Background: Perineural local anaesthetic and steroid injections around ilioinguinal (II), iliohypogastric (IH), and genitofemoral (GF) nerves are often performed to treat chronic refractory neuropathic pain in the lower abdomen and groin, but there is a lack of published data on outcomes of these interventions.Aims: The objective of this retrospective study was to evaluate analgesic outcomes of ultrasound-guided II, IH, and GF nerve blocks in patients with chronic neuropathic pain in the lower abdominal wall and groin.Methods: Analgesic outcomes were assessed at 6 weeks after injections and patients were classified as “responders” if the numerical rating scale for pain score reduced by 30% or more. Variables analyzed for impact on outcomes included demographics, intensity of pain and duration, etiology, dose of opioid, presence of anxiety, depression, and diabetes mellitus.Results: In this cohort of 54 patients with severe baseline pain who had failed to receive analgesic benefit from recommended first- and second-line medications for neuropathic pain, 30 patients had history of surgery and 24 had pain secondary to visceral inflammatory pathologies. Twenty-five (46.3%) patients were identified as responders. A majority of the patients in this cohort had pain for more than one year. There was a higher incidence of diabetes mellitus in nonresponders compared to responders but the difference was not significant .Conclusions: Ultrasound-guided perineural steroids can ameliorate chronic refractory abdominal wall and groin neuropathic pain in patients who have failed to respond to conventional medical management at 6 weeks after the procedures. Antecedent surgery is commonly associated with the development of persistent pain in the abdomen and genital regions.3 Macrae defined chronic postsurgical pain (CPSP) as pain developed after surgery and of at least two months duration, after exclusion of other caused of pain including the pre-existing pain.4 Hernia repairs are the most frequently performed operations worldwide and it is accepted that CPSP is the most common and serious long-term complication after inguinal hernia repair.7 Damage to ilioinguinal (IL), iliohypogastric (IH), and genitofemoral (GF) nerves during the intraoperative period can contribute to neuropathic pain after lower abdominal surgeries8 and a neuropathic component of pain is estimated to be present in up to 50% of patients with chronic pain after inguinal hernia repair.9 Inflammatory conditions of the abdomen and pelvis  can also be a cause of neuropathic pain in the abdominal wall and groin from entrapment of nerves innervating the overlying skin in fibrosis and scar tissue.1,11Chronic lower abdominal wall and groin pain is a frequent presentation in pain clinics and up to 25% of adults have abdominal pain at any one time.12 However, pharmacologic treatment with medications such as gabapentinoids and antidepressants to relieve neuropathic pain is often ineffective with responder rates varying from one in three to one in seven.13 These medications also have significant adverse effects, including sedation, drowsiness, edema, and cognitive impairment, that limit increases in dose and compliance. In patients with refractory neuropathic pain, perineural injections of steroids around the II, IH, and GF nerves are often performed for diagnostic and therapeutic purposes.14 Prognostication of response to surgical neurectomy is another indication for these interventions.15 The rationale for this therapy is that steroids prolong the duration of analgesia by stabilizing neuronal membranes and also by inhibiting the synthesis and release of proinflammatory mediators.17Patients with chronic lower abdominal wall and groin pain should be managed with a multimodal approach based on the biopsychosocial model of pain, with pharmacological therapies often being the first step.21 Existing literature is unable to provide a reliable estimate of analgesic outcomes of these procedures. There is a paucity of reliable data about the nature of pain syndromes and characteristics of patients who undergo these interventions. This retrospective study on data from patients at our two clinics was conducted with the primary objective of evaluating analgesic outcomes of II, IH, and GF perineural injections with local anaesthetics (LAs) and steroids at 6 weeks after the interventions for patients with chronic refractory neuropathic pain in the lower abdominal wall and the groin. The secondary objective was to identify variables associated with responders to this intervention.There is some evidence for analgesic efficacy of injections of perineural steroids around IH and II nerves, but the published studies are heterogeneous with methodological limitations.22 Data were analyzed from a database of adult patients (aged ≥ 18 years) who received ultrasound-guided local anaesthetic and steroid injections around II, IH, and GF nerves between January 1, 2009, and July 31, 2013. Pain centers at both of these hospitals are large, tertiary-level multidisciplinary pain clinics. Around 450 patients receive ultrasound-guided injections of perineural LA and steroids per year for neuropathic pain secondary to various etiologies and refractory to noninterventional management . Patients with abdominal wall and groin pain are part of this cohort, and these patients are referred to the two clinics by surgeons, gynecologists, urologists, gastroenterologists, internists, and family physicians.This retrospective cohort study was conducted at the Interventional Pain Clinic of Toronto Western Hospital, University Health Network and the Wasser Pain Management Centre at Mount Sinai Hospital in Toronto, Canada, following research ethics board approvals . Strengthening of Reporting of Observational Studies in Epidemiology guidelines were followed during reporting of this retrospective study.4 on the 11-point Numerical Rating Score [NRS] for pain, pain persisting for four or more months after surgery, no analgesic benefit from a trial of at least one medication in each of the four main classes for treatment of neuropathic pain ) or adverse effects resulting in discontinuation of medications.23 All patients had undergone extensive assessment by medical and surgical specialists to rule out treatable etiologies for ongoing pain. Though patients in our study cohort had pain from surgical and nonsurgical etiologies, the presentation was distinctly neuropathic and we considered our cohort as homogenous in terms of the pathology (entrapment of the nerves in scar tissue), presentation, and treatment protocols. This therapeutic approach is also endorsed by others, including the International Association for Study of Pain’s Special Interest Group on Neuropathic Pain.13Patients were identified from the clinical database of perineural procedures and their details were cross-matched against procedural lists of the two pain clinics. Electronic medical records of patients eligible for inclusion in the study (as defined below) were also reviewed. All patients had a diagnosis of refractory neuropathic pain of moderate or severe intensity based on the following criteria: pain of new onset following surgery on the abdomen or groin or in association with inflammation of abdominal or pelvic viscera; pain present in distribution of II and IH nerves with or without pain in distribution of GF nerves; pain described using one or more neuropathic pain descriptors selected by patients from the Short Form McGill Pain Questionnaire; abnormal sensory findings on physical examination; and refractory pain defined on the basis of “rule-of-four” used at our centers  around IH and II nerves at locations superior and posterior to the anterior–superior iliac spine and 4 mL of 0.25% bupivacaine and 20 mg of methylprednisolone acetate around GF nerves in the spermatic cord or round ligament (in patients who had pain in distribution of this nerve). The steroid preparation was a single-dose, preservative-free vial and its use was off-label. Accuracy of the injections was confirmed by demonstration of numbness in the area of sensory distribution of targeted nerves 15 min after the procedure. None of the patients in this cohort had repeat injections prior to the follow-up visit at 6 weeks.All perineural IH, II, and GF nerve injections were performed by staff pain physicians experienced in ultrasound-guided perineural injections. Sonosite M-Turbo  with a linear transducer (13–6 MHz) was used for ultrasound guidance and injections were performed as per the technique described by others and our group.26 The primary outcome of interest was to establish the proportion of responders following the interventions. The secondary outcome of this study was to identify variables associated with analgesic response. Information was extracted from the database and medical records for variables to be analyzed, including age, sex, data related to pain , nature of surgery and any postoperative complications, nature of inflammatory visceral condition and related complications, psychological comorbidities ,27 diagnosis of diabetes mellitus, preprocedural pharmacological treatment , and any complication related to the perineural injections. Among these variables, based on clinical rationale, we considered the following likely to have an impact on our outcome of interest: