See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/326735297

CHAPTER THREE: Materials & Methods

Chapter · August 2018

CITATIONS READS
0 2,902

1 author:

Naseer Almukhtar
University of Babylon
248 PUBLICATIONS   93 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

neuro-immune toxicology View project

new Physio-anatomic Brain map View project

All content following this page was uploaded by Naseer Almukhtar on 01 August 2018.

The user has requested enhancement of the downloaded file.

 Chapter three------------------------------------- materials & methods 35

CHAPTER THREE
Materials & Methods
3.1 Materials :
3.1.1 Subject of the study :
The study was conducted in the middle Euphrates provinces ( Al-Hilla
General Teaching Hospital/Al-Hilla, Al-Hussain General Teaching Hospital/
Kerbala, Al-Sader Medical Teaching Hospital/Al-Najaf Al-Ashraf and Al-
Qadisiyah General Teaching Hospital/Al-Qadisiyha ); at hearing and speech units
of ENT department in the period from November /2011 to July / 2012 .

3.1.1.1 Patients : one hundred ten patients were enrolled in this study after
exclusion of CHI and mixed HI clinically assessed by a specialist ENT doctor. (53
male and 57 female). The range age of studied patients were: (>1 -12 ) years old.
The patient classified into five groups according to pediatric classification (Elaine ,
2011) .
1. Toddler (>1-3) years
2. Prekindergarten (>3-5) years
3. Kindergarten (>5-7) years
4. Children (>7-10) years
5. Preteen (>10-12) years

3.1.1.2 Control :This group consisted of 60 normal hearing children

clinically assessed by a specialist ENT doctor. (29male and31 female) . The range
age of the control children were: ( >1 – 12) years old. They also classified into the
same five groups of patients according to pediatric classification (Elaine , 2011) .
 Chapter three------------------------------------- materials & methods 36

3.1.2 Instruments :
3.1.2.1 Audiological instruments : Main instruments used in
audiological assessment as in table (3-1) .

Table (3-1) shows the main instrument used in audiological assessment & their
sources
No. Instruments Sources

1 ABR Madsen Octavus GN otometrics ,Denmark

2 Four electrode channel GN otometrics ,Denmark
3 Disposable electrodes Pcs size Ceracarta,India
45*42 mm(millimeter)
4 Bilateral headphones GN otometrics ,Denmark
5 Laptop(Octavus software) Dell, china
6 Printer Canon, china
7 PTA pure tone audiometry Interacoustics ,Denmark
8 Bone conductive measuring Interacoustics ,Denmark
vibrator
9 Air conductive measuring Interacoustics ,Denmark
headphone
10 Autoscope Welchellen, USA
 Chapter three------------------------------------- materials & methods 37

3.1.2.2 Hematological and biochemical instruments :The main

instruments used in hematological and biochemical work and their
sources as in table (3-2) :

Table (3-2) shows the main instruments used in hematological and biochemical
work and their sources.

No. Instruments Sources

1 Hematocrit centrifuge Hettich ,Germany
2 Incubator Memmert ,Germany
4 Water bath Memmert, Germany
5 Refrigerator Concord ,Lebanon
6 Outoanalyzer Sysmex ,Japan.
7 Spectrophotometer Cecil. England.
8 Auto ESR analyzer Humased, Germany.
9 EDTA tube Afma – Dispo, Jordan.
10 Micro – pipette 100 -1000 µl Oxford, USA.
11 Micro – pipette 50-100 µl Oxford, USA.
12 Disposal syringe Asia medical instrument.
14 Plain tube Afma – Dispo, Jordan.
15 ESR auto tube Humased, Germany.
 Chapter three------------------------------------- materials & methods 38

3.1.3 Chemicals: the chemicals used in this study & their sources as
shown in table(3-3) .

Table (3-3) shows the main chemicals &their sources

No. Chemical material Sources

1. Urea kit Biomerieux , France
2. Bilirubin kit Spinreact , Spain
3. Glucose Kit Spinreact , Spain
4. Total calcium kit Human, Germany
5. Potassium kit Human, Germany
6. Sodium kit Human, Germany
7. Cell bag Sysmex ,Japan.
8. Stromatolyser-WH Sysmex ,Japan.
9. Cell clean CL-50 Sysmex ,Japan.
10. formal tri-sodium citrate solution Crescent ,Saudi Arabia

3.2 Methods:
3.2.1 History of Patient (Questionnaire):
Every patient and control was interviewed in a private room in the ENT
department. A well-structured questionnaire was developed for the study and was
filled for each of the patient and control groups . The questionnaire covered the
most important aspects of medical history, relevant hearing impairment type and
hearing impairment, medical history, and finally drugs and social history as shown
in the following pages:
Chapter three------------------------------------- materials & methods 39

Questionnaire

• Name: * PATIENT NO.:( )

