Patients Receiving Botulinum Toxin Type Research Articles

Conversion to AbobotulinumtoxinA Increases Waning Time and Efficacy for Cervical Dystonia.

Symptom re-emergence before re-injection negatively impacts cervical dystonia (CD) patients receiving botulinum toxin type A (BoNT-A) therapy. Longer waning time is associated with abobotulinumtoxinA (abo-BoNT-A) as compared to onabotulinumtoxinA (ona-BoNT-A)/incobotulinumtoxinA (inco-BoNT-A) formulations. To compare waning time and treatment outcomes when chronically injected CD patients experiencing early waning despite being optimized on BoNT-A (ona-BoNT-A/inco-BoNT-A) were converted to abo-BoNT-A. Thirty-three chronically injected CD participants with a waning time of ≤8 weeks were converted to abo-BoNT-A (1:2.5 dose ratio) for three injections every 12-weeks. The second and third injection patterns were kinematically optimized. Participants were converted back to their original BoNT-A for the fourth injection (1:2.5) using the same third abo-BoNT-A pattern. Participant-perceived waning times were collected post-injections. Clinical scales (Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS)) and kinematic measures were collected 12-weeks post-injection and at three peak effect time-points. Compared to baseline, waning time (12-22 days) significantly increased following all abo-BoNT-A treatments (P < 0.005) but was not significantly different at the fourth injection (original BoNT-A reconversion). TWSTRS sub-scores significantly reduced following all abo-BoNT-A treatments (P < 0.0001) and at peak effect following the third injection compared to original BoNT-A. Dysphagia and muscle weakness were reported and comparable to safety of original BoNT-A formulations. Optimized patients experiencing waning had significant improvement in the peak benefit as well as the duration of effect when converted to abo-BoNT-A. This effect was toxin dependent as reconversion to the original BoNT-A using the kinematically optimized pattern failed to produce an improvement in waning.

A cross-sectional structured survey of patients receiving botulinum toxin type A treatment for blepharospasm

To characterize satisfaction with current standard-of-care botulinum neurotoxin type A (BoNT/A) treatment for blepharospasm, we performed a cross-sectional, structured survey in subjects with blepharospasm who had received ≥2 BoNT/A cycles. Subjects were interviewed immediately before re-injection to evaluate treatment satisfaction, time course of treatment effects, preferred injection intervals, Jankovic Rating Scale (JRS), and Blepharospasm Disability Index (BSDI).Subjects’ (n=114) last treatment was onabotulinumtoxinA (n=78), incobotulinumtoxinA (n=35), or abobotulinumtoxinA (n=1). The most frequent injection interval was 12weeks (46.5% subjects); 30.7% had an interval >12weeks. The main rationale for interval choice was “to maintain treatment efficacy” (44.7%). However, 36.6% reported that treatment effects usually declined within 8weeks; 69.6% within 10weeks. JRS and BSDI scores indicated re-emergence of symptoms before re-injection, with 70.2% and 73.7% of subjects reporting difficulties to drive and read, respectively. Overall, treatment satisfaction was high, but declined at the end of the cycle. Many subjects (52.3%) would prefer an injection interval of <12weeks; 30.6% of <10weeks.In conclusion, the survey results indicate that blepharospasm symptoms, such as difficulties to drive and read, re-emerge at the end of a BoNT treatment cycle and that flexible, individualized treatment intervals may improve treatment satisfaction and outcomes.

A comparison of outcome of medical and surgical treatment of migraine headache: In 1 year follow-up.

Background:This study was designed to compare the efficacy of the medical treatment versus the surgical treatment approach to decompression of trigger point nerves in patients with migraine headaches.Materials and Methods:Fifty volunteers were randomly assigned to the medical treatment group (n = 25) or the surgical treatment group (n = 25) after examination by the team neurologist to ensure a diagnosis of migraine headache. All patients received botulinum toxin type A to confirm the trigger sites. The surgical treatment group underwent surgical deactivation of the trigger site(s). The medical treatment group underwent prophylactic pharmacologic interventions by the neurologist. Pretreatment and 12-month posttreatment migraine headache frequency, duration, and intensity were analyzed and compared to determine the success of the treatments.Results:Nineteen of the 25 patients (76%) in the surgical treatment group and 10 of the 25 patients (40%) in the medical treatment group experienced a successful outcome (at least a 50% decrease in migraine frequency, duration, or intensity) after 1 year from surgery. Surgical treatment had a significantly higher success rate than medical treatment (P < 0.001). Nine patients (36%) in the surgical treatment group and one patient (4%) in the medical treatment group experienced cessation of migraine headaches. The elimination rate was significantly higher in the surgical treatment group than in the medical treatment group (P < 0.001).Conclusions:Based on the 1-year follow-up data, there is strong evidence that surgical manipulation of one or more migraine trigger sites can successfully eliminate or reduce the frequency, duration, and intensity of migraine headaches in a lasting manner.

