Prevalence of the Frank's sign by aetiopathogenic stroke subtype: A prospective analysis.

BACKGROUND AND
PURPOSE: The Frank's sign is a diagonal earlobe crease running from the tragus to the edge of the auricle at an angle of 45°. Many studies have associated this sign with coronary artery disease and some with cerebrovascular disease. The objective of this study was to analyse the prevalence of the Frank's sign in patients suffering from acute stroke with a particular focus on its prevalence in each of the five aetiopathogenic stroke subtypes. Special interest is given to embolic stroke of undetermined source (ESUS), correlating the sign with clinical and radiological markers that support an underlying causal profile in this subgroup.
METHODS: Cross-sectional descriptive study including 124 patients admitted consecutively to a stroke unit after suffering an acute stroke. The Frank's sign was evaluated by the same blinded member of the research team from photographs taken of the patients. The stroke subtype was classified following SSS-TOAST criteria and the aetiological study was performed following the ESO guidelines.
RESULTS: The Frank's sign was present in 75 patients and was more prevalent in patients with an ischaemic stroke in comparison with haemorrhagic stroke (63.9 vs. 37.5, p<0.05). A similar prevalence was found in the different ischaemic stroke subtypes. The Frank's sign was significantly associated with age, particularly in patients older than 70 who had vascular risk factors. Atherosclerotic plaques found in carotid ultrasonography were significantly more frequent in patients with the Frank's sign (63.6%, p<0.05). Analysing the ESUS, we also found an association with age and a higher prevalence of the Frank's sign in patients with vascular risk factors and a tendency to a high prevalence of atherosclerosis markers.
CONCLUSION: The Frank's sign is prevalent in all aetiopathogenic ischaemic stroke subtypes, including ESUS, where it could be helpful in suspecting the underlying cardioembolic or atherothrombotic origin and guiding the investigation of atherosclerosis in patients with ESUS and the Frank's sign.

Introduction

Although there is no consensus as to the definition of the Frank’s sign and different authors have used variations with regaards to the extent of ear crease, the Frank’s sign, or earlobe crease (ELC), is widely defined as a diagonal crease in the earlobe extending from the tragus across the lobule to the rear edge of the auricle at an angle of 45° with varying depths.

Although historically the sign can be seen in busts of the Roman emperor Hadrian, who is presumed to have died from cardiac disease, it was first described by Sanders T. Frank in 1973 who, in doing so, suggested a positive relationship between ELC and coronary artery disease (CAD) [1]. Several studies have considered a possible link between ELC and CAD, and many studies have shown that ELC can serve as a marker of atherosclerotic disease and as a sign of an elevated risk of coronary heart disease in asymptomatic individuals [2-7]. This sign has also been associated with HLA-B27, C3-F arteriosclerosis gene and chromosome 11 [8]. Although different studies have tried to identify the pathophysiology of the Frank’s sign, the mechanisms underlying the association between ELC and vascular disease remain unclear. In the early 1970s and the 1980s, it was suggested that ELC might be a result of local poor supply from arteries to the earlobe [9]. In a case report, a link is suggested between macrophage activity (which is involved in atherosclerosis), ageing and maintaining earlobe collagen [10], and it has been argued that the ELC and CAD are related to the loss of elastin and to the rupture of elastic fibres in patients with ischaemic heart disease [11]. In a recent histopathological study in 45 consecutive adult patients referred for autopsy, Stoyanov GS et al. analysed samples from both earlobes as well as cardiac samples from all four cardiac compartments. They found a significant correlation between the morphological changes of the myocardium and the presence of the earlobe creases with arterial myoelastofibrosis, Wallerian-like degeneration in peripheral nerves, and deep tissue fibrosis found in the base of the crease [12].

Some studies have described an association between Frank’s sign and the presence of diabetes, hypertension [11, 13], myocardial infarction and coronary artery disease [6, 7] in patients of both sexes. According to these studies, this easily identifiable sign could be valuable in the screening of patients at high risk of having silent coronary artery disease.

