Optimization and testing of dried antibody tube: The EuroFlow LST and PIDOT tubes as examples

Abstract

Within EuroFlow, we recently developed screening tubes for hematological malignancies and immune deficiencies. Pipetting of antibodies for such 8-color 12-marker tubes however is time-consuming and prone to operational mistakes. We therefore evaluated dried formats of the lymphocytosis screening tube (LST) and of the primary immune deficiency orientation tube (PIDOT). Both tubes were evaluated on normal and/or on patient samples, comparing the mean fluorescence intensity of specific lymphocyte populations. Our data show that the dried tubes and liquid counterparts give highly comparable staining results, particularly when analyzed in multidimensional plots. In addition, the use of dried tubes may result in a reduced staining variability between different samples and thereby contributes to the generation of more robust data. Therefore, by using ready-to-use reagents in a dried single test tube format, the laboratory efficiency and quality will be improved.

Reproducibility of Flow Cytometry Through Standardization: Opportunities and Challenges

“…One possible solution is the use of stabilized reagent mixtures that not only provide ease of use but also limit room for pipetting errors (14,51,82). One possible solution is the use of stabilized reagent mixtures that not only provide ease of use but also limit room for pipetting errors (14,51,82).…”

“…A fully standardized system including instrument setup, sample preparation and data analysis was built in a stepwise manner to aid in diagnostics and translational research in PID. This standardized system provides a first tier test of lymphocyte subsets (PID orientation tube; including naïve vs memory compartments) (51,52) and is a powerful approach that was confirmed to be applicable to PID with a large international cohort of 99 PID patients and 250 healthy controls (52). A further detailed panel that aims to dissect T-cell and B-cell subsets was assembled and used by the consortium (52).…”

“…Of course, pipetting accuracy and reagent shelf stability influence the staining pattern and intensity. One possible solution is the use of stabilized reagent mixtures that not only provide ease of use but also limit room for pipetting errors (14,51,82). These are available either as commercially supplied ready to use mixtures or dried reagents or can be custom made by several manufacturers.…”

Detection of Minimal Residual Disease in B Cell Acute Lymphoblastic Leukemia Using an Eight‐Color Tube with Dried Antibody Reagents

“…Finally, some authors tested the Duraclone technology for screening lymphoproliferative and myelodysplasic syndromes with a 10‐color 14‐antibody tube, proving this tube was useful in 60% of cases with significant time and cost savings . Another study aimed to compare dried tube immunophenotyping with liquid reagent method, assaying mean fluorescence intensities (MFI) for both methods in the context of lymphocytosis or immune deficiency. This study revealed comparable results for MFI obtained with dried and liquid tubes.…”

EuroFlow Standardized Approach to Diagnostic Immunopheneotyping of Severe PID in Newborns and Young Children

“…In this study, the EuroFlow PID group consortium has designed, developed and validated a standardized approach for flow cytometric evaluation of naïve, RTE and activated CD4+ and CD8+ T-cells that would offer a high sensitivity test toward disclosing (S)CID in line with the ESID diagnostic criteria in the settings of a multi-center collaboration study. The here developed 8 color "SCID-RTE tube" complements the recently published PIDOT tube (18,28) for orientation and screening of primary immunodeficiencies (PID) of the lymphoid system. The combination of the two 8-color tubes (or a single 12-color variant of both tubes) could readily be applied in routine diagnostic screening for patients clinically suspected for having (S)CID, as well as in follow-up diagnostics in NBS programs.…”