Example reports

We would like to provide Examples of MIATA-compliant Materials and Methods sections based on the finalized MIATA guidelines, for ICS, Elispot, and Multimer Staining. You can find editable Materials and Methods documents with fill-in options to further assist you in the Implementation section.

(Numbers in brackets refer to the Guideline Item as per Checklist and are listed for demonstration purposes only)

1. The Sample

Peripheral blood was obtained from 20 patients suffering from renal cell cancer (RCC) with the approval of the local ethics committee including written informed consent. All patients underwent surgery for RCC stages pT1 (n = 10), pT2 (n = 2), and pT3 (n = 8). Peripheral blood samples were additionally obtained from 20 consenting healthy donors or buffy coats provided by the local blood bank (n = 15 men and n = 5 women; mean age, 48 years) [1.1].

Heparinized [1.4] whole blood was collected from healthy donors or RCC patients [1.2] by venipuncture under a procedure approved by our Institutional Review Board [1.3].  It was stored at room temperature [1.5prior to isolation of PBMC via Ficoll density gradient separation (Ficoll-Paque) [1.6]. Processing was completed within <20h for all sample specimens [1.7].

Isolated PBMC were washed once with cRPMI-10 medium, then frozen in FCS 10% DMSO [1.9, 1.12] using a controlled-grade freezing device (SY-LAB 14S-B) [1.10] using a qualified freezing program [1.11], and transferred to liquid nitrogen storage after 8-16 hours, where the cells remained until use. Thawing-Medium was Iscove’s modified Dulbecco’s medium supplemented with penicillin/streptomycin, 50 μmol/L β-mercaptoethanol, and 10% heat-inactivated human serum (source)[1.12]. Cells were frozen in aliquots of 10 Mio viable PBMC per vial in 1 mL freezing medium.

Cells were counted after thawing using trypan blue staining and a microscope following an established SOP in the lab [1.20]. The median yield for the sample specimens thawed was 5.8 Mio cells per vial for the RCC patients and 6.1 Mio cells for the healthy control donors, respectively. The median cell viability was 89% for the RCC patients and 91% for the healthy control donors, respectively [1.15 and 1.17]. All samples met minimum acceptance criteria of >80% viability and >50% recovery[1.19].

2. The Assay

cRPMI-10 consisted of RPMI-1640 medium supplemented with 10% fetal bovine serum, antibiotic/antimycotic solution, and HEPES buffer (source[2.1]. Fetal bovine serum was from a single lot previously qualified for low background and high responses in ICS assays [2.2].

Thawed PBMC were resuspended at 2×107 viable lymphocytes per mL in cRPMI-10 medium. For stimulation, 1 Mio cells/well were plated in polypropylene U-bottom microtiter plates in a total volume of 200 µL of cRPMI-10.  They were stimulated for 8 h at 37oC, 5% CO2, using Flu Matrix Protein peptide58–66 [GILGFVFTL] as well as the three tumor antigen-specific peptides x,y,z… at 1 µg/mL/peptide, or SEB at 1 µg/mL, or an equivalent volume of DMSO [2.3].

For the staining, cells were treated with 2 mM final concentration of EDTA for 15 min at room temperature, followed by centrifugation and resuspension with 25 µg/mL LIVE/DEAD Aqua amine dye (company) in PBS.  After washing with PBS+2% BSA, surface staining antibodies (Table I) were added in 100 µL of PBS+2% BSA and incubated for 30 min at room temperature.  Cells were washed again and treated for 10 min at room temperature with 100 µL FACS Lysing Solution (manufacturer), washed and treated for another 10 min at room temperature with 200 µL FACS Permeabilizing Solution 2 (source). Cells were washed twice more, then resuspended in 100 microliters of PBS+2% BSA containing intracellular staining antibodies (Table I).  They were incubated for 60 min at room temperature, with vortexing every 20 min, then washed once more, and resuspended in PBS+2% BSA for acquisition.  Cells were kept cold and dark until acquisition, which was performed within 24 hours [2.4].


Lymphocytes stimulated with medium containing an equivalent volume of DMSO served as negative control and lymphocytes stimulated with SEB at 1 µg/mL served as positive control [2.5].

