MINOXODIL RESPONSE TEST to assess efficacy.pptx

to test the minoxidil efficacy

1. MINOXODIL RESPONSE TEST -DR. VIRAL DESAI

2. MRT Instructions for Use The MINOXIDIL RESPONSE TEST is a prescription In-vitro diagnostic (IVD) device to be used by a physician as an aid in assessing the likelihood that a patient suffering from pattern hair loss will not respond to 5% topical minoxidil . Intended Use MINOXIDIL RESPONSE TEST quantitatively measures sulfotransferase (SULT) activity in the bulb of a plucked human hair sample. When performed according to these instructions, the results of this assay may be used to rule out non-responders to the topical 5% minoxidil for the treatment of androgenetic alopecia. This device is for in vitro diagnostic use by a physician only. Federal law restricts this device to sale by or on the order of a physician.

3. MRT Summary and Explanation of the Test Sulfotranseferase (SULT) is an enzyme that catalyzes the transfer of a sulfo group from a donor molecule to an acceptor alcohol. The enzyme has wide human tissue distribution, including the liver and outer root sheath (ORS) of the hair follicle. Inter-individual variation in sulfonation capacity is important in determining an individual's metabolism to certain xenobiotics . Minoxidil is one drug that requires sulfonation to have efficacy. The sulfonation capacity of an individual’s ORS can be used to rule out non-responders to 5% minoxidil for the treatment of androgenetic alopecia. Principles of the Procedure MINOXIDIL RESPONSE TEST measures inter-individual variation in sulfonation capacity. Endogenous SULT enzymes in the ORS of plucked hair are reacted with the test solution by immersing the hair bulb and incubating. After a period of time, the color intensity of the solution is measured photometrically . The color intensity of the solution depends on the level of active SULT present in the ORS and can be used to rule out non-responders to the drug minoxidil .

4. Reaction Scheme

5. Materials Provided Minoxidil Response Test (MRT) Solution - One 50 mL tube (blue cap) containing a ready to use test solution

6. Materials Required but not provided A calibrated spectrophotometer device capable of reading absorption at a wavelength of 405nm with a resolution of at least ± 0.01 OD and a minimum range of 0.0 1.0 OD (e.g., Shimadzu UV 1700). ( Note: reacted MRT can be diluted with an optically transparent solvent (e.g., water) to facilitate filling of cuvettes, as long as the dilution does not reduce the signal (sample OD) below the limit of detection of the instrument. Additionally, if a solvent is used to dilute samples an appropriate scaling factor (dilution factor) must be applied to the raw OD reading.) Pipettes capable of accurately delivering 10 to 200μl. Disposable pipette tips. Clean 0.5 ml polypropylene sample tubes (e.g. Seal-Rite® 0.5 mL microfuge tube, USA Scientific) Laboratory timer to monitor incubation steps

7. Precautions For in vitro diagnostic use only. Normal precautions exercised in handling laboratory reagents should be followed. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Wear suitable protective clothing, gloves, and eye/face protection. Do not breathe vapor. Dispose of waste observing all local, state, and federal laws. Adherence to the specified time and temperature of incubations is essential for accurate results. MRT should be keep refrigerated when not in use. Dilution or adulteration of these reagents may generate erroneous results. Reagents from other sources or manufacturers should not be used. Avoid contact of reagents and patient specimens with skin and mucous membranes . Avoid microbial contamination in reagents. Incorrect results may occur. Cross contamination of reagents and/or samples could cause erroneous results. Do not use MRT beyond expiration date. Incorrect results may occur.

8. Quality Control & Storage Conditions Quality Control: The company has established a complete Quality System in accordance with 21 CFR Quality System Regulations. This includes maintenance of all records associated with the manufactures Device Master Record and Design History File. Storage Conditions: MRT Solution: Store between 2° and 8°C. Properly stored, MRT Solution is stable for 90 day Discard solution if it appears yellow. DO NOT FREEZE. Discard if frozen.

