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
Requiredbut
notprovided
 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. QualityControl
&
StorageConditions
 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. Limitationof
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
&
Precisionofthe
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. Resultsafter60
minutes
 A CHANGE IN THE SOLUTION COLOR TO YELLOW INDICATES
THAT A PATIENT WILL HAVE A POSITIVE RESPONSE TO
MINOXIDIL.