Dr. Min Hyung Kang Uses the Odyssey CLx Imager to Study Glaucoma

“I cannot imagine, without the machine (Odyssey® CLx Imager) in our lab, we cannot do anything.” – Dr. Min Hyung Kang

Dr. Min Hyung Kang and his lab at Case Western Reserve University are studying the causative mechanisms for glaucoma. Their focus is on primary open-angle glaucoma which is believed to be caused by elevated intraocular pressure (IOP). This elevated pressure pushes against the optic nerve and can eventually lead to blindness.


He and his lab are investigating the role of the matricellular protein Secreted Protein Acidic and Rich in Cysteine (SPARC) in elevating IOP. They’ve observed a relationship between extracellular matrix (ECM) protein upregulation and the trabecular meshwork becoming blocked, which may prevent the aqueous humor from draining.

This blockage is suspected to be the result of an accumulation of ECM proteins in the trabecular meshwork. His lab has been evaluating how SPARC regulates “the ECM levels in the human eyes. Especially, in the trabecular meshwork, where the outflow happens.”

Often Used and Always Appreciated

Dr. Kang’s research involves performing quantitative Western blots on a daily basis. He estimates that his lab gets 80 percent or more of their data from Western blotting. For the past 10 years, Dr. Kang has chosen the Odyssey CLx Imaging System as his preferred imager.

Using the Odyssey CLx Imager helps Dr. Kang maintain confidence in his data because “it’s very consistent.” When he moved to his lab at Case Western Reserve University in 2013, he specifically requested an Odyssey CLx Imager based on his prior experience. Since then, he has convinced at least one other lab to make the switch after letting them try out his machine.

Dr. Kang has moved to exclusively using the Odyssey CLx Imager for his Western blot experiments. He prefers this imaging system because it saves him time. He loves that using the Odyssey CLx seems to give him back extra hours every day, while acquiring the highest quality images. Then in Image Studio™ Software, he can quantify band intensity. There’s no need for him to run back and forth to the darkroom, meticulously tracking exposure times, to get the image just right.

“If you use the x-ray film, we have measured the time, 30 seconds, one minute, or five minutes, to get the best images. With this machine, I don’t have to. Just scan it.”

If you ask Dr. Kang where the darkroom for his lab is, he isn’t sure his department has one anymore because he hasn’t needed to use the darkroom. Dr. Kang made his feelings clear about chemiluminescent Western blots, in stating: “I don’t want to go back to that.” That may explain why he no longer performs any chemiluminescent Western blots at all.

We thank Dr. Kang for his contributions to science and are proud to call him an Odyssey CLx Expert.

Publications resulting from work on the Odyssey CLx Imaging System

  1. Kang, M.H., Oh, D.J., and Rhee, D.J. (2011). Effect of Hevin Deletion in Mice and Characterization in Trabecular Meshwork. Investigative Ophthalmology & Visual Science. Vol. 52, 2187-2193. doi: 10.1167/iovs.10-5428.
  2. Villareal, G. Jr., Oh, D.J., Kang, M.H., and Rhee, D.J. (2011). Coordinated Regulation of Extracellular Matrix Synthesis by the MicroRNA-29 Family in the Trabecular Meshwork. Investigative Ophthalmology & Visual Science. Vol. 52, 3391-3397. doi: 10.1167/iovs.10-6165.
  3. Haddadin, R.I., Oh, D.J., Kang, M.H., Villareal, G. Jr., Kang, J., Jin, R., Gong, H., and Rhee, D.J. (2012). Thrombospondin-1 (TSP1)-Null and TSP2-Null Mice Exhibit Lower Intraocular Pressures. Investigative Ophthalmology & Visual Science. Vol. 53, 6708-6717. doi: 10.1167/iovs.11-9013.
  4. Kang, M.H., Oh, D.J., Kang, J., and Rhee, D.J. (2013). Regulation of SPARC by Transforming Growth Factor β2 in Human Trabecular Meshwork. Investigative Ophthalmology & Visual Science. Vol. 54, 2523-2532. doi: 10.1167/iovs.12-11474.
  5. Oh, D.J., Kang, M.H., Ooi, Y.H., Choi, K.R., Sage, E.H., and Rhee, D.J. (2013). Overexpression of SPARC in Human Trabecular Meshwork Increases Intraocular Pressure and Alters Extracellular Matrix. Investigative Ophthalmology & Visual Science. Vol. 54, 3309-3319. doi: 10.1167/iovs.12-11362.
  6. Keller, K.E., Vranka, J.A., Haddadin, R.I., Kang, M.H., Oh, D.J., Rhee, D.J., Yang, Y., Sun, Y.Y., Kelley, M.J., and Acott, T.S. (2013). The Effects of Tenascin C Knockdown on Trabecular Meshwork Outflow Resistance. Investigative Ophthalmology & Visual Science. Vol. 54, 5163-5623. doi: 10.1167/iovs.13-11620.
  7. Chatterjee, A., Villareal, G. Jr., Oh, D.J., Kang, M.H., and Rhee, D.J. (2014). AMP-Activated Protein Kinase Regulates Intraocular Pressure, Extracellular Matrix, and Cytoskeleton in Trabecular Meshwork. Investigative Ophthalmology & Visual Science. Vol. 55, 3127-3139. doi: 10.1167/iovs.13-12755.
  8. Villareal, G. Jr., Chatterjee, A., Oh, S.S., Oh, D.J., Kang, M.H., and Rhee, D.J. (2014). Canonical Wnt Signaling Regulates Extracellular Matrix Expression in the Trabecular Meshwork. Investigative Ophthalmology & Visual Science. Vol 55, 7433-7440. doi: 10.1167/iovs.13-12652.

Odyssey CLx User, Dr. Pierre-Jacques Hamard Studies AML and Epigenetic-based Therapies

Dr. Pierre-Jacques Hamard is researching ways to put the brakes on acute myeloid leukemia (AML) and other hematopoietic diseases. As an Associate Scientist for the Nimer Lab in the Sylvester Comprehensive Cancer Center, at the University of Miami, he and his colleagues are researching the role of epigenetic factors in normal and malignant hematopoiesis.

In particular, Dr. Hamard’s research involves determining the effect protein arginine methyltransferase-5 (PRMT5) may have on DNA repair in hematopoietic cells. This line of research recently made it to the cover of the high-profile publication Cell Reports.1

Another facet of Dr. Hamard’s research has a more therapeutic slant, as he and his colleagues test the efficacy of various epigenetic-based therapies.