age, sex, etiology, intensity and duration of pain, dose of opioids, presence of anxiety, depression, and diabetes mellitus.Patients who had 30% or more reduction in the pain NRS score compared to preintervention baseline values were categorized as responders and the rest were nonresponders. This dichotomization was based on Initiative on Methods, Measurements, and Pain Assessment in Clinical Trials recommendations.Adverse effects over the 6-week follow-up period were also recorded. These included reports of thinning of skin or necrosis at injection site; reports of impaired glycemic or blood pressure control in patients with history of diabetes mellitus and hypertension, respectively; history of fractures during the postprocedure period; and evidence of impaired immunity as manifested by recurrent infections or impaired wound healing.t-tests, and those with nonnormal distributions were analyzed using Wilcoxon rank sum tests. All t-tests were two-sided and unpaired. For P-values of t-tests, pooled values were used for equality of variances and Satterthwaite values were used for unequal variances. Categorical variables were analyzed using chi-square test (or Fisher’s exact test when any cell had an expected count of less than five). Exploratory analyses were performed to analyze the influence of various domains of pain on the probability of analgesic response. Univariable logistic regression models were created to determine odds ratios and 95% confidence intervals for prespecified variables of interest that could impact analgesic response. Multivariable logistic regression models were created to address confounding and to examine variables affecting probability of analgesic response and their adjusted odds ratios. All analyses were conducted using SAS Version 9.3 .The data collected from the study contained continuous and categorical variables. Continuous data were examined for normality of distribution using the Kolmogorov-Smirnov test. Continuous data with normal distributions were summarized as means and standard deviations, and data with nonnormal distributions were summarized as medians and interquartile ranges. Categorical data were summarized as numbers and percentages. Variables with normal distribution were analyzed using We wanted to assess and compare characteristics of responders and nonresponders for the intervention of interest. This was an exploratory study, so we collected data on all patients who received ultrasound-guided local anaesthetic and steroid injections around II, IH, and GF nerves between January 1, 2009, and July 31, 2013 at our two pain centers. Data were included from records of patients who had neuropathic pain in the abdominal wall or groin following surgery or in association with inflammation of abdominal or pelvic viscera and where data on variables of interest were available for analysis.Sixty-three patients who received perineural injections around II and IH nerves at our clinics were identified but nine patients were excluded because of missing data points, lack of follow-up, and other procedures being performed in the interval between procedure and postprocedure follow-up at 6 weeks. Data from 54 patients  were included for analysis in this study .10.1080/12 Documented physical examination findings included evidence of allodynia, hyperalgesia, or hypoesthesia. Based on our criteria for analgesic response, 25 (46.3%) of these patients were identified as responders. Baseline characteristics of the cohort are presented in The mean intensity of pain was severe, with a median numerical rating score of 9 (interquartile range [IQR] = 8–10). All patients had described their pain using neuropathic descriptors .P = 0.049). However, there was no difference between proportions of responders and nonresponders who had pain for more than 5 years  had pain for more than one year and 50% of the patients reported pain for more than 5 years. A higher proportion of nonresponders than responders had pain for more than one year and up to 5 years  from the overall cohort had surgical procedures on the abdomen and pelvis prior to onset of persistent abdominal wall and groin pain  and the remaining 24 (36%) had pain in these locations secondary to abdominal or pelvic visceral inflammatory pathologies. Of the patients with CPSP, 32% of responders and 31% of nonresponders reported onset of severe pain in the immediate postoperative period, and the remainder reported an increase in intensity of pain after 3 months of surgery . OperatiP = 0.022). We screen for presence of anxiety and depression in our clinics by administering the Hospital Anxiety and Depression Scale .27 The prevalence of anxiety was 28% and the prevalence of depression was 19% in the overall cohort in this study. There was no significant difference between prevalence of anxiety and depression among responders and nonresponders  patients in the cohort had diabetes mellitus. There was a higher incidence of diabetes mellitus among nonresponders compared to responders , suggesting that the model fit was adequate, and by examining the c-statistic (= 0.73). This model was constructed in an attempt to provide exploratory insights and was not intended to be a robust explanatory model.28We attempted to evaluate impact of three independent variables  on the dichotomous outcome of presence or absence of analgesic response while controlling for confounding by performing multivariable logistic regression analysis. We used only three independent variables in our model to avoid overspecification and we chose these variables based on a combination of perceived clinical significance and results of our univariable analysis. The results of this analysis are presented in Data regarding pharmacological therapies and perineural injections are reported in Chronic neuropathic pain in the abdominal wall and groin secondary to operative interventions or inflammatory conditions of the viscera is often of a high intensity and unresponsive to conventional recommended treatments. Our retrospective case series on this patient population revealed that ultrasound-guided perineural injections of LA and steroids around the II and IH nerves with or without injection around GF nerves provided analgesic benefit in almost every other patient (46.3% of subjects) with severe refractory neuropathic pain who received this intervention. The impact of duration of pain prior to the procedures on analgesic efficacy of this treatment was unclear. Univariable analysis revealed a decrease in efficacy of the procedures after pain had been present for one year as evidenced by a significantly higher number of nonresponders (compared to responders) in our study who had persistent pain for a year or longer but with no impact of duration on efficacy if pain had been present for less than one or more than 5 years. However, the effect of duration was not analyzed in multivariable analysis due to the smaller number of patients in the categories for this variable. This is the first reported case series of outcomes of ultrasound-guided perineural injections of LA and steroid around II, IH, and GF nerves in patients with chronic abdominal wall and groin neuropathic pain secondary to surgery or inflammatory visceral conditions.23 identified four case series that reported analgesic efficacy of perineural steroids around II, IH, and GF nerves in patients with CPSP following inguinal herniorrhaphy.21 All of the studies reported an analgesic benefit with this intervention with a reduction in pain NRS scores by 55%–75%. However, there was heterogeneity in doses of steroids, number of injections (varying from one to seven), and interval between intervention and follow-up (one to 68 months). The analgesic benefit from these studies was higher than in our retrospective study. Possible reasons for this difference include higher doses of steroids, repeat procedures, and enrollment of patients with CPSP in these studies while excluding pain from inflamed viscera that can result in severe scarring/fibrosis around cutaneous nerves. All patients in our study cohort had no other injections until completion of the follow-up at 6 weeks after the study intervention.Our study cohort included patients with CPSP and those with chronic abdomino-pelvic visceral inflammatory conditions but there was no difference between these two groups in incidence of analgesic success  with the study intervention. A recent scoping review on perineural steroids for the treatment of chronic post-herniorrhaphy groin pain29 the diagnosis of neuropathic pain is essentially clinical.30 However, validated tools and investigations , can help establish this diagnosis with greater accuracy while also helping to delineate the mechanism and potential treatment.31In our study cohort, pain was diagnosed as neuropathic in its presentation by pain physicians at our clinic based on the descriptors used by the patients and the results of physical examination. Though it is ideal to use validated tools to screen for neuropathic pain,13 were ineffective for all of our patients who received perineural injections of LA and steroids. A high failure rate of pharmacological trials (number needed to reduce pain by 30%–50% varies from three to seven) has also been documented in other publications on this topic.5 The incidence of serious adverse effects with these medications  