title: An optimized protocol for adenosine triphosphate quantification in T lymphocytes of lymphopenic patients.
abstract: In several clinical contexts, the measurement of ATP concentration in T lymphocytes has been proposed as a biomarker of immune status, predictive of secondary infections. However, the use of such biomarker in lymphopenic patients requires some adaptations in the ATP dosage protocol. We used blood from healthy volunteers to determine the optimal experimental settings. We investigated technical aspects such as the type of anticoagulant for blood sampling, the effect of freeze and thaw cycles, the reagent and sample mixing sequence, and the optimal dilution buffer. We also shortened the incubation time to 8h, and even showed that a 30min incubation may be sufficient. To evaluate the ATP rise upon lymphocyte activation, the optimal dose of stimulant was defined to be 4μg/mL of phytohaemagglutinin. Lastly, we determined that the number of T cells needed for this measurement was as low as 50,000, which is compatible with the existing lymphopenia in clinical settings. This optimized protocol appears ready to be assessed in lymphopenic patients to further investigate the interconnection between T lymphocyte metabolism and impaired phenotype and functions.
authors.affiliation: EA 7426 (Université Claude Bernard Lyon 1 - Hospices Civils de Lyon - bioMérieux) Joint Research Unit "Pathophysiology of Injury-Induced Immunosuppression - PI3", Hôpital Edouard Herriot, Lyon, France; Hospices Civils de Lyon, Anesthesia and Critical Care Medicine Department, Hôpital Edouard Herriot, Lyon, France.
authors.forename: Thibaut
authors.initials: T
authors.lastname: Girardot
authors.affiliation: EA 7426 (Université Claude Bernard Lyon 1 - Hospices Civils de Lyon - bioMérieux) Joint Research Unit "Pathophysiology of Injury-Induced Immunosuppression - PI3", Hôpital Edouard Herriot, Lyon, France.
authors.forename: Julie
authors.initials: J
authors.lastname: Mouillaux
authors.affiliation: EA 7426 (Université Claude Bernard Lyon 1 - Hospices Civils de Lyon - bioMérieux) Joint Research Unit "Pathophysiology of Injury-Induced Immunosuppression - PI3", Hôpital Edouard Herriot, Lyon, France.
authors.forename: Estellie
authors.initials: E
authors.lastname: Idealisoa
authors.affiliation: EA 7426 (Université Claude Bernard Lyon 1 - Hospices Civils de Lyon - bioMérieux) Joint Research Unit "Pathophysiology of Injury-Induced Immunosuppression - PI3", Hôpital Edouard Herriot, Lyon, France.
authors.forename: Fanny
authors.initials: F
authors.lastname: Poujol
authors.affiliation: EA 7426 (Université Claude Bernard Lyon 1 - Hospices Civils de Lyon - bioMérieux) Joint Research Unit "Pathophysiology of Injury-Induced Immunosuppression - PI3", Hôpital Edouard Herriot, Lyon, France; Hospices Civils de Lyon, Anesthesia and Critical Care Medicine Department, Hôpital Edouard Herriot, Lyon, France.
authors.forename: Christelle
authors.initials: C
authors.lastname: Rouget
authors.affiliation: EA 7426 (Université Claude Bernard Lyon 1 - Hospices Civils de Lyon - bioMérieux) Joint Research Unit "Pathophysiology of Injury-Induced Immunosuppression - PI3", Hôpital Edouard Herriot, Lyon, France; Hospices Civils de Lyon, Anesthesia and Critical Care Medicine Department, Hôpital Edouard Herriot, Lyon, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Rimmelé
authors.affiliation: EA 7426 (Université Claude Bernard Lyon 1 - Hospices Civils de Lyon - bioMérieux) Joint Research Unit "Pathophysiology of Injury-Induced Immunosuppression - PI3", Hôpital Edouard Herriot, Lyon, France; Hospices Civils de Lyon, Immunology Laboratory, Hôpital Edouard Herriot, Lyon, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Monneret
authors.affiliation: EA 7426 (Université Claude Bernard Lyon 1 - Hospices Civils de Lyon - bioMérieux) Joint Research Unit "Pathophysiology of Injury-Induced Immunosuppression - PI3", Hôpital Edouard Herriot, Lyon, France; Hospices Civils de Lyon, Anesthesia and Critical Care Medicine Department, Hôpital Edouard Herriot, Lyon, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Textoris
authors.affiliation: EA 7426 (Université Claude Bernard Lyon 1 - Hospices Civils de Lyon - bioMérieux) Joint Research Unit "Pathophysiology of Injury-Induced Immunosuppression - PI3", Hôpital Edouard Herriot, Lyon, France; Hospices Civils de Lyon, Immunology Laboratory, Hôpital Edouard Herriot, Lyon, France. Electronic address: fabienne.venet@chu-lyon.fr.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Venet
mesh_terms: D000255:Adenosine Triphosphate / Q000097:blood*; D000925:Anticoagulants / Q000494:pharmacology; D015415:Biomarkers / Q000097:blood; D001800:Blood Specimen Collection / Q000379:methods*; D002021:Buffers; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D064368:Healthy Volunteers; D006801:Humans; D008213:Lymphocyte Activation / Q000187:drug effects; D008231:Lymphopenia / Q000097:blood* / Q000175:diagnosis / Q000276:immunology; D010835:Phytohemagglutinins / Q000494:pharmacology; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D013997:Time Factors
chemical_list: D000925:Anticoagulants; D015415:Biomarkers; D002021:Buffers; D010835:Phytohemagglutinins; D000255:Adenosine Triphosphate
keywords: ATP content; Lymphopenia; Protocol optimization; T lymphocyte