3. Data acquisition

The samples were directly acquired after finishing the staining using a (equipment, model, manufacturer) equipped with xx software version yy (source) [3.1]. The PMT voltages were adjusted using unstained cell for all parameters. The mean autofluorescence values of unstained cells were adjusted to approximately 100 for all fluorochrome channels [3.2].

The analysis was done on singlet living lymphocytes and gates were set as mentioned in the respective figure legends using (software, source). A representative example showing the full gating strategy and all applied gates is shown in suppl. Fig. x[3.3, 3.4]. The experiments were performed on a median PBMC cell count of 1.5 x 10cells with a minimum of 9.05 x 105cells and a maximum of 1.7 x 106 cells being acquired [3.5].

4. Results

The median background staining level observed in the medium control was 0.02% based on viable CD8lymphocytes for IL-2 and 0.008% for IFN-gamma, respectively [4.1].  The generated raw data can be provided per request [4.3].

A successfully detected antigen-specific response was defined as a response where the percentage of cytokine-positive CD8+ lymphocytes was at least 2-fold above the background observed in the medium control and at the difference between specific response and background level being > 0.05% [4.4] as defined prior to the start of the study [4.6].

5. General Lab Operation

While our laboratory has not established GLP conditions [5.1], all work was performed using SOPs that were qualified as referenced below. [5.2] The assays were performed by the same individual throughout the course of the study. The operator also participates in an ongoing external ICS proficiency testing program organized by society xx [5.2].

This study was performed using established laboratory protocols covering the processing, freezing, storage and thawing of cells as well as the staining procedure, data acquisition and gating strategy [5.4].

This study was performed using general research investigative but pretested assays [5.5].

(Numbers in brackets refer to the Guideline Item as per Checklist and are listed for demonstration purposes only)

1. The Sample

The study protocol was approved by the IRB of (the institution), and informed consent was obtained from all participating donors (n=xx) prior to their inclusion in this study. The donor characteristics are summarized in Table 1 (not shown in this example report, info will vary depending on study, see also – References for Module 1) [1.1].

Whole blood [1.2] was obtained by venipuncture [1.3] using Na-Heparin blood collection tubes at the time points indicated inFig x.  (not shown in this example report) [1.4]. In order to achieve processing within 8 hours [1.7], blood tubes were transported at ambient temperature via messenger to core labs located within 70 miles [1.5]. PBMC were isolated via Ficoll gradient centrifugation using 50mL Leucosep tubes (manufacturer) following the manufacturer’s instructions, but without dilution of whole blood [1.6].

The median PBMC number obtained per 1mL whole blood was 1.26×106 [1/15/1.16]. PBMC were frozen in 2mL cryogenic vials (manufacturer) at 1×107 PBMC per vial using a serum-free cryomedia kit (company) and Mr. Frosty freezing container (company) following the manufacturer’s instructions [1.9 – 1.12]. The freezing container was placed at -80oC, and vials were transferred into the vapor phase of liquid nitrogen after 24 hours [1.13].  PBMC vials were transported to the main testing core lab in the vapor phase of liquid nitrogen using cryoshippers (company) within 4 weeks of storage. Upon arrival cells were stored in liquid nitrogen tanks until further use (in average 3 months) [1.13].

For Elispot testing, 2 vials per sample were thawed. The median cell recovery was 9.1×106 PBMC per vial (range 8.1 – 10.1×106/vial) with a median viability of 93% [1.15/1.17]. The cut-off for acceptable viability was 70% [1.19]. Cell counts and viability was obtained using a Guava counter (model) and the Viacount kit [1.20]. The median degree of apoptosis assessed with this kit was 4.1% [1.21].

2. The Assay

The assay was carried out in AIM-V medium (source) without addition of serum [2.1]. The medium lot was pretested for Elispot performance in comparative testing with other media supplemented with and without serum [2.2].

On Day 1, PVDF plates (source, cat# …) were treated with 15µL of 70% Ethanol, washed with PBS and coated with xx µL per well of anti-IFNg antibody (concentration, clone, source). The plate was stored overnight at 4oC [2.4].

PBMC were rested in AIM-V medium at cell concentration of 2×106 /mL. For that, the PBMC suspension was split into 5mL aliquots and transferred into 50mL polypropylene tubes and incubated overnight (at least for … hours)  at 37oC, 5% CO2 [2.3]. On day 2, the coating antibody was discarded, the plate washed and blocked [2.4]. Rested cells were washed twice, re-evaluated for …. (Fill in appropriately: Cell counts, viability, and apoptosis degree), and resuspended at … cells/mL [1.18].

