Performing Suction Blister Skin Biopsies

Abstract

Traditional skin sampling methods include punch or shave biopsies resulting in a sample of solid tissue for analysis. These biopsy procedures are painful, require anesthesia, and leave permanent scars. This unit describes suction blister skin biopsy that can be used in place of traditional biopsy methodologies as a minimally invasive, non-scarring skin sampling technique. Induction of suction blisters uses an instrument with a chamber that applies negative pressure and gentle heat on the skin. Blister formation occurs within one hour, producing up to five blisters, each 10 mm in diameter per biopsy site. Blister fluid can be extracted and centrifuged to retrieve cells from the epidermis and upper dermis for flow cytometry, single cell RNA sequencing, cell culture, and more without the need for digestion protocols. In addition, the blister fluid can be used for measuring soluble proteins and metabolites. This unit describes the methods of preparation for supplies and subjects, suction blister biopsy procedure and formation, blister fluid extraction, and post-blistering care for subjects.

Basic Protocol 1: Preparation of Supplies and Subject

Basic Protocol 2: Suction Blister Biopsy Procedure and Formation

Basic Protocol 3: Blister Fluid Extraction

Basic Protocol 4: Post-blister Care and Clean Up

INTRODUCTION:

In dermatology, tissue samples are critical for both diagnosis and prognosis of skin conditions as well as for use in translational research. Skin biopsies are common procedures that provide dermatology researchers advantages not found in other organs or tissues: the skin is external and has a large surface area, allowing for multiple biopsies, and skin biopsies are less invasive than biopsies of internal organs. Depending on the specific clinical or research goals, different types of biopsies may be beneficial over others.

Punch and shave biopsies are the most common skin sampling techniques and both methods involve the removal of a small piece of solid tissue from the patient. While these types of skin biopsies are an effective diagnostic and research tool, there are many possible side effects and complications that patients experience such as bleeding, permanent scarring, infection, pain, and the need for the administration of a local anesthetic (Nischal et al., 2008). The advantage to these biopsy methods is quick implementation, and the structure of the skin is maintained, which is critical for understanding spatial expression of proteins, RNA and cell-cell interaction. Histology, immunohistochemistry and immunofluorescence are often used to measure protein expression with these biopsies, while newer technologies including SeqFISH+ and Merfish can be used on solid tissue skin biopsies to understand spatial RNA expression patterns. Alternatively, tissue can be digested enzymatically for single cell analysis such as RNA sequencing, flow cytometry, and other techniques (Katz & Harris, 2021). However, using punch biopsies after dissociation have disadvantages: enzymatic digestion of the tissue is stressful on the cells and can lead to transcriptional changes unrelated to the disease state, and enzymes often cleave cell surface proteins leading to challenges for flow cytometric analysis. Suction blister biopsies are an alternative, minimally invasive, non-scarring approach to skin sampling. Gentle heat and negative pressure on the skin leads to blister formation, with a thin epidermal skin covering the blister called the blister roof. The fluid inside the blister contains skin structural cells and immune cells from the epidermis and upper dermis.

Suction blistering was first described in 1964 as an approach to separate the epidermis and upper dermis on human volunteers (Kiistala & Mustakallio, 1964). Soon after, the authors reported the ability to harvest blister roofs to study the epidermis and analyze epidermal bacteria. Blister roof cells were also used for cell culture, while blister fluid was extracted to analyze immune cells in the skin (Mustakallio et. al, 1967; Ingemansson-Nordqvist et al., 1967; Kiistala et al., 1967). Later, the technique was developed into a treatment for vitiligo where both the blister roof and fluid were used for epidermal grafting on stable vitiligo lesions (Koga, 1988; Dellatorre et al., 2017). Recently, the suction blister methodology has been used as a source for cells and fluid that can be used in a variety of modern techniques. Blister fluid can be centrifuged to retrieve cells for flow cytometry, single cell RNA sequencing, cell culture, and more without the need for digestion protocols. This is advantageous because the time from biopsy to recording of data is significantly reduced and therefore more physiologically relevant. Studies have shown that the blister fluid is interstitial skin fluid, not intravascular fluid by profiling and comparing molecular weight and blister-to-serum concentrations of various proteins and lipids. These studies concluded capillary integrity is not compromised during suction blistering, meaning there is not an influx of blood during the blistering process (Vermeer et al, 1979; Rossing & Worm, 1981; Niedzwiecki et al, 2018). Therefore, the blister fluid can be used for accurately measuring soluble proteins and metabolites in the skin (Gellatly et al, 2021; Liu et al, 2017; Strassner et al, 2016; Richmond et al, 2018; Refat et al, 2023; Essien et al, 2022).