However, only a few studies have focused on the association between the presence of ELC and cerebrovascular disease [14]. The microvascular damage, the presence of perivascular cellular infiltration and the decreased nitroglycerine-induced vasodilation described in different studies [15] suggest that ELC is associated with endothelial dysfunction, which is known to represent an early stage of systemic atherosclerosis. Additionally, carotid artery intima-media thickness (IMT) and atherosclerotic plaques in the carotid arteries have also been described in association with ELC [16, 17]. These findings suggest that the presence of ELC could serve as a reliable marker of systemic atherosclerosis.

The term ESUS (embolic stroke of undetermined source) describes a type of ischaemic stroke defined as a non-lacunar infarction by neuroimaging, with no internal carotid artery stenosis or dissection of the respective brain supplying artery, and based on the exclusion of other aetiologies such as atrial fibrillation, vasculitis, drug abuse, or coagulopathies [18, 19]. To the best of our knowledge, no studies have analysed the prevalence of ELC stratified by stroke subtype and none has focused on the potential relevance of the presence or absence of ELC in ESUS as a potential marker of the subjacent stroke mechanism in this important and prevalent stroke subtype.

Material and methods

The study has been submitted and approved by the Ethics Committee of our center (Comitè d’Ètica d’Investigació amb Medicaments CEIm Girona. Approval number 2021.072). This was a cross-sectional descriptive study that enrolled consecutive patients admitted with an acute stroke at the Stroke Unit of our centre from 16th September to 16th December 2019. Transient ischaemic attacks were included and stroke mimics were excluded. Informed consent was obtained to participate in the study. ELC evaluation was performed by taking photographs of both ears of the patients that were then assessed by a member of the research team, who was blind to the clinical data with a previous informed consent. Patients with potential confounding factors for the evaluation of ELC, such as earrings that had markedly deformed the earlobe, earlobe injuries or tattoos, were excluded from the study.

For the purpose of this study, positive ELC was considered when subjects had a crease or wrinkle extending 45° diagonally from the tragus towards the outer border of the earlobe. We considered both the unilateral and bilateral presence of such a crease or wrinkle to be the Frank´s sign (Fig 1).

Fig 1

The Frank’s sign.

Diagonal earlobe crease.

All patients were submitted to the following examinations: medical history with particular interest in classical stroke risk factors and their treatment, clinical examination, blood tests, 12-lead ECG and continuous Stroke Unit monitoring (blood pressure, heart rate frequency and oxygen saturation), non-contrast CT scan, colour-coded duplex sonography of the supra-aortic trunks, basal colour-coded transcranial ultrasonography, and detection of right-to-left shunt if appropriate. Extensive investigations, including transthoracic echocardiography, were carried out on patients diagnosed as suffering from stroke of uncertain aetiology. Stroke severity was quantified by a certified neurologist using the NIHSS at admission and every 12 hours at the Stroke Unit [20]. CT scan or MRI was carried out at admission. Colour-coded transcranial ultrasonography was carried out systematically and transthoracic echocardiography was performed in all cases of cryptogenic stroke. The suspected cause of stroke was classified as (1) large-artery atherosclerosis, (2) cardioembolism, (3) small-vessel disease, (4) stroke of other determined aetiology, and (5) stroke of undetermined aetiology, in accordance with the Trial of Org 10172 in Acute Stroke Treatment (SSS-TOAST) criteria [21, 22]. The modified Rankin scale (mRS) was measured at hospital discharge and at 3 months, also by a certified neurologist [23].

Statistical analysis

Results are expressed as frequencies or percentages for categorical variables. Continuous variables are expressed as mean ± standard deviation (SD) and were compared using the Student’s t test, or median and quartiles and using the Mann-Whitney tests, depending on whether the distribution was normal or not. We evaluated the normal distribution of the variables using the Kolmogórov-Smirnov test. Proportions between groups were compared using the Chi-square test or t test as appropriate. The importance of ELC in all stroke subtypes and specifically in ESUS was assessed by binary logistic regression analysis controlling for independent variables that obtained statistical significance of p < 0.1 in the bivariate analysis. Results were expressed as ORs and 95% CIs. For all analyses, significance was taken as a p value of <0.05.

