Evaluation of Clinical Usefulness of Monocyte Gating Using CD14 and CD64 for Detecting PNH Clone By Flow Cytometry
Authors: Kwoun Woo Jae , Ahn Jeong-Yeal , Seo Ja Young , Lee Jae Hoon , Kim Hawk , Park Jinny , Yoo Kwai Han , Cho Sung-Ran , Kim Hye Ryoun 
Published: October 2019

Abstract
Introduction
Flow cytometry is the gold standard in diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) by detecting the absence of glycol-phosphatidyl inositol (GPI)-linked protein expression on red blood cell, granulocyte, and monocyte. The current assays are 4-color analyses of GPI-linked markers such as fluorescein-labeled proaerolysin (FLAER), CD24, CD14, CD59, and CD235a and the lineage markers for granulocyte (CD15) and monocyte (CD64) cells to detect PNH clones. We investigated the utility of CD14/CD64 monocyte gating by comparing with CD45/light scatter (LS) gating in PNH study of the patients with cytopenia and analyzed the types and cell lineages of PNH clone according to the disease groups.
Method
Total 138 cases were recruited in this study from July 2017 to February 2018 at Gachon University Gil Medical Center in Korea. Flow cytometric analysis was performed with EDTA blood by Beckman Coulter Cytomics FC500 cytometer using gating antibodies such as CD45, CD14, CD15, CD64, CD235a and GPI-linked antibodies such as CD59, CD14, CD24, FLAER. The proportion of monocyte was estimated by CD14/CD64 gating and compared with those using CD45/LS gating. The type of PNH clone was defined according to the size of PNH population. A PNH clone is defined as a PNH population exceeding 1% of the gated cells, a minor PNH clone as between 0.1 and 1%, and rare cells with GPI-deficiency defined as a PNH population less than 0.1%. The types and cell lineages of the PNH clone were analyzed according to the disease groups. Statistical analysis was done using SPSS 17.0 and MedCalc 15.2, and P<0.05 was considered statistically significant.
Results
Of the 138 cases, PNH clone was detected with 27 cases including 15 cases with a PNH clone and 12 cases with a minor PNH clone. PNH clone was observed in all 8 cases (100%) of PNH cases. Two PNH clone and 4 minor PNH clones were identified in 6 of 16 cases (38%) of acute myeloid leukemia. In 6 of 21 cases (29%) of aplastic anemia (AA) show 5 PNH clones and 1 minor PNH clone. In 5 of 78 cases (6%) of cytopenia(s) only minor PNH clone was observed. The CD45 plus LS gating in monocyte represents a sensitivity of 100%, a specificity of 40.2%, and 60% (73/89) false positive rate in detecting of PNH clone. McNemar test indicates a significant difference between CD14/CD64 and CD45/LS gating methods (P = 0.00). The Bland-Altman plot of monocyte proportion between the two gating methods revealed that CD45/LS gating method was tended to underestimate monocyte proportion and the larger the number of monocytes, the greater the difference in number of monocyte between the two gating methods. The trend of the size of PNH clone in each cell lineage was confirmed by follow-up in three patients with PNH clone. Two patients showed more abrupt changes of PNH clone in monocytes than in red blood cells or in granulocytes. However, in the other patient, a significant trend found in only PNH clone of RBC.
Conclusion
The types of PNH clone observed in each disease group showed different characteristics. PNH clone was identified in 5 of 6 PNH population detected AA cases, whereas minor PNH clones were observed in all 5 PNH population detected cytopenia cases. Four minor PNH clones and two PNH clones were discovered in 6 PNH population detected AML cases. However, all observed PNH clones observed in AML cases were monocyte. Monocyte gating with CD45 and LS not only underestimated the proportion of monocyte in total WBCs but also showed a high false positive rate of 60% in detecting PNH clone. In contrast, the CD14/CD64 gating method can accurately measure the monocyte population and avoid making a false positive measurement of PNH clone. In addition, in monitoring PNH patients, the measurement of the PNH clone in monocyte tends to be more sensitive to change of PNH clone size than those measured in RBC or granulocytes. In conclusion, the gating using CD14 and CD64 is significantly valuable in flow cytometric diagnosis for detecting the PNH clone in diagnosing new patents as well as monitoring of PNH patients.

Disclosures
No relevant conflicts of interest to declare.