One such therapy they are exploring is inhibiting the expression of the protein CARM1. Dr. Hamard and his colleagues have shown that CARM1 “is important for leukemia cells but not for normal cells.”2

Their approach is “to show that inhibiting these proteins could be a viable therapeutic approach for some of the diseases that we work on such as AML.”

An Advocate of Near-Infrared Imaging

The Nimer Lab isn’t Dr. Hamard’s first experience with an Odyssey® Infrared Imager. He had previously used the imaging system during his time at the Icahn School of Medicine at Mount Sinai in New York City. In fact, when he came to Miami Dr. Hamard explained the benefits of an Odyssey Imaging System to his principal investigator, Dr. Nimer. In Dr. Hamard’s opinion, a key feature of the imager is its capability, along with Image Studio™ software, to provide quantitative Western blot data.

“I like the quantification feature of the software. It’s one of the arguments that I usually use when I want to convince people that it’s the way to go with Western blots. For me, that’s the best thing about the instrument.”

Eventually, Dr. Hamard succeeded in acquiring an Odyssey CLx. Not only does he now do approximately 95% of his own Western blots using the Odyssey CLx, Dr. Hamard says others in his lab have come to appreciate the imager, as well.

Reliable Multiplex Western Blotting

Another feature of the Odyssey CLx that Dr. Hamard has come to rely on is multiplexing Western blots. In his research, the capability to multiplex his blots is crucial. A multiplex Western blot allows Dr. Hamard to detect and assess modifications PRMT5 or CARM1 have made to a histone in relation to that histone’s total protein, regardless of modification.

“We do a lot of multiplex Western blots where we use one color for the modification, be it methylation/acetylation and so on, and another color for the total protein.”

Multiplexing his Western blots has saved Dr. Hamard time by allowing him to test multiple conditions at once and ensuring he’s using the proper reagents for his research. It also allows him to better characterize the suitability of antibodies for the experiments he is performing.

We thank Dr. Hamard for his contributions to science and are proud to call him an Odyssey CLx Expert.

Publications resulting from work on the Odyssey CLx

  1. PRMT5 Regulates DNA Repair by Controlling the Alternative Splicing of Histone-Modifying Enzymes. Hamard PJ, Santiago GE, Liu F, Karl DL, Martinez C, Man N, Mookhtiar AK, Duffort S, Greenblatt S, Verdun RE, Nimer SD. Cell Reports. 2018 Sep 4;24(10):2643-2657. doi: 10.1016/j.celrep.2018.08.002. PMID:30184499
  2. CARM1 Is Essential for Myeloid Leukemogenesis but Dispensable for Normal Hematopoiesis. Greenblatt SM, Man N, Hamard PJ, Asai T, Karl D, Martinez C, Bilbao D, Stathias V, McGrew-Jermacowicz A, Duffort S, Tadi M, Blumenthal E, Newman S, Vu L, Xu Y, Liu F, Schurer SC, McCabe MT, Kruger RG, Xu M, Yang FC, Tenen D, Watts J, Vega F, Nimer SD. Cancer Cell. 2018 Jun 11;33(6):1111-1127.e5. doi: 10.1016/j.ccell.2018.05.007. PMID: 29894694

Optical Surgical Navigation Clinical Trials with IRDye Infrared Dyes

Recent blog posts have highlighted some of the most exciting clinical developments of IRDye® near-infrared fluorescent dyes as surgical aides. Beyond these examples, IRDye infrared dye products are being used in more than 20 clinical trials around the globe, many of which involve the deadliest and most common cancers.

Brain and Pancreatic Cancer

Glioblastoma and glioma brain and pancreatic adenocarcinoma tumors are particularly aggressive forms of cancer that are difficult to treat. IRDye dye-conjugated optical probes are currently being investigated as an alternative to traditional surgical treatments for these cancers.

Very recently in April 2018, Deling Li and colleagues announced the results their first-in-human study a novel 68Ga-IRDye 800CW-BBN positron emission tomography (PET) and near-infrared fluorescent (NIRF) dual modality optical probe in patients with glioblastoma multiforme (GBM) [1, 2]. The authors used preoperative PET and intraoperative fluorescence-guided surgery methods, demonstrating that the “novel dual model imaging technique is feasible for integrated pre- and intra-operative targeted imaging via the same molecular receptor improved intraoperative GBM visualization and maximum safe resection” [1]. GBM is the deadliest and most aggressive glioma type, and novel GBM therapies have the potential to impact many lives.

Learn More About Dual Imaging Modalities with IRDye Optical Probes.


Figure 1. Intraoperative images of glioblastoma multiforme resection with IRDye 800CW-BBN fluorescent dyes [1].

The Dartmouth-Hitchcock Medical Center is sponsoring an investigatory study utilizing IRDye 800CW for ABY-029 in patients with recurrent glioma to determine if proper tumor/background ratio is present for surgical resection [3]. This study is expected to conclude in September 2019. A similar study is being conducted by Eben Rosenthal (Stanford University) to evaluate the effectiveness of IRDye 800CW-panitumumab in glioma surgery for distinguishing tumor cells from other central nervous system tissue [5]. Rosenthal’s study is set to conclude in 2022. Rosenthal has also studied Cetuximab-IRDye 800CW intraoperatively in patients with malignant glioma [4].

Pancreatic cancer has one of the highest mortality rates of all cancers. G.M. van Dam at the University Medical Center Groningen is currently evaluating dosing of IRDye 800CW-bevacuzimab conjugates for pancreatic adenocarcinoma [7]. Similar studies set to conclude soon by Eben Rosenthal are also evaluating the intraoperative potential of IRDye conjugates in pancreatic cancer [6].

Breast and Colorectal Cancer

Breast and colon cancers are some of the most common cancers around the globe with millions of cases diagnosed each year. Two very recent clinical trials by G.M. van Dam at University Medical Center Groningen in collaboration with Martini Hospital Groningen and UMC Utrecht have evaluated the anti-vascular endothelial growth factor antibody-IRDye 800CW-bevacizumab conjugate in breast cancer study [8, 9]. van Dam’s studies are currently assessing dosing, uptake, quantification, and localization of the optical probe, as well as determining if the conjugate is appropriate for intraoperative breast cancer surgery [8, 9].

Learn More About Optical Probe Validation and Parameters.

