Reference intervals and sources of variation of pressure pain threshold for quantitative sensory testing in a Japanese population

Quantitative sensory testing (QST) is useful when analysing musculoskeletal pain disorders. A handheld algometer is most commonly used for pressure pain threshold (PPT) tests. However, reference intervals for PPTs are not elucidated. We assessed reference intervals of PPTs for QST in 158 healthy adult Japanese with no history of musculoskeletal or neurological problems. A handheld algometer was used to record PPT at five different assessment sites on the body: lumbar paravertebral muscle, musculus gluteus maximus, quadriceps, tibialis anterior muscle, and anterior talofibular ligament. Multiple regression analysis was performed to explore sources of variation of PPT according to sex, age, body mass index, UCLA Activity Level Rating, and Tegner Activity Score. Reference intervals were determined parametrically by Gaussian transformation of PPT values using the two-parameter Box-Cox formula. Results of multiple regression analysis revealed that age was significantly associated with PPT of lumbar paravertebral muscle and musculus gluteus maximus. In females, body mass index showed significant positive correlation with PPT of anterior talofibular ligament, and UCLA Activity Level Rating also showed significant positive association with tibialis anterior muscle and anterior talofibular ligament. Site-specific reference intervals of PPTs for Japanese are of practical relevance in fields of pain research using a handheld algometer.


Abbreviations
Pressure pain threshold at the area of musculus gluteus maximus PPT-TL Pressure pain threshold at the area of anterior talofibular ligament PPT-QC Pressure pain threshold at the area of quadriceps Need for partitioning PPT reference values by sex and age.The above source-of-variation analysis by MRA revealed that there were significant sex and age differences in PPT depending on the assessment sites.
To determine the need for partitioning reference values by sex and/or age, two-level ANOVA was performed.
The magnitudes of between-sex and between-age variation were each calculated as a standard deviation ratio (SDR), SDRsex and SDRage, respectively 52 .By adopting 0.4 as a threshold for SDR, SDRsex 50 was significant at the site of PPT-QC (0.520) and at that of PPT-TA (0.503), whereas SDRage was significant at the site of PPT-PVM (0.669) as shown in Table 2.
Derivation of assessment site-specific reference intervals for PPT.The reference interval of PPTs at each assessment site was calculated as the lower limit (LL: 2.5% point), median (Me: 50% point), and upper limit (UL: 97.5% point) as listed in Table 2.For partitioning of reference values by age, we arbitrarily set 40 years as a mid-age range for boundary.When SDRsex or SDRage exceeded the threshold of 0.40 49 , corresponding reference intervals (LL ~ Me ~ UL) were marked in bold.Otherwise, reference intervals without partitioning were shown in italics as the default.Notably, the range of reference intervals was generally low for PPT-QC, PPT-TA, and PPT-TL, implying that between-individual variations of PPTs are narrow.In contrast, the range was wider for PPT-PVM and PPT-MGM.These site-specific differences in the variability of PPT measurements are also apparent from Fig. 2.

