The NVivo Coding

proforma prepared for the study. In a standardized manner, relevant information were obtained from each of the patients. These include age, sex, addresses and telephone numbers of the patients. History of easy fatigability, dyspnoea on exertion, cough, paroxysmal nocturnal dyspnoea, orthopnoea, right hypochondrial pain, leg and abdominal swellings were also obtained. Patients’ functional class of HF was determined using the New York Heart Association Classification.²¹ Detailed drug history and chronic medical conditions such as CKD, liver disease or cardiopulmonary diseases was taken.

4. 13 ANTHROPOMETRIC MEASUREMENT: Weight was measured after removal of shoes and when wearing light clothing only, using a portable calibrated Bathroom scale and was recorded to the nearest 0.1 kg. Height was measured to the nearest 0.01metre (m) without

40

shoes, head scarf or cap using a height scale incorporated in the weighing scale or measuring tape calibrated on the wall. Body Mass Index was calculated as weight (Kg)/Height (m²).⁷³ Blood pressure was measured in sitting position after the subject had rested for at least five minutes, using the first and fifth Korotkoff sounds as systolic and diastolic BP respectively.

Accosson^® mercury sphygmomanometer with appropriate cuff (25cm x 12cm) was used to measure the BP. Three consecutive measurements were obtained five minutes apart and the average obtained.

A thorough physical examination was performed by the investigator with particular emphasis on the cardiovascular system. The pulse rate (PR), blood pressure (BP), jugular venous pressure (JVP), presence of third heart sound, tender hepatomegaly and basal lung crepitations were assessed. Clinical diagnosis of HF was based on the Framingham criteria of concurrent presence of two major criteria or one major with two minor criteria. ⁷⁴ The criteria are presented in Table 3 below.

41

Table 3: Framingham clinical criteria for HF diagnosis.⁷⁴

Major criteria Minor criteria

Paroxysmal nocturnal dyspnoea/ orthopnoea Ankle oedema

Neck vein distension, Night cough

S3 gallop Dyspnoea on exertion

Cardiomegaly Hepatomegaly

Lung rales Pleural effusion

Acute pulmonary oedema, Tachycardia (range of > 120/min) Increase venous pressure > 16cm H2O Vital capacity 1/3 from maximum Hepatojugular reflux.

42 4.14 ECHOCARDIOGRAPHY

Conventional trans-thoracic echocardiography (TTE) was utilized to evaluate the subjects using ALOKA sonogram machine, 2006 model SSD-4000. Two dimensional Echo was obtained at conventional position and views. For 2-D directed M mode measurement, parasternal long axis (PLAX) view was used. Measurements were taken using the American Society of Echocardiography convention of leading edge to leading edge methodology.^75-79 Three measurements were taken for each parameter and the average taken for recording on the proforma. All Echo studies were performed by the investigator under the watchful eyes of one of the supervisors. Specific M- mode measurements were appropriately made at end diastole and end systole using ECG as timing index for cardiac cycles. The parameters that were recorded include LV internal dimension in diastole (LVIDd), LV internal dimension in systole (LVIDs), fractional shortening (FS), EF, left ventricular mass (LVM), left ventricular mass index (LVMI), aortic and left atrial dimensions.^{75, 79.}

FS was calculated using the following formula⁷⁹

FS = LVIDd – LVIDs × 100%.

LVIDd

EF was obtained using the standard cubed formula as follows

The LVEF was evaluated with biplane TTE by the modified Simpson rule.⁷⁹ EF = (LVIDd)³ – ( LVIDs)³ x 100

(LVIDd)³ Where

LVIDd= LV internal dimension in diastole

43 LVIDs= LV internal dimension in systole LVEF= LV Ejection Fraction

In this study, depressed LV systolic function was based on both FS <30%, EF < 50%.^{2, 78, 79} Left ventricular mass index was calculated using the American Society of Echocardiography (ASE) cubed formula.^{75, 79}

LVMI = 1.04[(LVIDd + PWTd + IVSTd)³ – (LVIDd)³] BSA

Where;

LVMI= Left ventricular mass index

LVIDd= Left ventricular internal dimension in diastole PWTd= Posterior wall thickness in diastole

IVSTd= Interventricular septal thickness in diastole BSA= Body surface area

Echo LV hypertrophy (LVH) was defined as LVMI > 110g/m² in females and 134g/m² in males.^{75, 79, 80}

Doppler Echo was obtained according to the recommendations of quantification of Doppler Echo of the ASE.⁸¹ The LV diastolic flow pattern from the atria to the ventricles was assessed by Doppler studies using the Apical 4 chamber view and Apical 5 chamber view. The Doppler studies (continuous and pulsed wave) were used including Trans-mitral, aortic and pulmonary velocities. The mitral inflow E-velocity, A-velocity and E/A ratio was recorded.

The deceleration time (DT) and Isovolumetric relaxation time (IVRT) was obtained.

44 The LV diastolic function ⁸¹ was classified as:

Normal; - E/ A ratio 1-2, DT- 160- 240msec, IVRT 70-100msec Ea≥ 10cm/sec, E/ Ea <8

Impaired relaxation;- Reduced E velocity, increase A velocity, E/A ratio < 1, prolonged DT(> 240msec), prolonged IVRT (>120msec), Ea

<7cm/sec.

Pseudonormalization; - Normal DT (160- 240msec), E/ A ratio1- 2,

wide mitral valve E point septal separation EPSS (>5mm), depressed EF and left atrial enlargement on two-dimensional Echo.

Restrictive pattern; - Decrease LV compliance, markedly increase LA pressure, increased E-velocity, decreased A-velocity, E/A ratio >2, shortened DT (<160msec), shortened IVRT (<70msec), mitral annulus Ea is reduced <70cm/sec.