An additional advantage of the suction blister approach as compared to a traditional biopsy is that suction blistering does not require local anesthetic and is not typically painful, with only some patients reporting a mild itching sensation similar to a mosquito bite during the procedure. While suction blistering is considered non-scarring, it does commonly leave hyperpigmentation at the blister site, which can last up to 1 year post procedure. To reduce hyperpigmentation, we recommend that blister roofs are left intact on the blister sites post fluid extraction and to cover wounds with petroleum jelly for one week.

Previously published reports using suction blistering fail to provide step-by-step details of how to perform this technique. Here we detail our protocol, which yields reproducible results over time and across personnel. Included are 4 sequential protocols for the suction blister biopsy technique, allowing for the minimally invasive collection of cells from the epidermis and upper dermis without the need for digestion. These protocols describe the preparation of the blister machine and patient, the selection of appropriate blister sites, the induction of suction blisters using negative pressure and heat, the extraction of blister fluid, and post-blister care for subjects.

NOTE: All experimental protocols involving humans or human tissue samples should be approved by the appropriate Institutional Review Board(s) for your institution and location, and informed consent must be obtained from the individuals involved.

STRATEGIC PLANNING:

All experimental protocols involving human subjects or human tissue samples were approved by the University of Massachusetts Chan Medical School Institutional Review Board, Protocol #H-14848. Informed consent was obtained from all subjects prior to enrollment and sample collection. All methods were carried out in accordance with relevant guidelines and regulations, and subject samples remain de-identified throughout the entire study. While we have performed this protocol on vitiligo, psoriasis, contact dermatitis, lupus, dermatomyositis and healthy skin, the cohort described here consists of vitiligo subjects and healthy control subjects free from dermatological conditions.

BASIC PROTOCOL 1: Preparation of Supplies and Subjects

In this protocol, we consent the subject, prepare the Electronic Diversities Negative Pressure Instrument Model NP-4 blister machine and gather supplies for the suction blister biopsy procedure (Figure 1A-B). We then select suction blister sites with clinical relevance and preference from the subject. Subject is properly positioned and prepared for the procedure to begin (Figure 1C).

Figure 1: Preparation of Supplies and Subject.

Reagents needed for blistering procedure (A). Electronic Diversities Negative Pressure Instrument Model NP-4 with one chamber (B). Chamber head securely attached onto subject’s arm with provided velcro (C). Abdominal suction blister sites with chamber head securely attached with paper tape, blister site immediately after removal of chamber heads, and 1 year post blistering (D).

Materials:

Electronic Diversities Negative Pressure Instrument Model NP-4

Orifice plates (Included with Negative Pressure Instrument)

1.5mL microcentrifuge tubes

70% Isopropyl alcohol wipes

1mL 30G syringes

Velcro straps (Included with Negative Pressure Instrument)

Paper Tape

Petroleum jelly

Surgical pen

Transparent film dressings (1624W, 3M^™ Tegaderm)

Non-sterile gloves

Container of ice

Chair or bed

Pillow (optional)

Camera (optional)

1. Explain procedure to the subject, collect consent, and allow subject time to undress as needed.

2. Gather supplies including the Negative Pressure Instrument, orifice plates, sample collection tubes, alcohol wipes, 1mL syringes, Velcro straps, paper tape, petroleum jelly, surgical pen, transparent bandages, non-sterile gloves, a container of ice, and a pillow and camera, if using (Figure 1A).

