Autofluorescent Flavoprotein Imaging of Intraepidermal Nerve Fibers: a Pilot Study: Complete Statistical Analysis

1. Trial at a Glance

This page covers the registry results for Autofluorescent Flavoprotein Imaging of Intraepidermal Nerve Fibers: a Pilot Study (NCT02537951), a non-randomized na study. The primary outcome is AFI-intensity After Nociceptive Stimulation.

2. Clinical Question

The scientific motivation is that SFN can be challenging to diagnose, according to the study description, because neurological examination and nerve conduction studies are often normal. Small nerve terminals are present in the epidermis, and the study rationale notes that intraepidermal nerve fibers are greatly reduced in people with SFN. The investigators hypothesized that AFI intensity would be reduced in SFN, and proposed a pilot experiment as an initial step toward evaluating that idea.

The experiment is not a diagnostic-accuracy study in patients with SFN. Its participants were healthy volunteers, and the capsaicin condition was intended to mimic a selective reduction of small nerve fibers temporarily. Thus the primary clinical question is not whether AFI diagnoses SFN in clinical practice. It is whether fingertip AFI intensity can be measured and respond to the experimental conditions in a way that could support later evaluation.

3. Trial Design

This was a completed pilot study at Erasmus Medical Center, with a registry enrollment of 10. The study began in 2015-09 and its primary completion was 2016-07. Its phase is listed as not applicable. The allocation is non-randomized, masking is none, and the model is sequential. These features describe an exploratory measurement study rather than a randomized, masked comparison of treatment groups.

The registry lists three measurement conditions: AFI intensity in healthy volunteers, measurement 1hour after application of lidocaine/prilocaine cream, and measurement 1week after application of an 8% capsaicin patch. AFI intensity was measured at the third fingertip with an AFI microscope. The stated primary outcome timeframe also identifies Day 1, T=0h for AFI measurements, Day 1, T=6h after lidocaine/prilocaine, and Day 7 after capsaicin. The arm description and outcome timeframe use different timing descriptions for the lidocaine/prilocaine measurement; the record does not explain that difference.

The healthy-volunteer measurement provides the reference condition described by the registry. Lidocaine/prilocaine is identified as a negative control. The capsaicin patch is intended to induce a temporary selective reduction of small nerve fibers, mimicking SFN for the purpose of testing AFI. The record does not report a randomized sequence, washout procedures, or additional design details, so those should not be inferred from the word “sequential.”

The registry explicitly notes that this is not a randomized comparison and that the arms are not a like-for-like control comparison. The listed arm descriptions each refer to 10 healthy volunteers, but the record does not describe them as independent cohorts. The design should therefore be read as measurements across sequential conditions, not as evidence from three independently randomized groups. No participant-level flow, exclusions, or analysis population is reported in the registry record.

4. Participant Flow and Accounting

The registry reports how participants moved through each stage of the study. All counts below are exactly as posted; missing counts are not inferred from other stages.

Before assignment: all 10 healthy volunteers underwent 3 experiments, all 10 have completed these experiments: 1. AFI intensity measurements 2. lidocaine/prilocaine creme, one hour later followed by AFI intensity measurements 3. capsaicin patch (2 hours after lidocaine/prilocaine creme), one week later followed by AFI intensity measurements

Overall Study

5. Baseline Characteristics

The table reproduces the baseline characteristics posted in the registry, which describe the participants analysed at baseline and help show whether the randomized groups were comparable.

6. Primary Endpoint

The registry lists the primary outcome as AFI-intensity After Nociceptive Stimulation, measured over Day 1, T=0h (AFI measurements), Day 1, T=6h (AFI measurements after lidocaine/prilocaine), Day 7 (AFI measurements after capsaicin).

The primary endpoint is AFI intensity, reported as delta F/F, at the third fingertip directly after application of grading nociceptive stimuli. It is a continuous measure, and the planned summary is a mean with standard error. The registry specifies measurements at baseline on Day 1, after lidocaine/prilocaine on Day 1, and after capsaicin on Day 7.

The endpoint was chosen to assess whether an optical signal can quantify neuronal activity in the termination area of small nerve fibers. The study rationale connects this question to the epidermis, which contains a high density of small nerve terminals, and to the reduced number of intraepidermal nerve fibers described in SFN. The capsaicin condition was intended to create temporary small nerve fiber reduction, allowing the investigators to examine whether AFI intensity could reflect that experimental change. The negative-control condition was included to provide another context for interpreting AFI measurements.

