Factors Influencing Fluorescence-activated Cell Sorting for Multiple Myeloma Fluorescence in situ Hybridization: Real-world Experience

Abstract

Background: FISH is the standard method for detecting cytogenetic abnormalities (CAs) in patients with multiple myeloma, and pre-enrichment of plasma cells is recommended to increase detection rates. However, optimal strategies to ensure sufficient plasma cell re- trieval when standard enrichment techniques fail remain underexplored. We investigated factors influencing the success of fluorescence-activated cell sorting (FACS) and assessed the use of direct FISH in cases in which FACS failed. Methods: A retrospective analysis was conducted on 457 bone marrow samples submit- ted for FISH between November 2016 and May 2022. FACS was considered successful when plasma cells (CD38+ and CD138+ cells) constituted >1% of the total number of cells. Direct FISH was performed for samples with FACS failure. Results: FACS was successful in 70.9% of cases and had a high positivity rate (94.8%). Shorter sample transfer times significantly improved FACS success, with a 77.1% success rate for transfer times <2 hrs, compared with 67.8% for longer times (P =0.0388). Plasma cell percentage was a strong determinant of FACS success, with a median of 31.2% in suc- cessful cases versus 8.5% in failures (P <0.0001). Even when FACS failed, direct FISH de- tected CAs in 43.6% of cases. Conclusions: Plasma cell percentage and sample transfer time are critical factors influ- encing FACS success. While FACS-FISH demonstrates superior sensitivity in detecting CAs, direct FISH serves as a valuable alternative when FACS fails. These findings highlight the importance of optimizing sample handling and FISH protocols for accurate cytogenetic analysis of multiple myeloma.