Ethical clearance
The approval of the Ethical committee was obtained to conduct the study. The Departments of Otorhinolaryngology and Radiotherapy of the University College Hospital, Ibadan were informed before the study was conducted and permission to enrol their patients was obtained.
Informed consent:
The consent of the patients /guardians of patients taking part in the study were obtained. The details of the procedure were explained to all the participants in the language they understood.
4.6.2 Procedure
A proforma was used to collect data on the age, sex, occupation, educational status and average income of the patients and the participants in the control group. The clinical presentations (symptoms and signs) of the patients were obtained and recorded. This was aimed at identifying the location, clinical diagnosis and staging of the head and neck cancer
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(as developed by the American Joint Committee on Cancer [AJCC/UICC] based on the TNM system84). The diagnosis was confirmed with the histology reports of the patients.
The TNM classification of these Squamous cell carcinomas was then condensed into a convenient number of TNM stage groups as indicated below. This was to ensure that each group is more or less homogenous with respect to survival. This was applicable to all the anatomical sites of HNSCC except the nasopharynx whose stage grouping is slightly different as indicated in the second table below.
Stage grouping for anatomical sites of HNSCC except Nasopharynx and Thyroid84
Stage T N M
0 Tis N0 M0
I T1 N0 M0
II T2 N0 M0
III T1, T2
T3
N1 N0,N1
M0 M0 IVa T1,T2,T3
T4a
N2
N0, N1, N2
M0 M0
IVb Any T
T4b
N3 Any N
M0 M0
IVc Any T Any N M1
34 Stage grouping for Nasopharyngeal carcinoma84
Stage T N M
0 Tis N0 M0
I T1 N0 M0
IIa T2a N0 M0
IIb T1
T2a T2b
N1 N1 N0, N1
M0 M0 M0
III T1
T2a, T2b T3
N2 N2
N0, N1, N2
M0 M0 M0
IVa T4 N0, N1, N2 M0
IVb Any T N3 M0
IVc Any T Any N M1
NUTRITIONAL STATUS
The nutritional status of the patients and the controls were evaluated using the conventional means.
1. Anthropometric measurement:
a. Weight measurement: The weight was determined using a Tanita HD-314 digital weighing scale manufactured by Tanita from Tokyo, Japan that has been standardized. Prior to measurement, shoes, heavy clothes and ornaments were removed. The weight was measured in kilograms.
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b. Height measurement: The height was measured in standing position bare- footed using a Seca 213 portable Stadiometer manufactured by Seca from Hamburg, Germany. This was measured in meters.
c. The Body Mass Index (BMI): This was calculated by dividing the weight (Kg) by the square of the height in metres. The values obtained (Kg/m2) were recorded and compared to the reference values according to the World Health Organisation (WHO). The case and control were then categorised into underweight if values obtained were < 18.5kg/m2, Adequate weight if the values obtained were between 18.5 to 24.9kg/m2, Overweight if the value obtained was between 25 to 29.9kg/m2, Moderately obese between 30 - 34.9kg/m2, Severely obese: 35 - 39.9kg/m2 and Morbidly obese > 40kg/m2. d. Mid upper arm circumference: The arm of the patient was exposed.
Thereafter, I stood on the right side of the patient and palpated for the tip of the acromium process of the right shoulder. I then placed a measuring tape with the zero point of this tip at this point. I palpated for the tip of the olecranon process of the same arm. The tape was drawn to this point and the value was noted. This value was divided into two to obtain its midpoint which was marked. At the midpoint of the arm, a non-elastic measuring tape was applied to the circumference without pressure on the skin. The value obtained was recorded in centimetres (cm).
e. Waist circumference: The procedure was explained to the participant. All clothing and accessories were removed from the abdominal region of the patient this was to remove any pressure on the abdomen. Male participants were requested to remove their upper garment. Then, the participant was asked to stand with their feet together and cross their arms over their chest in
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a relaxed manner. I palpated for the most superior border of the iliac crest on both sides which was marked with a washable marker. The zero end of a non-elastic measuring tape was places on one of the points and then passed around the circumference of the abdomen, at the level of the umbilicus, without pressure on the skin through the other marked point on the contralateral iliac crest and aligned with the marked point. The value obtained was recorded in centimetres (cm).