is also high.12 Chemical, radiofrequency, or surgical neurectomy is an analgesic option, but long-term pain relief is not assured and morbidity associated with these procedures is also a concern.9 Ultrasound-guided delivery of perineural LA and steroids has a low cost and incidence of adverse effects, and it can relieve neuropathic pain through multiple mechanisms.16The success rate for analgesia reported in our study (25 responders out of 63 subjects exposed to the treatment) is encouraging because pharmacological therapies recommended for neuropathic pain18 this technique is unreliable in ensuring delivery of injectate around injured nerves.33 Imaging modalities that have been used for guiding these injections include computed tomography guidance and ultrasound.20 Ultrasound has been shown to enhance accuracy of perineural injections, it can be done in the pain clinic under sterile conditions, and it does not expose patients and health care providers to radiation. The ability to visualize muscle layers, blood vessels (the deep circumflex iliac artery is in close proximity to the II and IH nerves), peritoneum, and bowel enhances accuracy  while minimizing the risk of inadvertent injury to neural and nonneural structures.34Though anatomic landmarks have often been used in the past for guiding II and IH nerve injections,34 We chose to categorize duration of pain in three epochs  because chronic pain is usually accepted as the presence of pain for more than 3 months, patients with severe neuropathic pain are often referred to pain physicians after one year of onset of pain,35 and 5 years appears to be the inflection point for a sharp drop-off in incidence of positive analgesic outcomes following interventional treatments for neuropathic pain.36 This rationale is partially supported by our results that show that duration of pain longer than one year reduced the probability of analgesic benefit. Only 32% (8/25) of respondents compared to 59% (17/29) of nonrespondents had duration of pain of over a year (P = 0.049). However, we are unable to propose that duration of pain and probability of analgesic success of perineural interventions are inversely correlated because there was no difference in analgesic response in our subcohort of patients who had pain for more than 5 years. Further, the significance of lower incidence of response in subjects who had pain for more than one but less than 5 years is unclear because even a small change in the counts of responders and nonresponders would affect the analysis. More research with larger data sets is required to arrive at reliable conclusions regarding the impact of duration of pain to this intervention.Approximately half of the patients (53.7%) in our study cohort did not attain analgesic benefit from perineural steroids around II and IH nerves. A possible reason for failure to relieve pain in these patients is chronicity of pain because duration of pain is often inversely related with probability of analgesic success with perineural steroids, often due to the presence of dense fibrosis around the II and IH nerves in the fascial plane between the internal oblique and transrversus abdominis muscles or development of central sensitization.37 However, we do not have data on either the incidence of peripheral neuropathy or the severity of glycemic impairment in the diabetic patients in our study. Further, only 4 out of 29 patients among the nonresponders and none of the responders had diabetes. It is difficult to draw meaningful conclusions from these small numbers of patients and we do not recommend that perineural LA and steroid therapies should be withheld from patients with neuropathic pain who have diabetes. Finally, factors such as high grades of anxiety, depression, and nociceptive component of mixed peripheral pain syndromes can also reduce analgesic benefits from treatments for neuropathic pain,38 but we did not find these associations in our study.Another possible reason for analgesic failure with perineural steroids for peripheral neuropathic pain is the presence of diabetes mellitus, because diabetes can be associated with active axonal degeneration, ischemic injury, and microvasculitis.39 Though our clinical protocols mandate postintervention assessment, we were unable to extract data for nine patients who did not return for follow-up. There may be several reasons for these missing data points, but lack of efficacy of the intervention is a possibility. A conservative assessment of beneficial effects of perineural steroids would lower the analgesic success rate from 46% (25/54) to 40% (25/63). This is still a promising outcome for a cohort that had limited options for relieving severe refractory neuropathic pain. However, a prospective, randomized controlled trial on patients with chronic neuropathic abdominal wall and groin pain with blinding of participants and observers is required to estimate the analgesic potential of perineural steroids at multiple time points  after the interventions. Recommendations for sample size and methodology for trials have also been provided in recently published reviews by our group.41We divided the patients in our cohort into responders and nonresponders based on the analgesic outcomes at 6 weeks after the intervention. We assess our patients at this time point because the peak effect of long-acting steroids manifests around 4 to 6 weeks after administration.26 lack of information on absolute pain scores after the intervention and absence of longitudinal, periodic follow-ups are limitations of this study. Categorization of continuous outcomes is efficient for understanding the impact of variables on the outcome, but it also limits the usefulness of the available data. We did not report absolute pain scores in this study because the earlier versions of our database allowed us to record dichotomous outcomes for analgesic therapies while not listing the absolute pain scores. We also recognize the potential for a type 1 error (finding a significant difference when none exists) due to multiple comparisons conducted in our univariate analysis. However, we did not find any significant differences between responders and nonresponders except for the difference in proportion of subjects with duration of pain between one and 5 years. Unrecognized confounding, responder bias, and missing data are also potential limitations of this retrospective study. However, retrospective designs may be useful in analgesic research by taking advantage of samples of convenience for development of pilot data that provide the basis for conducting high-quality randomized and controlled prospective studies.40 We also acknowledge that comparison of our study cohort with a group of patients managed with conventional medical management would have allowed an informed assessment of benefits and harms of perineural interventions. However, the referral criteria for our two clinics and the long wait times mandate that only patients who have failed to respond to first-, second-, and third-line pharmacological treatments and physical therapies (desensitization) for neuropathic pain and who continue to have moderate or severe pain are accepted for a trial of interventional approaches. There is also a robust body of published literature regarding success and failure rates of conventional medical management in peripheral neuropathic pain.17Analgesic response, the main outcome of this study, was dichotomous because it was based on patients reporting 30% or more reduction in their pain or not at the follow-up 6 weeks after the intervention. Though this cutoff is based on recommendations for assessment of efficacy of therapies for pain,Our results suggest that administration of perineural steroids can ameliorate chronic postsurgical and visceral inflammation related peripheral, severe refractory neuropathic pain in the abdominal wall and groin 6 weeks after the interventions in a significant number of patients.


