We used analyte recoveries computed from our examinations of hematocrit effects, blood-volume-per-spot effects, and filter-paper serum-absorption volumes to predict expected differences between Phe recoveries from the EWS-Phe-01 and the AARM. To evaluate the reliability of the Phe concentration values that were predicted from measurements of matrix variables, we analyzed the EWS-Phe-01 and the AARM in duplicate in each of five HPLC runs and compared the mean values of the measured Phe concentrations with the predicted Phe concentrations.
The slope of the regression line, derived from measured blood volume per disk vs hematocrit, predicted that the EWS-Phe-01 materials, with a calculated hematocrit of 50.1%, should have a blood volume of 3.1 [micro]L per ~3-mm disk, whereas the AARM, with a 53% calculated hematocrit, should have a blood volume of 3.2 [micro]L per disk.
The mean serum-absorption volume per ~3-mm disk punched from the S&S Grade 2992 paper used to prepare the EWS-Phe-01 was 1.250 0.104 [micro]L, whereas that from the S&S Grade 903 paper used to prepare the AARM was 1.502 [+ or -] 0.188 [micro]L (P <0.01).
The controlled comparisons of hematocrits, blood volumes per spot, and filter-paper serum-absorption volumes showed, in all cases, that analyte recovery per DBS disk was lower for the conditions used in the preparation of EWS-Phe-01 than from those used in the preparation of AARM. By summing these observed differences, we projected (Table 1) that analyte recovery from EWS-Phe-01 and AARM could be expected to differ by 30.8% when the two sets of materials were analyzed in tandem.
The effects of the differences in the blood hematocrit, blood volume per spot, and filter-paper sources used to prepare the EWS-Phe-01 and AARM yielded analytic recovery differences of 3.1%, 10.9%, and 16.8%, respectively.
The EWS-Phe-01 and AARM were prepared from blood with hematocrits typical of newborns and with filter papers and blood-spot sizes that reflect newborn-screening practices in the regions in which they are used.
In an effort to demonstrate and quantify the impact that standardized calibration of DBS methods has on the measurement of amino acids, we will distribute sets of AARMs, along with a panel of test samples, to a group of manufacturers and screening laboratories that use different analytical methods.
The AARMs will be stored at CDC and distributed from CDC.
We thank Carol Bell, Trudy Dobbs, Hugh Gardner, Omar Henderson, Roberta Jensen, Joanne Mei, Nancy Meredith, and Francis Spierto, Newborn Screening Quality Assurance Program (NSQAP), CDC, for participation in the production of the AARMs. We thank Sherri Barker, Michael Brinson, and Steven Worthy, Duke Medical Center, for help in the MS/MS analyses, and Leslie T.
Endogenous amino acid concentrations of AARMs (y4ntercepts of weighted regression lines).
STOCK CALIBRATOR SOLUTIONS FOR AARM WHOLE-BLOOD POOLS
A portion of each amino acid-enriched whole-blood pool used to prepare the AARM blood spots was combined with 40 [micro]L of a solution containing deuterium-labeled amino acid calibrators to make a final volume of 5 mL per pool.