2. Determination of serum protein and Zinc levels.
Blood collection procedure: I explained the procedure of blood collection for the investigation to the participants. Thereafter, the patient was sat comfortably in a semi recumbent position. I wore a pair of sterile latex (disposable) gloves and also put on a facemask. A tourniquet was applied above the left or right antecubital fossa of the participants. The skin over the antecubital fossa was cleaned with a local antiseptic (90%
alcohol). The participants were occasionally required to make a fist on the hand through which blood sample will be collected so as to make the antecubital vein prominent. After identification of the vein, a 21guage sterile needle connected to a sterile 10 millilitres (ml) syringe was gently introduced to puncture the vein. About 10ml of blood was withdrawn into the syringe under negative pressure. The tourniquet was removed, the needle withdrawn and bleeding stopped by applying digital pressure to the needle prick point with a piece of dry sterile cotton wool for 2 to three minutes. About 5 ml of the blood was immediately transferred into propylene bottles that have been coated with potassium ethylene diamine triacetic acid (K+EDTA) as the anticoagulant for serum zinc assay and the remaining 5 ml was transferred into propylene bottles that have been coated with Lithium heparin as the anti-coagulant for serum protein and albumin assay.
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The bottles were labelled, kept under ice pack at temperature of -5 to 100 C and transported to the Chemical pathology laboratory of International Institute of Tropical Agriculture, Ibadan.
This is one of the world’s leading research partners in finding solutions to hunger, malnutrition and poverty. It was established in 1967 with its headquarters in Ibadan, south-western Nigeria.
Obtaining serum from the collected blood sample
Once the collected samples were in the laboratory, plasma was separated from blood cells by placing the specimen bottles in a centrifuge machine. This was then spun at 2000-2500 revolutions per minute (rpm) for 15-20 minutes. The resulting supernatant (plasma) was withdrawn with Pasteur pipettes and transferred into a clean container. Thereafter, the serum was separated from plasma by using a centrifuge with fractionating capillary tubes at 650 rpm for 5 minutes and transferred to another container using the Pasteur pipettes.
Serum protein and albumin levels determination
Total serum level of protein and albumin in each of the patients was determined using the Bio-Rad protein assay based on the method of Bradford. It involved adding a soluble dye to the protein solution (in this case serum) and subsequent measurement at 595 nm with a Spectrophotometer or micro plate reader. In comparism to a predetermined standard curve, the relative measurement of protein and albumin concentrations in the serum were determined. The values were measured in grams per decilitre. The reference value for the total serum protein was 6.0 to 8.0 grams per decilitre. The serum albumin level was considered adequate if the obtained value was between 3.5 and 5g/dl. It is mild depletion if
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value is from 2.7 to 3.4g/dl, moderate depletion if from 2.1 to 2.6g/dl and severe depletion if less than 2.1 g/dl.
Serum Zinc determination/assay
To determine the Zinc level, one millilitre (1ml) of serum was deproteinized with 9ml of 10%
(w/v) trichloroacetic acid (TCA) in 0.1% lanthanum solution.
I diluted the deproteinized serum with 4 parts of distilled water (1:4) and aspirated the serum into the Atomic Absorption Spectrophotometer (AAS [Buck Model 205 manufactured by Labcompar, South San Francisco, United States of America])
Standards and blanks were prepared by diluting with 5% glycerine using the recommended standard series of 1, 3 and 6 parts per million (ppm). The level obtained was recorded.
The level was then recorded in micrograms per decilitre (µg/dl).The reference value (range) was 70 to 120 micrograms per decilitre.
The above blood collection procedure and serum Zinc level determination were also carried out on the participants in the control group.
4.6.3 Financial implications
This study was done at no extra cost to the participants. The cost of assaying the zinc serum level was borne entirely by me.