Colorectal cancer is also a very common diagnosis around the world. Two recent clinical trials by W.B. Nagengast of the University Medical Center Groningen evaluated IRDye 800CW-bevacizumab for colorectal cancer diagnosis [10, 11]. Nagengast noted “there is a need for better visualization of polyps during surveillance endoscopy in patients with hereditary colon cancer syndromes like Familial Adenomatous Polyposis (FAP) and Lynch Syndrome (LS), to improve adenoma detection rate,” also stating that “optical molecular imaging of adenoma associated biomarkers is a promising technique to accommodate this need” [10]. In addition to detection, IRDye 800CW-bevacizumab is also being investigated as an aid for narrowing down specific colon cancer management surgeries and therapies [11].

Other Clinical Applications

While the focus of this blog post series has been on pre-clinical and clinical applications of IRDye conjugates for cancer, these are not the only applications. Currently, IRDye fluorophores are being evaluated in several trials for clinical use in non-cancer surgeries, like abdominal and urological. Ureters, the path for urine between the kidneys and bladder, and the urethra, often present difficulties in abdominal and urological surgery. By illuminating these pathways with fluorescent dyes, the anatomy is bright and clear, which may allow surgeons to more precisely navigate around them during surgery.

A study published in 2017 by T.G. Barnes et.al. in Techniques in Coloproctology demonstrated urethra illumination in cadavers with IRDye 800BK as a method for enhancing low rectal surgical navigation [15]. The authors concluding that “IRDye 800BK is a promising alternative to ICG [indocyanine green] in visualizing the urethra,” and that “Its greater depth of penetration may allow earlier detection of the urethra during surgery and prevent wrong plane surgery sooner” [15].


Figure 2: Intraoperative images of low rectal surgery demonstrating urethral fluorescence at various depths of incision. The first row of images shows how IRDye 800BK illuminates the urethra through layers of tissue to better guide further incisions [15].

LI-COR Biosciences is currently sponsoring a clinical trial being conducted at the University of Alabama-Birmingham evaluating the dose response and safety/toxicity of IRDye 800BK for ureter delineation, which is expected to conclude soon [12]. A similar study is being conducted at the University of Oxford, sponsored by the Oxford University Hospitals NHS Trust, and is evaluating the signal/background ratio of IRDye 800BK in the ureter [13]. This trial will likely conclude later this year.


Figure 3: Intraoperative laproscopic images showing ureter delineation with IRDye 800BK. In minimally invasive surgery (such as laproscopy) ureters may be difficult to see in white light without fluorescent illumination [16].

One final application of IRDye fluorescent dyes is in endometriosis surgery. G.M. van Dam at the University Medical Groningen is investigating the feasibility of IRDye 800CW-bevacizumab for endometriosis surgery [14]. van Dam noted that “incomplete resection of endometriosis lesions often results in recurrence of symptoms and the need for repeated surgery, with considerable associated morbidity” [14]. This is the first feasibility study for IRDye 800CW and endometriosis, and is expected to conclude in early 2019.

Conclusion

This blog series on optical surgery navigation has illuminated the potential of IRDye fluorescent dyes as surgical aids. From the deadliest cancers to routine, minimally-invasive gynecological surgeries, IRDye fluorophores present a valuable opportunity for visualizing, understanding, and ultimately treating various diseases.

Could your probe be our next clinical study? For questions regarding probe development services, contact LI-COR Custom Services.

More information about the studies mentioned can be found at ClinicalTrials.gov at the locations mentioned below or on our Optical Probe Development and Molecular Activity Measurement web pages.

IRDye fluorophores are only used for investigative purposes in clinical trials.

References

  1. Li, D., Zhang, J., Chi, C., Xiao, X., Wang, J., Lang, L., & Ali, I. (2018, April 3). First-In-Human Study of PET and Optical Dual-Modality Image-Guided Surgery in Glioblastoma Using 68Ga-IRDye800CW-BBN. Theranostics, 8(9), 2508-2520. doi:10.7150/thno.25599
  2. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT02901925, A Microdose Evaluation Study in Recurrent Glioma (ABY-029); 2016 December. Available from: https://clinicaltrials.gov/ct2/show/NCT02901925.
  3. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT02910804, IRDye800CW-BBN PET-NIRF Imaging Guiding Surgery in Patients With Glioblastoma; 2015 November. Available from: https://clinicaltrials.gov/ct2/show/NCT02910804.
  4. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT02855086, Cetuximab-IRDye 800CW in Detecting Tumors in Patients With Malignant Glioma Undergoing Surgery; 2016 October. Available from: https://clinicaltrials.gov/ct2/show/NCT02855086.
  5. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT03510208, Panitumumab-IRDye800 in Diagnosing Participants With Malignant Glioma Undergoing Surgery; 2018 May 14. Available from: https://clinicaltrials.gov/ct2/show/NCT03510208.
  6. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT02736578, Cetuximab-IRDye800CW and Intraoperative Imaging in Finding Pancreatic Cancer in Patients Undergoing Surgery; 2016 July. Available from: https://clinicaltrials.gov/ct2/show/NCT02736578.
  7. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT02743975, Near-Infrared Image Guided Surgery in Pancreatic Adenocarcinoma (PENGUIN); 2016 September. Available from: https://clinicaltrials.gov/ct2/show/NCT02743975.
  8. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT02583568, Fluorescence Guided Surgery in Breast Cancer (MARGIN); 2015 October. Available from: https:/clinicaltrials.gov/ct2/show/NCT02583568.
  9. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT01508572, VEGF-Targeted Fluorescent Tracer Imaging in Breast Cancer; 2011 October. Available from: https://clinicaltrials.gov/ct2/show/NCT01508572.
  10. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT02113202, Molecular Fluorescence Endoscopy in Patients With Familial Adenomatous Polyposis, Using Bevacizumab-IRDye800CW (FLUOFAP); 2014 March. Available from: https://clinicaltrials.gov/ct2/show/NCT02113202.
  11. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT01972373, Visualization of Rectal Cancer During Endoscopy, Using a Fluorescent Tracer (RAPIDO-TRACT);2013 October. Available from: https://clinicaltrials.gov/ct2/show/NCT01972373.
  12. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT03106038, Dose-Escalation Study of a Constrant Agent for Delineation of Urological Anatomy in Minimally Invasive Surgery; 2017 May 4. Available from: https://clinicaltrials.gov/ct2/show/NCT03106038.
  13. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT03387410, Ureter Identification with IRDye 800BK; 2018 April 6. Available from: https://clinicaltrials.gov/ct2/show/NCT03387410.
  14. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT02975219, Feasibility Study of Using Molecular Fluorescence Guided Surgery in Endometriosis (Endo-Light); 2017 May 1. Available from: https://clinicaltrials.gov/ct2/show/NCT02975219.
  15. Barnes, T. G., Volpi, D., Cunningham, C., Vonjovic, B., & Hompes, R. (2018, February 19). Improved Urethral Fluorescence During Low Rectal Surgery: A New Dye and a New Method. Techniques in Coloproctology, 22, 115-119. doi:10.1007/s1051-018-1757-6
  16. Al-Taher, M., van den Bos, J., Schols, R. M., Kubat, B., Bouvy, N. D., & Stassen, L. S. (2018, February 2). Evaluation of a Novel Dye for Near-Infrared Fluorescence Delineation of the Ureters During Laparoscopy. British Journal of Surgery BJS Open. doi:10.1002/bjs5.59

