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Newborn & Infant Nursing Reviews 13 (2013) 189–195

Contents lists available at ScienceDirect

Newborn & Infant Nursing Reviews


journal homepage: www.nainr.com

Neonatal Extravasation: An Overview and Algorithm for Evidence-based Treatment


Victoria Beall, BSN, CWOCN, RN a,⁎, Brent Hall, PharmD c,
James T. Mulholland, BSN, RN b, Sheila M. Gephart, PhD, RN b
a
Neonatal/Pediatric Wound, Ostomy and Continence Nurse, Diamond Children’s Medical Center at the University of Arizona, Tucson, AZ
b
The University of Arizona, College of Nursing, Tucson, AZ
c
Diamond Children's Medical Center at The University of Arizona, Tucson, AZ

a r t i c l e i n f o a b s t r a c t

Keywords: The peripheral intravenous (PIV) catheter is the most used vascular access device for the administration of
Neonatal medications in hospitalized neonates, however 95% of PIV catheters are removed due to complications.
Infiltration Infiltration and extravasation are one of the most destructive complications to the neonate's fragile skin. This
Extravasation article reviews multiple aspects of infiltration and extravasation injury. First, starting at the cellular level the
Vesicant role of vesicants in vascular injury and its role triggering inflammation will be discussed, followed by a
Peripheral intravenous (PIV) therapy
comprehensive review of vesicants and their mechanism of injury, by pH, osmolality or chemical composition,
Algorithm
then an overview of the NICU nurses knowledge and actions to prevent infiltration and ending with the use of
an evidence-based algorithm that was developed at one children's hospital to minimize injury caused by
extravasations through targeted, prompt treatment.
© 2013 Elsevier Inc. All rights reserved.

Many medications given to neonates have the potential to injure nal structures as additional fluid collects around the vein, and in severe
when an extravasation occurs. An extravasation is described by the cases can result in compartment syndrome.6 Extravasations have the
Infusion Nurses Society (INS) as the inadvertent administration of a potential to cause peripheral tissue injury depending on the type of
vesicant solution or medication into the surrounding tissues. 1 A vesicant, concentration of the vesicant, location, amount, and duration of
vesicant is defined as a solution or medication that causes the exposure to the vesicant. Damage from a vesicant may progress over
formation of blisters leading to tissue necrosis and sloughing. time and become evident 48–72 hours after the extravasation occurs.7,8
Extravasation can result in varying degrees of localized tissue injury
and can cause pain, infection, and partial to full thickness tissue loss Neonatal Vulnerability to Vascular Injury
involving muscles and nerves. If extravasation is severe and depend-
ing on the site, skin grafts, long hospitalization and high costs result. The preterm and sick neonate is more susceptible to skin injury
Not surprisingly, with disfigurement and loss of function, parents may and complications from extravasation injury than their mature,
try to recover payment for their loss by initiating lawsuits. 2,3 healthy counterparts. Their immature skin structures, flexible subcu-
taneous tissue, small blood vessels and poor venous integrity increase
Infiltration Is Common Among Neonates the risk of complication from venipuncture and IV infusions. 5,8 The
goal in neonatal care is to prevent skin breakdown whenever possible.
The peripheral intravenous (PIV) catheter is the most used vascular Similarly, attention to thermoregulation, pain and stress that infants
access device for the administration of medications in hospitalized endure as a result of repeated IV attempts or restarts, and infiltrations
neonates; however 95% of PIV catheters are removed due to com- and extravasations must be considered and managed. 9,10 Multiple
plications such as leaking, occlusion and infiltration.4 Infiltration rates tools are available to score pain responses and enable the NICU
among neonates are as high as 57%–70% with extravasation occurring in (neonatal intensive care unit) nurses to manage this appropriately.
11–23%.5 Both infiltration and extravasation are destructive, causing Nonpharmacologic measures to decrease pain include the use of a
localized injury to the neonate's fragile skin.6 Infiltration of non- pacifier, swaddling, or administration of sucrose during the insertion
vesicants can cause considerable tissue damage from pressure on inter- of an IV or for infiltration and extravasation injury. 10 Aside from non-
pharmacologic interventions, treating infiltration and extravasation
⁎ Address correspondence to Victoria Beall, RN, BSN, CWOCN, Neonatal/Pediatric pain with analgesics should be considered. 8
Wound, Ostomy and Continence Nurse, Diamond Children’s Medical Center at the
University of Arizona, 1501 N. Campbell Ave, P.O. Box 245116 Tucson, AZ 85724. Inflammation in the Premature Infant
Tel.: +1 520 694 7328.
E-mail addresses: victoria.beall@uahealth.com (V. Beall), brent.hall@uahealth.com
(B. Hall), mulhollj@email.arizona.edu (J.T. Mulholland), gepharts@arizona.edu The neonatal immune system is poorly regulated compared to
(S.M. Gephart). adults and dysregulation is magnified when neonates are born

