Posted on Onctober 16, 2009

Updated to  Version 1.0 and moved to archive in May, 2010

The reporting framework modules; Version 0.0

The initial publication guidelines of immune monitoring strategies are based on the premise that reporting of immune monitoring data from clinical studies will benefit by accurately including information for 5 “modules” that critically impact test results. Detailed annotation of published data for these 5 topics will be of additional benefit if accompanied by the appropriate reporting of raw data to allow for full evaluation of the value of published data sets.

Module 1: The Sample

A. Donor: Essential information on donor including age, gender, HLA type, diagnosis, disease status, treatment, prior relevant treatments.

B. Source: Collection methodology for specimens (e.g. heparin, EDTA, citrate, other). Time lapse between blood draw and PBMC isolation, and interim storage. Methodology for PBMC isolation. Was shipment of specimen necessary? If yes, under which conditions was shipment performed? [1,2]

C. Cryopreservation and Storage: Methodology for cryopreservation (controlled temperature device, Mr. Frosty, other) and storage. Elapsed time between cryopreservation and final storage and final storage temperature. [1,2,3,4,5]

D. Description of quality assurance criteria applied for specimen and cut-off values for sample acceptance (e.g. viability, apoptosis, function). [5]

Module 2: The Assay

A. Cell counting: Methodology applied to count cells. Were viability and apoptosis assessed? If so report values. [5,6]

B. Medium/serum: Source of medium/serum used for the assay. Description of any pretesting performed and performance characteristics of the chosen medium/serum (e.g. background reactivity levels). [5,7,8]

C. Assay: An overview of critical assay procedures including source of main reagents and materials used and crucial treatment procedures of cells (e.g. overnight resting, in vitro stimulation). Description of any published harmonization guidelines and recommendations followed. [6,9,10,11]

D. Description of internal (e.g. positive control antigens or peptide pools, irrelevant or negative controls, mitogens) and external controls (e.g. PBMC reference samples) used in assay? [12,13,14] Description of quality assurance criteria for the controls including used peptide or peptide mixes. [15]

Module 3: Data acquisition

A. Description of equipment and software versions used for data acquisition. Setup of the equipment. Description of any methodologies employed for instrument calibration and validation and operator training. [16]

B. List of controls applied during data acquisition (e.g. spot size, form and intensity settings in the case of Elispot, compensation and gating strategies, control beads in the case of flow cytometry analysis). [11,14]

C. Description of quality assurance criteria (e.g. were results audited?) for the results? [6,7,17]

Module 4: The (interpretation of) results

Description of how raw data were processed and interpreted, and how the threshold for “positive reactivity" was defined. [18] Description of how a vaccine-induced induction of a response between two time points was defined. A description of statistical tests or empiric rules applied and the reasoning behind these test choices. [19,20,21,22] A statement on whether any data was excluded from the analysis, and if so the reason for the exclusion.

Module 5: The lab environment

A. A statement of how laboratory operations were guided. Does the lab work under GLP or GLP-like conditions? [23,24].

B. A description of whether procedures were standardized in the laboratory and if SOPs were established and followed for the relevant procedures and assays. A description of any deviation from the SOP, and if so, how these were handled. [7,25,26].

C. A statement of whether SOPs are qualified or validated. [27] If SOP are qualified or validated, a specific description of how the relevant parameters (accuracy, specificity, precision, sensitivity, upper and lower limits of quantification, robustness) were determined, and how the assay specifications were defined and met. For each validated (or qualified assay if the data is available) the quality control elements for the assay (positive and negative reference sample identity and range), and pass/fail criteria for the assay should also be included. [7,28,29,30,31]

D. A statement of whether the laboratory regularly participates in external quality assurance programs (proficiency panels) and harmonization efforts. A statement of whether trained personnel performed the testing, and if so, a description of how personnel is monitored for assay performance? [6,10,32]

1. Bull M, Lee D, Stucky J, Chiu YL, Rubin A, Horton H, and McElrath MJ (2007) Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials. J Immunol Methods 322:57-69

