09 Chapter 3

Materials and
Methods
Materials and methods
This chapter deals with the materials and methods implemented for the present
study. Research methodology is a way to elucidate scientifically how research was
carried out. It includes not only the research methods but also considers the reason
behind the methods used. Details of the procedures implemented in this chapter are
given under following heading.
3.1. Study setting, type and duration
3.2. Ethical aspect
3.3. Selection of subjects
3.4. Subjects eligibility criteria
3.5. Data collection
3.5.1. Demographic information:
3.5.2. Anthropometric and Physiological measurement
3.5.3. Blood collection, preparation and Biochemical investigations
3.5.4. Hematological investigation
3.5.5. Pulmonary function tests
3.6. Pilot study
3.7. Statistical analysis
3.1. Study setting, Type and Duration:
The present cross-sectional study was conducted in the Department of
Physiology, M.P. Shah Medical College, Jamnagar, India. This study was carried out
for a period of three years from July 2011 to October 2014.The subjects of either sex
and different age groups (26 to 65 years) were randomly selected from the attending
diabetes clinic and out patient’s clinic of Department of Medicine, M.P. Shah
Government Medical College and Guru Gobind Singh Government Hospital
Jamnagar.
The present study was done on a total of three hundred subjects, including
200 subjects with metabolic syndrome and 100 healthy subjects. 200 subjects with
metabolic syndrome (MetS) who fulfilled the National Cholesterol Education
Program, Adult Treatment Panel-III (NCEP ATP-III) criteria were included in the
study group (MetS group). 100 healthy volunteers of either sex or different age (26 to
65 years) without previous history of diabetes; hypertension, dyslipidemia, thyroid
and respiratory disorders were considered as control subjects. They were chosen from
the staff working in Medical College and associated hospital and relative of patient’s.
All metabolic subjects (n=200) who fulfilled the NCEP-ATP III criteria, were further
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classified into subgroups according (after estimation of metabolic components,

thyroid function and pulmonary function) to following:
3.1.1 According to pattern of thyroid dysfunction:
All metabolic subjects were classified into the following subgroups based on
the American Thyroid Association guideline for detection of thyroid dysfunction.
Normal range for TSH was 0.27–4.2 μIU/ml, T3 was 0.86-2.02 ng/ml, and for T4 was
5.13-14.06 µg/dl. A high serum TSH level (4.2–10 μIU/ml) and normal T3 and T4
levels were required for the diagnosis of subclinical hypothyroidism. Patients with
high TSH (>10 μIU/ml) and low T3 and T4 levels were classified as being overt or
clinical hypothyroid and Subclinical hyperthyroidism was characterized by circulating
TSH levels below the reference range and normal serum thyroid hormone levels.
Clinical hyperthyroidism was defined as a TSH concentration of less than below
reference range with an elevated T3 and T4 levels. Patients with normal TSH, T3, and
T4 were considered euthyroid (265).
Subgroup-I: Euthyroid metabolic subjects (n= 117)
Subgroup-II: Subclinical hypothyroid metabolic subjects (n=54)
Subgroup-III: Clinical hypothyroid metabolic subjects (n=26)
Subgroup-IV: Sub clinical hyperthyroid metabolic subjects (n=3)
3.1.2 According to ventilatory pattern of pulmonary functions:
According to an American Thoracic Society (ATS), Obstructive lung
impairment was defined as an FEV1-to-FVC ratio < 70% and an FVC > 80% of the %
predicted value. Restrictive lung impairment was defined as an FVC < 80% of the
%predicted value and an FEV1-to-FVC ratio >70%. Mixed lung impairment was
defined as a FEV1-to-FVC ratio < 70% and FVC < 80% of the % predicted value. The
(266)
normal lung function was defined as FVC >80% and FEV1/FVC > 70% . All
metabolic subjects were classified into the three subgroups based on ATS guideline.
Subgroup-I: Metabolic subjects with normal pattern (n=100)
Subgroup-II: Metabolic subjects with restrictive pattern (n=66)
Subgroup-III: Metabolic subjects with obstructive pattern (n=26)
Subgroup-IV: Metabolic subjects with mixed pattern (n=8)
3.1.3 According to numbers of metabolic components:
All metabolic subjects were divided into three subgroups according to
presence of metabolic components (NCEP-ATP criteria, 2001) (31).
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Subgroup-I: It comprised of 97 subjects (61 male & 36 female) with presence of any
three metabolic components out of five.
Subgroup-II: It comprised of 67 subjects (21 male & 46 female) with presence of any
four metabolic components out of five.
Subgroup-III: It comprised of 36 subjects (7 male & 29 female) with presence of five
metabolic components.
3.1.4. According to individuals of metabolic components:
1. Obesity (BMI):
Obesity and overweight was known by body mass index criteria (BMI)
(267)
suggested by World Health Organization (WHO) (2004) . All metabolic subjects
were classified into the three subgroups based on WHO guideline.
Table 3.1: WHO global existing norms and modified norms for adult Asian population
BMI status Global existing norms* For Asian population

Under weight < 18.5 <18.5
Normal 18.5-24.9 18.5-23.0
Overweight 25.0-29.9 23.0-27.5
Obese class I 30.0-34.9 >27.5
Obese class II 35.0-39.9 >32.5
Obese class III 40+ >37.5
*Global existing norms were used for the present study.
Subgroup-I: Normal weight (n=32)

Subgroup-II: Over weight (n=50)
Subgroup-III: Obese (class I, II & III) (n=118)
All metabolic subjects were divided into following subgroups according to types of
metabolic components (NCEP-ATP criteria) (31)
2. Abdominal non-obese (n=111) & Abdominal obese (n=89)
3. Non-diabetes (n=45) & Diabetes (T2DM; n=155)
4. Eulipidemic (n=89) & Dyslipidemic (n=111)
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RESEARCH DESIGN
SUBJECTS
(Metabolic and Non-Metabolic Subjects Selected From
Population of Saurashtra Region, Gujarat)
SAMPL
(Total 300 Subjects by Random Sampling)
It’s Included, 200 Metabolic and 100 Non-Metabolic
Subjects-NCEP ATP-III Criteria
RESERCH TOOLS
(Socio-Demographic Information, Anthropometric
Measurement, Physiological, Biochemical, Hematological
and Pulmonary Functions Observation
DATA ANALYSIS
(Descriptive Statistics, Chi-Square Test, Unpaired Student -t
Test, One Way Analysis of Variance with Post Hoc Tukey
(HSD), Multiple Linear Regression Analysis)
OUTCOME
Association of Thyroid and Pulmonary Function with
Components of Metabolic Syndrome
Figure 3.1: Research Design
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3.2. Ethical aspect:

The Institutional Ethics Committee reviewed and approved the study protocol,
proforma and the consent form to be used for the present study. The study protocol
was also approved by research development committee of Saurashtra University,
Rajkot, in a meeting dated 11 April 2014.
The purpose of the study was explained to each subject and an informed
written consent was taken from all the subjects prior to the study. Copy of consent
form was available in English and Gujarati.
3.3. Selection of subjects:
All subjects were examined clinically and diagnosis of metabolic syndrome by
using the NCEP ATP-III criteria.
3.3.1. Definitions of metabolic syndrome (MetS) used in the study:
The components of the metabolic syndrome were defined in accordance with
the National Cholesterol Education Program, Adult Treatment Panel III criteria
(NCEP/ATP III 2001) (31). The metabolic syndrome was diagnosed in the presence of
any three or more of the following five components.
• Abdominal obesity, defined as a waist circumference: >102 cm (40 inch) in
men and >88 cm (35inch) in women.
• Blood pressure>130/85 mmHg or drug treatment for elevated blood pressure.
• Serum triglyceride concentration >150 mg/dL (1.7mmol/L) or drug treatment
for elevated triglycerides.
• Serum HDL-C concentration < 40 mg/dL (1.03 mmol/L) in men and <50
mg/dL (1.3 mmol/L) in women or drug treatment for low HDL-C.
• Fasting plasma glucose > 110 mg/dL (5.6mmol/L) or drug treatment for
elevated blood glucose.
3.3.2. Definition of thyroid disorders:
According to American Thyroid Association guideline (265), Normal range for
TSH was 0.27–4.2 μIU/ml, T3 was 0.86-2.02 ng/ml, and for T4 was 5.13-14.06 µg/dl.
A high serum TSH level (4.2–15 μIU/ml) and normal T3 and T4 levels were required
for the diagnosis of subclinical hypothyroidism. Patients with high TSH (>15 μIU/ml)
and low T3 and T4 levels were classified as being overt or clinical hypothyroid and
Subclinical hyperthyroidism was characterized by circulating TSH levels below the
reference range and normal serum thyroid hormone levels. Patients with normal TSH,
T3, and T4 were considered euthyroid
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3.3.3. Definition of ventilatory pattern of pulmonary functions:

According to an American Thoracic Society (266), Obstructive lung impairment
was defined as an FEV1-to-FVC ratio < 70% and an FVC > 80% of the % predicted
value. Restrictive lung impairment was defined as an FVC < 80% of the %predicted
value and an FEV1-to-FVC ratio >70%. Mixed lung impairment was defined as a
FEV1-to-FVC ratio < 70% and FVC < 80% of the % predicted value. The normal lung
function was defined as FVC >80% and FEV1/FVC > 70%.
3.4. Subjects eligibility criteria:
The subjects were selected based on the following inclusion and exclusion criteria.
3.4.1. Inclusion criteria for Metabolic Syndrome subjects:
1. Subjects of aged between 26 to 65 years, who fulfilled the criteria for metabolic
syndrome by NCEP-ATP III, were taken into study.
2. Readiness to participate in the study.
3.4.2. Inclusion criteria for Non Metabolic Syndrome subjects:
1. Clinically healthy subjects without previous history of components of MetS,
thyroid disorders, and respiratory disorders were included in the study.
2. Readiness to participate in the study.
3. Subjects were aged between 26 to 65 years.
3.4.3. Exclusion criteria:
Subjects with any of the following characteristics were excluded from the
study after detailed evaluated by a physician: subjects with a history of thyroid
dysfunction (hyperthyroidism or hypothyroidism), pulmonary diseases (lung cancer,
pleural surgery, pulmonary embolism, lung tuberculosis, suffered from COPD,
emphysema), significant chronic diseases (renal disease, hepatic disease or having a
myocardial condition), chronic drug usage (steroid treatment, antidepressant or anti
psychotic drug, oral contraceptives), immobile, smoking, subject with history of
workplace dust exposure. Subjects were also excluded if they exhibited endocrine
obesity, pregnancy and lactation, presence of ascitis and history of heart failure,
significant neurological or psychological illness (depression, epilepsy, schizophrenia),
subject with infectious disease or other diseases associated with systemic
inflammation (rheumatoid arthritis, connective tissue disorders, inflammatory bowel
disease) that will have an impact on high sensitivity C- reactive protein level, thyroid
and pulmonary function test.
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Table: 3.2 List of various measurements taken, instruments used with the respective units
Measurement Instrument used Units
Height Stadiometer with the least count of 1.0 cm. cm
Weight Electronic weighing scale the least count of 0.5kg kg
BMI Calculated kg/m2
WC Circumference Flexible non elastic tape with a least count of 0.1 cm cm
Hip Circumference Flexible non elastic tape with a least count of 0.1 cm cm
W/H Ratio Calculated
Blood pressure (SBP & DBP) Sphygmomanometer and stethoscope mmHg
Pulse rate Stop watch pulse/min.
Fasting blood sugar Biochemical Auto Analyzer mg/dl
Lipid profile Biochemical Auto Analyzer mg/dl
hs-CRP Spectrophotometer mg/L
Fasting Insulin ELISA µU/mL
Insulin Resistance(HOMA-IR) Calculated
Thyroid profile Cobas e411, Electrochemiluminescence (ECL)
TSH Cobas e41, Electrochemiluminescence (ECL), µIU/mL
T3 Cobas e411, Electrochemiluminescence (ECL) ng/mL
T4 Cobas e411, Electrochemiluminescence (ECL) µg/dL
TLC Hemocytometer Cells/cumm
DLC Hemocytometer %
TEC Hemocytometer Cell/cumm
Pulmonary functions Portable Helios 401 RMS spiromerte % Predicted
WC=Waist circumference, TLC=Total leucocytes count, DLC=Differential leucocytes count,
TEC=Total Eosinophils count
3.5. Data collection:

1. Appropriate questionnaires were prepared to collect qualitative data relating to:
• Demographic and basic information including age, sex, education status,
occupation, socio-economic status, individual & family profile etc.
• Smoking and alcohol consumption.
• Personal and family medical history of the subjects for the components of
metabolic syndrome.
• Dietary habits such as vegetarian and non-vegetarian or mixed
• Regular physical activity and exercise pattern.
• Sleep pattern.
2. Anthropometric measurements like height, weight, waist and hip circumference
were taken using standardized techniques.
3. Subjects were investigative for blood pressure.
4. Data relating to biochemical parameters like fasting blood glucose, Insulin, lipid
profile, hs-CRP and thyroid functions test were measured using commercial
diagnostic kit.
5. Data relating to hematological parameters like total leucocytes count, differential
leucocytes count and total eosinophils counts were measured using hemocytometer.
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6. Data relating to Pulmonary functions like FVC, FEV1, FEV1/ FVC ratio, FEV3,
FEV3/FVC, PEFR and FEF25-75% were performed using an automated flow-sensing
spirometer based on American Thoracic Society/ European Respiratory Society
guidelines (ATS/ERS 2005).
3.5.1. Demographic information:
Demographic information was collected from all the subjects meeting the
inclusion criteria. A total of 300 subjects were included in the present study. All
subjects were interviewed through structured Proforma which containing close ended
questions regarding demographic information, personal and family medical history,
were used to gather information on various socio-demographic variables such as
name, occupation, education status, age, sex, health status, marital status, dietary
habit, physical activity level, life style pattern-sedentary or non-sedentary, sleep
pattern etc. Subjects were also requested in relation to the history of other diseases
(Particularly thyroid disease, diabetes, hypertension, dyslipidemia, coronary artery
heart disease (CHD), Myocardial infarction (MI), stroke, pancreatitis, tuberculosis
and their treatment), smoking habit, alcohol consumption, family history of
components of metabolic syndrome in first degree relatives (like parents, siblings) and
coronary heart disease. Coronary heart disease (CHD) was defined as using
nitroglycerine; experiencing characteristic chest pain or having a history of previous
myocardial infarction.
3.5.2. Anthropometric and physiological parameters:
All anthropometric variables together with height, weight, waist & hip
circumferences, body mass index, waist to hip ratio as well as physiological
parameters arterial blood pressure and pulse rate were taken using standardized
measures. All instruments were standardized and the zero error was checked before
each measurement. Brief metaphors of all the measurements are specified underneath:
3.5.2.1. Body Weight Measurement
This is the most commonly used, easy and reproducible anthropometric
parameter for assessment of dietary status. Weight shows the body mass that is the
composite of whole body ingredients like water, minerals, fat, proteins and bone (268).
Body weight was taken with minimum possible clothing on and standard
electronic weighing scale was used for this reason and the reading was recorded to the
nearest 0.5 kg. The subject was asked to remove shoes, extra clothing and other
accessories and was requested to stand erect on the platform of the weighing machine
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and the weight was recorded when the reading become stationary. The instrument was
checked for zero error before obtaining the measurement. Care was taken that the
subject did not move while the measurement was taken. Weighing scale was
calibrated using standard weight after every fifth subject.
3.5.2.2: Measurement of Height
Height is the highest distance from the floor to the vertex of the head. The
vertex is defined as the highest point on the skull when the head is held in the
Frankfort plane (a line connecting the superior border of the external auditory meatus
with the infra-orbital margin).
Height was measured to the nearest 1.0 cm without shoes and socks using a
stadiometer. The subject was made to stand on a horizontal platform against
stadiometer with heels together, extended upwards to the fullest extent, aided by the
gentle upward pressure by the researcher on the mastoid process and by encouraging
the subject to stand tall, to make a deep breath and relax. The subject’s back was kept
as straight as probable. Each subjects stood with heels, buttocks and shoulders resting
lightly against the backing board so that the Frankfurt plane (a line connecting the
superior border of the external auditory meatus with the infraorbital rim or margin)
was maintained. At same time placed the horizontal arm or headboard firmly down on
the vertex, crushing the hair as much as possible. It was made sure that the heels did
not leave the ground in an attempt to stand tall.
3.5.2.3. Body mass index (BMI):
Body mass index was defined as weight in kilograms over the square of the
height in meters (kg/m2) (WHO, 2000). BMI gives a consistent marker of body
fatness for most people and is used to screen for weight categories that may lead to
health problems. This is a measure of the relative body fatness to estimate risk factors
related with obesity. It is based on weight (in kg) with minimal clothing and height (in
meters) without shoes (269).
For adults, WHO (2004) defined cut off points has been used which are as follows:
(267)
Table 3.3: WHO global existing norms and modified norms for adult Asian population
BMI status Global existing norms For Asian population
Under weight < 18.5 <18.5
Normal 18.5-24.9 18.5-23.0
Overweight 25.0-29.9 23.0-27.5
Obese class I 30.0-34.9 >27.5
Obese class II 35.0-39.9 >32.5
Obese class III 40+ >37.5
Global existing norms were used for the present study.
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3.5.2.4. Waist circumference (WC):

Waist circumference (WC) gives a unique indicator of body fat distribution,
which can recognize patients who are at elevated risk of obesity associated cardio-
metabolic disease, over and ahead of BMI. WC assessment can from time to time give
extra information to help the physician decide which patients should be estimated for
the presence of cardio-metabolic risk factors, such as dyslipidemia, and
hyperglycemia. Furthermore, evaluating WC can be helpful in watching a patient's
response to diet and exercise management, because usual aerobic exercise can cause a
reduction in both WC and cardio-metabolic risk, without a change in BMI (270).
Waist circumference should be calculated at a level halfway between the
lower rib margin and iliac crest with the tape all around the body in parallel position.
The tape should be wobbly sufficient to permit the observer to put one finger between
the tape and the subject's body. Subjects are requested to take away their clothes,
except for light underwear. If this is not feasible, for instance due to cultural reasons,
the option is to determine the circumference on the subject without heavy external
clothes and record this fact in the data collection Proforma. Tight clothing, as well as
the belt, should be relaxed and the pockets emptied; Subjects were ready to stand with
their feet quite close together (about 12-15 cm) with their weight evenly circulated on
each leg; Subjects were requested to breathe usually; the reading of the measurement
was obtained at the finish of mild exhaling to avoid subjects from contracting their
abdominal muscles or from holding their breath; The measuring tape was held
resolutely, ensuring its horizontal position.
3.5.2.5. Hip circumference:
It measures the circumference of the hips at their widest part. The subject was
requested to stand straight, the tape was moved around the buttocks and the reading
was noted. Whereas obtaining the measurement care was in use that the subject was
wearing least feasible clothing and steel tape was not pulled very hard and was kept
horizontal.
3.5.2.6. Waist to hip ratio (WHR):
The ratio of waist circumference to hip circumference has been observed to be
a more receptive index of metabolic abnormalities in obese person than the use of
neck, bust, waist, hip circumference alone. The ratio is find out as the ratio of waist
circumference (WC) to hip circumference (HC).
136
The waist to hip ratio (WHR, according to the WHO criteria, for males normal was <
1 and for females < 0.9 and according to the adapted Asian definition normal for
males is < 0.90 and for females it was < 0.85 was designed as a calculate of fat
allocation (central obesity) while BM1 was measured a measure of adiposity (271).
3.5.2.7. Physiological measurement:
Blood Pressure
The systolic as well as diastolic blood pressures were evaluated. Blood
pressure is the lateral pressure exerted by column of blood on vessel walls. Arterial
blood pressure of subjects was measured using sphygmomanometer on the arm in
sitting position at heart level.
Systolic blood pressure (SBP):
It measures the blood pressure exerted by the column of blood against the wall
of the blood vessels, during the systole of the heart. The subject was request to sit
comfortable on a chair. The pressure or Riva-rocci cuff was applied intimately to the
upper arm with its lower border about one inch above the elbow joint. The cuff was
quickly inflated to a pressure more than 180 mmHg. The chest piece of stethoscope
was then put lightly over the brachial artery in the cubital fossa, and the mercury
column in manometer was permitted to fall at the rate of around 2 mmHg per second.
The SBP was decided by the starting of Korotkoff sound. Three readings were
obtained and mean of that was taken as the final reading.
Diastolic blood pressure (DBP):
It measures the blood pressure exerted by the column of blood against the wall
of vessels during the diastole of heart. The procedure implemented for measuring
systolic blood pressure was followed. After recording the systolic blood pressure the
mercury column was permitted the drop more till the sound ceased to be tapping in
quality, become fully muffled and finally disappeared. The level where it disappeared
was taken as diastolic blood pressure. While taking blood pressure measurement, care
was taken that the subjects were comfortable and had not eaten or smoked at least an
hour earlier to the recording.
Table3.4: Categories for Blood Pressure Levels in Adults (In mmHg)
Category Systolic blood pressure Diastolic blood pressure
Normal Less than 120 Less than 80
Pre hypertension 120–139 80–89
High blood pressure
Stage 1 140–159 90–99
Stage 2 160 or higher 100 or higher
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Table 3.4 shows normal blood pressure values for adults. It also shows which values
put an individual at higher risk for health problems (272).
3.5.2.8. Pulse rate:
This was considered by palpating the radial artery against solid radial bone at
the wrist in lateral semi flexed. The subject was requested to keep his/her arm in
supine position on the arm of the chair and the pulse rate was recorded with the help
of stop watch.
3.5.3. Blood sample collection, preparation & Biochemical Investigations:
All subjects were instructed, not to do any exhausting exercise for at least 24
hours earlier to collection of blood samples. The blood samples were withdrawn
between 09:00 to 11:00 AM subsequent to an overnight fast, from anti-cubital vein of
all the subjects by using aseptic method. The blood sample (5 ml) was collected in
plain; fluoride and Disodium salt of Ethylenediamine tetra acetic acid (EDTA) tubes.
Blood sample (3ml) which was collected in plain dry sample tubes and incubated at
37˚C or room temperature for 25-35 minutes. Following incubation, blood clot was
removed and remaining sample was taken in centrifuge test tube. Samples were
centrifuged at 3000 rpm (Revolution per minute) for 6 to 12 minutes to separate out
the serum. Supernatant (serum) transferred in clean serum micro-tube and stored at -
25°C to-35°C in the refrigerator until required for analysis of lipid profile (total
cholesterol, HDL-C, triglycerides), thyroid hormone (TSH, T3 and T4) and insulin as
well as inflammatory marker hs-CRP. Part of blood sample (1ml) was collected in
fluoride tube used for fasting plasma glucose estimation and reaming portion of blood
(1ml) was placed into EDTA tube used for hematological parameters like total white
blood cells count (WBC), differential leukocyte counts (DLC) and total eosinophils
counts (TEC).
Biochemical Investigations:
Biochemical parameters, fasting blood sugar and lipid profile (total
cholesterol, triglycerides and HDL-C) were estimated by biochemical analyser in a
commercially available kit according to manufacturer instructions. Low-density
lipoprotein cholesterol (LDL-C) and very low-density lipoprotein cholesterol (VLDL-
C) were calculated by Friedewald formula (273).
3.5.3.1 Estimation of serum glucose-ERBA diagnostic Mannheim GmbH (274):
 Method (Enzymatic end point-Trinder’s)
 Principle: Glucose oxidase (GOD) oxidizes plasma glucose to gluconic acid and
hydrogen peroxide then hydrogen peroxide is bind with phenol or 4-Hydroxy
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benzoic acid (4-HBA) and 4-Aminoantipyrine (4-AAP) to formed pink color

quinoneimine dye, in presence of enzyme peroxidase.
Glucose oxidase
Glucose + O2 + H2O Gluconic acid + H2O2
Peroxidase
H2O2+ 4 HBA + 4-AAP Quinoneimine dye + H2O
(4-AAP: 4-Aminoantipyrine, Phenol or 4-HBA: 4- Hydroxy benzoic acid)
The intensity of the pink color formed is proportional to the glucose

concentration in blood and can be measured photo-metrically between 500 to 540 nm.
 Reagent composition: blood glucose reagents are ready to use.
Glucose oxidase 20000 IU/L
4-Hydroxy-benzoic acid 10 m mol/L
Peroxidase 3250 I U/L
4-Aminoantipyrine 0.52 m mol/L
Phosphate buffer, pH 7.4 110 m mol/L
Glucose Standard: 100 mg/dl (5.55 mmol/L); Sample- Plasma
 Procedure: -
Mode of reaction End point method
Wavelength 505 nm
Cuvette 0.6 cm light path
Reaction Temperature 37°C or Room Temperature
Measurement -Blank Reagent blank
Sample Volume 5/10 μl
Depends on sample volume reagent Volume 500/1000 μl
Incubation 15 minutes at 37°C
Linearity (mg/dl) 500 mg/dL
Glucose Standard Concentration 100 mg/dL
Absorbance Limit (Max) 0.3
Reagent and Sample Blank Standard Test