Visualizing Excised Tumor Samples with IRDye Fluorescent Dyes and the Pearl Trilogy

For cancer surgery to be considered fully successful, a tumor must be completely removed with no diseased tissue left behind. Tumor margin analysis in excised tissue samples is a widely-used assessment of whether a tumor was fully removed. Traditionally, margin analysis has been based on subjective evaluations of tissue differences in white light. In many cases, however, differences in cancerous and non-cancerous tissue are very difficult to discern in white light. For some cancers like head and neck squamous cell carcinoma, “positive margin” rates – rates of cancerous tissue (likely) being left behind – are as high as 40% [1].

Novel methods are needed to reduce positive margin rates and provide better clinical outcomes. Fluorescent dyes conjugated to tumor-specific monoclonal antibodies are emerging as a visual aid for tumor margin analysis and are proving to be particularly useful in cancers where positive margins still dominate clinical outcomes.

Evaluating Tumor Margins in Excised Tissue Samples With IRDye® 800CW-Cetuximab Fluorescent Images

Previous blog posts (Optical Probe Specificity and Dual-Modality Labeling with IRDye Near-Infrared Fluorescent Dyes and The Pivotal Role of Validation in Optical Probe Development) have highlighted in vivo and in situ clinical applications of IRDye 800CW-cetuximab. In an article published Rosenthal et.al. in Clinical Cancer Research, fluorescent contrast agents were shown to also improve visualization of cancer margins in excised tissue samples.

In this first-in-human study, 12 patients scheduled to have squamous-cell carcinoma tumors removed from the head or neck were given an infusion of IRDye 800-cetuximab prior to surgery. Fresh tumor tissue sections were imaged ex vivo with the LI-COR Pearl® Impulse imager to “determine the ability of tumor fluorescence to differentiate tumor from normal tissue and identification of positive margins” [1]. Like the intraoperative in situ results, the histopathological ex vivo results were also promising. The authors noted that “Fluorescence in histologically confirmed tumor tissue was significantly greater (P<0.001) than negative epithelial margins, muscle, and skin for each dose” [1].

Rosenthal and colleagues also utilized the Odyssey® imaging platform (LI-COR Biosciences) to quantify fluorescence in slide-mounted tissue sections after imaging with Pearl Impulse imager within the surgery suite. The fluorescent images taken in the Odyssey imager were correlated with routine H&E (hematoxylin and eosin) stains to compare IRDye 800CW-cetuximab against established pathological standards, corroborating the results of the Pearl Impulse scans.

The authors concluded “Here we demonstrate for the first time that … cetuximab-IRDye 800CW can be safely administered as a tumor-specific contrast agent,” and that “The use of real-time fluorescence imaging during ablative procedures to delineate tumor margins has the potential to reduce morbidity, improve locoregional control and reduce operative time “[1].

Cetuximab-IRDye 800CW in the Clinic Part 2: Enhanced Pathological Assessment with Fluorescent Probes

In a 2016 article published in The Journal of Pathology: Clinical Research, Warram et.al. utilized IRDye 800CW-cetuximab to address the lack of “tools to consistently discriminate tumor and normal tissue in real-time” for pathological assessments of tumor margins in head and neck squamous cell carcinoma (HNSSC) [2]. In this proof-of-principle study, the authors tested fluorescent assessment of diseased tissue margins against standard histological methods. 80 tumor margin assessments were collected from post-resection wound beds of 20 mice with SCC1-luc tumors after administration of IRDye 800CW-cetuximab.

The results were significant: fluorescent images improved pathologist prediction of positive tumor margins from 21/39 (49%) to 33/39 (85%), or a 36% increase in sensitivity in positive tumor margin predictions. The authors noted false negative margin predictions lead to a 90% 5-year post resection mortality rate, demonstrating the magnitude of impact that fluorescent-guided tumor margin analysis may have on patient outcomes.


Figure 1: Fluorescent analysis of primary tumor specimen [2]. Circles represent positive or negative biopsy-confirmed cancer cells, showing the distribution and specificity of IRDye 800CW to diseased cells.
Figure 2: Demonstration of how specificity translates into margin classification in excised tissue samples [2].

The authors ultimately concluded that “The ability of fluorescence assessment to localize diseases in these margins was sensitive and specific with a NPV of 87%, which was superior to both surgical assessment (58%) and pathological assessment (66%)” [2]. The authors also noted that “This report provides evidence that tumor-specific fluorescence can be used by the surgeon or pathologist to guide sampling for frozen sections” [2]. Although the current research does not suggest that fluorescence is a bona fide replacement for current methods, “Fluorescence-guided pathology can … be easily implemented into the clinical care workflow and used in adjunct to fluorescence-guided surgery to help guide the pathologist when assessing margins for both intraoperative assessment and staging” [2].

Conclusion

In certain cancers like head and neck squamous cell carcinoma, even the most effective treatment still has relatively high rates of failure. Novel methods are needed to reduce the failure rate and provide better clinical outcomes.

Fluorescent dyes conjugated to tumor-specific monoclonal antibodies are emerging as a promising visual aid for tumor analysis. Rosenthal et.al. and Warram et.al. showed how fluorescent dye-antibody conjugates can enhance tissue assessments, also demonstrating the versatility of fluorescent probes for both in situ and in vitro assessments.

For more exciting clinical applications of IRDye probes and conjugates, visit the Optical Probe Development and Molecular Activity Measurement web pages.

Do you think IRDye infrared dye-labeled probes could be used in your research? Let us help! Contact LI-COR Custom Services.