   1. Decide on the number of blister sites based on study objectives. For example, if looking to compare blisters in lesional compared to non lesional skin, two sites are needed.
   2. Select orifice plates based on the number of desired blisters. Orifice plates range from 1–5 blisters. Each blister yields on average about 75µL of fluid.
3. Plug in the Negative Pressure Instrument Model NP-4 blister machine and place it on a table next to the subject (Figure 1B).

   1. Turn the vacuum valve fully counterclockwise to set the pressure to 0 inHg.
   2. Screw orifice plates tightly onto the chamber heads to ensure no air will leak out during the blistering process.
      The orifice plates should be sterile. Orifice plates can be reused after each procedure and cleaned in an autoclave.
   3. Insert chamber head and attach tubing into the Negative Pressure Instrument and turn the power switch on.
   4. Allow the metal orifice plates to preheat for a minimum of 10 minutes to reach 40°C. Do not turn on the pressure switch. Chamber lamps will cycle on and off to signal the instrument is heating.
      It is safe to utilize the machine on a subject without waiting to preheat, however this adds more time to the procedure and possibly compromises sample quality. The heat is an essential component to the process, and without turning on the heat apparatus the blisters will not form in a reasonable time frame.
      If chamber lamps do not illuminate, or if chamber lamps do not cycle off after 10 minutes, this can be a sign that the instrument is malfunctioning, either failing or overheating above 40°C.

4. Pre-label 1.5mL collection tubes with subject ID and required information for study objectives such as skin phenotype.

5. Select suction-blister site(s) with the subject and mark with a surgical ink pen.

   1. The suction blister biopsy procedure heals within one week but can leave hyperpigmentation. Hyperpigmentation occurs directly under the 10 mm blister orifice in orifice plates as seen in Figure 6.
      Sun exposed areas are more prone to hyperpigmentation, so subjects may prefer to be blistered in areas that are not sun exposed. Sites in sensitive areas (under arm, inner thigh, abdomen, etc.) are more likely to react to the heat of the machine and may be uncomfortable during the blistering process. Sites with fewer hairs are preferred because hair can prevent tight suction. Alternatively, the area can be shaved to avoid disruption to the negative pressure.
      Typically, it is advised to avoid blistering on the face because hyperpigmentation on the face may be burdensome for subjects. It is also advised to avoid hands and feet because it can be difficult to achieve a tight seal for suction, and it is also difficult for subjects to keep those sites covered for 7 days with the dressings.

6. Photograph blister site(s) (optional).

   Photography is not needed for the success of the suction blister procedure, but it is encouraged for documentation. Photos can be used to retrospectively evaluate site selection to confirm presence or absence of skin disease and to best document anatomical location of blister sites.

7. Position subject to remain in a comfortable position for up to 2 hours.

   It is best practice with medical procedures to position the subject tilted, reclined, or lying down to ensure they are relaxed and comfortable, and to reduce any possible risk of fainting.

   Subject positioning also depends on selected blister site(s). If blistering on the back of the subject, it is best to have the subject lying face-down on an exam table or stretcher for the duration of the procedure. If blistering on extremities, position the subject reclined in a chair. Subject may need to be supported with a pillow as seen in Figure 1C (optional).

   Subject should be placed in the middle of the room for the procedure so that all blister sites are easily accessible during blister fluid extraction.

8. Sanitize hands and put on gloves. Use an alcohol wipe to clean all blister site(s) and orifice plate(s).

9. Warn subjects that the device will be warm to the touch and then attach blister chamber(s) to the subject using velcro (provided with the machine) or paper tape so that they are securely fastened to the skin Figure 1C).

