Transdermal Microneedle Patch To Enhance Topical Anaesthesia Before Intravenous Line Insertion for Blood Transfusion In Paediatric Thalassemia Patients
Phase: PHASE2
Conditions: Thalassemia in Children
Sponsor: Universiti Kebangsaan Malaysia Medical Centre

 
 
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Research Protocol 
Transdermal Microneedle Patch to Enhance Topical 
Anaesthesia Before Intravenous Line Insertion for Blood 
Transfusion in Paediatric Thalassaemia Patients 
 
Date of Document: 12th November 2021 
 
PRINCIPAL INVESTIGATORS: Prof. Dr Cheah Fook Choe 
Prof. Dr Azrul Azlan Hamzah 
 
CO-INVESTIGATORS: Assoc. Prof. Dr Dee Chang Fu 
Dr Muhammad Irfan Abdul Jalal 
Chua Xin Yun 
Ooi Kai Shen 
 
COLLABORATORS: Assoc. Prof. Dr Low Teck Yew 
Dr Muhamad Ramdzan Buyong 
Dr Ooi Poh Choon 
Dr Goh Chee Seong 
Dr Doris Lau Sie Chong 
Prof. Dr Zarina Abdul Latiff 
 
 
 

 
 
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CHAPTER I 
 
INTRODUCTION 
 
1.1 Transdermal drug delivery 
Venepuncture causes pain and is regarded as the one of the more frequently encountered traumatic 
experiences among hospitalised children. The distress and fear toward needle puncture may further be 
aggravated in children with chronic diseases which require them to undergo regular venous 
cannulation or venepuncture. The pain perception in the long term may affect the psychological well-
being and potentially adversely affect personal self-esteem and relationships.  
 
Healthcare is constantly evolving as providers aim to deliver holistic care to patients. Local anaesthesia 
before venepuncture aligns with Good Clinical Practice and is considered a quality standard of care in 
children. More recently, transdermal drug delivery is rapidly evolving as a less invasive way of 
administering such medications in a cost-effective manner. 
 
When it comes to transdermal drug delivery (TDD), the traditional low-cost hypodermic needles and 
topical creams are the most common approach to be considered. However, both have their drawbacks. 
For the hypodermic needle, the downsides associated include significant pain, anxiety, distress, and, 
worst of all, needle phobia.1,2 Hence, it could be challenging in children as the needle was the least 
favourable option among the paediatric population and the parents.3,4 There are also other state-of-the-
art modalities to enhance transdermal delivery of local anaesthetic agents such as phonophoresis 
(utilising ultrasound to enhance local anaesthetic agents penetration through the skin), 
magnetophoresis (using magnetic field), iontophoresis (utilising electrical current to boost electrically-
charged or ionic local anaesthetic agents penetration into the deeper layer of dermis) and jet injection 
(J-tip) (using pressurized gas to enhance subcutaneous delivery of local anaesthetic agents).5-8 
However, the large size and the exorbitant prices of these devices warrant the utilisation of extra power 
sources and satisfactory training of operators before such complex devices could be handled 
effectively.9 
 
Topical route hence appeared to be an attractive choice to be focused on. As such, topical anaesthetic 
creams, for example EMLA Cream (lidocaine 2.5% and prilocaine 2.5%), provide a non-invasive and 
convenient means of administering anaesthesia. As the drug passively diffuses across the skin, it results 
in a slower time of onset of effective analgesia which is around 60 minutes.10,11 This delayed onset of 
full analgesia is impractical and infeasible in a busy clinical setting. Therefore, such limitations of all 
available TDD methods are attributed to the human skin anatomy. 
 
1.2 Skin structure and transdermal drug delivery 
The human skin, particularly the outermost epidermal layer, is responsible for the mechanical barrier 
function. Unfortunately, this barrier function impedes the percutaneous entry of beneficial chemical 
compounds for a successful transdermal delivery of drugs. 
 
The skin is histologically made up of 3 main layers, i.e., the outer epidermis, middle dermis, and inner 
hypodermis.12,13 For the epidermis, this avascular layer is composed of 5 keratinocytes layers and is 
generally 50–150 μm thick.12 The full epidermis is constituted of the outermost stratum corneum (SC) 

 
 
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and its beneath viable epidermis (VE), with a thickness spanning from 10 to 15 μm and from 50 to 100 
μm, respectively.13,14 In between the epidermis and dermis lies the undulating epidermal-dermal 
junction. The dermis is the thickest component of the skin, measuring 2 – 4 mm thick in depth.12 The 
papillary and reticular dermal layers consist of active cells, connective tissues, blood vessels, 
lymphatics, and nerves.12,13 Lying underneath is the innermost hypodermis, primarily composed of 
subcutaneous adipose tissue as well as other histological architecture such as blood vessels, 
lymphatics, and nerves.13 Thereby, the sophisticated capillary network and nerve endings embedded 
in the dermis and hypodermis are substantial for systemic delivery of drugs through the transdermal 
route.12 Figure 1 illustrated the human skin structure. 
 
 
Figure 1. Human skin structure. The 3 skin layers composed of i.e., epidermis, dermis and hypodermis. 
The structural components in each layer were illustrated in relative to its anatomical position. Figure 
courtesy: Tomáš Kebert &amp; umimeto.org - Own work, CC BY-SA 4.0 
 
 
The stratum corneum (SC), made up of 15 to 20 layers of dead corneocytes, is primarily responsible 
for the skin barrier function.14 Hence, SC is implicated for the failure in effective systemic delivery of 
those lipophobic and high molecular weight active pharmaceutical ingredient (API).2  Nevertheless, 
the role of full epidermis should also be taken into account. Basement membrane and tight junctions 
located at the epidermal-dermal junction may confer certain resistance on molecular transportation of 
API across the epidermis.13 In the previous literature, it is also observed that drug compounds that 
managed to pass through the papillary dermis could subsequently reach systemic circulation and 
exhibit its systemic effect.13  
 
The evidence was further supported by the poor bioavailability from topical preparation, whereby only 
10–20% of total drug reach the circulation. In contrast, a hypodermic needle is able to penetrate the 
SC layer and delivers 90–100% of the loaded drug.1 However, it punctures deep into the dermis and 
may hit the pain receptors. 
 
1.3 Emergence of microneedle and its applications 
With the maturation of the microfabrication manufacturing technology, research on microneedle (MN) 
had been carried out extensively. MN is the mimic of hypodermic needle, composed of hundreds of 

 
 
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micron-sized, out-of-plane protrusions, typically arranged in arrays on a patch that can be applied to 
the skin.14,15 The mechanism behind is MN creates multiple transient microchannels across the skin 
and disrupt the SC in a minimally invasive manner. These micron-sized conduits increase the 
permeability for better API transport into the systemic circulation, enhancing the therapeutic effect. 
Here lie the benefits of MN whereby the needle specifications can be tailored to avoid stimulating the 
nociceptors and penetrating the blood vessels. The MN can be fabricated from a variety of material. 
Remarkably, the recent focus towards biocompatible polymer holds excellent promise over silicon or 
metal. Microneedle biocompatibility is the most crucial aspect to consider in the paediatric population 
as the skin barrier function is immature and rapidly evolving.12 A biocompatible MN dramatically 
eliminates the risk of biohazard wastage and possible complications if needle breakage occurs. 
Maltose, a natural carbohydrate, is preferred for its biodegradability and dissolubility within minutes.16 
Its safety profile is well-recognized, which makes maltose being extensively used in pharmaceutical 
formulations.1,16 Among the MN types, coated MN refers to the solid MN coated with drugs formulation on 
its surface. It has a coat-and-poke feature which means the drug coating will be continuously dissolved and then 
released into the skin along with the polymer.2  
So far, there is a paucity of prior studies investigating the effects of combined usage of microneedle 
and local anaesthetic application in paediatric patients requiring regular blood transfusion for their 
clinical conditions. There are three prior studies investigating the use of microneedle for insulin 
delivery and glucose monitoring in paediatric patients (children and adolescents) with type I diabetes. 
Gupta, Felner and Prausnitz (2011) compared the use of a single hollow microneedle as a means for 
intradermal delivery of insulin with the subcutaneous administration of insulin using a catheter.17 They 
found no significant differences in terms of insulin’s area under the curve (AUC) between those two 
modalities but faster absorption of insulin using the former (tmax = 27 ± 13 minutes (MN + insulin 
group) vs 57 ± 20 minutes (catheter + insulin group)). They also found out that pain associated with 
procedures was substantially lower in the MN + insulin group. However, a study by Norman et al. 
(2013) demonstrated higher pain scores in children and adolescents aged between 10 and 18 years old 
who received insulin via MN than their counterparts who received insulin subcutaneously via insulin 
pump.18 Nevertheless, the difference is not statistically significant. On the contrary, in a recently-
published study by Samant et al. (2020) investigating the safety and suitability of different glucose 
monitoring modalities in 15 paediatric patients (mean age (SD): 16.8 (3.2) years) with type I diabetes, 
the authors demonstrated similar VAS scores between those whose glucose levels were monitored via 
interstitial fluid (ISF) sampling using MN patch (MN+ISF) and fingerstick capillary blood glucose 
monitoring.19 Interestingly, the MN+ISF group reported a statistically-significant lower VAS score 
mean than those who went blood glucose monitoring using intravenous catheters.19 
Hence, based on these prior findings, the utilities of microneedle in alleviating pain perception 
associated with routine clinical procedures in paediatric patients are still inconclusive. Therefore, 
further investigations are obviously warranted to bridge the gap in our current understanding of 
microneedle utilities in paediatric patients. 
 