• Age:
• Sex:
• Residence: Urban ( ) ; Rural ( )
• Duration of hearing impairment:
• Occupation:
• Natal history
 - Prenatal: Term ( ); preterm( ); posterm ( )
• Complication with pg. : bleeding ( ) ; anemia( ) ;TORCH ( ) ; Others:

 Perinatal history: (At birth) :

o delivery: normal ( ) ; C/S ( ) : home( ); hospital( )
o Position: cephalic( ) ; breach( )
o Duration of delivery:
o Complication with delivery: RDS( ) ; cyanosis( ) ; need ICU( );
Wt( ) ; Jaundice( )
o Blood transfusion( ) ; Others:

 -postnatal: feeding : breast ( ); bottle ( ) ; mix ( )

 Vaccination:
 O.M: ; meningitis: ; Any disease :
 Drug history:
• Family history:
o No. of children
o Smoking history:
o History of disease: ; TORCH:
o Drug history for mother:
o Using any sedation:
o Father relative :( ) No( )
o Education state: mother( ); father( ).
o Mother age with pg.:
o Socioeconomic state:
o Any relative disorders & congenital abnormality:
o Relative similar disorder: 1st degree ( ) 2nd degree ( )
 Chapter three------------------------------------- materials & methods 40

3.2.2 Physical examination:

Patients and control groups were physically examined by specialist ENT
doctors . The examination of the ear and hearing should always started with a
thorough inspection of the auricle and its surroundings. Attention should be given
to the following :
• Changes in the shape of the auricle or ear canal
• Surgical scars
• Crusting in the external ear canal and discharge: cerumen, mucus, pus,
blood, cerebrospinal fluid (otorrhia)
• Redness and swelling of the auricle or surrounding areas.
Otoscopy is performed with a hand-held otoscope . The auricle is rotated gently
backward and upward for the examination avoiding excessive traction. This
maneuver straightens the external ear canal and brings the lateral cartilaginous part
of the canal in line with the medial bony part . The diameter of the ear speculum
should conform to anatomic constraints, keeping in mind that a broad speculum
provides better exposure and illumimounted magnifier. The examiner gently pulls
the auride backward and upward to introduce the scope. The largest possible
speculum should be used to provide good exposure and adequate illumination of
the ear canalnation. The speculum is slowly introduced into the ear canal under
visual guidance, inserting it past the vibrissae but without touching the bony and
pain-sensitive medial portion of the ear canal. This should afford a clear view of
the ear canal and tympanic membrane. The normal tympanic membrane has a
grayish color and variable transparency. The left hand holds the otoscope for the
left ear examination and vice versa as in Fig. (3.1) (Rudolf et al., 2006) .
 Chapter three------------------------------------- materials & methods 41

Fig.(3-1) otoscope for the left ear examination(basic otorhinolarygiology2006)

After otoscope is done to evaluate ear and T.M(Tympanic membrane) conditions.
Then the patients and control referred to Audiological assessment and laboratory
testing .

3.2.3 Audio logical assessment :

3.2.3.1 Free-Field testing:
This is performed with the patient facing forward and the examiner
stationed opposite the ear to be tested. The patient should not be able to see the
examiner's lips move. It is necessary to mask the non-test ear to prevent it from
hearing sounds presented to the test ear (Scott-Brown's,2008).
3.2.3.2 Pure-Tone Audiometry(Scott-Brown's,2008);
The described method are applicable to patients from seven to eight
years old and older
 The auditory system can be stimulated via sound energy that is sent through
air to the ear drum through earphones air conduction (AC) testing or by
 Chapter three------------------------------------- materials & methods 42

placing a bone vibrator against the skull through the bone conduction(BC)
vibrator testing.
 For AC testing, the standard test frequencies range from 250 Hz through
8000 Hz in octave intervals.
 For BC testing, the frequencies test 250 Hz through 4000 Hz .
 Maximum AC values generally should not exceed by 120 dB HI; maximum
bone conduction (BC) values rarely exceed 70 dB HI.
 A pure-tone threshold is established at each frequency for each ear using a
bracketing technique to vary the level of the tone. The testing technique uses
the following three simplified rules:
1. If the patient responds positively to the presentation, then the level is decreased
to 10 dB for the next tone presentation,
2. If the patient does not respond to the tone presentation, then the level is
increased to 5 dB for the next presentation; and
3. If there are two positive responses at one level , then that level is designated a
threshold for that frequency and the next frequency is tested.
 The standard symbols figure (2-3) are recommended by (ASHA).

Fig(3-2) The standard symbols(American speech-language- hearing

association 2007)
 The no response symbol is simply an arrow that is attached to any of the
symbols.
 Chapter three------------------------------------- materials & methods 43

An audiogram is a graph showing hearing threshold as a function of frequency.

