Standardization of neutrophil CD64 and monocyte HLA-DR measurement and its application in immune monitoring in kidney transplantation

BACKGROUND: Infections cause high mortality in kidney transplant recipients (KTRs). The expressions of neutrophil CD64 (nCD64) and monocyte HLA-DR (mHLA-DR) provide direct evidence of immune status and can be used to evaluate the severity of infection. However, the intensities of nCD64 and mHLA-DR detected by flow cytometry (FCM) are commonly measured by mean fluorescence intensities (MFIs), which are relative values, thus limiting their application. We aimed to standardize nCD64 and mHLA-DR expression using molecules of equivalent soluble fluorochrome (MESF) and to explore their role in immune monitoring for KTRs with infection. METHODS: The study included 50 KTRs diagnosed with infection, 65 immunologically stable KTRs and 26 healthy controls. The blood samples were collected and measured simultaneously by four FCM protocols at different flow cytometers. The MFIs of nCD64 and mHLA-DR were converted into MESF by Phycoerythrin (PE) Fluorescence Quantitation Kit. The intraclass correlation coefficients (ICCs) and the Bland-Altman plots were used to evaluate the reliability between the four FCM protocols. MESFs of nCD64 and mHLA-DR, nCD64 index and sepsis index (SI) with the TBNK panel were used to evaluate the immune status. Comparisons among multiple groups were performed with ANOVA one-way analysis. Receiver operating characteristics (ROC) curve analysis was performed to diagnose infection or sepsis. Univariate and multivariate logistic analysis examined associations of the immune status with infection. RESULTS: MESFs of nCD64 and mHLA-DR measured by four protocols had excellent reliability (ICCs 0.993 and 0.957, respectively). The nCD64, CD64 index and SI in infection group were significantly higher than those of stable KTRs group. Patients with sepsis had lower mHLA-DR but higher SI than non-sepsis patients. ROC analysis indicated that nCD64 had the highest area under the curve (AUC) for infection, and that mHLA-DR had the highest AUC for sepsis. Logistic analysis indicated that nCD64 > 3089 and B cells counts were independent risk factors for infection. CONCLUSION: The standardization of nCD64 and mHLA-DR made it available for widespread application. MESFs of nCD64 and mHLA-DR had good diagnostic performance on infection and sepsis, respectively, which could be promising indicators for immune status of KTRs and contributed to individualized treatment.