1527-3369/1304-0528$36.00/0 – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1053/j.nainr.2013.09.001
190 V. Beall et al. / Newborn & Infant Nursing Reviews 13 (2013) 189–195

early. 11–13 While intravenous therapy is necessary in this population, The Neonatal Intensive Care Unit (NICU) Nurse's Role
it is not without its risks. Vesicants can harm the endothelial lining of
the blood vessel, triggering production and release of oxygen NICU nurses monitor the PIV site with vigilance to aid in early
free radicals that spur inflammation. 14,15 The release of the locally identification of infiltration and extravasation and prevent this type of
induced free radicals into the circulatory system when not controlled injury whenever possible. Identifying an infiltration may be difficult,
can trigger a system-wide response to the stressor and further free even for the most experienced nurse. 10 The NICU nurse is aware of the
radical release. In a normal response, the infant's body mounts subtle changes in heart rate, oxygen saturations, apnea, and the more
an anti-inflammatory release of free radical scavengers. When this obvious change in behavior such crying and agitation that may
anti-inflammatory response is inadequate, which is common in indicate problems with the PIV therapy. 4 Neonatal nursing entails not
prematurity, or the inflammatory assault is severe, endothelial only basic knowledge of the anatomy and physiology of neonatal skin
dysfunction leads to programmed cell death (apoptosis). 16 The load and how to prevent iatrogenic injury from routine nursing care but
of oxidative stress in premature infants is especially of concern as also knowledge of vascular anatomy and infection control for the safe
it has been linked to various neonatal morbidities including delivery of infusion therapy. 4,8,10 Several organizations have devel-
necrotizing enterocolitis, 16,17 retinopathy of prematurity, 18–20 and oped standards and guidelines that assist nurses with the best practice
chronic lung disease. 16,21–23 in infusion therapy. 10 These evidence-based guidelines and grading
scales for infiltrations can aid with standardizing documentation and
protocols that guide nursing care and improve patient outcomes. 5,6
Pathophysiology of Vascular Injury

In the human body, vascular injury, oxidative stress, and Potential Origins of Infiltration
inflammation are intricately related. Infiltration and extravasation
are both symptoms of vascular injury. When vascular injury occurs, There is a supposition that an infiltration or extravasation is caused
the release of free radicals is stimulated and the energy producing by IV catheter dislodgement or puncture of the vein during insertion
ability of the cell becomes dysfunctional as mitochondria are or during handling of the infant. Chemical composition of medications
damaged. As free radicals are released and the capacity of the also impacts risk of vein rupture. 5 The vein's tolerance to an infusion is
damaged mitochondria to produce energy is limited, the balance affected by the osmolality and pH of the vesicant, the duration of the
between nitric oxide and superoxide is disrupted. This reactive exposure, and irritation to the endothelial cells. 4 An additional factor
oxygen species excess extends the damage. 24–26 A continual cycle of in causing a cannulated vessel to rupture and leak is the pressure in
free radical production and endothelial apoptosis occurs leading to which the medication is being delivered by the infusion pump. 3,5
injury of cell membranes and vessels. Symptoms of this process Janet Pettit describes the following 3 theories of the mechanism of
include redness, swelling (inflammation with increasing vascular infiltration and extravasations. The first theory states that the
permeability), and visible tissue injury. 25 The inflammatory process irritation of the venous endothelium from the infusate causes
involved in extravasation is depicted in Fig 1. Note how the vasoconstriction and diminished blood flow. 4 The second theory has
interaction of the biological response to chemical and non-chemical been confirmed by dye studies. It shows infiltration of the infusate
risk factors when exposed to the harmful drug directly affects healthy through the catheter insertion hole created with IV placement; this
endothelium, leading to vascular injury. The framework is a helpful can occur when the flow proximal to the catheter tip becomes
tool in understanding the biological aspects of extravasation and obstructed. 4 The third theory is that irritation of the venous
action targets to prevent injury. endothelium caused by the osmolality, pH, or chemical composition

Fig 1. Model of vascular injury.


