Abstract Background Detection of glucose-6-phosphate dehydrogenase (G6PD) deficiency provides important information for neonatal clinical management. G6PD newborn screening is recommended using umbilical cord blood specimens in vulnerable patient populations.1 Also, cord blood is used by many clinicians instead of peripheral blood for neonatal screening of G6PD.2 Use of cord blood is a modification to the FDA-approved Pointe Scientific G6PD kinetic-340 nm assay. The aim of this study was to validate cord blood as an alternate specimen type for the Pointe Scientific G6PD assay. Methods Hemoglobin (Hgb) results are from Sysmex XN 9100™ analyzers that use SLS methodology with photometric detection. G6PD results are from Roche cobas® c502 analyzers with Pointe Scientific kinetic method and spectrophotometric detection of activity, expressed as U/g Hgb. Neonatal blood specimens were obtained as paired samples from heel sticks and umbilical cord collected in EDTA lavender top tubes. Accuracy acceptance criteria was defined as Deming regression slope between 0.90 and 1.10, r-value of >0.90 and bias ±15% and mixing study recovery of ±10%. Precision (intra-assay and inter-assay), reportable range and linearity, carryover, and stability were established using cord blood samples or control materials sourced from Thermo Fisher Scientific or Sysmex. Reference intervals3, analytical sensitivity, specificity, and clinical validity were transferred to cord blood due to comparability with whole blood FDA-approved method. EP Evaluator software by Data Innovations® was used for calculations. Results Direct comparison of G6PD results (U/g Hgb) from 125 paired samples yielded a 0.9025 correlation coefficient, 1.024 Deming slope, and -0.25% bias. Mixing and recovery of admixed samples gave % recoveries of 102, 103, 101, 102, 100, and 100 for G6PD (U/g Hgb), and 102, 102, 101, 104, 99, and 105 for Hgb (g/dL). Intra-assay precision results were 2.4, 0.6, 0.8, 0.5, 0.4 % CV for G6PD and Hgb, respectively. Inter-assay precision results were 3.1, 3.3, 0.7, 0.7, 0.5 % CV for G6PD and Hgb, respectively. Reportable range and linearity evaluation resulted in a maximum deviation from target recovery of 4%, and a slope of 0.979 for a range of 50 - 2740 U/L, and reportable range for Hgb was confirmed as 0.1 - 26.0 g/dL using controls (1% maximum deviation and 0.991 slope). Carryover was -5.8 (mean concentration) for G6PD and 0% for Hgb after testing 21 replicates of pooled high and low samples in a defined order. Testing 3 samples in 3 replicates at each defined time interval defined stability as 1 week refrigerated or 72 hours room temperature for G6PD, and 3 days refrigerated or 1 day room temperature for Hgb. Conclusions Regression statistics and mixing-recovery studies demonstrated accuracy for detecting G6PD (U/g Hgb) and Hgb (g/dL) from EDTA cord blood. Additional performance characteristics were transferrable for this specimen type from the FDA-approved method for whole blood. Umbilical cord blood is an acceptable alternate specimen type for quantitative detection of G6PD (U/g Hgb) using Pointe Scientific with Sysmex XN-9100™ and Roche cobas® c502 instrumentation.