REFERENCE MANUAL FOR SKIN PUNCH BIOPSY PROCEDURE

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Protocol 42160443NPP2002 Reference Manual for Skin Punch Biopsy Procedure ‐ Version 2.0: 24‐Sep‐2009

Johnson & Johnson Pharmaceutical Research and Development

REFERENCE MANUAL FOR

SKIN PUNCH BIOPSY PROCEDURE

A Multicenter, Randomized, Double‐Blind, Placebo‐Controlled, Dose‐Ranging Study to Evaluate the Efficacy, Safety, and Tolerability of JNJ‐42160443 in Subjects With Diabetic Painful Neuropathy, Followed by a Double‐Blind Safety Extension and an Open‐Label Safety Extension

Protocol 42160443NPP2002

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TABLE OF CONTENTS Page 1.0 Introduction 3

1.1 Authorized Staff 3

2.0 Schedule 3

3.0 Skin Punch Biopsy Procedures 3

3.1 Equipment 3

3.2 Skin Punch Biopsy Procedure 4

3.2.1 Subject Position 4

3.2.2 Selecting the Biopsy Sites 5

3.2.3 Preparation of Biopsy Sites 6

3.2.4 Obtaining the Biopsies 7

3.2.5 Removing the Biopsy Specimen 8

3.2.6 Placing Biopsy Tissue into Fixative 9

3.2.7 Biopsy Site Care 10

3.2.8 Cryoprotecting the Biopsy Samples 10

3.2.9 Shipment of Cryoprotected Biopsy Samples 11 3.2.10 Unscheduled Biopsies 11

3.2.11 Common Errors 11

4.0 Contact Information 13 Attachment 1: Biopsy Template 14 Attachment 2: Biopsy Procedure Flow Diagram 15

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1. INTRODUCTION Rationale

The skin punch biopsy procedures outlined in this document provide a detailed description of the procedures and are intended to anticipate potential questions or problems. Skin punch biopsies will be processed for intraepidermal nerve fiber density (IENFD). Epidermal nerve fibers are C and Aδ small caliber unmyelinated nerve fibers that are particularly relevant to diabetic neuropathy. These nerves are not assessed by conventional testing measures such as nerve conduction velocity, amplitude, or most forms of quantitative sensory testing.

Although the skin punch biopsy procedures and epidermal nerve measures are sensitive and objective, they are also highly complex tasks that can be significantly influenced by technical error or tissue crush artifact. This manual provides detailed procedures for the collection of skin punch biopsies for this study. A training presentation will be provided that will further illustrate the specific procedures. We recognize that skin punch biopsies intended for other purposes, such as pathological assessment for dermatologic lesions, may be different. For the purposes of this study, this manual for performing and handling skin punch biopsies must be carefully followed.

1.1 Authorized Staff The person responsible for performing the skin punches must be a trained medical professional with experience in minor procedures such as blood draws or suturing. Medical Doctors (M.D.), physician assistants, nurse practitioners and, in some states, RNs may perform skin punch biopsies.

All study staff performing the skin punch biopsy procedure are required to complete a web‐based training module prior to performing the procedure.

2. SCHEDULE The scheduled time points for the skin punch biopsies are provided in the study protocol. Shipment instructions will be provided in the central laboratory manual.

The procedure for obtaining all skin punch biopsies throughout the study is identical.

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3.0 SKIN PUNCH BIOPSY PROCEDURES

3.1 Equipment The following items will be supplied by the central laboratory for the skin punch biopsies:

• 1 cc 28 gauge syringe • Sterile disposable 3mm skin punch • Sterile disposable forceps • Sterile disposable scalpel • Zamboni’s fixative solution (individual pre‐filled tubes)

*Note: Each sample will always remain in the same tube it is fixed in • Cryoprotective solution (individual pre‐filled tubes) • Phosphate buffer (individual pre‐filled tubes) • Labels for sample vials • Requisition form • Shipping materials (refrigerated shipper) The following items will be supplied by the study site for the skin punch biopsies: • Gauze pads • Disposable razors (for shaving the biopsy area, if necessary) • Large bandages • Measuring tape • Examination gloves • Alcohol swabs • Paper tape • Parafilm (for securely sealing the sample vial) • Marker (used to mark the skin biopsy locations on the skin) • 2% lidocaine with epinephrine • Clean container (for decanting solutions) • Gelfoam, or other sponge absorbent material (to be used if necessary for significant

bleeding) The following additional items will be supplied by the Sponsor for the skin punch biopsies: • Template for biopsy site locations (1 per subject) – Attachment 1 • Web‐based training presentation

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3.2 Skin Punch Biopsy Procedure Note: Attachment 2 provides a schematic, general overview of the skin punch biopsy procedure.

