CHAPTER THREE

MATERIALS AND METHODS

3.1 Research Design

An experimental and cross-sectional research design was adopted for this study.

3.2 Study Area

This research was carried out at the antenatal clinic in Rivers State College of Health Science and
Management Technology. The institution is located at Kilometre 4, Ikwerre Road, Rumueme in
Obio/Apkor Local Government Area, Rivers State.

3.3 Study Population

A total of pregnant women aged between years were used for the study. Also apparently
healthy non pregnant women were used as controls for the study. Informed and written consent
was duly obtained from each participant before the study. A structured questionnaire was used in
collection of selected demographic data which include name, age and trimester of pregnancy.
Women on menstruation and acute/chronic kidney disorder were excluded from the study.

3.4 Sample Collection

The specimen bottles were used for each subject, Anticoagulant bottles containing lithium heparin
was used for electrolyte assay. The subject were allowed to sit, gloves were put on and the blood
sample was collected by vein puncture. A tourniquet was tightened on the upper arm above the site
of the puncture and the site of puncture was cleaned with 70% alcohol and allowed to air dry, 21G
disposal needle and syringe was used to collect 3ml of blood and it was dispensed into a lithium
heparin bottle. The container was well labelled with the subject's name. The separation of Plasma
from the whole blood collected was done using centrifuge, it was spun for 5 minutes at 4,000
r.p.m. The plasma from the spun whole blood was separated carefully using a pasteur pipette into
plain bottles respectively. The samples were transported to for electrolytes estimation.

3.5 Method for Electrolyte Estimation

Ion Selective Electrode (ISE) Method of electrolyte estimation using ISE 4000 analyzer made by
the State Forest Research Institute (SFRI) was used for the determination of Sodium, Potassium,
Chloride and Bicarbonate concentration in the blood sample collected from the pregnant and non
pregnant women.

3.5.1 Principle of Ion Selective Electrode

This method measures the voltage that develops between the inner and outer surface of an ion
selective electrode when solution containing these electrolytes are mixed with high ionic strength
ISE electrolyte bugger and passed through the panel (flow cell) . it uses a wet electrode, one
electrode for each analyte. The electrode ( membrane) is made of a material that is selectively
permeable to the ion being measured. A potential is generated at the surface of the ion selective
electrode and thereby measuring the sodium, potassium, chloride and bicarbonate concentration in
the sample (D' Orazio & Meyerhoff, 2008).
3.5.2 Procedure for Electrolyte Estimation
The standard operating procedure for the ion selective electrode (ISE) 4000 analyzer is that, the
analyzer is switched on and cleaned using the cleaning solution. The ISE reagent is connected to
the packs and then to the analyzer. The analyzer is calibrated to ensure a pass for sodium,
potassium, chloride and bicarbonate. After the calibration, the screen displays the main menu. The
plasma sample in the plain bottle was placed under the probe of the ISE analyzer and the "aspirate
" button on the analyzer screen is pressed. The probe sucks in the sample from the plain bottle into
the ISE analyzer and on hearing a beep sound made by the analyzer, the sample in the plain bottle
was removed. The sample was analyzed and the results of the four electrolytes were displayed on
the screen.

3.6 Statistical Analysis

The statistical analysis of results obtained from the study was done using SPSS software
application. Comparison of groups was done by T-test and results were expressed as Mean ±
Standard Deviation (S. D). Values of P< 0.05 were considered statistically significant.