A mean describes the average measured signal for a condition. In general, the standard error describes the precision of an estimated mean; it is not a direct description of how widely individual measurements vary. No mean, standard error, between-condition estimate, or confidence interval is reported in the registry record. Consequently, the endpoint definition and intended measurement schedule are available, but the size and precision of the observed signal are not.

7. Why No Re-analysis Is Shown

8. Statistical Interpretation

9. Statistical Methods Explained

The endpoint is continuous: AFI intensity is measured as delta F/F and summarized using a mean and standard error. In general, a mean represents the central value of a set of measurements. The standard error describes the precision of an estimated mean, whereas a standard deviation describes variation among individual measurements. These quantities answer different questions, so a standard error should not be read as the range containing most individual observations.

What would an estimate show?

For a continuous endpoint, a condition-specific mean would describe the average AFI signal under that measurement condition. A contrast between conditions would describe how the estimated means differ under the specified analysis. A confidence interval, if reported, would show a range of values compatible with the data and the estimation method. In general, narrower intervals indicate greater precision, while wider intervals indicate greater uncertainty. The registry record does not report means, contrasts, or confidence intervals for this endpoint, so none of these features can be assessed.

How should the conditions be compared?

The study is described as sequential and non-randomized, and the record cautions that its arms are not a like-for-like control comparison. In general, measurements collected from the same people across conditions are dependent: a person’s measurements may be related across time. An analysis of repeated measurements should account for that dependence rather than treating every condition as a separate, unrelated group. The registry record does not report the statistical model or analysis method used, so it is not possible to determine how the dependence was handled.

Sequence and timing can also affect interpretation in a repeated-measurement design. Changes could reflect the condition of interest, but could also be influenced by factors associated with measurement order or elapsed time. This is a general design consideration, not evidence that such influences occurred in this study. The registry does not report randomization of condition order, a washout plan, or an adjustment for order or time. It also gives both a Day 1, T=6h outcome timeframe and a 1hour arm-description timing for lidocaine/prilocaine; the difference is not explained in the record.

What does the negative-control label mean?

The registry calls lidocaine/prilocaine a negative control. In general, a negative control is a condition expected to help assess whether a measurement changes when the target effect is not expected. Its usefulness depends on the scientific rationale and the design. The label alone does not demonstrate that the condition is an adequate control, nor does it state a result. The record does not provide a numerical AFI result for this condition or an analysis comparing it with the other measurements.

Capsaicin is described as a means to induce temporary selective reduction of small nerve fibers and mimic SFN. In general, this creates an experimental model of one aspect of a condition; it is not the same as observing the full condition in patients. The record does not report the magnitude of any induced change in nerve fibers or AFI intensity, so the extent to which the intended manipulation was achieved cannot be evaluated from the posted results.

What can planning calculations establish?

The registry describes a sample-size planning illustration using the observed effect, a two-sided significance level of 0.05, equal allocation, and a normal approximation. It says the effect to detect is a stated fraction of the observed effect. In general, planning calculations estimate the sample size needed under specified assumptions; they are not evidence that a study had a particular probability of detecting an effect after the fact. They should not be described as observed power. The registry record does not report the numerical planning results or a primary-outcome estimate on which to assess them.

The planning description also specifies that non-time-to-event calculations are expressed as participants per arm for the stated power target, while time-to-event calculations use a total event count. The primary endpoint is continuous, not time-to-event. No numerical sample-size illustration for this endpoint is reported in the registry record. Because the allocation is non-randomized and the study is sequential, the equal-allocation assumption in the planning description should not be mistaken for a description of the actual study design.

What information is absent?

The registry lists one primary outcome measure and zero analyses. It does not report an analysis plan, test statistic, probability value, confidence interval, missing-data approach, or analysis population. It also does not state whether the primary outcome was analyzed with a paired method, a repeated-measures model, or another approach. Such details matter because the design includes sequential measurements and because estimates can depend on which observations are included and how within-person relationships are represented. The absence of these details in the registry record does not establish whether analyses were conducted elsewhere; it limits what can be concluded from this record.

In general, statistical interpretation should keep the measurement definition, study design, estimation method, and uncertainty together. Here, the record supplies a clear endpoint description and intended measurement occasions, but not the numerical endpoint findings or methods needed to evaluate an estimated effect. Thus, a reader can understand the question and design, but cannot assess the size, direction, or precision of an observed AFI response from the registry record.

10. Confidence Intervals for Each Arm

No two-arm comparison is shown for this trial, but each arm's mean can be given with a t-based 95% confidence interval computed from the posted mean, spread and number of participants.