Working Reagent 1000µl 1000µl 1000µl
Distilled Water 10µl - -
Standard 10µl
Sample (Serum) - - 10µl
Mix well and incubate for 15 minutes at 37°C. Read the absorbance of standard and
each test tube against blank at 505 nm on biochemical analyzer.
Calculation:
Glucose concentration (mg/dl) =
Conversion factor: mg/dl x 0.0555 = mmol/L
mg/dl x 0.01= g/L
mmol/L x 18 = mg/dl
Reference Values: Fasting blood glucose: 70- 110 mg/dl (3.89- 5.83 mmol/L).
Post prandial: 90-130 mg/dl
139
3.5.3.2. Estimation of serum total cholesterol-Tulip diagnostic group India. (275):

Method: CHOD / PAP (End point)
Principle: Cholesterol esterase hydrolyses esterified cholesterols to free cholesterol
and fatty acid. The free cholesterol is oxidized in the presence of enzyme cholesterol
oxidase, to form hydrogen peroxide which further reacts with phenol and 4-
aminoantipyrine by the catalytic action of peroxidase to form a red colored
quinoneimine dye complex. Intensity of the color formed is directly proportional to
the amount of cholesterol present in the sample.
Cholesterol Esterase
Cholesterol ester + H2O Cholesterol + fatty acid
Cholesterol Oxidase
Cholesterol + 1/2O2 + H2O Cholesteone + H2O2
Peroxidase
H2O2+ Phenol + 4-AAP Red Quinoneimine Dye + H2O
Reagent composition and preparation: Reagents are ready to use

Sodium cholate 5 m mol/L
Phenol: 24 m mol/L
Cholesterol esterase > 180 IU/L
Cholesterol oxidase > 200 IU/L
Peroxidase > 1000 IU/L
4-Aminoantipyrine 0.5 m mol/L, pH 7.0
Procedure: This reagent can be used on most of biochemical analysers.
Wavelength 505 nm
Reaction Mode End Point
Sample Volume 10 μl or 0.01 ml
Reagent Volume 1000 μl or 1.00 ml
Measurement (Blanking) Against Reagent Blank
Incubation 5 minutes at 37°C
Linearity 750 mg/dL
Reaction Temperature 37°C or R.T.
Standard Concentration 200 mg/dL
Manual Procedure:
Standard 10µl
Sample type: Serum free from haemolysis and standard: cholesterol: 200 mg/dl
Mix and incubate for 5 minute at 37°C. Read the absorbance of standard and test
against reagent blank.
Calculation:
Reference values: 150-200 mg/dl
140
3.5.3.3. Estimation of serum triglycerides: Tulip diagnostic group India

Method: GPO/PAP (Enzymatic end point method) (276)
Principle:
Lipoprotein Lipase
Triglycerides + H2O Glycerol + Fatty acids
Glycerol Kinase
Glycerol + ATP Glycerol-3-phosphate + ADP
G -3-P-Oxidase
Glycerol-3-P + O2 Dihydroxyacetone -P + H2O2
Peroxidase
2H2O2 + 4-AAP + 4-Chlorophenol Quinonemine + 4 H2O2
Lipoprotein Lipase hydrolyses serum triglycerides to glycerol & free fatty

acids. Glycerol, in turn is converted to glycerol 3-phosphate in presence of ATP and
glycerol-kinase. Glycerol-3-phosphate is subsequently oxidized by glycerolphosphate
oxidase (G-3-P-oxidase) to form dihydro-oxyacetone as well as hydrogen peroxide
which is further reacts with 4-cholrophenol and 4-aminoantipyrine by the catalytic
action of peroxidase to give a red colored (quinoneimine dye) complex. The intensity
of colour is measured photometrically at 545 nm. The intensity of colour is directly
proportional to the triglyceride concentration in the sample
Reagent composition & preparation: Reagents are ready to use and to store at 2-6°C.
Lipoprotein lipase 2000 U/L
Glycerol Kinase 500 U/L
Potassium ferrocyanide 10 µmol/L
Amino-4- antipyrine 0.31 mmol/L
Glycerol-3- phosphate Oxidase 4000 U/L
Peroxides 500 U/L
Reagents: R1-Enzyme Reagent
Standard: Glycerol (triglycerides equivalent): 200 mg/ dl (02 g/L, 2.28 mmol/L)
Procedure: This reagent can be used on most of biochemical analyzers.
Wavelength 505 nm
Reaction Mode End Point
Cuvette 1 cm light path
Reaction Temperature 37°C or RT
Measurement (Blanking) Reagent Blank
Sample Volume 10 μl
Reagent Volume 1000 μl
Standard Concentration 200 mg/dL
Incubation 5 min.
Linearity 1000 mg/dL
Manual methods: Absorbance of standard and test read against reagent blank.
Standard 10µl
141
Mix well and incubate for 10 minute at 37°C. Read the absorbance’s of standard and
test against 505nm.
Calculation:
Reference values: Men: 60-165 mg/dL & Women: 40-140 mg/dL

3.5.3.4. Estimation of HDL-Cholesterol: Tulip diagnostic group India
Indirect precipitation Method: The estimation of serum HDL-C was performed by
precipitation method (277).
Principle: When the serum reacted with the polyethylene glycol (PEG) which contains
phosphotungstate/Mg+2, than all the VLDL-C, chylomicrons and LDL-C are
precipitated. High density lipoprotein (HDL-C) fraction remains unchanged in
supernatant. Cholesterol content of HDL fraction is assayed using autozyme
cholesterol.
Phosphotungstate
Serum HDL Fraction+ (LDL+VLDL+Chylomicrons)
Mg +2
(Supernatant) (precipitate)
Components and concentration of precipitating reagent:
Component Concentration
Phosphotungstic acid 2.4 mmol/l
Magnesium Chloride 40 mmol/l
Procedure:
HDL separation: Pre warmed at room temperature (25-300 C) the necessary amount of
precipitating reagent and autozyme cholesterol working solution before use. Done the
assay as the given below
Pipette as follows:
Serum/plasma 0.5 ml
HDL-Precipitating reagent 0.5 ml
Mixed thoroughly and centrifuged at 4000 r/m for 5-10 minutes to get a clear
supernatant.
High density lipoprotein cholesterol (HDL-C) determination:
Reaction type End Point
Reaction time 10 mins.at 370C
Wavelength 510 nm. (505-530 nm.)
Zero setting with Reagent Blank
Blank absorbance limit < 0.100 Abs.
Sample volume 0.05 ml (50 μl)
Reagent volume 1.0 ml
Standard concentration 25 mg/dl
Linearity 400 mg/dl
142
Manual assay procedure:

Test the supernatant for HDL-C after centrifugation using working solution of
autozyme cholesterol reagent.
Reagent and Sample Test Standard Blank
Supernatant 0.05 ml 0.05 ml -
Autozyme cholesterol working solution 1.0 ml 1.0 ml 1.0 ml
Incubation: Incubated the assay mixture for 5-10 minutes at 370C after completion of
the incubation the absorbance of assay mixture was taken against blank at 510 nm.
Calculation:
Where:
2 was the dilution factor due to the serum dilution during precipitating step
Reference value presented in table for male and female.
Reference value: Male Female
Low risk > 50 mg/dL > 60 mg/dL
Normal Risk 35 - 50 mg/dL 45-60 mg/dL
High Risk < 35 mg/dL < 45 mg/dL
3.5.3.5. Estimation of Very Low Density Lipoproteins Cholesterol (VLDL-C):

The estimation of VLDL-cholesterol was performed by following formula as shown
below:
Reference range: Up to 35 mg/dL

3.5.3.6: Estimation of Low Density Lipoproteins Cholesterol (LDL-C):
(273)
The estimation of LDL-C was determined by using formula of Friedwald
as shown below:
LDL-C= Total Cholesterol – (VLDL+ HDL-Cholesterol)
Reference range: Up to 100 mg/dL
TC/HDL-C and LDL-C/HDL-C ratio were there by also calculated
3.5.3.7. Estimation of fasting plasma insulin:
Method: Enzyme-Linked Immune-Sorbent Assay.
Fasting plasma insulin kit: IBL-International ELISA Kit, GmbH & Co. (278).
Principle of the Assay: Insulin ELISA diagnostic kit is a solid phase enzyme-linked
immune-sorbent assay based on the sandwich principle. The micro-titer wells are
coated with a monoclonal antibody directed towards a exclusive antigenic site on the
insulin molecule. An aliquot of patient sample containing endogenous insulin is
incubated in the coated well with enzyme conjugate, which is an anti-insulin antibody
143
conjugated with biotin. After incubation the unbound conjugate is washed off. During
second incubation step streptavidin peroxidase enzyme complex binds to the biotin-
anti-insulin antibody. The quantity of bound HRP complex is proportional to the
concentration of insulin in the sample. Having added the substrate solution, the
strength of color produced is proportional to the concentration of insulin in the patient
sample.
Reagents:
1. Microtiterwells Wells coated with anti-insulin antibody (monoclonal).
2. Zero standard Ready to use 0μIU/mL
3. Standard (1-5) Ready to use,Concentrations:6.25,12.5,25,50,100μIU/mL
4. Enzyme conjugate Ready to use, Mouse monoclonal anti-insulin conjugated to biotin
5. Enzyme complex Ready to use, Streptavidin-HRP complex
6. Substrate solution Ready to use, Tetramethylbenzidine (TMB)
7. Stop solution Ready to use, 0.5M H2SO4
8. Wash solution* 30ml (40X concentration)
Supplementary materials required: A microtiter plate calibrated reader (450±10 nm);

standardize variable precision micropipettes; absorbent paper; distilled or deionised
water; timer ; Graph paper for data reduction.
Carry all reagents and necessary number of strips to achieve room temperature earlier
to use. Wash Solution: Dilute 30 ml of concentrated wash solution with 1170 ml
deionised water to a make final volume of 1200 ml. The diluted wash solution is
stable for 2 weeks at room temperature.
Specimen collection and preparation: Serum or plasma (only heparin or citrate
plasma) can be used in this assay.
Specimen storage: All samples should be capped and may be stored for up to 5 days at
room temperature 2-8°C prior to assaying.
Specimen dilution: If in a primary assay, a sample is observed to contain more than
the maximum standard, the sample can be diluted 10-fold or 100 fold with zero
standard and re-assayed as explained in assay procedure. For the calculation of the
concentrations this dilution factor has to be taken in to explanation for instance (i&ii).
i) Dilution 1:10 = 10 µl serum + 90 µl standard Zero standard (mix carefully)
ii) Dilution 1:100 = 10 µl dilution a) 1:10 + 90 µl zero standard (mix carefully).
Subsequent steps were used for assay procedure:
1. Safe the desired numbers of microtiter wells in the frame holder.
2. Distribute 25µl of each standard, controls and samples with new disposable
tips into suitable wells.
144
3. Distribute 25 µl enzyme conjugate into every well.