Results

A total of 124 consecutive patients who were hospitalized with an acute stroke and were eligible to take part in the study were recruited. The mean age of the total population was 70.0 (+/- 14.0), 74.2% were males and 25.8% females. The Frank’s sign was present in 75 patients (60.5%). Of the total population, 108 patients (87.1%) had suffered an acute ischaemic stroke (12 of them had a transient ischaemic attack), and 16 (12.9%) a hemorrhagic stroke. There were no relevant differences in the prevalence of ELC by stroke subtypes.

As previously described in the literature [13, 14, 24], we found a strong and significant association between the presence of ELC and age, with ELC being particularly prevalent in patients who were older than 70 years (74.7%). In comparison with ischaemic stroke, we found a lower prevalence of the Frank’s sign in haemorrhagic stroke (63.9% vs. 37.5%, p<0.05). The prevalence of ELC had a similar distribution across each aetiopathogenic ischaemic stroke subtype: atherothrombotic (69.2%), cardioembolic (60.0%), lacunar (64.3%) and ESUS (65.2%) (Table 1). As expected, we found a significant association between some classical stroke risk factors, such as hypertension, diabetes, or ex-smokers and ELC, although we observed an inverse correlation in the particular cases of alcoholic habit (>20 g/day) and smokers. On analysing carotid ultrasonographic markers of vascular atherosclerosis, we found a significant relationship between the prevalence of ELC and the presence of non-stenotic atherosclerotic plaques (63.6% vs. 45.2%, p<0.05). Other markers such as stenosis or occlusion of carotid arteries or of other vascular cerebral territories showed an association with the presence of ELC, although these did not reach statistical significance (Table 1). In binary logistic regression analysis, only age and diabetes remain independently associated with ELC (Table 2).

Table 1

Bivariate analysis of the Frank’s sign by clinical variables included in the study.

	FRANK’S SIGN
	NO	YES	p value
	(n = 49)	(n = 75)
Male	38 (77.6%)	54 (72.0%)	n.s.
Female	11 (22.4%)	21 (28.0%)
Age			<0.001
<50	16 (32.7%)	5 (6.7%)
51–59	10 (20.4%)	6 (8.0%)
60–69	12 (24.5%)	8 (10.7%)
70–79	7 (14.3%)	29 (38.7%)
>80	4 (8.2%)	27 (36.0%)
Stroke subtype			<0.05
Ischaemic	39 (79.6%)	69 (92.0%)
Haemorrhagic	10 (20.4%)	6 (8.0%)
Ischaemic stroke			n.s.
Atherothrombotic	8 (30.8%)	18 (69.2%)
Cardioembolic	14 (40.0%)	21 (60.0%)
Lacunar	5 (35.7%)	9 (64.3%)
Cryptogenic (ESUS)	8 (34.8%)	15 (65.2%)
Arterial hypertension	28 (57.1%)	60 (80.0%)	<0.01
Diabetes mellitus	9 (18.4%)	27 (36.0%)	<0.05
Dyslipidemia	21 (42.9%)	37 (49.3%)	n.s.
Obesity (BMI>30) (n = 79)	9 (25.7%)	13 (29.5%)	n.s.
Smoker	22 (44.9%)	15 (20.0%)	<0.05
Ex-smoker	10 (20.4%)	23 (30.7%)
Non-smoker	17 (34.7%)	37 (49.3%)
Alcohol intake (>20gr/d)	18 (36.7%)	11(14.7%)	<0.05
Ischaemic heart disease	7 (15.6%)	8 (11.6%)	n.s
Atrial fibrillation	7 (15.6%)	16 (23.2%)	n.s
Previous ischaemic stroke	8 (17.4%)	12 (16.7%)	n.s.
Presence of non-stenotic atherosclerotic plaque	19 (45.2%)	42 (63.6%)	<0.05
Internal carotid artery stenosis			n.s.
No stenosis	34 (79.1%)	52 (76.5%)
Stenosis >50%	2 (4.7%)	7 (10.3%)
Stenosis >70%	4 (9.3%)	7 (10.3%)
Occlusion	3 (7.0%)	2 (2.9%)
Stenosis other arteries	11 (25.6%)	17 (25.0%)	n.s.
mRankin ≤ 2 prior to index stroke	47 (95.9%)	70 (93.3%)	n.s.
mRankin ≤ 2 at discharge	34 (69.4%)	38 (50.7%)	<0.05
mRankin ≤ 2 at 3 months	31 (83.8%)	32 (61.5%)	<0.05

Table 2

Adjusted odds ratios of ELC for significant clinical variables in bivariate analysis.