1.4 The Pharmacological properties of EMLA constituents 
Both constituents of EMLA cream, lidocaine and prilocaine, are amide-type local anaesthetics. They 
have similar pKa (7.9 (lidocaine) and 7.7-7.9 (prilocaine)), percentage of ionization at physiological 
pH (75-76%(lidocaine) and 67-76% (prilocaine)) and elimination half-lives (t1/2 = 1.6 hours).20-22 
However, prilocaine has a much smaller partition coefficient (129 vs 366) and percentage of protein 
bound (55% vs 65%) than lidocaine, resulting in a much higher volume of distribution (2.73 L/kg vs 
1.30 L/kg) and a higher clearance (2.03 L/kg per hour vs 0.85 L/kg per hour) than lidocaine.20,22 Most 

 
 
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amide-type local anaesthethic agents undergo hepatic metabolism and around 5% of their mass is 
removed renally without metabolic transformation, except for prilocaine which could also be 
metabolised in the kidney.22 Hence, liver or renal impairment may affect the elimination of both agents, 
resulting in an elevated risk of systemic toxicity. Prilocaine results in less vasodilation than lidocaine 
and therefore the use of epinephrine is not warranted to lengthen its duration of action, an advantageous 
feature for those whom epinephrine use is contraindicated.22 
Since both lidocaine and prilocaine are less lipophilic than other local anaesthetic agents such as 
bupivacaine and tetracaine, they cause less cardiotoxicity. However, prilocaine causes less direct 
neurotoxicity (e.g. transient neurologic symptoms (TNS) and cauda equina syndrome) than lidocaine 
due to its larger volume of distribution, expeditious lung update and swift metabolism.20,21 Hence, 
prilocaine seldom reaches toxic plasma concentration level.23 Nevertheless, prilocaine induces 
methaemoglobinemia formation due to its toxic metabolite, ortho-toluidine (o-toluidine).24 
Consequently, neonates have the highest risk of developing methaemoglobinemia since their red blood 
cells have insufficient methaemoglobin reductase.22 To treat this, methylene blue or ascorbic acid, 
could be administered intravenously to revert the ferric ion (Fe3+) in the ring-like non-protein heme 
group of haemoglobin to its original ferrous state (Fe2+).25 
 
EMLA (Eutectic Mixture of Local Anaesthetics) is a eutectic mixture of 2.5% lidocaine and 2.5% 
prilocaine. Due to its eutectic property, EMLA exists in an emulsified form at room temperature. Skin 
analgesia is induced by the diffusion of lidocaine and prilocaine transdermally, resulting in the 
stabilisation of neuronal membranes via the blockade of ion fluxes that are needed for action potential 
initiation and transmission.26 EMLA should not be used on mucous membrane since it is rapidly 
absorbed into systemic circulation. The speed of onset, duration and depth of skin analgesia are largely 
reliant upon the length of application time.26 Besides, the duration and onset of action are also 
influenced by the thickness of the skin at the application are and local blood flow.27 The effects of 
increasing the dosage of EMLA on the onset, duration and depth of analgesia are still unknown. A 
maximal analgesic effect (under occlusive dressing) is achieved 2 to 3 hours post application and this 
may last up to 1 to 2 hours post removal.26 
 
1.5 Pain assessments 
Pain is a subjective perception, rendering its assessment difficult.  Several pain assessments tools had 
been proposed, and each presented with specific strength properties. Ideal measurement tools should 
always possess good sensitivity, consistency, validity, and particularly easy to understand so that they 
can be straightforwardly applicable to the paediatric population.28,29 Self-assessment scales require 
certain levels of comprehension skills and cognitive function. Hence, self-assessment scales are only 
reliable in children aged 6 years and above.28 Visual Analog Scale (VAS) is considered to be the most 
validated tool and correlates positively with other self-measuring scales, such as Faces-pain scale 
revised (FPS-R) and Numerical Rating Scale (NRS). It has been further proven as an optimal tool to 
evaluate pain as its values can be utilised to monitor pain progression. Besides, VAS scores are also 
sensitive to treatment effect, and represent real difference in pain intensity at 2 different timepoints.29 
Figure 2 shows a child-friendly VAS pain score device, which further gives the children a more 
convenient and visual way to measure pain. 
 

 
 
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Figure 2. The child-friendly VAS pain score. 
 
Nevertheless, scientists had set eyes on the objective approach that best measures the pain intensity. 
With the recent help in technology advancement, a pain monitoring device had been developed based 
on stress induced sweating. The device relies on the firing rate of the nociceptive nerve, mirrored by 
the skin conductance peaks, which is then translated to the pain stimuli perceived.30 The stress induced 
sweating is activated through skin sympathetic nerve and is independent of external environment.   
 
1.6 Problem statements and justifications of the research 
Based on our brief review of literature, there are two problem statements summarising the identified 
research gaps that justify the conceptual significance of this research: 
i) The combined effects of microneedle usage reduce the dose and hasten the action of EMLA Cream 
on skin anaesthesia in paediatric patients requiring venous cannulation for regular blood transfusion 
have not been properly elucidated in previous studies. This study aimed to demonstrate a better care 
for paediatric patients that frequently receive blood transfusion as part of the management of their 
clinical conditions. 
ii) The agreement between VAS score and the physiological skin conductance algesimeter index 
obtained using medical devices has not been previously investigated in paediatric patients. This study 
aimed to highlight the potential of physiological pain monitoring in critical and semi-critical care 
where the patient is often either semi-conscious or unconscious. 
Hence, it is anticipated that our research findings may satisfactorily provide the answers to the problem 
statements above, resulting in significant application of microneedle in TDS of drugs such as EMLA 
Cream, and the use of physiological monitoring of pain in paediatric patients. The conceptual 
framework (Figure 3) diagrammatically summarizes our research underpinnings, postulates and 
justifications. 
 

 
 
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1.7 Conceptual framework 
Thalassemic paediatrics patients requiring 
venous cannulation for regular blood transfusion 
Pain associated with venipuncture and IV 
cannulation 
Skin sympathetic nervous system 
activation, release of acetylcholine acting 
on muscarinic receptors, resulting in 
palmar sweat gland filling up. 
Reduced skin electrical resistance, skin 
electrical conductance increases 
Skin conductance peak is created which is 
dependent on the strength of sympathetic 
nervous firing 
Variable pain intensity as measured by VAS 
score resulted in variable skin conductance 
peaks per second (skin conductance 
algesimeter (SCA) index)  
Good agreement between VAS scores and 
SCA index 
Making SCA index useful for pain monitoring 
in critical and semi-critical care settings  
Microneedle (MN) enhances transdermal 
EMLA delivery 
EMLA is applied to reduce pain sensation 
associated with venipuncture and IV 
cannulation for blood transfusion 
Higher analgesic intensity in MN+EMLA 
recipients compared to those EMLA-only 
recipients. Faster onset of action associated 
with EMLA, thus requiring shorter 
application time.  
Lower VAS score and skin conductance 
algesimeter index in group receiving 
combined MN and EMLA than the EMLA-
only group and similar VAS scores 
regardless of longer (30 minutes) or shorter 
(15 minutes) application time 
Better pain perception associated with 
procedures related to blood transfusion 
Improved care and treatment compliance in 
thalassemic paediatrics patients 

 
 
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CHAPTER II 
 
STUDY OBJECTIVES 
 
2.1 General Objective 
To evaluate if the pain scores are different during venous cannulation through a pre-
anaesthetized skin patch, with and without the use of microneedle.  
 