4. Thoroughly mix for 10 seconds. It is important to have a complete mixing in
this step.
5. Incubate for 25-30 minutes at room temperature exclusive covering the plate.
6. Quickly shake out the contents of the wells.
7. Rinse the wells 3 times with diluted wash solution (400µl per well). Sock the
wells sharply on absorbent paper to remove residual droplets.
8. Add 50 µl of enzyme complex to each well.
9. Incubate for 25-30 minutes at room temperature.
10. Quickly shake out the contents of the wells. Rinse the wells 3 times with
diluted wash solution (400 µl per well). Sock the wells piercingly on absorbent
paper to take away remaining droplets.
11. Add 50 µl of substrate solution to each well.
12. Incubate for 10-15 minutes at room temperature.
13. Stop the enzymatic reaction by adding 50 µl of stop solution to every well.
14. Read the optical density at 450 ± nm with a microtiter plate reader within 10
minute following adding the stop solution.
Calculation of Results:
1. Determine the average absorbance values for every set of standards, controls
and patient samples.
2. Using liner graph paper, make a standard curve by plotting the mean
absorbance achieved from each standard against its concentration with
absorbance value on the vertical (y) axis and concentration on the (x) axis.
3. Using the mean absorbance value for every sample find out the corresponding
concentration from the standard curve.
4. The concentration of the samples can be read directly from this standard curve.
Samples with concentrations elevated than that of the maximum standard have
to be additional diluted. For the calculation of the concentrations this dilution
factor has to be taken in to description. Below is listed a typical example of a
standard curve with the Insulin ELISA.
Standard Optical Units (450 nm)
Standard 0 (0 µIU/ml) 0.03
Standard 1 (6.25µIU/ml) 0.07
Standard 2 (12.5µIU/ml) 0.14
Standard 5 (100µIU/ml) 2.05
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Expected values: It is sturdily suggested that each laboratory should determine its own
normal and abnormal values. In a study conducted with apparently normal healthy
adults, using the insulin ELISA the following values are found: 2-25 µIU/ ml.
Sensitivity: The analytical sensitivity of the Insulin ELISA was considered by adding
two standard deviations to the mean of 20 replicate analyses of standard A and was
observed to be 1.76 μIU/ ml.
Assay Dynamic Range: The range of the assay is between 1.76-100 μIU/ml.
3.5.3.8. Measurement of insulin resistance by HOMA:
Homeostasis Model Assessment measures insulin resistance (HOMA-IR) and
(279)
beta cell function which was intended by Matthews in 1985 to recognize and
estimate insulin resistance from fasting glucose and fasting insulin levels. This is a
computer-solved model used to predict the homeostatic concentrations which occur
from varying degrees of beta-cell deficiency and insulin resistance. This model
compares subjects fasting values with the models predictions, which permits a
quantitative measurement of the contributions of insulin resistance and deficient beta-
cell function to the hyperglycemia. The correctness of this model has been compared
with independent measures of insulin resistance and beta-cell function using
hyperglycaemic and euglycaemic clamps and an intravenous glucose tolerance test.
Insulin resistance values were derived using the homeostasis model assessment
(HOMA) method, utilizing the equation below.
IR = (Fasting plasma insulin micro units/L) × (Plasma fasting glucose mmol/L) / 22.5
IR = (Fasting plasma insulin micro units/L) × (Plasma fasting glucose mg/dL) / 405
1 mmol/L= 0.0555 mg/dL
Formula for calculation of mg/dl from mmol/L: mg/dl = 18 × mmol/L
Formula for calculation of mmol/L from mg/dl: mmol/L= mg/dl / 18
For instance, if plasma level of glucose is 5 mmol/l, recalculation to mg/dl is done as
follows:
18 × 5 mmol/dl = 90 mg/dl
The normal HOMA-IR value of healthy human ranges from 1.7-2.0.
Category HOMA-IR Score

Normal insulin resistance <3
Moderate insulin resistance Between 3 and 5
Severe insulin resistance >5
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3.5.3.9: Estimation of high sensitivity C-reactive protein (hs-CRP)-Tulip

diagnostic India (280).
C-reactive protein (CRP) is a pentameric acute phase reactant that is produced
by the liver. Its manufacture is regulated primarily by interleukin-6. The serum CRP
concentration may rise by up to 1000-fold with infection, trauma, surgery, and other
acute inflammatory events. Chronic inflammatory disorders such as autoimmune
diseases and malignancy can create constant high levels of serum CRP.
Conventionally, CRP has been used clinically for the diagnosis and monitoring of
auto-immune and infectious disorders. Latest studies have reported that chronic
inflammation is a significant component in the development and succession of
atherosclerosis. As a result, elevated serum CRP concentrations are positively related
with the risk of prospect coronary events.
Method: Turbidimetric Immunoassay
Principle: In this technique determination of C-reactive protein in human serum is
based on the principle of agglutination reaction. The serum is mixed with activation
buffer (R1), Turbilyte-hs-CRP latex reagent (R2) is after that added and permitted to
react. Presence of CRP in the test specimen results in the production of an insoluble
complex producing a turbidity, which is calculated at 546 nm wavelength. The
increase in turbidity corresponds to the concentration of hs-CRP in the test specimen.
Reagent:
1. Turbilyte-CRP Activation Buffer (R1): Prepared to use.
2. Turbilyte-CRP latex reagent (R2): Prepared to use consistent suspension of
polystyrene latex particles coated with anti-CRP antibody.
3. Turbilyte-CRP Calibrator: A lyophilized preparation of serum equal to the stated
amount of CRP on an mg/dl basis, when hydrated suitably. The Turbilyte-CRP
Calibrator is noticeable to the WHO international reference standard (85/506) for
human C-reactive protein.
Reagent storage and stability:
1. Store the reagents at 2-8°C.
2. The ledge life of the reagent, activation buffer and the calibrator is as per the expiry
date revealed on the respective vial label.
3. The reconstituted calibrator is stable for 7 days at 2-8°C and 48 hours at 25°C-30°C
room temperature.
147
4. The working reagent for Turbilyte-CRP can be ready by mixing R2 and R1 in the
ratio 1:10.
5. The mixing stability of the working regent (R1+R2) is 7 days when stored at 2-8ºC.
Specimen and preparation:
Only serum should be used for testing. Should a delay testing arise, store the
samples at 2-8ºC. Samples can be stored for up to three days at 2-8ºC, Do not use
hemolysed, icteric, or highly turbid sera, centrifugation at 2000 rpm for 15 minutes.
Use the clear supernatant for testing.
Procedure: Bring reagent and sample to room temperature before use.
Wavelength 546 nm
Reaction mode 2-point
Reaction Temperature 37ºC
Unit mg/dL
Detection limit (mg/dl) 0.3
Pipette into the cuvette:
Reagent and sample For calibration For sample
R1 500 µl 500µl
Calibrator 5 µl -
Sample - 5 µl
Mix well & incubate for 5 minutes and read absorbance A1.
R2 50 µl 50 µl
Add R2 and mix well, Read absorbance A2 at the end of exactly five minutes.
Calculations:
1. Calculate ΔA = (A2-A1)
Δ
2. Concentration of CRP in sample = Δ
Method for preparation of CRP calibration curve:

Test tube no. 1 2 3 4 5 6
Calibrator dilution no. D1 D2 D3 D4 D5 D6
Volume of Saline in μl - 100 100 100 100 100
Volume of Calibrator (S) in μl 100 100 100 100 100 100
Conc. of CRP in mg/dl 10 5 2.5 1.2 0.6 0.3
The calibrator must be reconstituted precisely with 1.0 ml of distilled water,

wait for 10 minutes quietly whirl the vial till the solution attains homogeneity. Once
reconstituted it is ready to use for preparing the hs-CRP calibration curve. The
Concentration of hs-CRP (S) in the reconstituted calibrator is as mentioned at the end
of the package insert. Dilute the calibrator successively as mentioned below for
preparation of calibration curve.
148
Five dilutions of the calibrator including the maximum 10 mg/dl (D1) and
lowest 0.3 mg/dl (D6) concentrations of measuring range must be used for the
preparation of the calibration curve. Select any three dilutions from D2 to D4 in
addition to D1 and D6 to prepare the calibration curve.
Test method for preparation of calibration curve
1. Zero the instrument with distilled water (Blank).
2. Pipette 500 µl of activation buffer (R1) and 50 µl of standard D1 from tube no.
1 in the measuring cuvette. Mix well and incubate for 5 minutes at 37°C.
3. Read optical absorbance (A1).
4. Add 50 ml of Turbilyte-CRP latex reagent -CRP reagent (R2) (pre incubated
at 370°C), mix quietly and begin the stopwatch at the same time.
5. Read absorbance (A2) at the end of accurately five minutes.
6. Repeat steps No. 2-5 for each diluted calibrator selected for preparing the
calibration curve.
7. Instrument calculates ΔA (A2-A1) for each diluted calibrator for preparing the
calibration curve and plots a graph of ΔA versus concentration of hs-CRP.
Interpolate ΔA for every sample on calibration curve and get hs-CRP
concentration.
Test procedure for specimen
1. For determination of hs-CRP concentration in the test specimen follows steps
2 -5 using the test specimen in place of the calibrator.
2. Calculate ΔA (A2 – A1) for the test specimen.
Reference value:
The American Heart Association and U.S. Centers for Disease Control and
Prevention have defined risk groups as follows (American Association for Clinical
Chemistry) (281).
Normal (Adult) <0.6mg/dL
Low risk <1.00 mg/L
Average risk 1.00-3.00mg/L
High risk >3.00 mg/L
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3.5.3.10. Determination of Thyroid Hormones:

Thyroid hormone (T3, T4) and thyroid stimulating hormone (TSH) were
estimated by the electrochemiluminescence (ECL) technique using commercially
available kits from Roche Diagnostics (Mannheim, Germany) with cobas e 411
analyzer. The reference ranges for thyroid parameters is given in table 3.4.
Table 3.5: Laboratory specific normal ranges of thyroid parameters
Thyroid parameters Reference ranges
FT3 (pM/L) 2.8 – 7.1
FT4 (pM/L) 12 – 22
TSH (μIU/ml 0.27 – 4.2
T3 (ng/ml) 0.86-2.02
T4 (µg/dl) 5.13-14.06
Electrochemiluminescence Assay Principles (282):

Electrochemiluminescence (ECL) processes are identified to arise with several
molecules including compounds of ruthenium, osmium, rhenium or other elements.
ECL is a process in which very reactive species are produced from stable forerunners
at the surface of an electrode. These very reactive species react with one another
generating light. The development of ECL immunoassays is based on the use of a
ruthenium chelate as the complex for the development of light. The chemiluminescent
reactions that lead to the emission of light from the ruthenium complex are initiated
electrically rather than chemically. This is achieved by applying a voltage to the
immunological complexes (including the ruthenium complex) that are attached to
Streptavidin-coated micro particles. Streptavidin, isolated from Streptomyces avidinii
is preferred to avidin in this biotin- mediated immunoassay since it has an affinity for
biotin analogous to that of avidin, is less basic and had no carbohydrate residues,
therefore limiting non-specific reactions with acidic groups and lectins. The
advantage of electrically initiating the chemiluminescent reaction is that the whole
reaction can be exactly controlled (282).
The following chief elements and substances in the ECL methods are:
Measuring cell; Voltage; Platinum electrode; Magnet; Photomultiplier; Antigen/
Antibody; Biotin; Paramagnetic micro beads coated with streptavidin; Pro Cell
solution (TPA-Tripropylamine with a phosphate buffer); Clean Cell solution (KOH
cleaning solution).
The Basic Principle (283): The core of the recognition unit is the flow through ECL
measuring cell. Three processes steps are done in the cell:
150
1. Bound/ free separation

