The SOLVO MDQ Kit™

 

SOLVO MDQ Kit™ is the first commercially available CE-IVD approved clinical diagnostic kit for the detection of MDR protein function (MDR1, MRP1, and BCRP) by quantitative flow cytometry.

Learn more about how your patients can benefit from MDR protein function determination.

 

Autoimmune Diseases

Learn more about the application of SOLVO MDQ KitTM in Rheumatoid Arthritis

Hematologic Malignancies

Learn more about the application of SOLVO MDQ KitTM in Hematologic Malignancies

MDQuest Ltd.

Learn more about us by visiting our main website!

 

For laboratory specialists

 

Page content:

I. Novelty

II. Clinical relevance

III. Features

IV. Principle of the test

V. Availability

 

I. Novelty

SOLVO MDQ Kit™ is the first biomarker-based diagnostic kit for the detection of MDR protein function by flow cytometry. It is designed to determine the functional activity of the three clinically most relevant drug efflux proteins: MDR1, MRP1, and BCRP.


II. Clinical relevance


• MDR protein function is an independent negative prognostic biomarker in AML
• MDR protein function can be correlated with disease activity in autoimmune diseases
• The incidence of MDR in previously untreated cancer cases is approximately 40%
• Conventional anticancer drugs (e.g.: Doxorubicin, Gefitinib, Imatinib, Irinotecan, Methotrexate, Mitoxantrone, Paclitaxel, Tamoxiphen, Topotecan, etc.) are substrates of MDR transporters
• Elevated MDR protein activity can be correlated with prognosis, drug efficiency and disease activity
• MDR protein activity determination is a safety measure for patients on highly demanding cytotoxic/immune suppressant drugs

 

III. Features


The SOLVO MDQ Kit™ is designed to maximize the benefits of quantitative flow cytometry. Literature data suggests that functional determination of MDR provides fast and more accurate results for the clinical lab than other methods such as quantifying the expression of the transporters at mRNA or protein level:
• The kit contains two proprietary assays and measures the three clinically most relevant transporter activities selectively
• Uses highly selective inhibitors and different probe substrates for MDR1/MRP1 and BCRP
• Compatible with cell surface markers
• Contains ready-to-use reagents
• 10 independent MDR1/MRP1 and BCRP measurements could be carried out in triplicates
• Specimen: cell suspension, blood, bone marrow etc.: 6 hours stability before testing
• The first test results can be expected within 90 minutes

 

Figures 1-5. MDR activities in Acute Myleoid Leukemia on CD45+ cells

AML graf 1 crop

AML Oldal 2

             AML Oldal 4

AML Oldal 3

           AML Oldal 5

 

IV. Principle of the test


For quantitative measurement of MDR1 and MRP1 activities in viable cells, SOLVO MDQTM Kit applies the proprietary Calcein-assay technology. This assay utilizes the fluorogenic dye calcein-acetoxymethyl ester (calcein-AM), which is a hydrophobic, non-fluorescent compound that readily penetrates the cell membrane. After entering the living cell, calcein-AM is rapidly hydrolyzed by endogenous esterases. As a result of the cleavage, highly fluorescent free acid derivative of the dye is formed, which becomes trapped in the cytoplasm due to its hydrophilic character. Since calcein-AM is an excellent substrate of both MDR1 and MRP1, the activity of these efflux transporters results in a lower cellular accumulation of the fluorescent calcein (Figure 2, Left Panel). Addition of selective inhibitors of MDR1 and MRP1 in excess blocks the dye extrusion activity of the relevant transporter and increases calcein acccumulation in the cells. Activities of MDR1 and MRP1 transporters are reflected by the difference between the amount of calcein accumulated in the presence or absence of the selective inhibitors. This difference is normalized to the dye uptake measured in the presence of the inhibitor and the results of the test are expressed in MDR activity factor (MAF) values. Thus, the result of the test becomes independent from factors influencing the cellular accumulation of calcein other than the activity of the multidrug transporters. These variables include the differences in the cellular properties (membrane composition, intracellular esterase activity, cell size, cell surface, etc.); and the methodological differences (e.g. use of different equipment, amplification, and individual variables). Since the influence of these factors is diminished by the simple normalization approach mentioned above, the intra- and inter-laboratory comparison of MAF values is possible.