V. Beall et al. / Newborn & Infant Nursing Reviews 13 (2013) 189–195 191

of the infusate damages the lining of the vein and allows transmission increased to 493–520 and thus increase potential damage to tissue. 4 It
of the infusate into the tissue, without creating a puncture in the is generally felt that solutions with an osmolarity b900 mOsm can be
vein. 4 These three theories demonstrate how infiltration and administered peripherally. This is based on an American Academy of
extravasation can occur; the remainder of the damage is thought to Pediatrics Committee on Nutrition recommendation governing the
be caused by the irritants and vesicants. use of peripheral parenteral nutrition (PPN). 4 There is some
controversy with this recommendation due to a lack of supporting
evidence. The Infusion Nurses Society recommends limiting the
Irritants and Vesicants Given to Neonates osmolarity of peripherally administered solutions to b 500 mOsm.
This is based on evidence that more concentrated solutions are more
Intravenous medications can be divided into three major sub- likely to result in chemical phlebitis. 4
categories: 1) non-vesicants, 2) irritants, and 3) vesicants. In order for The measurement of the concentration of hydrogen ions, pH, is
an infiltration to be a true extravasation, the offending agent, by another important consideration when evaluating a medication's risk
definition, must be a vesicant. There are a number of different of infiltration or extravasation. A normal pH of blood is between 7.35
qualities that affect the potential for a medication to result in tissue and 7.45 (arterially). As the pH of a medication moves away from this
damage. These include, but are not limited to: osmolarity, pH, direct normal range, the risk of tissue injury is higher. As the number of
medication effects and solubility. 27 See Table 1 for medications hydrogen ions increase, the solution will become more acidic (pH
categorized by their mechanism of injury. b7.35). The majority of medications have an acidic to neutral pH. Once
Osmolarity describes the number of particles which are suspended medications move out of the range of 5–9, risk of inflammation and
in solution. Normal serum osmolarity of a neonate is approximately vascular injury increases significantly. Vancomycin, for example, has a
280 mOsm. When a solution has a higher osmolarity, it is considered pH of 2.5–4.5, which helps to explain its potential for extravasation.
to be hyperosmolar. The effect causes cells to shrink as fluid shifts Medications with alkalotic pH (N7.45) can be just as damaging to
from inside to outside of the cell. This response occurs in order to tissue. One example is phenytoin with a pH of 10–12, approximating
increase the osmolarity inside the cell to equilibrate with the elevated that of household bleach.
osmolarity of the serum. The opposite of this effect would be a Medications have specific mechanisms of action that may result in
solution that has a lower osmolarity or is hypoosmolar. In this cellular damage when an infiltration/extravasation occurs. Vasoactive
situation, fluid would enter the cell in order to decrease the medications such as dopamine have effects on alpha-receptors. If this
osmolarity of the cell to equal that outside. At the most extreme, product is introduced into tissue, this alpha stimulation will result in
this can result in cell lysis as the volume of the cell exceeds its constriction of capillary beds, which subsequently will decrease local
capacity. Osmolarity may be altered by how a medication is diluted. blood flow. Local tissue will be deprived of oxygen and ischemic injury
Ampicillin is a good example of this. If 50 mg of ampicillin is mixed will ensue. 4 Another group of medications that may have direct effects
with 1 mL of sterile water, the osmolarity is 243. If 50 mg of ampicillin on tissues are electrolytes. Calcium is necessary for depolarization and
is mixed with 1 mL of normal saline, the osmolarity would be contraction of smooth muscle. If a concentrated calcium solution is

Table 1
Medication Mechanisms of Vascular Injury.