There are three variables to know whenever an audiogram is performed:
1. the frequency of sound that is being presented (Hz),
2. the intensity of sound that is being presented (dB HI), and
3. the method of sound presentation (AC [head set] or BC).

Fig. (3-3) shows the normal auditory thresholds (in dB HI) from 250 to 8000 Hz. Both air
conduction and bone conduction thresholds are represented. Normal hearing, on the dB HI scale,
is 0 dB.( Cumming’s 2010)

3.2.3.3 Auditory Brainstem Response testing:

General Procedures :
Patient and control were sedated with chloral hydrate (50 mg/kg,
administered orally) and were monitored for oxygen saturation, respiratory rate,
and heart rate throughout the procedure by a nurse from the otolaryngology clinic.
Testing took place in the audiology clinic.
First, electrodes will be placed on the forehead, scalp and mastoid bone. Small
headphones are put on each ear, red on right ear while blue on left ear. The
electrodes measure brain waves. After all equipment’s are in place, a series of
 Chapter three------------------------------------- materials & methods 44

clicks, hisses, and other sounds will be played. The brain's response to these
sounds is recorded and used to determine the level of hearing .
The electrical activity from the inner ear and auditory nerve is picked up by
electrodes and later analyzed on the computer or lap top. The resulting recording is
a series of vertex positive waves of which I through V are evaluated. These waves,
labeled with roman numerals in Jewett and Williston convention, occur in the first
10 milliseconds after onset of an auditory stimulus. The ABR is considered an
exogenous response because it is dependent upon external factor ( Burkard et
al.,2007; Hall, J.W. III 2007).
The auditory structures that generate the auditory brainstem response are believed
to be as follows(DeBonis et al., 2008).
Wave I – generated by the peripheral portion of cranial nerve VII
Wave II – generated by the central portion of cranial nerve VIII
Wave III – generated by the cochlear nucleus
Wave IV – generated by the superior olivary complex/lateral lemniscus
Wave V – generated by the lateral lemniscus/inferior colliculus
Our Procedures use: After a child sedated and made a sleep put him in
comfortable place and position then connect him with electrode and headphones to
instrument and start to give stimulation .Fig.(3-4) usually started at 70 db if there is
a response and wave V appear we decrease stimulation 10db until the wave
disappears (and this point consider degree of HI) or reachs 10db (and this point
consider normal degree of hearing) fig.(3-5). But if there is no response at 70db
we increase stimulation 10db until the wave V appears (and this point consider
degree of HI) or reach 100 db (and this point consider profound degree for HI).
Chapter three------------------------------------- materials & methods 45

Fig(3-4) shows an Iraqi patient child during ABR test examination in audiology
department at alhussain teaching hospital

Fig.(3-5) Normal latency intensity(audiology department at alhussain teaching

hospital )
Chapter three------------------------------------- materials & methods 46

Fig.(3-6) Normal ABR report audiology department at alhussain teaching hospital

L:Left ear hearing level (HL) ; R:Right ear hearing level
 Chapter three------------------------------------- materials & methods 47

Fig.(3-7) Normal numerical ABR value( audiology department at alhussain

teaching hospital )

3.2.4 Laboratory studies:

3.2.4.1 Blood collection:
The collection of blood was done in laboratory units of teaching hospitals
at 9 A.M. Five ml. of blood were drawn for each hematological and biochemical
studies . Two groups of labeled tubes were used ; the first tubes contained EDTA
as anti- coagulants to prevent clotting of blood to be used for hematological studies
.The second group tubes were without anti-coagulant as plain tubes, for blood to be
used for preparing sera for subsequent biochemical tests( Bishop et al., 2000 ).Each
sample was labeled and given a serial number together with the patient name. The
serum samples were frozen at -20°C for biochemical analysis (Lewis et al., 2006).
3.2.4.2 Hematological Studies:
3.2.4.2.1 Erythrocyte Sedimentation Rate (ESR) measurement: The
diluent solution for ESR measurement used was Trisodium citrate (3.8 %) (3.8 ml
of sodium citrate +100 ml of distilled water) ( Lewis et al., 2006) 0.4ml of diluent
was added to 1.6ml of the EDTA blood. The blood was mixed thoroughly then it
was withdrawn into Plus-Sed auto tube. The tube was put in auto ESR analyzer for
one hour.as shown in fig.(3-8) .
 Chapter three------------------------------------- materials & methods 48

Fig.(3-8) show auto ESR analyzer and it is Plus-Sed auto tubes at al sader teaching
hospital
3.2.4.2.2 Red blood cells count (RBCs count); Estimation of hemoglobin (Hb);
White blood cells count (WBCs count); And the platelets count:
The EDTA tube was filled with 2.5ml of blood (Lewis et al., 2006) .The
content was mixed then the tube inserted into the auto analyzer as in fig.(3-9)
which calculated and analyzed the content of blood sample within one minute and
the result came out as a tap.