 Streptavidin microbeads coated with antigen-antibody complex are aspirated.
 The magnet is activated.
 The antigen-antibody complex is captured by the magnet onto the working
electrode.
 A TPA solution (Pro Cell) is aspirated to rinse the microbeads on the working
electrode.
 TPA (Pro Cell) is used to flush out the surplus reagent and sample material.
2. ECL reaction
 The ruthenium complex and TPA (Pro Cell) are concerned in the reactions.
 They remain stable as long as no voltage is applied.
 Voltage is applied between the working and counting electrodes and an
electrical field is generated.
 An ECL reaction of ruthenium-tris (bipyridyl)2+ and TPA occurs on the
surface of the electrode.
 TPA is oxidized at the electrode.
 This liberates an electron and forms a TPA radical-cation.
 This reacts by releasing a proton (H+) to form a TPA radical (TPA).
 The ruthenium complex also releases an electron.
 The ruthenium complex then oxidizes to form the ruthenium cation
 Ru(bpy)33+ followed by the chemiluminescent reaction with the TPA radical.
 The ECL reaction is started.
 Peak light emission occurs for a short time interval (0.20-0.60 sec).
 A photomultiplier detects and converts the ECL signal into an electric signal.
 The corresponding signals are used for the calculation of findings.
 Each measuring cycle requires a total of 42 seconds for the subsequent two
stages: (1) Preconditioning - 2 seconds (around); Bead capturing/ BF
separation: 22 seconds (around); (2) The magnet is deactivated before the
measurement is started, to avoid any interference. Measuring- 2 seconds;
Cleaning-14 seconds; Re-conditioning: 2 seconds
3. Release of microbeads and cell cleaning:
 Clean-up solution is aspirated to the measuring cell.
 Microbeads are washed away from the electrode.
 TPA (Pro Cell) is aspirated.
151
 The surface of the measuring cell is renewed by varying the electrode voltage.
 The measuring cell is prepared for another measurement.
Test Principles:
Three test principles are used for the evaluation of test substance (analytes) and
antibodies in the samples:
1. Competitive principle: This principle is applied to test substance of low molecular
weight such T3, T4, FT3, FT4, cortisol, testosterone and estradiol.This type of test is
based on the competition between the test substance of interest and an enzyme-
conjugated version of the same test substance for a limited number of specific
antibody binding sites. The measurement is inversely proportional to the
concentration of sample, High signal=low concentration and Low signal = high
concentration (282).
2. Sandwich principle: This principle is applied to high molecular weight antigens
such as thyroid stimulating hormone, Follicle stimulating hormone, Luteinizing
hormone. This test involves two antibodies which sandwich the test substance
between them. The measurement is directly proportional to the sample concentration-
Low signal=low concentration; High signal=high concentration (282).
3. Bridging principle: This principle is similar to the sandwich principle, except that
the test is designed to detect antibodies, not antigens in the sample (for instance IgE,
IgG, IgA and IgM). This is completed by including bio-tinylated and ruthenium-
labeled antigens in the reagents for which the targeted antibody has the affinity. The
measurement is directly proportional to the sample concentration. Low signal=low
concentration: High signal = high concentration (282).
3.5.3.10.1: Estimation of Total triiodothyronine (T3)
The estimation of T3 was done by electrochemiluminance technique on Roche
cobas e 411 instrument. (T3-Operator’s manual for cobas e 411 analyzer, Roche
Diagnostics GmbH) (284).
Principle: The Elecsys T3 assay employs a competitive assay principle with
polyclonal antibodies specially bound for against T3. Endogenous T3, produced by the
action of 8-anilino-1- naphthalene sulfonic acid (ANS), competes with the added
biotinylated T3-derivative for the binding sites on the antibodies labeled with the
ruthenium complex (Tris(2,2'-bipyridyl)ruthenium(II)-complex (Ru(bpy)3+2.
Reagents - working solutions: The cobas e 411 pack is labeled as T3.
(M): Streptavidin-coated microparticles 0.72 mg/mL-preservative.
152
(R1): Anti-T3-Ab-Ru(bpy)32+ + contains polyclonal anti-T3-antibody (sheep) labeled

with ruthenium complex 75 ng/mL; ANS 0.8 mg/mL; phosphate buffer 100 mmol/L,
pH 7.4; preservative.
(R2): T3-biotin contains biotinylated T3 3ng/mL; ANS 0.8 mg/mL; phosphate buffer
100 mmol/L; pH 7.4; preservative.
Test procedure: Competition principle. Total duration of assay: 18 minutes.
 1st incubation: 30 µL of sample and a T3-specific antibody labeled with a
ruthenium complex; bound T3 is released from the binding proteins in the
sample by ANS.
 2nd incubation: After addition of streptavidin-coated microparticles and
biotinylated T3, the still-free binding sites of the labeled antibody become
occupied, with formation of an antibody hapten complex. The whole complex
becomes bound to the solid phase through interaction of biotin and
streptavidin.
 The reaction mixture was shucked into the measuring cell where the
microparticles were magnetically captured onto the surface of the electrode.
Unbound substances were subsequently removed with procell. Application of
a voltage to the electrode then stimulates chemiluminescent emission which
was measured by a photomultiplier.
 Results were established through a calibration graph which was instrument
particularly produced by 2-point calibration and a master curve or graph
supplied via the e cobas 411 link (reagent barcode).
Calculation: The analyzer automatically calculates the analyte or test substance
concentration of each sample (either in nmol/L, ng/mL or ng/dL). Conversion factors:
nmol/L x 0.651 = ng/mL; nmol/L x 65.09998 = ng/dL ng/mL x 1.536 = nmol/L.
Measuring range: 0.3-10 nmol/L or 0.195-6.51 ng/mL (defined by the Limit of
Discovery and the maximum of the master curve). Values below the limit of detection
are reported as < 0.3 nmol/L or < 0.195 ng/mL. Values above the measuring range are
reported as > 10 nmol/L or > 6.51 ng/mL.
3.5.3.10.2. Total thyroxine (T4): The estimation of T4 was done by
electrochemiluminance technique on Roche cobas e 411 instruments (T4 Operator’s
manual for cobas e 411analyzer, Roche Diagnostics GmbH) (285).
Principle: The Elecsys T4 assay employs a competitive assay principle with an
antibody particularly bound for against T4, Endogenous T4, produced by the action of
153
8-anilino-1 - naphthalene sulfonic acid (ANS), competes with the added biotinylated
T4-derivative for the binding sites on the antibodies labeled with the ruthenium
complex
(Tris(2,2'-bipyridyl)ruthenium(II)-complex (Ru(bpy)3+2.
Reagents - working solutions
(M) Streptavidin-coated microparticles 0.72 mg/mL; preservative.
(R1) Anti T4-Ab-Ru(bpy)3+2 contains Polyclonal anti-T4-antibody (sheep) labeled
with ruthenium complex 100 ng/mL; ANS 1 mg/mL; phosphate buffer 100 mmol/L,
pH 7.4; preservative.
(R2) T4-biotin contains biotinylated T4 ng/mL; ANS 0.8 mg/mL; phosphate buffer
100 mmol/L; pH 7.4; preservative.
Test procedure
Competition principle: Total duration of test: 18 minutes.
 1st incubation: 15 µL of sample and a T4-specific antibody labeled with a
ruthenium complex; bound 14 is released from binding proteins in the sample
by ANS.
 2nd incubation: After addition of streptavidin-coated microparticles and
biotinylated T4, the still-free binding sites of the labeled antibody become
occupied, with production of an antibody-hapten complex. The whole
complex becomes bound to the solid phase through interaction of biotin and
streptavidin.
 The reaction mixture was aspirated or sucked into the measuring cell where
the microparticles were magnetically, captured onto the surface of the
electrode. Unbound substances were then removed with procell/ procell M.
Application of a voltage to the electrode then stimulates chemiluminescent
emission which was calculated by a photomultiplier.
 Results were established via a calibration curve or chart which was instrument
specially generated by 2-point calibration and a standard or master curve
supplied via the reagent barcode.
Calculation: The e cobas411 automatically calculates the test substance or analyte
concentration of each sample (either in nmol/L, μg/dL or ng/L).Conversion factors:
nmol/L x 0.077688 = μg/dL; μg/dL x 12.872 = nmol/L; nmol/L x 0.77688 = μg/L.
Measuring range: 5.40-320.0 nmol/L or 0.420-24.86 μg/dL (defined by the lower
and upper detection range of the master curve). Values below the lower detection
154
limit are reported as < 5.40 nmol/L or < 0.420 μg/dL. Values above the measuring
range are reported as > 320.0 nmol/L or > 24.86 μg/dL.
3.5.3.10.3. Thyroid stimulating hormone (TSH):
The estimation of TSH was done by electrochemiluminance technique on Roche
cobas e 411. (TSH Operator’s manual for cobas e 411 analyzer, Roche Diagnostics
GmbH) (286,287).
Principle: The Elecsys TSH test employs monoclonal antibodies particularly bound
for against human TSH. The antibodies labeled with ruthenium complex consist of a
chimeric build from human and mouse-specific components. As a result, interfering
effects due to HAMA (human anti-mouse antibodies) are largely removed.
Reagents-working solutions:
 Streptavidin-coated microparticles 0.72 mg/mL, preservative.
 R1: Anti TSH-Ab-biotin contains biotinylated monoclonal anti-TSH antibody
(mouse) 2.0 mg/L; phosphate buffer 100 mmoVL, pH 7.2; preservative.
 R2: Anti-TSH-Ab-Ru(bpy)3+2contains monoclonal anti-TSH antibody labeled
with ruthenium complex 1.2 mg/L; phosphate buffer 100 mmol/L, pH 7.2;
preservative.
Procedure: Sandwich principle and the total duration of test were 18 minutes.
 1st incubation: 50 uL of sample, a biotinylated monoclonal TSH specific
antibody and a monoclonal TSH-specific antibody labeled with a ruthenium
complex react to make a sandwich complex.
 2nd incubation: After addition of streptavidin-coated microparticles, the
complex becomes bound to the solid phase through interaction of biotin and
streptavidin.
 The reaction mixture was sucked into the measuring cell where the
microparticles were magnetically captured on to the surface of the electrode.
Unbound substances were then removed with procell. Application of a voltage
to the electrode then stimulates chemiluminescent emission which was
calculated by a photomultiplier.
 Results were determined via a calibration curve which was instrument
specially produced by 2-point calibration and a master curve supplied via the
reagent barcode.
155
Measuring range: 0.005-100 µIU/mL (defined by the lower and upper detection range
of the master curve). The functional sensitivity is 0.014 µIU/ mL.Values of serum
TSH below the lower limit are reported as < 0.005 µIU/mL and values above the
measuring range are reported as > 100 µIU/mL (or up to 1000 µIU/mL for 10-fold
diluted samples).
Elecsys TSH test characteristics
Testing time 18 min
Test principle One-step sandwich assay
Calibration 2 point
Serum, Li-, Na-, NH4+-heparin plasma
Sample material
K3-EDTA, Na-citrate, NaF, K-oxalate plasma
Sample volume 50 μL
Detection limit 0.005 μIU/mL
Functional sensitivity 0.014 μIU/mL
Measuring range 0.005 - 100 μIU/mL
Traceability 2nd IRP WHO Reference Standard 80/558
Total imprecision (NCCLS) cobas e 411 analyzer, E2010: 1.8 - 8.7 %
3.5.4. Hematological Investigation.