A cross-sectional structured survey of patients receiving botulinum toxin type A treatment for blepharospasm

A cross-sectional structured survey of patients receiving botulinum toxin type A treatment for blepharospasm

Quality of life assessment in facial palsy: validation of the Dutch Facial Clinimetric Evaluation Scale.

This study aimed at validating an existing health-related quality of life questionnaire for patients with facial palsy for implementation in the Dutch language and culture. The Facial Clinimetric Evaluation Scale was translated into the Dutch language using a forward–backward translation method. A pilot test with the translated questionnaire was performed in 10 patients with facial palsy and 10 normal subjects. Finally, cross-cultural adaption was accomplished at our outpatient clinic for facial palsy. Analyses for internal consistency, test–retest reliability, construct validity and responsiveness were performed. Ninety-three patients completed the Dutch Facial Clinimetric Evaluation Scale, the Dutch Facial Disability Index, and the Dutch Short Form (36) Health Survey. Cronbach’s α, representing internal consistency, was 0.800. Test–retest reliability was shown by an intraclass correlation coefficient of 0.737. Correlations with the House–Brackmann score, Sunnybrook score, Facial Disability Index physical function, and social/well-being function were −0.292, 0.570, 0.713, and 0.575, respectively. The SF-36 domains correlate best with the FaCE social function domain, with the strongest correlation between the both social function domains (r = 0.576). The FaCE score did statistically significantly increase in 35 patients receiving botulinum toxin type A (P = 0.042, Student t test). The domains ‘facial comfort’ and ‘social function’ improved statistically significantly as well (P = 0.022 and P = 0.046, respectively, Student t-test). The Dutch Facial Clinimetric Evaluation Scale shows good psychometric values and can be implemented in the management of Dutch-speaking patients with facial palsy in the Netherlands. Translation of the instrument into other languages may lead to widespread use, making evaluation and comparison possible among different providers.

Nurses' views about caring for patients receiving botulinum toxin type A for focal spasticity

Background Few research studies have explored the role of nurses in caring for patients receiving botulinum toxin type A (BTX-A) injections for focal spasticity. This lack of research provided the impetus for the present study, which was carried out in a rehabilitation centre. Aim The aim of the study was to explore and describe nurses' views about their role in caring for patients treated with BTX-A for focal spasticity. Design A qualitative descriptive design was used. Methods Data were collected using focus groups, and volunteer sampling was used to recruit 12 nurses who met the inclusion criteria. Results Thematic analysis illustrated that although the participants felt nurses play an important part in caring for patients receiving BTX-A treatment, clarification is required on the role of the nurse, the efficacy of the nursing role, and the education and training required. Conclusion Although the literature has reported that nurses have an important role in the multidisciplinary team caring for patients receiving BTX-A treatment, it has not specifically identified what that role is. In this study, nurses reported that, although they feel they have an important role in caring for these patients, there is ambiguity about the specifics of that role. Furthermore, the participants felt that further education and training were important for preparing nurses to care for these patients adequately. Relevance to clinical practice In view of the relative newness of BTX-A in the treatment of focal spasticity and the paucity of research about the role of the nurse in caring for patients, the findings from this study make a contribution to evidence-based practice by raising awareness about the role of the nurse.