   Using the provided velcro straps can be easier than paper tape because there is a tighter seal against the skin. However, velcro straps can only be used on extremities. If there are challenges getting the chamber heads securely fastened to the skin with tape, then use tape as best as possible and hold chamber heads by firmly pressing into the skin as you proceed to Basic Protocol 2, step 1, to turn the vacuum on. The additional suction from the machine should help the chamber heads stay in place. Apply more tape as needed afterwards as seen in Figure 1D.

BASIC PROTOCOL 2: Suction Blister Biopsy Procedure and Formation

In this protocol, suction blister biopsies are induced. This procedure is used to isolate cells and fluid from the epidermis and upper dermis in the skin. Blister formation occurs within approximately 1 hour (Figure 2). This protocol is performed immediately after following Basic Protocol 1, step 9.

Figure 2: Suction Blister Biopsy Procedure and Formation.

Visual time course of blister formation. 0 minutes of negative pressure (A). 10 minutes of negative pressure (B). 15 minutes of negative pressure, early signs of vesicle formation in the center and bottom right orifices. Yellow arrows point to vesicle formation (C). 20 minutes of negative pressure, blister vesicle formation and enlargement in 3 orifices, as indicated by yellow arrows (D). 35 minutes of negative pressure, vesicle formation observed in all 5 orifices (E). 45 minutes of negative pressure, top left blister beginning to leak, shown by yellow arrow, but other sites not yet fully expanded to 10 mm. Yellow bars indicate space between blister and plate (F). 55 minutes of negative pressure, top left blister continuing to leak, all other sites fully expanded (G). 5 fully formed blisters immediately after cup removal at 55 minutes (H).

Materials:

Negative Pressure Instrument Model NP-4

1. Turn on the vacuum power switch and press firmly down on the chamber head(s) to achieve suction on the skin.

   The application of negative pressure may cause an itching sensation, similar to a mosquito bite that can persist throughout the procedure. The procedure should be stopped if the subject feels any extreme discomfort.

2. Continue to apply manual pressure to chamber head(s) while turning the vacuum valve clockwise until reaching approximately 5–7 inHg of negative pressure. Subjects over the age of 65, subjects with thin skin, or subjects prone to bleeding should start at 5 inHg.

   1. Record start time of negative pressure.

   In skin conditions that have a compromised skin barrier, such as atopic dermatitis, using a lower pressure and a longer blister induction time can prevent leaking of blisters.

   If unable to reach 5–7 inHg, check that all chamber heads and tubing are securely tightened and attached. Press down on chamber heads to check for suction. If there is still no increase in negative pressure with applied manual force, unplug the vacuum on the blister channel that is suspected to be causing issues (typically the subject will not feel suction at that site and/or there are no visible signs of suction). If the negative pressure increases after disconnecting that channel, you will need to change the tubing on that channel only. Start by turning off the pressure on the machine, unscrew the orifice plate, screw the plate onto new tubing, and plug into the Negative Pressure Instrument. Resume from Basic Protocol 1, step 8.

3. Closely monitor blister formation for the first 15 minutes of the procedure. If there is no sign of local hemorrhage, increase the pressure an additional 2 inHg.

4. After 20–30 minutes, early signs of blister formation will start to appear. This may include small raised fluid filled vesicles or blisters as seen in Figure 2A, typically centered around a hair follicle. If signs of blisters are observed by 30 minutes, remain at a constant pressure.

   If you do not see signs of blister formation, increase the pressure an additional 2 inHg every 5–10 minutes. Do not increase the pressure above 17 inHg.

5. After 45 minutes, blisters will expand to fill the entire 10 mm orifice. If the blisters are not fully formed, increase the pressure an additional 2 inHg continue suction for up to one hour from the recorded start time of negative pressure. Do not increase the pressure above 17 inHg.

