochsmann B, Rojewski M, Schrezenmeier H. Ann Hematol. 2011;90:887-899. 5. Sutherland DR, Kuek N, Azcona-Olivera J, et al.Am J Clin Pathol. 2009;132:564-572. 6. Brodsky R, Schrezenmeier H, Muus P, et al. Blood. 2009:114: Abstract 3007. 7. Brodsky RA. How I treat paroxysmal nocturnal hemoglobinuria. Blood. 2009;113:6522-6527. 8. Sharma VR. Paroxysmal nocturnal hemoglobinuria: pathogenesis, testing, and diagnosis. Clin Adv Hematol Oncol. 2013;11:1-11. 9. Sutherland DR, Keeney M, Illingworth A. Practical guidelines for the high-sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry. Cytometry Part B. 2012;82B:195-208. 10. Hochsmann B, Rojewski M, Schrezenmeier H. Paroxysmal nocturnal hemoglobinuria (PNH): higher sensitivity and validity in diagnosis and serial monitoring by flow cytometric analysis of reticulocytes. Ann Hematol. 2011;90:887-889. 11. Sutherland DR, Kuek N, Azcona-Olivera J, et al. Use of a FLAER-based WBC assay in the primary screening of PNH clones. Am J Clin Pathol. 2009;132:564-572. 12. Data on file, Alexion Pharmaceuticals, Inc.; 2009.