9. Specimen Collection & Preparation for Analysis Pluck 10 hairs from the area just outside (~1 inch) of the balding region. It is best to use tweezers and pluck one hair at a time. Visually inspect the hairs to make sure they have the bulb (the bulbs are larger in diameter than the hair shaft). Plucked hair samples containing bulb (ORS) can be stored at room temperature in clean sample tube or bag for up to 6 days. Samples should be processed or stored within 6 days of plucking. Samples may be stored at -80° C for up to 5 years if stored within 6 days plucking. Samples may be stored at -20° C for up to 1 month if stored within 6 days plucking. Hairs should be visually inspected for the presence of hair bulb prior to performing the test as hairs without bulbs may produce an erroneous result. If bulbs are too difficult to see, a dissecting microscope (40X) may be used.

10. General Procedure Trim plucked hairs containing bulb to a length of ~1cm with scissors. Place 2 hairs containing bulbs at the bottom of a clean 0.5 mL sample tube. Pipette 100 μl of MRT Solution into the tube so that the hair bulbs are immersed. Repeat steps 1-3 to produce a total of 4 sample replicates per patient (8 hairs total, 2 hairs per sample) Pipette 100 μl of MRT Solution into a clean tube. This will serve as a negative control (blank). Close tubes and spin down insure hair bulbs are immersed Incubate samples and control (blank) for 16-24 hours at room temperature (20-25°C). Remove and discard hairs. Read samples at a wavelength of 405 nm and measure the optical density (OD) of each sample. Determine the average of the 4 replicate samples. Read the negative control (blank) sample at a wavelength of 405 nm and measure the optical density (OD). Subtract the OD of the negative control from the average of the 4 replicate samples.

11. General Procedure Record results in a data processing log similar to below

12. General Procedure Calculate these data points using this equations.

13. Limitation of the Procedure

14. Interpretation of Results The final result of the Minoxidil Response Test is interpreted as follows:

15. Performance Characteristics The final result of the Minoxidil Response Test is interpreted as follows : Clinical studies of the Minoxidil Response Test showed test values can range from 3 % - 300% of the 0.4 OD cut-off (min=0.012 and max=1.348, respectively). In a clinical study, the biological (within-subject) variability of the sulfotransferase enzyme in plucked hair samples was determined to be 0.2 OD using 1 replicate of 2 hairs. Four (4) replicates of 2 hairs are required to achieve the positive predictive value (PPV) and negative predictive value (NPV) reported in this document.

16. Linearity & Precision of the method Linearity: The Minoxidil Response Test is linear around the cut-off value of 0.4 OD. It is not suitable for quantitative measurement of sulfotransferase . Precision of the method: Analytical method validation was performed following CLSI EPO5-A3 guidelines and precision was assessed at 25%, 50%, 75%, 100%, 125%, 150%, 175%, & 200% of the 0.4 OD cutoff value. One hundred percent (100%) precision was shown at ≤ 75% and ≥ 125% of the 0.4 OD cutoff (≤ 0.3 OD and ≥ 0.5 OD respectively).

17. Directions GENTLY TAP LID SO LIQUID COALESCES. PLUCK TWO HAIRS FROM SCALP WITH TWEEZERS. CHECK TO MAKE SURE EACH HAIR SAMPLE HAS A VISIBLE HAIR BULB. REMOVE CAP AND INSERT HAIRS. ENSURE HAIR BULBS ARE COMPLETELY SUBMERSED INTO LIQUID. TRIM ANY HAIR PROTRUDING FROM THE TOP OF THE TUBE WITH A CLEAN PAIR OF SCISSORS. REPLACE CAP, KEEPING VIAL IN AN UPRIGHT POSITION. STORE AT ROOM TEMPERATURE FOR 60 MINUTES .

18. Directions

19. Results after 60 minutes A CHANGE IN THE SOLUTION COLOR TO YELLOW INDICATES THAT A PATIENT WILL HAVE A POSITIVE RESPONSE TO MINOXIDIL.