Study Setting, design and population
An institution-based cross-sectional study was conducted from February 1 to May 30, 2019, at the gynecology and obstetrics department of University of Gondar comprehensive specialized hospital, Northwest Ethiopia. The hospital is located in Gondar town Amhara regional state, northwest Ethiopia. The town is located 2,133 meters’ elevation above sea level. The hospital provides both teaching and referral center in the region, which services more than five million people. Currently, the hospital holds more than 550 beds, and it handles approximately 8000–9000 deliveries per year. The hospital has a range of specialties including pediatrics, surgery, gynecology, psychiatry, human immunodeficiency virus (HIV) care, and an outpatient clinic.
The newborns were selected based on the following criteria: Birth at full-term (39-42 weeks of gestation), and absence of any congenital anomalies. All selected newborns were physically examined at birth and found normal and apparently healthy. The premature newborn (delivered less than 37 weeks of gestation), twin newborns, the pregnancy complicated with diabetics, preeclampsia, hypertension, HIV/AIDS, chronic kidney, liver disease, malaria, anemia, and hematological malignancy were excluded from the study. On the other hand, a mother who had bleeding during pregnancy, maternal drinking of alcohol during pregnancy, cigarette smoking during pregnancy, and no antenatal care also were excluded from being sampled. A systematic random sampling technique was employed to select study participants. The sample size is determined based on national Committee for Clinical Laboratory Standards, International Federation of Clinical Chemistry and Clinical Laboratory Standards Institute guideline recommendations, a minimum size of 120 observations is required for determination of reference intervals (15). Study participants were selected every three intervals based on flow of delivery to give equal allocation of study participants. The excluded study participants were substituted with the next consecutive study participants. A total of 202 study participants were selected during the study periods. However, 43 were excluded due the presence of maternal anemia, 5 were excluded due to twins and the remained 3 newborns were still birth. Finally, a total of 151 newborns with their respective mothers were included and their results were analysis in SPSS.
Data collection method
Socio-demographic and clinical data collection
A pre-tested structured questionnaire prepared in English and translated to the local language (Amharic) was used to obtain newborn gender, birth weight, and presence of bleeding during pregnancy, alcohol consumption habits, and cigarette smoking during pregnancy via face-to-face interviews. The presence of maternal complications like malignancy, hypertension, diabetics, tuberculosis, HIV/AIDS, chronic kidney, and liver disease were retrieved from maternal medical records with the aid of data extraction sheet.
Blood collection and laboratory analysis
About 3ml of UCB specimen were obtained from each study participants after delivery from the clamped umbilical cord. The two trend Midwifery professionals collected the cord blood sample from the clamped cord through excluding of the placenta. The collected sample was immediately poured into tri-potassium ethylene diamine tetra acetic acid (K3-EDTA) test tube and gently mixed to prevent blood clotting. In addition, 3ml of venous blood was collected from the mother after delivery with a sterile and disposable syringe. Hematological parameters: total white cell count (WBC), differential white cell count (neutrophils, lymphocytes and mixed which contains eosinophils, monocytes. and basophiles), platelet count, red blood cell count (RBC), Hgb, hematocrit (%), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), and red cell distribution width (RDW) were determined by using the using Sysmex KX-21N (Sysmex Corporation Kobe, Japan) automated hematological whole blood analyzer based on direct current principle. Three experienced laboratory technologist performs the complete blood count (CBC) by strictly adhering standard operating procedures.
Data quality control
In order to increase the reliability of data, training was given for the data collector prior to data collection. During laboratory data collection standard operating procedures were strictly followed and implemented from prior to specimen collection up to recording and interpreting of the laboratory results. The collected sample were immediately dispensed to the wall of the EDTA test tube slowly and properly mixed by inverting the tube gently 8-10 times to prevent hemolysis of blood samples. Then label of the sample and the request paper with the same identification number in order to avoid any mix up of errors. The expired date of reagent was checked before analysis of patient samples. Daily installations and background run were done to minimize any background errors. Repeated analysis of randomly selected samples for reproducibility check (delta check) was carried out three times a week to evaluate instrument performance consistently and precisely.
The data were cleaned, edited, checked for completeness, and entered into Epi-info version 188.8.131.52. Then it was exported into SPSS version 20 for analysis. Outliers were detected by using Z score and Tukey method. Before any analysis, normal distribution of numerical data was checked using the Kolmogorov-Smirnov and Shapiro-Wilk test. Since all data are not Gaussian distributed we used non-parametric tests to determine the hematological RI as recommended as the clinical laboratory standard institutes (12). Median with interquartile range (IQR), and 95% confidence interval were computed. The 97.5th percentile and 2.5th percentiles were the upper and the lower reference limit of the study populations, respectively. Mann-Whitney U test also used to test for the mean difference of variance between gender (male versus female), and delivery type (vaginal versus cesarean section). According to the guideline of clinical laboratory standard institutes, we determine the 95% hematological RI by computing 97.5th percentile and 2.5th percentile.