References

  1. Rosenthal, E.L., et al. Safety and Tumor-specifity of Cetuximab-IRDye800 for Surgical Navigation in Head and Neck Cancer. Clin Cancer Res 2015, Aug; 21(16):3658-3666. doi: 10.1158/1078-0432.CCR-14-3284.
  2. Warram, J. M., de Boer, E., van Dam, G. M., Moore, L. S., Bevans, S. L., Walsh, E. M., & Young, E. S., et.al. (2016, March 2). Fluorescence Imaging to Localize Head and Neck Squamous Cell Carcinoma for Enhanced Pathological Assessment. Journal of Pathological Cancer Research, 2(2), 104-112. doi:10.1002/cjp2.40

The Pivotal Role of Validation in Optical Probe Development

Underlying every successful clinical application of fluorescent probes is a rigorous, strategic probe validation process. Previous blog posts have discussed the importance of probe specificity, binding affinity, and distribution, and the validation process is where these and other parameters are determined. Given the time and expenses involved in clinical translation, efficient and accurate probe validation is essential.

A Systematic Approach to Developing and Validating Optical Imaging Contrast Agents

In a foundational 2007 article published in Analytical Biochemistry, Kovar et.al. demonstrated the principal steps involved in developing fluorescent optical probes suitable for human clinical use. The authors began with a comprehensive review of NIR fluorochromes, such as IRDye® 800CW, and targets and ligands for fluorescent optical probes, including monoclonal antibodies, tumor surface proteins, peptides, and small molecules. Steps for development and validation described in the remainder of the article are “applicable to any dye-conjugated optical agent,” demonstrating the versatility of this systematic approach [1].

STEP 1: CONJUGATION

The first step in probe development is the conjugation of target and NIR fluorochrome. In this study, the authors conjugated IRDye 800CW to five commercial epidermal growth factor (EGF) sources in equivalent ratios and evaluated signal intensity via the In-Cell Western™ (ICW) assay method. ICW imaging demonstrated variation between the signal strength of each EGF source. Because “Variations in signal strength measured in this fashion have the potential to predict probe performance in vivo,” choosing the correct target for conjugation is a critical first step [1].

STEP 2: SPECIFICITY AND BINDING AFFINITY VALIDATION IN VITRO

Prior to animal imaging, probe specificity and binding affinity are validated in vitro. Here, the authors again chose the In-Cell Western (cytoblot) method to evaluate IRDye 800CW EGF for binding specificity. Specifically, PC3M-LN4 and 22Rv1 human prostate adenocarcinoma cells were cultured in microtiter plates and were “treated with serial dilutions of labeled EGF to verify a high affinity binding of EGFR-targeted dye” [1]. Specificity was then determined by blocking access of EGF to the EGF receptor with an anti-EGFR monoclonal antibody, and by competition with unlabeled EGF [1]. The authors concluded that “Characterization of the targeting agent in a cell-based assay can simplify probe development,” and that although “success in a cell-based assay format does not guarantee performance in vivo, failure at this step is generally predictive of failure in the animal” [1].

Learn more about the In-Cell Western Method.

STEP 3: SPECIFICITY, DISTRIBUTION, AND CLEARANCE VALIDATION IN VIVO

Next, the authors validated probe specificity and clearance in vivo in living mice. This is the process of determining probe uptake by the target vs surrounding tissues, the rate at which the probe is expelled from the organism, and the probe to background ratio in the body of mice. First, clearance kinetics of unconjugated IRDye 800CW were established. Then, clearance measurements for IRDye 800CW-anti-EGFR antibody conjugates were established in mice bearing PC3M-LN4 subcutaneous or orthotopic tumors to ensure the conjugate did not accumulate non-specifically in the mouse. This interaction between clearance kinetics and specificity can impact in vivo analysis by falsely indicating tumor tissue in pooled optical probe in the liver or kidneys.

STEP 4: EX VIVO VALIDATION IN EXCISED TISSUE SAMPLES

Lastly, the PC3M-LN4 tumors were excised and injected intravenously with IRDye 800CW EGF or pre-injected with C225 anti-EGFR monoclonal antibody prior to dosing with IRDye 800CW EGF for ex vivo analysis. After imaging, the distribution of the IRDye probe was assessed and fluorescence signal area was determined against a control, optical agent only, and C225 competition.

The authors ultimately concluded that “Fluorochrome-labeled molecular probes are valuable tools for non-invasive longitudinal study of tumorigenesis and metastasis, preclinical studies of the effects of therapeutic agents, and pharmacokinetic and pharmacodynamic studies of drug-target interactions” [1]. Since this paper was published over a decade ago, IRDye labeled molecular probes have been featured in more than 20 clinical trials around the world.

EGFR-Specific Optical Probes Improve EGFRvIII-Targeted Molecular Imaging

In a 2014 study published in Cancer Biology & Therapy, Gong et.al. demonstrated an application of structured probe validation. In this investigative study, the authors created and validated the specificity, binding affinity, distribution, and clearance of three EFGRvIII-targeted fluorescent optical probes. An EFGR-specific affibody, the therapeutic antibody panitumumab, and an EGF ligand were conjugated with IRDye 800CW to create three probes: Aff800, Pan800, and EGF800. The experimental target was rat glioma cell line F98, a known over-expresser of EGFR. A control assay contained EGFR expression-devoid F98 parent (F98-p) cells, and two experimental assays contained F98-derived transgenic cells expressing EGFR or EGFR-vIII. Each probe was compared with each cell-based assay and imaged for comparison, creating a total of nine experimental conditions.

Comparison of specificity and binding affinity between the experimental conditions was performed in cell-based assays using the In-Cell Western method. All three probes successfully bound to F98-EGFR, and Pan800 and Aff800 bound to F-98vIII. Signal intensity was also compared in the nine conditions to assess if binding was dose-dependent. The authors concluded “Little signal was detected when Aff800 and Pan800 were incubated with F98-p [the expression-devoid parent cells], indicating that their interactions with F98-EGFR and F98-vIII is highly specific” [2].

Next, probe target specificity to EGFR- and EGFRvIII-expressing tumors and clearance profiles were assessed in vivo. Mice with F98-p, F98-EGFR, and F98-vIII xenograft tumors were injected with the three probes and imaged with the Pearl® Impulse Small Animal Imaging System (LI-COR Biosciences). Fluorescent signal to background ratio for each of the nine probe-tumor conditions were assessed, again revealing highly specific interactions between Aff800 and Pan800 with F98-EGFR and F98-vIII expressing tumors. EGF-800 signal was high in F98-EGFR tumors, corroborating cell based assay results.

