Neurocognitive Deficit After Paediatric Transplantation: Understanding the Role of Environment and Physical Function

Scores on the clinical neurocognitive tests (Bayley-III: Bayley Scales of Infant and Toddler Development - Third Edition, WPPSY-IV: Wechsler Preschool and Primary Scale of Intelligence 2.6-7.7 years of age, WISC-V: Wechler Intelligence Scale for Children Fifth edition for participants 7-15.9 years of age, WAIS-IV: Wechler Adult Intelligence Scale Fourth edition for participants 16 years of age or older).

Tested domains: Non-verbal reasoning, verbal reasoning, Working memory, Processing Speed Scaled scores range from 0-19, higher = better performance.

Excecutive functioning will be tested using D-KEFS (Delis-Kaplan Executive Function System) for all participants >8 y of age.

Sustained attention will be tested using CPT-3 (Conners Continuous Performance Test, 3rd Edition) for for all participants >8 y of age

Visuomotor functions will be tested using Beery VMI (Beery-Buktenica Developmental Test of Visual-Motor Integration)

Verbal learning will be tested using TOMAL-2 >6 y of age

Isometric knee strength will be measured with a handheld dynamometer (CITEC handheld dynamometer, type 3002, CIT Technics, Haren, Netherlands). Maximum isometric contraction will be evaluated using the "break technique" (Beenakker et al, 2001). The highest value out of three repetitions will be counted as the maximum strength and kept for analysis. The handheld dynamometry method has shown high validity (r = 0.74 and r = 0.98) and moderate intertester reliability (r = 0.42 to r = 0.73) in healthy and chronically ill children and adolescents (Brussock CM et al. (1992)), (Stuberg WA et al (1988)) and is considered a reliable and valid instrument for muscle strength assessment in a clinical setting compared to the gold standard isokinetic dynamometry (Stark T et al (2011)).

Handgrip strength will be measured using a hand-held dynamometer. Participants are placed in a seated position with the elbow flexed at 90°, with three attempts performed for each hand. During testing, the participant will be encouraged to exhibit the best possible force, and the best measure in the strongest hand will be used as the test score. Handgrip strength is also used as a surrogate measure for upper-body physical function (Bohannon RW et al (2001)), Adam C et al (1988))

Sit-to-Stand 30 sec: The participant sits on a chair allowing the participant to flex the legs at a 90 degree angle. The participant is instructed to stand up straight, and return to sitting, as many times as possible in 30 sec. The arms should be crossed in front of the body or hanging by the side. Number of repititions is the outcome.

Sit-to-Stand 60 sec: The participant sits on a chair allowing the participant to flex the legs at a 90 degree angle. The participant is instructed to stand up straight, and return to sitting, as many times as possible in 60 sec. The arms should be crossed in front of the body or hanging by the side. Number of repititions is the outcome.

Participants will sit on a chair with both feet flat on the ground. Subsequently, participants will be instructed to stand up, walk 3 meters, turn around, walk back, and sit down as fast as possible. Each participant will perform the test three consecutive times and the fastest test will be used for the analyses. The timed-up-and-go test depends on lower body muscular strength and balance and reflects a person's ability to move in an everyday setting.

This four-condition test is designed to assess how well an individual is using sensory inputs to maintain balance, when one or more sensory systems are compromised. In condition one, all sensory systems (i.e., vision, somatosensory, and vestibular) are available for maintaining balance. In condition two, vision has been removed, and the participant must rely on the somatosensory and vestibular systems to balance. In condition three, the somatosensory system has been compromised, and the participant must use vision and the vestibular system to balance. In condition four, vision has been removed, and the somatosensory system has been compromised.

Each trial is timed using a stopwatch. The trial is over when (a) the participant opens his/her eyes in an eyes closed condition, (b) raises arms from sides, (c) loses balance and requires manual assistance to prevent a fall or (d) maintains balance for 30 seconds.

The pre-school children will be evaluated with the Peabody Developmental Motor Scales, Second Edition (PDMS-2) Darrah J, et al (2007).

The PDMS-2 is a validated instrument to assess fine and gross motor skills in children from birth to 5 years of age (0-71 months).

performed on an electrical-break ergometer bicycle (Lode Corival Pediatric or Monark Ergomedic 839 E) using a modified Godfrey protocol. The participant will be instructed to keep a steady pace (70-80 rpm), while the workload increases gradually with 10-25 watts pr. minute until exhaustion. Oxygen uptake (VO2), heart rate (HR), minute ventilation (VE), respiratory exchange ratio (RER), breathing frequency (BF), and tidal volume (TV) will be measured continuously during the test using a transportable a portable Cortex Metamax 3B wireless spirometry system (Cortex, Leipzig, Germany). The participant will breathe into a Hans Rudolph Valve (2-wat NRBV, Hans Rudolph Inc., Kansas City, MO, USA). Heart rate and oxygen saturation will be measured during the test. Furthermore, two objective criteria need to be fulfilled before the test is valid: 1) heart rate >85% of estimated maximal heart rate 2) respiratory exchange ratio (RER)>1.1. The primary outcome will be the peak oxygen uptake(V02max)

These parameters will be assessed by an accelerometer. The accelerometers (ActiGraph™ model GT3X+, ActiGraph LLC, Pensacola FL, USA) measure accelerations of ±6 G. The sample rate will be set to measure raw signals at 100 Hz, translated into metabolic energy equivalents of light, moderate, and vigorous physical activity and sedentary time. The validity of the Actigraph accelerometer is good, with correlations of 0.65 between accelerometer assessed metabolic energy equivalents and indirect calorimetry.

