CHAPTER 4

METHODOLOGY
4.1 Introduction
The ANS functions can be studied by using HRV data of an individual is a well
known fact. The HRV analysis plays an important role in individuals with diabetes,
hypertension, physical fatigue, sleep apnea, coronary artery diseases and in addictions.
In the present work, stratification of subjects for HRV analysis has been done. It
was based on age, number of years of addiction (alcohol, smoking, stress). Age group
selected was basically young population (17 to 23 yrs.) as the identification in them helps
in prevention of diseases. A detailed questionnaire was administered to acquire the
personal information and consent was taken to analyze their ECG signals and
confidentiality was maintained.
In the present work, subjects selected age group between 17 to 23 years were
studied by stratifying the individuals based on the questionnaires administered for
acquiring patient data. Personal history consisted of;

I.

Name:

II.

Age:

III.

Sex:

IV.

Marital Status:

V.

Blood Group:

VI.

Height:

VII.

Weight:

VIII.

Smoking Since (in Years):

IX.

No. of Cigarettes per day:

X.

Alcoholic Since (in Years):

XI.

Diabetes Since (in Years):

XII.
XIII.

Hypertension Since (in Years)

Drug History (Mention if you are taking any regular medication):

4.2 HRV Extraction for identification of Addictions (Smokers and Alcoholics)

ECG
Electrodes
(3 Leads)

Bio-Amplifier

DAQ Card
6009

Computer

Preprocessing
Kubios
and peak
Software
detection using
Matlab
Figure 4.1: Block diagram used to analyze HRV by extractingsoftware
ECG

Analysis of
Heart Rate
Variability

Figure 4.2: Method employed for extracting HRV parameters

The block diagram of methods used in the present work is shown in figure 4.1.
Here we have used 3 leads ECG electrodes for acquiring data and bio amplifiers are used
to amplify the signals and through DAQ the signals are obtained and it is filtered. The
peaks of the filtered signals were obtained and is fed to Kubios software for extracting
HRV parameters. The flow chart of the present work is as shown in figure 4.2.
4.3 Algorithm for HRV Analysis using ECG
Step1: Start
Step2: Acquire ECG data using three lead electrodes
Step3: Read the ECG data
Step4: Remove the artifact in the ECG signal
Step5: Plot the artifact reduced ECG signal using Matlab software
Step 6: Detect positive peaks of the data by comparing 3 successive samples when
The second data is higher with respect to the first and third data or the first
And second data are equal and higher than the third data checked
Step7: Copy the detected samples to peak array else check successive samples
Step8: Find the maximum peak value of peak array and set 70% of it as threshold
Value
Step9: If array value is less then threshold then load 0 in that respective samples
Step10: If peak value is greater than 0 then suppress next successive 20 arrays
Values to 0
Step11: Store the peak value in an array
Step12: Read the peak value array and extract it to Kubios Software tool
Step13: Perform Time-domain analysis and non-linear analysis
Step14: Read the statistical values which are available on the tool
Step15: Stop.

4.3.1 Flow chart for the analysis of HRV using ECG

Start

Read original ECG

data artifact

Remove noise in ECG

Peak detection
Save peak data

Have
read ECG
data for 5
minutes.

NO

YES

Kubios Software
software
Time domain,
Frequency domain and
non-linear analysis
results
Analysis of HRV

Stop
Figure 4.3: Flow chart for Extracting HRV parameters using ECG Signal

The obtained ECG signals are filtered and the peaks of the signals were detected.
The detected peak were saved such that it has read for five minutes and is loaded to
kubios software for extracting HRV parameters. The flow chart is shown in figure 4.3 for
extracting HRV parameters.

4.3.2 Peak Detection of ECG Signal

Start

Read ECG
data into an
array

Plot ECG data

Initialize peak=[ ], Initialize i=1

B
a=file[i]-file [i-1], b=file[i+1]-file[i]

B
If(a>0)a
nd(b<0)

If(a==0)a
nd (b<0)
i=i+1
++1

YES

Peak [i]=file[i]

Peak[i]=0

If
I=length
(file)-1

Th=max (peak)*0.5

i-1

If peak
[i]<=Th

NO

YES

Peak [i]=0

i=i+1

N
O

If
i=length
(Peak)
YES

Plot peak

Stop
Figure 4.4: The flow chart for peak detection
The flow chart of the peak detection algorithm used in the present work is as shown in
Figure 4.4.

