Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring

Background Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intra- and interoperator variabilities in a multicentre project. Methods The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration. Results Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of cross-centre variability for naïve/memory subset frequencies particularly in the whole blood setting. Conclusion Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options.

Inter-assay repeatability of each operator for each cell subset for PBMCs after local and central anaysis (a and b respectively). In this case CV was first calculated for each donor-specific triplicate (3 rounds) and then the median value was determined on the 3 donors. Intra-assay repeatability of each operator for each parameter for WB samples after local (c) or centralized (d) analysis. Here the CV was calculated on the three experimental replicas (1 round) and then calculating the median on the 3 donors. Centralisation mitigated inter-assay variability for some cPBMC data, while it did not affect WB intraassay variability, which was already excellent in local analysis.    Warning: This protocol provides for the use of human blood derivatives. Operators should have received an appropriate training and they must work according to laboratory safety guidelines for hemoderivative products.
Purpose: This document describes the process for isolating and freezing stocks of human peripheral blood mononuclear cell (PBMC) samples from healthy donor blood bank buffy coat, to be used in the procedure described in SOP "PBMC thawing and counting .pdf" file.

Experimental plan
Procedure will be performed at ISS, main center of the harmonisation project, on 24-hour buffy coats released by the Policlinico Umberto I Transfusion Center, Rome, Italy. Gradient separation and freezing at -80°C must be performed consecutively, without pauses, on the same day. Liquid nitrogen freezing will be executed at least after 6 hours (even the day after).

PBMCs Isolation
Preliminary operations 1. Use 3 sets of 5 x 50 ml conical tube for each buffy coat bag, one for PBS dilution, one for Lymphoprep gradient and one for PBMC ring collection. 2. Label the conical tubes for each sample (PBMC1, PBMC2 and PBMC3). 3. Record sex, date of birth, date of withdrawal, bag identification code (or bar code). 4. Warm Lymphoprep and PBS at RT before use. For each buffy coat sample proceed as follows: 5. Thoroughly disinfect one of the exit tubes of the buffy coat bag with Ethanol 70°. 6. Cut it with sterile scissors. 7. Let the blood to flow into a conic 50 ml vial. 8. Squeeze the bag to recover as much blood volume as possible. 9. Record the collected blood volume (usually 50-60 ml). 10. Add 1 μl of heparin for each ml of buffy coat blood. 11. Mix blood and heparin by sterile pipetting. 12. Dispense 10 ml of blood into each of the 5 falcon conical tubes (first set). 13 33. Count both live and dead cells by using a 10x objective lens in an inverted microscope in 2 opposite quadrants of the chamber (Fig. 1). The amount of live cells must be between 35 and 100 in each quadrant: if this does not happen, repeat by correcting the dilution factor. Warning: This protocol provides for the use of human blood derivatives. Operators should have received an appropriate training and they must work according to laboratory safety guidelines for hemoderivative products.
Purpose: This document describes the process for the thawing of peripheral blood mononuclear cell (PBMC) samples to be used in the SOP described in "PBMC staining acquisition and analysis.pdf" file. It would be executed by all operators belonging to the harmonisation project.  -Turn on the thermostatic bath at 37 ° C.
-Warm up solution at room temperature.

Method:
1. Thaw quickly and incompletely by warming the cryovials at 37°C in the water bath. 14. Count both live and dead cells by using a 10x objective lens in an inverted microscope. 15. Count both live and dead cells by using a 10x objective lens in an inverted microscope in 2 opposite quadrants of the chamber (Fig. 1). The amount of live cells must be between 35 and 100 in each quadrant: if this does not happen, repeat by correcting the dilution factor.
16. Calculate number of PBMCs /ml (= "mean cell number of a 4x4 quadrant" x "10 4 " x "dilution factor"). Warning: This protocol provides for the use of human blood derivatives. Operators should have received an appropriate training and they must work according to laboratory safety guidelines for hemoderivative products.
Purpose: This document describes the process for staining, acquisition and analysis of thawed human PBMC samples with a 6 colour flow cytometry panel for naïve/memory T cell detection. PBMC thawing procedure is described in "PBMC thawing and counting.pdf" file. It would be executed by all operators belonging to the harmonisation project.

Experimental plan
For each round, the entire procedure (thawing, staining and acquisition) must be performed consecutively, without pauses, on the same day. Each operator will test 1 PBMC sample for each of the 3 donors, in 3 experimental rounds to obtain 3 replicas from each donor. The entire procedure will be carried out over a period of about twenty days, with a lapse of 6-7 days between one session and another, within the month of October 2017. First round is described in section a); second and third rounds in section b).

a) Method for the first experimental round (includes compensation)
Thawing and counting of the three donor cPBMCs (PBMC1, PBMC2 and PBMC3), is described in the SOP provided by Istituto Superiore di Sanità (thawing and counting.pdf).

Experimental plan
The entire procedure (collection, staining and acquisition) must be performed in 3 consecutive days:  Day 0: EDTA collection from 3 healthy donors (at ISS); distribution of the blood and reagent to the operators within 3 hours  Day 1: staining  Day 2: acquisition to the flow cytometer Each operator will have to test an aliquot of whole blood for each of the 3 healthy donors, in a single experimental round, in triplicate (to obtain 3 experimental replicas from each donor).   At least 2 lasers: 488 and 630/640 nm. It must undergo an internal quality control of alignment (required), sensitivity and linearity (highly recommended).