Serum levels of biomarkers related to severity staging of Raynaud’s phenomenon, neurosensory manifestations, and vibration exposure in patients with hand-arm vibration injury

Our aim was to explore possible relationships between serum levels of biomarkers in patients with hand-arm vibration injury in relation to the severity of the vascular, i.e., Raynaud’s phenomenon (RP), and neurosensory manifestations, the current exposure level, and the duration of exposure. This study was of case series design and involved 92 patients diagnosed with hand-arm vibration injury. Jonckheere’s trend test was used to assess any association between serum levels of biomarkers and RP as well as neurosensory manifestations, graded by the International Consensus Criteria. Generalized linear models with adjustment for possible confounders were also used for associations between serum levels of biomarkers and; (1) severity of RP recorded as the extent of finger blanching calculated with Griffin score, (2) vibration perception thresholds, (3) magnitude of current exposure as [A(8); (m/s2)] value, and (4) the duration of exposure in years. Serum levels of thrombomodulin, von Willebrand factor, calcitonin gene related peptide (CGRP), heat shock protein 27, and caspase-3 were positively associated with severity of RP. Serum levels of CGRP were positively associated with the neurosensory component. No associations with exposure were shown for these biomarkers. For Intercellular adhesion molecule 1 and monocyte chemoattractant protein 1, no associations were found with neither severity nor exposure. Levels of serum biomarkers associated with endothelial injury or dysfunction, inflammation, vasodilation, neuroprotection, and apoptosis were positively associated with the severity of hand-arm vibration injury.

Serum levels of TM, vWf, CGRP, HSP27, and caspase-3 were positively associated with severity of RP according to ICC vascular staging 22 (Table 2, Fig. 2).For several of the biomarkers, as shown in Fig. 1, the positive Table 1.Descriptive characteristics, severity of vascular (Griffin score) and neurosensory manifestations (vibration perception threshold, VPT) in the hand with most pronounced symptoms, and hand-arm vibration exposure, in 92 patients with hand-arm vibration injury.Data are median (range) or n (%). a Missing data in 7 patients.b Missing data in 10 patients.Duration of exposure (years) 20 (2.6-46)  trends were seen between grade 1 and 2, but not for grade 3.For ICAM-1 and MCP-1 there was no such trend (Table 2).There were linear associations between the log transformed levels of serum levels of TM, vWf and caspase-3 and the severity of RP, (Table 3), which remained after adjustment for possible confounding.For HSP27 the association was not linear.Serum levels of CGRP were positively associated with the neurosensory component, graded by ICC 0-3N (Table 4, Fig. 2).There were, however, no linear associations between the log transformed levels of any of the Table 2. Serum levels of biomarkers in 92 patients with hand-arm vibration injury according to the severity of vascular manifestations graded by ICC 0-3 V. a Median (interquartile ranges).b 10 individuals were excluded since they had not filled in hand diagram.c P values in boldface denotes statistically significant associations with Jonckheere-Tepstra trend test.d Limit of detection (LOD) 15 pg/ml.www.nature.com/scientificreports/biomarker and vibration perception thresholds (Table 3), nor the with the current exposure or duration of exposure (Table 5).