Discussion
QST is a formal variant of a time-honoured clinical examination technique in neurology.Currently, none of the neuropathic pain medications on the market have been developed based on prediction of treatment efficacy by QST.However, such an approach to the development of medicines has been encouraged by the European Medicines Agency 15,16 .
In the assessment of musculoskeletal conditions, clinicians often identify points of tenderness in superficial tissue 15,16 .The handheld algometer we used calculates PPT on superficial tissue that equate to one point of skin tenderness as found clinically when assessing a painful area [16][17][18][19][20] .It also includes measures of temporal summation by wind-up and documentation of dynamic mechanical allodynia [18][19][20][21][22][23] .When appropriate standards are applied, PPT can provide important and unique information about the functional status of the somatosensory system, which would complement already existing clinical methods [20][21][22] .Unfortunately, to our knowledge, reference intervals for the Japanese have never been reported using the handheld algometer for PPTs even though this measurement system is the most popular in the fields of pain research 3,5,[18][19][20][21][22]  the site of the most severe pain in patients with LBP was reported to be the most useful test in the assessment of hypersensitivity 24 .
In western countries, the data from healthy control subjects was reported at lumbar sites 5,28 .Lumbar PPT ranged from 299 to 628 kPa in the back area in these healthy control subjects 5 .However, the sample size was statistically too small to use the range as a reference interval for the PPT 5 .In the present study, we determined the reference intervals and sources of variation of the PPT for quantitative sensory testing by enrolling a larger number of healthy individuals, as shown in Table 2, which exceeded the minimum sample size of 120 recommended for determining reference intervals in the field of laboratory medicine 29 .The average values for all ages were as follows: PVM PPT-PVM, 539 kPa; PPT-MGM, 519 kPa; PPT-QC, 366 kPa; PPT-TA, 455 kPa; and PPT-TL, 368 kPa.We reviewed the previously published data in healthy control subjects measured by the instrument: The range of PPT at the lumbar area was 299-628 kPa, that at the gluteal areas was 535.9-863.97kPa, and that at the lower leg was 321.8-771.5 kPa 5,15,[23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] .There were some differences between the articles depending on the volunteers' backgrounds.However, these data may suggest differences in PPT in each race 37 : but more studies should be performed to confirm it.
The present study revealed the PPT at the PVM, MGM, QC, TA, and TL in healthy Japanese ranging in age from 19 to 59 years for the first time, to our knowledge (Table 2).It also revealed that the PPT at the PVM, MGM, QC, TA, and TL in healthy Japanese differed at each site, and these differences were influenced by sex, age, BMI, and ALR (Table 2).We show the characteristics of the PPT data for each site in Fig. 2. The variability of PPT-PVM and PPT-MGM was comparatively large, whereas that of PPT-QC, PPT-TA, and PPT-TL was small (Fig. 2, Table 2).These reference intervals should be used clinically with caution for patients with LBP.
Female participants showed higher pain thresholds for PPT-QC and PPT-TA compared to the male participants (Table 2).Females showed more tolerance than males for QST parameters, consistent with prior studies 1,[39][40][41][42] .These sex differences in pain thresholds are unlikely to be due to peripheral factors such as innervation density and different central processing 1,41,42 .
In previous reports, obese individuals were more sensitive to pressure pain than individuals within a normal range of BMI 43 .Pain response varied according to subcutaneous body fat at different body sites 43 .However, PPT-TL in the females correlated only with BMI in the present study.Most of our participants were within the standard range of BMI, and therefore, we thought that our data showed a weak correlation between PPT and BMI only in the females.
PPT-TA and TL in the females were associated with ALR (correlation coefficients: 0.319 and 0.361, respectively).This result indicates that Japanese females with higher daily activity are more tolerant of lower leg pain.In addition, there was no association between PPT and the TAS, which reflects sports activities.We did not detect any correlation of PPT with sports and daily activity level in the healthy Japanese male volunteers.Previous articles revealed that exercise-induced hypoalgesia occurred following exercise.In addition, endocannabinoid levels were found to be elevated following exercise [44][45][46] .Our results showed a correlation between PPT and the questionnaire on activities only in the females of our study.We could not directly compare people with low activity to those with moderate or high activity because the participants basically all participated in moderate to slightly high activity levels of sports and daily activities, and no people with low activity were included.
The limitation of this study was relatively small sample size of healthy individuals recruited: n = 158.It was regarded as acceptable for determining the RIs without partitioning by sex and age.However, despite statistically significant between-sex and between-age differences in PPT, we could not reliably determine the RIs specific for each subgroup.It is certainly necessary to expand the scale of the study for better clinical usage of the RIs.The other limitation was that inter-rater reliability was not examined in this study.Therefore, we could only show the reference intervals for PPT in this study.In summary, the present study determined the reference intervals of PPTs for the first time in healthy Japanese.In addition, we revealed that the PPTs at the PVM, MGM, QC, TA, and TL in healthy Japanese differed at each site and that these differences were influenced by sex, age, BMI, and ALR.Therefore, the above key points need to be taken into consideration when PPTs are measured in the assessment of patients with LBP.We hope that these data may become the reference intervals for the assessment of Japanese patients with LBP.

Conclusions
In this study, we determined test site-specific reference intervals for pressure pain thresholds in quantitative sensory testing in a healthy Japanese population.In addition, we showed that sex-and age-related differences in pressure pain thresholds also depend on the site of assessment.Consequently, in the clinical assessment of patients with low back pain, it is necessary to take into considerations that reference intervals of pressure pain thresholds differ according to the site of assessment and that the levels of pressure pain thresholds are influenced by sex, age, body mass index, and UCLA activity level rating.These findings may be of practical relevance in the fields of pain research using pressure pain thresholds measured by the handheld algometer in Japan.

Methods
Subjects.Participants in this study included 158 healthy Japanese subjects (73 females, 85 males; age, 35.2 ± 12.7 years [mean ± SD]; BMI, 22.3 ± 3.03 kg/m 2 ) with no history of musculoskeletal or neurological problems.Participants were medical staff, medical doctors, rehabilitation staff, medical students and their families in our institution.None of the participants had (1) ongoing pain problems, (2) circulatory disorders, (3) a history of metabolic disease or neuropathy, (4) current use of prescription medications, including analgesics, tranquilizers, antidepressants, or other centrally acting agents, (5) diagnosed mental health disorders, (6) current pregnancy, (7) liver or kidney disease, and (8) disorders involving the neuroendocrine system.The subjects were given a detailed written and verbal explanation of the procedures for measuring PPT, and all signed an informed consent form.This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Boards of Yamaguchi University (H2020-169) in May 2020.

