PB0040 - Assessing inter-operator variability in gating strategy of whole blood monocyte subsets using flow cytometry in patients with acute stroke suspicion

Sunday, June 25, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Evelyne HENG, Resident in haematology, PharmD, MSc

INSERM UMR-S 1140, Innovative therapies in Haemostasis, Université Paris Cité
Paris, Ile-de-France, France

Co-Author(s)

MN

Marie NEUWIRTH

MD, pathologist - hematology
INSERM UMR-S-1140, Innovative therapies in Haemostasis, Université Paris Cité
Paris, Ile-de-France, France
FM

Floriane MAS

Project Manager, Research & Dedevelopment
Biocytex, Marseille, France
Marseille, Provence-Alpes-Cote d'Azur, France
GC

Geneviève CONTANT

Prospective Research DRP Director
Diagnostica Stago
Gennevilliers, Ile-de-France, France
MM

Mikaël MAZIGHI

MD, PhD, neurologist
APHP, Neurology Department, Hôpital Lariboisière, Paris
Paris, Ile-de-France, France
BM

Bertrand MONTPELLIER

PhD
Biocytex, Marseille, France
Marseille, Provence-Alpes-Cote d'Azur, France
Georges JOURDI, PharmD, PhD (he/him/his)

Associate Professor in Hematology
Université Paris Cité and APHP. Nord
Paris, Ile-de-France, France
CB

Caren BRUMPT

MD, pathologist in hematology
Hématologie biologique, Hôpital Lariboisière (AP-HP) , Paris
Paris, Ile-de-France, France
EC

Emmanuel CURIS

PharmD, PhD, Ass. Professor in Statistics
UR 7537 BioSTM (Biostatistics), Université Paris Cité
Paris, Ile-de-France, France
VS

Virginie Siguret, PhD, PharmD

PharmD PhD, Professor in Hematology
INSERM UMR-S-1140, Innovative therapies in Haemostasis, Université Paris Cité, Paris
Paris, Ile-de-France, France

Background: Innovative tools to rapidly and reliably identify patients with acute stroke are needed. Peripheral monocyte subsets, i.e. classical (Mon1), intermediate (Mon2), and non-classical (Mon3) and corresponding activation marker expression analyzed using flow cytometry (FCM) could be interesting cell biomarkers candidates.

Aims: Given the complexity of monocyte subset gating, we sought to assess the extent to which inter-operator gating variability could affect results.

Methods: BOOST study (“Biomarkers algOrithm for strOke diagnoSis and Treatment resistance prediction”, NCT04726839) is a non-interventional prospective multicenter study. Patients aged 18 years or older with symptoms suggesting acute stroke within the last 24 hours were included following informed consent. Four mL of blood were collected on EDTA diK+upon admission to emergency unit before brain imaging. FCM analysis was performed on fresh blood using the six-color Becton-Dickinson FCM system and FlowJo-software. We explored inter-operator gating agreement comparing graphically the distribution of measures and computing, for each patient, the coefficient of variation (CV).

Results: In the absence of a clearly defined strategy for monocyte subset FCM identification, we developed a new strategy based on CD45, CD14, CD16, CD91 (panmonocytic), HLA-DR as well as CD62L, CD11b, CD86, CCR2, ICAM-1, CX3CR1 and TF using 4 tubes (Table).
Blood samples from 20 consecutive patients were analyzed by three experienced operators. The median coefficients of variation were below the pre-specified tolerance limits (based on CLSI recommendations), i.e. 10% for Mon1, 20% for Mon2 and Mon3 counts (Figure), and 15% for the median fluorescence intensities of activation markers; however, an inter-operator effect was observed. Inter-operator differences were less than 3% when percentages of monocyte subsets were analyzed.

Conclusion(s): Our original strategy allowed monocyte subset gating with an acceptable inter-operator variability, which was a pre-requisite for BOOST study. The inter-operator effect when present must be taken into account in data analysis.