Fig.(3-9) show auto analyzer for hemotological studies at al sader teaching hospital
 Chapter three------------------------------------- materials & methods 49

3.2.4.3 The biochemical studies:

3.2.4.3.1 Measurement of total serum bilirubin:

Working solution was prepared by mixing reagent 2 (R2) (sulfanilic acid,
hydrochIoric acid and dimethyl sulfoxide), with reagent 3(R3) (sodium nitrate)
.Mixed well and incubated exactly for 5 minutes at 37 C° and read wave length at
555 nm by using spectrophotometer (according to procedure recommended by the
bilirubin kit from the Spinreact company ,Spain) (Walter and Gerarde, 1970;
Burits and Ashwood , 1999).

3.2.4.3.1.1 Measurement of direct serum bilirubin :

Working solution was prepared by mixing reagent 1(R1) (sulfanilic acid and
hydrochIoric acid) with R3 ( sodium nitrate) .Mixed well and incubated exactly for
5 minutes at 37C°.After that read at wave length 555 nm by using
spectrophotometer (according to procedure recommended by the bilirubin kit from
the Spinreact company, Spain) ( WaIter and Gerarde, 1970; Burits and Ashwood ,
1999 ).

3.2.4.3.1.2 Determination of indirect serum bilirubin concentration:

It was obtained by the subtraction of the values of the direct bilirubin from
the values of total bilirubin (Walter and Gerard, 1970; Bruits and Ashwood , 1999).
3.2.4.3.2 Determination of serum urea :
Working solution was prepared by mixing the content of reagent 2 (urease)
to the bottle of reagent 3 (phosphate buffer sodium salicylate, sodium nitroprusside
and EDTA), and incubated for 5 minutes at 37C°, and read at 580 nm by using
spectrophotometer(according to procedure recommended by the urea kit from the
Biomerieux company , France) ( Fawcett and Scott,1960; Burits and Ashwood ,
1999).
 Chapter three------------------------------------- materials & methods 50

3.2.4.3.3 Determination of serum glucose:

The glucose is determined after enzymatic oxidation in the presence of
glucose oxidase. The formed hydrogen peroxide reacts under catalysis of
peroxidase with phenol and 4 –aminophenazone to a red – violet
quinoneimine dye as indicator. The absorbance of standards and samples were
measured against reagent blank at 505 nm (according to procedure recommended
by the glucose kit from the Spinreact company, Spain) (Barham and Trinder
,1972; Burits and Ashwood , 1999).
3.2.4.4 The electrolyte studies:
3.2.4.4.1 Determination of total serum calcium :
Calcium in the sample reacts with O-cresolphtaleine at alkaline pH. The
colored complex formed was proportional to the amount of calcium present in the
sample. The intensity of the color was measured photometrically by using
spectrophotometer at 570 nm wave length (according to procedure recommended
by the serum calcium from Human company, Germany) (Barnett,1973; Burits and
Ashwood , 1999).
3.2.4.4.2 Determination of serum sodium:
Sodium is precipitated with Mg - uranyl acetate ; the uranyl ions
remaining in suspension form a yellow - brown complex with thioglycolic acid
,according to procedure recommended by the sodium kit from the Human
company ,Germany ( Trinder ,1969 ; Burits and Ashwood , 1999).
3.2.4.4.3 Determination of serum potassium :
Potassium ions in a protein-free alkaline medium react with sodium
tetraphenylboron to produce a finely turbid suspension of potassium
tetraphenylboron. The turbidity produced is proportional to the potassium
concentration and read photometricaly , according to procedure recommended by
the potassium kit from Human company ,Germany (Hillman,1967; Burits and
Ashwood , 1999 ) .
 Chapter three------------------------------------- materials & methods 51

3.2.5 Radiological studies:

3.2.5.1 CT scan
Computed tomography (CT) provides excellent information for defining
differences between bony and soft tissue. In ENT it is used for looking for
problems affecting the temporal bone and the inner ear.
Procedure
The patient lied in supine position on the scanner bed after sedated with
chIoralhydrate (50 mg/kg, administered orally) and were monitored. The scan used
X-rays applied to a circumferential manner to acquire information. Information
was produced in the form of pixel cubes. These can be joined to produce images in
multiple planes. There was a significant radiation dose with this technique
(Corbridge et al., 2006).
3.3 Statistical Analysis:
Statistical analysis was performed using the SPSS programme version 18.
Categorical variables were presented as frequencies and percentages. Continuous
variables were presented as means with their 95% confidence interval. The
Pearson's chi-square test (x2) was used to determine the associations between
categorical variables. Independent sample t-test was used to compare means
between two groups. A p-value of < 0.05 was considered as statistically significant
(Daniel,1999).

View publication stats