3.2.1 Subject Position

Each skin punch biopsy procedure will usually require at least 15 minutes to perform therefore it is important for the subject to be in a comfortable position. The subject should lie on a hospital bed, stretcher, or examination table with the relevant side facing upwards. It often helps to place a pillow between the subject’s legs. The subject should keep the leg being biopsied as straight as possible (Figure 1).

Figure 1. Subject Positioning

3.2.2 Selecting the Biopsy Sites

• All biopsies in this study will be obtained from the proximal lateral thigh, 10cm distal to the greater trochanter (Figure 2).

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Figure 2. Location of proximal thigh biopsy sites

• The side for biopsies is not specified, but must be consistent throughout the trial for each subject. If the subject has had knee surgery, hip or other surgery proximal to the knee, use the contralateral leg, if possible. Likewise, areas with surgical scars, tattoos, burns, or other lesions or scars should be avoided when mapping out the biopsy area.

• At each scheduled timepoint, two skin biopsies will be performed at 1 location (proximal thigh). The skin biopsies should be performed on the same leg throughout the trial.

• At each scheduled timepoint, the biopsy locations will be marked on the subject’s template. There will be 1 template provided for each subject. An example of the template is provided in Attachment 1.

3.2.3 Preparation of biopsy sites

1. Using the tape measure, measure 10cm distal to the greater trochanter and place a small mark with the marker. After locating and marking the sites, the areas are shaved if necessary and cleansed with alcohol wipes.

2. A small intradermal bleb of 2% lidocaine with epinephrine is placed adjacent to the marked site using a 1cc syringe. Approximately 0.3 – 0.6 cc of lidocaine is injected at each biopsy site (Figure 3). As you will be obtaining 2 biopsies, inject in 2 areas to anesthetize a total area of about 1 to 1‐1‐1/2 cm.

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Figure 3. A bleb of 2% lidocaine with epinephrine is placed at each biopsy site.

3.2.4 Obtaining the biopsies

Once the site is numb, the biopsies may be performed. Waiting approximately 1 minute after injecting the 2% lidocaine with epinephrine optimizes hemostasis with the procedure.

It is important to avoid the needle track mark from the 2% lidocaine with epinephrine injection site, as well as any ink marks that may have been placed to mark the sites.

1. The sterile disposable 3mm skin punch should be placed gently, but firmly, against the skin and rotated in a single direction. The skin should not be pulled or stretched at the biopsy site (Figure 4).

2. The depth of the skin punch should extend at least ½ way up the bevel of the biopsy tool (approximately 3‐6mm) (Figure 5).

3. Using the same biopsy tool, complete the 2nd punch next to the first one, within the anesthetized area, 2‐3mm from the first punch.

4. If there is any bleeding at the sites, gentle pressure with sterile gauze pads is usually sufficient (Figure 6).

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Figure 4, 5, and 6 (Left to Right)

Figure 4. Biopsy tool is placed gently, but firmly, against skin adjacent to the marked area

Figure 5. Rotate tool until punch depth is at least ½ the length of the punch tool

Figure 6. Bleeding should be minimal

3.2.5 Removing the biopsy specimen

It is extremely important that the skin punch biopsies not be crushed when removing the tissue. Crushing the tissue will introduce artifact and may result in the biopsies not being interpretable.

Using the sterile disposable forceps, the dermis is gently lifted by one edge so that the connecting dermis can be cut using the sterile disposable scalpel, freeing the biopsy specimen.

Note the relationship of the position of the forceps to the scalpel in Figure 7: the scalpel should be placed below the forceps so that there is gentle traction of the tissue against the scalpel.

