High Sensitivity PNH assay - Cytometry B

EXBIO is proud to inform you, that recently a paper with title: "High-sensitivity 5-, 6-, and 7-color PNH WBC assays for both Canto II and Navios platforms." was published in Cytometry B.

One of authors of this paper is EXBIO Flow Cytometry Expert Miro Benko.

PAPER: High-sensitivity 5-, 6-, and 7-color PNH WBC assays for both Canto II and Navios platforms. Sutherland DR, Ortiz F, Quest G, Illingworth A, Benko M, Nayyar R, Marinov I. Cytometry B Clin Cytom. 2018 Jul;94(4):637-651. doi: 10.1002/cyto.b.21626. Epub 2018 Mar 5.
PMID:29381839

Fig4-1.jpgI’m glad I was given the opportunity to present this paper as a co-author in the name of EXBIO Praha a.s. I am a Director of Product Development Department and also Head of Flow Cytometry.

On ESCCA 2014 in Portugal I met Rob. Sutherland, an expert on hematopoietic stem cell analysis and counting and also an expert on high sensitivity PNH testing. Also a very nice guy and a friend of mine. We have discussed the possibility of PNH detection using only antibodies without the use of FLAER. This small gentleman talk ended with a published paper by I. Marinov[1].Dual GPI-anchored marker staining was utilized for PNH detection.

Further on, Rob Sutherland had the idea to put FLAER together with dual GPI-anchored marker PNH testing resulting in the paper "High-sensitivity 5-, 6-, and 7-color PNH WBC assays for both Canto II and Navios platforms"[2].

We have also developed and provided some antibodies specifically for data collection and publishing like CD24 APC (clone SN3), CD157 PE (clone SY11B5), CD235a FITC (clone JC159), CD15 PerCP-Cy™5.5 (clone MMA), CD15 PerCP-Cy™5.5 (clone MEM-158) and CD15 FITC (clone MEM-158).

I would like to give you a short introduction to PNH testing as Published in both papers[1,2].

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell disorder characterized by an inability to make Glyco‐Phosphatidyl‐Inositol (GPI)‐linked cell surface structures through a mutation on PIG-A gene[3]. Such mutation impairs membrane expression of complement regulators like CD55 and CD59 (both GPI-linked) causing chronic, complement-mediated, intravascular hemolysis resulting in anemia, hemoglobinuria, fatigue, blood clot formation and is common to appear in patients with aplastic anemia (AA) and rarely in patients with myelodysplastic syndrome (MDS).

For fast, highly sensitive detection, screening and monitoring of PNH Flow Cytometry is the method of choice. According to recent published guidelines[4] high sensitivity PNH testing consists of red blood cell (RBC) analysis and white blood cell (WBC) analysis.

RBC analysis consists of detection of Type III and Type II PNH clones using CD59PE and CD235a FITC antibodies. CD235a (non GPI-linked; clone JC159) is used to gate singlet erythrocytes. Type III PNH clones are cells with complete deficiency of GPI-linked proteins and Type II PNH clones are cells with markedly reduced level of activity; partial GPI-linked proteins deficiency[3] as shown in Fig. 1.

Fig. 1  CD59 expression on Normal (non-gated), Type II PNH clone RBC and Type III PNH clone RBC gated as CD235a positive events in normal and PNH specimens.

WBC PNH testing utilizes analysis of two GPI-linked molecules on both neutrophils and monocytes contrary to the CD59-only analysis on RBC[1,5]. Two markers provide more consistent results where variability in antigen expression may cause ambiguous results.

Fig. 2 shows PNH analysis on neutrophils using CD24 APC and CD157 PE; both molecules are GPI-anchored. CD15 FITC (non GPI-linked; clone MEM-158) is used to gate neutrophils.

Fig. 2 CD24 and CD157 expression on Normal (non-gated) and PNH specimens. PNH neutrophils are double negative while normal neutrophils are double positive.
FIG2.png

Fig. 3 shows PNH analysis on monocytes using CD14 APC-Cy™7 and CD157 PE; both molecules are GPI-anchored. CD64 PE-Cy™7 (non GPI-linked; clone 10.1) is used to gate monocytes.
Fig3.pngFig. 3 CD14 and CD157 expression on Normal (non-gated) and PNH specimens. PNH monocytes are double negative while normal monocytes are double positive.

CD14 provides somewhat heterogenous staining visible as CD157+CD14DIM and NEG events. These events are different monocytes which are mature macrophages and dendritic cells having weaker CD14 expression. In such cases use of FLAER - fluorescent Pro-aerolysin labeled with Alexa Fluor™ 488 is suggested for complete image as FLAER binds to GPI anchor with high affinity[2]. PNH monocytes or neutrophils will be FLAER negative.

Have a look on our portfolio of antibodies for PNH detection and don’t forget to titrate your antibodies and also don’t forget to exclude doublets and multiplets from RBC PNH analysis.

Miroslav Benko
Product Development Director and Head of Flow Cytometry


References:
1 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, Marinov I, Illingworth AJ, Benko M and Robert Sutherland, D.  Following Published PNH Guidelines. Cytometry Part B 2018; 94B: 257–263.
2 High-sensitivity 5-, 6-, and 7-color PNH WBC assays for both Canto II and Navios platforms. Sutherland DR, Ortiz F, Quest G, Illingworth A, Benko M, Nayyar R, Marinov I. Cytometry B Clin Cytom. 2018 Jul;94(4):637-651. doi: 10.1002/cyto.b.21626.
3 Rollinson, S., Richards, S., Norfolk, D., Bibi, K., Morgan, G., & Hillmen, P.(1997). Both Paroxysmal Nocturnal Hemoglobinuria (PNH) Type II Cells and PNH Type III Cells Can Arise From Different Point Mutations Involving the Same Codon of the PIG-A Gene. Blood, 89(8), 3069-2861.
4 Sutherland DR, Illingworth A, Marinov I, Ortiz F, Andreasen J, Payne D, Wallace PK
and Keeney M. ICCS/ESCCA Consensus Guidelines to detect GPI-deficient cells in Paroxysmal Nocturnal Hemoglobinuria (PNH) and related Disorders Part 2 – Reagent Selection and Assay Optimization for High-Sensitivity Testing. Cytometry Part B 2018; 94B: 23–48.
5 Sutherland, D. R., Keeney, M. and Illingworth, A. (2012), Practical guidelines for the high‐sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry. Cytometry, 82B: 195-208. doi:10.1002/cyto.b.21023