Lastly, tumor-containing organs were dissected and imaged ex vivo, validating the previously-measured fluorescence signals and assessing probe distribution in targets. This last step in validation was consistent with in vitro scans, again demonstrating Aff800 and Pan800 affinity to F98-EGFR and F98-vIII tumors. Based on these results, Aff800 and Pan800 may be valuable in “imaging of heterogenous tumors containing both versions of receptors” (EGFR, EGFRvIII) [2]. Alternatively, due to optimal clearance kinetics, Aff800 EGF800 is preferable in scenarios where imaging must be performed within a short time after probe administration” [2].

Conclusion

This example from Gong et.al. demonstrates how different optical probes may be used to assess different tumor properties. Additionally, the authors showed how structured approach to optical probe validation successively builds proof of probe parameters and provides several spots for go/no-go decision-making. Proof of probe parameters are critical for clinical application, and clear decision points provide efficiency and allow for early determination if a probe is worth exploring further.

Do you think IRDye dye-labeled probes could be used in your research? LI-COR Custom Services include chemistry and probe conjugation, biological assay services, translational services, and manufacturing, including cGMP manufacturing. Request a free project evaluation today.

REFERENCES

  1. Kovar, J. L., Simpson, M. A., Geschwender, A., & Olive, D. M. (2007, August 1). A Systematic Approach to the Development of Fluorescent Contrast Agents for Optical Imaging of Mouse Cancer Models. Analytical Biochemistry, 367(1), 1-12. doi:10.1016/j.ab.2007.04.011
  2. Gong, H., Kovar, J. L., Cheung, L., Rosenthal, E. L., & Olive, D. M. (2014, February). A Comparative Study of Affibody, Panitumumab, and EGF for Near-Infrared Fluorescence Imaging of EGFR- and EGFRvIII-expressing Tumors. Cancer Biology & Therapy, 15(2), 185-193. doi:10.4161/cbt.26719

Optical Probe Specificity and Dual-Modality Labeling with IRDye® Near-Infrared Fluorescent Dyes

Complete tumor resection is a critical aspect of cancer treatment. For many cancers, though, complete resection is very difficult to achieve. One major hinderance is the inability to distinguish diseased from non-diseased tissue. Surgery guided by near-infrared fluorescent optical probes is emerging as a promising advancement in surgical methodology, enabling surgeons to better visualize diseased tissue, resect tumors completely, and ultimately improve patient outcomes.

For a probe to be an effective surgical aide, it must be highly specific to the target. In other words, the probe must bind only to diseased tissue and should bind to it with a high affinity. This creates a highly-contrasted tumor-to-background ratio, which enhances both intraoperative in situ tumor visualization and ex vivo histopathological evaluations of tissue sections.


Figure 1: Hip tumor imaged with IRDye 800CW EGF dye and Pearl® Impulse showing target specificity.

Figure 2: Tissue section imaged with the Odyssey® CLx demonstrating the sharp contrast of the IRDye probe.

Mode of image capture is another important consideration for near-infrared (NIR) fluorescent optical probes as surgical aides. Several imaging modalities exist, and each serves a unique purpose. Positron Emission Tomography (PET) and Single-Photon Emission Computed Tomography (SPECT) allow for imaging of deep tissues and organs, bones, and inside the ribcage, but cannot be used intraoperatively. NIR fluorescence, on the other hand, is most effective for shallow tissue and organ imaging, and can be used real time during surgery to guide tumor resection. IRDye fluorescent dyes can be conjugated to compounds used in various modalities, and combining modalities can be very powerful for probe validation.

Dual Modality Tagging with IRDye 800CW-NHS Stained Monoclonal Antibodies and Radioactive Tracers Provide Greater Insight into Tumor Characteristics

A 2011 study published in the Journal of Nuclear Medicine by Terwisscha van Scheltinga et.al. demonstrates the power of combining imaging modalities with IRDye conjugates. This pre-clinical mouse study investigated the potential ability of targeted monoclonal antibodies to deliver fluorophores to specific tumors for surgical resection.

The antibodies in question were bevacizumab and trastuzumab. Bevacizumab is an anti-vascular endothelial growth factor (VEGF) monoclonal antibody and trastuzumab is an anti-human epidermal growth factor (HER) 2 monoclonal antibody. Each was stained with IRDye 800CW NHS ester for NIR fluorescent imaging, and were then combined with 89Zr-labeled radioactive tracers for PET imaging. Tumor uptake of the IRDye dye-conjugated antibodies was compared against uptake in the 89Zr-labeled counterparts in mice with VEGF- and HER2-overexpressing tumors. Tumor-background ratio (TBR) was assessed for specificity, and the results were promising:

“The excellent selective tumor uptake” of the 89Zr-labeled IRDye tracers imaged with PET “was also observed for the same antibodies labeled with a fluorescent dye” [1].

With the specificity of the dye-conjugated antibodies validated in tissue analysis, the authors performed intraoperative imaging of the same VEGF- or HER2-expressing tumor lesions, concluding that “In a preclinical setting, NIR fluorescence-labeled antibodies targeting VEGF or HER2 allowed highly specific and sensitive detection of tumor lesions in vivo” [1]. Lastly, this study demonstrates how combining PET and NIR fluorescence imaging may be used to validate the specificity of a dye-antibody conjugate ex vivo prior to intraoperative imaging.

Cetuximab-IRDye 800CW Conjugated Probes Target EGFR for Intraoperative Surgical Navigation of Head and Neck Squamous Cell Carcinoma

A 2015 study published in Clinical Cancer Research by Rosenthal, et.al. demonstrates the clinical potential of IRDye 800CW as a tumor-specific contrast agent in cancer surgery. Twelve patients participated in a dose-escalation study of cetuximab, an anti-EGFR monoclonal antibody, conjugated to IRDye 800CW. Over 90% of head and neck squamous cell carcinoma tumors overexpress EGFR, which presents an opportunity for EGFR-targeted antibodies as a vehicle for IRDye fluorophores [2]. Wide-field NIR imaging was used intraoperatively and multiple tissue sections were collected and imaged in the Pearl Impulse imaging platform (LI-COR Biosciences). These results were also promising:

  • Fluorescence imaging of the primary tumor in situ demonstrated high average tumor to background ratio. The authors noted that “fluorescence imaging provided robust contrast between tumor and surrounding tissue” [2].
  • Fluorescence in confirmed tumor tissue imaged ex vivo was significantly greater (P<0.001) than non-cancerous tissues, validating the preferential uptake of IRDye 800CW conjugated monoclonal antibodies in diseased tissues [2].
  • No Grade 2 or higher treatment emergent adverse effects occurred, and IRDye 800CW-cetuximab was reported to be “well tolerated” by participants, corroborating the positive results of IRDye 800CW pre-clinical toxicity studies [2].