The LV diastolic dysfunction was classified as impaired relaxation, pseudonormalization and restrictive pattern as defined above.

Pulmonary venous flow (PVF) velocity recording was obtained from apical 4 chamber view with the pulsed wave Doppler cursor placed over the right upper pulmonary vein in the roof of the right atrium close to the interatrial septum. The peak systolic S, diastolic D and atrial recoil (AR) flow velocities and ratio of S/D was calculated.

45 ECHO SESSION

Figure 2: The investigator in one of the Echo sessions

46

PARASTERNAL LONG AXIS VIEW OF ECHO IN VALVULAR HEART DISEASE

Figure 3: Mitral and aortic stenosis, dilated LV (62mm) and dilated LA (42mm)

47 APICAL 4 –CHAMBER VIEW OF ECHO IN HF

Figure 4: A restrictive diastolic dysfunction with mitral E/A ratio= 3.4

48 Echo conclusions:

Echo conclusions were drawn on the basis of one of the several combinations as highlighted below

Normal Echo study- Those participants with normal systolic functions (FS > 30%, EF

>50%), normal diastolic function (E/A ratio 1-2, DT= 160-240msec, IVRT=70- 100msec)

Systolic dysfunction- FS<30%, EF<50%, normal diastolic functions as above

Diastolic dysfunction- FS>30%, EF>50% but abnormal diastolic functions (impaired

relaxation, pseudonormalization and restrictive diastolic dysfunction as explained above

Systolic and diastolic dysfunction- FS<30%, EF<50%, Normal systolic, abnormal diastolic functions (impaired relaxation, pseudonormalization and restrictive diastolic dysfunction) as explained above 4.15 LABORATORY INVESTIGATIONS

BNP ASSAY: On the day of echocardiography, 10mls of peripheral venous blood of both HF group and healthy controls was collected into sample bottles containing ethylenediaminetetraacetic (EDTA) as anti- coagulant, centrifuged and frozen at -70^oC until further determination. In this study, BNP was analysed using the Phoenix BNP 32 EIA kit.

This immunoassay kit allows for the in vitro quantitative determination of human BNP concentration in serum, plasma and other biological fluids.

49

ASSAY PROCEDURE: The procedure followed international best practices.

1) The protocol was thoroughly read before performing an assay and the kit component was allowed to return to room temperature before use. Figure 5 shows the kit at a glance.

2) The assay buffer concentrate was diluted 20x with 950ml of distilled water. The assay buffer was used to dilute or reconstitute all other reagents in this kit and samples

3) The peptide standard was centrifuged and diluted with 1ml of 1x assay buffer as follows;

Standard No Std volume 1x Assay buffer Concentrations

Stocks 1000µl - 1000ng/ml

#1 100µl of stock 900µl 100ng/ml

#2 100µl of #1 900µl 10ng/ml

#3 100µl of #2 900µl 1ng/ml

#4 100µl of #3 900µl 0.1ng/ml

#5 100µl of #4 900µl 0.01ng/ml

4) The primary antibiotic was rehydrated with 5ml of 1x assay buffer and was allowed to sit for 5 minutes to completely dissolve.

5) The biotinylated peptide was rehydrated with 5ml of assay buffer and was allowed to sit for 5 minutes to dissolve completely.

6) The positive control was centrifuged and rehydrated with 200µl of 1x assay buffer and was allowed to stand for 5 minutes to dissolve completely.

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7) Wells A-1 and A-2 were left empty as BLANK, 50µl of 1x assay buffer into B-1 and B-2 as total binding, 50µl of prepared peptide standards from #5 to #1 ( in reverse order of serial dilution) into wells C-1 and C-2 to G-1 and G-2 respectively and 50µl of positive control into wells H-1 and H-2. Figure 6 shows the well before the processing.

8) Then 50µl of prepared samples was added into their designated wells as labelled and 25µl of rehydrated primary antibody was added into each well except the BLANK well. 25µl of rehydrated biotinylated peptide was also added into each well except the BLANK well.

9) The immunoplate was sealed with acetate plate sealer (APC), incubated at room temperature for 2hrs and 300- 400rpm orbital shaking during the incubation.

10) The streptavidin-horseradish peroxidase (SA-HRP was centrifuged and 12µl of it was added with 12ml of 1x assay buffer to make SA-HRP solution.

11) APS was removed from the immunoplate and the well’s contents were discarded. The wells were washed with 1x assay buffer, while the plate was blot-dried.

12) Then 100µl of SA- HRP solution was added into each well, resealed by APS and incubated at room temperature for 1 hour.

13) APS was removed from the immunoplate, washed and blot dried with 1x assay buffer as described in step 11.

14) Then 100µl of 3,3,5,5 tetramethylbenzidine (TMB) substrate solution was added into each well, again covered with APS and incubated at room temperature for 1 hour.

15) APS was removed from the immunoplate and 100µl of 2N HCl was added into each well to stop the reaction. The colour change was noticed from blue to yellow as observed in figures 7 and 8 respectively.

51 THE PHOENIX EIA KIT

Figure 5: The various components of the kits at a glance

52 THE IMMUNOPLATE WELL

Figure 6: The two immunoplate well before analysis

53 THE IMMUNOPLATE WELL

Figure 7: Showing the initial colour changed to blue.

54 THE IMMUNOPLATE WELL

Figure 8: Showing the final colour changed to yellow.

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16) The immunoplate was then loaded onto a microtiter plate reader with read absorbance optical density (O.D) at 450nm and respective O.D for each sample were obtained for calculation of BNP values for each sample.