A Study Comparing Survival of Hair Follicles Stored Cold and at Room Temperature

MATERIALS AND METHODS

Three male subjects ages 41-64 (two with bald frontal areas, one with Norwood Class VI balding) were recruited to examine the effects of storing follicles (5 hours) in either cold Plasma-Lyte (4°C) or room-temperature Plasma-Lyte (21°C). The focus of this study was to compare whether grafts under normal conditions (no stress) survived better in cold or in room-temperature storage solution (660 hair follicles divided evenly between the two temperature conditions).

I additionally evaluated whether the temperature of the holding solution in surgery had any effect on survival of grafts that received a stress treatment (an additional 800 hair follicles; Table 1). The idea was to ascertain if temperature interacted with trauma conditions to improve survival (e.g., if a cold temperature might rescue a follicle treated with 3% hydrogen peroxide). The description of each treatment condition is as follows:

• None: These follicles did not receive a “stressor” treatment condition, only a temperature storage condition.

• Hydrogen peroxide (1%): I dunked each follicle into 1% hydrogen peroxide for 80 seconds, followed by temperature storage conditions.

• Bulb crushed (hard): I crushed the bulb of the hair follicle using needle-nose pliers, followed by temperature storage conditions.

• Bulb crushed (soft): I crushed the bulb of the hair follicle using needle-nose pliers with a soft rubber cover around the tip (Figure 1), followed by temperature storage conditions.

  

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  FIGURE 1.

  Needle-nose pliers with a soft rubber cover was used for “soft” crush treatments (bulb and bulge). Photo: Michael L. Beehner

• Bulge crushed (soft): I crushed the bulge of the hair follicle using needle-nose pliers with a soft rubber cover around the tip, followed by temperature storage conditions.

• Stored dry (gauze): I stored the hair follicle on dry gauze for 18 minutes, followed by temperature storage conditions.

• Hydrogen peroxide (3%): I dunked each follicle into 3% hydrogen peroxide for 80 seconds, followed by temperature storage conditions.

Patient 1 received all stressor treatment conditions and both temperature storage conditions (cold, room temperature); patient 2 received one stressor treatment condition (3% hydrogen peroxide) and both temperature storage conditions; and patient 3 received no stressor treatment conditions and both temperature storage conditions (Table 1). All patients provided written informed consent for the procedures indicated here. The 3-year study was conducted sequentially on one patient at a time. The original research focus was to examine a combination of stress conditions and temperature storage, but when it became clear that the stress conditions did not interact with temperature, I shifted the focus almost entirely to temperature storage (patients 2 and 3).

For the placement of the grafts, the study boxes were 1.1 × 1.1cm for patients 1 and 2, and 1.2 × 1.2cm for patient 3. Each study box was marked with a tiny tattoo dot at each of the four corners. I left a 1.5mm-wide “moat” of bald scalp around each study box to facilitate easier and faster counting of follicles (Figure 2). I used 1mm-wide lateral slit blades to make sagittal recipient sites. All follicular unit (FU) grafts in the study were 2-hair FUs. The study grafts were out of the body for approximately 4-5 hours for each procedure. All follicles (1,460 in total) were left in their recipient sites to be counted for the presence of a growing hair approximately 1 year later.

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FIGURE 2.

For all patients, a 1.5mm-wide “moat” of bald scalp was left around each study box to facilitate the counting of follicles.

The hair counts on the three patients were performed respectively at 8-, 12-, and 13-months post-surgery. The density of planting these 2-hair FUs were as follows:

• Patient 1 – 25 FU/1.1 × 1.1cm

• Patient 2 – 25 FU/1.1 × 1.1cm

• Patient 3 – 45 FU/1.2 × 1.2cm

When conducting final hair counts, fine vellous hairs were not included in final counts but the slightly “wispy” ones were (these accounted for ~5-6% of the final counts). These percentages were nearly equal for all study boxes counted. All remaining hairs in all three patients were full terminal hairs.

Although each FU contained two hairs, I treated each hair follicle separately for assigning “survival” or “no survival” at the time of the final count. I calculated percentages for survival based on the number of hair follicles that survived/number of hair follicles that were transplanted (Table 2). To ascertain statistical significance, I constructed a logistic regression model (a general linear model in the binomial family) with these data. The outcome variable was Survival (whether each hair follicle survived or not when counted nearly 1 year later). The predictor variables were Patient (1, 2, 3), Treatment Condition (hydrogen peroxide [1%], bulb crushed [hard], bulb crushed [soft], bulge crushed [soft], stored dry [gauze], and hydrogen peroxide [3%]), and Temperature Storage (cold, room temperature). Because in this first model all treatment conditions except hydrogen peroxide (1%) showed lower survival (regardless of temperature condition), I conducted a second model where I collapsed all treatment conditions except hydrogen peroxide (1%) into one treatment group called “Stress” (i.e., the treatment groups in this model were “None,” “Hydrogen Peroxide [1%],” and “Stress”). For this second model, an interaction term was added between Temperature Storage and Treatment Condition to see if temperature storage could rescue the negative effects of a stressor. Results for the second model only are reported here. All statistics were run in R (4.0.5).