Description of Event or Problem · 1
NONLINEAR ANALYTICAL RECOVERY IN THE LIAISON 25 HYDROXY VITAMIN D CHEMILUMINESCENT IMMUNOASSAY BACKGROUND: IN (B)(6) OF 2010, WE DISCOVERED A MARKED UNDER-RECOVERY OF 25OHD IN A PT SAMPLE THAT WAS RANDOMLY SELECTED AND SERIALLY DILUTED FOR THE PURPOSE OF VERIFYING LINEARITY/RECOVERY. IN VIEW OF THIS INITIAL FINDING, WE BEGAN TO ROUTINELY ANALYZE TWO-FOLD DILUTIONS -MANUFACTURER'S SPECIFIED DILUENT- FOR ALL PT SPECIMENS THAT HAD INITIAL RESULTS THAT WERE > 58 NG/ML. WHEN THE RECOVERY AFTER DILUTION WAS < 85% OF THE RESULT FOR THE UNDILUTED SPECIMEN, WE SUSPECTED THAT THE SPECIMEN CONTAINED AN INTERFERING SUBSTANCE THAT CAUSED THE INITIAL RESULT TO BE FALSELY ELEVATED. METHODS: SUBSETS OF THE SAMPLES WITH NONLINEAR DILUTION PROFILES -NLD SAMPLES- WERE RETESTED BY THE LIAISON ASSAY TO SEE IF THE POSITIVE INTERFERENCE COULD BE ELIMINATED BY: EXTRACTION/DEPROTEINIZATION WITH ACETONITRILE, TREATMENT WITH A HETEROPHILE BLOCKING REAGENT -(B)(6), OR SUPPLEMENTATION WITH GOAT IGG OR RABBIT GAMMA GLOBULINS PRIOR TO ANALYSIS. A SUBSET OF THE NLD SPECIMENS WAS ALSO TESTED FOR TOTAL 25OHD BY (B)(6). RESULTS: OVER AN 86 DAY STUDY PERIOD - (B)(6) 2010 TO (B)(6) 2011, WE TESTED A TOTAL OF 5305 SPECIMENS FOR 25OHD. ONE HUNDRED FIFTY TWO SPECIMENS HAD INITIAL RESULTS THAT WERE GREATER THAN 58NG/ML, AND 31 OF THESE -20%- HAD FRACTIONAL RECOVERIES OF < 0.85 AFTER DILUTION. THE MEAN FRACTIONAL RECOVERY -SD- OF 25 OHD FOLLOWING ACETONITRILE EXTRACTION OF FIVE SAMPLES WITH LINEAR DILUTION PROFILES WAS 0.94-0 - 0.08 - AS COMPARED TO THE ORIGINAL SPECIMEN. IN CONTRAST, THE MEAN RECOVERY FOR 6 NLD SPECIMENS WAS 0.36 - 0.13-. THIS DIFFERENCE WAS HIGHLY SIGNIFICANT -P<0.001, UNPAIRED T-, AND SUGGESTED THAT THE INTERFERING SUBSTANCE WAS A PROTEIN. ONLY 1 OF 6 NLD SPECIMENS SHOWED A SIGNIFICANT DECREASE - >15 %- IN THE 25 OHD RESULT AFTER TWO SUCCESSIVE TREATMENTS WITH THE HBT REAGENT. THREE OF THESE 6 NLD SPECIMENS SHOWED A SIGNIFICANT DECREASE IN THE 25 OHD RESULT AFTER THE ADDITION OF GOAT IGG. THE TOTAL 25 OHD RESULTS FOR A GROUP OF 15 UNDILUTED NLD SPECIMENS WERE, ON AVERAGE, 2.6-FOLD ELEVATED - 95% CI:2.0 TO 3.2; RANGE: 1.6 TO 5.0- BY THE LIAISON METHOD AS COMPARED TO THE TOTAL 25OHD RESULTS DETERMINED BY (B)(6). CONCLUSION: OUR STUDIES DEMONSTRATE THE PRESENCE OF PROTEIN - POSSIBLY HETEROPHILE ANTIBODY- INTERFERENCE IN THE LIAISON ASSAY. THE MAGNITUDE OF THE INTERFERENCE AND ITS HIGH PREVALENCE AMONG SAMPLES IN THE UPPER HALF OF THE METHOD'S AMR INDICATE THAT IT IS AN IMPORTANT SOURCE OF CLINICALLY SIGNIFICANT ANALYTICAL ERROR IN 25 OHD ASSAYS PERFORMED BY THE LIAISON METHOD.