Uso de toxina botulínica en neurología: Experiencia en el Hospital Guillermo Almenara Irigoyen, EsSalud

Objetivo: Identificar las principales utilidades de la toxina botulínica en las enfermedades neurológicas. Lugar: Hospital Nacional Guillermo Almenara Irigoyen, EsSalud, Lima,Perú. Periodo 2001-2003. Material y Métodos: Se estudió 122 pacientes a quienes se les infiltró toxina botulínica tipo A a nivel de los músculos comprometidos, según el tipo de patología. Se les hizo una evaluación posterior a la aplicación de la toxina. Resultados: El espasmo hemifacial fue el diagnóstico más frecuente de uso (63,1%) seguido de la espasticidad (16,4%) y distonía cervical (11,5%). El espasmo hemifacial se presentó con más frecuencia entre los 60 y 69 años y en el lado izquierdo. En la distonía cervical, el tipo más frecuente de uso fue en la tortícolis y laterocolis. La evolución fue favorable, según el puntaje de la escala de TWSTRS aplicada antes y 3 meses después del tratamiento. Los efectos secundarios encontrados con más frecuencia fueron el ojo seco (15,6%), equimosis (9,1%) y dolor en el sitio de aplicación (7,8%), de naturaleza reversible. Conclusión: La toxina botulínica tipo A tiene múltiples aplicaciones en las enfermedades neurológicas, con buen resultado y pocos efectos secundarios.

Eficácia do resfriamento da pele no alívio da dor desencadeada pela injeção de toxina botulínica tipo A nas distonias faciais

OBJETIVO: Avaliar a eficacia do resfriamento da pele com gelo no alivio da dor desencadeada pela injecao de toxina botulinica tipo A na regiao periocular em pacientes portadores de distonia facial. METODOS: Neste estudo prospectivo, 13 pacientes receberam injecao de toxina botulinica tipo A em regiao glabelar (m. procero) e periocular (m. orbicular) para tratamento de distonia facial. Antes das aplicacoes, um lado da regiao glabelar foi resfriado com gelo durante 5 minutos, enquanto no outro lado foi aplicada pomada Epitezan®, funcionando como placebo. A aplicacao foi feita primeiramente no lado resfriado. Apos a aplicacao em cada um dos lados os pacientes foram instruidos a dar uma nota para a dor desencadeada pela injecao, em uma escala de 0 a 10 onde 0 era ausencia de dor e 10 a dor mais intensa. RESULTADOS: A media das notas dadas pelos pacientes a dor desencadeada pela injecao no lado onde foi aplicado placebo foi 3,92 ± 3,28. No local onde foi aplicado gelo a media das notas foi de 2,92 ± 2,18 (p < 0,0166). A media da diferenca entre notas dos dois grupos foi de 1,0. CONCLUSAO: No presente estudo o resfriamento da pele com gelo foi eficaz no alivio da dor desencadeada pela aplicacao de toxina botulinica tipo A na regiao periocular em pacientes portadores de distonia facial.

Effectiveness of Switching Therapy from Complexing Protein-containing Botulinum Toxin Type A to a Formulation with Low Immunogenicity in Spasticity after Stroke: A case report

Some patients receiving botulinum toxin type A therapy develop immunological resistance due to the production of neutralizing antibodies against the neurotoxin, thus partially or completely reducing the therapeutic effect. We report here neurophysiological and clinical findings for a 58-year-old man treated with botulinum toxin type A for spasticity after ischaemic stroke, who became a secondary non-responder patient. Subsequent treatment with a different preparation of botulinum toxin type A had a great therapeutic effect on his spasticity. The muscles injected and the dosages were the same for each treatment, but evaluation with the Modified Ashworth Scale after treatment with the second preparation showed a reduction of approximately 2 points compared with the first examination. The clinical results were also supported by extensor digitorum brevis testing of the right muscle, which showed a reduction in compound muscle action potential, whereas it was unchanged in the non-injected muscle. No side-effects were reported, and after 1 year of treatment with this formulation clinical benefits were still evident. The neurophysiological and clinical results obtained in this patient suggest that switching therapy from a complexing protein-containing product to a product potentially free of complexing proteins, which has low immunogenicity, may be a viable therapeutic option in secondary non-responder patients.