The authors conclude “this optical labeling technique could be safely applied to other protein-based therapeutics to confirm successful targeting or assess off-target activity during early phase trials” [2].

Conclusion

Near infrared fluorescent optical imaging with dye-conjugated tumor-specific antibodies is quickly emerging as a viable intraoperative tool for cancer surgery. For an optical probe to be successful, it must be highly specific to the target tumor and bind strongly to create high tumor to background ratio. In recent pre-clinical studies, IRDye 800CW NHS ester has shown equal or better tumor to background ratio than established PET methods with nuclear-tagged probes. In similar clinical studies, IRDye 800CW has demonstrated high tumor to background ratio in humans intraoperatively for tumor excision.

For more exciting clinical applications of IRDye, please visit the Optical Probe Development and Molecular Activity Measurement web pages.

Do you think IRDye infrared dye-labeled probes could be used in your research? Let us help conjugate your optical probe! If interested, please contact LI-COR Custom Services.

References

  1. Terwisscha van Scheltinga, A.G.T., et al. Intraoperative Near-Infrared Fluorescence Tumor Imaging with Vascular Endothelial Growth Factor and Human Epidermal Growth Factor Receptor 2 Targeting Antibodies J Nucl Med 2011; 52:1778–1785. doi: 10.2967/jnumed.111.092833.
  2. Rosenthal, E.L., et al. Safety and Tumor-specifity of Cetuximab-IRDye800 for Surgical Navigation in Head and Neck Cancer. Clin Cancer Res 2015, Aug; 21(16):3658-3666. doi: 10.1158/1078-0432.CCR-14-3284.
  3. Ahn BC. Sodium Iodide Symporter for Nuclear Molecular Imaging and Gene Therapy: From Bedside to Bench and Back. Theranostics 2012; 2(4):392-402. doi:10.7150/thno.3722.

Use of IRDye Infrared Dye-Labeled Optical Probes for Intraoperative Tumor Visualization

A major challenge in cancer surgery is being certain that all the tumor has been removed, including the residual cancer cells not immediately identified with the naked eye after resection. Surgeons need intraoperative methods of imaging tumors to assist them in identifying healthy and diseased tissue. These methods need to be safe and effective. Near-infrared (NIR) fluorescent optical probes may provide a viable solution.

Near-infrared fluorescent optical probes have been used intraoperatively in clinical trials. These NIR dye-conjugated compounds offer several advantages for use in the operating room. NIR probes can be used safely, unlike other imaging modalities that require radiation (such as CT, PET, and SPECT).

IRDye® dye-conjugated optical probes have been shown to be sensitive and biomarker-specific and their fluorescent signal correlates with tumor location observed by other imaging methods and traditional pathology. Because fluorescence from NIR optical probes is invisible to the human eye, visualization of the surgical field of view with white light is unimpeded.

Fluorescence from tissue excised during surgery can be visualized while in the operating room and used to assess whether resection of the tumor is complete. Traditional pathologic examination can then be done for confirmation. Specialized NIR imaging equipment, such as the Pearl® Imaging System, has been used successfully to image tumor sections during an operation.
The following two studies involved the intraoperative use of near-infrared fluorescence optical probes.

IRDye 800CW Dye-Conjugated Probes Provide Verification of Tumor

In this study by van Driel, et al., investigators evaluated the Artemis imaging system, developed in collaboration with the Center for Translational Molecular Medicine. The goal of the study “was to evaluate the Artemis camera in two oncological procedures in which real-time NIR fluorescence could be of added value: (a) radical tumor resection; and (b) detection of sentinel lymph nodes. . .” [1]
For the evaluation of the Artemis imaging system, the investigators used ICG and two IRDye® 800CW infrared dye-conjugated nanobodies. “IRDye 800CW (LI-COR, Lincoln, NE, USA, λex=774 nm, λem=789 nm) was chosen because it is one of two novel fluorophores in the process of clinical translation.” [1] The study assessed the sensitivity and utility of the Artemis system for intraoperative detection of head-and-neck tumors and sentinel lymph nodes in xenograft mouse models. [1]

Fluorescent images were concurrently acquired with the Pearl® Impulse Small Animal Imager (LI-COR). [1] “The Pearl system is expected to be an order of magnitude more sensitive than the Artemis, and therefore, these images serve as a ground truth comparison.” [1]

IRDye 800CW Dye-Labeled Probes Target VEGF and HER2

Research performed by Terwisscha van Scheltinga, et al. used IRDye 800CW dye-labeled antibodies to investigate their use in targeting certain tumors for optical surgical navigation [2]. The group concluded that “NIR fluorescence-labeled antibodies targeting VEGF or HER2 can be used for highly specific and sensitive detection of tumor lesions in vivo. These preclinical findings encourage future clinical studies with NIR fluorescence–labeled tumor-specific antibodies for intraoperative-guided surgery in cancer patients.” [2]

In this preclinical mouse study, fluorescent optical imaging with IRDye 800CW NHS ester coupled to bevacizumab was compared to PET imaging with 89Zr (5 MBq)-labeled bevacizumab or trastuzimab along with a non-specific antibody control, 111In-IgG (1 MBq). [2]

The researchers of this study stated, “IRDye 800CW is a NIR fluorophore with optimal characteristics for clinical use, allowing binding to antibodies when used in its N-hydroxy-succinimide (NHS) ester form. A preclinical toxicity study with IRDye 800CW carboxylate showed no toxicity in doses of up to 20 mg/kg intravenously or intradermally.” [2] They concluded that “In a preclinical setting, NIR fluorescence–labeled antibodies targeting VEGF or HER2 allowed highly specific and sensitive detection of tumor lesions in vivo.” [2]

IRDye 800CW dye-conjugated optical probes are currently involved in over a dozen clinical trials for a wide range of different cancers. These studies demonstrate the use of IRDye probes for optical surgical navigation. Several studies have employed the use of dual-labeled probes showing the strength of combining near-infrared fluorescence with other imaging modalities.

Examples of optical probe applications are detailed on Optical Probe Development and Molecular Activity Measurement web pages.

Do you have questions about how IRDye infrared dye-labeled probes could be used in your research or need help conjugating your optical probe? If so, please contact LI-COR Custom Services.