Experimental protocol.
As the QST, the PPT was recorded 5 times at each of five pressure points (see below), and the average of the middle 3 scores, excluding the top and bottom score, was used for the data of pressure thresholds measured at each point (Fig. 1).A handheld algometer (Somedic, Hörby) mounted with a 1-cm 2 probe (covered by a disposable latex sheath) was used to record the PPTs at 10 different locations on the body (Fig. 1).The investigator placed the handheld algometer on a site to be inspected and pressed against the tester in a vertical direction (Fig. 1).The investigator instructed the subjects to push the button by themselves when they felt slight pain.An interval of at least 20 s was kept between each assessment of a PPT.The PPT was defined for the subjects as "the time point at which the pressure sensation changed into pain." Pressure was increased gradually at a rate of 30 kPa/s until the pain threshold was reached and the subject pressed a button 3,10,11,47 .
The assessment sites were as follows: (1) right and left lumbar paravertebral muscle (PVM) at the level of iliac crest, 4 cm away from the lateral side of spinal process; (2) right and left musculus gluteus maximus (MGM), 5 cm below from iliac crest and 4 cm posterior to the posterior edge of the greater trochanter; (3) right and left quadriceps (QC), 10 cm proximal of the top of the patella; (4) right and left tibialis anterior (TA), 5 cm distal and 3 cm lateral of the tibial tuberosity; and (5) right and left anterior talofibular ligament (TL).( 1) and (2) assessments were in a prone position.(3), ( 4) and (5) assessments were in the supine position.
Questionnaires.We evaluated daily activity with the UCLA ALR 12 , which is a single-item 10-level scale, ranging from level 10, representing a highly physically active person, to level 1, a person who is dependent on others and unable to leave home.
We evaluated sports activity with the TAS, which was developed by Tegner and Lysholm in 1985 13 .An activity level of 10-6 corresponds to participation in competitive and/or recreational sports, 5-1 corresponds to participation in recreational sports and heavy/moderate/light labour, and 0 is recorded for a person on sick leave or with a disability pension 13,14 .
Statistical analyses.Summary values for numerical parameters are presented as the mean and standard deviation (SD).To explore sources of variation of PPTs, MRA was performed, separately for each sex.The PPT at each location was set as an objective variable and age, BMI, ALR, and TAS as the explanatory variables.The contribution of each variable to prediction of the level of PPT was expressed by the standardized partial regression coefficient (r p ), which corresponds to a partial correlation coefficient and takes a value between − 1.0 and 1.0.The practical significance was set to |r p | ≥ 0.30 guided by the medium effect size of Cohen for correlation coefficients (0.3) 48 .To evaluate the need for partitioning PPT values by sex and/or age, two-level nested ANOVA was performed, in which age was partitioned at 40 years.Variations of PPT by sex and age were computed as SDs, SDsex and SDage, respectively.The SDR was computed by dividing each by between-individual (residual) SD (SDind) as SDRsex = SDsex/SDind and SDRage = SDage/SDind, respectively.The threshold for SDR was set to 0.4 47 .Reference intervals were determined parametrically after Gaussian transformation of the PPT by use of the two-parameter Box-Cox formula 49,50 :

Figure 1 .
Figure 1.Handheld algometer and assessment of paravertebral muscles.(A) The handheld algometer used to assess pressure pain thresholds (PPTs).(B) Assessment of the paravertebral muscle.(C) Schematic of the body areas used for the analysis of PPTs.The assessment sites marked with red dots are as follows: (Top row) right and left lumbar paravertebral muscle at the level of iliac crest, 4 cm away from the spinal process; (Row 2) right and left musculus gluteus maximus, 5 cm below from iliac crest and 4 cm posterior to the posterior edge of the greater trochanter; (Row 3) right and left quadriceps, 10 cm proximal of the top of the patella; (Row 4) right and left tibialis anterior muscles, 5 cm distal and 3 cm lateral of the tibial tuberosity; (Bottom row) right and left of anterior talofibular ligament.

Figure 2 .
Figure 2. Sex and age-related changes in pressure pain thresholds (PPTs) at the five testing sites.Reference values (RVs) of PPTs recorded at the five sites (lumbar paravertebral muscle [PVM], musculus gluteus maximus [MGM], quadriceps [QC], tibialis anterior [TA], and talofibular ligament [TL]) were subgrouped by sex and age at 40 years.The box in the centre of each scattergram indicates the mid 50% range of RVs, and its central vertical bar represents the median.The data size of each subgroup is shown at the right bottom of the group labels.On the top of each graph, the magnitudes of the between-group differences by sex or age are shown as the SD ratio (SDR), SDRsex or SDRage, respectively.SDRs that exceeded the threshold of 0.4 are marked in bold.The background green shading indicates the reference interval determined from all RVs without partition by sex or age.The x-axis range was fixed at 0-1400 kPa for all graphs to show the test site dependency of the PPTs.

. In addition, detection of PPT atTable 1 .
Sources of variation of PPTs evaluated by multiple regression analysis.Significant values are in bold.PPT pressure pain threshold, Exp para experimental parameter, BMI body massindex, ALR UCLA Activity Level Rating, TAS Tegner Activity Score, PVM lumbarparavertebral muscle, MGM musculus gluteus maximus, QC quadriceps, TA tibialisanterior muscle, TL anterior talofibular ligament.