2.2 Specific Objectives 
2.2.1  To compare Visual Analogue Scale pain scores among Microneedle with 1 Finger Tip 
Unit EMLA Cream (experimental group) applied for 30 minutes, Microneedle with 0.5 
Finger Tip Unit EMLA (experimental group) applied for 30 minutes, Microneedle with 
1 Finger Tip Units EMLA applied for 15 minutes, and 1 Finger Tip Units EMLA Cream 
only applied for 30 minutes (controls) groups. 
 
2.2.2  To compare skin conductance algesimeter index among Microneedle with 1 Finger Tip 
Units EMLA Cream (experimental group) applied for 30 minutes, Microneedle with 
0.5 Finger Tip Unit EMLA (experimental group) applied for 30 minutes, Microneedle 
with 1 Finger Tip Units EMLA applied for 15 minutes, and 1 Finger Tip Units EMLA 
Cream only applied for 30 minutes (controls) groups. 
 
2.2.3 To evaluate the agreement between the Visual Analogue Scale pain scores obtained and 
the skin conductance algesimeter index obtained via PainMonitor™ machine 
 
2.3 Research Questions 
2.3.1 Is there any difference in terms of VAS pain score means among the MN with 1 Finger 
Tip Unit (FTU) EMLA Cream (30-minute application), MN with 0.5 FTU EMLA (30-
minute application), MN with 1FTU EMLA cream (15-minute application), and 1 FTU 
EMLA Cream only (30-minute application, controls) groups? 
 
2.3.2 Is there any difference in terms of skin conductance algesimeter index means among 
the MN with 1 FTU EMLA Cream (30-minute application), MN with 0.5 FTU EMLA 
(30-minute application), MN with 1FTU EMLA cream (15-minute application), and 1 
FTU EMLA Cream only (30-minute application, controls) groups? 
 
2.3.3 Is there any agreement between the VAS pain score and the skin conductance 
algesimeter index obtained via PainMonitor™ machine? 
 
 
 

 
 
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2.4 Research Hypotheses 
2.4.1 There is at least a difference in terms of the VAS pain score means among the MN-
with-1-FTU-EMLA (30-minute application time), MN-with-1FTU-EMLA (15-minute 
application time), MN-with-0.5 FTU-EMLA (30-minute application time), and 1-FTU-
EMLA-only (30-minute application time) groups. 
 
2.4.2 There is at least a difference in terms of the skin conductance algesimeter index means 
among the MN-with-1-FTU-EMLA (30-minute application time), MN-with-1FTU-
EMLA 
(15-minute 
application 
time), 
MN-with-0.5-FTU-EMLA 
(30-minute 
application time), and 1-FTU-EMLA-only (30-minute application time) groups. 
 
2.4.3 There is agreement between the VAS pain score and the skin conductance algesimeter 
index obtained via PainMonitor™ machine.  
 
 
 

 
 
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CHAPTER III 
 
RESEARCH METHODOLOGY 
 
3.1 Research Design 
This is a prospective, phase II, single-centre, single-blind, cross-over, randomized negative-
controlled (without microneedle (MN)) trial at Hospital Canselor Tuanku Muhriz (HCTM), 
Universiti Kebangsaan Malaysia. Eligible participants will be randomized in a cross-over 
fashion to one of 24 treatment sequences as shown in Figure 4. 
 
This crossover design is chosen over the more conventional parallel-group randomized trial 
since the lesser intra-patient variability than the inter-patient variability, thus minimising the 
required sample size. The study investigators also do not anticipate the inherent disadvantages 
associated with this trial design (greater attrition rate, prolonged carry-over effects and the 
inconsistency of participant’s condition) will be present in this study. 
 
3.2 Time period/ Duration 
From August 2021 until August 2022 (52 weeks). 
 
3.3 Study site 
Paediatric day-care centre at level 4 of UKM Medical Centre. 
 
3.4 Study population and sampling method 
Thalassaemic patients who came for regular blood transfusion at HCTM who fulfil the 
inclusion and exclusion criteria and voluntarily provide the voluntary written informed consent. 
Convenient sampling will be used to obtain the study subjects to prevent insufficient 
recruitment caused by the paucity of eligible study participants. 
 
3.5 
Inclusion Criteria 
i) 
Patient aged at least six years to below eighteen years old 
ii) 
Patients requiring venous cannulation for blood transfusion 
 
3.6 
Exclusion Criteria 
i) 
Patient with a previous history of sensitization or allergy to EMLA Cream 
ii) 
Patient with a previous history of allergy to materials used in the study i.e., plaster, 
electrodes, maltose, Polyvinyl Alcohol (PVA), and Polyethylene Terephthalate (PET)  
ii) 
Patient exposed to analgesic usage within 24 hours prior to the procedure 
iii) 
Generalized skin disorder/ rash 
iii) 
Agitated/ fretful patient 
 
 

 
 
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3.7  
Ethical issues 
Voluntary written informed consent will be obtained from each study participant. This study 
will be conducted in accordance to the principles of ethics in human research laid as stipulated 
by the Declaration of Helsinki (18th World Medical Association General Assembly, 1964) and 
the Good Clinical Practice (GCP) guidelines.31 Ethical approval will be obtained from the 
UKM Research Ethics Committee (Human) (JEPUKM). 
To aid the transparency of reporting, the trial will be registered at the Clinical Trials.gov 
registry (https://clinicaltrials.gov/). The full trial protocol will be made available in the same 
trial registry and prepared according to the Consolidated Standards of Reporting Trials 
(CONSORT) guideline for crossover design and the 2013 Standard Protocol Items: 
Recommendations for Interventional Trials (SPIRIT) statement.32,33 The trial data with study 
participant identification numbers removed (Hospital RN, Identity Card (IC) numbers, 
subject’s identification number (SIDNO) will be made available to the public via Harvard 
Dataverse repository for research data (https://dataverse.harvard.edu) to ensure the transparent 
dissemination of study findings and fulfil the requirements for the future publication of the 
findings of this research. 
 
3.8 
Randomization procedure 
For random allocation, simple randomization with random allocation rule (RAR) will be used 
by which a list of random numbers will be generated in a balanced 1:1 ratio (based on 24 
different treatment sequences) using the the randomizeR package version 20.0 executed on R 
platform.34 The list of generated random numbers will be used to allocate the study participants 
to either intervention or control branch. The allocation sequence generated will be kept in a 
password-protected document that is only accessible to the statistician to maintain allocation 
concealment. To further guarantee allocation concealment is satisfactory, randomisation code 
will not be revealed until the participants have been definitively recruited into the trial, which 
is after all baseline measurements are made and all eligibility criteria are deemed fulfilled. 
Besides, allocation concealment is further assured by ensuring the information on the 
intervention allocation is only given to the person who will be administering the allocation via 
secured phone calls (central randomization). Consecutive recruitments will be made until the 
final intended sample size is reached. 
For this study, the study participants, outcome assessors, care providers and data handlers will 
be blinded to the study interventions. Only the statistician and the person who will be 
administering the interventions will be unblinded to the study interventions. Besides, unique 
code to indicate each treatment sequence assignment will be utilised to ensure that the 
unintentional / intentional unblinding of one trial participant will not compromise the integrity 
of blinding for the rest of study participants. The main unblinded trial persons (the statistician 
and the key person who will be administering the intervention) are instructed not to divulge the 
identity of the allotted treatments to other blinded trial persons. The success of blinding will be 
determined by asking the blinded trial persons (e.g. study participants, care providers, outcome 
assessors) to guess the interventions received and then compare the results obtained with what 
would be anticipated by chance. Blinding indices such as James’ Blinding Index or Bang’s 
blinding Index could also be computed to objectively evaluate the success of blinding.35,36 
 

