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.