The purpose was to determine the efficacy of deep dry needling (DDN) applied on an active myofascial trigger point (MTrP) versus a latent-MTrP versus a non-MTrP location, on pain reduction and cervical disability, in patients with chronic neck pain. A randomized, double-blind clinical trial design was used. A sample of 65 patients was divided into non-MTrP-DDN, active-MTrP-DDN and latent-MTrP-DDN groups. The visual analog scale (VAS), reproduction of the patient’s pain, number of local twitch responses, pressure pain threshold (PPT) and Neck Disability Index (NDI) were assessed before, during and after the intervention and up to 1 month post-intervention. The active-MTrP-DDN-group reduced pain intensity more than non-MTrP-DDN-group after a week and a month (P < 0.01), as well as showing the greatest improvement in tibialis muscle PPT. The treatment of both Active and Latent MTrPs was associated with the reproduction of the patient’s pain. The application of DDN on an active-MTrP in the upper trapezius muscle shows greater improvements in pain intensity after 1 week and 1 month post-intervention, compared to DDN applied in latent-MTrPs or outside of MTrPs in patients with neck pain. Although most cases of acute neck pain seem to resolve with or without treatment, but up to 50% of people who have it may continue feeling pain or have frequent pain episodes4. In addition, there are several important factors that are related to the appearance of neck pain, especially those related to work, stress and depression1. In relation to work, neck pain appears more frequently in those jobs which involving prolonged static postures and repetitive movements of the upper limbs5.Neck pain is the fourth leading cause of disability, with an annual prevalence rate exceeding 30%. In the world, around 70% of the population will have neck pain throughout their lives, while the prevalence in Spain is 19.5% and is higher in women than men6. The prevalence of MTrPs in patients with myofascial pain was 93.75% for the upper trapezius muscle, being the most prevalent muscle for MTrPs presence. Within the same, the prevalence of active MTrP was higher on the right side with 82.1% while on the left side it was 79%4. The reason why the upper trapezius is the muscle that most frequently presents MTrPs of the whole body is due to its permanent activity and micro-trauma7.It has also been proposed that myofascial trigger points (MTrPs) could be the responsible for the development of pain in patients with mechanical neck pain; therefore, some studies include MTrP therapy for the management of these patients9. MTrPs are clinically classified as active or latent. Active MTrPs are those that cause spontaneous pain or pain during movement, stretching or compression. Latent MTrPs are usually asymptomatic, but they reproduce pain or discomfort when they are compressed9. Other typical symptoms associated with MTrPs in addition to local and referred pain is the appearance of muscle weakness and a restricted range of motion. In the case of active and latent MTrPs, both elicit a local twitch response (LTR) and pain that reproduces the patient's symptoms10. The combination of these symptoms could have a large impact on the quality of life, mood, and health status5.Myofascial trigger point is defined as a hypersensitive spot in a taut band of a skeletal muscle and referred pain while myofascial pain syndrome (MPS) can be diagnosed by the presence of one or more MTrPs11. Within the types of DN: the most commonly used to the MTrPs treatment is deep DN (DDN), which involves inserting a thin filiform needle directly into the muscle and through the MTrP13. Traditionally, the effectiveness of dry needling has been related to the provocation of the local twitch response (LTR). The LTR is characterized by a visible contraction of part of the taut band in the involved muscle upon mechanical stimulation with needling to a sensitive site in the MTrP area. Although there are different points of view about this, some authors suggest that MTrP DN is most effective if a LTR is elicited during the procedure14. To carry out the intervention, the needle is moved up and down into the muscle tissue to elicit LTRs15. Although there is a lack of knowledge about the mechanisms of dry needling, some studies support that LTR is crucial to achieve the elimination of pro-nociceptive and pro-inflammatory substances. However, in clinical practice, even when LTR is induced, there is not always a positive effect16. Some studies have shown the benefit of applying DN to an active MTrP in the trapezius muscle producing greater improvements in pain measured with visual analogue scale (VAS), pressure pain threshold (PPT) and levels of disability produced by neck pain17.Among the treatment of myofascial pain, this is based on the inactivation of MTrPs through manual techniques or applying the invasive technique known as dry needling (DN)19. Additionally, there is a sympathetic interaction with myofascial tension that demonstrates the facilitating contribution of sympathetic hyperactivity to mechanical sensitization, and the possible underlying mechanisms of local and referred muscle pain20. Moreover, vasoconstriction, cold skin, sweating, and pilomotor response in patients with pain due to active MTrPs may be related to sympathetic hyperactivity. The vasoconstrictor activity of the sympathetic nervous system (SNS) was activated by nociceptive stimulation of latent MTrPs21. Finally, another study identifies the neuromuscular junction as a target of the SNS and shows that sympathetic input is crucial for the maintenance and function of the synapse22.In relation to the DN mechanisms of action, some factors were involved in the activation of the central nervous system: complex endogenous pain modulating mechanisms and the activation of the autonomic nervous system to produce electromyographic changes in the MTrPs. This supposes a possible action on the etiopathogenesis of MTrPs, which could justify motor effects, beyond the purely sensitive ones24. However, another study indicates that performing DN without eliciting LTRs has superiority over the DN eliciting LTR in relation with the treatment long-term effects7.It is also important to decide the dose of DN treatment. On this matter, some studies explain that the greater the number of “in-and-outs” with the needle in the muscle, the greater the post-needling pain appears, but, in turn, the better results seem to be found when six or more LTRs are achievedTherefore, the aim of this study was to determine the efficacy of DDN applied on an active MTrP versus a latent MTrP versus a non-MTrP location, on pain reduction, mechanical hyperalgesia and cervical disability, in patients with chronic neck pain attributable to myofascial pain syndrome. The second aim was to evaluate the association between number of LTR and improvement and the third aim was to evaluate the association between reproduce the patient’s pain during dry needling with the improvement of pain and function following dry needling.25.A randomized, double-blind clinical trial design was used for the study. Patients were randomly allocated to one of three different intervention groups  performed by a computer-generated random-sequence table created before the start of the study with Epidat 4.2 software  with the support of the Pan American Health Organization (PAHO-WHO) and the CES University of Colombia)All subjects signed an informed consent, and the study was approved by the Ethics Committee for Animal Research and Experimentation at the University of Alcalá with the number identification CEID/HU/2018/41.The study was guided by the Consolidated Standards of Reporting Trials (CONSORT) statement and included the CONSORT checklist.This study was registered at NIH U.S. National Library of Medicine ClinicalTrials.gov under identifier (NCT number): NCT03345238. The study start date was registered on 13/03/2017 and the study completion date was registered on 10/08/2019.Participants were recruited from a Primary Health Care Centre in Alcalá de Henares  and the University community from the University of Alcalá. Patients with neck pain of muscular origin were referred and screened for possible eligibility criteria.26 and Patients were between 18 and 65 years of age27.In this research we defined neck pain as mechanical pain in the cervical muscles, which can be provoked both by maintained postures and by movements. Patients were selected if they met all of the following criteria: Non-specific neck pain, unilateral or bilateral, Neck pain ≥ 3 months of duration, Presence of active and latent MTP in the upper, left, right or bilateral trapezius muscle, in relation to the patient's neck pain28, such as taut band palpable, a hypersensitive spot at the pressure of a taut band, referred pain and the reproduction of any of the symptoms experienced by a patient and the recognition of pain will identify as active MTrP. It is considered positive when 2 of the clinical criteria are found8.The recent clinical criteria recommended identifying active and latent MTrPsIn order to meet the criteria to participate in the study, patients had to pass an initial physical examination performed by a single investigator to rule out the exclusion criteria: Unsurpassed fear of needles, Coagulation disorders, Specific alterations of the cervical region in the clinical history, Infiltration of corticosteroids or local anaesthetics during a year before the study, Surgical intervention of the cervical region or previous shoulder, Skin lesions in the area, as well as infection or inflammation, Taking analgesic, anti-inflammatory or anticoagulant medication the week before the study, Treatment of MTrP or DDN in the neck region in the 6 months prior to the intervention, Cognitive deficit in the medical history.Each participant received a thorough explanation about the content and purpose of the treatment before signing an informed consent form relative to the procedures.Participants were randomized into each intervention group without knowing which of the three groups they belonged to. For the masking of the practitioner physiotherapist, the evaluator therapist marked on the skin of each participant the place where the physiotherapist had to perform the DN without the latter knowing which group each of the participants belonged to.The primary outcome measure was neck pain intensity.Neck pain intensity was measured before, during, immediately after intervention and the follow up was at 1, 6, 12, 24, 48, 72 h after intervention and also 1 week and 1 month after intervention.29.A visual analogue scale (VAS) was used, which is a well-established and validated self-report to measure the intensity of