Discussion
Serum levels of TM, vWf, HSP27, CGRP and caspase-3 were positively associated with the severity of Raynaud's´ phenomenon according to ICC vascular staging.TM, vWf and caspase-3 showed a linear association, while for HSP27, there was also a positive association, though not linear.In addition, increased serum levels of CGRP were associated with increased severity of neurosensory manifestations according to ICC staging.No linear associations were, however, observed between log transformed levels of any serum biomarker and VPT, current exposure level or duration of exposure.
A limitation of the study was that the episode of finger blanching was assessed based only on medical history.Apart from this, RP was graded according to guidelines in the ICC and established on patients' history of number of phalanges affected on a "typical" episode of finger blanching.This gives an estimate of severity that does not depend on climate or whether the patient works indoors or outdoors, which makes the estimate more reliable.Another possible limitation could be that we did not include patients using serotonin or noradrenalin reuptake inhibitors and statins, but with no other treatment for cardiovascular disease, in our definition of patients with concurrent disease.No power calculation was performed prior to the study since it was not possible to predict an effect size since reference values for the selected biomarkers are vaguely defined.The question is relevant for ICAM-1 and MCP-1, where we did not find any associations with severity.However, for these biomarkers, data did not show a pattern that could indicate an association with either ICC V or ICC N. Thus, we do not think that the lack of power calculations hampered the study.To get a complete representation of the nerve injury, it is advised to perform a test battery of different sensory modalities that captures both small and large nerve fiber dysfunction 30 .Such dysfunctions are included in ICC N. A strength of the study is that we used both vibration perception thresholds and ICC.Vibration perception thresholds gave continuous data for severity of neurosensory manifestations, but an isolated small nerve fiber neuropathy was not captured by this test.
For vibration exposure, we did frequency weighting according to the standard ISO 5349-1, which underestimates the harm of higher frequencies or the use of impact tools 28,31 .This warrants further investigation in a prospective study.
Serum levels of TM were positively associated with the severity of RP, which is in line with a previous study on vibration-injured individuals, where a combination of SWS SN and SWS V was used 20 .Plasma levels of TM have also been shown to be elevated in hand-arm vibration injured patients compared to controls 20,32,33 .TM is an endothelial surface protein, normally expressed at low levels in serum, thus, this elevation might reflect a pathological condition in the endothelial cells due to vibration exposure.Elevated blood levels of TM have been associated with endothelial injury, e.g., in cardiovascular -, inflammatory-, infectious -, and metabolic diseases 34 .The association between serum levels of vWf and the severity of RP contrasts with a previous study, where vibration exposed workers with RP showed significantly lower blood concentration of vWf than vibration exposed controls without RP.This finding might reflect an ongoing mechanism with endothelial dysfunction, inflammation, and intimal hyperplasia.Hypothetically, increased levels of vWf could be caused by vibrationinduced changes in shear stress and cyclic stretch to the vessel wall 35,36 .Increased vWf levels have been reported to shear stress in a dose-response relationship to intimal hyperplasia 37 .Furthermore vWf might reflect a state of inflammation, since individuals with RP, that later on developed a systemic connective tissue disease, showed elevated levels of vWf compared to individuals with only the primary form of RP 38 .
Interestingly, serum levels of CGRP were positively associated with both the vascular and the neurosensory components of hand-arm vibration injury.Hypothetically, injury can start in the small nerve fibers innervating blood vessels (nervi vasori) or in the small blood vessels supplying nerves (vasa nervorum) 39 , a pathophysiological mechanism described in diabetes neuropathy 40 .Structural changes in both blood vessels and nerves have been observed in relation to vibration exposure e.g., disrupted endothelial cell lining 9 and loss of CGRPcontaining nerve fibers 41 .A loss in CGRP-containing nerves have also been shown in patients with RP 42 .CGRP, a neuropeptide and very potent vasodilator, is released from unmyelinated small C-fibres and myelinated small Aδ-fibres 43 .CGRP exerts its effect mostly locally, but studies have shown that serum can be used in biomonitoring, e.g., elevated levels in serum have been shown in individuals in relation to episodes of migraine 44 .
Associations with HSP27 and caspase-3 in hand-arm vibration injury has to our knowledge not been presented earlier.In the present material, serum levels were also elevated compared to controls 3 .HSP27 act as chaperone to protect nerve structures under stress [45][46][47] and increased levels of caspase-3 have been measured as an apoptotic response in Schwann cells, to balance the proliferative response after a nerve injury 48 .
Among the present patients, the prevalence of RP increased with rising severity of the neurosensory component and there was a great overlap between the three component; RP, small-, and large fibre neuropathy 49 .Thus, whether these biomarkers reflect an injury related to the vascular and/or the neurosensory component cannot be further elucidated in this study.
Several of the biomarker levels did not seem to be more elevated in ICC 3 V compared to ICC 2 V, indicating mechanisms where dysfunctional or injured endothelial cells first leads to unregulated release, measured as an increase of substances or compensatory mechanisms (stage 2) 43 .After repeated trauma, the endothelial cells or neuropeptidergic release from nerve fibres is diminished due to loss [41][42][43]50 and might not be able to produce more substance, reflected by the levels at stage 3. Finally, a possible interplay between the observed biomarkers could further be a fact to take into account, e.g., treatment with CGRP has been shown to significantly decreased the expression of caspase-3 51 . Tale 5. Associations (β with 95% confidence intervals) between log transformed values in serum biomarkers and severity of hand-arm vibration exposure, estimated by Generalized Linear Models in 92 patients with hand-arm vibration injury.Statistically significant associations in bold face.Adjustment for possible confounders with an association to the biomarker.For confounders with interaction, data was dichotomised according to that factor: a Adjusted for current smoking and previous frostbites.b Adjusted for age and concurrent disease.c Adjusted for sex.d Adjusted for age, concurrent diseases, and sex.e Adjusted for concurrent diseases. ICM-1 (ng/mL) − 0.01 (− 0.03 to 0.02) 0.00 (− 0.00 to 0.00) MCP-1(pg/mL) − 0.06 (− 0.12 to 0.01) − 0.07 (− 0.14 to − 0.01) a 0.00 (− 0.01 to 0.01) 0.00 (− 0.01 to 0.01) a TM (ng/mL) − 0.03 (− 0.12 to 0.05) 0.00 (− 0.01 to 0.01) vWf (µg/mL) − 0.06 (− 0.12 to − 0.01) − 0.04 (− 0.09 to 0.01) b 0.01 (0.01 to 0.02) 0.01 (− 0.00 to 0.02) b HSP-27 (ng/ml) 0.02 (− 0.07 to 0.10) 0.04 (− 0.05 to 0.12) d 0.01 (− 0.00 to 0.02) − 0.01 (− 0.02 to 0.01) d Caspase-3 (ng/ml) 0.01 (− 0.14 to 0.17) − 0.00 (− 0.02 to 0.02) − 0.00 (− 0.02 to 0.02) e Without concurrent diseases − 0.24 (− 0.46 to − 0.03) With concurrent diseases 0.20 (0.00 to 0.40) A previous study reported elevated plasma levels of ICAM-1 in individuals with rising severity of hand-arm vibration injury 20 , but this was not shown in the present study.For MCP-1 there was neither any association with the severity of hand arm vibration injury, nor the exposure.

