Forums

ELISPOT results interpretation after counting with IRIS

  • GV
    Guest Vivian2025-03-13T03:04:38Z
    Hello, may we request technical support for our ongoing CMV Elispot assay?
    We are conducting a study on CMV-specific IFN-γ detection using Mabtech's Elispot Path (Product code: 3420-2AST-P2-1). Our current setup includes:
    • Strong positive control: PHA stimulation (instead of kit-provided CD3)
    • Test group: CMV antigen peptide stimulation
    • Negative control: Cells only
    We have four technical questions:
    1. Blank control requirement: Must we include a "blank control" (reagents only, no cells) in addition to our current negative control?
      (Note: "Blank control" here refers to wells with culture medium + assay reagents but no PBMCs)
    2. Replicate optimization (cost consideration):
      ①We plan 3 replicates for test groups. Can we reduce negative/positive controls to 2 replicates?

       ②If only 2 replicates are used, DFR (2×) method cannot be applied for result interpretation. Are there alternative methods for spot counting validation?

         3.IRIS plate reader issue:

      ①In our pilot experiment, the IRIS reader identified unexpected "spots" that we suspect may be background smudges or un-dispersed cell clusters.

             ②Can we adjust reader settings to filter these? Or are these actually unlysed cell aggregates requiring technical optimization?

              As shown in the attached plate image, our pilot experiment results reveal:

                           Strong positive controls (A10/B10): PHA-stimulated wells (expected high IFN-γ spots)

                           Test group (C10/D10/E10): CMV peptide-stimulated wells from a patient with clinically suspected poor CMV-specific immunity (based on medical history)

                            Naked-eye observation: C10-E10 showed fewer discrete spots compared to A10/B10

                           IRIS reader output: Test group spot counts seem to have no statistically significant difference from positive controls.（A10-B10：1254spots/well,712spots/well，C10-E10：865 spots/well， 753spots/well, 882spots/well）

           4.   Inter-patient variability in strong positive controls (PHA-stimulated wells): All 3 patients showed robust spot formation, but inter-patient spot counts varied significantly (Patient 1: A1-B1, Patient 4: B4-C4, Patient 7: A7-B7).

    •   Experimental conditions (consistent across 3 patients): Identical cell input (4×10^5 PBMCs/well, via automated cell counter)；Fresh blood samples (processed within 8 hours of collection)；Standardized PHA concentration ；Fixed incubation time

            ① Biological interpretation: Do these differences reflect genuine variations in global T cell immunity (consistent with our study population's known immune heterogeneity)?
    ② Data analysis strategy:     Can we analyze strong positive control results individually to characterize baseline T cell function? Should CMV-specific responses (test group) be interpreted relative to each patient's own PHA response (e.g., calculate stimulation index = test spots / PHA spots)?

     We greatly value your expertise and look forward to your guidance. Thank you for your time!

    ELISPOT Result_1_20250313110208.pdf

  • R
    Renata2025-03-20T15:18:42Z

    Hi Vivian,

    Thanks for reaching out with your questions! Here are my responses to your questions:

    1. Blank control is always recommended! This allows you to control from false positive spots that are caused by the reagents. For example, sometimes aggregation of detection antibody, can lead to false positive spots, so this is a good control to include to make sure you are not getting any background due to reagents.

    2. I would recommend sticking to three replicates for all conditions, if possible. For negative controls especially! I recommend reading chapter 7 from this book about why it's important to have at least three replicates: S. Janetzki, Elispot for Rookies (and Experts Too), Techniques in Life Science and Biomedicine for the Non-Expert, DOI 10.1007/978-3-319-45295-1_7 

    3. Unfortunately, I am not able to open the file that you shared with us, could you please email the file to renata.varnaite@mabtech.com? The reader settings can definitely be adjusted to exclude spots that you don't want to count. We also have an Edit mask function where you can remove artifacts manually. Edit mask function can be found on the left side of the software under Tools (make sure you are in the well view though).

    4. It's pretty normal for different samples to have different spot numbers in PHA controls. This can be due to inherent differences between donors and how they respond to PHA, but it can also be to do with sample quality. Did all of your samples have high viability before being analyzed by ELISpot? In general, not all T cells will respond to PHA and secrete IFN-g, so normalization based on PHA counts may not be the most accurate approach. I would anticipate some T cells responding to PHA but not CMV, and vice versa.

    We would be happy to discuss your data and questions in more detail over email or Teams!