3.5.4.1. Total Leukocyte Count (TLC):
The total leukocyte count was determined by haemocytometry (288).
Principle: When blood mixed with WBC diluting fluid then glacial acetic acid
(GAA) lyses the membrane of all blood cells. In addition, crystal violet or gentian
violet slightly stains the nuclei of all WBC. The blood sample is diluted to 20 times
(1:20) in a white blood cell pipette with the help of Turks fluid as well as the total
leucocytes are counted under low power (10-X with reduced light intensity) advanced
microscope by using an improved neubauer counting chamber, The records of WBC
in blood are accounted as the total numbers of white blood cells per cubic millimeter
of whole blood. The extra requirements are the following: WBC pipette and WBC
diluting fluid or Turks fluid-it consist of 1% glacial acetic acid, gentian violet stain
both are dissolved in100mL distilled water. GAA assists the haemolysis of Red blood
cells and gentian violet stains the nuclei of white blood cells.
Procedure: Turks fluid was taken in a watch glass and anticoagulant blood sample
(EDTA) used for total white blood cells counting.
WBC diluting fluid was taken in a watch glass. EDTA blood was used for total WBC
counting. Blood was sucked upto 0.5 mark and further, diluting fluid was drawn equal
to 11 marks of the WBC pipette. The diluting fluid and blood were mixed well in the
bulb of WBC pipette subsequently the first 2-4 drops of fluids (present in the stem of
pipette) were discarded from WBC pipeette. The counting chamber was charged with
156
a drop of blood mixed with diluting fluid. The chamber was permitted to reconcile
white cells at the bottom of the neubauer chamber for 2-3 minutes. The four corners
of the chamber were focused under a low power (10 x) objective and the cells were
counted in all the four WBC corner squares with cells counting rule.
Calculation:
Where:
 Dilution factor = 1:20
 Area counted = 1/4 sq.mm
 Depth of fluid = 1/10mm (0.1mm)
 Number of white cells counted = N
Therefore
3.5.4.2. Differential Leucocytes Count (DLC):

The differential count of leucocytes was established by haemocytometry. The
Leishman’s stain contains both basic (methylene blue) and acidic dye (eosin). The
both (methylene blue and eosin dyes) produce multiple colors when applied to cells.
In addition, Acetone frees methyl alcohol (methanol) acts as fixative and also as a
solvent. The fixative does not permit any further change in the cells (immobilized
cells) and makes them adhere to the glass slide. The basic parts of white blood cells
(cytoplasm, eosinophils granules) are stained by eosin and they are expressed as
eosinophilic or acidophilic. The acidic parts (nucleus with granules of basophils) are
stained by methylene blue and they are illustrated as basophilic. The neutral
components of the cell (granules of neutrophils) are stained by both the dyes. The
necessities are the following:
Leishmann’s stain: It consists of Leishman’s stain and acetone free methyl alcohol.
Procedure:
A thin blood smear was prepared by spreading a small drop of blood evenly on
a glass slide by using a spreader at an angle of 45° or less than 45 degree. The dry
smear was stained by Leishman stain by placing the slide on a stain rack. It is
permitted to react with the stain for 2 to 3 minutes for fixation; subsequently put in
buffer solution on glass slide, and allowed 8-10 minutes for staining the smear then
tap water was used to wash the excess stain on the slide. It was allowed to dry for 7-
10 minutes and observed under high power objective (oil immersion) microscope. The
film was examined by moving from one field to the next methodically. About 100
157
leucocytes were counted to give high degree of accuracy. The WBCs were classified
as follows (a) Eosinophil: 1-4%, (b) Neutrophil: 20-70%, (c) Basophil: 0-1%, (d)
Lymphocyte: 20-40%, (e) Monocyte: 2-8%
The number of cells was calculated and the percentage of cells was recorded in table
3.5.4.3. Total Eosinophils Counts (289):
Principle: A sample of blood is diluted with a solution that selectively stains the
eosinophils and removes all other WBC and RBC from view. Subsequent mixing, the
specimen is introduced into the counting chamber and the number of eosinophils in a
recognized volume of blood is counted.
Composition of pilot’s solution: Each 100 ml solution contains:
 Phloxine: 10 ml (stains only eosinophilic granules)
 Propylene glycol: 50 ml (solvent for stain and lyses RBCs)
 Sodium carbonate (1% aqueous solution): 1 ml (to enhance the staining of the
eosinophils granules)
 Heparin: 100 units (Anticoagulant)
 Distilled water: As required to make 100 ml.
It lyses all WBCs except eosinophils which seems more resistant than other WBCs in
this respect.
Procedure:
 First suck the blood in the WBCs pipette accurately up to 0.5 mark and then
suck pilot’s fluid up to mark 11 (dilution factor 1:20)
 Gently shake the pipette well between the palms for 2-3 minutes.
 Place a moistened filter paper in a petridish and keep the filled pipette on it.
 Allow the pipette to remain in the moistened atmosphere for 15 minute.
 Remove the pipette from the petridish, shake it than discard first a few drops
of fluid in its stem and charge the counting
 Place the counting chamber in mosit surroundings atmosphere again. This
allows the staining to occur without the evaporation of the fluid.
 Finally counts the eosinophils (seen as pink cells with nuclei) in the four WBC
corner squares preferably under high power.
Determine absolute eosinophils counts per µl of blood (Nx50).
Calculation:
 Dilution factor = 1:20
158
 Area counted = 1/4 sq.mm

 Depth of fluid = 1/10mm (0.1mm)
 Number of eosinophils counted = N
Therefore
Normal range: (In Adult) =150-300 cells/cumm.

3.5.5. Pulmonary function tests:
Pulmonary functions were tested using the instrument ‘Spirometer Helios 401’
Recorders & Medicare Systems Pvt. Ltd Chandigarh, India (a self calibrating
windows based computerized spirometer that fulfills the criteria for standardized lung
function testing ATS/ERS, 2005).
3.5.5.1 Spirometer Helios 401:
Helios 401 is a type of open system and flow sensing spirometer which is used
in combination with a window based computer. This is a low cost high performance
instrument able of giving extremely precise and repeatable test outcomes and
represents the main improvement in computerized pulmonary function testing. It is
used to establish the dynamic lung function by measuring the Forced Vital Capacity
(FVC), Slow Vital Capacity (SVC) and the Maximum Ventilatory Volume (MVV).
Its development stems from the wants to regularly screen many subjects without
calculation and to standardize testing measures prediction. For screening applications,
where speed, correctness and repeatability are of highest importance the equipments
arrangement is ideal. The testing procedures in this instrument are fairly easy from the
subject’s point of view. It has a hand piece which contains an electromechanically
turbine transducer which is attached to the mouthpiece to sense when air flow through
it. The electronic circuit (hardware in hand piece) then converts the raw signal into
actual volume and flow rates. This hand piece is connected to a computer through a
USB interface cable. The software given along with the system is used to record
spirometry maneuvers and to recommend a diagnosis. The computer monitor is used
to show the spirometry variables, the device parameters, information messages and
user guide messages. A printer attached to the computer can be used to get a hard-
copy record of the maneuver and the associated parameter values.
Description of the Device
Specifications:
Speciﬁcations were according to ATS/ERS recommendations.
159
Hand piece and transducer:

Volume Range 0-8 Liters
Flow Range 0-16 Lit/Sec.
Overall Accuracy Within ± 1%
Transducer Turbine Type Cartridge
Flow Detection Volume Differential
Calibration syringes 3 Liters
Minimum computer configuration required
Monitor 15” colour and 800x600 pixels
Printer DeskJet or LaserJet
Processor: 866Mhz or above, RAM:64 MB,
FDD:1.44 MB, HOD:20 GB, AGP Card or on board,1
Computer USB2 serial, 1parallel port, Keyboard
Mouse and mouse pad.
Helios 401 Parts:

With state to the below figure 3.2 the standard accessories which include the
spirometer Helios 401 are detailed. 1 Spirometer hand piece; 2 Reusable Mouthpiece;
3 Transducer, turbine type; 4 Air filters; 5 Nose clip; 6 Software installation-CD, 7
USB-Serial connector, to be connected to the hand piece; 8 USB connector, to be
connected to the computer; 9 USB-Serial cable.
Figure 3.2: Spirometer Helios 401 Standard Accessories.
Figure 3.3: Spirometer hand piece.
160
The hand piece is shown in figure 3.3 described in details follows:

1. Turbine housing, 2. Device label 3. Socket to USB-Serial cable
The transducer used with the RMS Helios 401 spirometer is a turbine type
transducer. A close outlook of the transducer is revealed in Figure 3.4 below. The
transducer converts the flow of air, breathed by the patient against a frictionless
rotating vane, into an electrical signal which is used to produce the relevant plots.
First is a turbine dust cap and second is a turbine unit.
Principle of Turbine Spirometer:
A turbine built into the flow tube (house) characterizes this sensor, which is
also called a digital volume transducer. When gas flows via a turbine flow meter it
makes a vane turn; flow through the tube is proportional to the number of revolutions
per unit of time, the rotating elements (turbine) interrupt or reflect the light from a
light-emitting diode (LED). Photodiodes register the rotations, returning an electrical
impulse frequency proportional to flow, whereas the total count is proportional to
volume. A benefit of this system is that: It is insensitive to turbulent flow, gas
composition, and water vapor and gas temperature. Additional, results are consistent
and reproducible and these spirometers need no calibration and no thermostat if the
turbine is made of carbon or kevlar. Fruthermore, there is no influence of pressure or
humidity on the spirometry results. A disadvantage of the system is its inertia, which
needs to be minimized by using a very lightweight vane and applying a deflector.
Figure 3.4: Turbine transducer
Figure 3.5: Several calibration (known volume) syringes
161
Figure 3.6: Spirometer Hand Piece and Turbine with Separated Mouthpiece .
Figure 3.6 shows a hand piece with a transducer fixed into its housing. The
mouthpiece is fixed over the transducer.
Figure 3.7: Spirometer in use by a patient.

Calibration of the Instrument:
The system is calibrated by using 3 liters Jones syringe. By calibrating with a
known volume syringe, the correctness of the flow sensor and integrator can be
checked with one input. A correction factor is considered based on measured volume
of air divided by expected values (known volume of air).
The accuracy of Spiro meter is tested as follows
The highest acceptable error according to American thoracic society (ATS)

recommendations is 3% or exceeds ±50ml, whichever is larger. If the percentage of
error which ever a carefully examination of the Spiro meter, recording device and
162
(117)
testing device has to be carried out . In our study instrument calibration was
carried out once in 3 weeks using a 3 liters syringe.
Procedure:
Pulmonary functions assay were performed by using helios-401computerized
spirometer (RMS) as per suggestion of recent guidelines published by the American
Thoracic Society-European Respiratory Society (ATS-ERS-2005) Task Force on
(182)
Standardization of Lung Function Testing . The subjects were comfortable with
the set up and comprehensive commands and demonstrations were specified to our
fulfillment. The essential personal and anthropometric data (subject-ID, name, age,
gender, temperature, weight, height, date, smoker or nonsmoker, and position) were
feed into the software. The software was loaded to the computer and provided by
RMS with spirometer, it calculates the predicted value and also gives the actual value
and the percentage predicted value for the individual. All the tests were carried out at
the same time of the day, between 9.30 AM to 12.30 PM to evade probable variations.
The subjects were made to breathe out forcefully following deep inspiration into the
mouthpiece attached to the turbine transducer. Expiration was maintained for a
minimum period of 3-4 seconds. 3 to 4 trials of maximal inspiratory and expiratory
efforts were made and only the maximum reading was taken for data processing. All
recordings were performed at the BTPS (in respiratory physiology lung volumes and
flows are standardized to barometric pressure at sea level, body temperature, saturated
with water vapor: body temperature and pressure, saturated) as suggested by ATS, all
the readings were taken in sitting position. The tests were performed in a silence room
in order to lessen the emotional and psychological stresses. During the tests,
maximum effort from the subjects was ensured by sufficiently motivating them to
perform at their optimum level. A normal PEFR value of 3-5 L/sec ensured highest
effort by the subject while performing the test. Nose clips were used during the
performed test maneuver. For maximum voluntary ventilation, mouth piece was
placed into the subject’s mouth and was instructed to breathe quietly. When the
subject settled, he was requested to breathe in and out as quickly and deeply as
possible for 10-15 seconds. 3 readings were taken for data processing. In both the
groups (MetS and non MetS) subjects were highly motivated and supportive. They
done the tests with care and maximum efforts. The parameters under the Forced Vital
Capacity (FVC), slow vital capacity (SVC) and Maximum voluntary ventilation
(MVV) were studied.
163
Spirometry Tests: Following steps are used during pulmonary function test:
a. Assure spirometer has been checked for correctness (calibration check)
b. Assure proper patient preparation: Clarify the test, determine any
contraindications, get age, height, and weight; properly position the patient.
c. Clarify and demonstrate the maneuver: Keep explanation easy and brief,
demonstrate attaching to mouthpiece, correct chin and neck position, and the
maneuver, and reemphasize key points.
d. Done the maneuvers: Attach nose clip, place mouthpiece in mouth, and seal lips
(closed circuit), Have patient inhale entirely and quickly, train patient to blast the
air out as hard and fast as possible with minimal pause; provide enthusiastic
coaching, provide feedback to patient on quality of each maneuver, repeat test to
get at least three satisfactory maneuvers, assess repeatability and perform extra
maneuvers, if necessary
Figure 3.8: Spirometer standardization steps (182)