   Blisters will form at different rates depending on skin thickness (thicker skin such as legs take longer than abdomen). Monitor all blisters carefully and proceed to Protocol 3 step 1 once the blisters are fully formed at one site. Not all sites will finish at the same rate; some chambers will stay attached to the skin with negative pressure, while others will finish and need to be extracted as described in Basic Protocol 3 with the fluid immediately placed on ice.

   If one blister begins to leak, but the other blisters at that site are not fully formed yet, the procedure can continue as needed. In Figure 2, fluid can be seen leaking from the top left blister at 45 minutes. The other blisters did not expand yet to fill the 10 mm orifice, so the procedure was continued for an additional 10 minutes. When evaluating if the procedure should continue, consider the total fluid volume across all blisters in the chamber instead of only accounting for fluid loss. If possible, recover leaked fluid off orifice plates after completing Basic Protocol 3, step 5.

   If blisters are not fully formed by one hour, it is safe to continue up to 30 minutes longer for a maximum of 90 minutes assuming no signs of local hemorrhage occur across multiple blisters. However, cells in the blister fluid may begin to die if the procedure continues for extended periods, potentially compromising sample quality. Hemorrhagic blisters are usually excluded from sample analysis because interstitial fluid can not be distinguished from blood. Balancing size of blisters without inducing blisters to hemorrhage is needed.

BASIC PROTOCOL 3: Blister Fluid Extraction

In this protocol, we aspirate fluid from suction blister biopsies using an insulin needle (Figure 3A). Blister fluid can then be used for flow cytometry, single cell RNA sequencing, cell culture, and more, without the need for digestion protocols.

Figure 3: Blister Fluid Extraction.

Extraction of fully formed suction blister using 1-mL syringe with bevel face up (A). Blister site before and after extraction (B).

Materials:

Non-sterile gloves

Camera (optional)

1mL 30G syringes

1.5mL microcentrifuge tubes

Container of ice

1. Once blisters are fully formed, turn off the vacuum and note the end time of the negative pressure.

   If blistering in multiple sites and only one of the sites is ready for extraction, temporarily decrease the negative pressure on the machine to about 7 inHg. Carefully disconnect the vacuum directly from the Negative Pressure Instrument on only the site that is finished. Detaching this chamber may cause an unwanted increase in negative pressure for the remaining site(s) leading to localized hemorrhage. Adjust the machine back to the desired pressure and monitor the remaining site(s) until blister formation.

2. Wearing gloves, detach the tubing from the machine. Unstrap any velcro and remove tape from the subject. Hold the top of the chamber head and gently peel off the chamber head from the skin starting at one side. Be careful not to rupture the blisters.

3. Photograph blister site(s) pre-extraction (optional).

   Blister sites and the surrounding area may appear red from the heat and negative pressure. This redness should resolve within one hour.

   Photography is not needed for the success of the suction blister procedure. Photos taken after the procedure can document blister formation outcomes. Photos can also be used to document adverse events such as local hemorrhage or redness.

4. Open the 1mL syringe. Test the syringe by pulling the plunger to the end of the barrel and pushing it back into the syringe. This helps ensure the syringe is functional and able to extract fluid.

   If you do not test the syringe, it may get stuck when inserting into the blister leading to a loss in fluid.

5. Align the needle at a 30° angle from the surface of the skin and insert the needle into the center of the blister with the bevel up as described in Figure 3A. Simultaneously, pull back on the syringe plunger to extract fluid. Slowly aspirate the fluid and place it in the pre-labeled 1.5mL collection tube on ice. Keep the needle near the roof of the blister and continue to aspirate until completely removing all blister fluid. The blister will appear deflated as shown in Figure 3B.

   Keeping the needle along the roof of the blister will prevent pain for the subject and promote removal of the epidermal cells for collection. If the needle touches the blister base at the dermis, it may cause localized bleeding, pain, and possibly compromise sample integrity.

   Simultaneously inserting the needle into the blister while pulling back on the plunger prevents fluid loss. If you do not do this simultaneously you risk rupturing the blister causing fluid to leak and not be collected.

