We carried out a retrospective, cross-sectional study employing review of primary hospital data of term and near term neonates born (>35 weeks of completed gestation) in a tertiary care center from January to December 2008. King Abdullah International Medical Research Center (KAIMRC), Ministry of National Guard Health Affairs (MNGHA), International Review Board (IRB) has approved this study protocol (RC09/106), and all patients were provided with written informed consent through their guardian/parent.
Cord blood is defined as a specimen collected from the umbilical artery at the time of delivery and peripheral blood is the blood obtained from any other site of the body within one week of age. Unless otherwise specified, we define G6PD deficiency as a G6PD quantitative assay by spectrophotometric analysis of ≤5.7 U/g Hb (unit of enzyme activity/g hemoglobin), as per laboratory recommendation.
Cord and venous blood samples were collected from each patient using conventional techniques into Vacutainer (BD Plymouth, PL6 7BP, U.K.) or Microtainer (Becton, Dickinson and Co., Franklin Lakes, NJ 07417, USA) tubes with K2EDTA as anticoagulant at a concentration of 1.8 mg/ml. Samples were accessioned into the Laboratory Information System (LIS), then their hemoglobin (Hb) levels were determined on same time and day as the G6PD analyses using Cell-Dyn Sapphire blood analyzers (Abbot Diagnostics Division, Abbot Park, IL 60064, USA). All samples were stored at 2-8C, batched and analyzed for G6PD enzymatic activity within 4–6 h of collection. Sadly, three infants died before the peripheral blood sample could be obtained for measurement of the G6PD.
One hundred microliters of well-mixed whole blood was pipetted in a test-tube containing 400 μl of a proprietary lysing reagent, mixed well and let stand for 5 min. An aliquot of this was poured into a sample cup using the only G6PD assay kit designed for both newborn and adults, which then was placed in the Udilipse Random Access Analyzer (United Diagnostics Industry, P.O. Box 9466, Dammam 31,413, Kingdom of Saudi Arabia). Once hemolysates were made, analysis was carried out immediately and strictly within an hour.
The principle of the test involves the catalysis of glucose-6-phosphate to 6-phosphogluconate by G6PD and reduction of NADP to NADPH in the following reaction  (Glucose-6-phosphate + NADP ➔ 6-Phosphogluconate + NADPH + H
The activity of G6PD was proportional to the rate of production of NADPH which possesses a peak Ultraviolet (UV) light absorption at 340 nm. Results from the Analyzer were automatically transmitted to LIS permitting access to patient’s previously estimated blood hemoglobin level, computed and reported results in units/g (of hemoglobin).
In carrying out the study, we collected the names and medical record numbers (MRN) of all newborns with G6PD quantitative assays (cord or peripheral) for the specified time interval (January to December 2008). This was the year that our institution began implementing universal G6PD deficiency neonatal screening. We then selected the neonates with cord G6PD assays and from this pool, the overall incidence and the gender distribution of G6PD deficiency was computed. However, due to the unavailability of G6PD molecular genotyping in our institute DNA analysis was not possible. There is a future plan to include DNA genotyping in a forthcoming study.
From among the newborns with cord blood G6PD assays, we picked out those who also had peripheral samples taken (presumably within one week of age). A subgroup analysis was carried out on this particular subset of patients (n = 1, 253) comparing the cord and peripheral G6PD values.