 
 
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3.9  
Clinical protocols 
3.9.1 Fabrication of solid maltose microneedle 
The MN array patch size is measured 1 cm x 1 cm, containing 36 microneedles with 1 mm 
needle gap in between. The maltose (Hayashibara, Okayama, Japan) MN dimension is designed 
to be around 400 μm in height, base width 100 μm, with a 3-μm tip radius. The standard 
deviation of needle heights within the patch is controlled to be less than 3%.  The total patch 
thickness is therefore 0.8 mm. The MNs are grown on a soft cushion on top of Polyvinyl 
Alcohol (PVA) material (Kanto Chemical, Tokyo, Japan), with a Polyethylene Terephthalate 
(PET) patch (Acrysunday, Tokyo, Japan) to support the soft PVA patch as below. The base 
patch spans 125% larger than the array MN patch, with an estimated patch size of 1.5 cm x 1.5 
cm. The overall size of the microneedle device is thus 1.7 cm (width) x 4 cm (length) x 0.5 cm 
(height). Figure 3 illustrates the schematic representation of microneedle prototypes that will 
be used in this study.  
 
 
Figure 3. The schematic representation of microneedle prototypes. 
Since the MN patch will be manually applied to the skin with a normal thumb force, various 
studies reported the hole diameter should be around 1/3 of MN length.37,38 Therefore, this 
research's specification is speculated to achieve the desired penetration depth around 160 μm 
where the epidermal-dermal junction lies. To ensure a uniform force is applied all over the MN 
patch, the interventionist will stick a pillar handler of a size of 1 cm x 1cm x 4.9 cm on the 
bottom surface of MN patch with a double sellotape stuck onto it. The application of MN to 
the skin thus mimics a stamping action. 

 
 
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The maltose dissolving time is about 15s at a temperature between 25 and 30 °C and a humidity 
of more than 60%. Hence, in normal condition, the maltose MN is able to dissolve into the skin 
within 1 minute. For storage, the maltose MN is recommended to be stored at room temperature 
(upper limit not exceeding 40 °C) and at room humidity of less than 60% to avoid melting. The 
shelf time can last for more than 8 years, provided that the storage temperature is maintained 
at 25 °C and a room humidity of less than 10%. 
 
3.9.2 Administration of interventions / controls 
Prior to the administration of intervention / control, relevant clinic-demographic profiles (age, 
gender, ethnicity, anthropometric measurements, presence of comorbidities, thalassemia types 
etc) will be recorded and entered in the case report forms (CRFs) that are specifically designed 
for this study. This research study uses EMLA Cream (lidocaine 2.5% and prilocaine 2.5%) as 
the topical anaesthetic agent. EMLA Cream is a eutectic emulsion mixture of lidocaine and 
prilocaine at 1:1 ratio (i.e. each gram of EMLA cream contains lidocaine and prilocaine, 25 mg 
each). A eutectic mixture has a lower melting temperature than each constituent’s melting 
temperature. The anaesthetic efficacy of EMLA cream will be assessed via pain induced by 
intravenous cannulation and the primary endpoint is the participant’s VAS score measured after 
applying EMLA Cream (with and without MN application) for 15 and 30 minutes.  
 
The window period given to EMLA Cream for its effect to work will be based on the usual 
clinical practice observation where it is usually applied for 30 minutes prior to intravenous 
catheterization. The rationale behind it is due to logistical issues and for the day care's 
operational convenience. Nevertheless, in a busy clinical setting, the application time is 
sometimes shortened to 15 minutes for slight anaesthetic effect.  Thus, the study investigators 
postulate that, with the aid of microneedle, the time to onset of action for EMLA Cream could 
be greatly reduced, thus requiring less time to achieve its maximal effects. 
 
According to the routine hospital protocol, all study participants received their blood 
transfusion based on the Good Clinical Practice (GCP) guidelines. For each participant, the 
individual will be randomized to one of the 24 treatment sequences (Figure 4 and Table 1) 
and there will be a minimum of 3-weeks washout period before administering the next 
intervention. Figure 5 illustrated the complete clinical flow for the first and subsequent visits, 
which includes the preliminary recruitment and randomization phases. 
The investigator identified and drew a grid of 1cm × 1cm at the dorsum hand, which served as 
an ideal site for cannulation. The administrator of intervention (procedurist) will apply either 
1 FTU of EMLA Cream (approximately 0.68 g/cm2) or 0.5 FTU (approximately 0.369 g/cm2) 
over the preparation area.  If the patient is subjected to MN patching at his/her visit, the MN 
patch will be applied by thumb force and pressed firmly against the hand surface for 5 seconds 
to patch MN to the skin entirely before applying EMLA cream. Otherwise, an empty (i.e 
without MN) PVA-containing PET sham patch will be applied instead. Besides, the height-to-
base ratio (4:1) used for MN will also optimally minimise its adverse effects (pain, redness), 
thus preserving the masking (blinding) of study participants from knowing the types of 
interventions received. The preparation area will be covered with an adhesive dressing 
(Tegaderm™; 3M, Maplewood, Minnesota, USA) after EMLA cream application. After the 
allocated application time (15 or 30 minutes), the attending medical officer will set up the 
transfusion line with a 22-gauge hypodermic needle inserted into the dorsum hand. Throughout 
the process, the parents/guardians will be allowed to stay by the patient's side at all times.  

 
 
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3.9.3 Pain assessment 
After a random treatment sequence was assigned to the study participants, the study 
participants will be guided on the operating manual for a 10-points, 100mm VAS pain score. 
The children will be presented with a ruler that contains 100-mm slots with “No Pain” written 
on the left side and “Worst Pain” on the opposite right side.  
Before applying MN patch and EMLA Cream, the patients will be attached with the 
PainMonitor™ (Med-Storm Innovation AS, Oslo, Norway) device whereby the electrodes will 
be attached to the hypothenar eminence of the opposite hand not receiving the blood 
transfusion. The skin conductance peaks (in microSiemens (μS) and the average rise time (in 
microSiemens per second (μS/s)) will be recorded. Those parameters indicate the skin’s 
sympathetic nerve block induced by the applied EMLA cream.  
After each procedure, the children then will be asked to move and place the slider in the slot 
that accurately describes his/her pain at the following time points: 1) 1 minute after application 
of MN/ sham patch and before EMLA Cream application (baseline VAS score); 2) 1 minute 
after IV cannulation. The investigator will record the location of the slot where the slider is 
placed in (millimetres (mm), clearly printed on the ruler’s backside) and this will be the 
participant’s VAS score. Throughout the process, there will be a trained nurse standing by at 
the day-care to assist the verification of the pain scale and to aid the participants who require 
additional assistance.  
 
3.10 Interventional safety assessment  
3.10.1 Adverse Events (AEs) / Serious Adverse Events (SAEs): Definitions 
For this study, the study investigators define adverse events as “an abnormal sign, symptom, 
laboratory test, syndromic combination of such abnormalities, untoward or unplanned 
occurrence (e.g. an accident), or any unexpected deterioration of concurrent illness”.39 For 
serious adverse events (SAEs), they are defined according to the USA Code of Federal 
Regulations Title 21, Section 312.32 which is “adverse events result in the following outcomes: 
1) death; 2) life-threatening AEs; 3) inpatient hospitalization or prolongation of existing 
hospitalization; 4) a persistent of significant incapacity or substantial disruption of the ability 
to conduct normal life functions, or a congenital anomaly / birth defect”.40 Other significant 
medical events are also considered as SAEs if the events compromise the study participants 
and result in the requirement of medical or surgical intervention to prevent one of the outcomes 
above. As an example, allergic bronchospasm requiring intensive treatment in an emergency 
setting to prevent the outcomes above. 
3.10.2: The likelihood of AEs / SAEs: Classification of causality 
The likelihood of AEs and SAEs are classified based on the modified Naranjo et al. 1981 
recommendations as follows:41 
a) Unrelated: The AEs / SAEs are unlikely related to microneedle or EMLA application. 
b) Possible: The connection between AEs / SAEs with microneedle or EMLA cannot be 
excluded with absolute certainty: 