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Figure 7. Grasping dermis gently by its edge with forceps, the tissue is cut using the scalpel.

If the sample is not freed with a single cut, the scalpel should be gently moved back and forth against the tissue until the sample is freed. Do not lift the scalpel away from the tissue while cutting, as this action results in multiple cuts in the tissue, possibly rendering the sample uninterpretable.

If a portion of the sample remains in the punch tool, it is important to carefully remove and send this sample, as it contains the epidermal nerve fibers.

3.2.6 Placing biopsy tissue into fixative

1. Once the biopsy is removed from the subject, it is immediately placed into the properly labeled sample vial containing the fixative. At each scheduled time point, each of the biopsy samples should be placed in a separate sample vial (i.e., 2 vials per time point).

The biopsy should be completely submerged in order to ensure proper fixation. Samples initially have a tendency to float on the surface and this could result in poor fixation.

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Care should be taken not to catch the sample in the cap when closing the vial. After attaching the cap, the vial should be gently rotated until the biopsy sample sinks to the bottom.

2. The sample vial containing the biopsy sample placed in fixative solution should be immediately placed at 4°C for 6 to 24 hours. It is important that biopsy samples not be fixed for less than 6 hours or longer than 24 hours before placing in cryoprotectant (see 3.2.8).

3.2.7 Biopsy site care

If there is any bleeding, gentle pressure should be applied to the sites with sterile gauze.

Once hemostasis is accomplished, the biopsy sites are bandaged with sterile bandages.

If there is significant bleeding, a small amount of Gelfoam may be placed in the wound and a pressure dressing applied consisting of gauze and a small bandage covered by a larger elastic bandage.

It is normal for the biopsy sites to express serosanguanous fluid for several days, though they should never express pus.

Bandages should be replaced daily until a scab forms, usually 7 to 10 days.

Subjects should be advised not to immerse fresh biopsy sites in whirlpools, bathtubs, pools, etc until a scab is present.

With these precautions, the infection rate for skin biopsy is very low, even in neuropathic populations, and is about 1:500 biopsies.

3.2.8 Cryoprotecting the biopsy samples

1. After at least 6 hours, but no more than 24 hours, the sample vial will be removed from storage (at 4°C).

2. The fixative solution will be gently decanted from the vial into a clean container. The biopsy should remain in the vial. It is advised that the solution be decanted into a clean container to prevent the loss of a biopsy sample in the event a biopsy sample is accidentally poured out of the vial with the solution. In the event the biopsy sample is poured out, it should be gently picked up with forceps and placed back into the vial.

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3. The biopsy sample is then briefly washed twice with the phosphate buffer provided. This is accomplished by filling the vial containing the biopsy with the phosphate buffer and, after a few seconds, pouring the buffer off into a clean container. Repeat. The biopsy sample should remain in its original vial at all times. It is advised that the phosphate buffer be poured into a clean container to prevent the loss of a biopsy sample in the event a biopsy sample is accidentally poured out of the vial with the phosphate buffer. In the event the biopsy sample is poured out, it should be gently picked up with forceps and placed back into the vial.

4. Fill the vial completely with the cryoprotectant solution provided.

5. Once filled with cryoprotectant solution, tightly seal the vial without crushing the biopsy sample. Ensure the biopsy sample is submerged in solution and not stuck on the dry top of the vial. It is important to record the time the sample was placed in cryoprotectant.

6. Place a strip of parafilm over the cap.

7. Once cryoprotected, the biopsy should be immediately stored at 4°C until shipment to the central laboratory. Do not freeze the biopsy samples.

3.2.9 Shipment of cryoprotected biopsy samples Refer to the central laboratory manual for information regarding the shipment of the skin punch biopsy samples.

3.2.10 Unscheduled biopsies

In the event a biopsy sample(s) is rejected by the laboratory (e.g, considered damaged), the subject may be asked to agree to have a repeat skin punch biopsy procedure performed within a specified timeframe.

3.2.11 Common Errors In order to ensure the viability of the biopsy samples, below is a list of errors commonly encountered and the best means of avoiding them. For detailed information, please refer to the web‐based training module of the skin punch biopsy procedure.