A Simplified Technique for Botulinum Toxin Injections in Children With Neurogenic Bladder

Botulinum toxin type A has revolutionized the treatment of neurogenic bladder dysfunction. The original injection technique used a rigid cystoscope and a flexible collagen needle. To date botulinum toxin type A injection techniques have not been standardized. We present our experience in pediatric patients using a new flexible injection system. We treated 24 patients 3.8 to 17.5 years old who had neurogenic bladder dysfunction with botulinum toxin type A bladder and/or sphincter injection using a rigid cystoscope and the new N-DO™ endo-injector needle system. Another 24 patients 3.6 to 17.8 years old were treated with a 3.7Fr standard flexible needle and served as controls. Operative time, hospital stay, complications and efficacy were considered. Selection criteria and treatment were the same in the 2 groups. The 10 IU/kg dose was determined according to European Association of Urology guidelines. All patients received botulinum toxin type A bladder injection while 11 patients in the endo-injector group and 5 controls also received urethral injection. In the endo-injector needle and control groups average operative time was 12.4 and 17.3 minutes for the bladder, and 5.1 and 10.1 minutes for the urethra, respectively (each p <0.05). All patients were discharged home the day after the procedure. No complications were observed. Urodynamics revealed an average maximum detrusor pressure decrease of 25 and 21 cm H(2)O, and an average bladder capacity increase of 75 and 80 ml in the endo-injector and control groups, respectively (p not significant). While retaining efficacy, the endo-injector needle technique appears to be more rapid than the standard procedure for botulinum toxin type A injection for neurogenic bladder dysfunction. Whether patients may be treated with sedation only remains to be clarified.

Headache Prophylaxis With BoNTA: Patient Characteristics

To assess the characteristics of patients receiving botulinum toxin type A (BoNTA; BOTOX((R))) in the treatment of headache (HA) disorders. The following observational epidemiologic data and baseline patient characteristics were prospectively collected from eligible patients treated with BoNTA at 10 US HA specialty centers: demographics; HA diagnoses and characteristics (frequency, severity, and disability); prior and current HA treatments and response; clinical response to BoNTA; Migraine Disability Assessment (MIDAS) questionnaire; and adverse events. Patients maintained a daily HA diary and were evaluated at each follow-up visit. Of 703 patients enrolled (mean age 43.1 years, 78.5% females, 95.4% white), nearly 66% had a diagnosis of chronic migraine (CM), with or without medication overuse. Approximately 75% had severe disability (MIDAS grade IV), and the mean pain rating was 6.5 (where 0 = no pain, 10 = pain as bad as it can be). More than 90% of patients had >or=1 prophylactic HA treatment failure; median number of failures was 4. Significant association was observed between HA frequency and MIDAS grade (P < .001). Approximately 80% of patients with CM had severe (grade IV) disability. The median number of monthly medication days was higher in the group with MIDAS grade IV (P < .001). HA frequency and severity, failed prophylactic therapies, and greater number of coexisting medical conditions were all negatively associated with measures of health-related quality of life. Majority of patients treated with BoNTA in a specialty HA center presented with a CM diagnosis. HA disability was correlated with measures of frequency and treatment utilization.

The Use of Botulinum Toxin for Spasticity After Spinal Cord Injury

To describe the use and effects of botulinum toxin (BTX) injections in persons with spinal cord injury (SCI) and focal spasticity. Chart review of patients with SCI receiving their first injection of BTX for spasticity control at a freestanding urban rehabilitation hospital. Charts were reviewed for history and level of SCI, one of five self-identified goals (ambulation, positioning, upper-extremity function, hygiene, and pain control) before and after injection; site and doses of BTX used; and self-reported outcome on clinical follow-up. Charts of 28 adults receiving BTX were reviewed. All patients received BTX type A. Dosages of BTX ranged from 10 to 119 units per muscle. Improvement was noted for 56% in ambulation and 71% in positioning. Overall, upper-extremity function improved in 78%, hygiene improved in 66.6%, and pain decreased in 83.3%. Early use of BTX injections (less than a year after onset of symptoms) vs. late use of BTX injections did not influence effectiveness. BTX seems to be an effective treatment for focal spasticity and for reducing disability in persons with SCI. Randomized trials are needed to confirm the value of this treatment in the setting of SCI.

