Edta vacuette tube

Up to 50 mL of venous blood were collected in EDTA vacuette tubes (Greiner Bio-One International, Kremsmünster, Austria) from patients and controls. Blood was taken from MS patients prior to and 2, and 6 months after their first dose of CladT. Concurrently, the same numbers of blood tubes were also taken from age- and sex-matched non-MS control subjects at baseline, and then 2, and 6 months later. PBMC were then isolated from the blood using a Ficoll-Paque Plus (GE Healthcare, Chicago, Illinois, US) density separation gradient. Samples were cryopreserved in 5% DMSO/FBS for storage in liquid nitrogen prior to mass cytometry staining. Thawed cryopreserved Ficoll-isolated PBMCs were labelled with isotope-conjugated antibodies and examined by mass cytometry using the CyTOF2 system, Helios.

Peripheral blood mononuclear cells were isolated from blood and collected in EDTA vacuette tubes (Greiner Bio‐One International, Kremsmünster, Austria) using a Ficoll‐Paque Plus (GE Healthcare, Chicago, Illinois, US) density separation gradient. Samples were cryopreserved in 5% dimethyl sulfoxide/foetal bovine serum for storage in liquid nitrogen prior to mass cytometry staining.

Individuals were recruited to the trial on the basis of blood smear examination and gametocyte quantification, but ring stages and gametocytes were subsequently quantified by highly sensitive qRT-PCR, as described [14 (link)]. Briefly, EDTA blood (EDTA VACUETTE tube, Greiner Bio-One, Kremsmünster, Austria) was aliquoted into RNA protect cell reagent (Qiagen, Hilden, Germany) and stored at − 80 °C until temperature tracked shipment on dry ice to Radboud University Medical Center (Nijmegen, Netherlands) for assay performance. Total nucleic acids were extracted using a MagNAPure LC automated extractor (Total Nucleic Acid Isolation Kit-High Performance; Roche Applied Science, Indianapolis, IN, USA). Male and female gametocytes were quantified in a multiplex reverse transcriptase quantitative PCR (RT-qPCR) assay as described [14 (link)]. Asexual stages were quantified by quantification of SBP1 transcripts, as described elsewhere [19 (link)]. Samples were classified as negative for a particular gametocyte sex if the qRT-PCR quantified density of gametocytes of that sex was less than 0·01 gametocytes per μL (i.e. one gametocyte per 100 μL of blood sample).

All participants were informed to avoid strenuous exercise and alcohol consumption within 24 h before blood collection. Peripheral blood samples were drawn from cubital vein in fasting state in the morning before 9 a.m. We collected blood samples in EDTA VACUETTE tube (Greiner Bio-one, Thailand) and gently shook the tubes 4–5 times to mix up blood sample with anticoagulant. Then the tubes were centrifuged at 4,000 r/min for 10 min at 4°C. Plasma was immediately separated and stored at −80°C refrigerator. Samples were thawed on ice before preparation. Four hundred micro liter of cold methanol (containing internal standard) was added to 100 μL of plasma sample and vortexed for 60 s. After 10 min at room temperature, they were centrifuged at 14,000 g for 15 min to precipitate the protein. And the supernatant was then transferred and collected for the analysis in positive electrospray ionization (ESI+) mode and negative electrospray ionization (ESI-) mode, respectively. To evaluate the stability of the analysis, quality control (QC) samples were prepared by mixing equal volumes of each plasma sample and evenly injected at regular intervals throughout the analytical run.