Conclusion
Levels of several serum biomarkers were associated with the severity gradings of hand-arm vibration injury.No associations with exposure were shown for these biomarkers.These findings give insight into pathophysiological mechanisms that can be useful in developing prospect tools for diagnostics, grading and treatment of hand-arm vibration injury.

Study design
The study has an observational case series design.Data presented in this study has been collected in previously described studies 3,49 .The scheme of the studies is presented in Fig. 1.All participants gave their informed consent to participate in the study before taking part.The study was conducted in accordance with the Declaration of Helsinki, and approved by the Regional Ethics Board in Lund, Sweden (No. 2018/15).

Study group
Data were collected from patients consecutively referred to the Clinic for Occupational and Environmental Medicine in Lund, Sweden (enrolment from August 2018 until February 2020), following a standardized assessment for hand-arm vibration injury.When 100 patients had agreed to participate in the study, eight patients (seven men and one woman) declined to participate), one woman and seven men were considered by the physician not to have the diagnosis hand-arm vibration injury, and were therefore excluded, resulting in a study group of 92 patients, six of which were women 3,49 .

Clinical assessment
A questionnaire was to be filled in before their visit and then the complete consultation and examination by a nurse, a physician and an occupational hygienist took approximately four hours 49 .

Questionnaire
The questionnaire included six questions on symptoms, where the responses (Yes) or (No) were indicated separately for the right and left hand: Do you experience: (a) numbness or tingling?,(b) numbness or tingling during the night?, (c) pain/discomfort in fingers/hands during cold exposure?,(d) white fingers when exposed to cold or dampness?, (e) poor grip strength?, (f) poor fine motor skills or clumsiness?.
Patients who reported that they experienced episodes of finger blanching were asked to fill in a hand diagram marking affected phalanges on both hands.