pain. It consists of a 100 mm line in which 0 indicates no pain and 100 indicate the worst pain imaginable by the patient. The patients should indicate where their pain is generally located within the measure described above. The VAS presented a moderate to good reliability (intraclass correlation coefficient 0.60 to 0.77) for the assessment of disability in patients with chronic musculoskeletal painThe two factors that were assessed during the intervention were patient’s neck pain recognition and number of local twitches responses.The association between the DDN site and the provocation of the patient’s neck pain was assessed by asking the subjects of each group if their pain was reproduced and recognized during the intervention and the answers were collected as a dichotomous variable (yes/no).24.The LTRs number was evaluated by the physiotherapist who performed the intervention on an “n” number of up to 12 dry needling in and outs in the upper trapezius muscle (n/12)The secondary outcomes were neck disability and pressure pain threshold.30.The validated Spanish version of the Neck Disability Index (NDI) was used to assess the degree of disability before, 1 week and 1 month after the intervention. The NDI is a valid tool for the measurement of pain and self-assessment of cervical disability. The NDI is composed of 10 questions related to daily functional activities. Each of the sections  offers 6 possible answers that represent 6 levels progressive functional capacity, and it is scored from 0 to 5 . The total score is expressed as a percentage of the maximum possible. A score between 5 and 14 represents a mild disability, whereas a score between 15 and 24 is interpreted as a moderate disability. Neck Disability Index scores > 25 reflect a severe disability and the maximum score is 50. NDI presents an acceptable reliability with an intraclass correlation coefficient (ICC) ranging from 0.50 to 0.982 rubber disc. The results serve as a reference for the clinical diagnosis of abnormal sensitivity and for documentation of treatment results in the MPS. Pressure is applied at a rate of 1 kg/s. Three consecutive tests were performed on the active, latent and non MTrP of the upper trapezius muscle at 30 s intervals before and immediately after the intervention. Intra-evaluator reliability is high in the upper trapezius muscle (ICC = 0.94–0.97)31. PPT was also assessed in the tibialis anterior muscle in order to assess mechanosensitivity outside of the painful region, which showed an excellent test–retest reliability over tibialis anterior muscle (ICC = 0.71–0.91) in patients with chronic neck pain32. Furthermore, PPT expansion was calculated by the summation of the described pressure pain thresholds, including upper trapezius PPT as the painful region measurement and tibialis anterior PPT as the measurement outside of the painful region32.PPT was evaluated using a digital algometer. Pressure pain is defined as the minimum pressure that induces pain or discomfort with a 1 cmParticipants were randomly assigned to the non-MTrP group or to the active MTrP group or to the latent MTrP group using randomization. Each participant was informed about the study and filled in the informed consent sheet as well as the data sheet and the NDI. After collecting the information from the participant, the evaluating therapist performed the physical evaluation of the patient and determined the major dry needling site according to the assigned group where the practitioner therapist performed dry needling intervention. Participants did not know which group they belonged to. Next, the practitioner therapist performed the dry needling intervention without knowing the group to which each participant was assigned, and again the evaluating therapist performed the post-intervention evaluations. One session of intervention were performed in each patient.Patients were followed up for up to 1 month according to each measurement variable.17 and then the practitioner therapist inserted the needle through the skin. Consecutively, the taut band was needled forward and backward until the exact punction point. Precise needling of the MTrPs elicited a brief contraction followed by relaxation of the muscle fibers; this is known as a LTR. Needling of the MTrP or the non-MTrP was repeated until 12 in and outs at a frequency of 1 Hz24.Patients were asked to lie in a supine position with the arms next to the body. Dry needling was performed with a 0.30 × 30 mm DN needle (APS Regular Agupunt) with a guiding tube. The evaluator therapist palpated the MTrPs in experimental groups and non-MTrPs group, for this group the non-MTrP site, without any type of symptomatology, a 2 cm of distance to any other latent MTrP was selected in the upper trapezius muscleData were analyzed using SPSS Statistics for Windows, version 25.0  was used for statistical analysis. A Kolmogorov–Smirnov test was used to test normality and no statistically differences were founded. A descriptive analysis was selected to summarize the outcomes in the 2 measurements in all groups  and were included the mean, SD. A one-way ANOVA with the factor group  was performed.2) coefficient was used.Repeated-measures analysis of variance (ANOVA) was performed considering the significance of the Greenhouse–Geisser correction when the Mauchly test rejected the sphericity to compare the differences between the 3 groups in the baseline. To analyze the contrast between the three treatment groups we used the ANOVA of repeated measures with 2 factors, 3 (groups) × 2 times (pre and post), only the pain intensity variable was analyzed in the interaction with the group factor the 11 times  to include the calculation of the effect size, the partial eta  were considered as dependent variables. Independent variables were descriptive data, local twitch responses (LTR), patient’s pain recognition, group and the other outcome measurements at baseline. F probability was considered according to a P in = 0.05 and P out = 0.10.All analyses were carried out “per intent-to-treat”. Moreover, a P-value lower than 0.05 was considered as statistically significant for a 95% confidence interval (CI).A total sample size of at least 18 subjects per group had to be recruited based on an a priori power analysis (G*Power 3.1.9.2 from the University of Düsseldorf). This power analysis was performed for the within-between interaction in a repeated measures analysis with 3 groups, 11 measurements , a minimum power of 0.95, an effect size of 0.3 and an α level of 0.05. However, to cover possible losses, the sample size was increased by another 11 participants (n = 65).The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the University of Alcalá (CEID/HU/2018/41).Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patient(s) to publish this paper.Sixty five participants were assessed for eligibility Fig. . All subThere were not statistically significant differences between the 3 groups in terms of the demographic and clinical characteristics at baseline Table .Table 1Dp2 = 0.834) and demonstrated a significant interaction between group and time  for changes in intensity of the neck pain, data presented in Table The ANOVA showed a significant effect for time .p2 = 0.482) but not statistically significant interaction between group and time were found  for changes in PPT in the neck, data presented in Table The ANOVA showed a significant effect for time  and demonstrated a significant interaction between group and time  for changes in PPT of the tibialis muscle, data presented in Table The ANOVA showed a significant effect for time  but not statistically significant interaction between group and time were found  for changes in PPT summation expansion, data presented in Table The ANOVA showed a significant effect for time  but not statistically significant interaction between group and time were found  for changes in disability, data presented in Table The ANOVA showed a significant effect for time  but we found a statistically significant association that in the patients with the higher number of LTRs, it is also higher the percentage of subjects in whom their neck pain is reproduced (P < 0.05).A greater association of a greater number of LTRs was found in the active and latent trigger point dry needling groups (P < 0.0001), than in the non-MTrP group.In addition, we found an association of a higher percentage of subjects in which their neck pain is reproduced when dry needling was done at the active trigger point (77.3%) and in the latent (81.8%) (P < 0.001). However, no model was found in the prediction that producing their neck pain during dry needling produces a greater improvement in any of the variables measured.2 = 0.218; β = − 2.356; F[0.051] = 4.061; P < 0.05), indicating a prediction of higher mechanical hyperalgesia over tibialis muscle difference after versus before DN under the bleeding and site of dry needling (latent or active) difference. The rest of independent variables were excluded from the prediction model according to F probability .PPT over tibialis muscle after DN with respect to baseline showed a statistically significant predicted model by bleeding after DN and place of dry needling , indicating a prediction of lower difference at 1 week after DN versus baseline under less LTRs and under side treated. The rest of independent