Forced Vital Capacity (FVC) and flow volume loop:
Forced Vital Capacity (FVC) was the primary test performs with a spirometer.
It finds out how much and how fast a patient can exhale air. The Forced Expiratory
Volume (FEV) test refers to the highest volume of air that can be expelled in a
particular time during the presentation of the forced vital capacity maneuver. For
instance, FEV1 is the volume of the air expelled in the first second of the FVC.
From this test alone, one can determine how the patient can move air in and
out of his lungs, as compared with a healthy person of the same age, height, sex and
race; whether there is proof of a restrictive disorder, an obstructive disorder or a
combination of both, and if thus, the degree of severity.
164
3.5.5.2. Following steps were taken for conducting FVC, SVC and MVV tests:
Performed FVC tests:
(a) Spirometer was ready and calibrated. (b) Patient was recognized according to
inclusion criteria. (c) Proper training and demonstration was given to the patient about
the test maneuver. (d) All personal and anthropometry information were feed into the
spirometer software. It was loaded in the computer. (e) The subject was sitting upright
with feet flat on the floor. (f) Subject's chin was upright with the neck slightly
extended. (g) FVC button was clicked on the toolbar of spirometer software. (h) A
nose clip was attached to the patient nose. (i) Requested to the patient to breathe
normally several times. (j) Pre- medication test was chosen from the begin button in
spirometer software. (k) The pre-medication test graph appeared in red color (while
the post-medication test graph was blue). The Stop button was also displayed on the
screen. (l) Requested the patient to a highest inspiration. (m) Mouth piece (attached to
turbine transducer) was positioned firmly in the patient’s mouth. (n) The subject was
to breathe in extremely to his full extent. The subject then put the transducer to the
mouth and expelled the air in their lungs as rapidly as probable. Once all the air in the
lungs has been expelled, the subject was breathing in as rapidly as possible, still with
the transducer to the mouth, until the lungs were full. (o) For a best outcome, the
patient done above maneuvre at least 3 times to have reproducible findings. (p) The
test automatically (or manually stop by clicked the stops button) stops when flow rate
falls below a minimum rate. (q) Spirometer transducer removed from the subject
mouth. The FVC and FEV graphs were shown on the computer screen. Spirometer
gives a comparison of previous and the latest maneuver in terms of various
parameters thus we can accept or reject the last maneuver. It also point out which of
the two maneuvers is superior. The good FVC maneuver (greatest value of
FVC+FEV1) was chosen and save from the clicked the save button on the toolbar.
FVC Specific Features:
The FVC screen, figure 3.9, illustrates space to get the flow/volume and
volume/time graphics. The all maneuver being exhibited is indicated by the blue
arrow in the centre column of the screen. All, numerous, or a single maneuver can be
shown by clicking explain all maneuvers, also situated in the centre column of the
screen.
165
Figure 3.9: FVC Screen. Figure 3.10: All FVC Maneuvers.

Slow Vital Capacity (SVC):
The slow vital capacity (SVC) assay was performed to establish various lung
capacities. The SVC maneuver is tidal breathing followed by a slow maximal
inspiration after that flow maximal expiration and subsequently again tidal breathing.
Slow vital capacity was the volume of air which can be exhaled slowly from
maximum inhalation (TLC) to maximum exhalation (RV). The clinical use of SVC
test is when the measured FVC is decreased. This frequently happens with the
moderate to severe airflow limitations because forceful exhalation causes airway
collapse of premature airway closure. This traps air in the alveoli and diminishes the
volume of capacity in this situation can be taken by a slow exhalation maneuver.
Performed SVC assay: The subject was requested to breathe regularly through the
mouth piece. The subject was after that taken a deep breath followed by a deep
exhalation. Both inhalation and exhalation were done to the maximum extent. After
this slow maneuver the subject was taken a few gentle and normal breaths.
MVV test: It was conducted to assess the maximal voluntary ventilation capacity.
The subject was asked to breathe deeply and quickly through the transducer for 15
seconds. Breathing was as constant as possible during test maneuver.
Figure 3.11: SVC maneuver. Figure 3.12: MVV maneuver.

Test parameters were calculated by RMS Helios 401
Table 3.6: RMS Helios 401 is estimates the following parameters of pulmonary function
166
FVC Test Parameters

FVC* Forced Vital Capacity
FIVC Forced Inspiratory Vital Capacity
FEV.5 Forced Expiratory Volume in 0.5 sec.
FEV1* Forced Expiratory Volume in 1 sec.
FEV3* Forced Expiratory Volume in 3 sec.
PEFR* Peak Expiratory Flow Rate
PIFR Peak Inspiratory Flow Rate
FEF 25-75* Mean Forced Expiratory Flow during the middle of the FVC
FEF 75-85 Late Expiratory Flow Rate.
FEF .2-1.2 Mean Forced Expiratory Flow rate from 0.2 to 1.2 liters of volume expired.
FEF 25% Forced Expiratory Flow after 25% of the FVC has been expired.
FEV.5/FVC
FEV1/FVC* Ratio of Forced Expiratory Volume (at 0.5s, 1s, 3s) to Forced Vital Capacity
FEV3/FVC expressed as a percentage
FET Forced Expiratory Time
Expl Time Extrapolated Time
SVC Test Parameters
SVC Slow Vital Capacity (also known as Vital Capacity, VC)
ERV Expiratory Reserve Volume
IRV Inspiratory Reserve Volume
VE Volume expired per minute during normal breathing
Rf Breaths per minute (Respiration rate)
Ti Inspiration time
Te Expiration time
VT Volume, Tidal
VT/Ti Tidal Volume to Inspiration time ratio
Ti/Ttot Inspiration to Total breath time ratio
IC Inspiratory Capacity = IRV+VT
VC Vital Capacity = IRV+VT+ERV
MVV Test Parameters
MVV* Volume Expired per minute
MRf MVV maneuver breaths per minute
MVT MVV maneuver Tidal Volume
In the present study, pulmonary function variables* FVC, FEV1, FEV1/FVC, FEF25-
75% and PEFR are mainly studied.
3.5.5.4: The interpretation of the flow-volume loop (175):
Figure 3.13: Forced Vital Capacity - Flow-Volume Loop (175)
167
The flow volume loop can have four distinguishing shapes or morphology that
is associated to many lung function anomalies: obstructive lung disease, restrictive
lung disease, mixed lung disease and upper airway obstructions.
Normal Spirometry:
A normal expiratory flow-volume loop has a triangular morphology which is
show in figure 3.14. The inspiratory segment of the loop is a half circle shape
(normal). The values of the parameters (FVC & FEV1) are higher than 80% of the
predicted values, while the Tiffeneau index (FEV1/FVC x100) is higher as compared
to normal 70%.
Obstructive Lung Disease: Obstructive lung disease is the most frequent diagnosis
of pulmonary disease when using a spirometer. The Tiffeneau index is below 70%,
which outcomes in a concave expiratory portion of the flow-volume loop. When
obstructive lung disease is observed, frequently a post-medication test is done after
administration of a bronchodilator. In case the obstruction is reversible (FEV1 in post-
medication test significantly higher than FEV1 premedication test), the patient is
probable to suffer from asthma.
Normal Spirometry Obstructive Lung Disease Restrictive Lung Disease Mixed Lung Disease
Figure 3.14: Flow-Volume Loop interpretation (175)
Restrictive Lung Disease: Restrictive lung disease cannot be diagnosed with a

spirometer because spirometers can not measure residual volume. A spirometry test
can though be suggestive for restrictive lung disease when FVC is too low (less than
80% of the predicted value). Peak expiratory flow and FEV1 can be normal in
restrictive lung disease, but are often low as well.
Mixed Lung Disease: Individual with mixed lung disease shows both signs of
obstructive and restrictive lung disease: Tiffeneau index and FVC are very low.
168
Upper Airway Obstructions: With these pathologies the flow volume loop is
flattened and three forms are most common which are shown in figure 3.15:
 Variable extra-thoracic obstruction: flattening of the expiratory part of the
flow-volume loop.
 Variable intra-thoracic obstruction: flattening of the inspiratory part of the
flow-volume loop.
 Fixed obstruction: flattening of both the expiratory and the inspiratory part of
the flow-volume loop.
Normal spirometery Extrathoracic Obstruction Intrathoracic Obstruction Fixed Obstruction
Figure 3.15: Flow-Volume Loop shows upper airway obstruction.
3.5.5.5. ATS/ERS Task Force, (2005): Standardisation of Lung Function Testing:

The ATS first published suggestions for spirometer standards in 1979 and
updated them in 1987 and 1995. The ERS published like recommendations in 1983
and 1993. The ATS and ERS selected a task force to join these spirometry
suggestions, which were published in 2005. Manufacturers are very much aware of
these standards and design their instruments to follow with them. If the spirometer
measures FVC, it should be capable to accumulate volume for at least 15 seconds and
measure volumes of at least 8 liters, and it should be capable to measure flow rates
between 0 and 14 liters/sec. The total resistance to air flow at 14 liters/sec must be
less than 1.5 cm H2O/liter/sec. This total resistance must be measured with any
tubing, valves, and filters that might be inserted between the patient and the
spirometer (182).
169
Minimal Recommendations for Spirometers:-