The ECG signals were taken in real time from individuals in the age group of
17-23 at RVCE Health center. The ECG signals were obtained by 3 lead ECG electrodes.
The obtained ECG data was acquired in Lab VIEW. The file was saved in .txt format and
.dat format which was read to the Matlab software.
The hearts electrical activity is obtained through ECG. ECG signals are acquired
mainly through two types namely invasive methods and noninvasive methods. Invasive
methods are accurate but are not pragmatic. Non invasive methods are popular and
commonly used while measuring ECG. In the present work three lead surface electrodes
have been used. The signal obtained will be corrupted by various noises like baseline
shifts, power line interference and motion artifacts. Hence processing of the signals is
very important before they are used for analysis. The base
Line wanderance in the ECG signal and its removal is showed in figure 4.5(a) and figure
4.5(b) respectively.

Figure 4.5(a):ECG signal with baseline

Figure 4.5(b):ECG signal after removing

wanderance

baseline shift

Figure 4.6: ECG signals corrupted with trend

There is a baseline drift in figure 4.6 and hence its amplitude cannot be
considered as the original amplitude. Hence we must detrend the above signal before its
amplitude is used for measurements. The ordered polynomial is used to detrend the above
signal as shown in figure 4.7.

Figure 4.7: Detrended ECG Signal

4.4 Peak Detection of ECG Signal

The important part of the ECG signal is the QRS complex which denotes the
ventricular depolarization of the heart. The peaks of the signal are detected in Matlab
software which can be used for feeding the signals to Kubios Software. The peak
detection of the ECG signal is as shown in figure 4.8.

Figure 4.8: RR Peak Detected Signal

4.5 Acquisition of PPG Signal for HRV Analysis to identify stress and its associated
diseases
PPG has been applied in many different clinical settings, including clinical
physiological monitoring, vascular assessment and autonomous function and other
cardiovascular variability assessments. In contrast to ECG, pulse wave can be recorded
with a single sensor without electrode and indeed, pulse wave equipments are popular
and widely used not only in hospital cares but also in health sciences and clinical
homecare practices.
Fingertip
of the
subject

Analysis of
Heart Rate
Variability

PPG
Sensor

Kubios
Software

Acquisition
of PPG
Signal

Pre
processing
and peak
detection in
Matlab
software

Figure 4.9: Block Diagram for Analyzing HRV by extracting PPG

The block diagram of data acquisition and HRV analysis by using PPG signals is
shown in figure 4.9. PPG signals are taken from individual persons using bio kit at a
sample rate of 256 samples / second. The frequency response for PPG is 0.05-10Hz. It is
obtained by using reflection type PPG sensor, these signals are amplified using a PPG
amplifier and are interfaced with the PC using DAQ card 6009. The peaks of the PPG
signals are detected and the peaks are loaded to kubios software for HRV analysis.
4.6 Algorithm for HRV Analysis using PPG
Step1: Start
Step2: Acquire PPG data in Lab VIEW
Step3: Read the PPG data
Step4: Plot PPG data with artifacts in Matlab software
Step5: Remove the artifacts in the PPG signal
Step6: Plot the artifact free PPG signal
Step7: Detect positive peaks of the data by comparing 3 successive samples when the
Second data is higher with respect to the first and third data or first and second
data are equal and higher than the third data
Step8: Copy the detected samples to peak array else check successive samples
Step9: Find the maximum peak value of peak array and set 70% of it as threshold
value
Step10: If array value is less then threshold then load 0 in that respective samples
Step11: If peak value is greater than 0 then suppress next successive 20 array values
to 0
Step12: Store the peak value in an array
Step13: Read the peak value array and extract it to Kubios Software tool
Step14: Perform Time-domain analysis and non-linear analysis
Step15: Read the statistical values which are available on the tool
Step16: Stop.

4.6.1 Flow Chart of Present Study for HRV Analysis using PPG Signal
Start

Read original PPG

data artifact

Remove noise in PPG

Peak detection
Save peak data

Have
read 5
min.
data?