1. Biopsy of denervated or obscured site

• Do not select sites for biopsy with obvious scars from wounds , surgeries, burns, or tattoos

• The skin punch should not be placed directly over or touching the mark placed on the skin to locate the biopsy site (Figure 8)

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Figure 8. Common Error

2. Crushed biopsy sample

This typically occurs during excision of the sample due to crushing of the sample by the forceps. To decrease the likelihood of this, remember the following:

• Do not remove the skin punch until only the upper ½ to 1/3 of the bevel is visible. This ensures a deep enough core and will be easier to excise.

• When excising the sample, grasp it with the forceps as deeply and as close to its edge as possible while still ensuring you have a good grasp.

• OBSERVE ALL SAMPLES after they have been placed in the tube. Each biopsy should have a rounded appearance and not look crushed at any border.

• Use care not to crush the sample when capping the tube.

3. The sample does not easily dislodge from the forceps

• Often the sample adheres to the tip of the forceps and can be quite difficult to dislodge. If this occurs, try tapping the forceps edge gently against the rim of the tube. If this does not release the sample, GENTLY nudge it with the blunt edge of the scalpel or with the tip of the needle used to inject the local anesthetic. Do not dip the forceps into the fixative solution to dislodge the sample if the forceps will be in contact with the subject again (i.e., to remove the 2nd sample).

4. Multiple slices in biopsy sample

• This may occur during excision of the sample. Pay close attention to placement of the scalpel when excising the sample. It should be placed directly at the lowest end of the core

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near its attachment point, and moved in the opposite direction of the forceps as they gently tug on the sample. Try to excise using only 1 to 2 cuts with the scalpel.

• Make sure you have a firm grasp on the edge of the sample before attempting to excise it.

5. Overfixing

• The sample should remain in the fixative solution for at least 6 hours, and NO LONGER THAN 24 hours before it is buffered and placed into cryoprotectant. Make sure subjects are scheduled and staff is available to process the specimens in a timely fashion.

6. Not performing Quality Control on all samples

• Biopsies will be shipped directly to the central laboratory; the central laboratory will be sending the samples to the Johns Hopkins Cutaneous Nerve Laboratory at regular intervals. If all samples are not QC’d by the individual sites, it is possible multiple unusable samples will have been obtained before the clinical site receives feedback regarding sample quality/errors from the Johns Hopkins Cutaneous Nerve Laboratory. It is very important for the clinical site to perform a QC of the samples prior to shipment to the central laboratory.

4.0 CONTACT INFORMATION For questions regarding the skin biopsy procedure (e.g., collection, processing, handling, storage):

The Johns Hopkins Cutaneous Nerve Laboratory at 410‐955‐3254.

Please contact your Site Manager for the following:

• Questions regarding the biopsy sample shipment procedure

• Any other protocol‐related questions (unrelated to the skin biopsy procedure)

• For additional skin biopsy kit(s) or skin biopsy kit materials

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ATTACHMENT 1. TEMPLATE

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ATTACHMENT 2. SKIN PUNCH BIOPSY PROCEDURE

Subject to lie down with the relevant side facing upward

Using template,locate and mark biopsy sites

Prepare biopsy sites (e.g., cleanse area, inject 2% lidocaine)

Obtain biopsy samples

(2 per scheduled time point)

At least 6 hours, but no more than 24 hours later, remove vial from 4°C storage and decant

fixative solution

(Biopsy samples remain in original vial at all times)

Wash biopsy samples twice with phosphate

buffer

(Biopsy samples remain in original vial at all times)

Fill vial completely with cryoprotectant solution and tightly seal the vial with parafilm

Perform a QC check of the biopsy samples

Immediately store at 4°C until shipment to central

laboratory (Do not freeze)

Complete the requisition form and

ship the vial to central laboratory

within 72 hours after cryoprotection is

complete

Place each biopsy sample in aseparate sample vial

containing fixative (1 biopsy sample per vial) and

immediately place at 4°C for 6 to 24 hours

REFERENCE MANUAL FOR SKIN PUNCH BIOPSY PROCEDURE

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