Inciting Agent

Hyperosmolar
(N10% dextrose,
mannitol,
potassium, propofol
Vesicants and sodium Vasoactive Highly-Lipophilic
(phenylephrine) Irritants Hypo-osmolar bicarbonate) Alkalotic Medication Medication Medications

Definition Medications capable Causes pain within Osmolarity Osmolarity High concentration Alpha-receptor Medications do not
of causing tissue the vessel or b280 mOsm N280 mOsm, of hydrogen ions stimulation dilute well in water,
damage9 surrounding tissue causing cells to causing cells to (high pH) causes constricts capillary making it difficult to
e.g. Calcium but does not result swell as fluid shifts shrink as fluid shifts inflammation and beds, decreases local flush out or wash
stimulates smooth in tissue necrosis into the cell. As the outside of the cell can lead to vascular blood flow and off, damage results
muscle to contract intracellular volume injury deprives local tissue from the high
capillaries, leading exceeds its capacity, of oxygen leading to concentration of the
to hypo-perfusion cells can lyse. ischemic injury5 medication in the
and ischemic injury tissue

Medication Acyclovir Aminophylline 0.2 % NaCl 3% sodium chloride Sodium Bicarbonate Dobutamine Diazepam
Examples Aminophylline Calcium Gluconate Sterile water5 Calcium chloride Phenobarbital Epinephrine Digoxin
Calcium Chloride Digoxin, Contrast media Sodium thiopental Norepinephrine Nitroglycerine
Dobutamine Erythromycin, Total parenteral Phenytoin Vasopressin Phenytoin
Dopamine Gentamicin, nutrition5
Epinephrine Theophylline3
Nafcillin
Norepinephrine
Oxacillin
Penicillin
Phenytoin,
Potassium Salts
Total parenteral
nutrition (TPN)
Peripheral
parenteral nutrition
(PPN)
Vancomycin3
192 V. Beall et al. / Newborn & Infant Nursing Reviews 13 (2013) 189–195

infused into tissue, this may result in capillary constriction via of the injury, type of IV infusing, duration of exposure and location. 8
stimulation of smooth muscle, which will also result in ischemic Protocols and algorithms can be used to assist nurses in the steps
injury to hypoperfused tissue. 4,7 Finally the solubility of a medication needed to minimize the potential damage and start the treatment
can influence its contribution to local tissue damage. The more process. Stopping the infusion and elevation of the extremity is the
lipophilic a medication, the less water-soluble it is. This makes first actions followed by placement of a saline soaked gauze or
washing out the site impractical and leads to high concentrations of prepackaged normal saline pad. The saline draws out the vesicant, and
medications located in relatively small areas of tissue that can lead to impedes a scab from forming to allow fluid to leak out. Gently
concentration dependent direct tissue damage. squeezing the fluid from the open insertion site can also help to
remove the offensive agent. 8 While the saline soaks are held in place,
assistance with the various tasks that are required for treatment may
Nursing Actions to Prevent Vascular Injury
require additional personnel. A new PIV may need to be inserted
immediately, the physician or nurse practitioner (NP) will be called to
The best method to decrease complications of PIV therapy is to
assess the site and plan for pharmacologic treatment if indicated. For
prevent them in the first place.2 Serious complications are not entirely
an extravasation, treatment should be determined prior to catheter
preventable, but following recommended standards of IV therapy is the
removal. 8 The administration of antidotes or enzymes such as
best approach for avoiding complications.3 The decision to place a
hyaluronidase is the method of choice to manage IV extravasation. 6
peripherally inserted central catheters (PICC) or central venous lines
Treatment that improves tissue perfusion and prevents progressive
(CVL) might be needed if vascular access is difficult or long-term
tissue necrosis has been shown to be most effective if initiated within
parenteral therapy is planned. However, this may be difficult in low birth
1 hour. 8 Ongoing assessment of the neonate and measures to improve
weight (LBW) neonates when access for a CVL is limited, central line
comfort or alleviate pain may be indicated. Compassionately reporting
infection prevention initiatives are paramount, or a variety of incompat-
the situation to the family as soon as possible and keeping them up to
ible infusions are indicated.2,6 The risks and benefits of PICC and CVL are
date on the plan of action are required. 8 Prior exposure to standards of
evaluated by the medical and surgical teams. If the risks are reasoned to
care, protocols and algorithms and staff education could aid the
be too great for PICC or CVL, responsibility will be on the nurse to manage
process of administering antidotes, as this could be time consuming
the PIV and when vesicants are being infused vigilance needs to heighten.
“on-the-job-training” for an inexperienced NICU nurse, when timely
Evidence-based guidelines for infusion therapy start with the insertion.
treatment provides better patient outcomes. 8 An algorithm based on
See Table 2 for recommendations to prevent peripheral vascular injury.
the work of Sawatzky-Dickson and colleagues (2006) (see Fig 2) was
On insertion, attention needs to be paid to the selection of the appropriate
developed to streamline the described process and improved patient
catheter, site selection, skin antisepsis and stabilization once the device is
outcomes. 2
in place.10 NICU nurses learn to leave the neonates' extremity out of the
The quality improvement initiatives implementing the algorithm
swaddling or sleep sacks for easy visualization of the PIV site; so that
have not been formally evaluated. However, physicians, pharmacists
hourly assessments (and more frequent assessments if a known vesicant
and nurses are managing infiltration and extravasations promptly as a
is infusing) do not interrupt the neonate's developmental sleep. Warning
team. The administration of hyaluronidase or the appropriate antidote
signs of possible complication warranting further investigation include a
without the WOCN's assistance, as well as a decline in referrals for
PIV that is difficult to flush or an infant who responds to the flush with
long-term wound management for extravasation injuries are now the
crying or flinching.3 Assessments might reveal swelling, redness, and
NICU's norm.
coolness of the skin. Most infiltrates resolve spontaneously after the IV
catheter is removed, however in severe cases blistering and skin necrosis,
Pharmacologic Options to Treat Extravasations
prolonged capillary refill and decreased pulses and movement of the
extremities can result. Infiltration scales can be useful to nurses in
Currently there are multiple medications that are used to treat
identifying the severity of the assessment findings.3
extravasations. These include hyaluronidase, phentolamine, and
topical nitroglycerin 2% ointment. 8 Sodium thiosulfate, dimethyl
Key Actions to Minimize Injury When Extravasation Occurs sulfoxide (DMSO) and dexrazoxane are antidotes primarily associated
with treatment of chemotherapy extravasations. As the scope of this
Once an infiltration or extravasation is discovered, immediate article is directed at the neonatal population, it will focus on the first
treatment is the key to preventing progressive damage from the three non-chemotherapeutic extravasation medications: hyaluroni-
vesicant. 2 Treatment decisions are based on the size and appearance dase, phentolamine and nitroglycerin. 7,8,26,27