NO

YES

Kubios Software
software
Time domain,
Frequency domain and
non-linear results

Analysis of HRV

Stop
Figure 4.10: Flow chart for extracting HRV parameters with PPG Signals

The PPG signals are obtained and it is filtered. The filtered PPG signals peaks are
detected and by using Kubios software HRV parameters are obtained. The flow chart for
obtaining HRV parameters is as shown in figure 4.10.

4.6.2 Peak Detection in PPG Signal

Start

File=Read
PPG data

Plot PPG data

Initialize peak=[ ], Initialize i=1

B
a=file[i]-file [i-1], b=file[i+1]-file[i]

B
If(a>0)a
nd(b<0)

If(a==0)a
nd (b<0)
i = i+1

YES

Peak [i]=file[i]

Peak[i]=0

If
I=length
(file)-1

Th=max (peak)*0.7

i-1

If peak
[i]<=Th

NO

YES

Peak [i]=0

i=i+1

N
O

If
i=length
(Peak)
YES

Plot peak

Stop

Figure 4.11: Flow Chart of obtaining peaks in PPG Signals

The flow chart for detection of peaks in PPG signals are shown in figure 4.11.
The PPG signals were taken in real time from individuals from age group of 17-23 years
at RVCE Health center. The PPG signals were obtained by fingered type reflective PPG
sensor. The obtained ECG data was acquired in Lab VIEW. The file was saved in .txt
format and .dat format which was read to the Matlab software.
The figureure 4.12 refers to the PPG signal which was corrupted by motion
artifact and motion artifact noise is reduced using SVD (Singular Value Decomposition).
The results showed that the SNR (Signal to Noise ratio) values of SVD were higher than
other filtering techniques. After removal of baseline drift, the motion artifact was
removed by using simple 200 orders moving average filter in Matlab Software. The
power line interference was removed by using notch filter. Figure 4.13 shows the
obtained PPG signals and the peaks in the PPG signals were obtained as shown in figure
4.14.

Figure 4.12: Filtering the signal using SVD

The PPGs peaks were detected by Matlab software which was required for
further processing of HRV in Kubios Software.

Figure 4.13: Obtained PPG signal

Figure 4.14: Detection of PPGs peak

4.7 Kubios Software Tool for analysing HRV
Kubios Software is sophisticated software for analyzing HRV. The changes in
RR intervals were studied in Kubios Software tool. The peak plot from the Matlab
software was loaded to the system.
4.7.1 Time-domain analysis
It is also called the statistical analysis and is a common approach for studying
HRV because of its simplicity in application. The results of time-domain are the mean
RR intervals, Standard deviation of RR interval, NN50, PNN50, TINN values and
Triangular index values. Figure 4.15 shows the results of time-domain analysis of the
HRV signal.

Figure 4.15: Time domain parameters GUI

4.7.2 Frequency-domain Analysis
Frequency-domain methods, Discrete Fourier Transforms were applied to the RR
interval values in time domain to obtain a power spectrum density (PSD) estimate. The
frequency domain option on top of the results view segment was selected to get the

results of frequency domain. The frequency-domain results are shown in figure 4.16

Figure 4.16: Frequency-domain parameters GUI

4.8 Nonlinear Techniques
The nonlinear technique was computed using Detrndeed Fluctuation Analysis
(DFA), Poincare Plot, Recurrence plots and Approximate Entropy. The nonlinear results
were obtained pressing nonlinear option in the result segment. Nonlinear results view is
as shown in figure 4.17

Figure 4.17: Nonlinear parameters GUI

Kubios Software is a user friendly tool for extracting HRV parameters. Also,
Kubios Software is an open source and its results are obtained with considerable accuracy
.Kubios Software assists for HRV analysis with both linear and nonlinear parameters, the
GUI in the software is designed such that it is simple and easily accessible. GUI includes
both the graphical representation and displayed values. When compared to other open
source software for HRV analysis Kubios Software tool was found to be much advanced.
The methodology mentioned above gives the algorithms, flowchart and GUIs of
the software used for the present study of HRV analysis. Kubios Software was found to
be advanced and sophisticated software compared to other open source softwares
available. Hence the above methodology was adopted for the present study.