Briefly about PNH - EXBIO DryFlowEx PNH High Sensitivity Kit (CE IVD) Coming soon!

Among normal hematopoietic stem cells a somatic mutation of PIG-A gene may arise causing the cells to lose the ability to synthesize glycosyl phosphatidyl-inositol (GPI) anchor forming a PNH clone. All blood cells originating from a PNH clone lack surface GPI‑anchored proteins causing multiple negative health effects.
One of them is complement-mediated attack on red blood cells that are not protected by presence of GPI-anchored CD55 and CD59.
A side effect of the above mentioned mutation is, unfortunately, a growth advantage over normal cells, resulting in propagation of PNH clones.
Negative health effects include intravascular hemolysis, activation of coagulation system with increased risk of thrombosis and cytopenias related to bone marrow failure. PNH severity depends on number of PNH cells among normal healthy blood cells. The most serious disorder (>25% of PNH cells) happens only to 2-5 people among one million, but the less severe forms with small PNH clones are much more frequent and need to be identified soon to prevent aggravation of the disease. Although PNH (paroxysmal nocturnal hemoglobinuria) was called after nighttime hemoglobinuria with paroxysmal character, the name is misleading, as the typical hemolysis does not need to be observed at diagnosis of some forms of PNH and when there is a chronic and symptomatic red blood cell destruction, the resulting hemoglobinuria usually is not paroxysmal and occurs not only during nighttime.
  
Fig. 1: An example of GPI-anchored proteins.
 
 
Analysis of white blood cell PNH clones:
CD45, a general white blood cell marker, helps to exclude  debris and non-lysed erythrocytes. CD15 (neutrophil granulocytes) together with CD64 (monocytes) serve as backbone markers for easy gating. Although CD15 is expressed in lower quantity also on monocytes and CD64 in case of infection also on neutrophils, the neutrophil and monocyte gates can be easily set when plotted with SSC. The remaining four markers, namely GPI anchor, CD157, CD24, and CD14, serve to distinguish between PNH clones and normal cells. Proaerolysin binds to all surface sites expressing GPI anchor, hence it cannot bind to surface of a PNH clone. CD157, CD24, and CD14 are particular GPI-anchored proteins, that are expressed on healthy cell types (see fig. 2), and are not present on PNH clones.
 
Analysis of red blood cell PNH clones:
As a general red blood cell backbone marker not-bound through GPI anchor, glycophorin A or CD235a is used. GPI-anchored CD59 serves to distinguish between Type III PNH erythrocytes (negative with total loss of CD59), Type II PNH erythrocytes (weak CD59 expression) and Type I erythrocytes (CD59 positive healthy cells). CD71 is also a backbone marker identifying immature red blood cells (reticulocytes) and distinguishes them from erythrocytes. Reticulocytes are then distinguished by their expression of CD59 into Type III, Type II or Type I cells. This allows precise erythrocyte PNH clone analysis in patients receiving blood transfusions.

PNH WBC (white blood cell) 7 color panel:

 
PNH RBC (red blood cell) 3 color panel:

 
Fig. 2: Table of reactivity of PNH panel components with particular blood cells relevant for PNH analysis.  x = signal on both healthy and PNH clones, xx = higher signal (x) = signal on healthy, but not on PNH clones, (xx) = higher signal  * = signal in case of infection  
 
 
DryFlowEx PNH High-Sensitivity Assay Kit:
The above mentioned panels were used within DryFlowEx PNH High-Sensitivity Assay Kit (coming soon, cat. no. ED7750), which enables high sensitivity detection and enumeration of PNH clones in human peripheral blood. The following parameters are evaluated:

• Percentage of PNH cells (CD59 dim or CD59-) from all erythrocytes (CD235+CD71-)

• Percentage of PNH cells (CD59 dim or CD59-) from all reticulocytes (CD235+CD71+)

• Percentage of PNH cells (CD14-CD157-Proaerolysin-) from all monocytes (CD45+CD64+)

• Percentage of PNH cells (CD24-CD157-Proaerolysin-) from all neutrophils (CD45+CD15+)

 
SingleFlowEx Proaerolysin Alexa Fluor® 488  (Cat. No.: ED7051, already available from stock...)

• SingleFlowEx Proaerolysin Alexa Fluor® 488 is a fluorochrome-labeled recombinant protein single reagent for use in laboratory-developed multicolor flow cytometry tests in order to examine GPI deficiency in human clinical samples. SingleFlowEx Proaerolysin Alexa Fluor® 488 can be used in various multicolor combinations. Laboratory shall establish its performance characteristics in conjunction with other reagents in normal and pathological samples.
• Contents 1 vial of monoclonal antibody reagent. The content (1 ml) is sufficient for min. 200 tests.

Further reading:
Sutherland DR, Kuek N, Davidson J, Barth D, Chang H, Yeo E, Bamford S, Chin-Yee I, Keeney M: Diagnosing PNH with FLAER and multiparameter flow cytometry. Cytometry B Clin Cytom. 2007 May;72(3):167-77.
 
Borowitz MJ, Craig FE, Digiuseppe JA, Illingworth AJ, Rosse W, Sutherland DR, Wittwer CT, Richards SJ: Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Cytometry B Clin Cytom. 2010 Jul;78(4):211-30.
 
Sachdeva MU, Varma N, Chandra D, Bose P, Malhotra P, Varma S: Multiparameter FLAER-based flow cytometry for screening of paroxysmal nocturnal hemoglobinuria enhances detection rates in patients with aplastic anemia. Ann Hematol. 2015 May;94(5):721-8. doi: 10.1007/s00277-014-2267.
 
Marinov I, Illingworth AJ, Benko M, Sutherland DR. Performance Characteristics of a Non-Fluorescent Aerolysin-Based Paroxysmal Nocturnal Hemoglobinuria (PNH) Assay for Simultaneous Evaluation of PNH Neutrophils and PNH Monocytes by Flow Cytometry, Following Published PNH Guidelines. Cytometry B Clin Cytom. 2018 Mar;94(2):257-263. doi: 10.1002/cyto.b.21389. Epub 2016 Jul 6. PMID: 27294344.

Sutherland DR, Ortiz F, Quest G, Illingworth A, Benko M, Nayyar R, Marinov I. High-sensitivity 5-, 6-, and 7-color PNH WBC assays for both Canto II and Navios platforms. Cytometry B Clin Cytom. 2018 Jul;94(4):637-651. doi: 10.1002/cyto.b.21626. Epub 2018 Mar 5. PMID: 29381839.
 
Brando B, Gatti A, Preijers F: Flow cytometric diagnosis of paroxysmal nocturnal hemoglobinuria: Pearls and pitfalls - a critical review article. EJIFCC. 2019 Nov 25;30(4):355-370.
 
Illingworth AJ, Marinov I, Sutherland DR: Sensitive and accurate identification of PNH clones based on ICCS/ESCCA PNH consensus guidelines-A summary. Int J Lab Hematol. 2019 May;41 Suppl 1:73-81.

Sutherland DR, Richards SJ, Ortiz F, Nayyar R, Benko M, Marinov I, Illingworth A. CD71 improves delineation of PNH type III, PNH type II, and normal immature RBCS in patients with paroxysmal nocturnal hemoglobinuria. Cytometry B Clin Cytom. 2020 Mar;98(2):179-192. doi: 10.1002/cyto.b.21853. Epub 2019 Nov 8. PMID: 31705743.