2. Kierstead LS, Dubey S, Meyer B, Tobery TW, Mogg R, Fernandez VR, Long R, Guan L, Gaunt C, Collins K, Sykes KJ, Mehrotra DV, Chirmule N, Shiver JW, and Casimiro DR (2007) Enhanced rates and magnitude of immune responses detected against an HIV vaccine: effect of using an optimized process for isolating PBMC. AIDS Res Hum Retroviruses 23:86-92

3. Kreher CR, Dittrich MT, Guerkov R, Boehm BO, and Tary-Lehmann M (2003) CD4+ and CD8+ cells in cryopreserved human PBMC maintain full functionality in cytokine ELISPOT assays. J Immunol Methods 278:79-93

4. Maecker HT, Moon J, Bhatia S, Ghanekar SA, Maino VC, Payne JK, Kuus-Reichel K, Chang JC, Summers A, Clay TM, Morse MA, Lyerly HK, Delarosa C, Ankerst DP, and Disis ML (2005) Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT. BMC Immunol 6:17-

5. Smith JG, Joseph HR, Green T, Field JA, Wooters M, Kaufhold RM, Antonello J, and Caulfield MJ (2007) Establishing acceptance criteria for cell-mediated-immunity assays using frozen peripheral blood mononuclear cells stored under optimal and suboptimal conditions. Clin Vaccine Immunol 14:527-537

6. Janetzki S, Panageas KS, Ben-Porat L, Boyer J, Britten CM, Clay TM, Kalos M, Maecker HT, Romero P, Yuan J, Kast WM, and Hoos A (2008) Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI). Cancer Immunol Immunother 57:303-315

7. Janetzki S, Cox JH, Oden N, and Ferrari G (2005) Standardization and validation issues of the ELISPOT assay. Methods Mol Biol 302:51-86

8. Martinuzzi E, Scotto M, Enee E, Brezar V, Ribeil JA, van Endert P, and Mallone R (2008) Serum-free culture medium and IL-7 costimulation increase the sensitivity of ELISpot detection. J Immunol Methods 333:61-70

9. Britten CM, Gouttefangeas C, Welters MJ, Pawelec G, Koch S, Ottensmeier C, Mander A, Walter S, Paschen A, Muller-Berghaus J, Haas I, Mackensen A, Kollgaard T, thor SP, Schmitt M, Giannopoulos K, Maier R, Veelken H, Bertinetti C, Konur A, Huber C, Stevanovic S, Wolfel T, and van der Burg SH (2008) The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays. Cancer Immunol Immunother 57:289-302

10. Britten CM, Janetzki S, Ben Porat L, Clay TM, Kalos M, Maecker H, Odunsi K, Pride M, Old L, Hoos A, and Romero P (2009) Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium. Cancer Immunol Immunother Epub ahead of print:

11. Maecker HT and Trotter J (2006) Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69:1037-1042

12. Currier JR, Kuta EG, Turk E, Earhart LB, Loomis-Price L, Janetzki S, Ferrari G, Birx DL, and Cox JH (2002) A panel of MHC class I restricted viral peptides for use as a quality control for vaccine trial ELISPOT assays. J Immunol Methods 260:157-172

13. Maecker HT, Dunn HS, Suni MA, Khatamzas E, Pitcher CJ, Bunde T, Persaud N, Trigona W, Fu TM, Sinclair E, Bredt BM, McCune JM, Maino VC, Kern F, and Picker LJ (2001) Use of overlapping peptide mixtures as antigens for cytokine flow cytometry. J Immunol Methods 255:27-40

14. Maecker HT, Frey T, Nomura LE, and Trotter J (2004) Selecting fluorochrome conjugates for maximum sensitivity. Cytometry A 62:169-173

15. Currier JR, Galley LM, Wenschuh H, Morafo V, Ratto-Kim S, Gray CM, Maboko L, Hoelscher M, Marovich MA, and Cox JH (2008) Peptide impurities in commercial synthetic peptides and their implications for vaccine trial assessment. Clin Vaccine Immunol 15:267-276

16. Perfetto SP, Ambrozak D, Nguyen R, Chattopadhyay P, and Roederer M (2006) Quality assurance for polychromatic flow cytometry. Nat Protoc 1:1522-1530

17. Janetzki S, Schaed S, Blachere NE, Ben-Porat L, Houghton AN, and Panageas KS (2004) Evaluation of Elispot assays: influence of method and operator on variability of results. J Immunol Methods 291:175-183

