Detection and characterization of estradiol (E2) and unconjugated estriol (uE3) immunoassay interference due to anti-bovine alkaline phosphatase (ALP) antibodies

Anu S Maharjan; Sara P Wyness; Julie A Ray; Tanya L Willcox; Jonathan D Seiter; Jonathan R Genzen

doi:10.1016/j.plabm.2019.e00131

Detection and characterization of estradiol (E2) and unconjugated estriol (uE3) immunoassay interference due to anti-bovine alkaline phosphatase (ALP) antibodies

Pract Lab Med. 2019 Aug 29:17:e00131. doi: 10.1016/j.plabm.2019.e00131. eCollection 2019 Nov.

Authors

Anu S Maharjan¹, Sara P Wyness², Julie A Ray², Tanya L Willcox³, Jonathan D Seiter³, Jonathan R Genzen^{1

2

3}

Affiliations

¹ Department of Pathology, University of Utah, 500 Chipeta Way, Salt Lake City, UT, 84108, USA.
² ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT, 84108, USA.
³ ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT, 84108, USA.

Abstract

Objectives: Competitive immunoenyzmatic assays for estradiol (E2) and unconjugated estriol (uE3) on UniCel DxI 800 Access immunoassay systems (Beckman Coulter) utilize bovine alkaline phosphatase (ALP) for amplification. In these assays, rare 'IND' error flags indicate that a relative light unit (RLU) raw result is past the high or low end of the calibration curve but cannot be differentiated from an instrument error or analytical interference. The present studies were conducted to establish a protocol to identify analytical interference and to characterize its mechanism when present.

Design and methods: Matrix and recovery studies were conducted to establish a protocol for interference identification. Spiking experiments with inactivated calf intestinal ALP were performed to determine whether interference could be blocked. Commercial anti-ALP antibodies (Abs) were spiked into human serum to model assay interference. Three E2 immunoassays which do not include ALP as a reagent component (cobas e602, Roche; Centaur XP, Siemens; ARCHITECT i2000_SR, Abbott) were tested for comparative purposes.

Results: 1:2 dilution of specimen into Access Sample Diluent A (Beckman) differentiated IND error flags due to true low results (e.g. less than the analytical measurement range; AMR) from those due to assay interference. Interferences were reduced by pre-incubation with inactivated ALP and could be replicated by spiking with commercial anti-ALP Abs.

Conclusions: Patient anti-bovine ALP Abs can cause interference on DxI 800 E2 and uE3 assays. This model can be used to investigate interference risk with other ALP-dependent assays.

Keywords: ALP, alkaline phosphatase; AMR, analytical measurement range; Alkaline phosphatase; Analytical systems; CLIA, Clinical Laboratory Improvement Amendments; E2, estradiol; Endocrinology; IND, indeterminate ‘no value’ error flag; Immunoassay; Interference; MoM, multiple of the median; PBS, phosphate buffered saline; RLU, relative light unit; ddH2O, demineralized distilled water; uE3, unconjugated estriol.