Determination of Reference Values of MDR‐ABC Transporter Activities in CD3+ Lymphocytes of Healthy Volunteers Using a Flow Cytometry Based Method

Abstract

Background: MDR transporters are important biomarkers of drug resistance in cancer and in autoimmune conditions. We determined the MDR1, MRP1 and BCRP activity in CD3+ lymphocytes using a flow cytometry based method from 120 healthy volunteers in order to describe normal reference values of the activity of these transporters. The effects of gender and age were also determined.Methods: The Solvo MDQ Kit™ was used for measurements. In this assay, fluorescent reporter substrates (Calcein-AM for MDR1 and MRP1 and mitoxantrone for BCRP, respectively) are trapped in the cytoplasm and pumped out by MDR proteins depending on the presence or absence of specific inhibitors (verapamil for MDR1 and MRP1, indomethacin for MRP1 and KO134 for BCRP, respectively), allowing for quantitative, standardized assessment. Cell surface staining was applied to select CD3+ cells.Results: MAF values of MRP1 and BCRP are independent from age. MAFC and MAF of MDR1 show negative correlation with the age of the studied subjects (P = 0.003, r = −0.27 and P = 0.0001, r = −0.34, respectively). No difference was detected in any of the four MAF values between men and women. Gender does not affect the presence or lack of correlation between MAF values and age.Conclusions: The determination of the functional activity of MDR-ABC transporters is achievable using a flow cytometry based standardized method. Having established the normal range of MAF values on CD3+ lymphocytes of a healthy population, our results allow for the development of novel flow cytometry based diagnostic tools.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

“…Apply sequential gating strategy for the analysis (an example is provided in ref. ). Within the desired cell subset gate, determine the corresponding geo‐MFI values of the reporter substrate applied.…”

“…In case of hematological malignancies, the MAF C values in tumor cell populations are usually found between 0 and 50, but in extreme cases, values can be as high as 70. Reference values of MAF values in CD3 + lymphocytes were determined in a healthy population of 120 individuals . Importantly, MAF values are independent from gender.…”

Sat0541 multidrug Transporters of Peripheral Lymphocytes Serve as Biomarkers to Bdmard Response in Rheumatoid Arthritis (Ra)

“…At later time points, MAF C values above 20.3 and MAF MRP1 above 6.0 and MAF MDR1 values above 13.9 in CD3 + cells also predict unfavourable treatment response. Of note, these cut-off values are all below the respective reference ranges in healthy individuals established in our earlier study [3]. Conclusion: Importantly, little is known about the relation of MDR activities and therapeutic success to bDMARDs.…”

Comparison of P‐glycoprotein function in peripheral blood mononuclear cells ex vivo in stable Black and White male and female kidney transplant recipients

“…Studies in healthy individuals and participants with rheumatologic disease or lung transplant evaluated P-gp function using only a single timepoint which limits the ability to detect pharmacodynamic differences over time from these data. 10,[24][25][26][27] Although past investigations have reported mixed outcomes while studying P-gp expression and sex, 15,16,18,19 our study observed that White male and female KTRs exhibited greater P-gp function in PBMCs at time 0 h and with the composite parameter, AUC 0-12h % ΔMFI, compared to Black men and women. 12,25 The pair-wise comparison for each significant P-gp parameter was noted between White and Black women which reinforces the combined race-sex association.…”

“…11,20,21 Further investigations are needed to examine race and sex differences in P-gp function in PBMCs and lymphocyte subpopulations in stable transplant recipients and healthy participants as well as the application of this research blood tests as a routine liquid biopsy to guide drug therapy adjustments post-transplant. 12,25,26 Recently, liquid biopsies have been utilized to quantify proteins, such as P-gp, and efforts have been made to correlate such expression to activity (e.g., function) with patient response. Liquid biopsies have successfully detected variability in P-gp expression, and expression has been moderately and strongly correlated to P-gp activity.…”