Practical guidelines for the high‐sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry

Abstract

Background:Paroxysmal nocturnal hemoglobinuria (PNH) is a life‐threatening disorder caused by an inability to make glyco‐phosphatidyl‐inositol (GPI) anchors. While flow cytometry is the method of choice to detect the loss of GPI‐linked proteins, the development and validation of sensitive, standardized, methodologies have been hampered by the rarity of this disease and by technical difficulties in the accurate identification of PNH cells.Methods:Guidelines for the diagnosis and monitoring of PNH by flow cytometry were recently published by the International Clinical Cytometry Society (ICCS). However, specific reagent cocktails, and associated detailed analytic strategies were not directly addressed therein. In this supporting document based on the ICCS guidelines, we provide concise practical protocols for the high‐sensitivity detection of PNH RBCs and WBCs (both granulocytes and monocytes).Results:The CD235aFITC/CD59PE assay described was capable of detecting as few as 20 Type III PNH RBCs per million cells. Frequencies of Type III PNH cells in 10 normal samples were in the 0–6 per million RBCs. The high‐resolution granulocyte/neutrophil assays described in this study could detect PNH phenotypes consistently at a level of 0.01% sensitivity. Frequencies of PNH phenotypes in normal individuals were in the 0–10 per million granulocytes/neutrophils range.Conclusions:The careful screening and selection of specific antibody conjugates has allowed the development of reagent cocktails suitable for high‐sensitivity flow cytometric detection of PNH RBCs and PNH WBCs. The reagent cocktails described herein can be used on a variety of clinical flow cytometers equipped with four or more photo multiplier tubes. © 2012 International Clinical Cytometry Society

Monochromatic gating method by flow cytometry for high purity monocyte analysis

“…Building upon the ICCS recommendations for PNH testing, Sutherland et al elucidated the clones and antibody dilutions that comprise a ''perfect cocktail'' for high sensitivity PNH testing, incorporating CD45 PECy7 and CD64PECy5 into their WBC cocktail (13). The utility of CD64 monocyte gating in PNH was also recently reported to be superior to CD33 also on the basis of its high purity of monocyte in cells having moderate expression (14).…”

“…There was no statistically significant difference between the CD163 PE þ CD45 FITC, CD64 PE þ CD45 FITC, and CD64 FITC þ CD45 FITC gating methods, demonstrating one color is equally effective as two color gating. One practical use of this finding is that a leukocyte screening tube for PNH that can be designed to examine both a PI linked protein, such as CD157, and FLAER TM , while gating on monocytes using one color (CD45þ, CD64þ) and neutrophils in another color with CD15 (13). Thus, PNH leukocytes can be studied in a single tube assay using instruments restricted to only four-color detection and avoiding the use of more complex fluorochrome combinations.…”

Validation of cell‐based fluorescence assays: Practice guidelines from the ICSH and ICCS – part IV – postanalytic considerations

“…Early guidelines in PNH have been published (9)(10)(11). For the identification of small PNH clones, it is recommended that 250,000 events be collected in order to identify clusters of at least 25 events for a desired sensitivity of 0.01%.…”

Issue Highlights—January 2013

“…It is caused by a mutation in the phosphatidyl inositol glycan-class A gene leading to partial or complete loss of the cell membrane molecule called glycosyl phosphatidyl inositol (GPI) (15,16 (14), data analysis has not been standardized. The majority of current analytical methods use manual gating to enumerate PNH cells (14)(15)(16)(17)(18)(19) although new automated analysis techniques that improve the objectivity of operator input have also been recently developed (19). Gating strategies imply the recognition of the various cell types (monocytes, neutrophil granulocytes, and red blood cells) based on the expression of various phenotypic markers.…”