	Odds Ratio	95% CI	p
Age	1.07	1.01–1.12	0.01
Stroke subtype	0.51	0.04–5.96	n.s.
Arterial hypertension	2.33	0.68–8.10	n.s.
Diabetes mellitus	5.8	1.15–29.49	<0.05
Smoker	1.3	0.34–4.90	n.s.
Alcohol intake	0.39	0.12–1.29	n.s.
Presence of atherosclerotic plaque	0.71	0.17–2.95	n.s.
mRankin ≤ 2 at discharge	1.24	0.29–5.38	n.s.
mRankin ≤ 2 at 3 months	2.28	0.41–12.70	n.s.

Age was included as a continuous variable and so the 7% increased association risk is by every 1 year of age increase. Categorical variables were included as 0 = no or 1 = yes. ELC, or earlobe crease (the Frank’s sign).

The mRS was measured at hospital discharge in 124 patients and the outcome at 3 months was evaluated in 89 patients. Thirty-five patients failed to attend the appointment scheduled for 3 months after stroke. ELC is associated with a poor outcome (mRS >2) at discharge and at 3 months (76.9% at 3 months) in bivariate analysis (Tables 1 and 3). However, in binary logistic regression analysis only age remains as a predictor of mRankin >2 both at discharge and at 3 months (analysis not shown).

Table 3

Bivariate analysis of modified Rankin Scale at 3 months by baseline clinical variables included in the study.

	mRS at 3 months
	mRS ≤ 2	mRS > 2	p value
	(n = 63)	(n = 26)
Male	47 (74.6%)	20 (76.9%)	n.s.
Female	16 (25.4%)	6 (23.1%)
Age			<0.001
<50	17 (27.0%)	0 (0%)
51–59	12 (19.0%)	1 (3.8%)
60–69	8 (12.7%)	7 (26.9%)
70–79	19 (30.2%)	6 (23.1%)
>80	7 (11.1%)	12 (46.2%)
Stroke subtype			n.s
Ischaemic	56 (88.9%)	21 (80.8%)
Haemorrhagic	7 (11.1%)	5 (19.2%)
Ischaemic stroke			n.s.
Atherothrombotic	12 (23.5%)	8 (40.0%)
Cardioembolic	17 (33.3%)	6 (30.0%)
Lacunar	10 (19.6%)	4 (20.0%)
Cryptogenic (ESUS)	12 (23.5%)	2 (10.0%)
Frank’s sign	32 (50.8%)	20 (76.9%)	<0.05
Arterial hypertension	41 (65.1%)	22 (84.6%)	n.s
Diabetes mellitus	18 (28.2%)	8 (30.8%)	n.s
Dyslipidemia	28 (44.4%)	15 (57.7%)	n.s.
Obesity (BMI>30) (n = 79)	15 (29.4%)	5 (38.5%)	<0.05
Smoker	24 (38.1%)	6 (23.1%)	n.s
Ex-smoker	13 (20.6%)	10 (38.5%)
Non-smoker	26 (41.3%)	10 (38.5%)
Alcohol intake (>20gr/d)	15 (23.8%)	8 (30.8%)	n.s
Ischaemic heart disease	8 (12.7%)	5 (19.2%)	n.s
Atrial fibrillation	9 (14.3%)	6 (23.1%)	n.s
Previous ischaemic stroke	9 (14.3%)	8 (30.8%)	n.s.
Presence of non-stenotic atherosclerotic plaque	28 (45.9%)	18 (85.7%)	<0.05
Internal carotid artery stenosis			<0.05
No stenosis	52 (85.2%)	12 (60.0%)
Stenosis >50%	2 (3.3%)	4 (20.0%)
Stenosis >70%	4 (6.6%)	3 (15.0%)
Occlusion	3 (4.9%)	1 (5.0%)
Stenosis other arteries	12 (19.7%)	8 (40.0%)	n.s.