BCRP activity is measured using a similar principle: intracellular accumulation of the fluorescent BCRP-specific reporter substrate is measured in the presence and absence of the selective BCRP-inhibitor (Figure 2, Right Panel). However, the BCRP-specific reporter substrate is directly fluorescent and does not require cleavage by the intracellular esterases.

labspec formazott

Figure 2. Principle of the dye efflux assays applied in the SOLVO MDQTM kit

 

The more MDR proteins are active in the cell membrane, the less calcein is accumulated intracellularly. In MDR-expressing cells, the addition of selective inhibitors MDR1 and MRP1 in excess blocks the dye extrusion activity of the relevant transporter and increases calcein accumulation in cells. Activities of MDR1 and MRP1 transporters are reflected by the difference between the amount of calcein accumulated in the presence or absence of the selective inhibitors. This difference is normalized to the dye uptake measured in the presence of the inhibitor, and the results of the test are expressed in MDR activity factor (MAF) values. Thus, the result of the test becomes independent from factors influencing the cellular accumulation of calcein other than the activity of the multidrug transporters. These variables include the differences in the cellular properties (membrane composition, intracellular esterase activity, cell size, cell surface, etc.); and the methodological differences (e.g. use of different equipment, amplification, and individual variables). Since the influence of these factors is diminished by the simple normalization approach mentioned above, the intra- and inter-laboratory comparison of MAF values is possible.

Using selective inhibitors, the transport activity of P-glycoprotein (MDR1) and MRP1 can be distinguished. The kit component, verapamil blocks both MDR1- and MRP1-mediated dye effluxes providing a dye accumulation rate that can be used for standardization, while indomethacin selectively blocks the activity of MRP1. After a short, simple calculation, separate measures of multidrug resistance for both MDR1 and MRP1 can be obtained.


BCRP activity is measured using a similar principle: intracellular accumulation of the fluorescent BCRP-specific reporter substrate (mitoxantrone) is measured in the presence and absence of KO134, a selective BCRP-inhibitor (Figure 1, Right Panel). However, the mitoxantrone is directly fluorescent and does not require cleavage by the intracellular esterases.

 

Compatible fluorochromes for gating the cell populations of interest


Fluorescent-labeled antibodies used for immunophenotyping enable the gating of cell populations of interest during measurements. However, some antibodies might interfere with the transporter assays and not all of the fluorescent conjugates are compatible with the dye efflux assays of the SOLVO MDQ KitTM.
The SOLVO MDQ KitTM provides functional tests that require living cells. Thus, the antibodies used for immunophenotyping should be directed to extracellular epitopes, which do not require the fixation or permeabilization of the cells. Furthermore, some of the antibodies may interfere with the transporter functions. Therefore, the labelling of the cells with conjugated antibodies should always be carried out following the calcein/mitoxantrone assay. In addition, testing the antibodies for possible interferences is strongly recommended.

Compatible fluorescent conjugates are tabulated below:Compatible fluorescent conjugates are tabulated below:

Transporter(s)

Dye/Substrate

Detection channel

Compatible Fluorochromes

MDR1, MRP1

Calcein

~515 nm

PerCP, PerCP Cy5.5

BCRP

Mitoxantrone

~684 nm

FITC, PE

Since technical protocols for using the different antibodies vary, each investigator should follow the manufacturers’ instructions to obtain optimal results. 

 

V. Availability

 

PRODUCT SIZE CAT. NO.
SOLVO MDQ Kit™ 10 assays MDQ0101D

 

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References

1. Homolya Br J Cancer 1996

2. Sonneveld J Intern Med 2000

3. Karaszi Br J Haematol 2001

4. van den Heuvel-Eibrink Leukemia 2002

5. Jansen Scand J Rheumatol 2003

6. Benderra Clin Cancer Res 2004

7. Aleskog Anticancer Drugs 2005

8. Damiani Haematologica 2006

9. van der Heijden Nat Clin Pract Rheumatol 2007

10. van den Heuvel-Eibrink Ann Hematol 2007

11. Mansilla Biochem Pharmacol 2007

12. Kis Ann Rheum Dis 2009

13. van der Heijden Arthritis Rheum 2009

14. Marie  J Clin Oncol 2010