References:

  1. van Driel, P.B.A.A., et al. Characterization and Evaluation of the Artemis Camera for Fluorescence-Guided Cancer Surgery Mol Imaging Biol (2015) 17:413Y423. doi: 10.1007/s11307-014-0799-z
  2. Terwisscha van Scheltinga, A.G.T., et al. Intraoperative Near-Infrared Fluorescence Tumor Imaging with Vascular Endothelial Growth Factor and Human Epidermal Growth Factor Receptor 2 Targeting Antibodies J Nucl Med 2011; 52:1778–1785. doi: 10.2967/jnumed.111.092833.

What Type of Western Blotter are You?

How would you describe your Western blotting abilities and results?

Do you know how to get yourself out of pretty much any Western blotting jam? Do your blots always look perfect like this?

Now, THAT’S a nice blot. And, it looks like you are a star at multiplexing with near-infrared fluorescence.

Just be sure you are detecting in the combined linear range for your target and your internal loading control. (Not sure what a combined linear range is? Check out this protocol.)

Or, do you wish SOMEONE would just tell you what you are doing wrong because your blots look more like this?

You are PRETTY sure that a Western blot is not supposed to look like a splat of paint or a Rorschach test (although. . . .).

Wherever you are at in your Western blot experience journey, find out your Western blotting type by taking this quiz.

Let’s see if you are really a Western Blotting All-Star or a Western Blotting Newcomer in need of some friendly (and helpful) expert advice!

Create a Customized List of Journal Articles that Reference LI-COR Imaging Systems

Finding out how other researchers have used LI-COR® imaging systems and reagents can really help when you are trying to decide on which system is best for your lab. With over 10,000 journal citations, the Odyssey® Imaging Systems have a long, proven track record in life science research.

There is now a tool that you can use to customize a list of peer-reviewed references specific to your research and application interests. You can access this new Publications Database through pages on our website, such as Products > Imaging System pages (for example, Odyssey CLx > Who’s Using it) and Application pages (for example, Quantitative Western Blots > Publications) or through the link in the footer, which is on the bottom of all web pages.

You can filter results by four categories. Select at least one filter. Each filter you select narrows the search by making the resulting set a combination of all filter parameters.

Let’s go over the various options you can use to create your customized publications list.

You can filter by Research Area. The list of research areas includes 72 different categories. Choose a single, or multiple, area(s). Remember, the more you choose, the narrower your search will be.


You can filter by Instrument.


You can filter by Application.

You can filter by Country. This is the country of the corresponding author’s email address.

For example, if you select “Apoptosis” in Research Area, “Odyssey CLx” in Instrument, and “Germany” in Country, you will receive a list of three publications that specifically reference apoptosis, the Odyssey CLx, and publications where the corresponding author is from Germany.

You can sort the columns in the results by clicking on the corresponding header. You can also show 10, 25, 50, or all entries. If the list does not suit your needs, you can Clear All Filters and start over.

If the number of publications returned is large, you can refine the set using keywords, such as your protein or disease of interest, separated by a space. Results displayed will contain all the terms in the search field. You can also use the digital object identifier (DOI) prefix unique to each publishing group to search for publications in specific journals. For example, use 10.1074 for the Journal of Biological Chemistry and other ASBMB journals.

We regularly add publications to the database, so check back frequently to see who is using LI-COR imaging systems to get published, and how they are using the instrument in their research.

Create your own customized publication list and, if you have feedback on how to improve this tool, please click on the Feedback button and let us know!

Technical and Biological Replicates are Critical for Quantitative Western Blot Success

Replicates improve the reproducibility and accuracy of experimental findings. They are important because they confirm the validity of observed changes in protein levels. Without replication, it is impossible to know if an effect is real or simply an artifact of experimental noise or variation, which can directly affect conclusions made about experimental findings.

There are two types of replicates: biological and technical. Each type addresses different questions1,2,3. Peer-reviewed journals, such as the Journal of Biological Chemistry, have specific guidelines in regards to replicates.

“Authors must state the number of independent samples (biological replications) and the number of replicate samples (technical replicates) and report how many times each experiment was repeated.”
Instructions for Authors. The Journal of Biological Chemistry

Technical vs. Biological Replicates: Which Do You Need to Include?

Technical Replicates

Technical replicates are repeated measurements used to establish the variability of a protocol or assay, and determine if an experimental effect is large enough to be reliably distinguished from the assay noise1. Examples may include loading multiple lanes with each sample on the same blot, running multiple blots in parallel, or repeating the blot with the same samples on different days.

Figure 1. Technical replicates help identify variation in technique. For example, lysate derived from a mouse and treated under a set of experimental conditions (A, B, C), then run and measured independently three times, will help identify variation in technique.

Technical replicates evaluate the precision and reproducibility of an assay, to determine if the observed effect can be reliably measured. When technical replicates are highly variable, it is more difficult to separate the observed effect from the assay variation. You may need to identify and reduce sources of error in your protocol to increase the precision of your assay.

Technical replicates do not address the biological relevance of the results.

Biological Replicates

Biological replicates are parallel measurements of biologically distinct and independently generated samples, used to control for biological variation and determine if the experimental effect is biologically relevant. The effect should be reproducibly observed in independent biological samples. Demonstration of a similar effect in another biological context or system can provide further confirmation. Examples include analysis of samples from multiple mice rather than a single mouse, or from multiple batches of independently cultured and treated cells.

Figure 2. Biological replicates derived from independent samples help capture random biological variation. For example, lysates derived from 3 mice and treated under the same set of experimental conditions (A, B, C), will help identify variation resulting from the biology.

To demonstrate the same effect in a different experimental context, the experiment might be repeated in multiple cell lines, in related cell types or tissues, or with other biological systems.

An appropriate replication strategy should be developed for each experimental context. Several recent papers discuss considerations for choosing technical and biological replicates1,2,3.

This protocol, Quantitative Western Blot Analysis with Replicates, will guide you in choosing and incorporating technical and biological replicates in your experimental design for reproducible data. It includes calculations for replicate analysis as well as how to interpret the data you obtain.

Additional Resources to Help You Get the Best Data

LI-COR has additional resources that you can use as you plan your quantitative Western blot strategy.

References:

  1. Naegle K, Gough NR, Yaffe MB. Criteria for biological reproducibility: what does “n” mean? Sci Signal. 8 (371): fs7 (2015).
  2. Blainey P, Krzywinski M, Altman N. Replication: quality is often more important than quantity. Nat Meth. 11(9): 879-80 (2014).
  3. Vaux DL, Fidler F, Cumming G. Replicates and repeats – what is the difference and is it significant? EMBO reports 13(4): 291-96 (2012).