Early physiotherapy after injection of botulinum toxin increases the beneficial effects on spasticity in patients with multiple sclerosis

Objective : To determine whether additional physiotherapy increases botulinum toxin type A effects in reducing spasticity in patients with multiple sclerosis. Design : A single-blind, randomized, controlled pilot trial with a 12-week study period. Subjects : Thirty-eight patients with progressive multiple sclerosis affected by focal spasticity and who were observed at the Multiple Sclerosis Centre operating in the S. Andrea Hospital in Rome. Interventions : For intervention all patients received botulinum toxin type A; the treatment group also received additional physiotherapy to optimize management through passive or active exercise and stretching regimens. Main measures : To measure objective and subjective level of spasticity, patients were assessed at baseline, 2, 4 and 12 weeks post treatment by Modified Ashworth Scale and visual analogue scale. Results : When compared with the control group, we found a significant decrease of spasticity by Modified Ashworth Scale (P < 0.01 by t-test) in the treatment group at week 2 (2.73 versus 3.22), week 4 (2.64 versus 3.33) and week 12 (2.68 versus 3.33). The mean (%) difference in Modified Ashworth Scale score between baseline and the end of follow-up was —0.95 (26.1) in the treatment group and —0.28 (7.7) in the control group (P < 0.01). The combined treatment proved also to be more effective by visual analogue scale (P < 0.01) at week 4 (6.95 versus 5.50) and at week 12 (7.86 versus 6.56) but not at week 2 (5.18 versus 5.50; P = 0.41). Conclusions : Our data suggest that physiotherapy in combination with botulinum toxin type A injection can improve overall response to botulinum toxin.

Formation of neutralizing antibodies in patients receiving botulinum toxin type a for treatment of poststroke spasticity: A pooled-data analysis of three clinical trials

Objective: The purpose of this study was to investigate the incidence of neutralizing antibody (NAb) formation in patients with poststroke spasticity treated with a specific formulation of botulinum toxin type A (BoNTA). Methods: Data from 3 previous clinical trials of BoNTA in patients with upper and/or lower limb spasticity were pooled and evaluated. Study 1 was a randomized, double-blind, placebo-controlled, multicenter trial of BoNTA in patients aged ≥21 years who had experienced a stroke >6 months before the initiation of the study. Study 2 was an open-label extension of study 1. Study 3 was a randomized, double-blind, multicenter trial of a specific BoNTA formulation in patients aged ≥ 21 years who had experienced a stroke ≥6 weeks before study entry. Patients with a fixed contracture of the studied limb were excluded from participation in studies 1 and 2. Serum samples were obtained from each patient before each BoNTA treatment and at the end of each study. The mouse protection assay (MPA) was used for detection of NAbs to BoNTA in serum. Results: A total of 235 individual patients with post-stroke spasticity were enrolled in the 3 trials, including 126, 111 (all of whom participated in study 1), and 109 in studies 1, 2, and 3, respectively. Study 1 had an equal (50.0%) distribution of male and female patients (63/63). The distribution of male and female patients was 56 (50.5%) and 55 (49.5%), respectively, in study 2, and 55 (50.5%) and 54 (49.5), respectively, in study 3. The mean (SD) ages of patients in studies 1, 2, and 3 were 61.4 (13.8), 61.5 (14.1), and 58.5 (13.9) years, respectively. The MPA was used for detection of NAbs to BoNTA in the serum samples of 191 patients, including 64 from study 1, 111 from study 2 (55 of these patients were placebo recipients and 56 received their first BoNTA injection in study 1), and 72 (a sample was not obtained for 1 patient who had not received an injection) from study 3. The median number of BoNTA treatments received by these patients was 2 (range, 1–4 treatments) over a period lasting from 12 to 42 weeks. The mean dose of BoNTA was 241 U (range, 100–400 U), with a maximum dose of 960 U in any 1 patient. NAbs to BoNTA were detected in the serum sample of 1/191 (0.5%) patient who had participated in studies 1 and 2. Based on muscle-tone scores (3 and 4 for wrist and fingers, respectively) on a 5-point Ashworth Scale (0 = none to 4 = severe), the patient did not appear to exhibit a clinical response to BoNTA at any time during the studies. Conclusion: Formation of NAbs was rare (1/191) in this group of adults with poststroke spasticity from three 12- to 42-week clinical trials who received ≥1 treatment with a specific BoNTA formulation at doses ranging from 100 to 400 U.