Clinical examination
Quantitative (monofilament and vibration perception threshold for large nerve fibers) and qualitative (temp rolls and pin prick test for small fibers) sensory tests were applied.Details on the outcome assessments are presented in the former study 49 .Before starting the tests, finger skin temperature was measured in second and fifth fingers bilaterally with a Testo 845 infrared measuring instrument.If the finger temperature was below 28 °C, the patient was asked to perform a shortened submaximal cycle ergometer test.Perception of vibration was tested with a VibroSense Meter I ® .The patient was asked to wear earmuffs as high amplitudes at high frequencies generate an audible sound.Vibration perception thresholds were recorded at seven frequencies from 8 to 500 Hz 52 .The equipment automatically plots the data and generates a sensibility index (SI) defined as the ratio between the area under the test curve and that under an age-specific reference curve.Patients with a sensibility index below 0.8 were considered to have impaired vibrotactile sense 53 .Test of two-point discrimination (2PD) was used to assess tactile gnosis, and Purdue pegboard test was used to test manual dexterity.

Grading of severity of injury
The vascular manifestations were graded according to the ICC 0-3 V 22 and were assessed using the Griffin score to grade finger blanching 23 .Blanching of the distal phalanx on the index, long, ring and little fingers corresponded to one point, middle phalanx: two, proximal phalanx: three points, and thumbs: four and five points for the distal and proximal phalanges.Scoring was done separately for the right and left hand.No episodic blanching of phalanges corresponded to stage 0 V, score 1-4: stage 1 V, score 5-12: stage 2 V, and score > 12: stage 3 V.Ten individuals reported episodes of finger blanching but had not filled in the hand-diagram.These were not included in the evaluation.
Severity of the neurosensory manifestations was graded separately for the hands, according to ICC 0-3N 22,49 .Stage N0: exposed to vibration but no numbness or tingling; stage 1N: numbness and/or tingling; stage 2N: as in 1N and impairment in two out of three sensory modalities; perception of touch, vibration, or temperature; stage 3N: as in 2N and symptoms of impaired dexterity and impaired manipulative dexterity.

Biomarkers
Blood samples were collected after clinical examination, before noon, in 7 ml serum separation tubes with gel.After 30 min, serum was removed by centrifugation at 2000×g for 10 min, and the samples were stored at − 80 °C.Serum concentrations of TM, vWf, CGRP, HSP27 and caspase-3 were determined using commercially available

Table 4 .
Serum levels of biomarkers in 92 patients with hand-arm vibration injury according to the severity of neurosensory manifestations graded by ICC 1-3N.Median (interquartile ranges).Stage 0N is not included (n = 2).a P values in boldface denotes statistically significant associations, with Jonckheere-Tepstra trend test.b Limit of detection (LOD) 15 pg/ml.

Figure 2 .
Figure 2. Distribution of serum levels of (a) thrombomodulin (TM), (b) von Willebrand factor (vWf), (c) and (f) calcitonin gene-related peptide (CGRP), (d) heat shock protein 27 (HSP27), (e) caspase-3 in relation to severity of vascular (blue boxes, 82 patients) and sensorineural manifestations (green boxes, 92 patients), as graded by the International Classification Criteria ICC.Medians are indicated with a line.Boxes represent interquartile ranges.Values that are more than 1.5 (circles) or 3 times (stars) higher than the third quartile are indicated as outliers.Error bars indicate min-max values that are not outliers.One patient with S-CGRP 3700 pg/mL; ICC 2 V and 1N, one with S-HSP27 45 ng/mL; ICC 0 V, one with caspase-3 85 ng/mL; ICC 0 V and one with caspase-3 61 ng/mL; ICC 3 V, are not shown as the axes are cut.

.007 Figure 1. The
scheme of the studies.

Table 3 .
Associations (β with 95% confidence intervals) between log transformed values in serum biomarkers and severity of hand-arm vibration injury, estimated by Generalized Linear Models in 92 patients with handarm vibration injury.Statistically significant associations in bold face.a Adjusted for current smoking and frostbites.b Adjusted for age and concurrent diseases.c Adjusted for age.d Adjusted for age, concurrent diseases, and sex.e Adjusted for concurrent diseases.