Twenty-three patients (25% of ischaemic strokes) had suffered an ESUS. Among these, we found a high prevalence of patients with ELC (65.2%). In this group, there was also a significant association between ELC and age; whereas only 6 (42.8%) patients under 70 years old presented the Frank’s sign, all of the patients over 70 years old (9 patients) had the sign. In this subgroup, ELC is more frequent in patients with anterior circulation strokes (81.8% vs. 18.2%). A higher prevalence of ELC was observed in those patients with arterial hypertension, diabetes, dyslipidemia and obesity in the subgroup of ESUS, although none of these was statistically significant (Fig 2). Similarly to the total population analysis, smoking and alcohol habits seem to be related to the absence of ELC (33.3% and 13.3% respectively), and this was significant in non-smokers (p<0.05).

Fig 2

Frank’s sign by clinical variables in ESUS.

Age and prevalence of the main atherosclerotic risk factors in ESUS patients by the presence or not of ELC. Except in the case of age, the X axis represents the percentage of patients with the risk factor recorded on the Y axis. Age is expressed as mean (SD).

Discussion

The association between ELC and CAD has been reported several times since Sanders T. Frank noticed that many patients with ischaemic heart disease had an earlobe crease [3, 6, 7]. However, the prevalence of ELC in cerebrovascular disease has been less widely studied and there are no systematic studies evaluating its presence across each aetiopathogenic stroke subtype. The first retrospective preliminary report analysed the prevalence of the Frank’s sign in ischaemic stroke patients classified as large-vessel vs. lacunar stroke based on plain CT scans, describing an association between the Frank’s sign and non-lacunar ischaemic stroke [25]. Another prospective study that analysed the prevalence of ELC in patients with acute ischaemic stroke established that ELC could be a predictor of cerebrovascular events [13].

To the best of our knowledge, this is the first systematic prospective study evaluating the prevalence of the Frank’s sign in a consecutive series of acute stroke patients admitted to a stroke unit using an updated aetiopathogenic stroke classification.

In agreement with previous studies, we have found an association between the Frank’s sign and age, with it being more prevalent in patients over 70 years. A possible explanation for this is that ELC reflects the ageing processes of skin and arteries [12, 14]. We also found an association of ELC with classical vascular risk factors involved in atherosclerotic disease, especially with hypertension, and diabetes, as well as the presence of non-stenotic carotid plaques. An inverse association with smoking and alcohol intake was found, where non-smokers and patients without alcohol intake presented more ELC. This could be explained by the fact that smokers and patients with alcohol intake of more than 20 gr/day were younger than the global population. In spite of these previously described associations, the logistic regression analysis performed in our series showed that only age and diabetes mellitus remain significantly and independently associated with the presence of ELC. No relevant differences were found between unilateral (25% of patients) and bilateral (75% of patients) Frank’s sign.

An interesting finding of our study, not previously referred to in the literature, is that whereas the distribution was similar in atherothrombotic and cardioembolic aetiologies, the prevalence of the Frank’s sign was low in haemorrhagic strokes. This may be due to the fact that patients suffering from haemorrhagic strokes are younger, and that the underlying mechanism in this stroke subtype is suspected to be different from that of lacunar or atherothrombotic strokes.

Although ELC has been traditionally associated with atherosclerosis, we found a similar distribution of the Frank’s sign in both atherotrombotic and cardioembolic strokes. A possible explanation for this is that the underlying aetiopathogenic mechanism in some cardiac sources of embolism, such as left ventricular akinesia, dilated cardiomyopathy and reduced ejection fraction, is the atherosclerotic disease of coronary arteries [2, 3, 12, 15]. Furthermore, similarly to the prevalence of the Frank’s sign, atrial fibrillation is known to be more frequent in older patients. This may be the reason for the higher prevalence than expected of ELC in patients with cardioembolic strokes.