 
 
15 
 
i) There is a temporal relationship between the microneedle / EMLA application with the 
occurrence of AEs / SAEs. 
ii) However, there is an alternative factor (e.g. characteristic of patient’s disease / clinical state) 
that can possibly or likely explain such a relationship or a significant uncertainty exists over 
the cause of AEs / SAEs 
c) Probable: The connection between AEs / SAEs with microneedle or EMLA has a high 
degree of certainty 
i) There is a temporal relationship between the microneedle / EMLA application with the 
occurrence of AEs / SAEs 
ii) AEs / SAEs disappear or decreases upon the withdrawal of microneedle or EMLA 
application but do not reappear upon subsequent microneedle / EMLA application 
iii) Alternative causes cannot reasonably explain the relationship between microneedle / EMLA 
application with the occurrence of AEs / SAEs. 
e) Definite: The AEs / SAEs are obviously linked to microneedle / EMLA application with the 
occurrence of AEs / SAEs 
i) There is a temporal relationship between the microneedle / EMLA application with the 
occurrence of AEs / SAEs 
ii) AEs / SAEs disappear or decreases upon the withdrawal of microneedle or EMLA 
application and reappears on subsequent microneedle or EMLA application. 
iii) No other alternative causes. 
 
3.10.3 Methods for assessing and recording of AEs / SAEs 
The study protocol will be halted at any moment of the study period if the study participants 
develop any sudden (expected or unexpected) severe complications / adverse events. The 
withdrawal of a study participant from the trial will be documented on the adverse event page 
of the CRF and the participant will be further followed up for the study outcomes and included 
in the analysis as per the original randomization group (intention-to-treat analysis). All AEs / 
SAEs will be recorded and graded according to the Common Terminology Criteria for Adverse 
Events (CTCAE) version 5 and US FDA’s Toxicity Grading Scale Healthy Adults and 
Adolescents Volunteers Enrolled in Preventive Vaccine Clinical Trials criteria as shown in 
Appendix  III and IV.42,43  
3.10.4 Local skin reaction  
Prior to the IV cannulation and after blood transfusion, the adhesive dressing will be removed 
and skin antisepsis will be performed. The investigator will visually examine the tested site for 
any evidence of skin irritation. Photos will be taken for further documentation. Among local 
AEs / SAEs that will be solicited and documented are as follows: 
• Pain 
• Erythema (redness) 

 
 
16 
 
• Ecchymosis (bruising) 
• Swelling 
• Pruritus (itchiness) 
• Tenderness (pain upon touching the cannulation site) 
The patients will be followed up via phone call within 24 hrs after the procedure to obtain any 
subsequent evidence or occurrence of skin irritation. Any local skin reaction will be described 
and documented in terms of its maximum intensity, duration of reaction, subsequent action 
taken to mitigate any adverse event, potential causal link with the study intervention / control 
and the evolution of the skin reaction: (1) continues; 2) recovered with sequel; 3) recovered 
without sequel; 4) unknown. 
3.10.5. Systemic Reaction 
Among systemic AEs / SAEs associated with study interventions that will be solicited and 
documented are as follows: 
• Fever 
• Irritability 
• Tiredness 
• Anorexia 
• Vomiting 
• Tachycardia 
• Seizure 
• Hypotension 
Similar safety protocol will be adhered to as in 3.10.4 with adjustments made for systemic 
reactions. 
3.10.6 Reporting of AEs / SAEs to the Institution Review Board (IRB) 
All AEs will be recorded in the CRFs. AEs of grade 3 and above will be reported to the UKM 
Human Ethics Committee within 5 business days. All SAEs (including Serious Unexpected 
Suspected Adverse Events (SUSARs)) will be reported within 24 hours of occurrence 
(expedited reporting), to the UKM Human Ethics Committee. The reporting procedures for 
SAEs should adhere to the following steps: 
i) The SAEs initial report form should be filled in by the assessors (medical officers) and 
reviewed and signed by the PI (Prof Cheah Fook Choe) or the co-investigator (Dr Doris Lau). 
ii) The SAEs initial report should then be submitted to the UKM Ethics Committee in an 
expedited fashion. 
iii) SAEs should be reviewed and followed to resolution by the medical officers / PI / co-
investigator in charge of study participant 
iv) A subsequent follow-up report will be submitted that documents any additional important 
information, including the resolution of SAEs. 
3.10.8 AEs / SAEs follow-up at the end of study period 

 
 
17 
 
If AEs / SAEs occur or are still ongoing at the end of the study, the study participants will still 
be continuously followed up until the resolution of AEs / SAEs occur, except the PI deems that 
no further follow-up is necessary. The follow-up may take the form of 1) additional subject 
visits to the trial centre / hospital; 2) telephone calls to the subjects; 3) additional reporting in 
the form of letters from the treating physicians. 
3.10.7 Study Halting Criteria 
Participant enrolment and allocation and institution of interventions will be stopped if one of 
the following occur: 
a) Death related to microneedle or EMLA application 
b) Any participant experiences bronchospasm, laryngospasm or anaphylaxis within 24 hours 
post microneedle or EMLA application 
c) Any SAE related to microneedle or EMLA application 
d) Any AE of grade 3 and above or any SAE that cannot obviously be implicated on other 
causes 
e) Any study participant who develops abscess / ulceration / erosion at the site(s) of 
microneedle or EMLA application 
 
3.11 
 Quality Control 
To ensure the quality of data collection, the following procedures will be followed: 1) all parties 
involved in this study (including outcome assessors) will be required to attend a training session 
to standardize procedures and  data collection; 2) the CRFs will be continuously checked for 
data accuracy and consistency, missing data and other data collection-related issues on 
fortnightly basis; 3) the biostatistician (Dr Irfan) will be notified on missing data or any data 
inaccuracy and tasked with formulating remedial measures; 4) the principal investigators and 
the biostatistician will conduct a final review of the detailed reports on screening, enrolment, 
follow-up and any data completeness / accuracy; 5) upon the clearance obtained from the 
principal investigators and biostatistician, the data from each CRF will be eventually entered 
into the SPSS  spreadsheet. 
 
3.12 Sample size calculation 
The determination of sample size is carried out using G*Power software, version 3.1.9.6 
(Universitat Kiel, Germany; February 2020) using the F-test family, Repeated Measure (RM) 
ANOVA within and within-between interaction sub-options. The type I error (α) and the study 
power (1 – β, where β = type II error) are fixed at 0.05 and 0.80, respectively.  
 
Due to scarcity of information from previous literature on paediatric population, we could not 
use the parameter estimates (standard deviation of the differences, mean differences between 
pairs) from prior studies as guides for our sample size calculation. However, to circumvent 
this, we utilise Cohen’s guideline on choosing the effect size for this study.44 We assumed that 
the Cohen’s d is large (d = 0.8) and used this as our effect size. It had been demonstrated by a 

 
 
18 
 
previous study on the efficacy of microneedle as a delivery method for 5% lidocaine dental gel 
(Septodent, UK) that the effect size could be that large, thus justifying our choice of effect size 
in general.45 For objectives 1 and 2, we used the RM-ANOVA within-group comparison sub-
option and fix the number of groups and measurements to 1 and 4, respectively. Based on prior 
guidelines, moderate-to-high correlations between the within-group measurements (r = 0.7) are 
assumed.46,47 For non-sphericity correction (ε), a value of 0.75 is assumed since the sphericity 
assumption is rarely fulfilled in practical and actual trial setting due to unequal correlations 
between measurement pairs and the chosen ε value is midway between its lower bound of 0.5 
and maximal value of 1 to fairly preserve the RM-ANOVA test against inflated type I error 
rate (α) when non-sphericity occurs.48-51 Hence, for objectives 1 and 2, we require a sample 
size of 23 subjects. Assuming a 10% attrition (drop-out) rate, the total sample size is 26 (ntotal 
= 26). 
 