R code and output

arm <- c("AFI Intensity in Healthy Volunteers", "Negative Control 1: Lidocaine/Prilocaine", "Negative Control 2: 8% Capsaicin")
n   <- c(10, 10, 10)
m   <- c(0.6, 0.25, 0.5)
sp  <- c(0.25, 0.25, 0.5)
sd  <- sp * sqrt(n)      # standard errors converted to SDs
for (i in seq_along(n)) {
  se <- sd[i] / sqrt(n[i]); h <- qt(0.975, n[i] - 1) * se
  cat(sprintf("CARM|%s|%d|%.3f|%.3f|%.3f|%.3f\n", arm[i], n[i], m[i], sd[i], m[i] - h, m[i] + h))
}

CARM  AFI Intensity in Healthy Volunteers  10  0.600  0.791  0.034  1.166
CARM  Negative Control 1: Lidocaine/Prilocaine  10  0.250  0.791  -0.316  0.816
CARM  Negative Control 2: 8% Capsaicin  10  0.500  1.581  -0.631  1.631

11. Multiplicity, Missing Data and Analysis Populations

The registry lists one primary outcome measure, AFI intensity after nociceptive stimulation, and zero analyses. It does not report a multiplicity strategy, such as how inference would be handled across multiple time points, conditions, or other outcomes. The single listed primary measure does not establish that only one statistical comparison was considered. The statistical analysis plan is not reported in the registry record.

Missing-data handling is also not reported in the registry record. There is no description of whether any outcome measurements were missing, how incomplete measurements were treated, or whether any participants were excluded from an analysis. The registry provides an enrollment figure and condition descriptions but no participant flow or analysis-population definition. It is therefore not possible to determine from the record whether all planned measurements contributed to a reported estimate.

In general, missing observations can affect an estimate when their absence is related to the measured outcome or study conditions. The consequences depend on the amount and pattern of missingness and on the analysis method. Those details are not reported, so no assumptions about completeness or the effect of missing data should be made. Similarly, the record does not specify an intention-to-treat or per-protocol population, and those labels should not be inferred for this sequential study.

12. Safety Results

The registry reports serious adverse events as affected participants divided by participants at risk.

Deaths

Other (non-serious) adverse events

Participants with at least one non-serious event at or above the registry's 0% reporting threshold.

The registry lists serious adverse events, other adverse events, and deaths for each of the three conditions. For AFI intensity in healthy volunteers, the counts of serious adverse events and other adverse events are both zero, and deaths are zero. The lidocaine/prilocaine condition also lists zero serious adverse events, zero other adverse events, and zero deaths. The 8% capsaicin condition lists zero in each of these categories as well.

These entries are counts in the registry record. They do not provide event descriptions, severity details beyond the categories, or additional follow-up information. The record lists 10 at risk for each category in each condition; it does not explain how safety observation periods were defined. A count of zero recorded events should not be interpreted as proof that an intervention has no risk, or as a precise estimate of risk in other settings. The registry record does not report further safety analyses.

Because the study is sequential and the condition descriptions refer to healthy volunteers, the listed groups should not be assumed to be independent cohorts. The event entries are best read as the safety counts recorded for each condition, without drawing a comparative safety conclusion from them.

13. What the Result Does, and Does Not, Mean

This registry record describes an exploratory study of AFI intensity at the third fingertip in healthy volunteers. Its conditions include measurement without the listed control applications, measurement after lidocaine/prilocaine cream, and measurement after an 8% capsaicin patch. The intended scientific contribution was to assess whether AFI could provide a useful signal in epidermal small nerve terminals, including after temporary small nerve fiber reduction.

The record does not report the primary outcome values or an estimate comparing conditions. It therefore does not establish whether AFI intensity changed, how large any change was, or how precisely it was estimated. It also does not report results that would establish measurement reliability or validity. Completion status means the study is recorded as completed; it does not supply missing endpoint estimates.

The design is non-randomized and sequential, and the registry warns that the arms are not a like-for-like control comparison. The study is not described as enrolling patients with SFN. Consequently, the record does not show how AFI performs as a diagnostic test in people with SFN, nor does it report sensitivity, specificity, or other diagnostic-accuracy measures. The capsaicin manipulation was intended to mimic a temporary reduction of small nerve fibers, which is narrower than evaluating SFN as a clinical disorder.

The safety entries contain zero recorded serious adverse events, other adverse events, and deaths for each condition. These counts should be understood as registry entries, not as proof of absence of risk or as a basis for conclusions beyond the recorded study conditions.

