Utility of the GPI-Linked Antigen CD157 in the Diagnosis of Paroxysmal Nocturnal Hemoglobinuria (PNH) by Flow Cytometry: Advantages over CD14 for Analysis of Monocytes
Pramoda Challagundla, Jeffrey L Jorgensen. UT MD Anderson Cancer Center, Houston, TX
Background: PNH is a rare form of acquired hematopoietic stem cell disorder, characterized by partial or complete deficiency of glyco-phosphatidylinositol (GPI) linked molecules. This results in intravascular hemolysis, which may lead to complications from fatigue to life-threatening thrombosis. The standard assay for PNH is flow cytometric analysis of GPI-linked surface antigens, in order to demonstrate loss of two such antigens on at least two cell lineages (typically granulocytes, monocytes, and/or erythrocytes). CD14 is commonly used to assess monocytes, but normal monocytes often include a small subset of CD14 dim/negative cells, which may reduce the sensitivity to detect small PNH+ clones. We studied CD157, an alternative GPI-linked molecule which is brightly positive on monocytes.
Design: 11 normal PB samples, along with 2 QA send-outs from UK NEQAS, were stained with a 6-color single-tube PNH panel for granulocyte and monocyte analysis, which included either CD14-phycoerythrin (PE) or CD157-PE plus fluorochrome-conjugated aerolysin (FLAER), CD15, CD24, CD45, and CD64. Antibodies were from BD Biosciences (BDB, San Jose, CA) and FLAER was from Cedarlane Diagnostics (Burlington, NC). Data were acquired on FacsCanto II cytometers (BDB), and analyzed using FlowJo (TreeStar, Ashland, OR), with statistical analysis using GraphPad Prism (GraphPad Software, La Jolla, CA). CD64 bright+ monocytes were analyzed on CD14 vs. FLAER and CD157 vs. FLAER plots.
Results: In 11/11 normal PB, monocytes were uniformly brightly positive for both CD157 and FLAER (Fig. 1A), with rare cells slightly dim for CD157 (median 0.14%, 0.08-0.26%). While most monocytes were also CD14 bright+ (Fig. 1B), there was a subset of FLAER+ monocytes with dim to absent CD14 expression (median 0.73%, 0.44-3.2%). These were on average five-fold more frequent than CD157 dim cells in each specimen (p=.006, paired t-test). The PNH+ UK NEQAS samples showed events distinctly dim for both FLAER and CD157, as expected for PNH-phenotype cells.
Conclusions: CD157 provides more uniform positive staining of normal monocytes than CD14, with a lower background of dim cells, and thus is a superior marker for analysis of monocytes for PNH by flow cytometry.
Tuesday, March 20, 2012 11:45 AM
Platform Session: Section C, Tuesday Morning