For objective 2, we could not calculate the sample size since we don’t have the estimate of the 
standard deviation of the differences (denoted by (s) henceforth) between the two methods for 
pain evaluation (the VAS score and the skin conductance algesimeter index obtained via 
PainMonitor™). Information on (s) is required since the sample size formula for evaluating 
limit of agreement require s parameters to be explicitly specified. However, we could roughly 
assess the precision of the limit of agreements (as per (s) magnitude) based on the sample size 
proposed for objective 1 and 2 using the formula recommended by the original authors.52 Based 
on Bland and Altman (1986), the 95% CI of limit of agreement is given by: 
 
± 1.96* sqrt(3/n)*s52, 
 
where s = standard deviation of the differences between VAS score and the skin conductance 
algesimeter index. If we assume a sample size of 24, then the precision of the limit of agreement 
is: 
 
± 1.96* sqrt(3/26)*s = ±0.67*s, 
 
which we deem a satisfactory precision in our case.  
 
However, due to absence of an estimate of (s) from prior studies, the actual precision could not 
be accurately estimated, thus highlighting the importance of (s) in the actual sample size 
calculation. Thus, it is hoped the estimate of (s) obtained from this study will be used as a guide 
for calculating a sample size for a future follow-up study that can answer this study objective 
more reliably. However,  
 
Hence, the biggest overall sample size required for this study is 26 (ntotal = 26). 
 
3.13 Operational definitions of study variables 
a) Independent variables 
i) Age: The age of a study participant at the first study visit. The variable will be measured in 
years and month and modelled as a continuous numerical variable and will not be categorised 
into separate age groups. 

 
 
19 
 
ii) Gender: A categorical variable that will be recorded in the SPSS data frame as 0 = female 
(base category) and 1 = male. Missing data shall be recorded as 999 
iii) Ethnicity: A categorical variable that will be recorded in the SPSS data frame as 0 = Malay 
(base category); 1 = Chinese; 2 = Indian; and 3 = Other ethnicity. Missing data shall be recorded 
as 999. 
iv) Body mass index (BMI): A continuous numerical variable that is calculated using the 
standard body mass index formula; BMI = kg / m2. This variable will be categorised according 
to Kuczmarski et al. (2000) classification53: 
<5% percentile = underweight (SPSS code: -1) 
5% - <85% percentile = normal BMI (SPSS code: 0; base category) 
85% - 95% percentile = Overweight (SPSS code: +1) 
>95% percentile = Obese (SPSS code: +2) 
 
The categorized variable will then be used as a predictor variable for statistical modelling 
purposes. Missing data will be recorded as 999 in the SPSS spreadsheet. 
iv) Intervention groups: A categorical variable representing the types of interventions 
received by the study participants in each study visit. This variable will be recorded in the SPSS 
data frame as 0 = 1-FTU-EMLA only group (control, base category); 1 = MN + 0.5FTU EMLA 
(30 minutes), 2 = MN + 1FTU EMLA (15 minutes), 3 = MN + 1FTU EMLA (30 minutes). 
v) Baseline VAS score: A continuous numerical variable that will be measured before the 
administration of intervention. This will be used as a predictor variable to control the 
confounding effect of heterogenous baseline VAS scores among study participants. Missing 
data will be recorded as 999 in the SPSS spreadsheet. 
vi) Baseline pain score obtained via PainMonitor™  device: A continuous numerical variable 
that will be measured before the administration of intervention. This will be used as a predictor 
variable to control the confounding effect of heterogenous baseline VAS scores among study 
participants. Missing data will be recorded as 999 in the SPSS spreadsheet. 
b) Dependent (outcome variables) 
i) VAS score (15- and 30-minutes post EMLA application during IV cannulation): A 
continuous numerical variable that will be measured during each visit; 15 minutes after EMLA 
application and 30 minutes after EMLA application. Missing data will be recorded as 999 in 
the SPSS spreadsheet. 
ii) Pain score from PainMonitor™ device (15- and 30-minutes post EMLA application): A 
continuous numerical variable that will be measured during each visit: 15 minutes after EMLA 
application and 30 minutes after EMLA application. Missing data will be recorded as 999 in 
the SPPS spreadsheet. 

 
 
20 
 
3.14 Planned Statistical Analysis  
The data will be initially checked and cross-examined with the information in the data 
collection sheet for accuracy, consistency and the amount of missing data. Missing data will 
be treated as Missing At Random (MAR) and multiple imputation will be considered to impute 
the missing values in order to reduce the biasness of the parameter estimates. Any variable with 
serious missing data problem (more than 40%) will be dropped from further analysis.54,55 All 
patients will be analysed according to the original randomization group, based on the intention-
to-treat (ITT) analysis. 
 
All data will be descriptively summarized as mean and standard deviation (SD) for the 
normally-distributed numerical variables (or median and interquartile range (IQR) for the non-
normally distributed numerical variables) and frequency and percentage for categorical 
variable. For numerical data, the normality assumptions will be evaluated subjectively using 
the histogram with overlying normal distribution curve, box and whisker plot and steam-and-
leaf diagrams and objectively using the Kolmogorov-Smirnov and Shapiro-Wilks tests (p>0.05 
indicates normal distribution assumption is met). Fisher’s coefficient of skewness will be 
subsequently utilized to assess the severity of skewness in non-normally distributed numerical 
variables using the following formula: 
 
Fisher’s coefficient of skewness: Skewness / standard error of skewness 
Any value outside ± 2 range will indicate extreme skewness.56  
 
For the first and second objectives, repeated measure ANOVA and generalized mixed effect 
model (GLMM) with an identity link function will be used to assess and estimate the 
differences in terms of VAS score means between the periods of interventions, considering 
intra-subject correlations. For repeated measure ANOVA, Mauchly’s test of sphericity will be 
used to assess the sphericity assumption. If sphericity assumption is violated, the Greenhouse-
Geisser correction will be used if  ε < 0.75 or the Huynh-Feldt correction if ε > 0.75.57 Within-
group  analysis will be used to assess the significance of mean VAS score differences between 
treatment pairs. To assess the presence of carryover effect, the within group-between treatment 
sequence interaction effect will be assessed for statistical significance.  
 
For GLMM, the Restricted Maximum Likelihood (REML) estimator will be used to estimate 
the model parameters. The unadjusted and adjusted estimates of the parameters obtained using 
the GLMM will be examined, with and without the consideration of periodic effects. Random 
intercept model and robust variance estimators will be used for the calculation of standard 
errors. Likelihood ratio test will be utilized to compare the nested models and Akaike 
Information Criteria (AIC) and Bayesian Information Criteria will be used for comparing non-
nested models. Multicollinearity between fixed effects (age, sex and treatment effects) will be 
evaluated using bivariate correlations. Besides, interaction effects (effect modification) 
between significant fixed predictors will also be assessed by evaluating the significance of 
interaction terms.  Residual diagnostics will also be performed to ensure the validity of model 
assumptions and the presence of outliers and influential points will be investigated to ensure 
that they do not influence the model parameter estimates. The presence of any influential point 
will then be removed or corrected if these are due to measurement errors or wrong data entry 
or retained if the values are authentic. We will also carry out sensitivity analyses to investigate 
the effects of missing data under the Missing At Random (MAR) framework. 
 

 
 
21 
 
For the third and fourth objectives, the correlation between VAS pain score and PainMonitor™ 
device is tested using Pearson correlation coefficient if both VAS pain scores and 
PainMonitor™ device are normally distributed (Gaussian) and Spearman coefficient will be 
used if at least one pain scores (obtained by either measuring modality) are not normally 
distributed. Intraclass correlation coefficient (ICC) will be computed to assess the agreement 
between the VAS scores obtained using the two different modalities (VAS pain score vs 
PainMonitor™ device) and the results will be interpreted according to the Koo-Li criteria.58 
The Bland and Altman (B&A) plot will be obtained to further assess the agreement between 
the two scores. The limits of agreement obtained will be assessed to ensure there is an 
acceptable agreement between the pain measurement modalities. 
 
For each analysis, the significant threshold for p value is fixed at 0.05 and 95% confidence 
interval (CI) will be obtained for each parameter estimates. Data analysis will be performed 
using Statistical Package for Social Science (SPSS™) (IBM Corp. Released 2020. IBM 
Statistics for Windows, Version 27.0, Armonk, NY: IB Corp) and STATA™ version 15 
(StatCorp. 2017. Stata Statistical Software: Release 15. College Station, Texas: StataCorp LP).
 