We will investigate whether international guidelines for physical activity are met (Troiano RP et al, 2008) according to established cut-off levels . Measurements during hospitalization will cover the waking hours of as many days as possible.

These parameters will be assessed by an accelerometer. The accelerometers (ActiGraph™ model GT3X+, ActiGraph LLC, Pensacola FL, USA) measure accelerations of ±6 G. The sample rate will be set to measure raw signals at 100 Hz, translated into metabolic energy equivalents of light, moderate, and vigorous physical activity and sedentary time. The validity of the Actigraph accelerometer is good, with correlations of 0.65 between accelerometer assessed metabolic energy equivalents and indirect calorimetry.

We will investigate whether international guidelines for physical activity are met (Troiano RP et al, 2008) according to established cut-off levels . Measurements will be conducted continuously for the waking hours for 7 consecutive days

The cumulative dose of morphine-equivalents during the transplantation will be retrieved from medical records.

The level of mucositis will be graded daily during the period of neutropenia by the nursing staff, and subsequently retrieved from patient files. The CTCAE classification is used: Grade 1: asymptomatic or mild mucositis; intervention not indicated. Grade 2: moderate pain or ulcer that does not interfere with oral intake; modified diet indicated. Grade 3: severe pain; interfering with oral intake. Grade 4: life-threatening consequences; urgent intervention indicated

• Inflammatory parameters including C-reactive protein levels will be retreived from patients' files

• The number of days until neutrophile recovery, defined as neutrophile count >0,5 for two consecutive days, will be registered from patient files.

• Time to immune reconstitution, defined as CD4 of 0.10 x 10(9)/L will be registered from the patient files.

• Information on Acute GvHD will be retrieved from patients' files.

The Inclusion of Nature in Self (INS) Scale is a validated single-item visual measure of children's connectedness to nature and is available in a Danish translation. Nature connectedness has been linked to better psychological well-being, lower distress, and improved emotional regulation, supporting its relevance as a protective factor. The INS can be used both as a baseline measure and to assess changes over time. Its simple visual format makes it particularly suitable for children. Children aged 8-18 years will complete the survey themselves, while parents/caregivers will respond for children aged 0-7 years

Nature Connectedness Index (NCI) is a brief, validated 6-item questionnaire designed to assess an individual's emotional and cognitive connection to the natural environment. The items capture aspects such as enjoyment of nature, feeling part of nature, and interest in engaging with the natural world. Responses are rated on a Likert scale, with higher scores indicating greater connectedness to nature. In this study, the NCI will be administered as a self-report for children and adolescents aged 8-18 years. For younger children aged 0-7 years, the questionnaire will be completed by a parent or primary caregiver on behalf of the child, based on their observations of the child's behaviors and attitudes toward nature.

The Children's People and Nature Survey (C-PANS) is a brief 6-item questionnaire designed to capture children's experiences with nature, including outdoor time, enjoyment, and environmental concern. Although formal validation is still underway, recent Danish reference data from a nationally representative sample provide a useful benchmark, making C-PANS relevant in a Danish context. In this study, children aged 8-18 years will complete the survey themselves, while parents/caregivers will respond for children aged 0-7 years. Two items overlap between the NCI and C-PANS; these will be asked only once.

The PedsQL 4.0 Generic Core Scales assess health-related quality of life in children across four domains: physical functioning, emotional functioning, social functioning, and school functioning. The instrument uses a 5-point Likert scale (0 = never a problem; 4 = almost always a problem) - lower score meaning better outcome.

The PedsQL Multidimensional Fatigue Scale assesses fatigue in children in three domains: general fatigue, sleep/rest fatigue, and cognitive fatigue. It uses the same 5-point Likert scale (0 = never a problem; 4 = almost always a problem) - lower score meaning better outcome.

The PROMIS® Cognitive Function Short Form 8a is an 8-item self-report questionnaire assessing perceived cognitive functioning in the past 7 days. It evaluates aspects such as memory, mental clarity, and concentration. Each item is scored on a 5-point Likert scale ranging from 1 ("very often") to 5 ("never") - higher scores indicate better subjective cognitive functioning.

The PROMIS® Physical Activity Short Form 4a is a 4-item self-report questionnaire that assesses the frequency and intensity of physical activity during the past 7 days. Items are rated on a 5-point Likert scale, with higher scores indicating higher levels of physical activity

Patient and parental distress will be assessed using the validated Distress Thermometer, which has previously been applied in pediatric HSCT and other cancer populations. The instrument consists of a visual analogue "thermometer" indicating the level of distress from 0 to 10, and an accompanying problem checklist, allowing respondents to identify relevant sources of distress.

The Distress Thermometer will be administered to patients aged ≥10 years and to at least one parent per patient, specifically the parent primarily present during the patient's hospitalization.

Assessments in both the patients and parents will be conducted on the day of hospital admission, five days prior to HSCT (day -5), on the day of transplantation (day 0), and subsequently on day +7, +14, +21, +28, +35, +42, +50, and +60, as well as on the day of discharge. If discharge occurs before the planned assessment schedule, the final assessment will be conducted on the day of discharge.