variables were excluded from the prediction model according to F probability .Pain intensity difference at 1 week after DN with respect to baseline showed a statistically significant prediction model which was predicted by local twitch response (LTR) and side treated , the group with dry needling on the active trigger point had the least improvement.17 have studied the importance of treating the specific site that produces pain. In the case of the present investigation, our objective was to achieve the maximum number of LTRs as it was found previously by the authors to be associated with a better clinical response. In those cases, better short-term results can be achieved for our patients as we have observed in our study. However, in contrast to the above, some studies indicate that performing DN without eliciting LTRs presented superiority over the DN along with eliciting LTR while the treatment aimed to receive long-term effects7.Some previous works by Pecos Martín et al.33, showing that a local twitch response during dry needling is not always a synonym for successful treatment.Our results are consistent with another study in which dry needling was applied to the lumbar multifidus muscles in patients with low back pain. The presence of LTR did not produce a greater improvement in pain, disability, function and pain threshold to pressure at 1 week, although immediately the patients who had a LTR did have a greater improvement in function and pain threshold to pressure at 1 week14. Although there are certain methodological limitations to this study, other researchers also suggest that the LTR produces a greater improvement34. Additionally, in the results obtained, we have been able to observe how this type of intervention produces not only an improvement in pain perception, but also in the pressure pain threshold and neck disability at short term. According to these findings, we found several articles that, making other approaches, but evaluating the same and other variables, also found improvements until the short term38. These findings are in part supported by the results of a study that found that pain relief correlated with LTR, but only when pain intensity was high39.One of the initial studies comparing DN versus lidocaine infiltration found different results to ours, showing that mechanical hyperalgesia, pain intensity and range of motion improved more in the group that presented local twitch responses than the group that did not42. This study showed a significant effect for time but not statistically significant interaction between group and time were founded for changes in PPT in the upper trapezius. On the other hand the DDN active group obtains a lesser improvement in PPT in the tibialis muscle, compared to performing DDN in latent trigger points or outside of trigger points in patients with neck pain. It has relation with other previous studies where researchers showed that PPT levels increase when DDN is done in an active MTrP40 and it could be a greater increase indeed after 2 days of the intervention43. The results obtained from mechanical hyperalgesia are in accordance with the conclusions of a recent systematic review which concluded that dry needling produces an increase in the pain threshold to pressure44. In a previous study by Llamas-Ramos et al. found that 2 sessions of dry needling produced a 73% increase in the pain threshold to pressure which was maintained for 2 weeks during follow-up45 and in a study of Pecos-Martin et al. the research found that dry needling on the active trigger point resulted in an increase in PPT of 53.5%, whereas dry needling outside the trigger point did not produce the same increase17. One limitation that we have found in our study in relation to the PPT in the tibialis anterior muscle is that in the group of DN in latent MTrP group the PPT was already higher than the rest of the groups and that is what may have influenced the statistically significant differences between the groups and therefore cannot be taken into account, although in reality what we have reported is the intra-group analysis, and in which we have seen that the group of DN in active MTrP group is the one that obtained the least change over time, regardless of whether that group had a higher or lower pain threshold in the tibialis anterior muscle. Pain intensity difference at 1 week after DN versus baseline was predicted by LTRs and side treated with a R2 coefficient of 0.157, indicating a significant prediction model but suggestion a low percentage of prediction. Lower pain intensity difference at 1 week after DN was predicted by lower production of LTRs during the DN procedure in accordance with a recent systematic review and meta-analysis suggesting a low level of evidence for immediate effects of obtaining LTRs during DN interventions on pain intensity46.In relation with the pain intensity results, there is a greater soreness decrease after 1 week and 1 month, especially in the active MTrP group, as has been observed in previous studies, even over 2 points on the VAS scale when needling is done in the upper trapezius muscle47. A possible explanation why other studies have found changes with a greater clinical effect may be due to the number of sessions or in the case of Pecos-Martin, because the dry needling was performed on the lower trapezius muscle despite only one session17. Lower PPT over tibialis anterior muscle after DN with respect to baseline was predicted by the presence of bleeding after DN and the place of DN with a R2 coefficient of 0.218, determining a significant prediction model although representing a low percentage of prediction. Bleeding was reported to be the most common minor adverse effect presented in the 16% of dry needling procedures48 but, to the authors’ knowledge, our study may be considered as the first trial claiming that the presence of bleeding may predict a lower PPT outside of the painful region after DN in patients with chronic neck pain.Cerezo Tellez et al. found a clinically relevant improvement of 4 points in PPT that was maintained for 6 months. In their research, they applied 3 sessions of dry needling in patients with neck pain. In our study we only obtained an improvement of 4.87%24.The Neck Disability Index showed that all groups improved equally; therefore we can say that for the improvement of disability it does not matter where you perform the dry needling. The three groups have an improvement after 1 week and 1 month. This results are comparable to other studies where no differences were found between groups23. In our study, we found that there is a statistically significant association in relation to the greater the number of LTRs, the more frequently the symptoms of the patients are reproduced during the dry needling intervention (P < 0.05), although, on the other hand, we did not found an association that the more LTRs we achieved a greater clinical improvement. We have also obtained a greater number of LTRs when performing DDN on active and latent MTrPs, but that is not related to a major clinical improvement either. Other research also shown that the production of one or multiple LTRs during muscle MTrP DDN appeared to have a poor short-term correlation with pain and disability outcomes in patients’ neck pain49. The LTR during dry needling may be clinically relevant, but should not be considered necessary for successful treatment33. Relative to long-term interventions, DDN without causing LTRs has superiority over DDN with provocation of LTRs7. A greater association of a higher number of LTRs was found in the active and latent MTrP DN groups (P < 0.0001), than in the non-MTrPs group. The association was also found between the site where the dry needling was performed and the provocation of neck pain in the patients, finding a higher percentage of subjects in whom their neck pain was reproduced when the dry needling was applied to the active (77.3%) and latent (81.8%) (P < 0.001) MTrP. This finding is related to one of the recommended clinical criteria to identify active and latent MTrP, which is the appearance of LTR, in this case, during the intervention10. However, there was no model in predicting that producing your neck pain during dry needling would produce a greater improvement in any of the measured variables.So far, there are still studies showing that obtaining a LTR produces more immediate and lasting pain relief than not inducing LTR, as in a study where more patients were found with improvements in neck pain greater than those moderate clinically important differences observed when obtaining 6 LTRs and LTRs to exhaustion compared to not eliciting LTRs51 the formation of the MTrP and the taut band (TB) is controlled by the central nervous system through a “MTrP circuit” and, thus, stimulation with a needle can transmit a strong signal to the central nervous system to induce the powerful reflex of the LTR that will help to reorganize the control that the central nervous system exercises over the MTrP and the tight band, breaking the vicious circle of the “MTrP circuit”. However, some recent study’s findings show that needling the MTrP of the soleus muscle specifically decreased stiffness measured at the MTrP but without any changes at an adjacent point within the same TB. However, when the TB was needled, no changes were observed