BASIC PROTOCOL 4: Post-blister Care and Clean Up

In this protocol, we cover the blister biopsy sites with petroleum jelly and a protective dressing (Figure 4). Subjects are instructed how to care for sites for the following 7 days post procedure. The blister machine is disassembled, and supplies are sanitized.

Figure 4: Post-blister Care and Clean Up.

Petroleum jelly and transparent film dressing covering two separate blister sites after fluid extraction.

Materials:

Non-sterile gloves

Petroleum jelly

Transparent film dressings (1624W, 3M^™ Tegaderm)

1. With new, clean gloves, use your finger to cover deflated blisters with a thin layer of petroleum jelly followed by a protective transparent dressing.

   Avoid applying a thick layer of petroleum jelly to the wound, as it will leak out of the transparent dressing.

   Warn subject that the petroleum jelly application may temporarily sting.

2. Instruct the subject to leave the wounds covered in transparent dressing for 24 hours. After 24 hours, the subject can remove the bandage and shower, letting soap and water run over the area, and then reapply petroleum jelly and a new bandage. Continue for 7 days.

3. Advise subject to consult a medical professional if any signs of local or systemic infection are observed such as abnormal redness, swelling, drainage, or fever.

   Infections from procedure are rare but the subject should monitor the blistering site. 0 of the 40 participants in this study reported infection.

4. Allow subject time to re-dress and ask any remaining questions. Hand subject at home wound care supplies as they leave their procedure.

   Provide subjects with enough petroleum jelly and dressings to cover blister site(s) for 7 days.

5. Disassemble the Negative Pressure Instrument.

   1. Power off the machine and unplug from the outlet.
   2. Remove all tape or velcro from chamber heads.
   3. Unscrew orifice plates.
      Orifice plates can be reused after each procedure. Sterilize orifice plates in an autoclave.
   4. Disinfect all tubing, chamber heads, and machine with a disinfectant wipe.

6. Dispose of waste in appropriate containers. Sanitize the procedure room with a disinfectant wipe.

   Account for each 1-mL syringe and dispose of them in a sharps container.

7. Keep blister fluid on ice and begin processing immediately.

COMMENTARY:

Background Information:

Skin biopsies are common procedures that provide dermatology researchers advantages not found in other organs or tissues: the skin is external and has a large surface area, allowing for multiple biopsies, and skin biopsies are less invasive than biopsies of internal organs. Depending on the specific clinical or research goals, different types of biopsies may be beneficial over others.

To study the skin, traditional punch and shave biopsy sampling techniques have been developed to remove a small piece of solid tissue from patients. These biopsies are common procedures used for the diagnosis and prognosis of skin diseases as well as for research. While punch and shave biopsies are effective, there are many possible side effects including bleeding, permanent scarring, infection, pain, and the need for the administration of a local anesthetic (Nischal et al., 2008). This suction blister biopsy technique is a minimally invasive and non-scarring alternative to skin sampling.

Using a negative pressure instrument that applies gentle heat and negative pressure, suction blister biopsies form on the skin in approximately 60 minutes. Fluid inside the blister contains skin structural cells and immune cells from the epidermis and upper dermis. The thin epidermal layer covering the fluid is called the blister roof. The described suction blister biopsy procedure is an efficient, non-scarring, and low-risk technique for researchers to obtain a cell suspension from skin samples.

Critical Parameters and Troubleshooting:

The protocols described in this unit have been used to successfully induce suction blister biopsies yielding reproducible results both over time and across different personnel performing the biopsy (Gellatly et al, 2021; Liu et al, 2017; Strassner et al, 2016; Richmond et al, 2018; Refat et al, 2023; Essien et al, 2022). However, there are several critical parameters worth discussing.