The ELC is prevalent in those patients who have suffered an ESUS. We found that patients who have suffered an ESUS and have the Frank’s sign show a more atherosclerotic profile than those without ELC. Patients with an ESUS and with the Frank’s sign have a greater prevalence of hypertension, diabetes and dyslipidemia, as well as a greater presence of carotid atherosclerotic plaques and more stenosis of other intracranial arteries (Fig 2). These results suggest that the presence or absence of ELC in ESUS could be of utility in the search for the underlying cause in ESUS (e.g. high-resolution-MRI techniques looking for aortic or intracranial vulnerable atherosclerotic plaques as an embolic source in ESUS with ELC or prolonged cardiac rhythm monitoring in ESUS without ELC). Further studies are needed focusing on this particular topic.

The main limitation of our study is the small population size, particularly in the ESUS subtype, although it is one of the largest prospective series focused on consecutive stroke patients, using a validated classification. An additional potential limitation is that the assessment of the Frank’s sign was carried out through photographs. In fact, an initial appreciation of ELC was made by a vascular neurologist during the visit to the Stroke Unit, usually on the day of admission and photographs of both ears were taken. After this, a neurologist who was blinded to the neurological symptoms and aetiology of the patient made the evaluation of ELC based exclusively on the photos, which had the additional advantages of permitting the evaluation of the inter-intra-observer variability and of being able to discuss any difficult cases.

(SAV)

Click here for additional data file.

9 Aug 2021

PONE-D-21-20627

Prevalence of the Frank’s sign by aetiopathogenic stroke subtype: a prospective analysis.

PLOS ONE

Dear Dr. Serena,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Sep 23 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Juan Manuel Marquez-Romero, M.D., M.Sc.

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2.  In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

3. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well.

4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ.

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: It was an honour to review your manuscript. Unfortunately, it requires many corrections, including major changes in data analysis and interpretation. All my comments and explanations were provided in the attached file "Comments".

Reviewer #2: Dear Dr Joaquin Serena:

Thank you for allowing us to read your interesting manuscript. This is a cross-sectional descriptive study that analyzed the prevalence of the Frank’s sign in patients suffering from acute stroke with focus on its prevalence in each aetiopathogenic stroke subtypes. This study concluded that Frank's sign was significantly associated with age, particularly in patients older than 70 with vascular risk factors and that is prevalent in all aetiopathogenic ischemic stroke subtypes, including ESUS. It is a great effort, congratulations. Please consider the following comments.

1.- It should be important to mention in the discussion whether the presence of the unilateral or bilateral Frank's Sign has the same clinical translation according to previous reports, since in the current study both were taken without distinction

.

2.- In the paragraph: As previously described, we found a strong and significant association between the presence of ELC and age, with ELC being particularly prevalent in patients who were older than 70 years (74.7%). Does the term “as previously” mean in previous studies? Because it is the first time that this idea was mentioned in the results of the body of the text.

3.- In the paragraph: On analysing carotid ultrasonographic markers of vascular atherosclerosis, we found a significant relationship between the prevalence of ELC and the presence of atherosclerotic plaques (63.6% vs. 45.2%, p<0.05). Are you referring to non-stenotic plaques? It should be important to clarify.

4.- In the limitations of the study, it should be important to included that the assessment of Frank's sign was carried out through photographs.

5.- It should be important to improve the quality of the tables, especially Table 2, which has disordered information ("Internal carotid artery stenosis")

6.- I think that the conclusion could be improved if the relevance of investigating atherosclerosis in patients with ESUS and Frank's sign is pointed out.

Thanks,

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Vanessa Cano-Nigenda

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: Comments.docx

Click here for additional data file.

27 Sep 2021

Dear Prof

We are grateful for your comments and suggestions that will clearly improve the final version of the manuscript.

A point-by-point response to the comments is attached. We would be pleased to provide additional information if requested and to consider further modification of the text if it should be thought necessary.

Submitted filename: Response to Reviewer #1 and #2.docx

Click here for additional data file.

24 Nov 2021

Prevalence of the Frank’s sign by aetiopathogenic stroke subtype: a prospective analysis.

PONE-D-21-20627R1

Dear Dr. Serena,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Juan Manuel Marquez-Romero, M.D., M.Sc.

Academic Editor

PLOS ONE

6 Dec 2021

PONE-D-21-20627R1

Prevalence of the Frank’s sign by aetiopathogenic stroke subtype: a prospective analysis

Dear Dr. Serena:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Juan Manuel Marquez-Romero

Academic Editor

PLOS ONE