 

 
 
22 
 
3.15 Study flow 
 
 
 
                                  
                                                      
 
 
 
 
                                                                                       
 
 
 
 
                                                                                       
 
 
 
 
                                                                                       
 
 
               
Figure 4. The flowchart of this randomized crossover trial showing 4 random intervention sequences out of 24 
permutations generated. Intervention A:  MN + 1 FTU EMLA 30 minutes application; Intervention B: MN + 0.5 FTU 
EMLA 30 minutes application; Intervention C: MN + 1 FTU 15 minutes application; Intervention D: 1 FTU EMLA 
Only 30 minutes (controls) 
ABCD 
BACD 
CADB 
DABC 
ACBD 
BADC 
CABD 
DACB 
ABDC 
BDAC 
CBAD 
DBAC 
ACDB 
BDCA 
CBDA 
DBCA 
ADBC 
BCAD 
CDAB 
DCAB 
ADCB 
BCDA 
CDBA 
DCBA 
 
Table 1. The exhaustive list of permutations of intervention sequences that will be randomized to the participants of 
this trial. Intervention A:  MN + 1 FTU EMLA 30 minutes application; Intervention B: MN + 0.5 FTU EMLA 30 
minutes application; Intervention C: MN + 1 FTU EMLA 15 minutes application; Intervention D: 1 FTU EMLA Only 
30 minutes (controls)
RANDOMIZED TO SEQUENCE (n=26) 
INTERVENTION A 
INTERVENTION B 
INTERVENTION C 
INTERVENTION D 
ASSESSMENTS (VAS SCORES) (NUMBER OF DROPPED OUT SUBJECTS + REASONS MENTIONED) 
PERIOD 1 
EXCLUDED (n =) & REASONS 
STATISTICAL ANALYSES (NUMBER OF SUBJECTS EXCLUDED FROM ANALYSES + REASONS MENTIONED) 
PERIOD 2 
INTERVENTION B 
INTERVENTION A 
INTERVENTION D 
INTERVENTION C 
PERIOD 3 
INTERVENTION C 
INTERVENTION D 
INTERVENTION A 
INTERVENTION B 
PERIOD 4 
INTERVENTION D 
INTERVENTION C 
INTERVENTION B 
INTERVENTION A 
ASSESSMENTS (VAS SCORES) (NUMBER OF DROPPED OUT SUBJECTS + REASONS MENTIONED) 
ASSESSMENTS (VAS SCORES) (NUMBER OF DROPPED OUT SUBJECTS + REASONS MENTIONED) 
ASSESSD FOR ELIGIBILITY 

 
 
23 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 5. Schematic flow of the clinical trial. 
On 1st day-care (preliminary) visit 
Informed consent from the parent/guardian  
On the 1st day-care (preliminary) visit 
 
Randomization of treatment sequences  
 
Briefing/Education on what the study arms involve 
 
Introduction & explanation on VAS score & Painmonitor™ 
 
Baseline scoring of VAS & Painmonitor™ for MN application 
 
Baseline skin condition assessment of MN application  
(pre- & post-cannulation) 
On subsequent (experimental) visit: 
 
Identification of ideal site for venous cannulation. 
 
PainMonitor™ probe application at the opposite hand 
 
Application of EMLA with or without MN patch 
 
VAS scoring for MN application (if applicable) 
• 
Post-cannulation VAS scoring  
• 
PainMonitorTM data extraction 
• 
Post-cannulation skin condition assessment after blood 
transfusion is complete  
• 
Skin antisepsis 
• 
Pre-cannulation skin condition assessment 
• 
Venous cannulation to the patient for blood transfusion 
Allocated window period at 15-/ 30-minutes 
Data analysis and interpretation 
Follow-up phone call skin condition assessment 

 
 
24 
 
 
GANTT CHART 
 
Project Activities 
Timeline of event 
2021 
2022 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
1 
2 
3 
Proposal preparation 
• 
• 
• 
• 
• 
 
 
 
 
 
 
 
 
 
Ethics approval 
 
 
 
 
• 
• 
 
 
 
 
 
 
 
 
Microneedle fabrication 
 
 
 
 
• 
• 
• 
 
 
 
 
 
 
 
Purchasing of PainMonitor 
 
 
 
 
• 
• 
• 
 
 
 
 
 
 
 
Patients’ recruitment 
 
 
 
 
 
• 
• 
• 
 
 
 
 
 
 
Launching of clinical trial 
 
 
 
 
 
• 
• 
• 
• 
• 
• 
• 
• 
• 
Data analysis and interpretation 
 
 
 
 
 
 
 
• 
• 
• 
• 
• 
• 
• 
Manuscript preparation and submission 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
End of research project 
 
 
 
 
 
 
 
 
 
 
 
• 
• 
• 

 
 
25 
 
 
BUDGET 
 
No. 
Description 
Unit price (RM) 
Quantity 
Price (RM) 
1 
RESEARCH MATERIALS & SUPPLIES 
1.1 
Schlenker VAS Pain Scale Rulers 0-100mm 
with Slider 
40 pcs 
4 
160 00 
1.2 
EMLA Cream 5%, 5G, 1s x 5 
160 Box 
12 
1920 00 
1.3 
Hansaplast Sensitive Kids Plaster 20's 
6 box 
7 
42 00 
 
 
  
 
  
2 
PROFESSIONAL SERVICE 
  
 
  
2.1 
Educational souvenir to patient 
10 pcs 
26 
260 00 
 
 
  
 
  
3 
RENTAL 
 
 
 
 
 
3.1 
PainMonitorTM  
33850 pcs 
1 
33850 00 
3.2 
Microneedle machine 
  
 
  
 
 
 
 
 
 
 
 
Total 
 
 
36232 00 

 
 
26 
 
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29 
 
APPENDIX I 
 
PATIENT INFORMATION SHEET 
 
Research Title:  
 
Transdermal Microneedle Patch to Enhance Topical Anaesthesia Before Intravenous Line Insertion for 
Blood Transfusion in Paediatric Thalassaemia Patients 
 
Introduction:  
 
You are invited to participate in a research study. Before participating in this study, it is crucial that you 
thoroughly read and understand the information provided in this sheet. However, before you take part 
or agree to continue in this research study, the study will be verbally explained to you, and you will be 
allowed to ask questions. After you are adequately satisfied that you understand this study and you wish 
to take part or continue to participate in this study, you must sign this informed consent form. You will 
be given a copy of these patient information sheet and consent forms to take home with you. 
 
Purpose of Study: 
Venepuncture (cannulation of blood vessel) elicits much pain and is regarded as the most frequently 
traumatic experience encountered among children. The pain may be reduced by applying topical 
anaesthetic drugs. For example, EMLA Cream (lidocaine 2.5% and prilocaine 2.5%) is a non-invasive 
and convenient means of administering anaesthesia. The recommended duration of time taken for 
EMLA Cream to work is around 60 minutes. However, in a busy clinical setting, the time is often 
expedited to 30 minutes. As such, microneedles had been introduced to enhance the delivery of topical 
anaesthesia by puncturing the skin at a very superficial level to create multiple tiny tracts for faster 
action. The microneedle is a device containing multiple small needles made of sugar that will dissolve 
into the skin. 
 
What will the study involve? 
In this study, your child is about to undergo venepuncture for regular blood transfusion in 4 consecutive 
day-care visits. Depending on the sequence of interventions we have determined, your child will receive 
application of the topical anaesthetic drug i.e., EMLA Cream, with or without the introduction of the 
microneedle to the skin. This will result in numbness on the site of his/her hand intended for the blood 
transfusion, which then reduces pain. The incidence and severity of pain will be recorded after the 
cannula for blood transfusion has been inserted.  
 
For this study, we would like to compare whether EMLA Cream alone or in combination with 
microneedle results in better anaesthetic effect. Therefore, there will be a maximum additional 30 
minutes imposed to your child normal routine for this medicine to be applied, and for us to record the 
findings. 
 
Risks:  
This investigation poses minimal risk to participants and is unlikely to cause side effects.  
Nevertheless, the possible common side effects from the EMLA Cream application include:  
• 
pallor,  
• 
redness,  
• 
alterations in temperature sensation over the application area.  
 