Overall, the registry gives a rationale and a measurement plan, but not the numerical findings needed to judge the primary question. Statistical conclusions about effect direction, magnitude, uncertainty, or evidence against a null hypothesis are not supported by the posted results.

14. Why This Trial Matters Statistically

The study raises a measurement question that is statistically important for biomarker development: can an optical signal be quantified at a small, anatomically defined site and respond to a controlled change in small nerve fibers? The primary endpoint is continuous AFI intensity at the third fingertip, which makes the definition of the measurement and its precision central to the question. The registry identifies a mean and standard error as the summary approach, but does not report the resulting values.

The design also highlights why repeated measurements require careful interpretation. Baseline and post-application measurements may be related within a participant. General statistical methods for repeated observations account for this dependence, but the registry does not state which method was used. The non-randomized, sequential design further means that the conditions cannot automatically be treated as equivalent groups differing only in the application being studied.

The distinction between an experimental model and a clinical population matters statistically as well as clinically. A capsaicin-induced temporary reduction of small nerve fibers can address a focused measurement question, but it does not establish diagnostic performance in people with SFN. A later study would need evidence in the population and context relevant to the intended diagnostic use. The record does not report such evidence.

Finally, an exploratory pilot can help define what should be measured and what design questions deserve attention, but its value cannot be assessed from a rationale alone. Without endpoint estimates, uncertainty measures, and analysis details, readers cannot judge the observed signal or its reproducibility from this record. The study’s statistical importance therefore lies in the question and proposed measurement framework, while the registry record leaves the empirical result unavailable.

15. Limitations

• The posted summary statistics are the only data these results rest on; patient-level data, full protocols and statistical analysis plans are not available.
• Recalculated results are simple, unadjusted calculations from posted summaries. They do not replace the sponsor's prespecified analysis.

The main limitation for interpreting the registry record is that no numerical primary outcome result is reported. AFI means, standard errors, condition contrasts, confidence intervals, and test results are absent. The record therefore cannot support an assessment of effect magnitude, direction, or precision, despite providing the endpoint definition and measurement schedule.

The study enrolled healthy volunteers rather than people described as having SFN. Capsaicin was intended to model temporary selective small nerve fiber reduction, but the record does not report whether that manipulation was achieved or quantify it. The findings, even if numerical results were available, would need to be distinguished from evidence about diagnosis in patients.

The design is non-randomized and sequential. The registry specifically cautions that the arms are not a like-for-like control comparison. It does not report the order-assignment process, washout procedures, or an approach to within-person dependence. The arm description gives a 1hour timing after lidocaine/prilocaine, whereas the primary outcome timeframe states Day 1, T=6h; the registry does not reconcile the descriptions.

Other relevant details are not reported in the registry record, including participant characteristics, measurement reliability procedures, missing-data handling, analysis populations, and the statistical analysis plan. The record lists zero analyses, so the intended or completed inferential approach cannot be evaluated. Safety counts are reported, but detailed event information and safety follow-up are not. These omissions limit both assessment of the measurement process and interpretation of the study’s broader relevance.

16. Related Tutorials

17. Related Statistical Calculators

18. Sources

• ClinicalTrials.gov: Autofluorescent Flavoprotein Imaging of Intraepidermal Nerve Fibers: a Pilot Study — NCT02537951.
• Registry record last updated 2019-06-11; status last verified 2019-02.
• Results first posted 2019-06-03.
• Data retrieved from ClinicalTrials.gov on 2026-11-09.
• Group sizes, means, event counts and sponsor-reported p-values are reproduced as posted. Effect estimates, confidence intervals and p-values not attributed to the sponsor are independently calculated from the posted summary statistics.

19. Record Summary

NCT02537951 is a completed pilot study titled “Autofluorescent Flavoprotein Imaging of Intraepidermal Nerve Fibers: a Pilot Study,” sponsored by Erasmus Medical Center. The registry lists enrollment of 10, non-randomized allocation, no masking, and a sequential model. The study began in 2015-09 and primary completion was 2016-07.

The primary outcome is mean AFI intensity, expressed as delta F/F, at the third fingertip after grading nociceptive stimuli. Measurements were planned at baseline, after lidocaine/prilocaine, and after an 8% capsaicin patch. The registry describes the participants as healthy volunteers and presents capsaicin as an experimental mimic of temporary small nerve fiber reduction.

The record lists one outcome measure and zero analyses. Numerical primary outcome findings and the statistical analysis plan are not reported in the registry record. Safety entries list zero serious adverse events, zero other adverse events, and zero deaths in each condition.