in the TB or the MTrP. Such findings emphasize the importance of needling precision into the MTrP area to modify myofascial mechanical properties, although real effectiveness and effect are unknown in a clinical context52.According to HongThe rest of the mechanisms could explain the decrease in pain and the elevation of the pressure pain threshold that is usually observed in studies with DDN techniques as well as this current study. However, the known capacity of the autonomic nervous system to produce electromyographic changes in the MTrPs supposes of a possible action on the etiopathogenesis of MTrPs, which could justify motor effects, beyond the purely sensitive ones.For all these reasons, it is essential to expand knowledge in basic research in order to continue making new findings in the experimental and clinical field that make us take not only a step, but also a leap in improving the treatment of our patients.32. Nevertheless, future studies should consider reliable and useful measurements such as the temporal summation and conditioned pain modulation53.The current study has some limitations. We suggest that this study could be done on a larger sample size to provide more insight regarding the variables effects. The second is that we assess and treat the upper trapezius muscle in a single session, although other muscles may be involved in neck pain. Furthermore, the trapezius is one of the most intolerant to puncture and this prevents the results obtained from being extrapolated to dry needling in other muscles. Third, our study measured short-term effects; therefore, future studies should investigate the medium and long-term effects of deep dry needling by performing several treatment sessions on various muscles or by adding different locations of the same. The last possible limitation is the reliability of the identification of trigger points, as there is still a lot of controversy in the diagnosis and this may contribute to the fact that we do not find differences between the different groups of trigger points. Finally, PPT expansion was added in the present study in order to determine mechanosensitivity of both local and remote regions with respect the painful region, supposing that the summation may be a reliable measurement as the sum of PPT measurements of the upper trapezius and tibialis anterior musclesThe present study offers an idea of how to manage better the treatment of MTrPs using the DDN technique. Knowing a little more about the behavior of pain in relation to the place where we perform the technique, we can address the problems of our patients with more efficient treatments.According to this study, patients have grater improvement if the physiotherapist applies the DDN technique in an active MTrP in relation with pain intensity but only for the first week, but not for a month, which means that in the short term it doesn't matter where you do the dry needling.A greater contribution is necessary in this line of work in which the sample of participants is increased, as well as the number of sessions to be carried out, the muscles treated and a longer-term follow-up.Deep dry needling of the trapezius muscle, regardless of whether it is on a trigger point, latent or non-trigger point area, produces the same positive effects in improving pain intensity, discomfort and local mechanical hyperalgesia. In terms of mechanical hyperalgesia at a distance , the group with dry needling on the active trigger point had the least improvement. There is no association between the number of LTR and improvement and reproducing the patient’s pain during dry needling with the improvement of the patient.It seems that precise location is not necessary when applying dry needling in clinical practice, but more studies need to be carried out with larger sample sizes, applying more sessions and with longer follow-ups.


Oral food challenge using gluten and cofactors is the gold standard to diagnose wheat‐dependent exercise‐induced anaphylaxis (WDEIA), but this procedure puts patients at risk of an anaphylactic reaction. Specific IgE to ω5‐gliadins as major allergens and skin prick tests to wheat may yield negative results. Thus, we designed a proof‐of‐principle study to investigate the utility of the basophil activation test (BAT) for WDEIA diagnosis.+ basophils, the ratio of %CD63+ basophils induced by GPT/gluten to %CD63+ basophils induced by anti‐FcεRI antibody, area under the dose‐response curve and test sensitivity and specificity.Different gluten protein types  and gluten were used in different concentrations to measure basophil activation in 12 challenge‐confirmed WDEIA patients and 10 control subjects. The results were compared to routine allergy diagnostics. Parameters analyzed include the percentage of CD63+ basophils and for %CD63+/anti‐FcɛRI ratio in a dose‐dependent manner in patients, but not in controls . BAT performance differed from acceptable (0.73 for LMW‐GS) to excellent (0.91 for ω5‐gliadins) depending on the specific GPT as evaluated by the area under the receiver operating characteristic curve. Patients showed individual sensitization profiles. After determination of the best cut‐off points, ω5‐gliadins and HMW‐GS showed the best discrimination between patients and controls with a sensitivity/specificity of 100/70 and 75/100, respectively.GPT and gluten induced strong basophil activation for %CD63+ basophil values for ω5‐gliadins and HMW‐GS.This study shows the alternative role of BAT in better defining WDEIA and the causative wheat allergens. The best BAT parameters to distinguish WDEIA patients from controls were %CD63 All GPT together constitute gluten, the storage proteins in wheat flour.WDEIA diagnosis is challenging, because of the variety of possible allergenic wheat proteins and the combination with a cofactor. Skin prick tests (SPTs) and specific IgE (sIgE) to wheat may be negative. Even sIgE to ω5‐gliadins are only positive in about 80% of WDEIA patients, indicating that other GPT also play a role in WDEIA. Wheat product and exercise challenge failed to induce symptoms in the majority of patients despite a clear history.A new approach to complement WDEIA diagnosis is the basophil activation test (BAT) combined with florescence‐activated cell sorting. Stimulation with an allergen‐containing solution  induces upregulation of the expression of cell surface proteins, such as CD63 or CD203c. BAT‐derived parameters such as the percentage of basophils that respond to a given dose of the ATS or the area under the curve (AUC) of a dose‐response curve have been shown to be sensitive biomarkers corresponding to the clinical severity of anaphylactic reactions.The aim of this proof‐of‐principle study was to investigate the utility of the BAT to improve WDEIA diagnosis and to determine individual sensitization profiles in WDEIA patients to different isolated and well‐characterized GPT and gluten.22.1The following exclusion criteria were considered in the selection of participants to avoid potential confounding factors and/or health risk to any of the participants: Pregnancy/lactation; systemic intake of corticosteroids (cortisone) 3 weeks and/or antihistamines (anti‐pruritic drugs) 1 week before the start of the test; intake of laxatives, anti‐diarrhea drugs, thyroid hormone preparations, antibiotics, immunosuppressive drugs, analgesic drugs  taking psychotropic drugs and certain blood pressure medications ; serious internal diseases ; bronchial asthma.A total of 23 participants were consecutively recruited from the medical center . Twelve of them were patients with a history of WDEIA based on positive oral food challenge, SPT, sIgE, and clinical history  was used as positive and isotonic sodium chloride solution (Fresenius Kabi Deutschland GmbH) as negative control.2.3Dermatophagoides pteronyssinus (d1), timothy grass (g6), birch pollen allergen , wheat flour (f4), rye flour (f5), gluten (f79), gliadin (f98), ω5‐gliadin , and lipid‐transfer protein .Serum sIgE and total IgE levels were measured by ImmunoCap and Phadia 250 (Thermo Fisher Scientific). Serum sIgE levels of the following allergens were determined: 2.4Gliadins were extracted from wheat gluten using 60% aqueous ethanol. After dialysis and lyophilization, the gliadin fraction was separated into ω5‐, ω1,2‐, α‐, and γ‐gliadins by preparative reversed‐phase high‐performance liquid‐chromatography. The glutenins were extracted from the residue after gliadin removal using 50% aqueous propanol, 60°C and reducing conditions. The HMW‐GS and LMW‐GS were obtained by sequential precipitation with 40% and 80% acetone, respectively.GPT or gluten (15 mg) and 0.6 ml pepsin solution  were incubated for 120 min at 37°C. The digest was stopped by adjusting the pH value to 7.0 with sodium hydrogen carbonate solution (50 mg/ml). The solution was filtered (0.45 μm) and the protein/peptide concentrations were measured at 205 nm by a micro volume UV/VIS spectrophotometer NanoDrop One (Thermo Fisher Scientific). If necessary, the sample solution was diluted with water to a concentration of 4 mg/ml. Further dilutions were made, to receive the following concentrations: 2.0, 0.8, 0.4, and 0.08 mg/ml. A pepsin‐control was prepared in the same way, but without gluten proteins. ATS were prepared and stored at −20°C in aliquots until use in BAT.2.5g for 5 min. The