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  How should PNH flow cytometry assays be reported?

The following components are recommended for a PNH (paroxysmal nocturnal hemoglobinuria) flow cytometry report:

  1. Report if a PNH clone present or absent. It is important to be clear and to avoid potentially misleading or ambiguous terminology. A report stating that a CD59 test is negative may imply to some providers that the target population is negative for the glycosylphosphatidylinositol (GPI)-linked marker CD59 (thus indicating a PNH clone) or that no CD59 absence is seen (thus indicating the absence of a PNH clone).
  2. Report the PNH clone size in the red blood cells (RBCs), including both total PNH clone size as well as the percentages for type II and type III PNH populations. There is clinical significance associated with the proportion of type II and type III RBCs. Type I RBCs are normal red blood cells with bright CD59 expression and a lifespan of approximately 120 days. Type III PNH RBCs have complete CD59 deficiency, which results in no protection from complement-mediated lysis and a shortened lifespan of 10-15 days. Type II PNH RBCs have partial CD59 deficiency resulting in partial protection from complement mediated lysis. Their lifespan is intermediate between type I and type III PNH RBCs and depends on the level of CD59 deficiency, often as a result of multiple mutations. Since the clinical significance of type II versus type III PNH RBCs is well established, it is recommended to report them both as individual and combined percentages.
  3. Report the PNH clone size in both lineages for the white blood cells (WBCs; including neutrophils/granulocytes and monocytes). The PNH monocyte clone is often larger than the granulocyte/neutrophil PNH clone, and thus reporting only the PNH granulocyte/neutrophil clone may underestimate the PNH clone size in the WBCs. Granulocytes/neutrophils and monocytes may also show the presence of Type II populations but the clinical significance of these populations has not been established at this time. It is therefore recommended to report only the total PNH clone size in the neutrophils/granulocytes and monocytes.
  4. Interpretive terminology of reporting PNH clones:
    1. PNH population >1%: "PNH clone"
    2. PNH population 0.1% to 1%: "minor population of PNH cells" or "minor PNH clone"
    3. PNH population <0.1%: "rare cells with GPI deficiency" or "rare cells with PNH phenotype
  5. List all gating and diagnostic markers used for the PNH assay
  6. State the sensitivity for the neutrophil/granulocyte assay and the RBC assay on the report. It is important to include this information to the provider. The recommended sensitivity for RBCs is 0.01% and for granulocytes/neutrophils a sensitivity of 0.01%-0.05%. A sensitivity of 1% means that the possibility of a minor clone (less than 1%) cannot be excluded based on this sensitivity.
  7. Histograms or dotplots: These are optional as many laboratory information systems are not able to include images. However, the dotplots may provide powerful visual supportive evidence of the PNH clone, and show evidence of the quality of the assay.


  1. Borowitz MJ, Craig FE, DiGiuseppe JA, Illingworth AJ, Rosse W, Sutherland DR, Wittwer CT, Richards SJ. On behalf of the Clinical Cytometry Society. Guidelines for the Diagnosis and Monitoring of Paroxysmal Nocturnal Hemoglobinuria and Related Disorders by Flow Cytometry. Cytometry Part B 2010; 78B:211-230.
  2. Sutherland DR, Keeney M and 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.
  3. Davis BH, Keeney M, Brown R, Illingworth AJ, King MJ, Kumpel B, Meier ER, Sandler SG, Shaz BH, Sutherland DR. 2014. Red Blood Cell Diagnostic Testing Using Flow Cytometry; Approved Guideline - Second Edition. Clinical and Laboratory Standards Institute. Document H52-A2 (ISBN Number: 1-56238-957-2).
  4. Sutherland, D.R., Illingworth, A., Keeney, M., and Richards, S.J. 2015. High-sensitivity detection of PNH red blood cells, red cell precursors, and white blood cells. Curr. Protoc. Cytom. 72:6.37.1-6.37.29.

Sample report for patient with no PNH clon

Sample report for patient with PNH clone

Sample report for patient with follow-up of PNH clone


Author: Andrea Illingworth