Table 3.7: Range and accuracy recommendations specified for forced expiratory maneuvers (182).
Flow
Resistance and Back
Test Range/ accuracy (BPTS) range Time Test signal
pressure
Ls-1
3-L
0.5–8 L, ±3% of reading or
VC 0-14 30 Calibration
±0.050 L, whichever is greater
syringe
24 ATS
0.5–8 L, ±3% ofreading or <1.5 cmH2O.L-1.s-1 waveforms,
FVC 0-14 15
±0.050 L, whichever is greater (0.15 kPa.L-1.s-1) 3-L Cal
Syringe
0.5–8 L, ±3% of reading
<1.5 cmH2O.L-1.s-1 24 ATS
FEV1 or±0.050 L, whichever is 0-14 1
(0.15 kPa.L-1.s-1) waveforms
greater
Time The time point from which all
0-14 Back extrapolation
zero FEVt measurements are taken
Accuracy: ±10% of reading Mean resistance at 200,
or±0.30 L.s-1 (20 L.min-1), 400,
26 ATS
whichever is greater; 600 L.min-1 (3.3, 6.7,
PEF 0-14 flow
repeatability: ±5% of reading 10 L.s-1) must be <2.5
waveforms
or±0.15 L.s-1 (10 L.min-1), cmH2O.L-1.s-1 (0.25
whichever is greater kPa.L-1.s-1)
Instantaneous Accuracy: ±5% of reading Data from
<1.5 cmH2O.L-1.s-1
flows (except or±0.200 L.s-1, whichever is ±14 manufactur
(0.15 kPa.L-1.s-1)
PEF) greater ers
7.0 L.s-1, ±5% of reading
±14 24 ATS
FEF25–75% or±0.200 L.s-1, whichever is 15 Same as FEV1
(±3%) waveforms
greater
250 L.min-1 at VT of 2L
<1.5 cmH2O.L-1.s-1 Sine wave
MVV within±10% of reading or ±15 12-15
(0.15 kPa.L-1.s-1) pump
L.min-1,whichever is greater
BTPS: body temperature and ambient pressure saturated with water vapour; VC: vital capacity; FVC: forced vital capacity;
ATS: American Thoracic Society; FEV1: forced expiratory volume in one second; FEVt: forced expiratory volume in t
seconds; PEF: peak expiratory flow; FEF25–75%: mean forced expiratory flow between 25% and 75% of FVC; MVV:
maximum voluntary ventilation; VT: tidal volume
3.6. Pilot Study: The pilot study is a small trial which was conducted in preparation
for the main study. The pilot study enables researchers to find the feasibility of the
tool and technique then to create the necessary amendments and modifications in the
tool. The data from 30 subjects was collected for pilot study during May-June, 2011.
The analysis was done after pilot study, the tool and technique was found to be
feasible, workable and satisfactory.
3.7 Statistical analysis
Statistics is the science which deals with statistical methods and their
application in an investigation. Statistical method is techniques to collect, classify,
tabulate, analyze and interpret the qualitative and quantitative data. The aim of
statistical method is to present the data in a comprehensible and intelligent way. For
the present study the following statistical method were taken into consideration for
analyzing the data.
170
3.7.1: Mean ( x )
Mean, also known as arithmetic average, is the most common measure of
central tendency and may be defined as the value which we get by dividing the total
of the values of various given items in a series by the total number of items.
x1 + x2 + x3 +........... xn
x =  X/N (Mean) x =
N
  X = sum of all the vales of the variable x
 N = number of observations
3.7.2: Standard deviation (SD)
Standard deviation is the most widely used measure of dispersion of a series. It
gives the numerical expression to the extent of derivation of the observed data from,
the arithmetic mean. Therefore, it is helpful in studying the dispersion. It can be taken
by taking the square roots of the sum of the squares of deviations from the mean. It
can be mathematically represented as follow:
SD (σ) =
 = is the sum of the squares of deviations from arithmetic mean.
 N = total number of observations
A high value of standard deviation indicates a more dispersed or variable population
and a lower value indicates a less variable population/ Standard deviation is useful in
comparing the variability of groups with regard to some characters.
3.7. 3: Test of significance:
Student‘t’ test is considered an appropriate test for judging the significance of
a sample mean or judging the significance of difference between the means of two
sample. The relevant test statistic, t, is calculated from the sample data and then
compared with its probable values based on t distribution at a specified level of
significance for concerning degrees of freedom for accepting or rejecting the null
hypothesis.
It was calculated as:
t=
Where,
 = mean of the sample
 = mean of the second sample
 S.E. of = standard error of mean.
 S.E. of = standard of the second mean
171
3.7.4: Chi square test (χ2)

It is an important test amongst the numerous test of significance developed by
statisticians. It is representatively written as χ2 (pronounced as Ki square), is a
statistical measure used in the context of sampling analysis for comparing a variance
to a theoretical variance. This test of significance is observed useful in computing the
difference in regards to non parametric data. The conventional formula for the
computation of chi square test is as follows:
χ2 =∑
The probability table of Fisher and Yates (1953) is used for finding out level of
significance against the degree of freedom (df) which is obtained by multiplying
(column number-1) (row number-1).
3.7.5: ANOVA (Analysis of Variance)
A statistical analysis tool that separates the total variability observed within a
data set into two parts: random and systematic factors. The random factors do not
have any statistical influence on the given data set, whereas the systematic factors
perform. The ANOVA test is used to decide the impact independent variables have on
the dependent variable in a regression analysis. It is used to establish whether there
are any significant differences between the means of two or more independent groups.
In the present study,
3.7.6: ANOVA Post - hoc test
Post hoc tests are considered for situations in which the researcher has already
achieved a significant collection F-test with a factor that consists of three or more
means and further examination of the differences among means is required to give
specific information on which means are significantly different from each other. The
most widely used post hoc test is Tukey's honestly significant difference or HSD test.
There are several types of post hoc tests all based on different assumptions and for
different aims. Tukey's HSD is an adaptable, simply calculated technique.
3.7.7: Coefficient of Correlation:
Correlation refers to the relationship between two variable in which with alter
in the value of one variable the value of other variable also changes. Therefore the
two values are supposed to be related in such cases. Its values range from -1 (an ideal
negative relationship) to +1 (an ideal positive relationship). It is denoted by ‘r’. Karl
Pearson’s method popularly known as Pearson’ coefficient of correlation is
extensively used in practices.
172
It is calculated as follows:
r =
Where, and are the respective means of and variable and r is coefficient of correlation.
3.7.8: Regression:
Regression analysis is a statistical tool for the investigation of associations
between variables. Data regression analysis is a method used for the modeling and
analysis of numerical data consisting of values of a dependent variable (response
variable) and of one or more independent variables (explanatory or independent
variables). The dependent variable in the regression equation is modeled as a function
of the independent variables. Multiple regressions are a statistical technique that
allows the prediction of someone’s score on one variable on the basis of their scores
on numerous other variables. There are different ways that the relative contribution of
each predictor variable can be estimated. In the synchronized method (or enter
method as used in IBM-SPSS), the set of predictor variables are specified that build
up the model. The of this model in predicting the criterion variable is then assessed.
3.7.9: Graphs:
Graphs are basic abets in conversion of complex statistical tabulation into a
figure, which can be simply grasped. For this and other reasons, the attention getting
power of visual presentation is utilized, and at the same time, numerical facts often
abstract and difficult of presentation is translated into actual and understandable form.
3.7.10: Computer application in statistical analysis:
The data collected was subjected to both qualitative and quantitative analysis. The
data was fed into defined groups and was analyzed using IBM-SPSS 20.0 (Statistical
Package for Social Sciences) which is among the most widely used programs for
statistical analysis in social science, in which all socio-demographic, anthropometric,
physiological, biochemical, hematological, pulmonary functions data as well as other
relevant variables which directly or indirectly may be associated to present study were
analyzed statistically. The responses obtained on the questionnaire (qualitative data)
were transformed to master code sheets for easy interpretation.
In the present study, the baseline characteristics of participants were expressed
in mean ± SD. Independent Student’s-t test was used to find out significance
differences in the mean values of thyroid and pulmonary function variables,
components of metabolic syndrome, hematological and inflammatory variables
173
between the metabolic and the non-metabolic group. It was also used to compare the
mean difference in thyroid and pulmonary functions variables between abdominal
non-obese and obese, non-diabetes and diabetes, eulipidemic and dyslipidemic
subjects or subgroup of metabolic syndrome. Analysis of variance (ANOVA) with
post hoc analysis according to Tukey was used to find out the significance difference
in the mean values of thyroid and pulmonary function variables, components of
metabolic syndrome, hematological and inflammatory variables among thyroid
dysfunction, ventilatory patterns and numbers of metabolic components subgroups
(Multiple groups comparison). Chi-square test was used for the comparison of
qualitative data between metabolic and non-metabolic groups.
Pearson’s correlation coefficients were calculated to determine the strength of
the relationship between two variables. In the present study, it was used to evaluate
the strength of the relation between thyroid function variables and metabolic
components, pulmonary function variables and metabolic components among
metabolic group, thyroid dysfunction subgroups and ventilatory pattern subgroups of
metabolic subjects.
Multiple linear regression analysis was performed to assess the independent
association of thyroid and pulmonary functions variables to components of metabolic
syndrome among metabolic group, thyroid dysfunctions and ventilatory patterns
subgroups of metabolic subjects. Some values were log transformed to improve the fit
of the linear regression models. P< 0.05 was considered statistically significant.
174

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Materials and

classified into subgroups according (after estimation of metabolic components,

BMI status Global existing norms* For Asian population

Subgroup-I: Normal weight (n=32)

Figure 3.1: Research Design

3.2. Ethical aspect:

3.3.3. Definition of ventilatory pattern of pulmonary functions:

3.5. Data collection:

3.5.2.4. Waist circumference (WC):

benzoic acid (4-HBA) and 4-Aminoantipyrine (4-AAP) to formed pink color

The intensity of the pink color formed is proportional to the glucose

Reagent and Sample Blank Standard Test

3.5.3.2. Estimation of serum total cholesterol-Tulip diagnostic group India. (275):

Reagent composition and preparation: Reagents are ready to use

Reference values: 150-200 mg/dl

3.5.3.3. Estimation of serum triglycerides: Tulip diagnostic group India

Lipoprotein Lipase hydrolyses serum triglycerides to glycerol & free fatty

Reference values: Men: 60-165 mg/dL & Women: 40-140 mg/dL

Manual assay procedure:

3.5.3.5. Estimation of Very Low Density Lipoproteins Cholesterol (VLDL-C):

Reference range: Up to 35 mg/dL

Supplementary materials required: A microtiter plate calibrated reader (450±10 nm);

3. Distribute 25 µl enzyme conjugate into every well.

Category HOMA-IR Score

3.5.3.9: Estimation of high sensitivity C-reactive protein (hs-CRP)-Tulip

Method for preparation of CRP calibration curve:

The calibrator must be reconstituted precisely with 1.0 ml of distilled water,

3.5.3.10. Determination of Thyroid Hormones:

Electrochemiluminescence Assay Principles (282):

1. Bound/ free separation

(R1): Anti-T3-Ab-Ru(bpy)32+ + contains polyclonal anti-T3-antibody (sheep) labeled

3.5.4. Hematological Investigation.

3.5.4.2. Differential Leucocytes Count (DLC):

 Dilution factor = 1:20

 Area counted = 1/4 sq.mm

Normal range: (In Adult) =150-300 cells/cumm.

Hand piece and transducer:

Helios 401 Parts:

Figure 3.2: Spirometer Helios 401 Standard Accessories.

Figure 3.3: Spirometer hand piece.

The hand piece is shown in figure 3.3 described in details follows:

Figure 3.4: Turbine transducer

Figure 3.5: Several calibration (known volume) syringes

Figure 3.7: Spirometer in use by a patient.

The accuracy of Spiro meter is tested as follows

The highest acceptable error according to American thoracic society (ATS)

Figure 3.8: Spirometer standardization steps (182)

Figure 3.9: FVC Screen. Figure 3.10: All FVC Maneuvers.

Figure 3.11: SVC maneuver. Figure 3.12: MVV maneuver.

FVC Test Parameters

Figure 3.13: Forced Vital Capacity - Flow-Volume Loop (175)

Figure 3.14: Flow-Volume Loop interpretation (175)

Restrictive Lung Disease: Restrictive lung disease cannot be diagnosed with a

Normal spirometery Extrathoracic Obstruction Intrathoracic Obstruction Fixed Obstruction

Figure 3.15: Flow-Volume Loop shows upper airway obstruction.

3.5.5.5. ATS/ERS Task Force, (2005): Standardisation of Lung Function Testing:

Minimal Recommendations for Spirometers:-

3.7.4: Chi square test (χ2)

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