The following stimulators were added in a volume of 50µL per well [2.4]:

AIM-V (as negative control), CEFT peptide pool (concentration, source) as antigen-specific positive control [2.5], (experimental) peptide pool consisting of …(how many)  15mers overlapping by 11 amino acids (concentration/source), PHA-L (concentration, source) as mitogenic positive control  [2.5].

If PHA wells had less than 100 spots, the results of this test were not accepted, and the experiment had to be repeated. For the reported study, results from …  (number) of samples were excluded due to PHA responses <100 spots/well [2.6].

PBMC were added at 50µL per well (for final cell number of … per well) [2.4]

One PBMC reference sample (source) with known reactivity against the CEFT peptide pool was also tested (but not against the experimental antigens), following the same setup. Spot numbers were recorded to monitor assay performance over time. The CEFT reactivity of this sample during the study duration was recorded in a range of … to …. spots per well. The assay acceptance cut-off was … spots/well for this external PBMC control [2.7, 2.8].

Plates were incubated at 37oC, 5% CO2 overnight, at least for .., but not more than … hours [2.4].

On Day 3, cells were rigorously washed off plate with …., and … µL of biotinylated anti-IFNg detection antibody (concentration, source) were added. Plates were developed using a streptavidine-peroxide complex (source) and AEC substrate (source) [2.4].

3. Data acquisition

Plates were evaluated using the … (name, source) automated reader system, software …. (name, version) [3.1]. Spot parameters were established using the CEFT wells and negative control wells (cells plus medium only) obtaining an optimal Signal : Noise ratio. Artifacts and faint small background spots observed in the negative control wells were excluded [3.3]. A representative data set is shown in Fig xx [3.4]. All obtained counts were audited and reviewed. The counting strategy was reviewed by an independent scientist [3.7].

4. Results

The median background reactivity observed in this study was 4 spots per well (or # of PBMC), with a range of 0 – 15 spots per well [4.1]. Raw data is provided in Suppl. Data, Fig Y [4.3].

Positive reactivity to an experimental antigen was predefined [4.6] as a p-value of equal or smaller than 0.05 when applying the distribution free resampling method (DFR(2x) comparing the spot counts in the background control wells with the counts from the antigen specific wells using a web tool (link), reference [4.4, 4.5]. Responses were not considered positive if the antigen-specific reactivity level was below the Limit of Detection (LOD = 6 spots per x 100,000 PBMC) for this assay protocol [4.8, 5.6]. The DFR method was used because it controls the overall false positive rate and avoids parametric assumptions of the data [4.9]. A donor was defined as a responder to vaccination if at least at one time point post-vaccination there was a positive response to the experimental antigen(s) detected, if the donor was a non-responder at the pre-vaccination time point. If a donor had a response to the experimental antigen(s) at the pre-vaccination time point, the DFR(2x) method was used for determining a positive response to vaccination, comparing the antigen-specific counts (after background reactivity was subtracted) of each post-vaccination time point to the antigen-specific counts of the pre-vaccination time point [4.7].

5. General Lab Operation

These studies were conducted in laboratories that operate under GLP [5.1]. The core laboratory participates in external Elispot proficiency panels conducted by (name) [5.2]. The study was performed using standard operating protocols (SOPs)[5.4]. The Elispot assay was qualified [5.5].

(Numbers in brackets refer to the Guideline Item as per Checklist and are listed for demonstration purposes only)


1. The Sample

The phase I/II clinical study was reviewed and approved by the local ethics committee of the University Hospital. The forty patients (22 men and 18 women) all suffered from advanced colorectal carcinoma and were recruited on the basis of HLA-A*02 expression (HLA determination was performed by DNA typing at the local blood bank) and after giving written informed consent. Patients´ characteristics are listed in Table 1. [1.1]

EDTA-blood was obtained by venipuncture [1.2,1.3, 1.4], kept at room temperature (RT) [1.5] and processed within 8 hours after blood drawing [1.7]. Briefly, blood was diluted 1:2 with PBS and peripheral mononuclear cells (PBMC) were isolated at RT using a density gradient separation solution (company) [1.6], washed twice in PBS, and counted using trypan blue [1.20]. Viability was always > 90% [1.15/1.16]. Cells were frozen in cryovials at 6-8 x106 cells in 1ml cold fetal calf serum (FCS) (source) containing 10% DMSO [1.9,1.12]. A freezing container with a -1°C/minute cooling rate was systematically used for freezing at -80°C following the manufacturer´s instructions (company[1.10, 1.11], and cryovials were transfered in a liquid nitrogen tank within one week and until use [1.11, 1.13].