Additional risks associated with microneedle application might include possible mild-to-moderate skin 
irritation, especially in those with sensitive skin, such as 
• 
redness,  
• 
swelling,  
• 
itching.  
 
Should your child experience any problems, we will give your child medications to reduce the side 
effects of interventions accordingly. 

 
30 
 
 
Benefits:   
There will be skin anaesthesia effect from topical anaesthetic drug, i.e. EMLA Cream.  Furthermore, 
the information from this study may add to the medical knowledge about the use of dissolvable solid 
maltose microneedle in transdermally delivering local anaesthetic agents which may help future 
patients. 
 
Do you have to take part? 
Participation in this study is absolutely voluntary. Your child's medical care is not affected if you decide 
not to participate in this study. He/She will have the usual standard of care according to the day-care 
protocol.  
 
If you agree to participate, you will be asked to sign the "Informed Consent Form". You will be given 
a copy of the informed consent form and this Patient Information Sheet. Should you decide to 
participate, you are still free to withdraw from the study at any time without giving a reason and penalty. 
If you decide to cease from participating in this study, you must inform your study investigator and no 
new data will be collected from your child. The researcher may also remove your child from the study 
for various reasons. In this event, your child will not lose his/her rights as a patient and will still receive 
the usual standard of care. 
 
Data & Confidentiality: 
Participant’s confidentiality will be maintained throughout the investigation. The personal data will be 
anonymized. Hence your identity will be kept confidential. Data collected and entered into the Case 
Report Form remain the property of UKM. In the event of any publication regarding this study, your 
identity will remain confidential. 
 
By signing the Informed Consent form attached, you (or your legally acceptable representative, if 
relevant) are authorising such access to your study records. 
 
Payment and compensation: 
You do not have to pay, nor will you be paid to participate in this study. You do have to pay for the 
usual hospital charges.   
 
Whom can I ask about the study? 
If you have any questions about this study or your rights, please contact 
 
Principal Investigators:   
Prof. Dr Cheah Fook Choe 
Department of Paediatrics 
UKM Medical Centre 
Phone Number : 03-9145 5391/ 5380 
Co-investigator  
Dr. Doris Lau Sie Chong 
Department of Paediatrics 
UKM Medical Centre 
Phone Number :  03-9145 5387 
 
Signatures 
To be entered into this study, you or a legal representative must sign and date the signature page 
[APPENDIX II]   
  

 
31 
 
APPENDIX II 
Patient/Subject Information and Consent Form 
(Signature Page) 
 
Research Title: Transdermal Microneedle Patch to Enhance Topical Anaesthesia Before Intravenous Line 
Insertion for Blood Transfusion in Paediatric Thalassaemia Patients 
Researcher's Name: Prof. Dr Cheah Fook Choe / Prof. Dr Azrul Azlan Hamzah  
 
To become a part of this study, you or your legal representative must sign this page. By signing this page, I am 
confirming the following: 
• 
I have read all of the information in this Patient Information and Consent Form, including any information 
regarding the risk in this study and I, have had time to think about it. 
• 
All of my questions have been answered to my satisfaction. 
• 
I voluntarily agree to be part of this research study, to follow the study procedures, and to provide necessary 
information to the doctor, nurses, or other staff members, as requested. 
• 
I may freely choose to stop being a part of this study at any time. 
• 
I have received a copy of this Patient Information and Consent Form to keep for myself. 
 
 
 
Patient Name (Print or type) 
 
 
 
 
 
Patient Initials and Number 
 
 
 
Patient I.C No. (New) 
 
 
 
 
 
 
 
 
 
 
 
Signature of Patient or Legal Representative  
 
 
 
Date (dd/MM/yy) 
(Add time if applicable) 
 
 
 
Name of Individual  
Conducting Consent Discussion (Print or Type) 
 
 
 
 
Signature of Individual  
 
 
 
 
 
              Date (dd/MM/yy) 
Conducting Consent Discussion 
 
 
 
 
 
Name & Signature of Witness 
 
 
           
 
 
Date (dd/MM/yy) 
 
 
Note:  
i) 
All subject/patients who are involved in this study will not be covered by insurance.  

 
32 
 
APPENDIX III 
LOCAL 
SKIN 
REACTIONS 
GRADES 
 
0 (Nil) 
 
1 (Mild) 
2 (Moderate) 
3 (Severe) 
4 (Life Threatening) 
Pain 
 
Nil 
Does not interfere 
with activity 
Repeated use of non-
narcotic pain reliever 
for more than 24 
hours or interferes 
with activity 
 
Any use of narcotic 
pain reliever or 
prevents daily 
activity 
Emergency room (ER 
visit or 
hospitalization 
Erythema 
 
Nil 
2.5 cm – 5 cm 
5.1 cm – 10 cm 
>10 cm 
Necrosis or 
exfoliative dermatitis 
 
Ecchymosis 
 
Nil 
2.5 cm – 5 cm 
5.1 cm – 10 cm 
> 10 cm 
ER visit or 
hospitalization 
 
Swelling 
 
Nil 
2.5 cm – 5 cm and 
does not interfere 
with activity 
5.1 cm – 10 cm or 
interferes with 
activity 
>10 cm or prevents 
daily activity 
Necrosis 
Pruritus 
 
Nil 
Mild or localized, 
requires topical 
intervention 
Widespread and 
intermittent, limiting 
instrumental activity 
of daily living 
(ADL), requires oral 
intervention 
 
Widespread and 
constant, limiting  
self-care ADL, 
requires systemic 
corticosteroid or 
immunosuppressive 
therapy 
 
- 
Tenderness 
 
Nil 
Mild discomfort to 
touch 
Discomfort with 
movement 
Significant 
discomfort at rest 
ER visit or 
hospitalization 
 

 
33 
 
APPENDIX IV 
SYSTEMIC 
REACTIONS 
GRADES 
 
0 (Nil) 
 
1 (Mild) 
2 (Moderate) 
3 (Severe) 
4 (Life Threatening) 
Fever 
 
<38.0 oC 
38.0 – 38. 4 oC 
38.5 – 38.9 oC 
39.0 - <40.0 oC 
>40.0 oC 
Tachycardia (bpm) 
 
<100 
101-115 
116-130 
>130 
ER visit or 
hospitalization 
 
Hypotension 
(systolic; mm/Hg) 
 
90 and above 
85-89 
81-84 
<80 
ER visit or 
hospitalization for 
hypotensive shock 
 
Seizure 
 
Nil 
Brief partial seizure 
and no loss of 
consciousness 
 
Brief generalised 
seizure 
New onset seizures 
(partial or 
generalised). 
Multiple seizures 
despite medical 
intervention 
 
Prolonged repetitive 
seizures, life 
threatening 
consequences 
Irritability 
 
Nil 
Mild; easily 
consolable; No 
interference with 
activity 
Moderate; limiting 
instrumental ADL, 
increased attention 
indicated; medical 
intervention is not 
indicated 
Severe abnormal or 
excessive response; 
limiting self-care 
ADL; inconsolable; 
medical / psychiatric 
intervention is 
indicated 
 
ER visit or 
hospitalization 

 
34 
 
Tiredness / Lethargy 
 
Nil 
Mild symptoms; no 
interference with 
daily activity; 
reduced alertness and 
awareness 
Moderate symptoms; 
limiting instrumental 
ADL; no medical 
intervention is 
required 
 
Prevents daily 
activity; requires 
medical intervention 
ER visit or 
hospitalization 
 
Anorexia 
 
Nil 
Loss of appetite 
without alteration in 
eating habits 
 
Oral intake altered 
without significant 
weight loss or 
malnutrition; oral 
nutritional 
supplements 
indicated 
 
Associated with 
significant weight 
loss or malnutrition 
(e.g., inadequate oral  
caloric and / or fluid 
intake) 
Life threatening 
consequences / 
urgent intervention is 
required 
Vomiting  
 
Nil 
No interference with 
daily activity or 1-2 
episodes / 24 hours 
Some interference 
with daily activity or 
>2 episodes / 24 
hours 
Prevents daily 
activity, requires 
outpatient IV 
hydration 
ER visit or 
hospitalization due to 
hypotensive shock