Hyaluronidase
Table 2
Recommendations for Practice to Prevent Vascular Injury.
Hyaluronidase is an enzyme that breaks down hyaluronic acid, a
compound best described as the “glue” which holds cells together.
Peripheral IV Insertion and Maintenance When this extracellular glue is dissolved, cells are separated. This is
Use small enough plastic/silicone catheter to avoid restriction of blood flow
Avoid repeated use of a vein
helpful in extravasations as it allows for the medication to distribute
Avoid placing a PIV in an areas difficult to immobilize through a larger area by breaking down the walls that keep it
Use transparent tape to secure localized. This not only helps to decrease the concentration effects of
Cover the site with a sterile semi-permeable transparent dressing that will permit the extravasated product, but also exposes the medication to more
ongoing visualization of the insertion site
capillary beds that allows for reduction of edema via more rapid
Upper extremities less likely to infiltrate or leak compared with peripheral IV in
lower extremities or scalp veins reabsorption and removal of the product from the damaged area. The
Place tape loosely over boney prominences to avoid restricting blood flow to the overall effect is to minimize the local damage caused by the
extremity extravasation. This change is not permanent, as hyaluronic acid will
Infusion Maintenance be regenerated within a 24 to 48 hour period. Efficacy of hyaluron-
Limit PIV glucose to 12.5% idase is supported by an FDA subcommittee evaluation from 2009
Dilute medications as much as possible before administration are other solutions based on evidence from animal studies, human studies and clinical
to preventing extravasation case reports. Hyaluronidase has been used to limit extravasation
PIV= peripheral intravenous. injury since 1976. One study suggested that use of hyaluronidase may
V. Beall et al. / Newborn & Infant Nursing Reviews 13 (2013) 189–195 193