Shortly before staining, cells were thawed in 15ml polystyrene conic tubes (company) in Iscove’s modified Dulbecco’s medium containing 25mM Hepes, L-Glutamin, 100 units/mL penicillin/streptomycin, 10% heat-inactivated fetal calf serum and 3mg/ml DNAse-I (sources) [2.3]. Cells were washed once, resuspended in medium without DNAse-I and counted using trypan blue [1.20]. The recovery range was 4.1 – 8.2 x106per vial with a median of 6.5 x106 cells and viability for all sample samples was > 70% [1.15/1.17].

2. The Assay

Thawed PBMC were washed once in 6 ml staining buffer (PBS 2% FCS, 2 mmol/L EDTA, and 0.02% NaN3, FACS buffer)[2.1, 2.3] and distributed in an unsterile 96-well round botton plate (company) in 200 ml/ well. Between 0.5 × 106 and 2 × 106cells were used per test, depending on the donor [2.4]. Staining was performed in two consecutive steps on the cell pellets after centrifugation at 4°C. Cells were first incubated for 30 min at RT in the dark with 50ml of diluted HLA-A*0201 peptide multimers. All multimers were diluted at 5mg/ml in PBS 50% FCS, 2 mmol/L EDTA, 0.02% NaN3 [2.1] and centrifuged at 14.000 g for 5 min before use to eliminate aggregates, according to preliminary experiments. Biotinylated HLA-A*0201 peptide monomers folded with peptides X, Y (source protein, sequence, amino- acid positions) were produced as previously reported(reference), and 50mg aliquots stored at -80°C until use. HLA-multimers were generated by co-incubation with streptavidin-PE(company) at a molecular ratio of 4:1 (reference) and kept at 4°C for not more than one month [2.4]. After incubation with the HLA-multimers, cells were washed by adding 150ml of FACS buffer, then stained in 50ml FACS buffer containing CD3-FITC, CD8-PE-Cy7 and CD4-APC antibodies (companies) at pretested concentrations for 20 min at 4°C in the dark. After two washes, cells were fixed in 200ml FACS-buffer containing 1% formaldehyde and kept in the fridge until acquisition [2.1, 2.4].As control stainings, cells of each patient were stained with the antibody mix without HLA-multimer (FMO = fluorescence minus one) [2.5].

3. Data acquisition

Cell samples were acquired on the day following staining on a flow cytometer X (equipment, model, manufacturer) equipped with software X/ version Y (source[3.1]. The PMT voltages were adjusted for each fluorescence channel using unstained cells and compensations were set using a mixture of anti-mouse Ig/negative control beads (company) labeled with antibodies according to a local SOP [3.2].

All cells contained in the tests were acquired (range: approx. 0.3 x 106 to 1.8 x 106 counted events) [3.5]. The analysis was done on singlet, CD3+CD4 lymphocytes (software, version,source[3.3]. A representative example showing the full gating strategy and all applied gates is shown in suppl. Fig. X [3.4].

4. Results

All results are expressed as % of HLA-multimer (PE)+CD8+ cells among gated CD3+CD4– singlet lymphocytes. The median background obtained in the –PE channel for FMO stainings was 0.01% in the gated CD8+ lymphocyte populations [4.1]. Stainings were considered to be positive if at least 0.02% of the CD8+ subset was HLA-multimer+ and if the stained population was clustered, but not diffuse [4.4], as defined during the study design [4.6]. Dots-plots can be provided per request [4.3]. Stainings containing less than 20.000 analyzed CD8+ cells were excluded [4.8].

5. General Lab Operation

The study was conducted under exploratory research conditions [5.1]. Work steps for PBMC preparation (cell isolation, freezing, thawing) were performed using laboratory SOPs [5.4]. The cell staining protocol was established in previous experiments and also performed per SOP [5.4]. The studies performed used investigative assays [5.5]. The laboratory participates regularly in all HLA-multimer proficiency panels organized by Association X [5.2].