Basic Protocol 1

In this protocol, it is important that the orifice plates preheat for a minimum of 10 minutes before being applied to the skin (step 3d). We strive to be as efficient as possible to minimize the procedure time for subjects. Because of this, we typically obtain verbal consent in advance of the procedure to reduce total time so that we can start with Basic Protocol 1, steps 2–4, preparing the machine and supplies and allowing the machine to properly preheat prior to the subject’s arrival. When the subject arrives, we collect written consent and immediately proceed to Basic Protocol 1, step 5. This reduces the time of the procedure and ensures the subject does not need to wait for supplies to be prepared and or for the machine to preheat.

It is also essential to select blister sites with clinical relevance and guidance from the subject. The unique skin phenotype in the selected anatomical location will greatly impact blister formation and healing. Sun exposed sites such as arms, legs, and neck, are more prone to hyperpigmentation after the suction blister biopsy procedure. Sites in sensitive areas such as under arm and inner thigh may be uncomfortable during the blister process for subjects. Sites with large hairs can be difficult to achieve tight suction with. For best results for research objectives and subject well-being, it is advised to carefully select blister sites.

Basic Protocol 2

It is critical to follow this protocol closely to maximize blister fluid volumes. In the event that suction blister biopsies are unable to form, see table 1 for a troubleshooting guide for suction blister biopsy formation.

Table 1.

Troubleshooting Guide for Suction Blister Biopsy Formation

Problem	Possible Cause	Solution
Negative pressure won’t increase	Leak in blistering apparatus	Check that all chamber heads and tubing are securely tightened and attached. Press down on chamber heads to check for suction. If there is still no increase in negative pressure with applied manual force, unplug the vacuum on the blister channel that is suspected to be causing issues (typically the subject will not feel suction at that site and/or there are no visible signs of suction). If the negative pressure increases after disconnecting that channel, you will need to change out the tubing on that channel only. Start by turning off the pressure on the machine, unscrew the orifice plate, screw the plate onto new tubing and plug into the Negative Pressure Instrument. Resume from Basic Protocol 1, step 8.
Blisters are formed at one anatomical site but not all	Different skin phenotype in different anatomical location	If blistering in multiple sites and only one of the sites is ready for extraction, temporarily decrease the negative pressure on the machine to about 7 inHg. Carefully disconnect the vacuum directly from the Negative Pressure Instrument on only the site that is finished. Detaching this chamber may cause an unwanted increase in negative pressure for the remaining site(s) leading to localized hemorrhage. Adjust the machine back to the desired pressure and monitor the remaining site(s) until blister formation.
No sign of blister formation after 30 minutes	Subject has thick skin, scar tissue, or a condition resulting in plaques on the skin (i.e. psoriasis)	Assuming no sign of local hemorrhage, increase the negative pressure by 2 inHg incrementally every 5–10 minutes until reaching around 17 inHg. Some skin can withstand higher pressure, or some blisters may take longer to form.
Local Hemorrhage	Sudden increase in pressure	Decrease the pressure immediately. Check to see if signs of hemorrhage are observed in all blisters or just one. Continue the process at a low pressure (5–7 inHg) for the first 30 minutes and increase as needed assuming no other sites of hemorrhage. When extracting the blister fluid, keep hemorrhage samples separate to prevent contamination of blood cells in your analysis.
Leakage of fluid	While blisters occasionally leak on non compromised skin, it is more common while blistering skin with disrupted skin barriers (such as contact derm)	Blister as close to the disrupted skin as possible but not directly on it. To reduce likelihood of blisters leaking close to the disrupted skin, blister at a 5 inHg and do not increase past 7 inHg.

Basic Protocol 3

To properly collect all blister fluid from each blister site, it is important to follow Basic Protocol 3 slowly and carefully. Testing the syringe in step 4 is integral to collection. If the syringe is faulty, the blister will rupture once the needle is inserted, and the fluid will be lost because it will not be collected into the syringe. Additionally, the needle insertion and extraction by pulling on the syringe plunger must happen simultaneously. If this is not done simultaneously there is a risk rupturing the blister causing fluid to leak and not be collected.