18. Dubey S, Clair J, Fu TM, Guan L, Long R, Mogg R, Anderson K, Collins KB, Gaunt C, Fernandez VR, Zhu L, Kierstead L, Thaler S, Gupta SB, Straus W, Mehrotra D, Tobery TW, Casimiro DR, and Shiver JW (2007) Detection of HIV vaccine-induced cell-mediated immunity in HIV-seronegative clinical trial participants using an optimized and validated enzyme-linked immunospot assay. J Acquir Immune Defic Syndr 45:20-27

19. Cox JH, Ferrari G, Kalams SA, Lopaczynski W, Oden N, and D'souza MP (2005) Results of an ELISPOT proficiency panel conducted in 11 laboratories participating in international human immunodeficiency virus type 1 vaccine trials. AIDS Res Hum Retroviruses 21:68-81

20. Hudgens MG, Self SG, Chiu YL, Russell ND, Horton H, and McElrath MJ (2004) Statistical considerations for the design and analysis of the ELISpot assay in HIV-1 vaccine trials. J Immunol Methods 288:19-34

21. Lathey JL (2003) Preliminary steps toward validating a clinical bioassay - a case study of the ELISpot assay. Biopharm International - The applied Technologies of Biopharmaceutical development 16:42-50

22. Moodie Z, Huang Y, Gu L, Hural J, and Self SG (2006) Statistical positivity criteria for the analysis of ELISpot assay data in HIV-1 vaccine trials. J Immunol Methods 315:121-132

23. Ezzelle J, Rodriguez-Chavez IR, Darden JM, Stirewalt M, Kunwar N, Hitchcock R, Walter T, and D'souza MP (2008) Guidelines on good clinical laboratory practice: bridging operations between research and clinical research laboratories. J Pharm Biomed Anal 46:18-29

24. Kelley M (20008) Considerations while setting up cell-based assays. Validation of cell based assays in the GLP setting A practical guide John Wiley Chapter 1:1-9

25. Maecker HT, Rinfret A, D'Souza P, Darden J, Roig E, Landry C, Hayes P, Birungi J, Anzala O, Garcia M, Harari A, Frank I, Baydo R, Baker M, Holbrook J, Ottinger J, Lamoreaux L, Epling CL, Sinclair E, Suni MA, Punt K, Calarota S, El Bahi S, Alter G, Maila H, Kuta E, Cox J, Gray C, Altfeld M, Nougarede N, Boyer J, Tussey L, Tobery T, Bredt B, Roederer M, Koup R, Maino VC, Weinhold K, Pantaleo G, Gilmour J, Horton H, and Sekaly RP (2005) Standardization of cytokine flow cytometry assays. BMC Immunol 6:1-18

26. NCCLS (2004) Performance of single cell immuine response assays; approved guidelines. NCCLS document I/LA26-A

27. International Conference on Harmonization (ICH) (1997) Guidance for Industry:Q2B Validation of Analytical Procedures Methodology. http://www ich org/LOB/media/MEDIA417 pdf

28. Findlay JW, Smith WC, Lee JW, Nordblom GD, Das I, DeSilva BS, Khan MN, and Bowsher RR (2000) Validation of immunoassays for bioanalysis: a pharmaceutical industry perspective. J Pharm Biomed Anal 21:1249-1273

29. Maecker HT, Hassler J, Payne JK, Summers A, Comatas K, Ghanayem M, Morse MA, Clay TM, Lyerly HK, Bhatia S, Ghanekar SA, Maino VC, Delarosa C, and Disis ML (2008) Precision and linearity targets for validation of an IFNgamma ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides. BMC Immunol 9:1-9

30. Pifat D (2006) Assay Validation. www fda gov/cber/summaries/120600bio10 ppt

31. Tuomela M, Stanescu I, and Krohn K (2005) Validation overview of bio-analytical methods. Gene Ther 12 Suppl 1:S131-S138

32. Britten CM, Janetzki S, van der Burg SH, Gouttefangeas C, and Hoos A (2007) Toward the harmonization of immune monitoring in clinical trials: Quo vadis? Cancer Immunol Immunother 57:285-288