CD157 Can Replace CD24 and CD14 in a Single-Tube Flow-Cytometric Assay to Detect Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones on Both Neutrophils and Monocytes: A Prospective Study From North India

Abstract

Introduction As per current guidelines, detection of paroxysmal nocturnal hematuria (PNH) clones on leucocytes requires the demonstration of the loss of at least two glycosyl-phosphatidyl-inositol (GPI)-linked molecules on both neutrophils and monocytes by flow cytometry. CD24 and CD14 are GPI-linked molecules expressed on neutrophils and monocytes respectively, whereas another GPI-linked molecule, CD157, is expressed on both neutrophils and monocytes. This prospective study evaluated the ability of CD157 to replace both CD24 and CD14 in a single-tube flow-cytometric assay to detect PNH clones on both neutrophils and monocytes. Materials and methods PNH clones were newly detected in 52 patients by an existing "standard" single-tube six-color flow-cytometric method, which was routinely performed in our laboratory at the time of undertaking this study. Six antibodies (CD45/CD15/CD64/CD24/CD14/FLAER) were used in this "standard" technique. Subjects were divided into two groups: (i) PNH disease (n=10), and (ii) aplastic anemia/myelodysplastic syndrome (AA/MDS) (n=42). Diagnosis of PNH disease and AA/MDS were made as per standard literature and guidelines. Results were compared with a single-tube five-color "test" assay using the antibodies CD45/CD15/CD64/CD157/FLAER by flow cytometry. Samples from 20 healthy control subjects were used to calculate cut-off values for the "test" assay. Results By the "test" method, cut-off values for detecting PNH clones obtained from receiver operating-characteristic curve analysis were >0.4% for neutrophils (sensitivity=96.15%, specificity=95%), and >0.9% for monocytes (sensitivity=98.08%, specificity=95%). There was significant correlation between PNH clone sizes measured by both the "standard" and "test" assays in neutrophils (PNH disease: r=0.976, p<0.001; AA/MDS: r=0.980, p<0.001) as well as monocytes (PNH disease: r=0.806, p=0.005; AA/MDS: r=0.915, p<0.001). Bland-Altman analysis showed agreement between both assays in all the 52 patients and in individuals with AA/MDS. The cost of the test to the patients was about 15% less in the "test" method than the "standard" technique, with improved technical efficiency. Conclusion CD157 can replace both CD24 and CD14 in a single-tube flow-cytometric assay to detect PNH clones on both neutrophils and monocytes, with reduced cost to the patients and improved technical efficiency.

Keywords: cd14; cd157; cd24; flaer; flow cytometry; paroxysmal nocturnal hemoglobinuria; pnh.

Figure 1. Flow cytometry on a peripheral blood sample for detecting PNH clones - lineage-gating strategies for leucocytes

For neutrophils, initially, a broad gate P1 is put on CD45/SSC plot (A), followed by a lineage-specific gate P3 on CD15/SSC plot (B). For monocytes, an initial broad gate P2 is put on CD45/SSC plot (A), followed by a lineage-specific gate P4 on CD64/SSC plot (C). For determining PNH clone size, further analysis is done on the neutrophils gated on “P3” and monocytes gated on “P4” CD: cluster of differentiation; PNH: paroxysmal nocturnal hematuria; SSC: side scatter

Figure 2. Flow cytometry on a peripheral blood sample showing PNH clones on neutrophils and monocytes after lineage gating as per Figure 1

For the “standard” method, CD24/FLAER double negative PNH neutrophils are detected in quadrant Q3 (9.9%) (A), whereas CD14/FLAER double negative PNH monocytes are detected in quadrant Q3-1 (5.5%) (B). For the “test” assay, CD157/FLAER double negative PNH neutrophils are detected in quadrant Q3 (10.1%) (C), whereas CD157/FLAER double negative PNH monocytes are detected in quadrant Q3-1 (5.6%) (D). Note: data and plots are from the same sample; the percentages mentioned above are not displayed in the figure PNH: paroxysmal nocturnal hematuria; CD: cluster of differentiation

Figure 3. Receiver operating characteristic curve analysis for CD157/FLAER-based detection of PNH clone on neutrophils (A) and monocytes (B)

CD: cluster of differentiation, PNH: paroxysmal nocturnal hematuria

Figure 4. Correlation of PNH clone sizes measured on neutrophils and monocytes between the “test” method using CD157/FLAER, and the “standard” method using CD24/FLAER for neutrophils and CD14/FLAER for monocytes in patients with PNH disease (A, B) and in individuals with AA/MDS having PNH clone (C, D)

CD: cluster of differentiation; PNH: paroxysmal nocturnal hemoglobinuria; AA/MDS: aplastic anemia/myelodysplastic syndrome; R: correlation coefficient

Figure 5. Bland-Altman plots showing agreement between the “standard” and “test” methods in the detection of PNH clones on neutrophils (A) as well as monocytes (B) in all 52 patients enrolled in the study

PNH: paroxysmal nocturnal hemoglobinuria

Figure 6. Bland-Altman plots showing agreement between the “standard” and the “test” methods in the detection of PNH clones on neutrophils (A) as well as monocytes (B) in AA/MDS patients with PNH clone (n=42)

AA/MDS: aplastic anemia/myelodysplastic syndrome, PNH: paroxysmal nocturnal hemoglobinuria