Suction blister roofs can be removed after step 5 and used for experiments such as cell culture (Ingemansson-Nordqvist et al., 1967), but it is not recommended unless it is critical to study objectives. Removing blister roofs can be a painful process for subjects, increases risk of infection, prolongs healing time, and increases the risk of hyperpigmentation. All wound healing images shown in this protocol were taken with blister roofs still attached.

It is also important that blister fluid is placed on ice immediately after collection and remains on ice for the duration of Basic Protocols 3 and 4.

Basic Protocol 4

At the conclusion of the suction blistering procedure, follow the described Basic Protocol 4 to cover wound sites with petroleum jelly. Petroleum jelly can temporarily sting at blister sites (especially if blister roofs are removed), so warn subjects before applying. Do not cover wounds with excessive amounts of petroleum jelly or else it will leak out of the transparent dressing. Follow Figure 4 as a guide.

Troubleshooting:

For a list of problems, possible causes, and solutions, see Table 1.

Statistical Analysis:

All data are reported as mean and standard deviation of the mean. Statistical differences were tested using an unpaired t test. Prism10 software was used for statistical comparisons and article figures.

Understanding Results:

We performed suction blister biopsies in 36 vitiligo and 4 healthy control subjects (age 49.05±16.53) with 417 total blisters. Sample yield using this suction blister biopsy technique was 75.05±31.26µL (range= 16.6–156µL) per blister (Figure 5A). The average time for complete blister formation using the Negative Pressure Instrument was 54.01±12.64 minutes (Figure 5B). The pressure was generally kept between negative 7–11 inHg throughout the procedure.

Figure 5: Average blistering yields and outcomes.

Average time for blister formation for lesional vitiligo skin and unaffected skin. (A). Average volume of blister fluid collected from lesional vitiligo skin and unaffected nonlesional skin (B). Pie chart showing the percent of subjects willing to volunteer for a punch biopsy of those who consented for a suction blister biopsy (C).

One of the biggest advantages of using the blistering biopsy technique is that it is less invasive than a punch biopsy. We often have a difficult time recruiting subjects for punch biopsies but have greater success recruiting for suction blistering because of the reduced side effects. In our cohort who consented for blistering, 14 subjects (36.84%) declined an additional skin punch biopsy sample due to the invasiveness of the procedure and permanent scarring (Figure 5C). Therefore, we obtain more clinical samples because of use of this suction blister biopsy protocol unit.

5 of 417 blisters (1.19%) became hemorrhagic during the procedure. Hemorrhagic blisters were excluded from sample analysis because the interstitial fluid could not be distinguished from blood. Subjects who experienced hemorrhagic blisters did not report any adverse events during recovery. As seen in Figure 6B, hemorrhagic blisters fully healed along with other blister sites.

Figure 6: Hyperpigmentation.

Suction blister sites before the procedure, immediately after, and at different time points showing hyperpigmentation (A). Comparison between suction blister biopsy and a 4mm punch biopsy 1 year later (B).

None of the subjects reported signs of infection following the blistering procedure. One subject reported minor skin irritation from the transparent bandage applied during the one-week recovery period. The subject was instructed to stop using the bandages and apply over the counter topical steroid to reduce pain and swelling if needed.

Time Considerations:

The entirety of protocols 1–4 will take approximately 2 hours per subject. Protocol 1, Preparation of Supplies and Subjects, takes approximately 30 minutes with 10 minutes to prepare supplies, 10 minutes for the machine to pre-heat, and 10 minutes for subject consent and preparation. Protocol 2, Suction Blister Biopsy Procedure and Formation, takes approximately 1 hour for blister formation. Protocol 3, Blister Fluid Extraction, takes approximately 5 minutes per blister site, or 20 minutes total if using all 4 chambers on the NP-4 model. Protocol 4, Post-blister Care, takes approximately 10 minutes.

DATA AVAILABILITY STATEMENT:

The data that support the protocol are available from the corresponding author upon reasonable request.