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• Quantitative fit testing (QNFT) requires a sampling tube attachment. • Robust sampling tube stabilization is needed for QNFT of flat-fold type respirators. • Lanyard technique had significantly lower QNFT pass rates than hand-hold technique. • Fit testers should pay close attention to the sampling tube during QNFT. When performing quantitative fit testing (QNFT) on filtering facepiece respirators using an ambient aerosol technique, a twin sampling tube is connected between the condensation nuclei count machine and the probed respirator. To achieve high quality and repeatable QNFT results, robust sampling tube stabilization is required. In this prospective randomized crossover study, conducted in December 2021 to February 2022, we compared the commonly used hand-hold technique with the manufacturer-recommended lanyard technique in stabilizing the sampling tube during QNFT on a Halyard N95 respirator. Outcomes included QNFT pass rates, overall and individual fit factors, and concordance between the two techniques. A total of 228 out of 316 participants (72.2%) passed the QNFT with the hand-hold technique, compared to the lanyard technique (166/316, 52%, P <.001). The most significant drop in the fit factors with the lanyard technique occurred during head movement side-to-side and up-and-down. The concordance between the 2 techniques was fair (Kappa coefficient = 0.39). Our study demonstrates that the method of sampling tube stabilization during QNFT has a significant impact on fit test pass rates, with a potential for false negative fit tests due to inadequate tube stabilization. Further research is required to examine the generalizability of these results to other respirators and fit testing apparatuses. [ABSTRACT FROM AUTHOR]