In the field of biomedical research, multicolor fluorescence immunohistochemistry (mIHC) and multicolor flow cytometry (FCM) are two important technologies that play a key role in the detection and analysis of biological samples. Although they both involve fluorescent labeling and multicolor analysis, there are clear differences in their principles, sample selection, applications, and benefits. This article will break down the differences between these two technologies for you in detail.
Principles and features
Multicolor fluorescence immunohistochemistry (mIHC), also known as tyrosine signal amplification (TSA) technology, is a multiplex sequential immunostaining technique based on tyramide signal amplification. This technology utilizes horseradish peroxidase (HRP) for high-density in situ labeling of target proteins or nucleic acids, allowing simultaneous detection of multiple targets of interest on a single cell or tissue sample. The strength of mIHC technology lies in its dual ability to display in situ and quantify, enabling comprehensive studies of cell composition, function, and cell-cell interactions.
Multicolor flow cytometry, also known as flow cytometry (FCM), is a new high-tech cell analysis technology integrating laser technology, photoelectric measurement technology, computer technology, fluid mechanics, cell immunofluorescence chemistry technology, and monoclonal antibody technology. FCM uses a laser light source to excite the intensity and color of the fluorescent substances labeled on the cells and the intensity of the scattered light, and performs multi-parameter analysis of particles, including particle shape and size, cell cycle, intracellular cytokines, bacterial surface antigens, cellular DNA inclusions, etc.
Sample requirements
Multicolor fluorescence immunohistochemistry (mIHC) sample requirements:
1. Sample type: mIHC typically uses formalin-fixed wax blocks or slides, including large tissue blocks or tissue chips (TMA).
2. Tissue fixation: The tissue should be fixed with 10% neutral formalin/paraformaldehyde, and the normal fixation time is 18-24h.
3. Slice thickness: The slice thickness is about 4 microns, and anti-detachment slides are used.
4. Sample preservation: try to select relatively new samples (within one month), and the longest should not exceed half a year, so as not to cause poor staining effect due to target deletion or strong non-specificity.
5. Tissue treatment: After taking fresh tissue, it is quickly transferred to the fixative solution, and the temperature of wax immersion after fixation should be controlled at about 58~60 °C.
6. Slide requirements: The tissue needs to be close to the slide to avoid wrinkles, and the slide must not be damaged, scratched or stained.
Multicolor Flow Cytometry (FCM) Sample Requirements:
1. Sample type:FCM requires a single-cell suspension. Peripheral blood or suspension-grown cells can be prepared directly into a single-cell suspension, whereas adherent cells, solid tissues, or tumor tissues need to be prepared as a single-cell suspension first.
2. Cell viability:Samples for FCM testing need to be live cells, especially those that have been transported and stored for a long time, and dead cells are non-specifically stained for many antibodies, so cell viability testing is important.
3. Sample preservation: Ideally, samples should be processed and stained immediately after collection. Different types of anticoagulants can be stored for different periods of time, e.g., heparin-anticoagulated blood and bone marrow can be stored until 48 to 72 hours/room temperature.
4. Red blood cell depletion: red blood cells may need to be removed before FCM analysis, and commonly used methods include red blood cell lysis and density gradient centrifugation.
5. Cell-to-antibody ratio: The cell-to-antibody ratio needs to be adjusted according to different samples to obtain the most appropriate cell/antibody ratio.
Applications and benefits
mIHC technology has a wide range of applications in tumor microenvironment, tumor immune infiltration, cell senescence/apoptosis/ferroptosis/autophagy and other fields. It can obtain information on the types, components, and expression levels of various targets in situ in tissue cells, as well as the spatial localization, qualitative and quantitative information of each target and its interaction through quantitative pathological analysis.
1. Tumor microenvironment research: mIHC technology can simultaneously obtain tumor markers, cell status, immune cell typing, immune regulation, mesenchymal cells and other information of tissues, and analyze the overall picture of tumor immune characteristics. For example, in tumor immune infiltration and chemokine receptor expression studies, mIHC technology can demonstrate in situ interactions between different cells, which is particularly important for understanding the tumor microenvironment and evaluating drug efficacy.
2. Efficacy evaluation of tumor immunotherapy: mIHC has shown higher accuracy in evaluating anti-PD-1/PD-L1 therapy response. Compared with single-index IHC, TMB, and GEP, the mIHC method is more accurate in predicting the response to anti-PD-1/PD-L1 therapy and provides a higher area under the curve (AUC).
3. Reduce tissue sample loss: mIHC technology can label multiple markers in a single section to reduce sample loss, especially suitable for small tissue samples such as clinical rare samples or punctures.
FCM technology has a wide range of applications in many fields such as cell biology, molecular biology, immunology, hematology, oncology, and genetics. It can detect cell size, cell granularity, DNA and RNA content, protein content, cell-specific antigens, cell viability, intracellular PH value, cell cycle, apoptosis, cell function analysis, etc.
Applications of Multicolor Flow Cytometry (FCM):
1. Tumor cell parameter detectionFCM can detect the proliferative activity marker molecules and differentiation/apoptosis marker molecules of tumor cells, which can be used to study the pathogenesis of tumors, formulate personalized treatment plans, and make prognostic judgments. For example, measuring the DNA content of cells by FCM can be an important marker of cancerous or precancerous changes with malignant potential.
2. Minimal residual lesion (MRD) monitoringFCM has high sensitivity and specificity in the monitoring of MRD of hematologic malignancies, and has become an important basis for clinical efficacy observation and prognostic stratification.
3. Assessment of tumor immune status:FCM accurately assesses the immune status of cancer patients through TBNK analysis, which is helpful for early diagnosis of malignant tumors, and then provides guidance and basis for subsequent treatment of patients.
summary
Overall, mIHC and FCM play different roles in biomedical research. mIHC technology focuses on multi-target analysis at the tissue level, emphasizing in situ presentation and quantification, and is suitable for studying cell composition, function, and interactions. On the other hand, FCM technology focuses on the multiparametric analysis of single cells, which is suitable for research in fields such as cell biology and immunology. Understanding the differences between these two technologies can help researchers choose the right technology platform for their research needs.