Fig 2. Diamond Children’s IV Infiltration and Extravasation Algorithm (adapted from Sawatzky-Dickson and colleagues, [2006]). LIP = Licensed Independent Practitioner, MD =
Medical Doctor, WOCN =Wound, Ostomy, and Continence Nurse.
194 V. Beall et al. / Newborn & Infant Nursing Reviews 13 (2013) 189–195

decrease the development of skin ulcers by 50–60% and decrease skin and atraumatic for the neonate with minor skin breakdown or in the
ulcer size by as much as 50%. 7,28,29 neonate with full thickness wounds requiring wound care for weeks.
Hyaluronidase has been used in various ways and consensus is Hydrogel dressings are one of the moist wound healing treatments
lacking on which way is best. Most protocols advocate administration that have been found to be evidence-based and safe for neonates of all
of hyaluronidase within 1 to 2 hours of the initial injury but there are gestations. 8,9 Hydrogels consist of 80–90% water, which can be
case reports suggesting administration as far out as 10 days may be soothing and gentle to skin and keeps the wound moist to facilitate
beneficial. Concentrations of hyaluronidase used in studies ranges auto-debridement of wounds by rehydrating sloughing tissue and
from 15 unit/mL to 1500 unit/mL but standard practice for neonatal enhancing the rate of autolysis. 8 A section of hydrogel sheet cut to
and pediatric patients is to use a concentration of 15 unit/mL. Some cover the wound and secured with transparent dressing reduces
centers will have the practitioner pull back the infiltrated IV 1 to dressing changes to every 3 days, resulting in decreased handling and
2 mm to avoid IV injection and then administer 0.2 mL of a 15 unit/mL discomfort of the neonate, and less trauma to regenerated tissue, as
solution through the catheter to deliver hyaluronidase directly to the well as protects the tissue from outside oxygen tension and provides a
site of extravasation. The standard way to utilize hyaluronidase is to lower pH, which inhibits the growth of pathogens. 2,3 Full thickness
draw up 5 syringes of 0.2 mL of the 15 unit/mL concentrations and, injury may require surgical debridement and skin grafting, by a
after cleansing the site, inject these at 5 separate sites around the area pediatric or plastic surgeon. 8 Adhesive silicone foam can be used for
of extravasation. Again, the idea is to break down the barrier that is shallow wounds that may only require a small daily application of
holding the medication in that specific area and allow it to disperse to amorphous gel to maintain moisture to the site. The moisture of
a larger surface area. 26,28 Adverse effects are uncommon but hydrogels at times can macerate the periwound, in infants greater
potentially include tachycardia, hypotension, dizziness, chills, urti- than 30 days; an alcohol free skin barrier can be applied to protect the
carial erythema, angioedema, nausea and vomiting. fragile skin surrounding the wound. 8

Phentolamine Conclusions

Phentolamine is an antidote that will counteract the effect of Preventing and detecting early damage from vesicants are a
vasoactive agents such as dopamine, epinephrine, norepinephrine priority of every neonatal nurse. In this article we have described the
and phenylephrine. 8 These medications result in vasoconstriction via pathophysiology of vascular injury, the medications that incite it and
stimulation of alpha-receptors. Phentolamine acts to block the activity recommendations to prevent and treat infiltrations and extravasa-
of alpha-receptors and subsequently will help relax vascular smooth tions. Incorporating training on critical nursing assessments, signs of
muscle. This will improve circulation in the area of the extravasation injury and key actions to treat it are an important part of every NICU's
and thus decrease ischemia and cell death. Phentolamine can also be education plan. We recommend using an algorithm, like the one
utilized for vasopressin or dopamine extravasation. 28 Phentolamine presented here, to guide actions once extravasation occurs. Using
should be administered within 12 hours of initial exposure but non-pharmacologic approaches to treat the injury is a first step in the
administration should occur as soon as possible. Prepare a 0.5 to process of limiting damage. For certain vesicant-induced injury,
1 mg/mL solution and administer 0.1 mg/kg (to a max of 2.5 mg in pharmacologic treatment using hyaluronidase, in particular, has been
neonates, 10 mg in older children and adults) into the existing IV shown to limit the injury. If ineffective or the injury is extensive,
of the affected area. Neonates should be monitored closely for surgical treatment may be necessary and a plastic surgeon may need
potential development of hypotension, dysrhythmias and tachycardia to be consulted. Along the way, openly speaking with the parents
due to potential systemic absorption of phentolamine. 3,28 The about the treatment is necessary and important to maintain the
efficacy of phentolamine has been demonstrated in animal studies family-healthcare partnership.
and case reports. 30–32

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