Table 3.
Assessment of the Reagents and Instrument Set‐up in Different Centers by Evaluating the Relative Signal of Required Diagnostic Markers on Control Samples
| Antigen | CD19 | CD20 | CD5 | Kappa | Lambda | CD23 |
|---|---|---|---|---|---|---|
| Relative signal target value | ≥10 | ≥10 | ≥30 | ≥5 | ≥5 | ≥5 |
| Center 1 | 225 (123–479) HD37 RPE‐Cy5 | 127 (51.9–183) L27 FITC | 56.3b (16.2–5,892) DK23 APC | 24.4 (12.6–87.6) Polyclonal FITC | 100 (44.8–302) Polyclonal PE | 11 (7.4–17.9) MHM6 FITC |
| Center 2 | 5,462 (4,291‐6,393) LT19 APC | 64.8 (36.6–103) 2H7 APE‐eF780 | 41.1a (17.7–57.2) L17F12 V450 | 17.1a (4.9–37.6) G20–193 APC‐H7 | 2.9b (2.1–4.9) 1–155‐2 APC | 4b (3.1–6.8) Tu1 FITC |
| Center 3 | 12,126 (85.1–14,264) J3–119 PE‐Cy7 | 5.4b (2.5–7.1) L27 V450 | 44.2a (2.8–102) L17F12 PerCP‐Cy5.5 | 20.2 (7.1–55.5) Polyclonal PE | 35.8 (8.4–116) Polyclonal FITC | 43.2a (0.8‐1,670) MHM6 FITC |
| Center 4 | 17.9a (5.6–23.5) SJ25C1 PerCP‐Cy5.5 | 175 (102–306) L27 FITC | 237 (52.8–368) L17F12 PE | 35.6 (12.6–60) TB28‐2 FITC | 430 (148–612) 1–155‐2 PE | 49a (2.5–223) EBVCS‐5 PE |
| Center 5 | 16.5 (11.2–18.8) SJ25C1 PerCP‐Cy5.5 | 24.6 (16.7–30.2) L27 APC‐H7 | 42.9a (15.1–56.7) L17F12 PE‐Cy7 | 22.6 (10.3–65.1) G20–193 BV421 | 17.5 (10.3–24.2) JDC‐12 FITC | 18.7 (8.6–31.7) M‐L233 BV421 |
| Center 6 | 56.8 (32.8–81.9) J3–119 PE‐Cy7 | 2,812 (398‐5,030) 2H7 PacBlue | 37.2a (24.4–105) L17F12 PerCP‐Cy5.5 | 19.7 (11.4–65.6) Polyclonal FITC | 74.4 (13.6–317) Polyclonal PE | 15.4 (9.7–39.3) MHM6 FITC |
| Center 7 | 106 (89.9–175) SJ25C1 APC | 53.6 (41.2–67.4) L27 PerCP | 26.2b (17.9–39) L17F12 FITC | 22.1 (6.9–45.1) TB28‐2 FITC | 149 (72.2–287) 1–155‐2 PE | 16.9 (8.6–35) EBVCS‐5 PE |
| Center 8 | 217 (130–234) J3–119 PE‐Cy7 | 82 (58.8–145) 2H7 Pacific Blue | 88.6 (51–123) BL1a APC | 25.3 (10.7–80.1) Polyclonal PE | 19.6 (7.4–74.8) Polyclonal FITC | 10.3 (5.8–14.1) 9P25 FITC |
| Center 9 | 16.3a (5.5–130) J3–119 PE‐Cy7 | 29.9 (18.3–58.7) B‐Ly1 FITC | 5.4b (2.4–45.6) BL1a PE | 12.3a (4.7–29.7) Polyclonal FITC | 46.6 (6.5–75.5) Polyclonal PE | 19.1 (9.8–48.4) 9P25 FITC |
| Center10 | 31.6 (22.6–41.7) J3–119 ECD (Coulter) | 82.1 (38.4–119) B9E9 Pacific Blue | 16.6b (3–31.5) BL1a APC‐AF750 | 6.1a (1.7–11.2) Polyclonal FITC | 18 (12.3–37) Polyclonal PE | 9.1 (7.1–13.6) 9P25 APC‐AF700 |
| Center 11 | 142 (20.2–10,558) SJ25C1 APC | 99.5 (46.9–240) L27 FITC | 24.1b (9.7–45.8) L17F12 PerCP‐Cy5.5 | 87.1 (33.3‐3,398) TB28‐2 FITC | 160 (55.4‐2,821) 1–155‐2 PE | 70.6 (31.3–290) EBVCS‐5 PE |
The signal for each marker on the internal positive and negative controls was determined using a simple gating strategy applied to ten control cases (see Fig. 2). The table shows the median relative signal (range) for the cases above the clone and fluorochrome (supplier).
Indicates that the results were sub‐optimal in 1–3 of the 10 cases, typically reflecting issues with individual samples rather than instrument/reagent quality.
Indicates that the results did not meet the specified criteria in >3/10 cases due to one or more of several factors such as clone, fluorochrome, manufacturer, equipment, or operating procedures as well as factors related to the evaluation procedure such as a limitation in the proposed gating strategy with multiplex approaches (e.g. CD20 at center 3).