Patient‐Reported Outcomes with Botulinum Toxin Type A Treatment of Glabellar Rhytids

Global patient-reported outcomes do not evaluate specific aspects of treatment that are important to patients. The objective was to evaluate self-perception of age and specific outcomes that are important to patients receiving botulinum toxin type A or placebo for moderate to severe glabellar lines (using the Facial Line Outcomes Questionnaire to assess how much facial lines bother them, make them look older, detract from their facial appearance, prevent a smooth facial appearance, and make them look tired, stressed, or angry). In the double-blind phase of this 12-week study, 70 patients were randomly assigned to treatment with 20 U botulinum toxin type A (BOTOX Cosmetic) or placebo. At Week 4, those still with moderate or severe glabellar lines were offered open-label 20 U botulinum toxin type A. Median glabellar line severity was significantly lower after botulinum toxin treatment than after placebo. Compared with placebo, botulinum toxin also resulted in significantly superior patient assessments and a greater proportion of patients considering they looked younger than their current age. Botulinum toxin type A can achieve specific goals of treatment that are important to patients and help them feel that they look younger than their current age.

Camptocormia

Originally considered a psychogenic disorder, camptocormia, an abnormal posture with marked flexion of thoracolumbar spine that abates in the recumbent position, is becoming an increasingly recognized feature of parkinsonian and dystonic disorders. Prior reports were limited by sample size, short follow-up, and paucity of data on response to therapy. The authors reviewed 16 patients evaluated in their PD Center and Movement Disorders Clinic diagnosed with camptocormia. In addition to detailed neurologic assessment all patients were videotaped. The mean age was 64.9 +/- 17.4 years, mean age at onset of neurologic symptoms was 51.5 +/- 19.9 years, duration from onset of neurologic symptoms to development of camptocormia was 6.7 +/- 7.6 years, and the mean duration of camptocormia was 4.5 +/- 3.9 years. Of the 16 patients, 11 (68.8%) had Parkinson disease (PD); others had dystonia (n = 4) and Tourette syndrome (n = 1). Twelve patients received levodopa, with minimal or no improvement in the camptocormia. Nine patients received botulinum toxin type A injections into the rectus abdominus, with notable improvement in their camptocormia in four. One patient underwent bilateral subthalamic nucleus deep brain stimulation for PD, but there was no improvement in camptocormia. Based on this series and a thorough review of the literature of camptocormia, head drop, and bent spine syndrome, the authors propose etiologic classification of camptocormia and conclude that this heterogeneous disorder has multiple etiologies and variable response to systemic and local therapies.

Temporary Management of Upper Lid Ptosis, Lid Malposition, and Eyelid Fissure Asymmetry with Botulinum Toxin Type A

During the past 10 years the primary focus for the aesthetic use of botulinum toxin has been directed to the treatment of dynamic facial lines. This agent has been shown to be very effective for the improvement of facial shape. The use of botulinum toxin type A for the correction of a variety of presentations of facial asymmetry has also been well established. The general principles regarding the counter-effects of facial muscle protagonists and antagonists and their potential effects on the position of facial soft-tissue regions apply here as well. Twenty-two patients received botulinum toxin type A for the temporary treatment of mild to moderate unilateral upper eyelid ptosis and aesthetic improvement of lower eyelid position, with favorable results. Although commonly related to a rare yet feared adverse consequence from the inappropriate application of botulinum toxin, its application for the treatment of upper eyelid ptosis, eyelid position, and other lid fissure asymmetries for aesthetic improvement is presented.

Improvement of headache symptoms and reduction in headache medication usage in patients treated with botulinum toxin type A

SummaryThe charts of 132 consecutive patients receiving botulinum toxin type A (BoNT-A; BOTOX®) for headache prevention were reviewed retrospectively. During a follow-up visit, patients reported their overall improvement in headache symptoms. Headache medication utilisation and costs for the 6 months before and during treatment were compared using the Average Wholesale Price. The mean BoNT-A dose used was 101.9 units and 131/132 patients (99%) reported some improvement in headache symptoms. Patients reported an average of 77% improvement in theirBOTOX is a registered trademark of Allergan Inc, Irvine, CA, USAheadache symptoms from baseline. BoNT-A treatment resulted in a 47.2% reduction in the use of headache medications. The average monthly cost of all oral headache medications declined from $242.7 to $89.2, a significant reduction of 63.3% (p < 0.001). A total of 122/132 patients (92.4%) reported no side effects. The cost of BoNT-A treatment appears to be offset to a large degree by the reduction in headache medication usage. Interpretation of these results is limited by the retrospective, uncontrolled and open-label design of the analysis.