supernatant was decanted and the residue was resuspended in 200 μl of wash buffer by gentle mixing. Highly specific anti‐FcεRI mAb and N‐formyl‐methionyl‐leucyl‐phenylalanine were used as positive controls. To determine the background value, stimulation buffer alone was used. The flow cytometric analysis was performed using a FACSCalibur system (Becton‐Dickinson Immunocytometry System) with a 488 nm, 15 mW and a 635 nm, 10 mW argon laser. Basophils were gated as low side scatter CCR3/side scatterlow. CCR3 was used to identify basophils and CD63 as basophil activation marker, both marked with fluorescence‐dye‐labeled mAb. BD CellQuest (Becton‐Dickinson Immunocytometry System) was used for data analysis. In each measurement, ≥450 basophil granulocytes (BG) were counted. The upregulation of the basophil activation marker CD63 by the tested ATS reflects the induced basophil activation.For quantitative determination of in vitro basophil activation, Flow CAST (Buehlmann Laboratories AG) was used, as described previously.2.6+ basophils) was calculated by the percentage of CD63‐expressing BG relative to the total number of counted BG in each measurement. The %CD63+ basophils/anti‐FcεRI ratio is defined as the quotient of the maximum percentage of activated %CD63+ basophils, induced by an IgE‐dependent stimulus, and the percentage of activated basophils triggered by the anti‐FcεRI mAb as positive control.The basophil activation , diameter of wheals and erythema in SPT, severity  and sIgE values were analyzed using Spearman's correlation test.Statistical analysis was performed with SigmaPlot 14 (Systat Software GmbH) and Origin 19 (OriginLab Corporation). Statistical significance was tested by one‐way ANOVA and Dunn's post hoc test. Receiver operating characteristic (ROC) analyses were carried out to estimate the discriminatory ability of the investigated parameters. Therefore, the area under the ROC curve, was used as further characteristic. The optimized cut‐off of basophil activation (%) for best selectivity and specificity was determined from the ROC curve. Correlations between BAT results  with a clinical history of WDEIA and positive challenge test were included in the study Table . The con3.2p > 0.05).A SPT is classified as positive when the diameter of the wheal, caused by the test substance, is greater or equal than the diameter of the wheal of the negative control with 3 mm added. Patients did not show wheals for the negative control, and they all showed a distinct allergic reaction to the positive control. There were positive responses to gluten and gliadins in SPT in all patients Table . In caseDermatophagoides pteronyssinus, timothy grass, wheat flour, rye flour, gluten gliadins, and ω5‐gliadins in patients compared to controls (p < 0.05), respectively. No significant differences between patients and controls were found for sIgE against birch pollen allergen (Bet v 1) and lipid‐transfer protein (Tri a14) (p > 0.05). For details see Tables Significantly higher values were found for total IgE and sIgE against en Bet v  and lipi3.3+ basophils, %CD63+/anti‐FcɛRI ratio and AUC of dose‐response curves. The ATS made from gluten and GPT induced basophil activations in patients with WDEIA. The basophil activation was dose‐dependent up to a maximum of 71.3 %CD63+ basophils in case of ω5‐gliadins, 61.5% for gluten, 59.8% for LMW‐GS, 53.7% for γ‐gliadins, 50.7% for HMW‐GS, 49.2% for α‐gliadins, and 37.3% for ω1,2‐gliadins. Significant differences between patients and controls were found for each ATS at every concentration tested (p < 0.001)  and controls . The %CD63+/anti‐FcɛRI ratio was significantly higher for gluten and all GPT for patients compared with controls at most concentrations (p < 0.001)  and all tested doses in one. The AUC of ω5‐gliadins, α‐gliadins, and HMW‐GS were significantly higher in patients compared to controls (p < 0.001), but there were no significant differences for ω1,2‐gliadins, γ‐gliadins, LMW‐GS, and gluten (p > 0.05). For patients, median AUC values were 56.5 (range: 3.8–232.2) for ω5‐gliadins, 34.7 (range: 0.4–163.7) for α‐gliadins, and 24.0 (range: 3.7–102.9) for HMW‐GS. Table The induced allergenic response to the ATS was evaluated by three parameters used in BAT: %CD63) Figure . The dos3.4+ basophils as characteristic, for ω5‐gliadins (0.908), HMW‐GS (0.867), and gluten (0.850)  for %CD63+ basophils, when a basophil activation is classified as “allergen response” to a ATS, was determined for best sensitivity and specificity of concentration‐independent ROC curves  present in a soap in Japan. Significant enhancement of CD203c expression was observed with ω5‐gliadins in patients with classical WDEIA and with HWP_A in patients sensitized by the soap, but not vice versa.CD63 and CD203c are both in use as activation markers in BAT, while CD63 is the most common one. The upregulation of CD63 is closely associated with basophil degranulation induced by allergen stimuli.In BAT, only water‐soluble allergen solutions can be tested in patient's blood. Chinuki et al. used aqueous, ethanolic, and alkaline extractions to generate ATS for BAT, but without further protein characterization.In the present study, a well‐characterized representative gluten sample was used to isolate single GPT, α‐, γ‐, ω1,2‐, and ω5‐gliadins as well as HMW‐ and LMW‐GS. Detailed information about the basophil activation in patients to GPT were obtained.+ basophils and the %CD63+/anti‐FcεRI ratio after incubation with milk protein for prediction of the outcome of an oral challenge test. They reported a significant correlation between the %CD63+/anti‐FcεRI ratio and the outcome of the oral challenge test, depending on the ingested dose and reaction severity in patients with food allergy.The highly specific anti‐FcεRI mAb that imitates bridging of the receptor by an allergen has been used as a positive control in BAT for numerous years. Rubio et al. analyzed %CD63+/anti‐FcεRI ratio and the reaction severity during oral food challenge to peanuts.+ basophils, %CD63+/anti‐FcεRI ratio), diameter of wheals and erythema in SPT, severity of symptoms  and sIgE values  in our study.In addition, Santos et al. found a correlation between the %CD63+ basophils) and %CD63+/anti‐FcεRI ratio were significantly higher in patients for all tested GPT and gluten in most concentrations compared to controls. Additionally, a helpful characteristic is the AUC of the dose‐response curve, which combines basophil activation and sensitivity. In our study, the AUC of dose‐response curves of ω5‐ and α‐gliadins and HMW‐GS were significantly higher in patients compared to controls.Basophil activation , HMW‐GS (AUC ROC: 0.867) and gluten (AUC ROC: 0.850). Sensitivity and specificity of basophil activation to these substances at optimal cut‐off in WDEIA patients as compared to atopic and nonatopic control subjects were good for ω5‐gliadins , HMW‐GS , and gluten  respectively. The maximum %CD63+ basophils, concentration 4.0 mg/ml).Matsuo et al. recommended to determine sIgE against epitopes of ω5‐gliadins and HMW‐GS in combination for WDEIA diagnosis.One limitation of our study is the comparatively small number of WDEIA patients and controls. Due to the very low prevalence of WDEIA overall, a single‐center study such as ours can only include a certain number of individuals from the surrounding area. Our main intent was to identify the most suitable ATS for use in BAT from the panel of different gluten and GPT preparations tested. Now that we have identified ω5‐gliadins, HMW‐GS and gluten as most promising ATS, further work with more WDEIA patients from multiple centers is needed to put the cut‐off levels on a broader basis and include WDEIA patients with negative SPT, WDEIA patients with positive SPT, but positive challenge only with cofactors as well as individuals who are sensitized (wheat IgE‐positive), but clinically tolerant as proven by oral challenge.+ basophils as best parameter to discriminate between patients and controls and highlight the allergenicity particularly of ω5‐gliadins and HMW‐GS for WDEIA.According to the results of the proof‐of‐principle study we showed the potential of the BAT as alternative to routine SPT and sIgE measurements in WDEIA diagnosis. The BAT turned out to be promising to study the allergenicity of different GPTs, which becomes only possible after special preparation to increase water solubility, as required for BAT. Our findings indicate the use of %CD63B. Eberlein received methodological and technical support from Buehlmann Laboratories AG . The other authors declare no competing interests.Conceptualization: Angelika Miriam Gabler, Julia Gebhard, Bernadette Eberlein, Tilo Biedermann, Katharina Anne Scherf, and Knut Brockow; Formal analysis, investigation, and methodology: Angelika Miriam Gabler and Julia Gebhard; Data curation: Angelika Miriam Gabler, Julia Gebhard and Bernadette Eberlein; Funding acquisition, resources, and Supervision: Bernadette Eberlein, Tilo Biedermann, Katharina Anne Scherf, and Knut Brockow; Visualization: Angelika Miriam Gabler; Writing—original draft: Angelika Miriam Gabler; Writing—review & editing: Julia Gebhard, Bernadette Eberlein, Tilo Biedermann, Katharina Anne Scherf, and Knut Brockow. All authors read and approved the final manuscript.Supporting Information 1Click here for additional data file.