A-342 Performance evaluation of an automated test system for determining the soluble fms-like tyrosine kinase/Placental growth factor ratio

Background Preeclampsia is a hypertensive disorder that affects the mother and fetus and is a major cause of perinatal and maternal morbidity and mortality. The ratio of soluble fms-like tyrosine kinase (sFlt-1) to placental growth factor (PlGF) is used to predict the risk that a pregnant individual will develop preeclampsia. Measurement of sFlt-1 and PlGF is quantitatively determined in serum or EDTA plasma using the B·R·A·H·M·S sFlt-1/PlGF KRYPTOR Test System. The system calculates and reports the sFlt-1/PlGF ratio. The purpose of this study was to validate the KRYPTOR sFlt-1 and PlGF assays on the KRYPTOR Test System. Methods Performance characteristics evaluated included accuracy, linearity, precision, analytical sensitivity, analyte stability, carryover, and reference limits. Serum specimens from self-reported healthy volunteers and deidentified residual serum specimens were collected under University of Utah’s Institutional Review Board approved protocols. PlGF and sFlt-1 were measured according to the test kit manufacturer’s protocols. Results For sFlt-1, a method comparison study against an outside laboratory generated a slope of 0.927, r2 of 0.996, and bias of -9.1% (n=32, Deming regression). PlGF comparison demonstrated a slope of 0.962, r2 of 0.994, and bias of -6.3% (n=31). High and low-end sFlt-1 linearity was assessed by combining specimens with high, mid and low sFlt-1 concentrations at different ratios. Regression analysis produced a slope of 1.00, and r2 of 0.996, with percent recoveries ranging 93.7–100.0% for the high-end linearity (n=5, range 21,120–70,205 pg/mL). Low-end linearity for sFlt-1 demonstrated a slope of 1.00 and r2 of 1.000, with percent recoveries ranging 98.6–100.9% (n=5, range 318–20,925 pg/mL). For PlGF linearity, combinations of high and low concentration specimens produced a slope of 1.00 and r2 of 0.996, with percent recoveries ranging 87.5–100.0% (n=5, range 5.7–3871.0 pg/mL). The observed limits of detection were 22.8 and 2.0 pg/mL for sFlt-1 and PlGF, respectively. Precision was determined from two serum pools of differing sFlt-1 concentrations tested over 5 days, four replicates per pool per day. sFLt-1 repeatability and within-laboratory CVs were 0.4 and 1.6%, respectively, at a mean of 1461 pg/mL, and again 0.4 and 1.6%, respectively at a mean of 9847 pg/mL. For PlGF, two serum pools each produced CVs of 7.3% for repeatability and within-laboratory reproducibility, at a mean of 28.1 pg/mL, and 2.0 and 2.1%, respectively at a mean of 360.6 pg/mL. Both analytes were stable for minimums of 24 hours ambient, 31 days refrigerated, 31 days frozen (-20°C), and 3 freeze/thaw cycles. Carryover was insignificant. Testing of paired serum and K2 EDTA plasma from three pregnant volunteers resulted in differences ranging from -4.6 to -0.3%. Reference limits were verified using specimens from 20 healthy pregnant women (singleton pregnancies, 23 weeks – 34 weeks, 5 days gestation) with 100% of results having a sFlt-1/PlGF ratio of <40. Conclusion The B·R·A·H·M·S sFlt-1/PlGF KRYPTOR Test System demonstrates acceptable performance for quantifying both sFlt-1 and PlGF in human serum and plasma. Results demonstrate that the test system can be a tool for predicting the risk of pregnant women developing preeclampsia.